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119291197	pes2o/s2orc	v3-fos-license	The random walk of a low-Reynolds-number swimmer Swimming at a micrometer scale demands particular strategies. Indeed when inertia is negligible as compared to viscous forces (i.e. Reynolds number $Re$ is lower than unity), hydrodynamics equations are reversible in time. To achieve propulsion at low Reynolds number, swimmers must then deform in a way that is not invariant under time reversal. Here, we investigate dispersal properties of self propelled organisms by means of microscopy and cell tracking. Our system of interest is the micro-alga \textit{Chlamydomonas Reinhardtii}, a motile single celled green alga about 10 micrometers in diameter that swims with to two front flagella. In the case of dilute suspensions, we show that tracked trajectories are well modeled by a correlated random walk. This process is based on short time correlations in the direction of movement called persistence. At longer times, correlations are lost and a standard random walk characterizes the trajectories. Moreover, high speed imaging enables us to show how the back-and-forth motion of flagella at very short times affects the statistical description of the dynamics. Finally we show how drag forces modify the characteristics of this particular random walk. Cell motility [1] is crucial to many biological processes including reproduction, embryogenesis, infection, etc. Many microorganisms are able to propel themselves, bacteria, sperm cells, microalgae, etc. A quantitative understanding of the hydrodynamics of flagella and cilia is thus of great interest [2,3]. One of the peculiarities of the swimming of microorganisms is that it occurs at very low Reynolds numbers which is very different from our usual experience of swimming at our meter length scale [4,5]. Indeed when inertia is negligible as compared to viscous forces (i.e. Reynolds number Re is lower than unity), in order to achieve propulsion, swimmers must deform in a way that is not invariant under time reversal. This is known as Purcell's scallop theorem [4]. In living systems, several different strategies are used to achieve propulsion in such conditions: the E. coli bacterium uses a rotating flagellum at the "back" of its body, sperm cell propulsion relies on the asymmetry of their flagellar bending waves, the power and recovery strokes of the two front flagella of Chlamydomonas Reinhardtii (CR) algae are asymmetrical. Flagellar propulsion in CR induces complex swimming behavior of cells. Over short time scales, the cells undergo an oscillating movement with changes in velocity direction occurring at the same frequency as the beating frequency of flagella. On a time scale longer than the period of beating, average swimming behavior is directional. Eventually, on larger time scales, direction is lost and swimming trajectories resemble a random walk. CR is a 10µm motile bi-flagellated unicellular alga. The cell is spheroidal in shape with two anterior flagella [6]. It belongs to the puller type of swimmers as it uses its front flagella to propel itself, producing what a breaststroke-like movement. The swimming direction of the cells can be controlled by stimulus gradients, a phenomenon known as taxis, such as chemotaxis, rheotaxis or phototaxis. Gradients are not used in our experiments in order to avoid any external tropism on the motility. .k(t0 + t)] for different times t ranging from 0.05tc to 3.33tc. Here tc=2.3s and the viscosity of the medium is 2.4mPa s. Only few data points are displayed for the sake of clarity. (b) Correlation functions of direction C(t/tc) as defined in the text (Log-Lin scale). Time has been rescaled by the decaying time of an exponential. The different symbols correspond to the viscosities used by varying the concentration of dextran. Wild-type strains were obtained from the IBPC lab in Paris [7]. Synchronous cultures of CR were grown in a Tris-Acetate Phosphate medium (TAP) using a 12/12 hour light/dark cycle at 22 o C. Cultures were typically grown for two days under fluorescent lighting before the cells were harvested for experiments. We studied the swimming dynamics of this microor-ganism by means of bright field microscopy imaging on an Olympus inverted microscope coupled either to a CCD camera (Sensicam, Photon Lines) used at a frame rate of 10Hz or to a high speed CCD camera (Miro, Phantom) used at a frame rate of 400Hz. Long time experiments used a ×10 magnification lens whereas we used a ×64 lens for high speed imaging. Two hundred micrometer thick glass chambers were coated with bovine serum albumine to prevent cell adhesion. The imaged cells were located 30 to 60 micrometers from the glass walls. A red light filter was used in order to prevent phototaxis. Cell tracking [8] was performed using IDL (Interactive Data Language) with a submicron precision in the detection of hundreds of cells (high speed experiments) and thousands of cells for long time sequences. To quantify the effect of drag on the cell dynamics, small amounts of short chain dextran (Sigma Aldrich) were added to the culture medium. The chains were short enough for non-Newtonian effects to be absent and long enough to avoid damaging the cells with osmotic effects. This allowed the viscosity η of the medium to be varied between 1.5 and 3.7mPa s. The range of viscosity is restricted to this interval to ensure the viability of the cells. Let us first recall the global dynamics of swimming, i.e. over time scales of the order of a few seconds. Cell trajectories are found to be correctly modelled by a persistent random walk [9][10][11]. Cells swim in an almost fixed direction for a typical time of about one second. This stage corresponds to a ballistic regime characterized by a mean velocity V . The ballistic regime ends when the swimmers make a turn. A new direction of motion is then observed due to the desynchronization of the pair of flagella [12]. At long time scales, the dispersal properties of the swimmers are random-like [13,14]. To describe this specific random walk quantitatively, we measured different statistical quantities of interest. Let us first define a persistence angle θ(t) = arccos[k(t 0 ).k(t 0 + t)], wherê k(t) is a unitary vector in the direction of movement at time t. Thus, a value of θ close to zero reflects a certain persistence of the trajectory. We measured the probability distribution function of angles θ for different times t. At short time scales, angle distribution peaks at around zero, characterizing the directional persistence in swimming trajectories. Over longer times, we observed a broadening of the distribution and eventually we ended up with an equidistribution of angle values characteristic of a random walk ( figure 1(a). This phenomenon is even better quantified by looking at the mean value of angle distribution or equivalently at the correlation function of direction defined as C(t) =<k(t 0 ).k(t 0 + t) >, where <> is an average over time t 0 and over all tracked trajectories. Correlations with infinite decay time (C(t) = 1 for all t > 0) correspond to direction correlations preserved over arbitrarily long times, i.e. a purely ballistic regime; whereas a zero life-time (C(t) = 0 for all t > 0) corresponds to standard random walk behav- ior ( figure 1(b). The correlation functions decay exponentially over a characteristic time t c . This correlation time t c is related to the mean time of persistence over which the direction of swimming is preserved. The different symbols in figure 1.b correspond to experiments where the concentration of short chain dextran was varied, hence modifying the viscosity of the medium from 1.5mPa s to 3.7mPa s. As viscosity increases, correlation time t c increases (data not shown) from 1.5 to 3.9 seconds. The global dynamics of swimming of CR can thus be described as a correlated random walk characterized by a ballistic regime (with a mean velocity V ) and a decorrelation process (over a characteristic time t c ) due to the turns made by the cells. As a consequence, a persistence length L is naturally defined as the product V t c . From a statistical point of view, such a bahaviour is described by the mean square displacement of cells < r 2 (t) > which is linear for long times (t t c ) and quadratic at shorter times (t t c ) [13,14]. At even shorter times, the dynamics reflect the consequences of low Reynolds swimming, i.e. a non-reciprocal movement of flagella. This then leads to a zigzagging motion of cells due to the backand-forth motion of flagella [15]. In the present case, cells of diameter 2R ∼ 10µm are moving at a velocity V around 50µm/s in a wa- ter like medium (viscosity η ∼ 1mPa s and density ρ ∼ 10 3 kg/m 3 ). This represents a very low Reynolds number of the order of Re = ρV R/η ∼ 2.5 × 10 −4 . The propulsion strategy of CR consists in swimming in a kind of breaststroke where the pair of flagella are wide open during the forward movement and folded along the cell body during the backward movement [16]. Hence, viscous friction is high when the pair of flagella are fully extended during the forward movement and friction is lower during the backward movement. The symmetry under time reversal is thus broken and propulsion is ensured. However, because inertia has no role in this regime, this kind of propulsion leads to a back-and-forth movement of the cell in which the velocity is alternatively positive and negative. High speed imaging (400Hz) allows us to resolve the very short time dynamics due to flagella beating and thus to study the consequences of this back-andforth movement on the properties of the swimmers' random walk. The insets in figure 2 show typical cell trajectories imaged at 400Hz: the back-and-forth movement of swimmers due to the absence of inertia (Re << 1) together with the long time swimming behavior are visible. In these examples, the cells are swimming either in a nutritive medium of viscosity η = 1mPa s (top left inset) or in a dextran-rich medium of viscosity 3.7mPa s (bottom right inset). Cells have a net forward movement corresponding to the power stroke, followed by the recovery stroke that propels the cell backward. As the distance traveled forward is longer than the backward movement, the cells ultimately progress forward. However, these fluctuations in the direction of the velocity have consequences on the measured statistical quantities [17] that we will discuss now. The measured mean square displacement < r 2 (t) > shows a plateau region at very short time (t t c ) that reflects the transition between two quadratic regimes : on the one hand, a fast ballistic regime characterized by the instantaneous velocity u of swimmers and, on the other hand, a slower ballistic regime corresponding to the mean velocity V of swimming which is the resulting forward velocity over several back-and-forth movements. The position of the plateau therefore corresponds to the beating frequency f of the swimmer, which depends on the viscosity of the surrounding medium. To quantify the back-and-forth swimming motion of the cells, we measured the angle probability distribution function. Figure 3.a shows distribution functions for different times. For a given short time t, the distribution of angles θ(t) as defined earlier, peaks at around zero, reflecting a given direction at very short time. For longer time scales (close to 1/2f), anti-correlation in cell direction resulted in new distribution peaks at values of around ±π. When a new stroke is produced, the measured angle is again close to zero giving rise to a peak around zero. Hence, angle distributions have a periodicity which reflects the beating frequency. This is shown in figure 3.a as the distributions are very similar at times shifted by 1/(2f ), where the typical frequency of the beating f is deduced from the periodical nature of the correlation function of direction. Figure 3.b shows such correlation functions at varying f × t, the product of time multiplied by the fitted frequency of the signal. Data are well described by an exponentially attenuated cosine function. The different symbols correspond to different viscosities of the medium. The exponential decay of the correlation function should reflect the turns in direction the cells eventually perform within a characteristic time t c . However, due to the 3D nature of the trajectories and the 2D geometry of our setup, correlation was attenuated faster than that. The other consequence of the fact that swimming is produced at low Reynolds number is that propulsion requires non-zero drag forces. Viscous friction is thus crucial in the dynamics of microswimmers. By varying the viscosity of the medium, we were able to draw some conclusions about the effects of friction forces on the locomotion of microorganisms such as this micro-alga. Here, short time dynamics of swimming can be fully described by few mean quantities: flagella frequency beating f (deduced from a cosine fit in figure 3.b) and the mean modulus of instantaneous velocity u, which is the velocity achieved during a power or a recovery stroke. We studied the effects of viscous forces on these quantities. Velocities and beating frequency are found to be inversely proportional to the viscosity of the bath (figure 4). As viscosity increases, the beating frequency decreases, varying from 30Hz to 13 Hz with a viscosity variation from 1.5 to 3.7mPa s (figure 4(a) giving a slope ηf = 0.045 ± 0.01Pa. Accordingly, velocity decreases from 135 to 75µm/s ( figure 4(b) giving a slope ηu = 0.15±0.04Pa µm. These results supports the idea of imposed-force locomotion [14]. The corresponding stall force, which is proportional to the product η × u, is then constant. The velocity u can be related to the mean propulsion force on the cell body by Stokes' law. Let's now assume that a power stroke (respectively a recovery stroke) results from the friction length ξ ⊥ (resp. ξ ) of the flagella moving perpendicular (resp. parallel) to its long axis. Moreover, the beating frequency f can be related to the friction of flagella acting on a typical distance of one cell diameter: 6πηRu = 2Rf η(ξ ⊥ + ξ ). Using measurements of the slopes ηu and ηf , we can estimate a sum ξ ⊥ + ξ ∼ 32µm. Using the friction coefficient expressions of a cylinder given in [18], this leads to an aspect ratio of 200 for a 10µm long flagellum with a radius of 25nm. This is a reasonable estimate [6] considering that flagella are not exactly perpendicular and parallel to the flow during power and recovery strokes. In this work, we quantified the complex dynamics of swimming at short time scale using high speed microscopy imaging and particle tracking techniques. This study allowed us to analyze the breaststroke like swimming of a CR cell in a fluid at low Reynolds number and how this swimming is influenced by the viscosity of the ambient fluid. It showed how the friction acting on a CR cell can be qualitatively extracted from the backand-forth motion of a thin and elongated pair of flagella. This means that a description in terms of time averaged flows is not to be encouraged for such systems [3,19]. This work was financed by the Rhone-Alpes Region (Cible program). We also acknowledge ANR Mosicob for its partial support.	2011-02-16T14:19:05.000Z	2010-11-12T00:00:00.000	{ "year": 2010, "sha1": "9749c08b0e9d6e4f11b2ae80c23ce3fdc3556374", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "9749c08b0e9d6e4f11b2ae80c23ce3fdc3556374", "s2fieldsofstudy": [ "Biology", "Physics" ], "extfieldsofstudy": [ "Physics" ] }
119146434	pes2o/s2orc	v3-fos-license	From microscopic theory to macroscopic theory: symmetries and order parameters of rigid molecules We use density functional theory to describe the phase behaviors of rigid molecules. The construction of kernel function G(x, P, x, P) is discussed. Excluded-volume potential is calculated for two types of molecules with C_{2v} symmetry. Molecular symmetries lead to the symmetries of G and density function f(P), enabling a reduction of configuration space. By approximating G with a polynomial, the system can be fully characterized by some moments corresponding to the form of G. The symmetries of G determine the form of the polynomial, while the coefficients are determined by temperature and molecular parameters. The analysis of the impact of coefficients helps us to choose independent variables in the moments as order parameters. Order parameters for bent-core molecules are predicted. Introduction Non-spherical rigid molecules may show orientationally ordered phases, which are often referred to as liquid crystalline(LC) phases. Among all rigid molecules, (nonpolar) rod-like molecules have attracted most interests. Rod-like molecules have shown several LC phases experimentally, including nematic, smectic A and smectic C. The shape of rigid molecules can be more complex, which may induce richer phase behaviors. In the recent two decades, a novel type of molecules has occupied a place in the research of liquid crystals. This type of molecules can be represented by two rigid rods connected end to end with a fixed angle, thus called bent-core molecules. Bent-core molecules break the rotational symmetry of rod-like molecules, and have proven to exhibit numerous new liquid crystalline phases [16]. A few rigid molecules of other architectures have also been studied and the experimental results indicate more complex phases [5]. To describe LC phases, orientation-dependent variables are necessary to be included in the free energy. The statistical mechanics of nematic phase of rods gives the free energy (1.1) where f (m) is orientational probability density function, G(m, m ) is kernel function, and c is concentration. Two of the most well-known G are Onsager potential [12] for a rod of length L and diameter D, and Maier-Saupe potential [8] C\|m × m \| 2 = C − C(m · m ) 2 (1. 3) with C related to L, D and temperature T . The top eigenvalue of the second moment of m, is defined as the order parameter of the uniaxial nematic phase [2], which is the only spatially homogeneous phase found for rod-like molecules other than isotropic phase. The free energy (1.1) is a natural extension of virial expansion of spheres. When handling a generic rigid molecule, a three-dimensional rotation P ∈ SO 3 is necessary to describe its orientation. Thus we need to substitute m with P in (1.1). Kernel function G(P, P ) can be deduced from pairwise interaction of molecules. Different phases correspond to different local minima of the free energy. However, it is obscure to distinguish phases with the probability density function f . One always wants to seek a few order parameters to classify them, like the eigenvalues of mm for nematic phase of rods. In the existing approaches, order parameters are usually considered at first. Models at different levels are constructed about these order parameters. For example, in [7,9] Landautype free energies are constructed for molecules with C 2v and D 2h symmetries. In [15] a molecular theory of D 2h is dicussed. Four order parameters are proposed. The kernel function there is a polynomial of m i · m j . When solving the model, further assumptions are made to deduce the equations of the four order parameters. The purpose of this article is to present a procedure of reducing f to a few order parameters. In this procedure, symmetries of molecules play a key role. These symmetries will be inherited by kernel function G and probability density function f . The symmetries of G and f make it possible to reduce the configuration space. As an example, the reduction will derive (1.1) for molecules with axial symmetry. The next step is to look for a good approximation of G. Here we are partially inspired by some thoughts in [15]. We will prove that G is a function ofP = P −1 P = (p ij ) 3×3 , and approximate G with a polynomial of p ij . The advantage of polynomial approximation is that the Euler-Langrange equation of f could be replaced with self-consistent equations of several moments of m i that fully characterize the system. The symmetries of G determine the form of approximate kernel function. In other words, the symmetries of G determine the candidate moments. Truncation within the remaining terms is followed, which relies on intuitions from experiments and simulations. Maier-Saupe potential is obtained spontaneously after this step for molecules with D ∞h symmetry, and the form of approximation is derived for molecules with C 2v symmetry. The coefficients of polynomial approximation of G are determined by molecular parameters and temperature. The analysis of the impacts of these coefficients might further reveal some properties of the chosen moments. Analysis of this type has been done for rods [6,3,17] and polar rods [4]. We will present some results on molecules with C 2v . The analysis would enable us to find independent variables in these moments, which are chosen as order parameters. From the implications of the analysis and the experimental results, we predict that five order parameters are enough for bent-core molecules. The rest of this paper is organized as follows. Sec.2 describes the density functional theory of generic rigid molecules. The construction of kernel function G is discussed. Some simple properties are presented. Excluded-volume potential is derived for two types of molecules with C 2v symmetry. In Sec.3, we analyze the symmetric properties of G and f and describe the reduction of configuration space. Sec.4 shows the derivation of the equations of moments and the screening of the moments by symmetries of kernel function. Sec.5 is dedicated to the analysis of the impacts of the coefficients in polynomial approximation of G. In Sec.6, we make a conclusion and propose some prospective problems. Modelling of rigid molecules of arbitrary shape This section presents the density functional theory of rigid molecules. We start from a general formulation, then deduce the free energy for spatially homogeneous phases. A three-dimensional rigid molecule might be chiral, leading to two possible configurations that cannot coincide through proper rotation. In this work we simply deal with systems with single chirality. Systems with mixed chirality can be treated by regarding two kinds of chirality as different molecules. Representation of the configuration of rigid molecules We choose a reference pointÔ on the rigid molecule and a body-fixed orthogonal basis m 1 , m 2 , m 3 . The configuration of the molecule is determined by the position ofÔ and the orientation of m i . In a space-fixed orthogonal coordinate system (O; e 1 , e 2 , e 3 ), they can be expressed in terms of x 0 = − − → OÔ and a three-dimensional proper rotation P ∈ SO 3 . In the language of matrix, P is orthogonal such that (m 1 , m 2 , m 3 ) = (e 1 , e 2 , e 3 ) P. (2.1) The elements of P T = (m ij ) is given by The position of a fixed point on the molecule is represented by its coordinates in the bodyfixed coordinate system (O; m 1 , m 2 , m 3 ): Its location in space, expressed by its coordinates in (O; e 1 , e 2 , e 3 ), is Every P ∈ SO 3 has a representation by Euler angles α, β, γ: cos α − sin α cos γ sin α sin γ sin α cos β cos α cos β cos γ − sin β sin γ − cos α cos β sin γ − sin β cos γ sin α sin β cos α sin β cos γ + cos β sin γ − cos α sin β sin γ + cos β cos γ The uniform probability measure on SO 3 is given by dν = 1 8π 2 sin αdαdβdγ. Density functional theory We start from the extension of virial expansion that includes inhomogeneity both spatial and orientational. The probability density function f agrees with the concentration c: Virial expansion is appropriate for small concentration. Corrections for large concentration have also been discussed, such as in [1]. The kernel function in (2.3) is given by Mayer function [10] G(x, P, where U is pairwise interaction. Many types of interaction could appear in U . But here we model the molecule as a combination of spheres with the same diameter D and assume that U consists of the sum of interaction of every pairs of spheres. Suppose that the distribution of their centers is given by ρ(x) in the body-fixed coordinate system, then U can be written as where V 0 (r) is the potential of a single pair of spheres. It can take hardcore potential (2.6) or Lennard-Jones potential (2.7) In (2.7) σ is a function of D. Some other types of interaction between spheres can also be incoporated in V 0 , such as electrostatic potential for charged molecules. Independent of V 0 , kernel function G has the following properties: 1. G(x, P, x , P ) remains unchanged when switching (x, P ) and (x , P ): G(x, P, x , P ) = G(x , P , x, P ). (2.8) Now we deduce the free energy of spatially homogeneous phases, namely f (x, P ) = cf (P ) wheref (P ) is a density function on SO 3 . Define homogeneous kernel function as It is well-defined because the integration on the right side is invariant with x: Applying Proposition 2.1 toG, we have Proposition 2.2.G(P, P ) satisfiesG (P, P ) =G(P , P ) andG (P, P ) =G(T P, T P ). By setting T = P −1 , we know thatG(P, P ) is a function ofP = P −1 P , which is denoted bỹ G(P ). Proof. Using (2.8) and (2.10), we get The rest of paper will focus on spatially homogeneous phases. For convenience we use f (P ) and G(P, P ) instead off (P ) andG(P, P ). Then the free energy (2.3) becomes For rod-like and bent-core molecules, the sphere centers lie on a curve. They can be viewed as either discretely or continuously distributed on the curve, which means In the discrete version, a rod-like molecule is modelled bỹ and a bent-core molecule is modelled bỹ where N is even. In the continum version, a rod-like molecule is modelled bỹ and a bent-core molecule is modelled bỹ Both the discrete and continuous versions have the same symmetry: rod-like molecules have D ∞h symmetry, and bent-core molecules have C 2v symmetry. Another example of C 2v symmetry is isosceles spherotriangles (by sphero-A, we refer to the Minkowski sum of A and a sphere): where T is an isosceles triangle that lies in plane Om 1 m 2 . Spherocuboids, which possess D 2h symmetry, is considered in [15]. The distribution of sphere centers is given by where C is a cuboid with edges parallel to m i . The molecules mentioned above are drawn in Fig.1. Next we try to compute G(P, P ) when using hardcore potential (2.6). Notice that G(x, P, x , P ) equals to 1 if two molecules overlap and 0 elsewhere. Thus by (2.11) G(P, P ) is actually excluded-volume potential, and the problem has converted into finding this volume. Onsager potential (1.2) is an approximation of the excluded volume for rod-like molecules. Similar approach has been done for spherozonotopes by B. M. Mulder [11] based on Steiner formula [14]. Here we present the calculation of the excluded volume of spherotriangles. Denote the set of sphere centers of two molecules as T 1 and T 2 . The excluded region of two molecules is K + B D , where K = T 1 − T 2 and B D is a sphere of diameter D. When K is convex, the measure of K + B D is expressed by Steiner formula in a polynomial of D. Here we write down the three-dimensional case where V 3 is the volume and V 2 is the surface area of K(see p.210 of [14]). V 1 is the mean width of K (for the definition, see p.42 of [14]). For a polytope, V 1 is written as The sum is taken over all the edges of K. l(e) is the length of edge, and γ(e, K) represents the external angle at e. It is defined as where θ is the angle between outward normal vectors of two faces that share edge e. For a polytope K, each of the four terms in (2.20) represents a part of K + B D : K; parallelepipeds growing outward at each face; circular sector cylinders at each edge; corner regions at each vertex. We list V i for the excluded volume of two spherotriangles T 1 = OAB and −T 2 = O A B . Details are left to Appendix. Denote the edges of triangles as We have The expression of V 2 depends on the relative orientation of two molecules. Assume that Otherwise we could rotate the notations of the edges. If (c × c · a)(c × c · a ) > 0, then When K is not convex, V (K + B D ) does not have a general formula. In Appendix we give an expression for the excluded volume of bent-core molecules. Excluded-volume potential fails to contain temperature T in kernel function, which makes it insufficient to study thermotropic LC. In the next two sections we will present a systematic procedure of constructing an approximation of kernel function that does not eliminate temperature. Symmetries play an important role throughout the procedure. Symmetries of kernel function and reduction of configuration space In this section we study the symmetric properties of G and f inherited from molecular symmetries. That a rigid molecule is symmetric under T ∈ SO 3 means Denote by H a subgroup of SO 3 that leaves the molecule invariant. We start from two fundamental theorems. Proof. From (3.1) and (2.5), U is symmetric under T : Hence by (2.11), we get The other equality in (3.2) could be obtained similarly. If a molecule has a symmetry plane with unit normal vectork, then where J is the rotation aroundk by π. Proof. Assume that the symmetry plane containsÔ, otherwise we shift the body-fixed coordinate system to meet this requirement. Now we have Similar to Theorem 3.1, we have The local minima of the free energy (2.12) satisfy the Euler-Lagrange equation where λ is a Lanrangian multiplier to ensure the normalization of f . Denote The solution of (3.5) has the form Theorem 3.3. If T ∈ H , the solutions of (3.5) satisfy Proof. Substitute P with P T in (3.5). By Theorem 3.1, G(P T, P ) = G(P, P ), thus With the symmetries of G and f , the configuration space could be reduced. Theorem.3.1 and 3.3 indicate that G is a function of HP H , and f is a function of P H . Note that cosets of subgroup H form a partition of SO 3 . Thus it allows us to define Ω = {P H \|P ∈ SO 3 } as the new configuration space, where f and G are well-defined in Ω and Ω × Ω, respectively. If we denote the probability space on SO 3 as (SO 3 , F, ν), then the new probability space (Ω, F H , ν H ) is defined as follows: It can be proved that for any F H measurable function h, dν whereh is defined ash(P ) = h(P H ). Hence the free energy could be rewritten as The above process reduces the configuration space of molecules with C ∞ symmetry to S 2 . Theorem 3.4. For molecules with C ∞ symmetry, the configuration space is reduced to S 2 with the uniform probablity measure Proof. Because H consists of all the rotations around m 1 , So we could select P (α, β, 0) as the representative element of P H , and Ω becomes Hence any A H ∈ F equals to A if A consists of some P (·, ·, 0). The measure on the reduced configuration space is given by Thus dν H = sin α 4π dαdβ is the uniform measure on S 2 . Polynomial approximation of kernel function In this section we describe the construction of approximate kernel function. We aim to use a polynomial of nine elements ofP as the approximation. This form of approximation reduces the Euler-Lagrange equation (3.5) to a few self-consistent equations about moments. Suppose that G has a term It corresponds to a term in the free energy. And W (P ) must be of the form Denote by M the set of moments that appear in the free energy. This formula indicates that W (P ) is determined by the value of moments in M. On the other hand, the moments can be calculated with (3.7) by Notice that the right side is a function of the moments. Applying this formula to all the moments in M, we obtain a group of self-consistent equations about these moments. So we only need to solve the moments in M instead of f . Next we will deduce the form of polynomial approximation of kernel function from its symmetries. Maier-Saupe potential will be derived naturally from the analysis. The nine elements ofP are not independent. The third column is uniquely determined by the other two columns: Therefore G can be expressed by a function of six variables Proposition 4.1. If a molecule has reflection symmetry, and the symmetry plane is perpendicular to m 3 , then G depends only on the following four elements ofP : Proof. When p ij (i, j = 1, 2) are given properly, (p 31 , p 32 ) might take two possible pairs of value: (y 1 , y 2 ) and (−y 1 , −y 2 ), which satisfy p 2 11 + p 2 21 + y 2 1 = p 2 12 + p 2 22 + y 2 2 = 1, p 11 p 11 + p 21 p 22 + y 1 y 2 = 0. In the following theorem, m 1 always coincides with the rotational axis. 2. For a molecule with C ∞ symmetry, G is a function of p 11 = m 1 · m 1 . If the molecule has D ∞h symmetry, G is a function of \|p 11 \|. Proof. With the above discussion, we are able to construct polynomial approximations for molecules with different symmetries. We start from the approximate kernel function of molecules with D ∞h symmetry. In Theorem 4.3 we have proven that G = G(\|m 1 · m 1 \|). Its approximation should be a polynomial of m 1 · m 1 without odd-degree terms. Therefore it is at least quadratic, which coincides with the form of Maier-Saupe potential: The above form indicates that M = m 1 m 1 . When a molecule has only C ∞ symmetry, odd-degree terms of p 11 no longer vanishes. Quadratic approximation will be which is discussed in [4]. When this kernel is used, M = m 1 , m 1 m 1 . Now we turn to the approximations of kernel function for molecules with C 2v symmetry, including bent-core molecules and spherotriangles. By Proposition 4.1, an approximation of G is a polynomial of four variables p 11 , p 12 , p 21 , p 22 . Then by Proposition 2.2, it is symmetric with respect to p 12 and p 21 , which means G = G(p 11 , p 22 , p 12 + p 21 , p 12 p 21 ). Using (4.6), we are able to determine the form of polynomial. Quadratic approximation is written as G = c 0 + c 1 p 11 + c 2 p 2 11 + c 3 p 2 22 + c 4 (p 2 12 + p 2 21 ). From the above discussion we know that the form of polynomial approximation is determined by molecular symmetries. The coefficients c i can be calculated by projecting G to the space spanned by all the polynomials of the given form. If the approximation has the form i c i q i (P ), then the coefficients c i are determined by dν(P )G(P ; Θ)q j (P ). In the above, Θ is a set that consists of temperature and a group of molecular parameters. The formula reveals that these coefficients are functions of Θ. Generally speaking, as temperature is included in Θ, the approximate G is able to describe both lyotropic and thermotropic liquid crystals. The projection of Onsager potential to span{1, p 2 11 } gives As a constant difference in G does not affect the solution, c 0 is ignored. It is easy to see that c 2 is propotional to one effective parameter cL 2 D. The projection of the excluded-volume potential of spherocuboids is derived by R. Rosso and E. G. Virga in [13]. In Appendix, we discuss the projection of excluded-volume potential of isoceles spherotriangles to the space of quadratic approximations. Suppose that the top corner is θ and the length of lateral is L/2. And c 1 is proportional to cL 2 D with where K(θ) is a function of θ defined in (7.11). Further analysis and the choice of order parameters In the previous section we select some moments and reduce the density functional theory to a group of equations about them. Those equations usually imply some properties of the moments. They could help us to choose independent components of the moments as order parameters. Here we try to extract these properties. Some of them depend on the values of coefficients. As the coefficients are determined by molecular parameters and temperature, these properties would reveal the impacts of them. When G takes Maier-Saupe potential, the only moment in M is m 1 m 1 . It can be diagonalized by selecting axes along its eigenvectors. Its trace equals to 1, leaving only two degrees of freedom remained. These two degrees of freedom could be further reduced to one by the proof of uniaxial property [6,3,17]. Theorem 5.1 (Axially symmetry of the solution with Maier-Saupe potential). If G takes Maier-Saupe potential (1.3), every solution of (3.5) is axially symmetric When M has more than one moments, there are usually some relations between them. In [4] the following conclusion is shown, which reduces the number of order parameters for polar rods to 3. Proof. The self-consistent equation of m 1 yields If m 1 = 0, we substitute the above inequality into (5.2) and get \| m 1 \| 2 < \| m 1 \| 2 , which is a contradiction. 2. Select coordinate axes that diagonalize m 1 m 1 and m 2 m 2 . Now W 1 (P ) is of the form the form of W 1 (P ) indicates that This inequality violates the diagonalization of m 1 m 1 . 3. From c 2 4 = c 2 c 3 , we can write Without loss of generality, the sign on the right side is assumed positive. Because c 1 ≥ −1, we get m 1 = 0. Thereby W converts into This would enable us to select coordinate axes according to the axes of The zero values of other off-diangonal elements can be obtained similarly. Using W (P ) = W (JP ), we get We tend to believe that m 1 m 1 and m 2 m 2 can be diagonalized simutaneously. The results of Theorem 5.3 would reduce the degrees of freedom of order parameters of bent-core molecules to 5. We choose coordinate axes as eigenvectors of m 1 m 1 as well as those of m 2 m 2 , then m 1 parallels to one of the eigenvectors of m 1 m 1 . Because the trace of m 1 m 1 and m 2 m 2 equal to 1, both of the two second moments contribute two degrees of freedom. At last m 1 contributes one, making them five in total. Finally, we should point out that the set of order parameters should be decided by results of experiments and simulations so as to be able to distinguish dirrerent phases. It should also follow this criterion to determine where to truncate the polynomial approximation of G. Rodlike molecules exhibits only uniaxial nematics. As we have described, Maier-Saupe potential is a polynomial approximation of G truncated on the second order. With thorough analysis the number of order parameter is reduced to 1. Therefore Maier-Saupe potential is proven to be the most concise model of rod-like molecules that covers experimental results. Up to now, spatially homogeneous phases of bent-core molecule are restrained to uniaxial or biaxial nematics, without the observation of polar order. This seems to indicate the sufficiency to approximate G with quadratic polynomials, which contradicts with what is proposed in [7]. Also it will be interesting to see if any phases with polar order would appear. Conclusion and outlook A generic modelling procedure is proposed for rigid molecules of arbitrary shape. The modelling of kernel function incorporates pairwise interaction. We show that the symmetries of molecule determine the reduced configuration space and the form of polynomial approximations of G with its coefficients depended on temperature and molecular parameters. An approximate kernel is deduced for molecules with C 2v symmetry. By approximating G with polynomial, the system is reduced to a group of equations about moments of body-fixed axes. Some properties of these moments are studied for molecules with C 2v symmetry, and the number of order parameters is predicted for bent-core molecules. The prediction needs to be verified by results of simulations and comparison to experiments. Moreover, it remains unknown whether there are some general relationships between the moments. A clear understanding of them would help us to find out a minimal complete set of order parameters. Here we calculate the excluded-volume potential of two spherotriangles T 1 + B D/2 and T 2 + B D/2 . The excluded region can be represented by K + B D where K = T 1 − T 2 . By (2.20), we need to calculate V 3 , V 2 , V 1 for K. Denote the vertices of T 1 as OAB that lie in plane π, and those of −T 2 as O A B that lie in plane π . K is a polytope, for it is the convex hull of nine points The edges of two triangles are denoted as If π and π do not parallel, we can label the vertices properly such that the plane π+O −O seperates A and B , and the plane π + O − O seperates A and B, namely If the intersection of two triangles T 1 and T 2 + O − O is not empty, which indicates (m 3 · c )(m 3 · c) < 0, K is drawn in the left part of Fig.2; and if it is empty, which indicates (m 3 · c )(m 3 · c) > 0, Now we may assume that O = O , then K is drawn in the right part of Fig.2. The notion P AA represents the point located at O + −→ OA + −−→ O A , etc.. When π and π are parallel, we can label the vertices such that T 2 intersects with ∠AOB or its vertical angle. First we calculate V 3 (K). For the case on the left part of Fig.2, K can be divided into the prisms Thus For the case on the right, K can be divided into the prisms Thus For both cases, we have Next we calculate V 1 (K). Each edge of K parallels to one of the six edges of T 1 and T 2 . As an example, we describe the contribution to V 1 of edges parallel to a. As the faces contain one of those edges, the outward normal vectors lie in a plane perpendicular to a. For the case on the left, there are three edges parallel to a: A P AA , OA, B P AB . As their length equals to \|a\|, we only need to calculate the sum of external angles, which is 1 2π (∠ n A P AA P BA , n OAP AA A + ∠ n OAP AA A , n OAP AB B + ∠ n OAP AB B , n B P AB P BB ). Note that n A P AA P BA and n B P AB P BB are reverse, and the four vectors n A P AA P BA , n OAP AA A , n OAP AB B , n B P AB P BB are sequentially arranged. Thus the three angles add up to π, and the sum of the external angles equals to 1 2 . For the case on the right, there are two edges parallel to a: A P AA , B P AB . Again we only need the sum of the external angles: 1 2π (∠ n A P AA P BA , n A P AA P AB B + ∠ n A P AA P AB B , n B P AB P BB ) = 1 2 . Therefore the amount of the external angles at the edges parallel to a is always 1 2 . The above calculation can be done for the other five edges, leading to The expression of V 2 (K) is different for two cases in Fig.2. The faces of K always contain four triangles AP AA P AB , BP BA P BB , A P AA P BA and B P AB P BB . The other faces are some parallelograms. For the case in the left, they are For the case in the right, they are ABP BA P AA , ABP BB P AB , A B P AB P AA , A B P BB P BA . We point out that This means that one of T 1 − T 2 and T 1 + T 2 corresponds to the case in the left, while the other corresponds to the case in the right. Therefore (7.3) holds. The excluded volume of rods could be obtained for congruent OAB, O A B with ∠AOB = π. In this case, c = Lm, V 3 = 0 and V 1 = 2L. which is a constant different from Onsager's form. Quadratic projection of the excluded-volume potential The above derivation for excluded volume is valid for any pair of triangles. Now we suppose that T is isoceles with top corner θ and length of lateral sides L/2. Two triangles are given by T 1 = P T and T 2 = P T . The unit vectors along the edges of two triangles are written as follows. with \|a\| = \|b\| = \|a \| = \|b \| = L/2 and \|c\| = \|c \| = L sin θ 2 . We aim to project V onto the space spanned by Q = {1, p 11 , p 2 11 , p 2 12 , p 2 21 , p 2 22 }. Note that the following functions in span{Q} are mutually orthogonal: We focus on the even-order terms first. Let The even-order part of projection will be written as By comparing the coefficients, we have In the above, k 0 , k 1 , k 2 , k 3 can be evaluated analytically. We use the notation p ij (P ) to represent the (i, j) element ofP . First we point out that In fact, V 2 (P ) = V 2 (P T − P T ). Let J = diag(−1, −1, 1), then JT = −T . Thereby V 2 (P J) = V 2 (P T − P JT ) = V 2 (P T + P T ). By (7.3) we have Meanwhile p ij (P J) = p ij (P ), therefore (7.7) holds. We need to calculate the terms like 7.8) and We describe the strategy to compute integrals where e, e are unit vectors. The following formula is needed. Choose R 1 and R 2 such that R 1 e = m 1 , R 2 e = m 1 . The integral is rewritten as in which Q is a trigonometric polynomial of α, β, γ. When the cross product is replaced by dot product, \| sin α\| is substituted with \| cos α\|. We compute (7.8) as an example. Define R 1 and R 2 by Then we have Hence SO 3 dν(P )p 2 11 \|m 3 · (m 1 cos − sin α sin β cos θ 2 + sin θ 2 (cos α sin β cos γ + cos β sin γ) The other terms could be handled similarly. All the results are listed in By (7.4)-(7.6), we get (4.12)-(4.14). The computation of c 1 is complicated. Note that V 3 does not contribute to c 1 . In fact, it is obvious that V 3 (P J) = V 3 (P ) and p 11 (P J) = −p 11 (P ), which yield Denote I aa = \|e a × e a \|, I ab = \|e a × e b \|, I ac = 2 sin θ 2 \|e a × e c \|, Thus the excluded volume can be written as We have already known that So we only need to compute the volumes of the intersections above. When calculating the volume of a region U , we can write \|U \| = dxdym(Ω(x, y)) where m(·) denotes the measure of a set and Ω(x, y) = {z\|(x, y, z) ∈ U }. Because V ij is convex, Ω(x, y) is an interval [l ij (x, y), u ij (x, y)] for U = V ij . Thus \|V ij ∩ V i j \| = dxdy min{u ij , u i j } − max{l ij , l i j } + , \|V ij ∩ V i j ∩ V i j \| = dxdy min{u ij , u i j , u i j } − max{l ij , l i j , l i j } + , \|V 11 ∩ V 12 ∩ V 21 ∩ V 22 \| = dxdy min{u 11 , u 12 , u 21 , u 22 } − max{l 11 , l 12 , l 21 , l 22 } + , where x + = max{x, 0}. Now the problem turns into computing l ij (x, y) and u ij (x, y). Put one molecule in the plane xOy with the arrowhead at O and m 1 along −x. Then p 1,2 = L(cos θ 2 , ± sin θ 2 , 0). We describe how to compute u(x, y) of the spheroparallelogram Op 1 p 1 + B D , where p 1 /L = (p, q, r) is a unit vector. A spheroparallelogram consists of a parallelpiped, four half cylinders at each edge of parallelogram, and four corners, each of which is enclosed by two planes and a sphere. Classify u(x, y) into three cases by where (x, y, u(x, y)) lies: the parallelpiped; one of the four half cylinders; one of the four spheres. For the first case, the distance of (x, y, u(x, y)) to plane Op 1 p 1 equals to D. The normal vector of Op 1 p 1 is Hence For the second case, the distance equals to D bewteen (x, y, u(x, y)) and the axis of one of the four half cylinders. Thereby u(x, y) is the larger root of (x − x 0 ) 2 + (y − y 0 ) 2 + u(x, y) − z 0 2 − a(x − x 0 ) + b(y − y 0 ) + c(u(x, y) − z 0 ) 2 = D 2 . All remaining is to clarify the region of three cases. In Fig.3, they are coloured by red, white and blue respectively. The region contains all points whose distance to the central parallelogram (drawn in dotted line in Fig.3), which is spanned by where (x 0 , y 0 , z 0 ) is one of the vertices of Op 1 p 1 . By eliminating z, we get the equation of the curve: p(x − x 0 ) + q(y − y 0 ) 2 + r 2 (x − x 0 ) 2 + (y − y 0 ) 2 = r 2 D 2 . The blue regions are those enclosed by a line segment, an elliptical arc defined above, and a circular arc on the boundary.	2013-05-21T06:33:12.000Z	2013-05-21T00:00:00.000	{ "year": 2013, "sha1": "48ea95f5b316067d2aff6117d7907580e741d73e", "oa_license": null, "oa_url": "http://arxiv.org/pdf/1305.4726", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "48ea95f5b316067d2aff6117d7907580e741d73e", "s2fieldsofstudy": [ "Chemistry" ], "extfieldsofstudy": [ "Mathematics", "Physics" ] }
40673397	pes2o/s2orc	v3-fos-license	Generalized nil-Coxeter algebras over discrete complex reflection groups We define and study generalized nil-Coxeter algebras associated to Coxeter groups. Motivated by a question of Coxeter (1957), we construct the first examples of such finite-dimensional algebras that are not the 'usual' nil-Coxeter algebras: a novel 2-parameter type $A$ family that we call $NC_A(n,d)$. We explore several combinatorial properties of $NC_A(n,d)$, including its Coxeter word basis, length function, and Hilbert-Poincare series, and show that the corresponding generalized Coxeter group is not a flat deformation of $NC_A(n,d)$. These algebras yield symmetric semigroup module categories that are necessarily not monoidal; we write down their Tannaka-Krein duality. Further motivated by the Broue-Malle-Rouquier (BMR) freeness conjecture [J. reine angew. math. 1998], we define generalized nil-Coxeter algebras over all discrete real or complex reflection groups $W$, finite or infinite. We provide a complete classification of all such algebras that are finite-dimensional. Remarkably, these turn out to be either the usual nil-Coxeter algebras, or the algebras $NC_A(n,d)$. This proves as a special case - and strengthens - the lack of equidimensional nil-Coxeter analogues for finite complex reflection groups. In particular, generic Hecke algebras are not flat deformations of $NC_W$ for $W$ complex. Introduction and main results Throughout this paper, k will denote a fixed unital commutative ground ring. In this paper we define and study generalized nil-Coxeter algebras associated to Coxeter groups, and more generally to all discrete complex reflection groups, finite or infinite. These are algebras that map onto the associated graded algebras of (generic) Hecke algebras over complex reflection groups and of Iwahori-Hecke algebras over Coxeter groups. As we discuss, working with these algebras allows for a broader class than the corresponding reflection groups. We begin with real groups. Coxeter groups and their associated Hecke algebras play an important role in representation theory, combinatorics, and mathematical physics. Each such group is defined by a Coxeter matrix, i.e. a symmetric "integer" matrix M := (m ij ) i,j∈I with I finite and m ii = 2 m ij ∞ ∀i = j. The Artin monoid B 0 M associated to M is generated by {T i : i ∈ I} modulo the braid relations T i T j T i · · · = T j T i T j · · · for all i = j with m ij < ∞, with precisely m ij factors on either side. The Artin group (or generalized braid group) B M is the group generated by these relations; typically we use {s i : i ∈ I} to denote its generators. There are three well-studied algebras associated to the matrix M : the group algebra kW (M ) of the Coxeter group, the 0-Hecke algebra [Ca, No], and the nil-Coxeter algebra N C(M ) [FS] (also called the nilCoxeter algebra, nil Coxeter algebra, and nil Hecke ring in the literature). These are all free k-modules, with a "Coxeter word basis" {T w : w ∈ W (M )} and length function (T w ) := (w) in W (M ); in each of them the T i satisfy a quadratic relation. In a sense, the usual nil-Coxeter algebras N C(M ) are better-behaved than all other generic Hecke algebras (in which T 2 i = a i T i + b i for scalars a i , b i , see [Hum,Chapter 7]): the words T w have unique lengths and form a monoid together with 0. Said differently, the algebras N C(M ) are the only generic Hecke algebras that are graded with T i homogeneous of positive degree. Indeed, if deg T i = 1 ∀i, then N C(M ) has Hilbert-Poincaré polynomial i∈I [d i ] q , where [d] q := q d −1 q−1 and d i are the exponents of W (M ). We now introduce the main objects of interest in the present work: generalized Coxeter matrices and their associated nil-Coxeter algebras (which are always Z 0graded). Definition 1.1. Define a generalized Coxeter matrix to be a symmetric 'integer' matrix M := (m ij ) i,j∈I with I finite, 2 m ij ∞ ∀i = j, and m ii < ∞ ∀i. Now fix such a matrix M . (1) Given an integer vector d = (d i ) i∈I with d i 2 ∀i, define M (d) to be the matrix replacing the diagonal in M by d. where we omit the braid relation T i T j T i · · · = T j T i T j · · · if m ij = ∞. We are interested in the family of (generalized) nil-Coxeter algebras for multiple reasons: category theory, real reflection groups, complex reflection groups, and deformation theory. We elaborate on these motivations in this section and the next. 1.1. Tannaka-Krein duality for semigroup categories. In [Kho], the representation categories Rep N C(A n ) were used to categorify the Weyl algebra. For now we highlight two properties of generalized nil-Coxeter algebras N C(M ) which also have categorical content: (i) for no choice of coproduct on N C(M ) can it be a bialgebra (shown below); and (ii) every algebra N C(M ) is equipped with a cocommutative coproduct ∆ : T i → T i ⊗ T i for all i ∈ I. Viewed through the prism of representation categories, the coproduct in (ii) equips Rep N C(M ) with the structure of a symmetric semigroup category [ES,§13,14]. Note by (i) that the simple module k does not serve as a unit object, whence Rep N C(M ) is necessarily not monoidal. It is natural to apply the Tannakian formalism to such categories with "tensor" structure. We record the answer which, while not surprising once formulated, we were unable to find in the literature. Definition 1.3. A semigroup-tensor category is a semigroup category (C, ⊗) which is also additive and such that ⊗ is bi-additive. Theorem A. Let A be an associative unital algebra over a field k, C := Rep A, and F : C → Vec k the forgetful functor. (1) Any semigroup-tensor structure on C together with a tensor structure on F equips A with a coproduct ∆ : A → A ⊗ A that is an algebra map. Notice that generalized nil-Coxeter algebras are indeed examples of such triangular algebras, with a (cocommutative) coproduct but no counit. Such algebras are interesting in the theory of PBW deformations of smash product algebras; see Section 2. We also show below how to obtain an "honest" symmetric tensor category from each algebra N C(M ), via a central extension. As noted above, Theorem A is in a sense "expected", and serves to act more as motivation. That the algebras N C(M ) provide concrete examples of symmetric, nonmonoidal semigroup-tensor categories is novel. The main results below now focus on the algebras N C(M ) themselves. 1.2. Real reflection groups and novel family of finite-dimensional nil-Coxeter algebras. Our next result constructs a novel family of generalized nil-Coxeter algebras of type A, which are finite-dimensional. Classifying the finitedimensional objects in Coxeter-type settings in algebra and combinatorics has been a subject of tremendous classical and modern interest, including Weyl, Coxeter, and complex reflection groups, their nil-Coxeter and associated Hecke algebras; but also finite type quivers, Kleinian singularities, the McKay-Slodowy correspondence, simple Lie algebras, etc. A very recent setting involves the classification of finite-dimensional Nichols algebras. Some of the prominent ingredients in the study of these algebras are common to the present work. See [GHV,HV1,HV2] for more details. Another famous recent classification is that of finite-dimensional pointed Hopf algebras [AnSc], which turn out to arise from generalized small quantum groups. With these motivations, our goal is to similarly classify all generalized nil-Coxeter algebras, and our next result presents the first novel family of such examples. We remark that in equation (1.2), in generalizing the "order relations" from T 2 i = 0 to T mii i = 0 we were also motivated by another such setting: the classical work of Coxeter [Cox3], which investigated generalized Coxeter matrices M for which the group W (M ) is finite. Specifically, Coxeter considered the (type A) Artin braid group B An−1 , and instead of quotienting by the relations s 2 i = 1 to obtain the symmetric group S n , he worked with s p i = 1 ∀i ∈ I. Coxeter was interested in computing for which (n, p) is the quotient group W An−1 ((p, . . . , p)) a finite group, and what is its order. He showed (see also [As]) that W An−1 ((p, . . . , p)) is finite if and only if 1 n + 1 p > 1 2 , in which case the size of the quotient group is 1 n + 1 p − 1 2 1−n · n!/n n−1 . Coxeter's result was extended by Koster in his thesis [Ko] to completely classify the "generalized Coxeter groups" W (M ) that are finite. These turn out to be precisely the finite Coxeter groups and the Shephard groups. Parallel to the above classical works, we wish to understand for which matrices M is the algebra N C(M ) finitely generated as a k-module. If W (M ) is a Coxeter group, then dim N C(M ) = \|W (M )\|. Few other answers are known. For instance, Marin [Mar] has shown that the algebra N C A2 ((m, n)) is not finitely generated when m, n 3. However, apart from the usual nil-Coxeter algebras, to our knowledge no other finitely generated algebras N C(M ) were known to date. In the following result, following Coxeter's construction in type A above, we exhibit the first such finite-dimensional family of algebras N C(M ). Theorem B. Given integers n 1 and d 2, define the k-algebra In other words, N C A (n, d) is generated by T 1 , . . . , T n , with relations: In particular, for all l ∈ [1, n − 1], the subalgebra R l generated by T 1 , . . . , T l is isomorphic to the usual nil-Coxeter algebra N C A l ((2, . . . , 2)). Remark 1.8. We adopt the following notation in the sequel without further reference: let (1.9) w • ∈ S n+1 , w • ∈ S n , w • ∈ S n−1 denote the respective longest elements, where the symmetric group S l+1 corresponds to the k-basis of the algebra R l for l = n − 2, n − 1, n. The algebras N C A (n, d) have not been studied previously for d > 2, and we begin to explore their properties. When d = 2, N C A (n, d) specializes to the usual nil-Coxeter algebra of type A n . In this vein, we present three properties of N C A (n, d) akin to the usual nil-Coxeter algebras. Theorem C. Fix integers n 1 and d 2. (1) The algebra N C A (n, d) has a length function that restricts to the usual length function An−1 on R n−1 N C An−1 ((2, . . . , 2)) (from Theorem B), and (3) If k is a field, then N C A (n, d) is local, with unique maximal ideal m generated by T 1 , . . . , T n . For all k, the ideal m is nilpotent with m 1+l n,d = 0. We also study the algebra N C A (n, d) in connection to Khovanov's categorification of the Weyl algebra. See Proposition 5.6 below. 1.3. Complex reflection groups and BMR freeness conjecture. Determining the finite-dimensionality of the algebras N C(M ) is strongly motivated by the study of complex reflection groups and their Hecke algebras. Recall that such groups were enumerated by Shephard-Todd [ST]; see also [Coh, LT]. Subsequently, Popov [Pop1] classified the infinite discrete groups generated by affine unitary reflections; in the sequel we will term these infinite complex reflection groups. For more on these groups, see e.g. [BS,Hug1,Hug2,Mal,ORS,ReS] and the references therein. For complex reflection groups, an important program is the study of generic Hecke algebras over them, as well as the associated BMR freeness conjecture of Broué,Malle,and Rouquier [BMR1,BMR2] (see also the recent publications [Et, Lo, Mar, MP] and the thesis [Ch]). This conjecture connects the dimension of a generic Hecke algebra to the order of the underlying reflection group. Here we will study this connection for the corresponding nil-Coxeter algebras, which we define as follows given [Be,BMR2]. Definition 1.11. Suppose W is a discrete (finite or infinite) complex reflection group, together with a finite generating set of complex reflections {s i : i ∈ I}, the order relations s mii i = 1 ∀i, a set of braid relations {R j : j ∈ J} -each involving words with at least two distinct reflections s i -and for the infinite non-Coxeter complex reflection groups W listed in [Mal, Tables I, II], one more order relation R m0 0 = 1. Now define I 0 := I {0} for these infinite non-Coxeter complex reflection groups W , and I 0 := I otherwise. Given an integer vector d ∈ N I0 with d i 2 ∀i, define the corresponding generalized nil-Coxeter algebra to be where the braid relations R j are replaced by the corresponding relations R j in the alphabet {T i : i ∈ I}, and similarly for R 0 if R m0 0 = 1 in W . There is also the notion of the corresponding braid diagram as in [BMR2, and [Mal, Tables I, II]; this is no longer always a Coxeter graph. Note by [Pop1,§1.6] that in the above definition, one has to work with a specific presentation for complex reflection groups, as there is no canonical (minimal) set of generating reflections. See [Ba] for related work. There is no known finite-dimensional generalized nil-Coxeter algebra associated to a finite complex reflection group. Indeed, Marin mentions in [Mar] that a key difference between real and complex reflection groups W is the lack of nil-Coxeter algebras of dimension precisely \|W \| for the latter. This was verified in some cases for complex reflection groups in loc. cit. Our final result shows this assertion -and in fact a stronger statement -for all discrete finite and infinite, real and complex reflection groups. Even stronger (a priori ): we provide a complete classification of finite-dimensional generalized nil-Coxeter algebras for all such groups. Notice by [Pop1,Theorem 1.4] that it suffices to consider only the groups whose braid diagram is connected. Theorem D. Suppose W is any irreducible discrete real or complex reflection group. In other words, W is a real reflection group with connected braid diagram, or a complex reflection group with connected braid diagram and presentation given as in [BMR2,, [Mal, Tables I, II], or [Pop1, Table 2]. Also fix an integer vector d with d i 2 ∀i (including possibly for the additional order relation as in [Mal]). Then the following are equivalent: (1) The generalized nil-Coxeter algebra N C W (d) is finitely generated as a kmodule. (3) The ideal m generated by {T i : i ∈ I} is nilpotent. If these assertions hold, there exists a length function and a unique longest element in N C W (d), say of length l; now m 1+l = 0. In other words, the only finite-dimensional examples (when k is a field) are the usual nil-Coxeter algebras, and the algebras N C A (n, d). Note also that all of the above results are characteristic-free. A key tool in proving both Theorems B and D is a diagrammatic calculus, which is akin to crystal theory from combinatorics and quantum groups. 1.4. Further questions and Organization of the paper. To our knowledge, the algebras N C A (n, d) for d > 2 are a novel construction -and in light of Theorem D, the only finite-dimensional generalized nil-Coxeter algebras other than the 'usual' ones. In particular, a further exploration of their properties is warranted. We conclude this section by discussing some further directions. (1) Nil-Coxeter algebras are related to flag varieties [BGG, KK], categorification [Kho, KL], and symmetric function theory [BSS]. Also recall, the divided difference operator representation of the usual type A nil-Coxeter algebra N C A (n, 2) is used to define Schubert polynomials [FS, LS], and the polynomials the T i simultaneously annihilate are precisely the symmetric polynomials. It will be interesting to determine if N C A (n, d), d > 2 has a similar "natural" representation as operators on a polynomial ring; and if so, to consider the polynomials one obtains analogously. (See [Mar] for a related calculation.) We observe here that for d > 2, the algebra N C A (n, d) does not "come from" a finite reflection group, as it is of larger dimension than the corresponding generalized Coxeter group, by equation (2.1) below. (2) Given both the connection to Coxeter groups as well as the crystal methods used below, it will be interesting to explore if the algebras N C A (n, d) are connected to crystals over some Lie (super)algebra. (3) Our proof of Theorem D below involves a case-by-case argument, running over all discrete complex reflection groups. A type-free proof of this result would be desirable. The paper is organized as follows. In Section 2 we elaborate on our motivations and make additional remarks. In the following four sections we prove, in turn, the four main theorems above. Background and motivation In this section we elaborate on some of the aforementioned motivations for studying generalized nil-Coxeter algebras and their finite-dimensionality. First, these algebras are interesting from a categorical perspective, as their module categories are symmetric semigroup-tensor categories (see Definition 1.3) but are not monoidal. We will discuss in the next section a Tannaka-Krein duality for such categories, as well as a central extension to a symmetric tensor category. The second motivation comes from real reflection groups: we provide a novel family of finite-dimensional algebras N C A (n, d) of type A (akin to the work of Coxeter [Cox3] and Koster [Ko]). In this context, it is remarkable (by Theorem D) that the algebras N C A (n, d) and the usual nil-Coxeter algebras N C W ((2, . . . , 2)) are the only finite-dimensional examples. As Theorem C shows, the algebras N C A (n, d) for d > 2 are similar to their "usual" nil-Coxeter analogues for d = 2. Note however that these algebras also differ in key aspects. See Theorem 5.2 and Proposition 5.3, which show in particular that for N C A (n, d) with d > 2, there are multiple "maximal" words, i.e., words killed by left-and right-multiplication by every generator T i . A more fundamental difference arises out of considerations of flat deformations, which we make precise in the remarks around equation (2.1) below. Our third motivation comes from complex reflection groups and is of much recent interest: the BMR freeness conjecture, which discusses the equality of dimensions of generic Hecke algebras and (the group algebra of) the underlying finite complex reflection group. In this paper we study the associated graded algebra, i.e. where all deformation parameters are set to zero. As shown by Marin [Mar] in some of the cases, non-Coxeter reflection groups do not come equipped with finite-dimensional nil-Coxeter analogues. We make this precise in a strong way in Theorem D above, for all complex reflection groups W . In particular, Theorem D shows that generic Hecke algebras are not flat deformations of their underlying (associated graded) nil-Coxeter analogues for complex W . This is a property shared by the algebras N C A (n, d) for d > 2 (but not by Iwahori-Hecke algebras of Coxeter groups W = W (M ), which are flat deformations of N C(M )). Indeed, if M n,d denotes the generalized Coxeter matrix corresponding to N C A (n, d), then we claim that: The generic Hecke algebras discussed above fit in a broader framework of deformation theory, which provides a fourth motivation behind this paper (in addition to the question of flatness discussed above). The theory of flat/PBW deformations of associative algebras is an area of sustained activity, and subsumes Drinfeld Hecke/orbifold algebras [Dr], graded affine Hecke algebras [Lu], symplectic reflection algebras and rational Cherednik algebras [EG], infinitesimal and other Hecke algebras, and other programs in the literature. We also highlight the program of Shepler and Witherspoon; see [SW1,SW2] and the references therein. In all of these settings, a bialgebra A (usually a Hopf algebra) acts on a vector space V and hence on a quotient S V of its tensor algebra, and one characterizes the deformations of this smash-product algebra A S V which are flat, also termed the "PBW deformations". In this regard, the significance of the generalized nil-Coxeter algebras N C(M ) is manifold. First, the above bialgebra settings were extended in recent work [Kha] to the framework of "cocommutative algebras" A, which also include the algebras N C W (d). Moreover, we characterized the PBW deformations of A Sym(V ), thereby extending in loc. cit. the PBW theorems in the previously mentioned works. The significance of our framework incorporating A = N C W (d) along with the previously studied algebras, is that the full Hopf/bialgebra structure of A -specifically, the antipode or even counit -is not required in order to characterize the flat deformations of A Sym(V ). Coming to finite-dimensionality, it was shown in the program of Shepler and Witherspoon (see e.g. [SW2]), and then in [Kha], that when the algebra A with coproduct is finite-dimensional over a field k, it is possible to characterize the graded k[t]-deformations of A Sym(V ), whose fiber at t = 1 has the PBW property. For A = N C W (d), this deformation-theoretic consideration directly motivates our classification result in Theorem D. We conclude with a third connection to the aforementioned active program on PBW deformations. We studied in [Kha] the case when (A, m, ∆) is local with ∆(m) ⊂ m ⊗ m. In this setting, if m is a nilpotent two-sided ideal, then one obtains a lot of information about the deformations of A Sym(V ), including understanding the PBW deformations, as well as their center, abelianization, and modules, especially the simple modules. Now if A = N C W (d) then m is generated by the T i ; this explains the interest above in understanding when m is nilpotent. Theorem D shows that this condition is in fact equivalent to the generalized nil-Coxeter algebra being finite-dimensional. Proof of Theorem A: Tannakian formalism for semigroup categories The remainder of this paper is devoted to proving the four main theorems in the opening section. We begin by studying the representation category of N C(M ) for a generalized Coxeter matrix M . The first assertion is that this category can never be a monoidal category in characteristic zero, and it follows from the following result. Proof. Note there is a unique possible counit, ε : Note that m ⊗ m constitutes the terms of higher 'total degree' in ∆(T i ), in the Z 0 -grading on N C(M ). Now if ∆ is multiplicative, then raising (3.2) to the m ii th power yields: This is impossible as long as the image of T i in N C(M ) is nonzero; assuming this, it follows ∆ cannot be multiplicative, hence not a coproduct on N C(M ). Finally, N C(M ) surjects onto the usual nil-Coxeter algebra N C(M 2 ) with M 2 = M ((2, . . . , 2)). As N C(M 2 ) has a Coxeter word basis indexed by W (M ), it follows that T i is indeed nonzero in N C(M ). As a consequence of Proposition 3.1 and the Tannakian formalism in [ES,Theorem 18.3], for any generalized Coxeter matrix M the module category Rep N C(M ) is necessarily not a tensor category. That said, the map ∆ : T i → T i ⊗ T i is a coproduct on N C(M ), i.e. a coassociative algebra map. The cocommutativity of ∆ implies Rep N C(M ) is a symmetric semigroup category. We now outline how to show the first theorem above, which seeks to understand Tannaka-Krein duality for such categories (possibly without unit objects). Proof of Theorem A. The proof of part (1) follows that of [ES,Theorem 18.3]; one now ignores the last statement in that proof. The additional data required in the two braided versions in part (2) can be deduced from the proof of [ES,Proposition 14.2]. We conclude this section by passing from Rep N C(M ) to an "honest" tensor category -say with k a field. Alternately, via the Tannakian formalism in [ES,Theorem 18.3], we produce a bialgebra N C(M ) that surjects onto N C(M ). Namely, N C(M ) is generated by {T i : i ∈ I} and an additional generator T ∞ , subject to the braid relations on the former set, as well as Now asking for all T i and T ∞ to be grouplike yields a unique bialgebra structure on N C(M ): and hence a monoidal category structure on Rep N C(M ), as claimed. Proof of Theorem B: Distinguished basis of words We now prove our main theorems on the algebras N C(M ) -specifically, the family N C A (n, d) -beginning with Theorem B. Note that if d = 2 then the algebra N C A (n, d) is the usual nil-Coxeter algebra, while if n = 1 then the algebra is . Theorems B and C are easily verified for these cases, e.g. using [Hum,Chapter 7]. Thus, we assume throughout their proofs below that n 2 and d 3. We begin by showing the k-rank of N C A (n, d) is at most n!(1 + n(d − 1)). Notice that N C A (n, d) is spanned by words in the T i . We now claim that a word in the T i is either zero in N C A (n, d), or equal by the braid relations to a word in which all occurrences of T n are successive, in a monomial T k n for some 1 k d − 1. To show the claim, consider a word T := · · · T a n T w T b n · · · , where a, b > 0 and T w = T i1 · · · T i k is a word in T 1 , . . . , T n−1 . Rewrite T using the braid relations if required, so that w ∈ S n has minimal length, say k. We may assume k > 0, else we would be done. Now using the braid relations T i T n = T n T i for i n − 2, further assume that i 1 = i k = n − 1 (otherwise the factors may be 'moved past' the T n using the braid relations). Similarly, i 2 = i k−1 = n − 2, and so on. Thus, if T w = 0, then assume by the minimality of (w) that T w = T n−1 T n−2 · · · T m+1 T m T m+1 · · · T n−2 T n−1 , for some 1 m n − 1. We next claim that the following relation holds in the Artin braid group B n , hence in N C An (d) for any d: This is shown by descending induction on m n − 1. Hence, If max(a, b) = 1 then the claim follows; if a > 1 then the last expression contains the substring (T n T n−1 T n )T n−1 = T n−1 T n T 2 n−1 = 0; and similarly if b > 1. This shows the claim. GENERALIZED NIL-COXETER ALGEBRAS OVER COMPLEX REFLECTION GROUPS2981 We now prove the upper bound on the k-rank. Notice that T 1 , . . . , T n−1 generate a subalgebra R n−1 ⊂ N C A (n, d) in which the nil-Coxeter relations for W An−1 = S n are satisfied. Hence the map : N C An−1 ((2, . . . , 2)) R n−1 := T 1 , . . . , T n−1 is an algebra map. Now notice by equation (4.2) that every nonzero word in and hence T w , T w ∈ R n−1 . By a similar reasoning as above, assuming w of minimal length in S n−1 , we may rewrite T such that T w = T n−1 · · · T m for some 1 m n. Carrying out this operation yields T w T k n T n−1 · · · T m for some reduced word w ∈ W An−1 (i.e., such that T w is nonzero in N C An−1 ((2, . . . , 2))). Thus, As R n−1 has at most n! generators, it follows that N C A (n, d) has at most (1 + n(d − 1)) · n! generators, which shows the desired upper bound on its k-rank. The hard part of the proof involves showing that the words T w T k n T n−1 · · · T m form a k-basis of N C A (n, d). We will require the following technical lemma on the symmetric group and its nil-Coxeter algebra. A proof is included for completeness. Lemma 4.3. Suppose W = W An−1 = S n is the symmetric group, with simple reflections s 1 , . . . , s n−1 labelled as usual. Then every element w of W An−1 \W An−2 = S n \ S n−1 can be written in reduced form as w = w s n−1 · · · s m , where w ∈ S n−1 = W An−2 and m ∈ [1, n − 1] are unique. Given such an element w ∈ S n , we have in the usual nil-Coxeter algebra N C An ((2, . . . , 2)): otherwise. Note that equation (4.4) can be thought of as a statement on lengths in the symmetric group. Proof. We first claim that w ∈ W An−1 \ W An−2 has a reduced expression in which s n−1 occurs exactly once. The proof is by induction on n: clearly the claim is true for n = 2. Now given the claim for n − 2 2, consider any reduced expression for w that contains a sub-word s n−1 w s n−1 , where w ∈ W An−2 . By the induction hypothesis, w = w s n−2 · · · s m for some w ∈ W An−3 and m ∈ [1, n − 1]. Hence if m n − 2, then w = · · · s n−1 (w s n−2 s n−3 · · · s m ) s n−1 = · · · w (s n−1 s n−2 s n−1 )(s n−3 · · · s m ) · · · , and by the braid relations, this equals a reduced expression for w ∈ W An−1 , with one less occurrence of s n−1 . A similar analysis works if m = n − 1. Repeatedly carrying out this procedure proves the claim. We can now prove the uniqueness of w , m as in the lemma. By the previous paragraph, write w ∈ W An−1 \ W An−2 as w = w 1 s n−1 w 2 , with w 1 , w 2 ∈ W An−2 and w 2 of smallest possible length, say w 2 = s i1 · · · s i k for i 1 , . . . , i k n − 2. Using the braid relations, clearly i 1 = n−2, hence i 2 = n−3 (by minimality of k). Choose the smallest l 3 such that i l = n − 1 − l. We now produce a contradiction assuming that such an integer l exists. If i l < n − 1 − l, then we may move s i l past the preceding terms, contradicting the minimality of k. Clearly i l = n − l, else w 2 was not reduced. Thus i l > n − 1 − l, whence w 2 is of the form s n−2 · · · s i l s i l −1 s i l · · · . Now verify in W An−1 that w = w 1 s n−1 s n−2 · · · s i l +1 s i l s i l −1 s i l · · · = w 1 s n−1 · · · s i l +1 s i l −1 s i l s i l −1 · · · = w 1 s i l −1 · s n−1 · s n−2 · · · s i l +1 s i l s i l −1 · · · , which contradicts the minimality of k. Thus such an integer l cannot exist, which proves that w = w 1 s n−1 · · · s m for some m ∈ [1, n − 1]. We next claim that the integer m is unique for w ∈ W An \ W An−1 . We first make the sub-claim that if w ∈ W An−1 is reduced, then so is ws n s n−1 · · · s m . To see why, first recall [Hum, Lemma 1.6, Corollary 1.7], which together imply that if wα > 0 for any finite Coxeter group W , any w ∈ W , and any simple root α > 0, then (ws α ) = (w)+1. Now the sub-claim follows by applying this result successively to (ws n · · · s j+1 , α j ) for j = n, n−1, · · · , m. Next, define C m := W An−1 ·s n s n−1 · · · s m , with C n+1 := W An−1 . It follows by the sub-claim above that \|C m \| = \|W An−1 \| = n! for all m. Hence, This shows that W An = n+1 m=1 C m , which proves the uniqueness of m in the above claim. Now write w 1 in reduced form to obtain that w = ws m · · · s n−1 is also unique. Remark 4.5. Notice that equation (4.4) holds in any algebra containing elements T 1 , . . . , T n that satisfy the braid relations and T 2 1 = 0. In particular, (4.4) holds in N C A (n, d) for n > 1. Returning to the proof of Theorem B, we now introduce a diagrammatic calculus akin to crystal theory. We first write out the n = 2 case, in order to provide intuition for the case of general n. Let M be a free k-module, with basis given by the nodes in the graph in Figure 1 below. In the figure, the node 12 2 1 should be thought of as T 1 T 2 2 T 1 (applied to the unit 1 N C A (2,d) , i.e., to the generating basis vector corresponding to ∅), and similarly for the other nodes. The arrows denote the action of T 1 and T 2 ; all remaining 1)). Since M is generated by the basis vector corresponding to the node ∅, we have a surjection : to the corresponding basis vectors in the free k-module M . Now the result for n = 2 follows by the upper bound on the k-rank, proved above. The strategy is similar for general n, but uses the following more detailed notation. For each w ∈ S l with l n, let T w denote the corresponding (welldefined) word in the alphabet {T 1 , . . . , T l−1 }, and let R l−1 denote the subalgebra of N C A (n, d) generated by these letters. Now define a free k-module M of k-rank n!(1 + n(d − 1)), with basis elements the set of words We observe here that the basis vectors B(w, k, m), B(w) are to be thought of as corresponding respectively to the words (4.7) T w T k n T n−1 · · · T m , T w , w ∈ S n , k ∈ [1, d − 1], m ∈ [1, n]. Definition 4.8. An expression for a word in N C A (n, d) of the form (4.7) will be said to be in standard form. We now define an N C A (n, d)-module structure on M , via defining a directed graph structure on B (or more precisely, on B {0}) that we now describe. The following figure (Figure 2 below) may help in visualizing the structure. The figure should be thought of as analogous to the central hexagon and either of the two "arms" in Figure 1. We begin by explaining the figure. Each node (wkm) (or (w)) corresponds to the basis vector B(w, k, m) (or B(w)). Notice that the vectors {B(w, 1, m)} {B(w)} are in bijection with the Coxeter word basis of the usual nil-Coxeter algebra N C An ((2, . . . , 2)). Let V 1 denote their span, of k-rank (n + 1)!. Now given 1 m n and 1 k d − 1 =: d , define V k,m to be the span of the basis elements {B(w, k, m) : w ∈ S n }, of k-rank n!. Then M = V 1 ⊕ k>1,m V k,m . Note as a special case that in Figure 1, the central hexagon spans V 1 , the nodes 2 k 1, 12 k 1 span V k,1 , and 2 k , 12 k span V k,2 . We now define the N C A (n, d)-action: • Let V 1,n+1 denote the k-span of {B(w) : w ∈ S n }. Then for 1 m n + 1, each V 1,m has a distinguished basis in bijection with S n ; the same holds for each V k,m with k ∈ [2, d − 1] and m ∈ [1, n]. Now equip all of the above spaces V k,m with the corresponding module structure over the usual nil-Coxeter algebra of type A n−1 . Such a structure is uniquely determined, if given w = s i1 · · · s i l ∈ S n with all i j < n, we set T w · B(1, k, m) := B(w, k, m) and T w · B(1) := B(w). • We next define the action of T n on M . Via Lemma 4.3, write w ∈ S n as w s n−1 · · · s m with w , m unique. Now using the previous paragraph, it follows that B(w, k, m) = T w T n−1 · · · T m · B(1, k, m). Correspondingly, if w ∈ S n−1 (i.e., m = n), define It remains to show that the above graph structure indeed defines an N C A (n, d)module structure on M ; then a similar argument as above (in the n = 2 case) completes the proof. In the following argument, we will occasionally use Lemma 4.3 (as well as Remark 4.5) without reference. First notice that the algebra relations involving only T 1 , . . . , T n−1 are clearly satisfied on M as it is a R n−1 -free module by construction. To verify that the relations involving T n hold on M , notice (e.g. via Figure 2) that the k-basis B of M can be partitioned into three subsets: Recall by the opening remarks in Section 4 that n 2 and d 3. We first show that the relation T d n = 0 holds as an equality of linear operators on each vector b ∈ B, and hence on the k-module M . We separately consider the cases b ∈ B i for i = 1, 2, 3, as in (4.10). (1) If b = B(w, k, m) ∈ B 1 , then b lies in the "top rows" of Figure 2. It is easily verified that T d−k−1 n · B(w, k, m) = B(w, d − 1, m), and this is killed by T n , as desired. The same reasoning shows that T d n kills b = B(w) for w ∈ S n−1 . (2) Let b = B(w, k, m) ∈ B 2 . Then the relation holds on b since T n · B(w, k, m) = 0. (These correspond to vectors in V k,m for k 2, which do not lie in the "top rows" in Figure 2.) (3) Finally, let b ∈ B 3 ; thus w ∈ S n \ S n−1 , and we write w = w s n−1 · · · s m by Lemma 4.3. It follows from Remark 4.5 that T 2 n · B(w, 1, m) = 0 and T d n · B(w) = T d−1 n · B(w , 1, m ) = 0. We next show that the relation T i T n = T n T i holds on B for all i n − 2. We consider the same three cases as in (4.10). (1) Fix w ∈ S n−1 . If b = B(w, k, m) with k 1, then verify using the aforementioned action that both T i T n · B(w, k, m) and T n T i · B(w, k, m) equal B(s i w, k + 1, m) if (s i w) > (w) and k d − 2, and 0 otherwise. Similarly, (2) Let b = B(w, k, m) with w ∈ S n \S n−1 and k 2. Then T i T n ·B(w, k, m) = 0. To compute T n T i · B(w, k, m), since i n − 2, it follows that s i w ∈ S n \ S n−1 . If T i T w = 0 then we are done since B(w, k, m) = T w ·B(1, k, m). Else note that s i w ∈ S n \ S n−1 , whence T n T i · B(w, k, m) = T n · B(s i w, k, m) = 0 from above. (3) Finally, let w ∈ S n \ S n−1 and write w = w s n−1 · · · s m by Lemma 4.3. First suppose b = B(w, 1, m). If (s i w) < (w), then it is not hard to show that both T i T n · B(w, 1, m) and T n T i · B(w, 1, m) vanish. Otherwise both terms are equal to B(s i w s n−1 · · · s m−1 , 1, m ). A similar analysis shows that if (s i w) < (w), then T i T n · B(w) = T n T i · B(w) = 0, otherwise T i T n · B(w) = T n T i · B(w) = B(s i w , 1, m ). Next, we show that the braid relation T n−1 T n T n−1 = T n T n−1 T n holds on B. This is the most involved computation to carry out. We consider the same three cases as above. (1) Fix w ∈ S n−1 . If b = B(w, k, m) with k 2, then it is easily verified that both sides of the braid relation kill B(w, k, m). If instead k = 1, then T n T n−1 T n · B(w, 1, m) = T n T n−1 · B(w, 2, m) = 0. Now if the braid relation holds on B(w) for all w ∈ S n , then T n−1 T n T n−1 · B(w, 1, m) = T n−1 · T n T n−1 T n · B(ws n−1 · · · s m ) = T n−1 T n−1 T n T n−1 · B(ws n−1 · · · s m ) ∈ T 2 n−1 · M = 0, where the last equality follows from the definition of M as an R n−1 -module. It thus suffices for this case to verify that the braid relation holds on B(w) for w ∈ S n . This is done by considering the following four sub-cases. (a) If w ∈ S n−2 commutes with s n−1 , s n , then both T n T n−1 T n · B(w) and T n−1 T n T n−1 · B(w) are easily seen to equal B(s n−1 w, 1, n − 1). GENERALIZED NIL-COXETER ALGEBRAS OVER COMPLEX REFLECTION GROUPS2987 Notice this calculation shows the "braid-like" action of T n , T n−1 on strings of the type Similarly, one shows that T n T n−1 T n · B(w, 1, m) = 1(m < m < m)B(w s n−2 · · · s m−2 s n−1 · · · s m −1 , 1, m), which verifies that the last braid relation holds in the last case. Thus the algebra relations hold on all of M , making it an N C A (n, d)-module generated by B(1), as claimed. In particular, N C A (n, d) M as k-modules, by the analysis in the first part of this proof. This completes the proof of all but the last assertion in Theorem B. Finally, the nil-Coxeter algebra N C A l ((2, . . . , 2)) surjects onto R l , and R l R l · B(∅) ⊂ V 1 is free of k-rank (l + 1)! from above. Hence R l N C A l ((2, . . . , 2)), as desired. Proof of Theorem C, primitive elements, and categorification In this section we continue our study of the algebras N C A (n, d), starting with Theorem C. Proof of Theorem C. We retain the notation of Theorem B. Via the k-module isomorphism M N C A (n, d), we identify the basis element B(w, k, m) with T w T k n T n−1 · · · T m and B(w) with T w , where w ∈ S n , k ∈ [1, d − 1], and m ∈ [1, n]. Let : B → Z 0 be as in equation (1.10). We now claim that if T = T i1 · · · T i l is any nonzero word in N C A (n, d), then l is precisely the length of T when expressed (uniquely) in standard form (4.7). The proof is by induction on l. For l = 1, T i is already in standard form (and nonzero). Now given a word T = T i T of length l + 1 (so T has length l and satisfies the claim), write T via the induction hypothesis as a word in standard form of length l. Now the proof of Theorem B shows that applying any T i to this standard form for T either yields zero or has length precisely l + 1. This proves the claim. The above analysis shows (1) and (2). Now suppose k is a field. Then the algebra N C A (n, d) has a maximal ideal m = {T i : i ∈ I} ; in fact, m has k-corank 1 by the proof of Theorem B. Moreover, m is local because any element of A \ m is invertible. (In particular, one understands representations of the algebra N C A (n, d), e.g. by [Kha,§6.1].) The aforementioned claim also proves that m l+1 = 0, where l := An−1 (w • ) + d + n − 2. This is because any nonzero word can be expressed in standard form without changing the length. As an immediate consequence, we have: Corollary 5.1. If k is a field and T 1 , . . . , T n all have graded degree 1, the Hilbert-Poincaré series of N C A (n, d) is the polynomial The proof also uses the standard result that the Hilbert-Poincaré series of the usual nil-Coxeter algebra N C A (n, 2) is [n] q ! (see e.g. [Hum,§3.12,3.15]). Next, we discuss a property that was explored in [Kho] for the usual nil-Coxeter algebras N C A (n, 2): these algebras are always Frobenius. We now study when the algebras N C A (n, d) are also Frobenius for d 3. As the following result shows, this only happens in the degenerate case of n = 1, i.e., k[T 1 ]/(T d 1 ). Theorem 5.2. Suppose k is a field. Given n 1 and d 2, the algebra N C A (n, d) is Frobenius if and only if n = 1 or d = 2. One checks via equation (2.1) that these conditions are further equivalent to (the group algebra of) the "generalized Coxeter group" W (M n,d ) being a flat deformation of N C A (n, d). The proof of Theorem 5.2 crucially uses the knowledge of "maximal", i.e., primitive words in the algebra N C A (n, d). Formally, given a generalized Coxeter matrix M , say that an element x ∈ N C(M ) is left (respectively, right) primitive if mx = 0 (respectively, xm = 0), cf. Theorem C(3). Now x is primitive if it is both left-and right-primitive. Denote these sets of elements respectively by Prim L (N C(M )), Prim R (N C(M )), Prim(N C(M )). Proposition 5.3. Every generalized nil-Coxeter algebra N C(M ) is equipped with an anti-involution θ that fixes each generator T i . Now θ is an isomorphism : Prim L (N C(M )) ←→ Prim R (N C(M )). Moreover, the following hold. (1) If W (M ) is a finite Coxeter group with unique longest word w • , then (3) If N C(M ) = N C A (n, d) with n 2 and d 3, then: (a) Prim L (N C(M )) is spanned by T w• := T w • T n T n−1 · · · T 1 and the n(d − 2) words In all cases, the map θ fixes both Prim(N C(M )) as well as the lengths of all nonzero words. Proof. The first two statements are obvious since θ preserves the defining relations in k {T i : i ∈ I} . The assertion in (1) is standard -see e.g. [Hum,Chapter 7]and (2) is easily verified. We next classify the left-primitive elements as in (3)(a). Suppose for some k 2 and 1 m n that T = T w • T k n T n−1 · · · T m . Then clearly T i T = 0 for all i < n, and T n T = 0 since k 2, as discussed in the proof of Theorem B. Similarly, if T = T w• then T i T = 0 for i < n, and we also computed in the proof of Theorem B that T w• = T 1 · · · T n−1 T n T w • . Hence, To complete the proof of (3)(a), it suffices to show that no nonzero linear combination of the remaining words of the form T w T k n T n−1 · · · T m is left-primitive. Suppose first that there is a word w ∈ W An−1 such that the coefficient of T w is nonzero. In that case, choose such an element w of smallest length, and left-multiply the linear combination by T d−1 n T w • w −1 . As discussed in the proof of Theorem B, this kills all terms T w T k n T w with w , w ∈ W An−1 and k 1. Moreover, by [Hum,Chapter 7], left-multiplication by T w • w −1 also kills all terms of the same length that are not T w . Thus we are left with T d−1 n T w • w −1 T w = T d−1 n T w • = 0, so the linear combination was not left-primitive. The other case is that all words in the linear combination are of the form T w T k n T n−1 · · · T m with k 1. Once again, choose w ∈ W An−1 of smallest length for which the corresponding word has nonzero coefficient, and left-multiply by T w • w −1 . This yields a nonzero linear combination by the analysis in Theorem B, which proves the assertion about left-primitivity. We next identify the primitive elements in N C(M ) = N C A (n, d). The first claim is that T k := T w • T k n T n−1 · · · T 1 is fixed by θ. Indeed, we compute using the braid relations in type A that θ fixes T w • ∈ R n−1 and T w • ∈ R n−2 . Hence, (5.4) θ(T k ) = T 1 · · · T n−1 T k n T w • T n−1 · · · T 1 = T 1 · · · T n−1 T w • T k n T n−1 · · · T 1 = T k . Using this we claim that T k is right-primitive. Indeed, if i < n, then T k T i = T 1 · · · T n−1 T k n T w • T i = 0, while for i = n, we compute: We now claim that no linear combination of the remaining left-primitive elements listed in (3)(a) is right-primitive. Indeed, let m 0 denote the minimum of the mvalues in words with nonzero coefficients; then m 0 > 1 by the above analysis. Now right-multiply by T m0−1 · · · T 1 . This kills all elements with m-value > m 0 + 1, since T m0−1 commutes with T n T n−1 · · · T m , hence can be taken past them to multiply against T w • and be killed. The terms with m-value equal to m 0 are not killed, by the analysis in Theorem B. It follows that such a linear combination is not rightprimitive, which completes the classification of the primitive elements in (3)(b). Next, that Prim(N C(M )) is fixed by θ was shown in equation (5.4). Moreover, if N C(M ) equals N C A (n, d) or kW (M ) with W (M ) finite, then it is equipped with a suitable length function . Now θ preserves the length because the algebra relations are -homogeneous and preserved by θ. Remark 5.5. In light of Proposition 5.3, it is natural to ask how to write rightprimitive words in standard form. More generally, given w = w s n−1 · · · s m for unique w ∈ S n−1 and m ∈ [1, n] (via Lemma 4.3), we have: T m · · · T n−1 T k n T w = T w T k n T n−1 · · · T m , where w = s m · · · s n−1 w . With Proposition 5.3 in hand, we turn to the Frobenius property of N C A (n, d). The following proof reveals that N C A (n, d) is Frobenius if and only if Prim(N C(M )) is one-dimensional. Proof of Theorem 5.2. For finite Coxeter groups W (M ), the corresponding nil-Coxeter algebras N C(M ) are indeed Frobenius; see e.g. [Kho,§2.2]. It is also easy to verify that N C A (1, d) = k[T 1 ]/(T d 1 ) is Frobenius, by using the symmetric bilinear form uniquely specified by: σ(T i 1 , T j 1 ) = 1(i + j = d − 1). Thus, it remains to show that for n 2 and d 3, the algebra N C A (n, d) is not Frobenius. Indeed, if N C A (n, d) is Frobenius with nondegenerate invariant bilinear form σ, then for each nonzero primitive p there exists a vector a p such that 0 = σ(p, a p ) = σ(pa p , 1). It follows that we may take a p = 1 for all p. Now the linear functional σ(−, 1) : Prim(N C A (n, d)) → k is nonsingular, whence dim k Prim(N C A (n, d)) = 1. Thus n = 1 or d = 2 by Proposition 5.3. We conclude this section by discussing the connection of N C A (n, d) to the categorification by Khovanov [Kho] of the Weyl algebra W n := Z x, ∂ /(∂x = 1 + x∂). Namely, the usual type A nil-Coxeter algebra A n := N C A (n, 2) is a bimodule over A n−1 , and this structure was studied in loc. cit., leading to the construction of tensor functors categorifying the operators x, ∂. We now explain how the algebra N C A (n, d) fits into this framework. Proposition 5.6. For all n 1 and d 2, we have an isomorphism of A n−1bimodules: When d = 2, this result was shown in [Kho,Proposition 5]. For general d 2, using the notation of [Kho], this result implies in the category of A n−1 -bimodules that the algebra N C A (n, d) corresponds to 1 + (d − 1)x∂ (including the previously known case of d = 2). In particular, Proposition 5.6 strengthens Theorems B and C, which explained a left A n−1 -module structure on N C A (n, d) (namely, that N C A (n, d) is free of rank 1 + n(d − 1)). Proof of Proposition 5.6. From the proof of Theorem B, the algebra N C A (n, d) has a 'regular representation' ϕ : , and m ∈ [1, n]. Also recall the subspaces V k,m defined in the discussion following equation (4.7): V k,m = w∈Sn kB(w, k, m). By Theorem B, M k := n m=1 ϕ(V k,m ) is a free left A n−1 -module of rank one. It is also a free right A n−1 -module of rank one, using the anti-involution θ from Proposition 5.3 and Remark 5.5. In fact, the uniqueness of the standard form (4.7) shown in the proof of Theorem B, implies that for all 1 k d − 1, the map a ⊗ a → aT k n a , is an isomorphism of A n−1 -bimodules. Now the result follows from (the proof of) Theorem B. Remark 5.7. Notice that the proof of Proposition 5.6 also categorifies Corollary 5.1. Proof of Theorem D: Finite-dimensional generalized nil-Coxeter algebras We now prove Theorem D, which classifies the generalized nil-Coxeter algebras of finite k-rank. The bulk of the proof involves showing (1) =⇒ (2). We again employ the diagrammatic calculus used to show Theorem B, now applied to the five diagrams in Figure 3 below. We begin by assuming that W = W (M ) is a generalized Coxeter group, and classify the algebras N C(M ) that have finite k-rank. Following this classification, we address the remaining finite complex reflection groups W (and all d), followed by the infinite discrete complex reflection groups with their Coxeter-type presentations. Case 1. Suppose m ii = 2 for all i ∈ I. In this case W (M ) is a Coxeter group, so by e.g. [Hum,Chapter 7], N C(M ) has a k-basis in bijection with W (M ), which must therefore be finite. (The "+" at the head of an arrow refers precisely to the index increasing by 1.) It is easy to verify that the defining relations of N C(M ) hold in End k (M ), as they hold on each A r , B r , C r , D r . Therefore M is a module over N C(M ) that is generated by A 1 , but is not finitely generated as a k-module. As N C(M ) M , N C(M ) is also not a finitely generated k-module. GENERALIZED NIL-COXETER ALGEBRAS OVER COMPLEX REFLECTION GROUPS2991 This approach is used in the remainder of the proof, to obtain a k-basis and the N C(M )-action on it, from the diagrams in Figure 3. Thus we only mention the figure corresponding to each of the cases below. Case 3. Figure 3.1 is actually a special case of Figure 3.2, and was included to demonstrate a simpler case. Now suppose more generally that there are two nodes α, γ ∈ I such that m αα , m γγ 3. Since the Coxeter graph is connected, there exist nodes β 1 , . . . , β m−1 for some m 1 (in the figure we write m := m − 1), such that α ←→ β 1 ←→ · · · ←→ β m−1 ←→ γ is a path (so each successive pair of nodes is connected by at least a single edge). Now appeal to where each T i kills all basis vectors above, except for the actions obtained from Figure 3.2. Once again, M is generated by A 1 , so proceed as above to show that N C(M ) is not finitely generated. Case 4. The previous cases reduce the situation to a unique vertex α in the Coxeter graph of M for which m αα 3. The next two steps show that α is adjacent to a unique node γ, and that m αγ = 3. First suppose α is adjacent to γ with m αγ 4. Now appeal to Figure 3.3, setting (s, t, u) (α, α, γ), and define an N C(M )module structure on M := span k {A r , B r , C r : r 1}. Then proceed as above. Case 5. Next suppose α is adjacent in the Coxeter graph to two nodes γ, δ. By the previous case, m αγ = m αδ = 3. Now appeal to Figure 3.4 with m = 1, to define an N C(M )-module structure on M := span k {A r , B 1r , B 1r , C r , D r : r 1}, and proceed as in the previous cases. We now observe that if N C(M ) is finitely generated, then so is N C M ((2, . . . , 2)), which corresponds to the Coxeter group W M ((2, . . . , 2)). Hence the Coxeter graph of M is of finite type. These graphs were classified by Coxeter [Cox1,Cox2]. We now rule out all cases other than type A, in which case the above analysis shows that d = (2, . . . , 2, d) or (d, 2, . . . , 2). Case 6. First notice that dihedral types (i.e., types G 2 , H 2 , I) are already ruled out by the above cases. The same cases also rule out one possibility in types B, C, H, where we may now set n 3. For the remaining cases of types B, C, H, assume that the Coxeter graph is labelled α ←→ β 1 ←→ · · · ←→ β m−1 ←→ γ, with m αα 3, m γγ = 2, m βm−1γ 4. In this case we construct the N C(M )-module M by appealing to Figure 3.5; now proceed as above. Case 7. The next case is of type D n , with n 4. Notice that α is an extremal (i.e., pendant) vertex by the above analysis. First assume α is the extremal node on the "long arm" of the Coxeter graph. Now appeal to Figure 3.4 with m = n − 2, to construct an N C(M )-module M . The other sub-case is when α is one of the other two extremal nodes in the D n -graph. Define the quotient algebra N C (M ) whose Coxeter graph is of type D 4 (i.e., where we kill the n − 4 generators T i in the long arm that are the furthest away from α). Now repeat the construction in the previous paragraph, using Figure 3.4 with m = 2. It is easy to verify that the space M is a module for N C (M ), hence for the algebra N C D4 ((2, 2, 2, m αα )). This allows us to proceed as in the previous sub-case and show that N C (M ) is not finitely generated, whence neither is N C(M ). Case 8. If the Coxeter graph is of type E, then we may reduce to the D n -case by the analysis in the previous case. Hence it follows using Figure 3.4 that N C(M ) is not finitely generated. Case 9. If the Coxeter graph is of type F 4 , then we may reduce to the B n -case by the analysis in Case 7. It now follows from Case 6 that N C(M ) is not finitely generated. This completes the classification for generalized Coxeter groups W (M ). We now appeal to the classification and presentation of all finite complex reflection groups, whose Coxeter graph is connected. These groups and their presentations are listed in [BMR2,. In what follows, we adopt the following notation: if W = G m for 4 m 37, then the corresponding generalized nil-Coxeter algebras will be denoted by N C m (d). Similarly if W = G(de, e, r), then we work with N C (de,e,r) (d). In what follows, we will often claim that N C W (d) is not finitely generated (over k), omitting the phrase "unless it is the usual nil-Coxeter algebra over a finite Coxeter group". Case 10 (Exceptional types with finite Coxeter graph). If W = G m for m = 4, 8, 16, 25, 32, then its Coxeter graph is of type A. This case has been addressed above; thus the only possibility that N C W (d) has finite rank is that it equals N C An ((2, . . . , 2, d)) for d 2, as desired. Next if W = G m for m = 5, 10, 18, 26, then its Coxeter graph is of type B, which was also addressed above and never yields an algebra of finite k-rank. Now suppose W = G 29 . Then s, t, u form a sub-diagram of type B 3 , whence the quotient algebra N C 29 generated by T s , T t , T u is not finitely generated, by arguments as in Case 7 above. It follows that N C 29 (d) is also not finitely generated. The next case is if W = G m for m = 6, 9,14,17,20,21. In this case the Coxeter graph is of dihedral type, which was also addressed above. Case 11 (All other exceptional types). For the remaining exceptional values of m ∈ [4, 37], with W not a finite Coxeter group, we will appeal to Figure 3.3. There are three cases: first, suppose m = 31. In this case, set (s, t, u) (s, u, t) in Figure 3.3 and define an N C m (d)-module M that is k-free with basis {A r , B r , C r : r 1}. Now proceed as above. Next if m = 33, 34, then set (s, t, u) (w, t, u) in Figure 3.3 to define an N C m (d)-module M , and proceed as above. Case 12 (The infinite families). It remains to consider the six infinite families enumerated in [BMR2], which make up the family G(de, e, r). Three of the families consist of finite Coxeter groups of types A, B, I, which were considered above. We now consider the other three families. (a) Suppose W = G(de, e, r) with e 3. Then by [BMR2, Table 1], consider the quotient algebra N C (de,e,r) of N C (de,e,r) (d) which is generated by s, t := t 2 , u := t 2 , by killing all other generators T i . The generators of N C (de,e,r) now satisfy the relations Thus, use Figure 3.3 to define an N C (de,e,r) -module structure on M , and proceed as above to show that N C (de,e,r) (d) is not finitely generated. Table 2]. Apply a similar argument as in the previous sub-case, using the same generators and the same figure. (c) Suppose W = G(e, e, r) with e 2 and r > 2. If e = 2, then G(2, 2, r) is a finite Coxeter group, hence was addressed above. Next, if r > 3 then killing T s reduces to (a quotient of) the D n -case, which was once again addressed above. Finally, suppose r = 3 e. Setting s := t 3 , t := t 2 , u := t 2 , the generators of N C (e,e,3) satisfy Once again, use Figure 3.3 to define an N C (e,e,3) -module structure on M , and proceed as above. This completes the proof of (1) =⇒ (2) for finite complex reflection groups. Next, by e.g. [Hum,Chapter 7], for no infinite Coxeter group W is N C W ((2, . . . , 2)) a finitely generated k-module, whence the same result holds for N C W (d) when all d i 2. We now use the classification of the (remaining) infinite complex reflection groups W associated to a connected braid diagram. These groups were described in [Pop1] and subsequently in [Mal]. Thus, there exists a complex affine space E with group of translations V ; choosing a basepoint v 0 ∈ E, we can identify the semidirect product GL(V ) V with the group A(E) of affine transformations of E. Moreover, W ⊂ A(E). Define Lin(W ) to be the image of W in the factor group GL(V ), and Tran(W ) to be the subset of W in V , i.e., It remains to consider three cases for irreducible infinite complex reflection groups W . Case 13. The group W is noncrystallographic, i.e., E/W is not compact. Then by [Pop1,Theorem 2.2], there exists a real form E R ⊂ E whose complexification is E, i.e., E R ⊗ R C = E. Moreover, by the same theorem, restricting the elements of W to E R yields an affine Weyl group W R . Hence if N C W (d) is a finitely generated k-module, then so is N C W R ((2, . . . , 2)), which is impossible. Case 14. The group W is a genuine crystallographic group, i.e., E/W is compact and Lin(W ) is not the complexification of a real reflection group. Such groups were studied by Malle in [Mal], and Coxeter-type presentations for these groups were provided in Tables I, II in loc. cit. Specifically, Malle showed that these groups are quotients of a free monoid by a set of braid relations and order relations, together with one additional order relation R m0 0 = 1. We now show that for none of these groups W is the algebra N C W (d) (defined in Definition 1.11) a finitely generated k-module. To do so, we proceed as above, by specifying the sub-figure in Figure 3 that corresponds to each of these groups. There are three sub-cases: (1) Suppose W is the group [G(3, 1, 1)] in [Mal, Tables I, II], we appeal to Figure 3.3 as in Case 11 above, with three suitably chosen generators in each case. Case 15. Finally, we consider the remaining "nongenuine, crystallographic" cases as in [Pop1, Table 2]. Thus, E/W is compact and Lin(W ) is the complexification of a real reflection group. In these cases, verify by inspection from [Pop1, Table 2] that the cocycle c is always trivial. Thus W = Lin(W ) Tran(W ), with W := Lin(W ) a finite Weyl group, and Tran(W ) a lattice of rank 2\|I \|, where I indexes the simple reflections in the Weyl group W . We now claim that the corresponding family of generalized nil-Coxeter algebras N C W (d) are not finitely generated as k-modules. To show the claim requires a presentation of W in terms of generating reflections. The following recipe for such a presentation was communicated to us by Popov [Pop2]. Notice from e.g. [Pop1, Table 2] that Tran(W ) is a direct sum of two Lin(W )-stable lattices Λ 1 and Λ 2 = αΛ 1 (with α ∈ R), each of rank \|I \|. Thus, Λ 1 ∼ = Λ 2 as ZW -modules, with W = Lin(W ) a finite real reflection group as above. Moreover, for j = 1, 2, the semidirect product S j := W Λ j is a real crystallographic reflection group whose fundamental domain is a simplex; this yields a presentation of S j via \|I \| + 1 generating reflections in the codimension-one faces of this simplex. One now combines these presentations for S 1 , S 2 to obtain a system of \|I \| + 2 generators for W ; see in this context the remarks following [Mal,Theorem 3.1]. In this setting, it follows by [Pop1,Theorem 4.5] that each S j is isomorphic, as a real reflection group, to the affine Weyl group W over W , since the Coxeter type of S j is determined by the Coxeter types of W and Λ j . Thus W is in some sense a 'double affine Weyl group'. (For simply-laced W , it is also easy to verify by inspection from [Pop1, Table 2] that Λ j is isomorphic as a ZW -module to the root lattice for W , whence S j ∼ = W for j = 1, 2.) Equipped with this presentation of W from [Pop2], we analyze N C W (d) as follows. Fix a ZW -module isomorphism ϕ : Λ 1 → Λ 2 , and choose affine reflections s 0j ∈ S j , corresponding to µ 1 and µ 2 = ϕ(µ 1 ) respectively, which together with the simple reflections {s i : i ∈ I } ⊂ W generate S j ∼ = W . Then W W upon quotienting by the relation: s 01 = s 02 . Using the presentation of N C W (d) via the corresponding \|I \| + 2 generators {T i : i ∈ I } {T 01 , T 02 }, N C W ((2, . . . , 2)) N C W ((2, . . . , 2))/(T 01 −T 02 ) ∼ = N C W ((2, . . . , 2)), and this last term is an affine Weyl nil-Coxeter algebra, hence is not finitely generated as a k-module. Therefore neither is N C W (d), as desired. This shows that (1) =⇒ (2); the converse follows by [Hum,Chapter 7] and Theorem B. We now show that (2) and (3) are equivalent. Note from the above case-bycase analysis that if N C W (d) is not finitely generated, then either it surjects onto an affine Weyl nil-Coxeter algebra N C W ((2, . . . , 2)), or one can define a module M as above, and for each r 1 there exists a word T wr ∈ N C W (d), expressed using O(r) generators, which sends the k-basis vector A 1 ∈ M to A r . It follows in both cases that m is not nilpotent. Next, if W = W (M ) is a finite Coxeter group, then it is well-known (see e.g. [Hum,Chapter 7]) that m is nilpotent. Finally, if N C(M ) = N C A (n, d), then m is nilpotent by Theorem C. This shows (2) ⇐⇒ (3). The final statement on the length function and the longest element also follows from [Hum,Chapter 7] and Theorem C. Remark 6.2. If M is a generalized Coxeter matrix with some m ij = ∞, then we can similarly work with M the k-span of {a r , b r : r 1}, where T i : a r → b r , b r → 0, T j : b r → a r+1 , a r → 0, and all other T k kill M . It follows that N C(M ) once again has infinite k-rank.	2017-05-24T22:21:29.339Z	2016-01-29T00:00:00.000	{ "year": 2016, "sha1": "95fbf246a272ab882727a3aee85c0cd4ccf06e31", "oa_license": "publisher-specific, author manuscript", "oa_url": "https://doi.org/10.1090/tran/7304", "oa_status": "HYBRID", "pdf_src": "Arxiv", "pdf_hash": "5862c47cc2d6107099ece87669e0fa4b18b0b198", "s2fieldsofstudy": [ "Mathematics" ], "extfieldsofstudy": [ "Mathematics" ] }
259897241	pes2o/s2orc	v3-fos-license	Non-Specific Inhibition of Dipeptidyl Peptidases 8/9 by Dipeptidyl Peptidase 4 Inhibitors Negatively Affects Mesenchymal Stem Cell Differentiation DPP4 may play a relevant role in MSC differentiation into osteoblasts or adipocytes. Dipeptidyl peptidase 4 (DPP4) inhibitors (DPP4i), such as sitagliptin and vildagliptin, are used as antidiabetic drugs. However, vildagliptin is not a specific DPP4i and also inhibits DPP8/9, which is involved in energy metabolism and immune regulation. The aim of this study is to evaluate how sitagliptin, vildagliptin or 1G244 (a DPP8/9 specific inhibitor) may influence cell viability, as well as osteogenic and adipogenic differentiation in human mesenchymal stem cells (MSC). Viability, apoptosis, osteoblastogenesis and adipogenesis markers, as well as protein synthesis of β-catenin, were studied in MSC cultures induced to differentiate into osteoblasts or adipocytes in the presence or absence of sitagliptin, vildagliptin or 1G244. The two tested DPP4i did not affect MSC viability, but 1G244 significantly decreased it in MSC and osteoblast-induced cells. Additionally, 1G244 and vildagliptin inhibited osteogenesis and adipogenesis, unlike sitagliptin. Therefore, inhibition of DPP4 did not affect MSC viability and differentiation, whereas inhibition of DPP8/9 negatively affected MSC. To the best of our knowledge, these results show for the first time that DPP8/9 have an important role in the viability and differentiation of human MSC. This data can be considered for human clinical use of drugs affecting DPP8/9 activity. Introduction Dipeptidyl peptidase 4 (DPP4) or cluster of differentiation 26 (CD26; E.C. 3.4.14.5) is a protease ubiquitously synthesized. It has been detected in numerous organs and tissues such as the lung, kidney, pancreas, uterus, prostate, blood vessels, brain, thymus, bone, lymph nodes and spleen. It is found on the surface of different cells, including epithelial, endothelial and immune ones, among others. In addition, transmembrane-domain cleavage releases a soluble form of the enzyme that can be identified in blood plasma [1]. Through its protease activity, it can cleave various substrates such as chemokines, growth factors, fibronectin, neuropeptides like neuropeptide Y (NPY) and substance P, as well as incretin hormones [gastric inhibitory polypeptide or glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1)], thereby regulating their concentrations and functions. Due to the diversity of substrates on which it acts, its activity plays a role in regulating numerous cellular processes such as proliferation, adhesion, differentiation, immunomodulation and apoptosis [2,3]. 2 of 20 Among the main substrates of DPP4 are GLP-1 and GIP incretins. These hormones are secreted by gut L and K cells, respectively. Their physiological functions involve control of blood glucose concentration. This is accomplished by stimulating insulin secretion or decreasing glucagon production [4]. Circulating incretins are rapidly degraded by DPP4. Therefore, inhibition of this protease increases GLP1 levels and, thus, the ability to metabolize glucose. On this basis, numerous DPP4 inhibitor (DPP4i) molecules have been developed as antidiabetic drugs. These molecules belong to the gliptin family. Among others, they include sitagliptin, saxagliptin, linagliptin and vildagliptin. DPP4i increases GLP-1 and GIP levels, which inhibit glucagon release, increase insulin secretion and decrease gastric emptying, as well as blood glucose levels in diabetic patients [5]. The advantages of these drugs include that they have a very low risk of hypoglycemia, are very well tolerated and have a neutral effect on weight [6]. DPP4 is able to recognize different substrates involved in different physiological functions. Therefore, the generalized use of DPP4i for type-2 diabetes mellitus (T2DM) treatment has revealed other effects derived from DPP4 inhibition [7]. Thus, in recent years, the study of how DPP4 inhibition can affect other pathologies associated with cardiovascular or renal systems, as well as bone metabolism, for example, has increased; many of them are related to T2DM [8][9][10]. DPP4 activity modulates the physiology of different cell types and tissues. Among them are mesenchymal stem cells (MSC) [11][12][13]. MSC in adults are involved in organism homeostasis. They mainly participate in tissue regeneration under both physiological and pathological conditions [14]. MSC have been detected in different tissues, including bone marrow, fatty ones, periodontal ligament, synovium, umbilical cord, placenta and hair follicle, among others [15]. In vitro, they are characterized by their high proliferation capacity, adherence to plastic and ability to differentiate into osteoblasts, adipocytes or chondrocytes under different stimuli. In addition to their differentiation ability, the presence of surface markers, such as CD73, CD90 and CD105, as well as the absence of haematopoietic markers, like CD34 and CD45, have been used to define them [16]. They can be manipulated and cultured in vitro, exhibiting interesting immunomodulatory and differentiation capacities. Therefore, MSC are a tool with high potential for applications in regenerative medicine [17]. In bone marrow, during aging, MSC may differentiate more into adipocytes than osteoblasts. This increases adiposity in bone marrow, loss of bone mass and risk of fractures [18]. Some studies have associated high plasma DPP4 levels with increased bone turnover, as well as increased prevalence of osteoporotic fractures in postmenopausal women [19]. Furthermore, it has been found that in normoglycemic postmenopausal women, DPP4 activity is associated with an increased risk of osteoporosis, insulin resistance, inflammation and decreased GLP-1 levels [20]. In addition, high plasma DPP4 levels in type II diabetic patients have been linked with bone loss, as well as an increased risk of bone fractures [21]. Among the DPP4i used for the treatment of T2DM, there are some that are not specific for DPP4 and can also inhibit DPP8/9 [22]. Therefore, these DPP4i may have additional effects on patients besides their antidiabetic effects. DPP8/9, as DPP4, belong to the dipeptidyl peptidase 4 activity and/or structure homologues (DASH) family [23]. But unlike DPP4, they do not possess a transmembrane domain. Therefore, their localization is exclusively cytoplasmic [24]. The expression of genes encoding DPP8/9 in the organism is ubiquitous. They have been related to cell behavior, energy metabolism and immune regulation [25]. However, their putative roles in the viability and differentiation of MSC have not been studied so far. Therefore, the aim of this study is to evaluate how DPP4 and/or DPP8/9 inhibition may influence the viability and osteogenic/adipogenic differentiation of MSC. Such knowledge should shed new light on the development of new therapeutic strategies in regenerative medicine for the treatment of pathologies including T2DM, osteoporosis and obesity. It will also provide information on possible side effects of certain non-specific DPP4i drugs on patients with T2DM, allowing them to take appropriate precautions, if necessary. To achieve these goals, human bone marrow MSC were treated with 1G244 (a specific DPP8/9 inhibitor) or DPP4i drugs (vildagliptin and sitagliptin). The former can inhibit DPP8/9, in addition to DPP4, whereas the latter is specific for DPP4 [22]. Cells were detached with trypsin-EDTA (Gibco) when cultures reached near 90% confluence. After 3 or 4 passages, MSC were seeded in culture plates (Nalgene-Nunc-Thermo Fisher Scientific) at a density of about 1000 cells/cm 2 . Once a confluence between 60 and 80% was reached, they were induced to differentiate into adipocytes or osteoblasts. To induce adipocyte differentiation, culture media were supplemented with 5 × 10 −7 M dexamethasone, 50 µM indomethacin and 0.5 mM isobutylmethylxanthine. During differentiation, two stages were considered for collecting samples (preadipocytes at 6 to 7 days and mature adipocytes at 13 to 14 days). On the other hand, differentiation into osteoblasts was carried out by supplementing culture media with 10 −8 M dexamethasone, 10 mM β-glycerolphosphate and 0.2 mM ascorbic acid. Cultures induced into osteoblasts were grown for three to four weeks to allow extracellular mineralization. All inducers were from Sigma-Aldrich. Vildagliptin, Sitagliptin or 1G244 Treatments MSC cultures not differentiated or induced to differentiate into adipocytes or osteoblasts were treated with 10 µM of vildagliptin from Selleckchem (Houston, TX, USA), sitagliptin or 1G244 (both from Sigma-Aldrich). Cultures maintained under the same conditions but not treated with inhibitors were used as controls. MTT Assay for Cell Viability Cell viability was determined using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich). MSC were seeded in 96-well plates at a density of 4000 cells per well in culture media. They were incubated for 24 h prior to treatment. Subsequently, cells were treated with 10 µM of vildagliptin, sitagliptin or 1G244. After 48 h, media were removed and 100 µL of MEMα supplemented with 1 mg MTT/mL was added. After 2 h of incubation in culture conditions, solutions were removed. Insoluble formazan crystals produced were dissolved in isopropanol. Absorbance at 570 nm of resulting solutions was measured, using absorbance at 650 nm as reference, with a PowerWave XS microplate spectrophotometer from BioTek Instruments (Winooski, VT, USA). In MSC cultures induced to differentiate into adipocytes and osteoblasts in presence of vildagliptin, sitagliptin or 1G244, cell viability was measured on day seven after differentiation started, as described above. Apoptosis Assay Culture media were removed, and apoptotic cells were detected with 4 µM Caspase 3/7 reagent (CellEvent) from Thermo Fisher Scientific in phosphate-buffered saline (PBS) with 5% FBS. After incubation for 1 h at 37 • C, cells were fixed with 3.7% formaldehyde, and nuclei were stained with Hoechst stain from Sigma-Aldrich. Images were taken with a fluorescence microscope and analyzed with Image J software version from National Institutes of Health (NIH; Bethesda, MD, USA) <https://imagej.nih.gov/ij>. Caspaseactivity signals and the corresponding Hoechst-staining signals were quantified. Apoptosis was calculated as caspase/Hoechst staining ratios. Mineralized Extracellular-Matrix (Osteoblasts) and Lipid Droplet (Adipocytes) Staining Mineralization in cultures induced to differentiate into osteoblasts was evaluated by staining with alizarin red S. For this purpose, cultures were fixed with 3.7% formaldehyde for 10 min at room temperature. Then, they were stained for 10 min with a 40 mM solution of alizarin red S (pH 4.1) from PanReac AppliChem (Darmstadt, Germany) and then washed with 60% isopropanol. For quantification, stainings were eluted with 10% acetic acid and neutralized with 10% ammonium hydroxide. Absorbance of the resulting solutions were measured at 405 nm using a PowerWave XS microplate spectrophotometer. Formation of lipid droplets in cultures induced to differentiate into adipocytes was evaluated by oil red O staining. Cultures were fixed with 3.7% formaldehyde for 15 min and stained with a solution prepared by mixing six volumes of oil red O at 0.35% (w/v, in isopropanol) with four volumes of distilled water. After 20 min of incubation, cells were washed with distilled water and stained with hematoxylin. Optical microscopy pictures were then taken. For quantification of stainings, oil red O was eluted with 100% isopropanol for 10 min at room temperature, and absorbance was measured at 510 nm by spectrophotometry (PowerWave XS). Values of oil red O stain were normalized, considering the number of cells per well, as estimated by crystal-violet staining. Stains were eluted with 10% acetic acid for 20 min, and quantification was performed at 590 nm by spectrophotometry (PowerWave XS). Lipid-droplet formation in cell cultures was calculated as A 510 nm/A 590 nm. Additionally, images obtained by optical microscopy were analyzed by Image J software 1.53f51. Thus, formation of lipid droplets was quantified by both methods. Quantification of Gene Expression by Quantitative Real-Time PCR RNA was isolated using NZY total RNA isolation kit from NZYTech (Lisbon, Portugal), following the manufacturer's instructions. Nucleic acids were quantified with a NanoDrop ND-1000 Spectrophotometer from Thermo Fisher Scientific. Next, 900 ng of RNA were retrotranscribed into cDNA, using iScript cDNA Synthesis Kit from Bio-Rad (Hercules, CA, USA), according to the manufacturer's directions. Quantitative real-time PCR (qRT-PCR) was carried out in a LightCycler 96 Instrument from Roche Applied Science (Penzberg, Germany). Each PCR reaction was performed in a 10 µL volume containing 1 µL of cDNA, 10 pmol of each primer pair ( Table 1) and 1X of SensiFAST Sybr No-Rox Mix from Bioline (London, UK). The PCR amplification program included one cycle at 95 • C for 2 min (DNA denaturation) and 40-45 cycles of 95 • C for 5 s (DNA denaturation) and 65 • C for 30 s (primer hybridization and extension by DNA polymerase). Results were analyzed with LightCycler 1.1 software from the same manufacturer. Polymerase (RNA; Targeted DNA) II polypeptide A (POLR2A) was used as housekeeping gene. Relative gene expression respect control (value = 1) was calculated using the 2 −∆∆Ct method, where Ct is the cycle threshold. Western Blot Cells were lysed with Cell Extraction Buffer (Thermo Fisher Scientific) supplemented with 1 mM of phenylmethylsulfonyl fluoride (PMSF) and 50 µL of protease inhibitor cocktail (PIC)/mL (both from Sigma-Aldrich) for total protein isolation. Lysates, once collected, were incubated in ice for 30 min, with vortex agitation every 10 min. Finally, they were centrifuged for 10 min (13,000× g) at 4 • C. Supernatants were transferred into new tubes and stored at −20 • C until used. Protein concentrations were quantified with the DC Protein Assay kit (Bio-Rad) according to the manufacturer's directions. Subsequently, 15-20 µg of protein from each sample were loaded into 8-16% acrylamide nUView Tris-Glycine Precast Gels from NuSeP (Germantown, MD, USA) under denaturing conditions. Electrophoreses were carried out in a Mini-Protean (Bio-Rad) system. Then, proteins were transferred into polyvinylidene difluoride (PVDF) membranes (Bio-Rad) by a Trans-Blot Turbo Transfer System from the same manufacturer. Membranes were blocked with a 5% solution of skimmed milk in Tris-Tween-Buffered Saline (TTBS) buffer (20 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.05% Tween) for one hour at room temperature. Subsequently, membranes were incubated overnight at 4 • C, with primary antibody anti-β-catenin (1:1000) from Cell Signaling Technology (Danvers, MA, USA), in 1% skimmed milk in TTBS. After incubation, membranes were washed with TTBS and incubated with the secondary anti-Rabbit IgG H&L-HRP antibody (1:3000) from Abcam (Cambridge, UK) in 1% skimmed milk in TTBS for one hour. Finally, membranes were developed with Clarity Western ECL Substrate (Bio-Rad). Stain-free technology was used with nUView Tris-Glycine Precast Gels. In short, UV light was used to activate samples in the ChemiDoc XRS+ Gel Imaging System (Bio-Rad). Images of total protein loaded for each sample were generated. They were analyzed with Image Lab software version 6.0 from the same company. Total protein signals were quantified [28,29]. Values obtained were used for normalization of band intensity of β-catenin. Statistical Analyses GraphPad Prism 6.0 program from GraphPad Software (San Diego, CA, USA) was used to analyze data and generate plots. Comparison of different treatments was performed using analysis of variance (ANOVA) tests to detect significant changes. This was followed by a Tukey's test to identify significant differences between pairs of treatments. Differences were considered statistically significant when p < 0.05. At least three data per parameter studied were obtained. All graphs show mean plus standard error of the mean (mean ± SEM). Dipeptidyl Peptidase Activity in Culture Media DPP4i in the treatment of T2DM are mainly directed to the inhibition of soluble DPP4. As the aim of this study was to understand how DPP4i can affect MSC and their differentiation, we first determined whether the culture medium where the cells were grown and differentiated contained soluble DPP4 activity. Unsupplemented culture medium (α-MEM) consists of a well-defined solution of salts, vitamins, amino acids and sugars, with no proteins present. The possible source of DPP4 activity in the medium was FBS, with which it was supplemented. Therefore, dipeptidyl peptidase activity in α-MEM + 10% FBS medium supplemented or not supplemented with the differentiation inducers was first studied. As expected, α-MEM alone did not have dipeptidyl peptidase activity. However, the medium supplemented with FBS had this activity, which was inhibited in the presence of 10 µM vildagliptin or sitagliptin, indicating the presence of DPP4 activity in the culture medium. Furthermore, the use of 1G244 did not inhibit dipeptidyl peptidase activity, as expected, since there was no soluble DPP8/9 in the serum. The presence of inducers in the osteogenic (OM) or adipogenic (AM) medium did not modify results ( Figure 1). Therefore, culture media (α-MEM + 10% FBS), in which the different experiments were performed, represent the basal DPP4 conditions in which cells were grown. nUView Tris-Glycine Precast Gels. In short, UV light was used to activate samples in the ChemiDoc XRS+ Gel Imaging System (Bio-Rad). Images of total protein loaded for each sample were generated. They were analyzed with Image Lab software version 6.0 from the same company. Total protein signals were quantified [28,29]. Values obtained were used for normalization of band intensity of β-catenin. Statistical Analyses GraphPad Prism 6.0 program from GraphPad Software (San Diego, CA, USA) was used to analyze data and generate plots. Comparison of different treatments was performed using analysis of variance (ANOVA) tests to detect significant changes. This was followed by a Tukey's test to identify significant differences between pairs of treatments. Differences were considered statistically significant when p < 0.05. At least three data per parameter studied were obtained. All graphs show mean plus standard error of the mean (mean ± SEM). Dipeptidyl Peptidase Activity in Culture Media DPP4i in the treatment of T2DM are mainly directed to the inhibition of soluble DPP4. As the aim of this study was to understand how DPP4i can affect MSC and their differentiation, we first determined whether the culture medium where the cells were grown and differentiated contained soluble DPP4 activity. Unsupplemented culture medium (α-MEM) consists of a well-defined solution of salts, vitamins, amino acids and sugars, with no proteins present. The possible source of DPP4 activity in the medium was FBS, with which it was supplemented. Therefore, dipeptidyl peptidase activity in α-MEM + 10% FBS medium supplemented or not supplemented with the differentiation inducers was first studied. As expected, α-MEM alone did not have dipeptidyl peptidase activity. However, the medium supplemented with FBS had this activity, which was inhibited in the presence of 10 µM vildagliptin or sitagliptin, indicating the presence of DPP4 activity in the culture medium. Furthermore, the use of 1G244 did not inhibit dipeptidyl peptidase activity, as expected, since there was no soluble DPP8/9 in the serum. The presence of inducers in the osteogenic (OM) or adipogenic (AM) medium did not modify results ( Effect of DPP4 and DPP8/9 Inhibition on MCS Viability and Apoptosis In order to find out whether inhibition of DPP4 or DPP8/9 could affect the viability of MSC cultures, the effect of different doses of the inhibitors on undifferentiated MSC cultures were evaluated. Thus, cells were treated with 0, 1, 5 and 10 µM of vildagliptin, sitagliptin or 1G244 for 48 h. Then, cell viability was quantified by MTT assays. Results showed that viability decreased by about 35% in cultures treated with 10 µM 1G244. Yet, those treated with different concentrations of vildagliptin or sitagliptin showed no significant changes in cell viability compared to controls (Figure 2a). Effect of DPP4 and DPP8/9 Inhibition on MCS Viability and Apoptosis In order to find out whether inhibition of DPP4 or DPP8/9 could affect the viability of MSC cultures, the effect of different doses of the inhibitors on undifferentiated MSC cultures were evaluated. Thus, cells were treated with 0, 1, 5 and 10 µM of vildagliptin, sitagliptin or 1G244 for 48 h. Then, cell viability was quantified by MTT assays. Results showed that viability decreased by about 35% in cultures treated with 10 µM 1G244. Yet, those treated with different concentrations of vildagliptin or sitagliptin showed no significant changes in cell viability compared to controls (Figure 2a). Taking into account these results, a concentration of 10 µM of different inhibitors was chosen for subsequent studies. The reasoning was that vildagliptin, DPP4i and DPP8/9 partial inhibitors did not affect viability at such concentration. Therefore, it was of interest to check if it could affect MSC differentiation with respect to sitagliptin (specific DPP4i). To this end, we first studied the effects of treatments with vildagliptin, sitagliptin or 1G244 on the viability of MSC cultures (differentiated or not into adipocytes or osteoblasts for seven days) in the presence or absence of DPP4 or DPP8/9 inhibitors. Results showed a significant decrease in viability of not differentiated MSC and osteoblast-induced cultures treated with 1G244, mainly for the latter ones. Conversely, those treated with vildagliptin and sitagliptin showed no changes in cell viability. It is interesting to note that, in MSC cultures induced to differentiate into adipocytes, none of the treatments affected cell viability. However, a decreasing trend was observed in cultures treated with 1G244 ( Figure 2b). In addition, taking into account the above results, apoptosis was also measured in undifferentiated or induced-to-differentiate MSC cultures for seven days, treated as described above. Results were similar to those obtained for viability. Thus, apoptosis increased only with 1G244 treatment in undifferentiated and osteoblast-differentiated MSC, whereas in adipocytes, no significant changes were seen with any of the treatments (Figure 3). Taken together, the viability and apoptosis results showed that the role of DPP8/9 in adipocytes was different from the one in MSC and osteoblasts. Taking into account these results, a concentration of 10 µM of different inhibitors was chosen for subsequent studies. The reasoning was that vildagliptin, DPP4i and DPP8/9 partial inhibitors did not affect viability at such concentration. Therefore, it was of interest to check if it could affect MSC differentiation with respect to sitagliptin (specific DPP4i). To this end, we first studied the effects of treatments with vildagliptin, sitagliptin or 1G244 on the viability of MSC cultures (differentiated or not into adipocytes or osteoblasts for seven days) in the presence or absence of DPP4 or DPP8/9 inhibitors. Results showed a significant decrease in viability of not differentiated MSC and osteoblast-induced cultures treated with 1G244, mainly for the latter ones. Conversely, those treated with vildagliptin and sitagliptin showed no changes in cell viability. It is interesting to note that, in MSC cultures induced to differentiate into adipocytes, none of the treatments affected cell viability. However, a decreasing trend was observed in cultures treated with 1G244 ( Figure 2b). In addition, taking into account the above results, apoptosis was also measured in undifferentiated or induced-to-differentiate MSC cultures for seven days, treated as described above. Results were similar to those obtained for viability. Thus, apoptosis increased only with 1G244 treatment in undifferentiated and osteoblast-differentiated MSC, whereas in adipocytes, no significant changes were seen with any of the treatments (Figure 3). Taken together, the viability and apoptosis results showed that the role of DPP8/9 in adipocytes was different from the one in MSC and osteoblasts. Effect of DPP4 and DPP8/9 Inhibition on Osteoblastic Differentiation To evaluate whether inhibition of DPP4 or DPP8/9 could affect osteogenic differentiation of MSC, cultures induced to differentiate into osteoblasts were treated with 10 µM vildagliptin, sitagliptin or 1G244 during the differentiation process. After 21 days of differentiation and treatment, significant decreases in extracellular-matrix mineralization were observed in cultures treated with vildagliptin and mainly with 1G244, as shown by alizarin red S staining (Figure 4a). Expressions of osteoblastic gene markers in these cultures were studied on days 7 and 14 after the initiation of differentiation. Thus, the gene encoding runt-related transcription factor 2 (RUNX2) was downregulated on days 7 and 14, with vildagliptin or 1G244 being statistically significant in cultures treated with 1G244 ( Figure 4b). However, treatments did not produce significant changes in the expression of the gene encoding osterix (SP7) transcription factor. Nevertheless, a tendency to decrease was observed with vildagliptin or 1G244 on day seven (Figure 4b). Expressions of integrin-binding sialoprotein (IBSP) and collagen, type I, alpha 1 (COL1A1) genes, encoding extracellular matrix proteins, were also repressed in cultures treated with 1G244. No significant changes were observed in cultures treated with the two evaluated DPP4i. Although, in the case of IBSP, treatment with sitagliptin tended to increase its expression, mainly on day seven of differentiation ( Figure 4b). Thus, these results indicate that inhibition of DPP8/9 by 1G244 downregulated osteoblastic-marker gene expression, further preventing mineralization in osteoblastogenesis. Effect of DPP4 and DPP8/9 Inhibition on Osteoblastic Differentiation To evaluate whether inhibition of DPP4 or DPP8/9 could affect osteogenic differentiation of MSC, cultures induced to differentiate into osteoblasts were treated with 10 µM vildagliptin, sitagliptin or 1G244 during the differentiation process. After 21 days of differentiation and treatment, significant decreases in extracellular-matrix mineralization were observed in cultures treated with vildagliptin and mainly with 1G244, as shown by alizarin red S staining (Figure 4a). Expressions of osteoblastic gene markers in these cultures were studied on days 7 and 14 after the initiation of differentiation. Thus, the gene encoding runt-related transcription factor 2 (RUNX2) was downregulated on days 7 and 14, with vildagliptin or 1G244 being statistically significant in cultures treated with 1G244 ( Figure 4b). However, treatments did not produce significant changes in the expression of the gene encoding osterix (SP7) transcription factor. Nevertheless, a tendency to decrease was observed with vildagliptin or 1G244 on day seven (Figure 4b). Expressions of integrin-binding sialoprotein (IBSP) and collagen, type I, alpha 1 (COL1A1) genes, encoding extracellular matrix proteins, were also repressed in cultures treated with 1G244. No significant changes were observed in cultures treated with the two evaluated DPP4i. Although, in the case of IBSP, treatment with sitagliptin tended to increase its expression, mainly on day seven of differentiation ( Figure 4b). Thus, these results indicate that inhibition of DPP8/9 by 1G244 downregulated osteoblastic-marker gene expression, further preventing mineralization in osteoblastogenesis. Interestingly, the β-catenin pathway plays an important role in osteogenic differentiation. Thus, it was analyzed if inhibition of DPP4 or DPP8/9 could affect it during Interestingly, the β-catenin pathway plays an important role in osteogenic differentiation. Thus, it was analyzed if inhibition of DPP4 or DPP8/9 could affect it during osteogenesis. Therefore, expression of the gene encoding β-catenin (CTNNB1), as well as the ones encoding dickkopf Wnt signaling pathway inhibitor 1 (DKK1) and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6), involved in inhibition or activation of the β-catenin pathway were studied. This was carried out during osteoblastic differentiation, in the presence of vildagliptin, sitagliptin or 1G244, on day 14. Results showed that sitagliptin treatment increased CTNNB1 gene expression. However, inhibition of DPP8/9 by 1G244 significantly increased expression of the gene coding DKK1, which is an inhibitor of the β-catenin pathway (Figure 5a). On the other hand, LRP5 and LRP6 genes encode proteins that activate the β-catenin pathway. Thus, the relationship between DKK1 expression and one of the other genes is indicative of possible activation or inhibition of the β-catenin pathway by the different treatments used. DKK1/LRP5 and DKK1/LRP6 gene expression ratios are shown in Figure 5a. Results showed an increase of both with 1G244 treatments, although they were not statistically significant. These results are in line with those obtained by analyzing β-catenin protein synthesis by Western blot They showed that 1G244 decreased the synthesis of this protein on day 14 of osteoblastic differentiation (Figure 5b). osteogenesis. Therefore, expression of the gene encoding β-catenin (CTNNB1), as well as the ones encoding dickkopf Wnt signaling pathway inhibitor 1 (DKK1) and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6), involved in inhibition or activation of the β-catenin pathway were studied. This was carried out during osteoblastic differentiation, in the presence of vildagliptin, sitagliptin or 1G244, on day 14. Results showed that sitagliptin treatment increased CTNNB1 gene expression. However, inhibition of DPP8/9 by 1G244 significantly increased expression of the gene coding DKK1, which is an inhibitor of the β-catenin pathway (Figure 5a). On the other hand, LRP5 and LRP6 genes encode proteins that activate the β-catenin pathway. Thus, the relationship between DKK1 expression and one of the other genes is indicative of possible activation or inhibition of the βcatenin pathway by the different treatments used. DKK1/LRP5 and DKK1/LRP6 gene expression ratios are shown in Figure 5a. Results showed an increase of both with 1G244 treatments, although they were not statistically significant. These results are in line with those obtained by analyzing β-catenin protein synthesis by Western blot They showed that 1G244 decreased the synthesis of this protein on day 14 of osteoblastic differentiation (Figure 5b). Effect of Vildagliptin, Sitagliptin or 1G244 on Adipogenic Differentiation To study whether DPP4 or DPP8/9 inhibition could affect MSC adipogenesis, cultures of these cells were differentiated into adipocytes in the presence or absence of 10 µM of vildagliptin, sitagliptin or 1G244. Results obtained showed that treatment with 1G244, followed by vildagliptin during 14 days of adipogenic differentiation, significantly decreased lipid-droplet formation. This was made evident by oil red O staining quantification (Figure 6a). Interestingly, expressions of marker genes such as peroxisome proliferatoractivated receptor gamma 2 (PPARG2; encoding a master transcription factor in adipocyte differentiation), adipose triglyceride lipase (ATGL) and lipoprotein lipase (LPL) on day seven were not significantly affected by the presence of vildagliptin, sitagliptin or 1G244. However, the down-regulation of these genes involved in fatty-acid metabolism was mainly observed on day 14, with 1G244, followed by vildagliptin treatments. Decrease in mRNA levels were statistically significant for cultures treated with 1G244 (Figure 6b). Effect of Vildagliptin, Sitagliptin or 1G244 on Adipogenic Differentiation To study whether DPP4 or DPP8/9 inhibition could affect MSC adipogenesis, cultures of these cells were differentiated into adipocytes in the presence or absence of 10 µM of vildagliptin, sitagliptin or 1G244. Results obtained showed that treatment with 1G244, followed by vildagliptin during 14 days of adipogenic differentiation, significantly decreased lipid-droplet formation. This was made evident by oil red O staining quantification (Figure 6a). Interestingly, expressions of marker genes such as peroxisome proliferator-activated receptor gamma 2 (PPARG2; encoding a master transcription factor in adipocyte differentiation), adipose triglyceride lipase (ATGL) and lipoprotein lipase (LPL) on day seven were not significantly affected by the presence of vildagliptin, sitagliptin or 1G244. However, the down-regulation of these genes involved in fatty-acid metabolism was mainly observed on day 14, with 1G244, followed by vildagliptin treatments. Decrease in mRNA levels were statistically significant for cultures treated with 1G244 ( Figure 6b). On the other hand, because the β-catenin pathway also influences adipogenic differentiation of MSC, the expression of genes involved in this pathway and β-catenin synthesis were studied. Expression of CTNNB1, DKK1 and LRP6 genes did not show significant changes with any of the treatments. However, cultures treated with 1G244 significantly repressed LRP5 and induced DKK1/LRP5 and Dkk1/LRP6 gene expression ratios (Figure 7a). This suggests a possible inhibition of the β-catenin pathway. Indeed, quantification of β-catenin protein levels showed that on day 14 after adipogenic induction, cultures treated with vildagliptin or 1G244 had the lowest values (Figure 7b). On the other hand, because the β-catenin pathway also influences adipogenic differentiation of MSC, the expression of genes involved in this pathway and β-catenin synthesis were studied. Expression of CTNNB1, DKK1 and LRP6 genes did not show significant changes with any of the treatments. However, cultures treated with 1G244 significantly repressed LRP5 and induced DKK1/LRP5 and Dkk1/LRP6 gene expression ratios ( Figure 7a). This suggests a possible inhibition of the β-catenin pathway. Indeed, quantification of β-catenin protein levels showed that on day 14 after adipogenic induction, cultures treated with vildagliptin or 1G244 had the lowest values (Figure 7b). Discussion During aging and in certain pathologies, bone marrow adipocytes inhibit hematopoietic and bone regeneration. This is accomplished, in part, through the secretion of large amounts of DPP4 [30,31]. Indeed, such secretion takes place in bone marrow niches. This protease recognizes diverse substrates with important physiological functions [32]. In fact, its activity may affect the differentiation of osteoblast-and adipocyte-precursor cells. On the other hand, the use of DPP4i is generalized as antidiabetic. Thus, it is important to understand whether the DPP4 inhibition could affect another physiological process besides glucose metabolism. Among them are numerous ones related to MSC biology, as recently reviewed by our group [11]. Therefore, knowledge of how inhibition of DPP4 (or other DPP, as DPP8/9 affected by non-specific DPP4i) may influence MSC viability and differentiation is important. For instance, this kind of knowledge would be invaluable for developing MSC-based therapeutic strategies targeting T2DM patients consuming these drugs and, likewise, for the identification of new potential applications of DPP4i related to regenerative medicine. Our results indicate that inhibition of DPP4 by sitagliptin did not significantly affect MSC viability and differentiation. Yet, inhibition of DPP8/9 by vildagliptin, and mainly by 1G244, decreased viability and inhibited osteogenic and adipogenic differentiation of MSC. This suggests that DPP8/9 play key roles in maintaining the viability and differentiation capacity of MSC. Indeed, DPP8/9 have been implicated in different biological processes related to energy metabolism, inflammation, and cell behavior. The functions of these proteins can be independent of their enzymatic activity or be mediated by their peptidase activity. Thus, in the latter case, inflammatory proteins, chemokines and growth factors have been identified among their natural substrates [25]. To the best of our knowledge, our data show for the first time that DPP8/9 are also involved in the maintenance of human MSC viability, as well as in their differentiation into osteoblasts and adipocytes. Cell viability decreased when DPP8/9 activity was inhibited with 10 µM of 1G244 specific inhibitor, but not with the same concentration as vildagliptin. The latter inhibited DPP4, but with respect to 1G244, it had less power to inhibit DPP8/9. This showed that DPP4 inhibition did not affect viability, as was also shown by treatment with sitagliptin as a specific DPP4 inhibitor. These results suggest that DPP8/9 activity is important for the maintenance of cell viability in MSC. This may explain the in vivo effects in preclinical models identified by other authors. For instance, they showed that different doses of a specific DPP8/9 inhibitor produced alopecia, thrombocytopenia, reticulocytopenia, enlarged spleen, multiorgan histopathological changes and mortality in rats. Additionally, they produced gastrointestinal toxicity in dogs. However, DPP4 inhibition had no cytotoxic effects in these species [33]. Yet, there is controversy about whether the effects observed with the specific DPP8/9 inhibitors were due to inhibition of the activity of these peptidases or to some cytotoxic effects of the inhibitor used. Another study with vildagliptin showed that maintaining high plasma doses (that should inhibit DPP8/9) did not generate toxic effects in mice and rats. They concluded that loss of DPP8/9 activity had no adverse effects [34]. Others denied the in vivo toxicity of 1G244. Nevertheless, the possible contribution of some undesirable effects of high concentrations of this DPP8/9 inhibitor could not be excluded [35]. These studies were only carried out for 14 days. However, Longer periods might produce adverse effects due to possible deterioration of progenitor-cell populations. On the other hand, it has been shown that loss of DPP9 activity resulted in neonatal death 24 h after birth in dipeptidyl peptidase 9 (DPP9) knock-in mice. This was due to an increase in apoptosis of occipital somite-derived migratory tongue-muscle progenitors. This caused the loss of the sucking ability of those mice. This effect was specific to DPP9 activity. Furthermore, DPP8 activity in those cells was not able to compensate for the absence of DPP9 [36]. This study supports our results on the importance of DPP9 activity in maintaining progenitor-cell viability, like MSC. Moreover, it has been shown that 10 µM of 1G244 in skin-cell cultures of fibroblasts and human epidermal keratinocyte (HaCaT) cells decreased proliferation in vitro [37]. In monocytes, DPP9 activity has also been shown to be important for viability and differentiation into macrophages. Thus, DPP9 increased during differentiation into M1 and M2 macrophages. Furthermore, treatment with 1G244 inhibited M1 macrophage activation and induced apoptosis [38]. Also, in SiHa and HeLa tumor cells, DDP8 silencing by small interfering RNA (siRNA) inhibited proliferation, migration and invasion, in addition to inducing apoptosis [39]. All these data support our results obtained on MSCs when DPP8/9 activity is inhibited. Substrates of DPP8/9 are still largely unknown. Nevertheless, it has recently been shown that the activity of these peptidases is involved in controlling the processing of more than 100 mitochondrial proteins, most notably adenylate kinase 2 (AK2). Therefore, DPP8/9 should play an important role in energy metabolism [40]. AK2 may have an intermembrane localization in mitochondria, regulating adenine-nucleotide interconversion. But it can also localize in the cytoplasm, where it is cleaved by DPP8/9 (predominantly, the latter), being therefore marked for proteosome degradation. In this way, DPP8/9 prevent an increase in cytoplasmic AK2, favoring mitochondrial localization [40]. If DPP8/9 activity is inhibited, AK2 accumulates in the cytoplasm. Then, it becomes active, inhibiting cell proliferation and inducing apoptosis through interaction with dual-specificity phosphatase 26 (DUSP26) [41]. Therefore, these data may partly explain our results, showing that DPP8/9 inhibition decreases viability, further increasing apoptosis of undifferentiated MSC induced to differentiate into osteoblasts. However, it is interesting to note that, in our study, adipocyte-induced MSC were not significantly affected in terms of viability when treated with a DPP8/9 inhibitor, as 1G244. This may be related to the fact that undifferentiated human MSC, and those differentiated to osteoblasts, maintain a high degree of proliferation. Yet, those differentiated into adipocytes lose division capacity when exposed to adipogenic inducers, in contrast to mouse 3T3-L1 preadipocytes [42]. Thus, results suggest that activation of apoptosis after DPP8/9 inhibition occurs mainly in proliferative and mitotically active cells. In the case of osteoblastic differentiation of MSC, when cultures were treated with vildagliptin or 1G244, mineralization of the extracellular matrix was inhibited. This indicates that minimal levels of DPP8/9 activity are necessary for cell viability, as well as correct maturation of osteoblasts. Our results show that expression of genes encoding transcription factors RUNX2 and SP7 decreased with vildagliptin or 1G244 treatments. This was statistically significant in RUNX2 expression, with inhibition of DPP8/9 by 1G244. Both transcription factors are critical for the initiation of osteoblastic differentiation. Therefore, if the expression of their encoding genes remains repressed during the differentiation process, osteoblastic differentiation does not occur [43,44]. Also, the use of 1G244 downregulated IBSP gene expression, whereas sitagliptin upregulated it. This is in line with the results obtained since the protein encoded by this gene is important in extracellular matrix formation in osteoblasts. Furthermore, COL1A1 expression was repressed by 1G244. Collagen is the main bone-matrix protein and is essential for its mineralization [45]. Therefore, this result may explain the poor mineralization in these cultures. Recently, it has been described that some drugs, like anagliptin, trelagliptin and saxagliptin, promote osteoblastic differentiation and mineralization of MC3T3-E1 mouse preosteoblastic cell line. This is accomplished through increased expression of RUNX2 [46][47][48]. While anagliptin and trelagliptin are specific inhibitors of DPP4, saxagliptin also inhibits DPP8/9, as does vildagliptin [49]. Therefore, the results of the study with saxagliptin contradict what was observed in our case for vildagliptin. However, another study performed in vivo and in the same MC3T3-E1 cell type, as well as in rat-derived MSC with saxagliptin, concluded that, in rats, oral administration of this DPP4i alters long-bone microarchitecture. In the two cell types evaluated, saxagliptin treatment inhibited RUNX2 expression and mineralization [50]. Saxagliptin concentrations used in the latter study were 1.5 and 15 µM. Both concentrations inhibited osteoblastogenesis, being in the range of that used in the study, where they observed the opposite effect. The best response on osteoblast differentiation was obtained with 2 µM of saxagliptin. Because both saxagliptin and vildagliptin have a certain ability to inhibit DPP8/9 [49], our results support that this type of DPP4i can negatively affect osteoblast differentiation and mineralization. On the other hand, diabetes is a risk factor for osteoporosis [51]. Additionally, high plasma DPP4 levels in T2DM patients have been associated with bone loss and risk of fracture [21]. Thus, several studies have evaluated whether the use of DPP4i may impact bone health in humans. Currently, treatment with some DPP4i, like alogliptin, has been associated with decreased fracture risk. But in other cases, no such association has been reported [9,52]. In the case of vildagliptin, patients with T2DM treated for a year did not show any effect on bone metabolism [53]. The results of these studies have been mixed, so there are currently no conclusions on how DPP4i usage may affect bone metabolism. Moreover, recent meta-analyses suggest a neutral effect [54,55]. This supports our results obtained with sitagliptin, which did not significantly affect osteoblast mineralization. However, our data with respect to vildagliptin suggest that it may have negative effects on bone formation through inhibition of DPP8/9. However, it has been found that vildagliptin treatment improved trabecular-bone mineral density and microstructure in a model of obese, insulin-resistant, pre-diabetic rats [56]. Also, treatment with DPP4i promoted bone formation and reduced bone resorption by improving the microstructure of trabecular bones in a mouse model of T2DM [57]. These effects may be partly related to the improvement of glucose metabolism and glycemic control caused by DPP4i treatment, which may contribute to decreased bone loss [58]. With respect to adipogenesis, our results showed that the expression of PPARG2, ATGL and LPL genes was significantly repressed after 14 days with 1G244 treatment. The used cells are considered mature adipocytes at this stage. This may explain the decrease in lipid accumulation in these cultures. Therefore, these results suggest that DPP8/9 inhibition does not affect the initiation of adipogenesis but rather the maturation of adipocytes. It should be noted that inhibition of DPP4 by sitagliptin does not affect the differentiation of MSC into adipocytes. The 1G244 inhibitor has been shown to block adipogenesis in 3T3-L1 and 3T3-F422A preadipocytes, while DPP4 inhibitors had no effect. In addition, inhibition of DPP8/9 significantly affected the differentiation of preadipocytes into adipocytes. This highlights the relevance of DPP8/9 in adipogenesis, corroborating results obtained in this study [59]. However, the use of 1G244 does not allow us to determine which of the two-or if both-dipeptidyl peptidases (DPP8 or DPP9) is/are involved in adipogenic differentiation. Recently, however, a knockout mouse for DPP8 has been described as obese [60]. It is still unknown what role DPP8 may play in fat metabolism. Nevertheless, considering such data, our results suggest that DPP9 may have a relevant role in MSC differentiation into adipocytes. Indeed, although DPP8 and DPP9 present high homology between them, they do not share the same specificity for different substrate-cleavage sites. For example, DPP8 has lower specificity for Val-Ala sites than DPP9. Therefore, it has been suggested that each of these peptidases may have specific functions [61]. In adipocytes, the presence of DPP4 on the cell surface has been associated with a phenotype of high proliferation capacity and low differentiation potential. The opposite happened with DPP4 − preadipocytes [12,62]. However, soluble DPP4 synthesis increased during adipocyte differentiation, with adipocytes being one of the major sources of circulating DPP4 [63]. This indicates the different roles of DPP4 in adipogenesis. In our case, the use of specific DPP4i did not affect the adipogenic differentiation of MSC. This suggests that, as a whole, DPP4 activity is not determinant for MSC to reach adipocyte phenotype. Among the functions that have been assigned to DPP8/9 are those of regulating energy metabolism. Specifically, the loss of DPP9 activity in DPP9 gene knock-in mice (DPP9 gki) produced important metabolic alterations in the liver and intestine of neonates [64]. Differentiation of MSC into osteoblasts and adipocytes leads to important metabolic changes. In the case of osteoblasts, there was a significant activation of the glycolysis pathway [65] and fatty-acid metabolism in adipocytes [66]. Therefore, inhibition of DPP8/9 could alter those metabolic changes, negatively affecting the differentiation process. Therefore, it would be interesting to study that in the future. The Wnt/β-catenin signaling pathway plays an important role during the differentiation of osteoblasts and adipocytes [67]. In the case of osteoblastogenesis, activation of this pathway is necessary when MSC differentiate into preosteoblasts. Regarding adipocyte differentiation, the Wnt/β-catenin pathway must be inhibited [68]. Low-density lipoprotein (LDL) receptor-related proteins 5 and 6 (LRP5 and LRP6) participate in the activation of the canonical Wnt/β-catenin pathway. They are part of the LRP5/LRP6/Frizzled co-receptor group for Wnt proteins [69]. On the other hand, DKK1 is an antagonist of the Wnt/βcatenin signaling pathway that acts by sequestering LRP6 co-receptors. Thus, it cannot function together with frizzled receptors and thus activates the Wnt signaling cascade [70]. Therefore, the ratio of DKK1/(LRP5 or 6) expression is an indicator of the ability to activate or inhibit the Wnt/β-catenin pathway. Our results show that, in osteoblasts, sitagliptin increased mRNA levels of the βcatenin encoding gene. However, it did not produce significant changes in protein synthesis. Similarly, vildagliptin did not affect the protein production of β-catenin. This is consistent with the fact that neither of the two DPP4i used produced changes in the DKK1/(LRP5 or 6) gene expression ratio with respect to untreated cultures. In the case of sitagliptin, this result was expected because, as our results showed, it did not produce significant changes in osteoblastic phenotype through extracellular matrix mineralization. Interestingly, sitagliptin decreased β-catenin expression under conditions of high glucose in the medium in other cell types, such as normal rat kidney (NRK)-52E, which are immortalized renal proximal-tubule epithelial cells [71]. On the other hand, sitagliptin is protected from apoptosis, partly through induction of the β-catenin pathway, in vascular smooth muscle cells (VSMC) exposed to oxidative stress with H 2 O 2 [72]. This suggests that depending on cell type and culture conditions, this DPP4i may differently affect the Wnt/β-catenin pathway. This is probably related to the diversity of substrates that DPP4 can recognize. In the case of vildagliptin, the lower degree of mineralization of vildagliptin-treated cultures cannot be explained by a decrease in β-catenin protein synthesis. Interestingly, 1G244 did inhibit β-catenin expression, at least in part, by inducing DKK1 expression on day 14 of culture. Considering the role of the Wnt/β-catenin pathway on osteoblastogenesis, this correlates with the decrease in extracellular-matrix mineralization observed on day 21, when DPP8/9 activity was inhibited in osteoblast-induced MSC. Therefore, the possible effect of vildagliptin on DPP8/9 activity was not potent enough to repress β-catenin expression, suggesting the existence of another mechanism, explaining the negative effect of vildagliptin on mineralization. In adipocytes, gene expression of CTNNB1, DKK1, LRP5 and LRP6 genes was unchanged on day 14 of differentiation, in sitagliptin-and vildagliptin-treated cultures, as compared to controls. However, β-catenin protein levels showed a tendency to decrease with these treatments, mainly with vildagliptin. Furthermore, as in osteoblasts, inhibition of DPP8/9 activity with 1G244 increased the DKK1/(LRP5 or 6) ratio and decreased the β-catenin protein. Inhibition of the Wnt/β-catenin pathway is important in the early stages of adipogenic differentiation. Nevertheless, it has been described that activation of the canonical Wnt/β-catenin pathway by different components is critical for adipocyte maturation and lipid metabolism in the final differentiation process [73]. In our study, by day 14 of differentiation, adipocytes were in their late maturation phase, with a significant accumulation of lipid droplets. Therefore, a decrease in Wnt/β-catenin pathway activity by vildagliptin or 1G244 may affect the maturation process of adipocytes. This may lead to a decrease in lipid-droplet formation, as we have shown by the oil red O staining of these cultures. In conclusion, taken together, these results suggest that DPP8/9 activity, directly or indirectly, modulates the Wnt/β-catenin pathway in different physiological processes, including cell differentiation. Our results show that DPP8/9 activity plays an important role in maintaining the viability and differentiation of human MSC. This implies that non-specific DPP4i, such as vildagliptin, may have undesirable effects on MSC differentiation through its ability to partially inhibit DPP8/9. However, the effects produced by vildagliptin are not as potent as those generated by the specific DPP8/9 inhibitor, mainly in osteoblasts. This may explain why different studies have not observed significant clinical adverse effects in the use of this type of DPP4i [22]. Overall, our results suggest that specific DPP4i may have a higher degree of safety and that the use of potential inhibitors of DPP8/9 activity in human clinics should take into account their possible effects on MSC populations. Our results suggest that certain DPP8/9 substrates regulate the viability and differentiation of MSC. The accumulation of these substrates in cells, due to inhibition of DPP8/9 activity, must be partly responsible for the negative effects observed on viability and osteogenic and adipogenic differentiation of MSC. Therefore, in future studies, it will be important to identify these substrates to advance the knowledge of how DPP8/9 regulates these physiological processes of MSC. A limitation of this study is that it was performed in vitro. Thus, it would be interesting, taking into account our results, to study how the inhibition of DPP8/9 by non-specific inhibitors of DPP4 or by specific inhibitors of these enzymes can affect MSC populations at the level of viability and differentiation in preclinical models. This could provide more detailed information on possible repercussions that the clinical use of these inhibitors might have on the regenerative capacity of the organism and, in particular, on bone and fat metabolism. Supplementary Materials: The following supporting information can be downloaded at https://www. mdpi.com/article/10.3390/jcm12144632/s1: Figure S1. MSC cultures in passage 2 and at 80% confluence were raised with "Cell Dissociation Solution Non-enzymatic" (Sigma-Aldrich) and incubated with the anti-CD34-APC from Bio-Techne-Novus Biologicals (Minneapolis, MN, USA), anti-CD45-BB515 and anti-CD105-APC from Becton Dickinson Biosciences (BD; Franklin Lakes, NJ, USA), as well as anti-CD73-FITC and anti-CD90-PE-Cy5 (from eBioscience-Thermo Fisher Scientific) antibodies for 25 minutes in the dark. Cells were then washed with PBS (Sigma-Aldrich), 1% BSA from Tocris Bioscience (Bristol, UK) and 0.1% sodium azide (Sigma-Aldrich), and finally resuspended in CellFIX solution (Becton Dickinson Biosciences) before analysis on an LSRFortessa SORP flow cytometer from the same manufacturer. Results show that cells are positive for CD73, CD90 and CD105, but negative for CD34 and CD45 hematopoietic markers. Figure S2. Differentiation of MSC into adipocytes and osteoblasts. A: Light microscopy image of undifferentiated MSC, stained with oil-red O and hematoxylin. B: Same as in A, but MSC differentiated into adipocytes during 14 days. In this case, cells positive for oil-red O staining, specific for fatty vesicles, are observed. C: Well of P12 plate of undifferentiated MSC culture, stained with alizarin red. D: Same as in C. but MSC differentiated into osteoblasts for 21 days. Mineralization of extracellular matrix appears stained with alizarin-red S. Funding: This research was funded by grants PI18/01659 and CIBER "Fragilidad y Envejecimiento Saludable" (CIBERFES) of "Instituto de Salud Carlos III" (ISCIII), "Ministerio de Ciencia e Innovación", Spain, and the European Union (EU). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data that support the findings of this study are available from the corresponding authors upon reasonable request.	2023-07-15T15:12:37.342Z	2023-07-01T00:00:00.000	{ "year": 2023, "sha1": "21629622058970c1f92684ca2e711cf1e030313c", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2077-0383/12/14/4632/pdf?version=1689142363", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "3503764794acf1cca6b3cb4ef464a2957789195f", "s2fieldsofstudy": [ "Biology", "Medicine" ], "extfieldsofstudy": [] }
237418592	pes2o/s2orc	v3-fos-license	Effects of air-conditioning systems in the public areas of hospitals: A scoping review Almost all hospitals are equipped with air-conditioning systems to provide a comfortable environment for patients and staff. However, the accumulation of dust and moisture within these systems increases the risk of transmission of microbes and have on occasion been associated with outbreaks of infection. Nevertheless, the impact of air-conditioning on the transmission of microorganisms leading to infection remains largely uncertain. We conducted a scoping review to screen systematically the evidence for such an association in the face of the coronavirus disease 2019 epidemic. PubMed, Embase and Web of Science databases were explored for relevant studies addressing microbial contamination of the air, their transmission and association with infectious diseases. The review process yielded 21 publications, 17 of which were cross-sectional studies, three were cohort studies and one case−control study. Our analysis showed that, compared with naturally ventilated areas, microbial loads were significantly lower in air-conditioned areas, but the incidence of infections increased if not properly managed. The use of high-efficiency particulate air (HEPA) filtration not only decreased transmission of airborne bioaerosols and various microorganisms, but also reduced the risk of infections. By contrast, contaminated air-conditioning systems in hospital rooms were associated with a higher risk of patient infection. Cleaning and maintenance of such systems to recommended standards should be performed regularly and where appropriate, the installation of HEPA filters can effectively mitigate microbial contamination in the public areas of hospitals. Introduction The outbreak of the novel severe acute respiratory syndrome coronavirus 2 disease (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID- 19), has currently spread to almost all parts of the world. Available evidence indicates that the agent is transmitted via respiratory droplets and contact routes between humans [1]. Measures that hinder the spread of the virus include environmental control of indoor air flow [2]. However, relatively little attention has been paid to air-conditioning systems, which are one of the most common factors affecting indoor air flow. Some reports have implicated such systems in the transmission of SARS-CoV-2 [3,4], and norovirus [5]. Air-conditioning systems play an important role in maintaining indoor air temperature and humidity in public buildings and hospitals. In the latter, particularly, intensive care units (ICUs) and operating rooms, the systems are fitted with high-efficiency particulate air (HEPA) filtration and laminar flow design to reduce the risk of air-borne infections. Installation of air-conditioning systems can help prevent hyperthermia in critically ill infected patients in a heat wave, and may reduce the cost of blood cultures requested since the number of cultures taken increases in such patients if a high ambient temperature is sustained [6]. The systems have also proven effective in reducing mortality in heat-related illness in domestic homes [7], and hospitals [8]. However, air-conditioning systems represent a potential source of microbial contamination in hospitals, as accumulated dust and moisture increase the risk of contamination and associated infections. Indeed, several fungal genera have been demonstrated in air-conditioned ICU [9], and mould colonisation has been observed in HEPA filters, and in air-conditioning systems [10], as has the presence of SARS-CoV-2 on swab samples taken from surfaces of filters [11]. Likewise, contamination of air-conditioning systems has been implicated in some hospital-acquired infections [12][13][14]. The risk of proliferation of microbes from air-conditioning systems and their transmission to high-risk patients, in hospitals is greatly reduced if strict management and control practices are followed [15]. However, despite the several regulations covering the installation of these systems in hospitals, such as the HVAC Design Manual for Hospitals and Clinics published by the American Society of Heating, Refrigeration and Air-Conditioning Engineers, adherence to these standards is variable in routine practice. Indeed, epidemiological surveillance in a hospital in Paris found that only 32% of the patients diagnosed with invasive nosocomial aspergillosis were housed in rooms where an HEPA air filter system had been installed [16]. Studies on heating, ventilation and air-conditioning (HVAC) systems in hospitals have largely been conducted in restricted areas such as operating rooms and ICUs, and have focused on the impact of different airflow patterns, number of personnel, ventilation rates and other extrinsic factors [17][18][19]. However, in the public areas of hospitals (wards, clinics etc.) airflow may be suboptimal and result in a higher risk of microbial contamination. This study therefore focused on these areas in which highefficiency filters are rarely installed, and which have often been overlooked in other investigations. Our aim was to clarify the presence and nature of potential risks associated with the use of air-conditioning systems, through the systematic assembly and analysis of published evidence on the effect of air-conditioning systems on the transmission of pathogens and related infectious diseases. Further, we explored effective measures for the protection of patients, staff and visitors from the potential risks of exposure to microorganisms related to air-conditioning systems, and application of measures with potential to combat such transmission in the COVID-19 pandemic. Methods The guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Reviews (PRISMA-ScR) [20] were followed in this research study. The key stages of this framework [21] were: identifying the research question, identifying relevant studies, study selection, charting of data and collating, summarising and reporting the results. Research questions The study addressed the following questions: 1. Is there any association between air-conditioning systems and the presence of pathogenic microorganisms in public areas of hospitals? 2. Do air-conditioning systems increase the risk of infection in such hospital areas? Relevant studies The complete PubMed, Embase and Web of Science databases were explored for relevant studies in December 2020. The search strategy is outlined in 'Supplementary Material'. Papers published since the databases were established were included in the search, and relevant cited references. Inclusion and exclusion criteria All studies met the following criteria: published in English; intervention included different kinds of air-conditioning systems, such as unfiltered air or air-conditioning systems with HEPA filters; comparators were other areas without air-conditioning systems; assessment of the presence and measurement of pathogenic microorganisms in indoor air, ratios of viable microorganisms detected, incidence of infectious diseases, among others. The year of publication was not restricted in the literature search. The exclusion criteria included the following: air-conditioning systems located only in operating rooms or other restricted areas; additional interventions (e.g. ultraviolet germicidal irradiation) combined with air-conditioning but focused on outcomes irrelevant to air-conditioning; and studies lacking specific data or comparators. Study selection All articles identified in the databases were exported into Endnote (Version 9.3), and duplicates were removed on initial screening. Study titles and abstracts and web searches of citations of relevant studies were screened by two independent researchers (Han-Ting Wu and Rong-Chen Dai) to assess their potential relevance for full review. The same researchers also independently reviewed the full texts of candidate articles against the inclusion and exclusion criteria. Any discrepancies were resolved through discussion with a third reviewer. The reasons for exclusion during the screening of the full texts were recorded. Charting the data Data were extracted independently by two review authors and discrepancies were identified and resolved as above. They comprised: names of the authors, year, type of study, outcome of interest, bacterial or fungal pathogens, hospital locations tested, air-conditioning systems used and relevant results and study conclusion. Collating, summarising and reporting results Due to the heterogeneity of studies and difficulty of quantifying the data, we tabulated key information i.e. the kinds of air-conditioning systems, tested areas and study designs and described relevant parameters in detail. Quantitative and qualitative findings were summarised within each grouping of air-conditioning systems and related quantitative data such as the concentrations of microorganisms found in samples of indoor air were recorded. Associations are presented using the summary measures reported in individual studies with P-values where available Figure 1 presents the PRISMA diagram for the screening and selection of articles. A total of 1059 studies were retrieved, of which 299 duplicates and 688 irrelevant studies were excluded often because either they were not reported in English, did not meet the inclusion criteria, or their full texts were not available. As a consequence, 72 studies were assessed for eligibility; 51 were excluded as ancillary disinfection equipment was used along with air-conditioning systems, or only samples taken from air conditioners were tested, or evidence of the effect of airconditioning systems or comparators was lacking. This process left 21 articles for analysis . Seventeen were cross-sectional studies, three were cohort studies and one was a case−control study. All, but one, were published after 2000 and the other in 1975. Most of the articles were published in internationally recognised and specialised journals; an overview of the articles and their outcomes is presented in Table 1. Concentration of microorganisms in indoor air Of the 21 studies included, 16 reported the concentrations of microorganisms in air samples from rooms (wards, corridors, laboratories) with different air-conditioning systems; five of the 16 also sampled outdoor hospital sites. Seven studies [22,26,27,29,33,34,36] reported results of microbial concentrations between naturally ventilated, and areas with common airconditioning systems. In public areas of the hospitals, fungal loads in air-conditioned areas were considerably lower than those recorded in other indoor naturally ventilated environments [26,27,29,33,34]. Moreover, the average levels of bacteria were similar to those recorded for fungi [22,34,36], the latter being most probably derived from the outdoor environment of the hospital [27]. Air-conditioning systems were further classified into those with, or without HEPA filters in 10 studies. Compared to rooms without air-conditioning or with natural ventilation, indoor airborne fungal and bacterial concentrations were the lowest in rooms with HEPA filters, thus demonstrating their effectiveness for the reduction of bioaerosols [22,24,26,28,29,31,32,37]. Furthermore, the type of air conditioner used was considered crucial as central air conditioners proved to be more effective than non-centrally sited systems such as window, or single-split types [35,36]. Notably, one study identified that, compared with hybrid ventilation, the concentration of indoor bioaerosols was positively correlated with the type of ventilation system used (e.g., central air conditioners, P < 0.05) [23]. Ratios of viable microorganisms detected Four studies analysed associations between air-conditioning and the rates of viable microorganisms, mainly Aspergillus, detected on sampling [22,28,30,39]. The proportion of air samples positive for Aspergillus was consistently much higher in rooms in which the air-conditioning systems were not in use at the time of sampling [22,28,39]. However, the lowest mean recovery rate, and percentage of samples positive for Aspergillus were recorded in another study in areas with HEPA-filtered air-conditioning systems. In contrast, the samples collected from patient care areas without HEPA-filtered systems and the other reference samples The ward with no air-conditioning system (A) had the worst results on all three types of sampling carried out; the total bacterial load and the sedimented mycotic load were almost twice as high as the values recorded in the ward with the system without HEPA filters (B). The percentage of samples positive for air-borne Aspergillus was also twice as high in A as in B. Epidemiology and Infection Air-conditioning systems markedly reduce the concentration of aspergilli in the environment. While the number of microorganisms collected in hospital 2 before the disinfection process was higher than those after the disinfection process, this was reversed in hospital 1. In the latter, the air-conditioning system and the HEPA filters which were switched on before the disinfection process, were turned off during the weekend, and thus the number of airborne live microorganisms increased fivefold after the disinfection process. Microbial loads in the hospital air were effectively controlled due to use of HEPA filters in air-conditioning systems. Concentrations of particles ≥0.5 mm (P = 0.04) and ultrafine particles (P = 0.001) were significantly lower in workplace rooms with additional ventilation on and air-conditioning systems than in rooms without. In rooms without ventilation and air-conditioning systems bacterial concentrations (P ≤ 0.001) and particles ≥0.5 mm (P = 0.011) and ≥5 mm (P ≤ 0.001) were significantly higher with window ventilation (n = 10) than not (n = 6). Window ventilation leads to higher particle but not bacterial concentrations than HVAC systems. Concentrations of particles were significantly lower in air of rooms with additional ventilation and air-conditioning systems Tracer gas measurements of air changes per hour for natural ventilation, central air conditioning and window or wall-mounted air conditioning areas were respectively 31.0 (51.4; P < 0.001), 12.6 (51.4); P < 0.05 and 2.7 (9.1); P < 0.001). Ventilation rates in indoor work areas with air conditioning especially window or wall-mounted less efficient than natural ventilation. Inadequate ventilation is a major contributory factor to the spread of TB in nosocomial outbreaks. Air-conditioning systems (AC) used in each area were categorised as 'Common AC,' 'Natural ventilation,' or 'With HEPA filters' if other details of ventilation were not given. Specific parameter description such as the type of air conditioner was recorded if given. were almost indistinguishable in terms of mean counts of Aspergillus spp. [30]. Related infectious diseases Five studies (two cross-sectional, two cohort and a case−control) reported outbreaks of related infectious diseases in air-conditioned hospitals. Exposure to central air-conditioning (OR 8.59) had a higher probability of causing hospital-acquired infections (P < 0.05) [40]. In addition, 8.9% of the patients admitted to the emergency room with onset of respiratory symptoms had viral infections, and exposure to air-conditioned air was the only linking factor [41]. Moreover, nosocomial invasive pulmonary mycoses occurred more frequently during seasons in which the HVAC systems were not in use than when they were used [39]. Only one study reported that the occurrence of invasive aspergillosis (IA) and median time to onset of infection was not significantly different between groups of patients treated in areas with, and without, HEPA-filtered airconditioning [38]. However, another study indicated that HEPA filters were protective for highly immunocompromised patients with haematologic malignancies and were effective for removing most Aspergillus conidia from the ambient air [37]. Microbe species Only five studies reported on specific identification of microbial species, mainly fungi, in air-conditioned hospitals. A crosssectional study in a South Korean hospital [29] assessed the degree of fungal contamination in hospital air environments over the course of a year, and found that Aspergillus spp. were the most prevalent both inside (47.0%) and outside (62.0%) the hospital. Within the hospital, Penicillium spp. were the second most predominant fungi, accounting for 37.9% (n = 25) of the identified species and 8.9% (n = 14) of those found outside (P < 0.001). Overall, the third most common moulds were of the Alternaria genus [29]. Similar results were reported in another cross-sectional study in 10 hospitals by Perdelli et al. [26], which found that the mean concentrations of Aspergillus, Penicillium, Cladosporium and Rhizopus, which were implicated in patient's infections, were significantly higher in the kitchens than in other tested areas with HEPA filters in the air-conditioning systems. In another study, samples of airborne fungi at a tertiary university hospital were collected monthly over 10 years, and all Aspergillus isolates were further categorised into different species, namely, A. fumigatus, A. niger and A. flavus; the latter two species were the most prevalent [30]. Likewise, in another study, A. flavus and A. fumigatus were the most common species isolated in rooms with or without air conditioners. The average number of Aspergillus spp. isolated from the non-air-conditioned rooms was significantly higher than from air-conditioned areas (P = 0.013) [33]. Indirect factors Evidence of indirect factors influencing the effectiveness of airconditioning was provided through a cross-sectional survey of 323 patient care, and ancillary areas, in hospitals of Thailand. This found that indoor ventilation rates (air changes per hour) of areas with central air-conditioning (median, 12.6) were consistently lower than those of work areas with natural ventilation (median, 31.0) (P < 0.001). Furthermore, the ventilation rates of areas with window or wall-mounted air conditioners (median, 2.7) were significantly less than in centrally air-conditioned areas (P < 0.001) [42]. Patients in rooms with low ventilation rates might have a higher risk of getting infected by the spread of Mycobacterium tuberculosis [43]. Discussion Scoping reviews aim to show the primary resource and types of available evidence to provide key concepts for clinical practice, policy formulation and research, especially in an area which has not been reviewed systematically [21]. In this study, we reviewed the relevant literature to assess how air-conditioning systems affect the incidence and impact of pathogenic microorganisms in the public indoor areas of hospitals. Air-conditioning systems play a more important role than heating or cooling the air in hospitals and other healthcare environments. A hospital is a public setting visited by various kinds of patients from different places. Thus, the issue of microbial contamination related to the use of air-conditioning systems cannot be underestimated, especially given the ongoing COVID-19 pandemic. The review identified that, in public areas of hospitals, bacterial and fungal bioaerosol concentrations were generally higher in naturally ventilated rooms compared with the degerming effect of central air-conditioned systems which are proven to be effective in removing airborne microbes, although fungal spore levels may remain high in air-conditioned rooms [33]. The latter reinforces the need for periodical maintenance and disinfection of airconditioning systems to prevent environmental colonisation and dissemination of fungi [33,34]. Evidence suggests that patients exposed to air-conditioning systems had higher risks of acquiring a viral, or hospital-associated bacterial or fungal infection, the latter potentially causing invasive pulmonary mycoses. Moreover, when air-conditioning systems were in use, doors and windows were often closed to maintain a suitable temperature, which resulted in reduced ventilation rates [42]. Likewise, poor design and operation of air-conditioning systems can contribute to inadequate ventilation [44,45] and these factors may account for the increase in infection risks when exposed to air-conditioning systems in hospitals. Compared to window or split types of airconditioning systems, often used in single-patient rooms, recycled central air-conditioning systems were more often installed in multiple-patients' room in a study conducted in a certain hospital in India [40]. Contact between patients and increased movement of personnel may also contribute to higher risk of acquiring hospital infections when exposed to central air-conditioning systems. To the best of our knowledge, this is the first review in which the influence of high-efficiency filters in air-conditioning systems on the spread of microorganisms has been evaluated. Our key finding is that filters appear to be an indispensable part of airconditioning systems. Ten of the studies addressed the benefits of HEPA filters in these systems and clearly showed that the concentration of airborne microorganisms in areas with HEPA filters was lower than the concentration in areas without them. However, the included studies did not focus on the non-HEPA filters that are commonly installed inside air conditioners, and few provided details of the operating system, such as pressurisation, humidity, temperature etc. Two studies reported on the efficiency of their non-HEPA filters used in the areas tested [30,37]. Indeed, only one gave details of the mean temperatures and relative humidity of the natural ventilated areas and in the air-conditioned areas [29]. These factors may be the source of the heterogeneity of data noted in studies that simply classified areas based on the presence of an air conditioner or did not specify the type of air conditioners. In a workshop summary of the Institute of Medicine (US) Forum on Microbial Threats, HEPA was defined as a pleated mechanical air filter composed of mats of randomly arranged glass fibres that collects and traps particles greater than 0.1 μm by diffusing, intercepting and impacting the passage of particles [46]. A study conducted in two Wuhan hospitals showed that SARS-CoV-2 aerosols were mainly found in the submicrometer areas (aerosol size distributions between 0.25 and 1.0 μm) and supermicrometer areas (aerosol size distributions > 2.5 μm) [47]. Air filtration through HEPA can intercept most pathogens, including fungi, bacteria and encapsulated viruses, with an efficiency >99.97% [46]. Although direct studies for SARS-CoV-2 have not as yet been performed, the current study on HEPA filter functionality, and prior CDC guidelines for SARS-CoV-1 together suggest a theoretical efficacy for HEPA filters in eliminating airborne SARS-CoV-2 [48]. HEPA filters in air-conditioning systems are widely acknowledged to be highly effective for the removal of microorganisms from the air and protective for high-risk patients. However, owing to their high costs of installation and maintenance, it may prove difficult for healthcare facilities to fit air-conditioning systems with HEPA filters in isolated areas, let alone in public areas. Even in the United Kingdom, only a quarter of 203 hospitals surveyed had isolation facilities available in their emergency departments [49]. This situation could only be worse in lowincome and developing countries. Nevertheless, a costeffectiveness incremental analysis showed that for prevention of invasive aspergillosis, rooms with HEPA-filtered systems were better cost-saving interventions than antifungal (posaconazole) prophylaxis and environmental protection measures ($2665 vs. $ 42 531 vs. $4073, respectively) [50], and thus the economic benefits of such filters can exceed the costs of installation and maintenance. For areas where HEPA filters are currently not available, possible substitutes to improve air hygiene are: lamps with germicidal ultraviolet irradiation, increasing room ventilation rates, and less widely applied, generation of hydrogen peroxide mists stabilised with silver ions [51][52][53][54][55][56]. Microbial contamination of room air and risks of transmission can be reduced to a minimum by regular implementation of disinfection measures. For hospitals in poor areas or with inadequate external air quality, mobile airdecontamination units and portable HEPA filtration units are alternative options and are easy to maintain [57,58]. This scoping review has some limitations. First, all the included studies reported different descriptions of the airconditioning systems used, which may be responsible for differences in their conclusions. Second, although several studies provided seemingly detailed descriptions of the air sampling methods used, variables in the experimental set-up were not described. Details of the sampling time, and the position and height of the sampler when samples were taken, were generally imprecise or not reported. Third, locations of the hospitals, humidity, temperature and season have recognised impacts on microbial contamination of indoor air [24,29,59]; these factors were considered in relatively few of the studies. Lastly, as standard deviations of microbe concentrations were reported inconsistently, the data presented may therefore be an underestimation of reality since the sampled areas were not randomly selected. Further, specific microorganisms in various settings were assessed based on selective sampling and reliance on existing techniques; thus, other microbes in the air and on surfaces might have been overlooked. Nevertheless, we consider that these limitations do not affect the validity and conclusions of the study. In conclusion, this study focused on ventilation of hospital public areas, which are more likely to be overlooked relative to operating room and ICUs, and reviewed evidence regarding the risk of air-conditioning systems and hospital-acquired infections. The cleaning and maintenance of such systems should be done regularly according to existing standards as patients residing in contaminated air in rooms have a higher risk of exposure to pathogenic microorganisms. The universal installation of HEPA filters can effectively mitigate against microbial contamination and constitute a protective measure for patients. These findings may help improve management of air-conditioning systems during a pandemic. Future studies should attempt to assess multiple air-conditioning parameters during operational hours with quantitative and qualitative measurements of temperature, relative humidity and ventilation rates. Strengths and limitations of this study • Systematic methods were used to provide a comprehensive review of effects of air-conditioning systems and HEPA filters on the transmission of pathogenic microorganisms and related diseases. • This study focused on hospital public areas, which are more likely to be overlooked relative to areas such as the operating room and ICU. • Only articles published in English were included in this study.	2021-09-06T13:17:35.321Z	2021-08-27T00:00:00.000	{ "year": 2021, "sha1": "193b16092efd69976e32d3d4cc6d19c58176c119", "oa_license": "CCBY", "oa_url": "https://www.cambridge.org/core/services/aop-cambridge-core/content/view/CA24F8E12257DFFD400CCE74602BB8D6/S0950268821001990a.pdf/div-class-title-effects-of-air-conditioning-systems-in-the-public-areas-of-hospitals-a-scoping-review-div.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "193b16092efd69976e32d3d4cc6d19c58176c119", "s2fieldsofstudy": [ "Environmental Science", "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
201900629	pes2o/s2orc	v3-fos-license	Adaptive Dynamic Grid Partitioning for Reactive-Power/Voltage Control Based on Secondary Voltage Control Reactive power voltage partition control is an important part of AVC secondary voltage control. Aiming at the current status of secondary voltage control, based on the voltage/reactive sensitivity and modularity function, an adaptive dynamic partitioning method is proposed, which changes with the operating conditions of the grid. With each reactive power source partition center, the load nodes are mapped to the corresponding reactive power sources according to the voltage/reactive sensitivity, and the regional merging automatically forms the optimal partition by using the modularity function as a measure. Finally, the proposed method is verified by the IEEE-39 system. Introduction Reactive voltage control is an important means to ensure the safe and stable operation of the power system. For large-scale power grids, due to the differences between the parameters of the branches and the different access points of the reactive power source, the reactive voltage problem has obvious areas characteristic. At present, the layered voltage control scheme proposed by the French Electric Power Company (EDF) has been widely used [1]. Among them, the large-scale power grid is decomposed into several loosely coupled partitions, which decomposes the whole network voltage control problem into sub-problems of independent voltage control in several partitions, which is the main content of the secondary voltage control [2]. Partition control is based on the local characteristics of reactive voltage. Because of the electrical connection of the entire power grid, reactive voltage coupling between regions is present, so the effect of secondary voltage control depends on the degree of electrical coupling between regions. EDF adopts the fixed partition mode of offline tuning, that is, the "hard partition" mode. With the expansion of power system scale and the change of operation mode, the reactive voltage sensitivity between nodes changes in real time with the change of operating conditions, and the coupling between regions the degree changes, and the fixed partition mode is difficult to meet the needs of development. Therefore, the domestic adopted a layered voltage control mode based on "soft partitioning". The area REES2019 IOP Conf. Series: Earth and Environmental Science 300 (2019) 042109 IOP Publishing doi:10.1088/1755-1315/300/4/042109 2 division is completed online in the main station system. According to the current grid structure characteristics, the grid is divided into loosely coupled areas, load nodes and power supplies. The area to which the node belongs is not static, but is completed online by the online soft partition module according to the current topology and operation mode [3]. At present, the partitioning problem can be reduced to a typical clustering problem. Many clustering algorithms [4][5][6][7] have been applied to the division of reactive voltage regions, but the number of partitions needs to be given in advance regardless of the clustering algorithm, or give a parameter threshold to determine the number of partitions, it is difficult to achieve real-time dynamic partition without human intervention. In this paper, a new grid adaptive dynamic reactive voltage partition method is proposed. From the regional controllability, the reactive power source node is used as the center of each partition, and the load node is mapped to the load node voltage/reactive sensitivity according to the control node. Each reactive source forms an initial partition. Then, the modularity function is used as a measure to automatically determine the number of partitions for regional merging to form an optimal partition. Finally, the proposed method is verified by the IEEE-39 system. Partitioning Principle The grid reactive voltage partition needs to meet the following requirements: 1) Ensure strong electrical coupling between the internal nodes of each partition, and weakly couple the electrical coupling between the partitions to reduce the mutual influence of reactive voltage control between the regions; 2) Ensure the reactive power balance in each zone and leave a certain reactive reserve to realize the rational distribution of reactive resources; 3) Each node in the same partition maintains connectivity, that is, each node in the same partition is directly or indirectly connected, and does not need to be connected through nodes in other partitions; 4) There are no isolated nodes, that is, there are at least two nodes in the partition, and each partition cannot have duplicate nodes. Full-scale Voltage / Reactive Sensitivity When studying the voltage partition problem, the nodes in the power grid can be basically divided into two categories: load node and control node. The load node generally appears as a PQ node in the power flow equation, and the control node is a reactive power source node capable of providing reactive power support. In this paper, the generator node is used to replace the general reactive power source node, and an automatic excitation regulator (AVR) is installed. The generator node is generally represented as a PV node in the power flow equation. The voltage/Reactive sensitivity of each PQ node in the system can be obtained from the inverse of the Jacobian matrix in the power flow calculation. Based on the AC flow equation, the nonlinear power flow equation is linearized at the steady state solution to obtain a matrix expression: Where: ΔP and ΔQ are respectively injected into the active and reactive power deviation matrix; Δθ and ΔU are respectively the node voltage phase angle and amplitude variation matrix; J is the Jacobian matrix. Inverting equation (1) Where: the sensitivity factors S PU and S QU are the unit injection active and reactive power voltage amplitude changes respectively; the S Pθ and S Qθ are the unit injection active and reactive power voltage phase angle changes respectively. From equation (2), The relationship between the distribution node voltage variation ΔU and the active and reactive power variation sequences ΔP and ΔQ can be expressed as: In the formula: In addition to the influence of the active/reactive power variation, the node i voltage is also affected by the injection of other nodes ΔP j and ΔQ j , which is expressed as: Where: U 0 i is the steady-state voltage of node i; S PU,ij and S QU,ij are elements of S PU and S QU , respectively. The sensitivity factors S PU and S QU reflect the magnitude of the effect of active/reactive power on the node voltage, respectively. The PV node contains the control of the voltage, and the PV node needs to be dimensioned into the sensitivity matrix. In reference [8], the concept of reactive power control space is proposed, and the influence of reactive power failure on the voltage of each node is considered. Based on this idea, the literature [9] further constructs a node including the system except the balance node. Voltage/reactive full-dimensional sensitivity matrix. For n-node systems, nodes 1~m are PQ nodes, and nodes m+1~n-1 are PV nodes. According to the linearized power flow equation, the system PQ node voltage/reactive sensitivity can be expressed as: Each reactive source node is solved one by one in a "successive recursive" manner, and its corresponding physical meaning is the voltage response of other nodes in the case of only regulating the reactive power source node reactive. When solving the sensitivity of a reactive source node A, set node A as the PQ node, which is called the observation power supply, and the other reactive power source nodes are still PV nodes (if a reactive source node has insufficient reactive reserve, it is set to The PQ node) can obtain an augmented voltage/reactive sensitivity matrix X'. 11 1 The first m elements in the last column of matrix X' represent the voltage/reactive sensitivity of the observed power supply to other PQ nodes of the system; the first m elements in the last row indicate the voltage/reactive sensitivity of other PQ nodes to the observed power supply; the last element of the matrix To observe the voltage/reactive sensitivity of the power supply node itself; the other elements of X' are substantially equal to the X matrix. Before solving the sensitivity of the next reactive source node B, set A back to the PV node and B to the PQ node, so that each power node is successively listed as the power of the observation point, and the above process is repeated to obtain the full-width augmentation sensitivity matrix S., as in the formula (7), wherein Y is a diagonal matrix of n-m-1 order. Before and after the observation power is changed from the PV node to the PQ node, the system power flow changes little, and the power supply corresponding to the X' array in the upper left corner of the m-th order is fixed to be equal to the X array. ( 1 ) The full-dimensional sensitivity matrix contains the information of the load node and the power node, which can directly integrate the power node into the dynamic partitioning process of the system. Compared with the existing AVC secondary voltage control partition algorithm, only the load node is partitioned, and the power node is added to the geographic node. The practice in similar partitions is more reasonable. Modularity Function The community structure is an important attribute of a complex network. It is a set of nodes in the network with similarities between each other and less similarity with other nodes. The complex network consists of several communities [10,11]. Girvan and Newman et al. proposed the concept of modular Q function, which was extended to the weighted network to measure the structural characteristics of complex networks and determine the optimal number of partitions [12][13], as described below: In the formula: A ij is the weight of the sides connecting node i and node j, A ij =1 when node i and node j are directly connected, A ij =0 when they are disconnected; k i represents the weight sum of all the sides connected to node i; m represents the weight sum of all the sides in the network; and δ(i,j)=1 if node i and node j are in the same partition; otherwise δ(i,j)=0. According to the definition of module degree, the value of module Q is strictly less than 1. In a complex network, the modularity is positive if the sum of the weights of the internal links is greater than the sum of the weights of the random internal links. In practical applications, the higher the modularity, the closer the community inside, the more sparse the outside, the more reasonable the community structure of the network. Therefore, the optimal network partition can be determined by searching for the maximum modularity in all connections. As a kind of artificial complex network, power system has the characteristics of community structure, which can be studied by the theory of community structure. In this paper, in order to accurately describe the coupling coefficient between nodes, the weights of sides between nodes are mainly determined by the sensitivity of voltage and reactive power: Sensitivity between different nodes is related to the impedance between nodes, and the impedance between nodes is directly related to the geographical attributes between nodes. The sensitivity of voltage and reactive power between two nodes directly connected is higher, and the sensitivity between nodes not directly connected is lower. Therefore, the voltage sensitivity can be used as a partition evaluation index to ensure the connectivity and electrical coupling between nodes in a certain extent. Partition Method The partitioning method proposed in this paper is mainly divided into two parts: the first part is the initial partitioning; the second part is the merging of partitions. 1) Starting from the controllability of the reactive power source, the initial partition is carried out. Firstly, n-m-1 reactive power node is regarded as n-m-1 independent partition, and m load nodes are mapped to the reactive power node which is most sensitive to voltage/reactive power respectively, and the balanced node is added to the partition which is close to its geographical location. In this way, all nodes of the whole network are divided into n-m-1 regions, and each region contains reactive power nodes to ensure the controllability of the region. 2) The modularity function of the initial partition is calculated. Then, the modularity function is calculated by two sets of combinations of initial partitions. If the modularity function value of the original partition is higher than that before the combination, the combination scheme with the maximum modularity function value is selected to merge. Repeat the above process until the modularity function reaches its maximum to stop the merging of partitions, at which point the partition is the best partition. Most of the existing partitioning methods, which take modularity function as evaluation index, adopt hierarchical clustering. Each time, the two nodes with the largest modularity are merged to determine the optimal network partition. With the expansion of power grid scale, this global search method has higher time complexity and space complexity. In this paper, the initial partitions based on voltage/reactive power sensitivity can reduce the complexity of partition calculation to a certain extent and improve the efficiency of partition. Modularity index is used to merge partitions to ensure the strong electrical coupling of nodes within the partition and the weak electrical coupling of nodes between the partitions. The number of partitions can be automatically determined according to the current operating state of the system. Example Calculation and Analysis In this paper, IEEE 39 node system is used for simulation analysis. 1-29 nodes are load nodes (PQ nodes), 30-31 nodes and 33-39 nodes are control nodes (PV nodes), and 32 node is balance node. Table 1 is the result of the initial partition. 6 From the results of the initial partition in Table 1, it can be seen that the connectivity between the power nodes and the load nodes in each partition meets the requirements. Table 2~5 is the result of each region merging. Figure 2 shows the trend of the modularity function value with the number of partitions. As shown in Figure 1, the modularity function value increases first and then decreases as the number of partitions decreases. When the number of partitions is merged from 6 to 5, the modularity function value decreases. The optimal partition number is 6, and the partition scheme in Table 4 is the best partition scheme. The optimal partition diagram is shown in Figure 2. Conclusion Aiming at the problem that the existing reactive power and voltage partition schemes need to determine the number of partitions in advance, an adaptive dynamic partition scheme without manual intervention is proposed, which can automatically update the partitions according to the current operating conditions of the grid. In this paper, the partition scheme is proposed under the condition of sufficient reactive power capacity, but the actual reactive power reserve is constantly changing with the operating conditions. On this basis, considering the dynamic partition method to meet the constraints of regional reactive power reserve will be the main direction of future research.	2019-09-09T05:26:25.068Z	2019-08-09T00:00:00.000	{ "year": 2019, "sha1": "d4fde8badc761e1c8447392b404b4bb288bc4587", "oa_license": null, "oa_url": "https://doi.org/10.1088/1755-1315/300/4/042109", "oa_status": "GOLD", "pdf_src": "IOP", "pdf_hash": "b1ccfe365faef13de83df12f64449fc501065672", "s2fieldsofstudy": [ "Engineering" ], "extfieldsofstudy": [ "Physics", "Computer Science" ] }
118677880	pes2o/s2orc	v3-fos-license	The dark degeneracy and interacting cosmic components We study some properties of the dark degeneracy, which is the fact that what we measure in gravitational experiments is the energy momentum tensor of the total dark sector, and any split into components (as in dark matter and dark energy) is arbitrary. In fact, just one dark fluid is necessary to obtain exactly the same cosmological and astrophysical phenomenology as the LCDM model. We work explicitly the first order perturbation theory and show that beyond the linear order the dark degeneracy is preserved under some general assumptions. Then, we construct the dark fluid from a collection of interacting fluids. Finally, we try to break the degeneracy with a general class of couplings to baryonic matter. Nonetheless, we show that these interactions can also be understood in the context of the LCDM model as between dark matter and baryons. For this last investigation we choose two independent parameterizations for the interactions, one inspired in electromagnetism and the other in Chameleon theories. Then, we constrain them with a joint analysis of CMB and Supernovae observational data. I. INTRODUCTION Current cosmological observations indicate that about 96% of the total energy content of our Universe is made of yet unknown dark components and only 4% is made of particles of the standard model. Among them, there are precision measurements of anisotropies in the Cosmic Microwave Background (CMB) radiation [1][2][3], baryon acoustic oscillations [4,5], and Type Ia Supernovae [6][7][8]. For a recent review of the nowadays status of cosmology see, e.g., [9]. Usually this dark fluid is separated into two components: a clustering dark matter piece responsible for forming structure in the Universe and a nonclustering dark energy with a negative pressure that is responsible for the current accelerated cosmic expansion. Moreover, in the concordance model of cosmology -the so-called Lambda Cold Dark Matter (ΛCDM) model-the dark energy is of geometric nature and enters as a constant at the Lagrangian level of Einstein's gravitational theory. For gravity matters, the cosmological constant is indistinguishable from the vacuum contribution of quantum fields, and here appears an intriguing problem, the sum of both contributions is about 1 over 10 120 times the latter [10]. A second conceptual problem with the ΛCDM model comes out when one considers the ratio of dark energy over dark matter energy densities, which in spite of growing with the third power of the scale factor of the Universe, its value today is of order one. This is the so-called coincidence problem. As a consequence, a plethora of alternative proposals has appeared in the literature; as a short sample see [11][12][13][14][15][16][17][18][19][20]. * Electronic address: aviles@ciencias.unam.mx † Electronic address: jorge.cervantes@inin.gob.mx In this paper we argue that the separation of the dark fluid into dark matter and dark energy is arbitrary and is only favored for historical reasons and computational simplicity. After all, we define the dark fluid as our lack of knowledge as 1 where G µν comes from the observed geometry of the Universe and T obs µν from its observed energy content. In fact, the dark sector could be composed by a large zoo of particles with complicated interactions between them. Or, it could be even just one exotic unknown dark fluid. This property has been called dark degeneracy by M. Kunz [21]; see also [22][23][24][25][26][27][28]. In this paper we assume an astrophysical perspective to the dark fluid by defining it as a barotropic fluid with speed of sound equal to zero, as in [29,30] and more recently in [31]; for similar approaches see [32][33][34]. Making the speed of sound equal to zero ensures that dark fluid energy density perturbations will grow at all scales, but at the same time, it allows the fluid to have a nonzero pressure. This is quite in contrast with canonical scalar fields as quintessence, for which the speed of its perturbations is equal to one, a feature that makes dark energy perturbations to be quickly damped, so they do not grow inside the horizon. Interactions within the dark sector has been studied largely in the literature [35][36][37][38][39][40][41][42][43], mainly as a mechanism to solve the coincidence problem. On the other hand, interactions between dark matter and the standard model of particles are expected, and have been studied also on several occasions. In fact, the weakly interacting massive particles (WIMPs) paradigm has emerged as the predominant scenario to solve the missing mass problem [44][45][46][47][48]. Nevertheless, other alternatives have been proposed. Among them, special attention has been attracted by the strong interacting dark matter scenarios [49][50][51][52][53][54], in which dark matter interacts with itself and with baryons [50] through the strong force. It has been shown [49,55] that this alternative can alleviate the cuspy halos and overdensity of substructure problems that appear in N-body simulations based on the standard ΛCDM model [56]. In this paper we develop a general class of couplings of the dark fluid to the standard model of particles, and show that they can be also understood as interactions of dark matter to baryons. Then, we impose constraints by using CMB anisotropies and supernovae observations. This work is organized as follows: In Sec. II we define the dark fluid from the properties of the dark matter itself. In Sec. III we work out the cosmological background solutions for the dark fluid and show that they are identical to the ΛCDM model ones, leading to the dark degeneracy. In Sec. IV we show that the dark degeneracy is preserved under some assumptions when one goes beyond zero order in cosmological perturbation theory. In Sec. V we work a multifluid description of the dark sector, allowing interaction among its components. In Sec. VI we extend these interactions to baryons to try to break the dark degeneracy. Finally, in Sec. VII, we present our conclusions. II. THE SOUND SPEED OF THE DARK FLUID Considering Newtonian gravity for the moment, the overdensities of matter in an expanding Universe follow the equation in Fourier space where k phys is a physical wavelength (contrary to the comoving wavelength which we will use in the following sections) related to a physical length scale by k phys = 2π/l phys , c 2 s is the speed of sound of the fluid under consideration and prime means derivative with respect to cosmic time. In astrophysical scales it is safe to set H = 0 and then, the scale factor is equal to a constant that we make equal to one for this discussion matters. It follows that there is a threshold scale, the Jeans length, given by for which perturbations with physical length above it, l phys > l J , grow by gravitational collapse, and perturbations with l phys < l J develop acoustic oscillations. We expect to have dark matter structure at a wide range of scales: from the largest cosmological structure to galaxies. In fact even dwarf galaxies need to have dark matter halos in order to stabilize their disks and to account for the necessary gravity sources to obtain the observed flat rotation curves which follow the luminous matter in there. The only way to guarantee that dark matter perturbations grow at all scales is demanding that the Jeans length be equal to zero. For barotropic fluids the adiabatic speed of sound coincides with the speed at which perturbations propagate in the fluid. We define the dark fluid as a barotropic fluid with an adiabatic speed of sound equal to zero, and that, at a first approximation, does not interact with particles of the standard model. 2 Without lost of generality we can write its equation of state as where w is its equation of state parameter and is a function of the energy density ρ only. Thus we have c 2 s = (∂P/∂ρ) s = dP/dρ. Using Eq. (4) we obtain that the equation of state parameter is solved by the equation ρdw/dρ + w = 0, whose solution is with C a constant and the minus sign is set for later convenience. This means that the pressure is a constant Astrophysical observations constrain this value to be very small, \|P \| ≪ ρ A , where ρ A is the energy density of typical astrophysical scales where dark matter has been detected. Usually, it is assumed that dark matter is pressureless, but this is by not means necessary, for instance it could be the case that \|P \| ∼ ρ c0 , where ρ c0 is a typical cosmological energy density scale at present, without getting in contradiction with observations. In fact, this is the entrance that leads us to consider the dark fluid to be dark energy as well as dark matter. III. DARK FLUID AS DARK ENERGY Now, let us consider a Friedmann-Robertson-Walker description of the Universe at very large scales, filled with standard model particles (b, γ, ...) and with the above-defined dark fluid, from now on labeled by d. The evolution equations of such a Universe are and where H ≡ a ′ /a is the Hubble factor. Equations (9) and (10) give ρ b = ρ b0 a −3 and ρ γ = ρ γ0 a −4 , where a subindex 0 means that the quantity under consideration is evaluated at present time, and we have normalized the scale factor to be equal to one today, a 0 = 1. Integration of Eq. (11) gives, using Eq. (6), where we have defined the constant K = (ρ d0 − C)/C. This expression is what we expect for the evolution of a unified fluid: a piece that redshifts with the scale factor as a −3 , as a dark matter component does, and a piece that remains constant, as vacuum energy. This result has been found elsewhere in the literature, as in [34], which investigated the ΛCDM limit of a Chapliying gas, or as in the study of barotropic fluids with constant speed of sound [29][30][31][32]. In fact, using the language of E. Linder and R. Scherrer [32], in our case the term proportional to a −3 is the aether piece of a barotropic fluid. Now, the equation of state parameter of the dark fluid (6) becomes We want to stress that the energy density of the dark fluid is proportional to the inverse of its equation of state parameter and that its pressure, expressed in terms of the constant K instead of C, is In order to ensure the positivity of the energy density at all times, K must be a positive number. This implies that the pressure is negative, a quality that allows the dark fluid to accelerate the Universe, and as we have outlined in the last section it could take values of the order of the critical density (∼ 3H 2 0 /8πG) without affecting the behavior of the dark fluid as dark matter in astrophysical scenarios. The Friedmann equation (8) becomes (16) This is the same evolution equation for the scale factor as in the ΛCDM model. This is not an accident, if one assumes ΛCDM as the valid model and considers the total energy density of the dark components, ρ T = ρ DM + ρ Λ , the total equation of state parameter, w T , defined by where the subindex a runs over dark matter (DM) and cosmological constant (Λ), is given by where Ω DM = 8πGρ DM0 /3H 2 0 and Ω Λ0 = Λ/3H 2 0 . (Clearly, the equationρ T = −3H(1 + w T )ρ T is accomplished for the total energy density.) Then, comparing these results to Eqs. (13) and (16), we note that under the identifications and where Ω d = 8πGρ d0 /3H 2 0 , the resulting evolution cosmology in both models are exactly the same, at least at the background level. This property has been called dark degeneracy by M. Kunz in [21]. In fact, it is more general than for the single fluid case worked here: any collection of fluids whose total equation of state parameter is equal to Eq. (18) and that do not interact with baryons and photons will behave exactly as the composed dark matter-cosmological constant fluid, leading to a degeneracy with the ΛCDM model. IV. BEYOND THE BACKGROUND In this section we explicitly show that the degeneracy is preserved when one goes beyond the homogeneous and isotropic cosmology, but only under some general assumptions that have been taken for granted in previous works. We divide the discussion into linear and higher orders in cosmological perturbation theory. A. Linear order Let us consider cosmological perturbation theory in the conformal Newtonian gauge, the metric is given by (for details see [57]) (21) where τ is the conformal time related to the cosmic time by dt = adτ . As usual, we define the matter perturbation variables through the expressions of the energy momentum tensor where Π i j is the anisotropic (traceless) stress tensor. The energy density ρ and the pressure P denotes background quantities and are functions of the conformal time only. The vector v i is called the peculiar velocity and is related to the four velocity u µ of the fluid by the relation v i = u i /u 0 . In Fourier space we define the velocity θ = ik i v i and the scalar anisotropic stress σ = 2k i k j Π ij w/3(1 + w). We use the flat space metric (δ ij ) to raise and lower indices of intrinsic space geometrical objects like v i and Π i j . The hydrodynamical equations for a general fluid are where δP = P π L , δρ = ρδ, H =ȧ/a and a dot means derivative with respect to conformal time. For the dark fluid case, (1 + w d ) = 0 and these equations becomė while for baryons after recombination (when the coupling to photons can be safety neglected) The fluid equations are supplemented with the Einstein's equations and where the sum runs over all fluid contributions and is the rest fluid energy density [58]. To solve these equations, we need to add information about the nature of the dark fluid. The barotropic condition implies that δP = c 2 s δρ and thus δP d = 0, and because it is a perfect fluid, the anisotropic stress vanishes, Π i d j = 0. Accordingly, the space-space components of the perturbed energy momentum tensor are equal to zero, δT ij d = 0. Therefore, in the right-hand side (rhs) of Eq. (27) the last term vanishes, and in the rhs of Eq. (28) only the two first terms survive. If there are only baryons and dark fluid the two gravitational potentials are equal, Ψ = Φ. Figure 1 shows the evolution of a mode k = 0.05 Mpc −1 of the perturbations variables. The dashed lines shows results for the ΛCDM model, for which we have to use the dark matter perturbations equations, instead of Eqs. (27) and (28). In evolving the perturbations for both models, ΛCDM and dark fluid, we have imposed on the initial conditions the relations for the velocities, and we let α to take different values. These initial conditions are given at an initial time well after recombination, so we can use Eqs. (29) and (30) and the relation δ DM ≃ δ b holds. We note that the evolution of the density contrast of baryons for the specific mode we have chosen is indistinguishable for both models if we take α = 1. Then, although the cosmological observable is the baryonic matter power spectrum which includes a wide range of wavelengths, Fig. 1 suggests that indeed the results are the same for both models for any wavelength, as we show below. In the cosmological context, imposing these two initial conditions is equivalent to demand that at first order in perturbation theory, the time-time and time-space components of the perturbed energy momentum tensor of the dark fluid and ΛCDM models are equal at the given initial time τ i . It is straightforward to show that both conditions, in the case α = 1, will be preserved at all times. This means that in obtaining the α = 1 solutions in Fig. 1 we have imposed that and From now on, we will demand Eqs. (36) and (37) to hold for all considered fluids, staying in degeneracy with the ΛCDM model at first order in cosmological perturbation theory. Later on, in Sec. VI, we will try to break this degeneracy, but through couplings of the dark fluid to baryonic matter. From the results of the previous section it is straightforward to see that ρ DM /ρ d = 1 + w d , then Eq. (37) implies that θ DM = θ d , which explains the behavior of the curves in Fig. 1d. Moreover, from Eq. (31) it follows that the gravitational potentials Φ are the same for both models, so the behavior in Fig. 1a. Finally, inserting Eqs. (36) and (37) into Eqs. (34) and (35), respectively, and using Eq. (14), we obtain that the hydrodynamical perturbation equations for the dark fluid arė These equations are equal to (27) and (28) for a barotropic perfect fluid, confirming the dark degeneracy at first order level. It is interesting to note the behavior of the density contrast of the dark fluid (Fig. 1c), which initially grows as a dark matter component to later decay asymptotically to zero. In fact, this behavior is natural for all wavelengths modes as can be seen from Eqs. (27) and (28): At late times, when w d tends to -1, theδ d source becomes independent of θ d and is a negative quantity which tends to zero. Meanwhile at early times, for w d → 0, Hw d → 0, and the equations become equal to the cold dark matter evolution equations, (34) and (35). B. Beyond linear order To go beyond the linear order, let us make perturbation expansions to the dark fluid and ΛCDM energy momentum tensors about the (zero order) background cosmological fluids as If the total energy momentum tensors of both models are equal T d µν = T ΛCDM µν , clearly each of the terms in the expansion will be equal as well T This argument is outlined in [21] to argue that the degeneracy is preserved at all orders. Certainly, this is correct. Nonetheless, we want to stress a different approach: We are affected gravitationally by the total energy momentum tensor, but usually when comparing observations to models we expand it as in Eq. (40), and after this we assign values to each of the pieces. The fact that both energy momentum tensors are equal, say at zero order, does not imply that they will be equal at first order. In this situation, equations such as (36) and (37) are conditions of the theory and not consequences of it, and if not imposed, the degeneracy is broken, as seen in Fig.1 for α = 1. This approach, which we will adopt, is very close to that followed in some papers which investigate modified gravity theories that are indistinguishable in the background from the ΛCDM model but could differ in linear order, leaving imprints that are parametrized to compare to current and future observations (see [59] and references therein). In fact, if some of these imprints were detected, they could be a consequence of interactions within the dark sector (or even with baryons, as those we study in Sec. VI), instead of a deviation of General Relativity. Finally, there is a subtle point that is pertinent to assert at this moment. The fact that the Universe is so smooth at very large scales is what allows us to expand the energy momentum tensor of the cosmic fluids as in Eq. (40). The zero-order term is in fact a spatial average in hypersurfaces of constant cosmic time, T (0) µν = T µν , and thereafter we assume that general relativity holds in the form G µν (g (0) ) = T µν . This is not true in general due to the nonlinear character of gravity; it is well known that backreaction effects contributes to this last equation. For recent work in the averaging procedure in cosmology and its caveats see [60]. For the specific case that concerns us there is a related issue. Let us consider a fluid with equation of state P = P (ρ) that leads to a unified description of the dark sector. It has been noted that it is not necessarily true that P = P ( ρ ) [61]; in fact higher-order corrections enter into this equation. Therefore, when some scale grows and leaves the linear regime, the naive averaging procedure on the equation of state is no longer valid. This nonlinear effect has been investigated in [29,61,62], and it was shown that instabilities are expected to occur in unified dark models, even on large cosmological scales. Two exceptions are the cases in which the involved equations of state are P = constant and P = wρ, with w a constant. The former is the case of the dark fluid, and then, it does not suffer for this averaging problem. Nonetheless, it is worth emphasizing that in models that depart -although indistinguishable by current observations when calculations are performed using the average procedure-from the dark fluid or from the ΛCDM models these effects must be considered. V. MULTIPLE INTERACTING DARK FIELDS From the last section it is clear that the dark fluid, although not necessarily, could be the sum of a dark matter and a cosmological constant components. Nonetheless, if the interaction to the particles of the standard model is only gravitational (which is the ultimate definition of dark), the nature of the dark fluid is fundamentally impossible to elucidate, because of the universality of this force. In this section we explore the possibility that the dark fluid is composed of a collection of dark components with possible interactions between them. The equations that we obtain will be generalized in the next section to include interactions to baryons. Let us consider the Einstein field equations, where the subindex a labels the different dark components. If we forbid dark components to interact with standard model particles, the Bianchi identities imply ∇ µ T µν SM = 0 and where the energy momentum transfer vectors, Q ν a , obey the constraint a Q ν a = 0. Considering for the moment the homogeneous and isotropic cosmology, the continuity equation for each fluid isρ where q a denote the energy transfer between different dark components. This implies that at zero order, (ignoring the subindex a's for the moment) if we define Q ≡ −g µν Q µ Q ν , then Q (0) = q/a and Q ν (0) = a −1 (Q (0) , 0). Now, following [63,64], we split the interaction vector as where the momentum transfer, f ν , is first order and is orthogonal to the four velocity f ν u ν = 0. Thus, under spatial rotations Q transforms as a scalar and f i as a vector. Accordingly, we decompose f i as with f a scalar and ǫ i a transverse vector, ǫ i ,i = 0, and we define the perturbation δQ through where in the last equality we defined δq ≡ aδQ. From (46) it follows that up to first order Finally, from Eq. (43) one obtains (after restoring a's) the constriction equations a δq a = 0, a q a = 0, and a 2 a Q i a = a (q a v i a + f ,i a + ǫ i a ), which by taking the divergence and going to Fourier space become a q a θ a + k 2 f a = 0. Now, to first order in perturbation theory, the divergence of the energy momentum tensor -for each fluid, omitting the subindex a-is Note that if the interaction vanishes, both equations reduce to the usual ones. We take the divergence of Eq. (54) to isolate the scalar mode perturbations, then Eq. where we have restored the index a's and back to Fourier space. Also we have used the decomposition for Π ij in tensor, vector and scalar pieces. Then when hit with k i k j only the scalar anisotropic stress survives and the first term of the rhs of Eq. (56) becomes 2k 2 Π (s) P/3 = (ρ + P )k 2 σ. Defining the total energy density of these dark fluids as ρ T = ρ a , it follows that the total density contrast, δ T , is the weighted sum of the individual components and the total velocity is given by where w T is given by Eq. (18), and the index a runs over all dark components. Using these two last equations it is straightforward to show that the hydrodynamical equations for the total fluid arė where δP T = δP a . These are exactly the equations of a single noninteracting fluid, Eqs. (25) and (26). The adiabatic speed of sound of the total fluid is c 2 sT =Ṗ T /ρ T = ρ a c 2 sa /ρ T . Thus, by forcing the condition c 2 sT = 0 we obtain that w T = w d , and if each dark component has positive energy (ρ a > 0), it also implies that c 2 sa = 0 and δP a = 0. Therefore, we obtain that the hydrodynamical equations for the total fluid perturbations become the same as the dark fluid perturbations (Eqs. (38) and (39)), under the substitution T → d. Thus, the composed fluid we have constructed is just the dark fluid. If one considers the unphysical case in which not all dark components have positive energy density, the speed of sound of some components can be different from zero, and the total pressure perturbation is equal to is the relative entropy perturbation between fluids a and b [65]. Therefore, to obtain the dark fluid in the case in which some components have negative energy density we have to impose the pressure perturbations given by Eq. (61) to be equal to zero. This condition also implies that the total fluid is barotropic. VI. INTERACTIONS TO THE STANDARD MODEL OF PARTICLES. In this section we study dark fluid couplings to baryonic matter. We consider models in which the background cosmology is the same as in the ΛCDM model, accordingly we do not allow energy transfer (q = 0) between the cosmic components. Nevertheless, we allow a momentum transfer different from zero. Thus, the interactions affect the fluids only at first order in perturbation theory. In this work we will not consider interactions to electromagnetism. This is not only for simplicity, many theoretical models present conformal couplings, as the chameleon theories [66,67] (and in general, scalar tensor gravity theories [68]), or even direct couplings to the trace of the energy momentum tensor [27,[69][70][71][72]. Also, this is expected in scenarios like the strong interacting dark matter [49,50], where the couplings are through the strong force, for which photons are chargeless. In the previous section we found the hydrodynamical equations for a system of coupled dark components. It is straightforward to generalize those results to a coupling between the dark fluid and baryonic matter. The conservation equations for the perturbations are, for the dark fluid,δ and for baryonṡ . These equations are supplemented by the constrictions (51) and (52). The interactions to electromagnetism are considered, but not shown in Eq. (66). Note that we have not considered a term c 2 sd k 2 δ d in Eq. (64), in agreement with the definition of dark fluid. In the absence of a fundamental theory we parametrize the coupling. But before do it, let us make a comparison to electromagnetism. Under the formalism we developed in the last section, the hydrodynamical perturbation equations for Thomson scattering (neglecting photon moments beyond the quadrupole) can be obtained if we choose δq γ = 0 and k 2 f γ = −ρ γ (1 + w γ )ax e n e σ T c(θ b − θ γ ), where σ T = 6.65×10 −25 cm 2 is the Thomson scattering cross section, n e is the number density of electrons, x e is the ionization fraction, and c = 1 is the velocity of light Using the analogy to electromagnetism we choose the interactions terms to be and where the parameter Σ I has units of area times velocity, or thermalized cross section σv , which we identify with some, unknown, fundamental interaction. n d is the number density of dark particles that we set equal to where we use the proton mass, m p = 0.938 GeV, as an arbitrary mass scale and ρ d0 is the energy density of the dark fluid evaluated today. Here, we have not an analogous to the ionization fraction, in empathy to universal interactions. The constrictions f b = −f d and δq d = −δq b = 0, enter into the baryons equations. Because of the relation ρ DM = (1 + w d )ρ d the interaction term goes as (θ d − θ b )a −2 . Then, the greatest deviation from the standard model of cosmology will come from the early Universe. Accordingly we expect the interaction to be tightly bounded from CMB anisotropies tests. We have modified the public available code CAMB [73] to account for the interaction. In Fig. 2 we show the angular power spectrum for the CMB radiation. There several curves are shown for different values of Σ I , which is expressed in units of 10 −6 times the Thomson scattering cross section times the speed of light (c = 1). We note that the greatest differences with the ΛCDM model comes at high multipoles, which is not surprising because the interaction quickly decays and only modes which have entered into the horizon at early times in the Universe were affected by it. On the other hand, it is well known that interactions between the dark sector and baryons are tightly constrained from equivalence principle and solar system tests [74]. Nonetheless, such interactions could be originated by general predictions of string theory [75,76], leading to the necessity of some screening mechanism [66,67,77,78] to evade these experimental constraints. In chameleon Notes. theories [66,67], the range of this interaction depends on the energy density of the surrounding medium, leading to long-range forces when the density is low, and to short-range forces when it is high. Inspired by these theories, we give a k dependence to the hydrodynamical equations, such that the interactions respond to an effective wavenumber given by k 2 eff = k 2 g(ρ), where g is a monotonic growing function of the ambient energy density. By noting that ρ falls with the scale factor, we choose a potential law for the wavelength number, k 2 eff = k 2 /a n . In the following we specialize to the case n = 2. We use the substitution k → k eff in Eq. (68) and obtain 3 Clearly, we can also understand this interaction as a dependence of the cross section on the ambient energy density. In Fig. 3 we show the CMB angular power spectrum for different values of the parameter Σ II . We note that, in remarkable contrast to the Σ I interaction, here all scales are affected in a similar way. This is because the effective interaction remains constant while the scale factor grows and all modes feel it when they enter the horizon. It is possible to treat both parametrizations together if we choose The hydrodynamical equations becomė and for baryonṡ If one assumes the ΛCDM decomposition of the dark fluid in dark matter and cosmological constant, it is easy to see that instead of Eqs. (63) and (64), the following where we have used the conditions (36) and (37) Thus, although the degeneracy with the ΛCDM has been broken at first order in perturbation theory, there exist degeneracies to other models, as in this case to ΛCDM with the same interactions, which means that the general class of interactions given by Eqs. (63) and (64) does not help us to elucidate the actual decomposition of the dark fluid (if it exists). Accordingly we can treat the above described couplings as interactions between dark matter and baryonic matter, without loss of generality, as we will do for numerical purposes 4 . Thereafter, if desired, we can go back and forth between both models' results using Eqs. (19) and (20). To constrain the interactions, we perform a Monte Carlo Markov Chain (MCMC) analysis over the nine-parameter space (Model A) {Ω b h 2 , Ω DM h 2 , θ, τ, n s , log A s , A sz , Σ I , Σ II } using the code CosmoMC [81]. θ is the ratio of the sound horizon to the angular diameter distance at recombination, τ is the reionization optical depth, n s is the spectral index of the primordial scalar perturbations and A s is its amplitude at a pivot scale of k 0 = 0.05 Mpc −1 . We have imposed flat priors on the two interaction parameters: 0 < Σ I < 10 −7 × σ T and −11 × σ T < Σ II < 10 × σ T . For the CMB anisotropies and polarization data we used the Wilkinson Microwave Anisotropy Probe (WMAP) seven-year observations results [3]. For the joint analysis we use also Hubble Space Telescope measurements (HST) [82] to impose a Gaussian prior on the Hubble constant today of H 0 = 74 ± 3.6 km/s/Mpc, and the supernovae type Ia Union 2 data set compilation by the Supernovae Cosmology Project [8]. We also study two other models: Model B, only considering the interaction Σ I , it has an eight-parameter space {Ω b h 2 , Ω DM h 2 , θ, τ, n s , log A s , A sz , Σ I }; and Model C, which does not consider any interaction, a sevenparameter space {Ω b h 2 , Ω DM h 2 , θ, τ, n s , log A s , A sz }, corresponding to the standard ΛCDM model. The summary of the posterior one-dimensional marginalized probabilities is outlined in Fig. 4 and Table I. In Fig. 5 we show the contour confidence intervals for 4 Special care must be taken because numerical codes, as CAMB, use synchronous gauge and fix the residual gauge freedom by taking θ DM = 0. In cases in which there are sources in the θ evolution equation, like ours, this is not possible to do. the marginalized Σ I − Σ II space at 0.68 and 0.95 confidence levels (c.l.). There, the high degeneracy between both parameters is shown: while Σ II takes values closer to zero, Σ I also does. It is interesting that nonzero values of the interactions (when introduced) are consistent and preferred by the considered data at 0.95 c.l. We note that the addition of the Σ I and Σ II interactions to the theory produce remarkable differences between the parameters estimations of Model A and Model C. This is more evident for the baryonic matter energy density Ω b h 2 , as can be observed from Figs. 4, 6, and 7, or read directly from Table I. For this reason, we study Model B, which only considers the Σ I interaction. In this case, the tensions between the parameters estimations are alleviated. The discrepancies are also evident in the primordial power spectrum parameters A s and n s , which also can be seen from Figs. 4 and 8. Nonetheless, these are not alleviated when one considers Model B. Instead of using the proton mass as the scale in the interactions, we can use an arbitrary associated mass to the dark matter or dark fluid particles, m d . We obtain the following constraints at 0.68 c.l. on the ratio Σ/m d (we use c = 3 × 10 10 cm/s): For the case in which we consider both interactions (Model A) We only obtained bounds of m d and Σ in the combination Σ/m d , although it is possible to find constrictions of them separately, even to the cross section and the particles' thermal velocity, leading to constraints in the σ−m d plane. Nevertheless, to do this we have to allow c 2 s = 0, and moreover, make further assumptions on the thermodynamics of the dark matter (or the dark fluid) [52]. Note that the effective thermalized cross section for interaction II is a 2 Σ II , and this is equal to Σ I at about a redshift z ∼ 10 5 . Before this epoch interaction I dominates, and after that, interaction II starts to do it. Just before recombination, at z ≃ 1100, interactions I and II are smaller in strength than the Thomson interaction by about 9 and 5 orders of magnitude, respectively. After this time, the ionization fraction x e falls exponentially and Thomson scattering becomes subdominant very quickly. In Fig. 9 we plot the CMB power spectrum for the mean values estimated by the MCMC analysis. The reported difference is ∆ = l(l + 1) C Model C l − C l . We note that the three models are well inside the error bars of the binned measurements from WMAP seven-year observations results. Nonetheless, at higher multipoles (l > 1000) the three models start to have notable discrepancies. The higher differences appear in the region 1000 < l < 2000, just inside the window in which the primordial power spectrum parameters are expected to be estimated with higher precision (1000 < l < 3000). For l > 3000 secondary anisotropies, mainly the Sunyaev-Zeldovich effect, are expected to dominate the CMB spectrum, while for multipoles l < 1000 the spectrum is more sensible to the other parameters. These high-l power spectrum multipoles will be probed by the PLANCK mission [83], so we expect to obtain tighter constraints in the near future. In this work we have not considered the effect that interactions I and II could have on big bang nucleosynthesis. This is because our phenomenological model only includes the thermalized cross sections Σ I and Σ II of elastic collisions, whose strengths are at least 9 orders of magnitude weaker than the Thomson scattering at this epoch, and more important, do not annihilate baryons and maintain the baryon-to-photon ratio unaltered. Accordingly, we expect the effect over this process to be quite weak. VII. CONCLUSIONS In this paper we worked out some properties of the so-called dark degeneracy [21]: the fact that we can only measure the total energy momentum tensor of the dark sector and any split into different pieces (as in dark matter and dark energy) is artificial. Although it could be mathematically convenient. We start by defining the dark fluid as a barotropic fluid with speed of sound equal to zero, as in [29,30] and, more recently, in [31] (for similar approaches see [32,33]). This is motivated by astrophysical constraints in the dark matter besides its cosmological properties. Making the speed of sound equal to zero allows dark fluid energy density perturbations to grow at all length scales, as a cold dark matter component does, while it leaves room for a constant pressure different from zero. Astrophysical scenarios forbid this pressure to be very high, but it well could take values of the cosmological critical density today. From the positivity of the energy density of the dark fluid it follows that this pressure has to be negative. Thus, we conclude that the dark fluid could act as dark energy. Then we study the background cosmology for the dark fluid and show that, not surprisingly, the same evolution equations as in the ΛCDM model are obtained, leading to a degeneracy between both models. We conclude that any collection of fluids for which its total equation of state parameter is equal to Eq. (18) will lead us to the same result at the background cosmological level. This is not necessarily the case when we consider linear perturbations. In order to preserve the dark degeneracy we demand that the dark fluid be a perfect fluid. For a barotropic fluid, this last condition implies that the first order space-space part of the energy momentum tensor is equal to zero. Furthermore, one has to add that the energy momentum flux density is equal to that of the ΛCDM. Thus, we have demanded that the energy momentum tensor of the dark fluid be equal to the ΛCDM one at first-order in perturbation theory, otherwise the degeneracy is broken. When considering a collection of multiple interacting fluids and demanding that the adiabatic speed of sound of the total composed fluid be equal to zero, we obtain exactly the perturbation equations of the dark fluid. This shows that the degeneracy is present also for more complicated dark sector schemes. From the first five sections, we conclude that there exist an infinity of cosmological models that give exactly the same observational signatures. Accordingly, it is fundamentally impossible to elucidate the actual structure of the dark sector. Thus, it is a matter of taste to prefer the ΛCDM model over any one of the exposed in this paper. In fact, to economize it is better to take the one fluid choice (the dark fluid) as the correct model. The only hope we have to understand the actual nature of the dark sector, is that it interacts in some way with the particles of the standard model. Of course interactions would in general break the degeneracy, but this is not necessarily true. This subject is studied in Sec. VI, where we allow the degeneracy of the dark fluid and the ΛCDM model in the homogeneous and isotropic cosmology, and then, we try to break it by adding interactions to baryons at first order in perturbations theory. We show that a general class of interactions defined by Eqs. (63) and (64) also can be understood as between dark matter and baryons in the ΛCDM model context, leading to a new degeneracy, although not with the concordance model, but with a ΛCDM-plus-interactions model. As a consequence, these interactions does not help us to favor any decomposition of the dark fluid. For the latter investigation we have chosen two independent interactions: one that resembles the Thomson scattering between photons and baryons, regulated by a parameter Σ I , and a second, density-dependent interaction, inspired by chameleon theories and parametrized by Σ II . For the analysis we have used a combination of the seven-year results of WMAP, the Union 2 data set compilation of supernovae measurements, and a prior in the Hubble constant of the Hubble Space Telescope. When we consider both interactions together, we obtain that other parameters of the theory differ too much from its standard values, up to almost 10% in the case of the baryonic density parameter Ω b h 2 . Then, we study the case in which Σ II is equal to zero, allowing Σ I to vary (Model B). Here, we show that the tension between the parameters estimation is reduced. The introduction of the two parameterized interactions is allowed by current data, and if done, it is remarkable that nonzero values of them are preferred at 0.95 confidence level.	2011-10-14T19:13:52.000Z	2011-08-11T00:00:00.000	{ "year": 2011, "sha1": "b7b14eac2a2ee46eaca9974b6b374f945dff5821", "oa_license": null, "oa_url": "http://arxiv.org/pdf/1108.2457", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "b7b14eac2a2ee46eaca9974b6b374f945dff5821", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
125109046	pes2o/s2orc	v3-fos-license	IMUNNOLOGICAL AND STRUCTURAL ANALYSIS OF HPV-POSITIVE CERVICAL CARCINOMA CELL LINES AND BOVINE PAPILLOMAVIRUS VIRUS-LIKE PARTICLES (BPV-VLP). Dra Dra. Rachel Siqueira de Queiroz Simões and Dra. Ortrud Monika Barth. Laboratory of Morphology and Viral Morphogenesis, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Avenida Brasil 4365, Manguinhos, 21040-900, Rio de Janeiro, RJ, Brazil. E-mail: rachel.simoes@ioc.fiocruz.br ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Introduction Papillomaviruses (PVs) have closed circular double-stranded DNA genome (8kb) presenting icosahedral capsid structural proteins, composed of the L1 (major) and L2 (minor), which can spontaneously co-assemble to form virus like particles. Cell transfection method for efficient intracellular assembly of papilloma viral vectors have been developed using bovine papillomavirus type 1 (BPV1) and human papillomavirus type 16 (HPV 16) as model papillomaviruses (Buck et al., 2004). Previous studies have been described an infectious HPV pseudovirion (PsV), comprising an HPV capsid containing a reporter plasmid as a surrogate virus (Buck et al., 2004). New therapeutic targets to HPV vaccination have been described including DNA-based vaccines, recombinant proteins, nanoparticles, synthetic peptides, viral and non-viral vectors (Simões & Barth, 2015) and expressed chimeric proteins self-assembled into virus like particles (VLP) from L1 major capsid proteins to produce HPV vaccines (Huber et al., 2017). 1004 L1 protein self-assembled into VLP presented inside HPV vaccine is immunologically and morphologically similar to native virions. L2 structural proteins minor capsid also induced high-titer neutralizing antibodies as the main mechanism of vaccine efficacy. Both proteins are used as synthetic peptides in studies about immune responses in BPV-1 and HPV-16 (Huber et al., 2017). There are several phenotypes of antigen-presenting cells (Langerhans cell, Migratory LC, Langerin dendritic-cell populations, dermal macrophages) in the skin which are migratory in the epithelial tissue. Merad et al. (2008) described different cellular markers in the skin and skin-draining lymph nodes in mice and humans. The expression of genes and the role of proteins involved in DNA damage repair pathways in cell lines as primary human keratinocytes (PHK) and HPV-positive (SiHa -HPV-16 and HeLa -HPV-18) and HPV-negative (C33A) human cervical carcinoma cell lines, as also in immortalized keratinocyte cell lines (HaCaT, not tumor control) have been described as possible prognostic markers of cervical cancer. Some studies have investigated the ability of the cytokine to inhibit the proliferation "in vitro" of normal and HPV infected keratinocytes, as well as the expression of E6 and E7 oncogenes (Prati, 2014). Cytokines include the growth factor (TGF-β), tumor necrosis factor (TNF) and interferons type I (IFN-α and IFN-β), which are produced by epithelial cells. The cytokine TGF-β has proved to be an inducer and inhibitor growth of tumor cells not infected by HPV 16 and 18. This effect appears to be associated with inhibition of E6 and E7 expression (Braun et al, 1992). IFN-α inhibits transcription of E6 and E7 genes in HPV-18 HeLa cells and also inhibits the expression of the E7 protein of HPV- 16 (Khan et al, 1993;Perea et al, 1995) In contrast, few studies have investigated the cell pathomechanisms and morphological changes in the host cells. The present study report the presence of the bovine papillomavirus virus-like particles (BPV-VLP) and morphological alterations inside the host cells in previously PCR positive samples from bovines affected by cutaneous papillomatosis. Additionally, described the morphology of HPV-positive (SiHa and HeLa) cervical carcinoma cell lines detected by electron microscopy. Warts samples and cell lines: Warts lesions from bovines of a typical cauliflower-like appearance were collected by surgical procedures using disposable gloves and scalpels and immediately stored on dry ice and formalin according to each techniques used. HPV-positive human cervical carcinoma cell lines as SiHa (HPV-16) and HeLa (HPV-18) were cultivated in RPMI medium. About 3x10 6 cells were counted and selected to molecular and morphological studies. Histopathology Examination: For the histopathological observation, the tumor samples were fixed in 10% formalin, embedded in paraffin and stained with haematoxylin and eosin. The microscopical patterns observed were indicative of papillomavirus infection. Ultrastructure: For ultrastructural analysis, the specimens (warts and SiHa and HeLa cells) were embedded in epoxy resin, fixed in 1% glutaraldehyde and post-fixed in 1% osmium tetroxide. Later steps followed by washes in cacodylate buffer 0.2 M in sodium sucrose 0.7% and distilled water. The dehydration steps were performed and using 1% uranyl acetate in 70% acetone, followed by 90% and 100% acetone. Warts and lines cells were included in epoxy resin and kept at 60 0 C to complete polymerization. Ultra-thin sections, 50nm thick, were performed to electron microscopy observation. Semi-thin sections, 0.5µm thick, were performed to optical photonic microscopy observation. The ultra-thin sections were stained with uranyl acetate and lead citrate, the semi-thin sections with methylene blue (De Souza, 2001; Simões et al., 2014). Molecular: About 30mg of tissue were used for DNA extraction using the tissue kit (Qiagen) according to manufactures`s instructions. The primers amplified approximately 268bp fragment of the β -globin gene used to evaluate the quality of genomic DNA extracted from the samples: β-globin forward: 5`-AACCTCTTTGTTCACAACCAG-3`) and βglobin reverse: 5`-CAGATGCTTAACCCACTGAGC-3`) (Yaguiu et al., 2006). DNA was used for polymerase chain reaction (PCR) analysis using degenerate primers. The PCR FAP59/FAP64 was originally designed to amplify 1005 a 478bp fragment of the L1 open reading frame in the conserved regions. The PCR primer pair FAP59/FAP64 was originally designed to amplify a 478bp fragment of the L1 open reading frame in the conserved regions. DNA samples were amplified using two PCR primer pairs: FAP 59 (forward: 5`-TAACWGTIGGICAYCCWTATT-3`) and FAP64 (Reverse: 5`-CCWATATCWVHCATITCICCATC-3`) (Forslund et al., 1999). PCR was executed in the same conditions applying the same primers used to verify the quality of DNA by β-globin gene. The samples were inserted in the thermocycler and followed the protocol described by Ogawa et al (2004). PV DNA-specific bands were identified by electrophoresis on a 1% agarose gel in TBE buffer under UV light (Ogawa et al., 2004).PCR products were purified using GFX PCR DNA purification kit (GE Healthcare) and sequenced. In order to optimize the current cell lines, DNA of SiHa and HeLa cells naturally infected with HPV-16 and -18, respectively, were extracted using DNA blood mini kit (Qiagen) and used as positives controls. The amplification mixture without DNA was used a negative control. MY09/11 consensus primers that amplify a 450 bp DNA sequence specific for HPV L1 ORF were used to positive standard samples. Results In the clinical evaluations papillomas were observed on the haired skin of the face, pinna and forelimbs. Histological examination of haematoxylin and eosin stained sections demonstrated epithelial hyperplasia, acanthosis, hyperkeratosis and tissue proliferation. The histopathological examination confirmed the characteristic of papillomavirus infection. Intranuclear corpuscular inclusions were observed by photonic microscopy. Strong electron-dense cells presenting well-developed mitochondria and numerous vesicles and rough endoplasmic reticulum (rER) were detected by electron microscopy (Figure 1). By optical photonic microscopy we observed in both cell lines similar features such as presence of very large nuclei with highlighted nucleoli, reduced cytoplasm, cell membranes presenting filopodia, few vacuoles and interconnected cells by desmosomes. Very electrodense cells were detected by electron microscopy presenting well developed mitochondria and rough endoplasmic reticulum, many vesicles and ribosomes in HeLa and SiHa cell lines ( Figure 2). Furthermore, HPV-DNA was amplified by PCR method using different amount of template (1-5µL) (Figure 3). These preliminary results suggested a strong cellular activation in these cervical keratinocytes. 1006 At the National Institute of Infectious Diseases in Japan, a Japanese researcher showed typical structures of autophagosomes evidenciated by the entry of HPV pseudovirions (PsVs) in HeLa cells inoculated with 16 PsVs (Ishii, 2013). According to recent reports, the presence of large vacuoles in HeLa cells inoculated with 16PsVs as described by Ishii et al. (2013) has been reported. In our study, without inoculation of VLP in the cell lines, the same large vacuoles were observed. The human keratinocytes in vitro of the HeLa and SiHa cell lines derived from carcinomas were immortalized by the presence of E6 and E7 genes expressed in cervix cancer (Zur Hausen, 1996). So, the ability of the cytokine to inhibit the proliferation "in vitro" of normal keratinocytes and of HPV infected keratinocytes as well as the expression of E6 and E7 oncogenes has been demonstrated by Howley et al. (2001).Ours findings indicated high cellular activity in the keratinocytes of the HPV-positive in SiHa and HeLa (HPV-18) cell lines by ultrastructural analysis, suggesting that these cells can be possible prognostic markers of cervical cancer.	2019-04-22T13:06:13.121Z	2017-04-30T00:00:00.000	{ "year": 2017, "sha1": "2708381bee89b2911ec8ea45cac846c8198e8c91", "oa_license": "CCBY", "oa_url": "http://www.journalijar.com/uploads/188_IJAR-16979.pdf", "oa_status": "HYBRID", "pdf_src": "MergedPDFExtraction", "pdf_hash": "782044306f8286c5482250add7e00aa7dc6de29e", "s2fieldsofstudy": [ "Biology", "Medicine" ], "extfieldsofstudy": [ "Biology" ] }
220424712	pes2o/s2orc	v3-fos-license	Dynamic Spin Fluctuations in the Frustrated A-site Spinel CuAl2O4 We performed nuclear magnetic resonance (NMR) and muon spin relaxation ({\mu}SR) experiments to identify the magnetic ground state of the frustrated quantum A-site spinel, CuAl2O4. Our results verify that the ground state does not exhibit a long-range magnetic ordering, but a glass-like transition manifests at T=2.3 K. However, the Gaussian shape and the weak longitudinal field dependence of {\mu}SR spectra below T show that the ground state has dynamic spin fluctuations, distinct from those of conventional spin-glasses. I. INTRODUCTION The A-site spinel system, where magnetic ions occupying the crystallographic A-site form a diamond lattice, has been extensively studied over the last decade or so as a candidate for the novel frustrated magnetic system. When it is influenced under antiferromagnetic nearest (J1) and next-nearest (J2) neighbor exchange interactions, its magnetic ground state can have noncollinear magnetic structures. Depending on the strength of the quantum fluctuations [1] and the ratio of the exchange interactions (J2/J1) [2], the ground state of the A-site spinel shows a complex phase diagram. For instance, when S≤1 and J2/J1>1/8, there is no thermal phase transition, and the ground state has the superposed state of spiral states with propagation vectors q spanning a particular surface in momentum space [2]. For S>3/2, a quantum-to-classical and muon spin relaxation (μSR), we report that the ground state of CuAl2O4 does indeed not order or freeze completely. Still, it has significant spin fluctuations, which persist well below T. II. METHODS High quality polycrystalline CuAl2O4 samples were synthesized by a solid-state reaction method. Initial raw materials of CuO (99.995 %, Alfa Aesar) and Al2O3 (99.995 %, Alfa Aesar) were mixed in a 1:1 molar ratio, and the mixture was pelletized to be sintered at 1000 ºC in the air for 2-3 days with several intermediate grinding steps. Single-crystals of CuAl2O4 were grown using a flux method [12]. CuO and Al2O3 were mixed homogeneously in a 1:1 molar ratio with a sufficient amount of flux material, which is anhydrous sodium tetraborate (99.998 %, Alfa Aesar). The mixture was contained in a platinum crucible and melted using a ceramic tube furnace flowing O2 gas. After heating up to 1350 ºC, it was dwelled for 24 hours to make the batch molten homogeneously and then slowly (1 ºC/h) cooled down to 750 ºC. After removing the flux by applying diluted HCl (~17 %), we separated dark brown singlecrystals of CuAl2O4 (photo shown as an inset in Fig. 1b) from the flux. The crystal size obtained measures up to 1 mm 3 with typical octahedral morphology of spinel-type crystals. The purity of the polycrystalline and single-crystal samples was further checked by powder and singlecrystal x-ray diffraction (XRD), respectively. High-resolution powder diffraction experiment was performed (Fig. 1c) using a commercial powder diffractometer, D8 Discover (Bruker), with wavelengths λKα of 1.540590 and 1.544310 Å (Kα1 and Kα2, respectively). XtaLAB P200 (Rigaku) with Mo source (λKα=0.710747 Å) was used to identify the structure of the single-crystals. The number of total independent reflections given by the single-crystal diffraction experiment was 148 (Fig. 1b). The observed intensity, F 2 obs, and the calculated intensity, or square of structural factor, F 2 calc of each reflection points are well-matched with each other, and this leads to reasonably good agreement factors: RF2, RwF2, and RF. The whole analysis was done through the integrated intensity refinement method powered by FullProf software [13]. We measured the bulk DC magnetic susceptibility of the CuAl2O4 single-crystal using a commercial equipment (MPMS-3, Quantum Design). We applied an external magnetic field parallel to the crystallographic [0 0 1] direction of the crystal. The NMR and μSR experiments were conducted to identify the characteristics of the local moments. We measured the NMR spectra of a single-crystal CuAl2O4 under a constant external magnetic field of B0=20 kG, scanning around the Larmor frequency of 27 Al with a nuclear spin I=5/2. μSR experiments were done at the ISIS muon source (MuSR) with a polycrystalline sample. We identified the temperature and field dependence of the muon depolarization spectra by cooling down to 1.4 K and by changing the longitudinal magnetic field up to 3 kG. The μSR data [14] with high statistics, obtained for 0.1<t<32.29 μs, were analyzed using a μSR analysis package, Mantid [15]. Density functional theory (DFT) calculations were carried out using the plane-wave program QUANTUM ESPRESSO [16] and utilized the generalized gradient approximation for the exchange-correlation functional [17]. The ions were modeled with ultrasoft pseudopotentials [18], while a norm-conserving hydrogen pseudopotential was used to model the muon. The energy cutoffs for the wave function and the charge density were set to 60 and 600 Ry, respectively, and the integration over the Brillouin zone was carried out using a 3×3×3 Monkhorst-Pack k-space grid [19]. III-A. Crystal structure CuAl2O4 has a cubic spinel lattice with space group Fd3 ̅ m (#227) and the lattice parameter a=8.083 Å (see Fig. 1 , which can lead to disordered magnetic interactions in this system. Other detailed structural information obtained from the refinement, such as the fractional atomic positions and thermal parameters, is summarized in Table 1. III-B. NMR measurement The observed NMR spectra show the central peak around 22.19 MHz (Fig. 2a), which corresponds to the Larmor frequency of 27 Al, ω0=γB0=22.1929 MHz, with the gyromagnetic ratio of 27 Al, γ=1.109406 MHz/kG and the external magnetic field B0=20 kG. As the temperature decreases, the width of the spectra gets broadened. It is related to the enhancement of internal fields upon the reduction of thermal fluctuations and the onset of a correlated state. In particular, we could not observe any evidence of long-range magnetic ordering, such as peak splitting below T [20]. According to the 27 Al MAS (magic-angle spinning) NMR spectrum of a spinel analog MgAl2O4 [21,22], the asymmetric central peak close to ω0 of CuAl2O4 originates from Al in the B-site. Since the peak shifts within 0.04 % down to the base temperature, the information obtained via the central peak can be related to the internal fields in the B-site for the whole temperature range. The temperature dependence of the inverse of the full width at half maximum (FHWM) of the NMR spectra matches well with the inverse susceptibility (Fig. 2b). Since the width of NMR spectrum is related to the distribution of internal fields on the B-site and the field distribution is associated with the disorder between Cu 2+ and Al 3+ ions [23], the correspondence between the spectral width and the bulk susceptibility indicates that the bulk susceptibility originates mainly from magnetism induced by the disorder. Meanwhile, the peak shift, K (%) from the resonance frequency w0 is another quantity that directly probes the local moments free from defects [20,23]. The magnitude of K increases up to T=4.2 K and then drops down at T=1.4 K as the temperature decreases, indicating a change in the magnetic ground state across T. A noticeable difference between the fieldcooled (FC) and the zero-field-cooled (ZFC) spectra is seen to develop as the temperature goes down below T (Figs. 2c and 2d). It shows that the correlated ground state behaves somewhat like a spin-glass state. In the case of 27 Al with I=5/2, the Zeeman splitting and the first-order nuclear quadrupolar interactions [24] produce four additional satellite peaks, coming from the transitions between Iz=±1/2 to ±3/2 and ±3/2 to ±5/2 states, around the central peak at ω0 (transition between Iz=±1/2). The NMR spectra of other spinel compound CoAl2O4 shows a five-peak structure, and the detailed feature changes depending on the direction of the external magnetic field B0 [25]. The minimum width of the spectra coincides with the angular factor of the quadrupolar interactions (3cos 2 θ−1)/2 (where θ is the angle between B0 and the three-fold rotational symmetry (C3) axis of the B-site) becoming zero, which occurs, However, we barely observed such an angular dependence in our CuAl2O4 spectra. Figs. 2a and 3 show the spectra taken at 200 K of a sharp central and broad satellite peaks. The satellite peaks are too broad to be resolved into a four-peak structure in our spectrum. And the intensity of the satellite peaks does not vary visibly when the orientation of the crystal is changed. Moreover, there are still broad shoulder peaks, which should disappear when the field is parallel to [0 0 1]. These characteristics can be understood as the effect of site-disorder. NMR spectra are sensitive to local fields surrounding the Al-site, and the NMR peaks can become broadened when the local fields are deformed. As the Al-site forms a pyrochlore structure, there are six neighboring Al 3+ ions around one Al 3+ ion, where some of them are interchanged with Cu 2+ ions. It naturally produces a distribution of local fields, which broadens the peaks [26]. Since the local fields have no preferred orientation (as for a powder sample), then angularindependent broadening emerges in both the primary and satellite peaks. III-C. μSR measurement Muon spin relaxation (μSR) is another essential tool for the study of local magnetism. In particular, it can identify fast spin dynamics well outside of the detection window of the NMR technique [27]. Also, it is possible to study a ground state without applying an external magnetic field, which may modify the nature of a ground state. The μSR spectra can be normalized following a relation A(t)=A1P(t)+Ac, where A1 and Ac are a normalization factor and a constant background, respectively. Above 3 K, the depolarization spectra P(t) shows Gaussian decay (Fig. 4a), which can be fitted to the following decay function: where the term inside the square bracket indicates a static Gaussian Kubo-Toyabe function [28] of the nuclear dipole moments with a distribution of Δ. The exponential term denotes fast paramagnetic fluctuations of the electron moments of Cu 2+ ions with the relaxation rate of = 2 e 2 /( 2 + 2 ( 0 ) 2 ) [29,30], where Δe is the width of a distribution of local fields induced by the electrons, ν is the fluctuation frequency of the local field at the muon site, γμ is the gyromagnetic ratio of muon (85.1 MHz/kG), and HLF is the applied longitudinal magnetic field (equals to zero for the zero-field data). In the high-temperature region (6-15 K), paramagnetic fluctuations are too fast ( − ≈ 1). And the nuclear contribution dominates over the depolarization with a constant value of Δ≅0.2 MHz (Fig. 4b), which is similar to the nuclear field distribution of another Cu-based spinel, CuGa2O4 [31]. As the temperature decreases, however, the correlation of electron moments starts to develop, and it forms finite local internal fields that can be decoupled from the nuclear moments. This picture corresponds well with the measured temperature dependence of Δ(T), which starts to reduce below 5 K and finally vanishes at 3 K. Moreover, ( ) ∝ 1/ gets enhanced almost by two orders of magnitude as the paramagnetic fluctuations reduce. Below T=3 K and down to the base temperature 1.4 K, we could not observe an oscillating feature that is expected for a long-ranged magnetic order. We did several trials to fit the spectra using non-oscillating models, such as single stretched exponential and two exponential functions. Still, we failed to get a proper fitting with realistic physical parameters. The zero-field spectra could only satisfactorily be fitted to an empirical decay function, , which is composed of two different decay channels. The result of the fitting is shown as solid lines in Fig. 4a. As the temperature goes down, the portion of the first term x increases and dominates at the base temperature ( Fig. 5c) (this point is discussed later), with the exponent β leveling off to 2 from 1 (Fig. 4b). Such initial Gaussian decay below T* is different from conventional spin-glasses in the narrowing limit (ν ≫5Δe), where the polarization decays exponentially (β=1) for a dense system or root-exponentially (β=1/2) for a diluted one [32]. In addition to the first term in Eq. (2), which fits the primary feature of the relaxation spectrum at the base temperature, there is an additional exponential term necessary to fit the tail of the μSR spectra. The decay rates λ1 and λ2 seem to level off to finite values at 1.4 K (Fig. 4b), which is the characteristic behavior of a dynamic ground state with significant quantum fluctuations [32,33]. These decay rates are comparable to what we obtained from 1/T1, of which T1 is the relaxation time to depolarize down to 1/e, and it does not depend on a model of a decay function used. In the case of a slowly fluctuating limit, however, the relaxation rate is rewritten to = 2 /3 [30], and it decreases as temperature goes down. It makes a sharp peak at the transition temperature for a statically freezing state [34], which is different from our result. We further measured the muon spectra with applied longitudinal magnetic field HLF to elucidate the dynamics of the ground state [35]. In the case the spin fluctuations in a ground state are significant, compared with the static case, a larger HLF is needed to cause the decoupling of the muon spin from local moments. Fig. 5a shows the asymmetry below T* under various HLF. If the magnetic ground state of CuAl2O4 is frozen, a longitudinal field μ0HLF=10Δe/γμ≈10·(1/T1)/γμ≈320 G will be sufficient to decouple the muon spins [33,35]. However, the moments of CuAl2O4 cannot be entirely decoupled even up to 3000 G, which provides a clear evidence for a dynamic ground state. To analyze the data quantitatively (Fig. 5b), we firstly tried fitting the data using the dynamic Kubo-Toyabe (DKT) decay function [28]. For μ0HLF=30 G, which is large enough only to decouple the nuclear moment, the depolarization curve can be fitted with Δe=3. 28(3) μs -1 and ν=2.64(6) μs -1 (line 1). However, using this model with these fitting parameters, the calculated polarization should be decoupled entirely under fields above μ0HLF=1000 G (line 2). The conventional DKT model, therefore, cannot be adopted to demonstrate the observed depolarization spectra. To fit the data under the longitudinal fields, we used the decay function from Bono et al. [32,36], where is the DKT decay function with a scaling factor f. The first term originates from the model with 'sporadic field fluctuations,' where the muon can observe local moments only in the fraction f of the total relaxation time t and the dynamics of the state are not affected by a longitudinal field [32,36]. This model was introduced first to explain frustrated kagomé bilayer compounds SrCr9pGa12−9pO19 (SCGO) and Ba2Sn2ZnGa10−7pCr7pO22 (BSZGCO), where a singlet state occasionally breaks into propagating unpaired spins (spinons). At the same time, the local moments of the systems can be detected during the short time ft when the unpaired spins emerge from the dimer [32,36]. For the rest of the experimental time, the polarization relaxes following the exponentially decaying function (the second term in Eq. (3)) representing other electron spins, which fluctuate according to a conventional Markovian process [28,30]. Using this model, we can successfully reproduce the whole field-dependent data (solid lines in Figs. 5a and 5b) with the fitting parameters of f≈0.046, Δe≈117 μs -1 , and ν≈115 μs -1 . These parameters are comparable to other systems that adopt similar models [32,36,37]. Notably, the fluctuation rate is as fast as that of a quantum spin-liquid candidate herbertsmithite [38] and almost two orders of magnitude faster than the fluctuation rate, which can be detected by NMR [27]. The weight x saturates to the maximum under zero-field (inset of Fig. 5c), which indicates that the major spin dynamics of the ground state is demonstrated by the sporadic field fluctuation process. Moreover, the fact, (1-x)>0 denotes the coexistence of minor phase, having relatively slower spin fluctuations ν≈7 μs -1 [Appendix]. Now, we can understand that the Gaussian decay of muon depolarization by utilizing the dynamic model with the sporadic field fluctuations. According to the conventional Markovian modulation of internal fields, a rapidly fluctuating state shows exponential decay on the limit of ν≫Δe [28,30]. Despite its remarkable local field fluctuations (ν≈115 μs -1 ), depolarization of CuAl2O4 can show a Gaussian decay because its local fields are in a region of ν≤Δe [32,36,37]. Moreover, the sporadic emergence of the local fields leads to the relaxation of having an effectively reduced local field distributions fΔe. It makes the 'actual' longitudinal field necessary to decouple the local moments (10·Δe/γμ≈14 kG) much larger than the external field evaluated from the initial decay rate (10·(1/T1)/γμ≈320 G). In addition to the first term in Eqs. (2) and (3), which depicts the dynamic nature of the ground state, there is an additional exponential term necessary to fit the tail of the μSR spectra. Like some of the spin-glasses showing exponential decay with moderate spin fluctuations [39,40], the other term can be related to the glassy phenomena observed in the previous bulk magnetic measurements [8] and our NMR results. Therefore, the ground state of CuAl2O4 is composed of a major (x=0.7) dynamic phase with the sporadic spin fluctuations and a minor (1−x=0.3) phase, rendering its glassy nature. Interestingly, x at the base temperature and under zero-field corresponds to the ratio of Cu 2+ ions in the A-site (1−η=0.7). The coexistence of dynamic and glassy states also was observed in kagomé systems SCGO and BSZGCO [32,36]. As the temperature (or HLF) increases, x decreases (Fig. 5c), which indicates that the dynamic state transforms into the state, in which spins fluctuate following the classical Markovian process. To estimate the potential muon stopping sites in CuAl2O4, we employed DFT calculations to map out the electrostatic Coulomb potential within the unit cell. The maxima of such potential map correspond to likely locations for adding a positive charge, such as the muon [41,42]. This analysis leads to a single candidate muon site for 'ideal' CuAl2O4 (neglecting any site-mixing) with site symmetry 96g and fractional coordinates of around [0.04, 0.04, 0.91]. This site is approximately 1.4 Å away from an oxygen anion and about 1.9 Å away from two copper cations. We also repeated the calculations with configurations, which took into account site-mixing. And there was a clear energetic preference for the muon to choose a location close to a copper cation sitting on an octahedral B-site (occupied by aluminum in the ideal case). The site in this case (shown in Fig. 6) is closest to two oxygen anions but is only ~2 Å separated from each three copper cations (the B-site one that is due to site-mixing and two A-site ones). One might then expect that the muon-spin relaxation is dominated by correlated fluctuations on this triangle of copper spins. For our experiment, where the site-mixing is quite significant (η=0.3), we expect that muons will preferentially locate close to B-site copper anions, as in Fig. 6. Note that this site is only approximate, as the electrostatic potential approach does not consider local distortions caused by the muon itself. However, we can estimate the extent of the likely induced distortion based on experience with pyrochlore oxides [42], and this will not alter the conclusion that the muon will be dominated by fluctuations from a triangle of copper spins. IV. DISCUSSION Our NMR spectra, at first glance, show several features related to a normal spin-glass state: the absence of a long-ranged order, the onset of metastability separating the ZFC and FC spectra, and the unresolvable satellite peaks. These are most likely to originate from the distribution of crystal electric fields and the randomized exchange interactions coming from the finite amount of disorder in the system. The excellent matching of the width of NMR spectra and the bulk susceptibility (Fig. 2b) indicates that the bulk magnetic properties relating to the glassy nature mainly reveal an effect of the disorder. Meanwhile, our μSR measurements, as a faster local probe to observe spin dynamics, also verified notable dynamics below T, which is not seen in a conventional spin-glass: the initial Gaussian-like decay, the saturation of the relaxation rate, and the much weaker HLF dependence than a frozen state. We carefully examined the reports for other spinel analogs to understand further the effect of the site-disorder on the frustrated diamond lattice. For example, [Co1-ηAlη]A[Al2-ηCoη]BO4, having a classical spin-3/2 in the diamond lattice, has an antiferromagnetic collinear ordering [5] for the minimum η because the ratio J2/J1=0.11 [6] is below 1/8. When the disorder is introduced, the long-ranged order transforms into a short-ranged order to induce a spin-glass phase [7,43]. Notably, up to η=0.36, the critical temperature T, at which FC and ZFC magnetic susceptibility curves start to bifurcate, reduces as η increases [7,43]. However, in the region of 0.36<η<0.77, T* increases closely following the trend of η. This different disorder-dependency can also be interpreted as follows. The magnetic susceptibility data [43] and the η-T phase diagram [7] of the low-disorder system correspond to those of a diluted diamond lattice, such as Co1-xZnxAl2O4 [44], where the number of Co ions in A-site is variable but that in the B-site is controlled. It indicates that the low-disorder system can be approximated as the diluted diamond system [45]. In the low-disorder region, the reduction of T* occurs because the nonmagnetic ion (Zn 2+ ), introduced in the A-site, hinders the magnetic interactions between Co ions in the A-site. On the other hand, the interactions between the A-sites becomes negligible in the high-disorder region. And the dominant interactions exist between the Aand B-sites, and the B-sites. The increased number of magnetic ions (Co 2+ ) in the B-site, which reduces the average distance between the magnetic atoms in the B-sites, eventually leads to the rise of T* [43]. Following the above example, our system [Cu1-ηAlη]A[Al2-ηCuη]BO4, η=0.3 can be understood as a diluted diamond lattice-like [Cu1-ηAlη]A[Al2]BO4. The nature of the diluted diamond lattice with the quantum spin-1/2 was previously studied in Cu1-ηZnηRh2O4 [46,47]. Contrary to the collinear ordering of cobalt spinel such as CoAl2O4 and CoRh2O4, the ground state of CuRh2O4 is reported to have an incommensurate helical order [46]. Its average J2/J1=0.125 meets the condition [1] of being proposed to induce the spiral spin-liquid phase without the thermal phase transition into a magnetically ordered ground state. However, as CoRh2O4 has a distorted tetragonal lattice, the exchange interactions become separated into the in-and out-of-plane terms, which produces the ordered ground state [1,46]. Moreover, the ordering of Cu1-ηZnηRh2O4 becomes weaker [47], and fast spin fluctuations emerge as η increases above 0.44. It is because that the slight local distortion induced by the doping leads to modulations of the interactions, stabilizing adjacent other helical order, and eventually it induces a competition between the phases. From the similar Curie-Weiss temperature of CuRh2O4, θCW=−132 K [46], we anticipate CuAl2O4 to be close to the Lifshitz point J2/J1=1/8 [3]. Unlike tetragonal CuRh2O4, the cubic lattice of CuAl2O4 [48] can, moreover, host rather isotropic J1 and J2 interactions, which is a favorable condition to induce the spiral liquid state. We note that the fast spin fluctuations observed in CuAl2O4 cannot be explained by the two previous reports. One is the ground state of [Cu0.7Zn0.3]Rh2O4 with an order at 9 K [47]. Another is on [Co0.6Zn0.4]Al2O4, where the heat capacity shows a nearly linear temperature dependence of a canonical spin-glass state [49] below T~5 K [44]. The fluctuations are known to become more substantial when the 'stiffness' of the bulk spiral vanishes on approaching the Lifshitz point [3]. Therefore, we can ascribe the dynamic term of CuAl2O4 to the liquid state. The reduction of x depending on the external magnetic field in the inset of Fig. 5c is then naturally related to breaking the degeneracy of the superposed manifold of the liquid state by applying the field. The sitedisorder, moreover, can also lift the degeneracy of the state, inducing a finite region around it, in which the spins are deformed from an ideal spiral pattern [3]. It can then stabilize the frozen or ordered ground state. Furthermore, by comparing our result with a mostly disordered case [Cu1-ηGaη]A[Ga2- ηCuη]BO4, η=0.75 [31,50], we can comprehend how the magnetic ions in the B-site work. As magnetic ions present in the B-site outnumbers those in the A-site, the magnetic interactions between the Aand B-sites (J), and the B-sites (K) play a dominant role in determining the ground state. For instance, G. A. Petrakovskii et al. [31] assumed CuO6 octahedra in their sample as being tetragonally distorted in random directions due to Jahn-Teller distortion. They explained its glassy nature using a model with antiferromagnetic J=12 K (with modulation Δ(J)=1.2 K due to the distortion) and K=6 K. Moreover, below T=2.5 K, the local magnetism probed using μSR method captures electronic spins ( e~8 MHz) fluctuating at the rate =3.7 MHz. This feature is comparable to the term in CuAl2O4, depicting spins fluctuating in a relatively slower rate =7(1) μs -1 (Fig. A1). It implies that site-disorder is the reason why it can induce the observed glass-like properties in CuAl2O4. However, e =151(66) MHz is much larger than that of CuGa2O4 because it is due to the low concentration of the B-site in CuAl2O4, which leads to a broader distribution of local fields. Under the high longitudinal field of 3 kG, where the second term of Eq. (3) dominates, the partially decoupled shape of CuAl2O4 depolarization spectrum (solid line 6 in Fig. 5b) originates from the large e . Due to the large ν≈115 μs -1 , moreover, the depolarization curve under the low field of 30 G (solid line 5 in Fig. 5b), where the first term of Eq. (3) prevails, does not have an upturn found in CuGa2O4 (dotted lines 3). It shows instead that the dynamic term in CuAl2O4 behaves differently from the relaxation of the glass phase in CuGa2O4. All these considerations given above reinforce our view that the dynamic contribution of the ground state of CuAl2O4 arises from the possible liquid state of the frustrated diamond lattice, and the glass-like contribution emerges from the site-disorder. V. CONCLUSIONS We have studied CuAl2O4, a material which realizes the quantum limit of the frustrated A-site spinel system with the finite disorder (η=0.3). To observe the nature of the ground state, we used two local magnetic probes, nuclear magnetic resonance (NMR) and muon spin relaxation (μSR) methods. The absence of peak splitting in NMR spectra and the non-oscillating feature in μSR spectra verify a ground state with only short-ranged order below the correlation temperature T*. Instead of completely freezing, the ground state shows the dynamic spin fluctuations characterized by the Gaussian decay in the μSR spectra. The field dependence of λ2ʹ in Eq. (3) is given in Fig. A1. To estimate the distribution of local fields and fluctuation frequency, firstly, we used the relation 2 ′ = 2 e 2 /( 2 + 2 ( 0 ) 2 ) to fit the data. We can roughly fit the data (dashed line) with Δe=3.0(2), ν=14(2) μs -1 , but this fails for fields above 0.5 kG. This relation is based on the simple exponential form for the spin dynamic autocorrelation function. Instead of the simple exponential form, we can use a more general form of the autocorrelation function [51], and this leads to a relaxation rate with which we can fit all the data (solid line) with Δe=151(66) and ν=7(1) μs -1 . The smaller value of fluctuation frequency ν comparing with that of the sporadic fluctuation term suggests that the local field corresponding to the second term of Eq. (3) has slower dynamics, more comparable to the value ν=3.7 MHz found for CuGa2O4 [31]. FIG. 6. The local environment around the muon based on a DFT calculation of the electrostatic potential and including site mixing. The muon lies between two oxygen anions (its nearest neighbors) but will be sensitive to fluctuations on three copper cations (its next-nearest neighbors), two on the usual A-site, and one on the site-mixed B-site.	2020-07-10T01:01:01.320Z	2020-07-09T00:00:00.000	{ "year": 2020, "sha1": "b0ce01ad4e137f3755564cda44b7514cad7f0f1a", "oa_license": null, "oa_url": "https://ora.ox.ac.uk/objects/uuid:a2068db1-0472-490c-91d2-99b98a1ae344/download_file?safe_filename=Cho_et_al_2020_dynamic_spin_fluctuations.pdf&type_of_work=Journal+article", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "b0ce01ad4e137f3755564cda44b7514cad7f0f1a", "s2fieldsofstudy": [ "Materials Science" ], "extfieldsofstudy": [ "Physics" ] }
215764720	pes2o/s2orc	v3-fos-license	Current challenges in content based image retrieval by means of low-level feature combining – The aim of this paper is to discuss a fusion of the two most popular low-level image features - colour and shape - in the aspect of content-based image retrieval. By combining them we can achieve much higher accuracy in various areas, e.g. pattern recognition, object representation, image retrieval. To achieve such a goal two general strategies (sequential and parallel) for joining elementary queries were proposed. Usually they are employed to construct a processing structure, where each image is being decomposed into regions, based on shapes with some characteristic properties - colour and its distribution. In the paper we provide an analysis of this proposition as well as the exemplary results of application in the Content Based Image Retrieval problem. The original contribution of the presented work is related to diﬀerent fusions of several shape and colour descriptors (standard and non-standard ones) and joining them into parallel or sequential structures giving considerable improvements in content-based image retrieval. The novelty is based on the fact that many existing methods (even complex ones) work in single domain (shape or colour), while the proposed approach joins features from diﬀerent areas. Introduction Computer vision has been explored for many years in the scientific society. This specific domain of computer science is related mainly to pattern recognition, image analysis, and image processing. One of its most promising applications is contentbased image retrieval, which focuses on managing large sets of visual data, e.g. images and video streams. The problem of retrieving an image based on its content by a computer is not trivial since it requires to model the human visual system (HVS). The most important are the way the images are represented in the database and the way they are being compared. The automatic recognition of the objects, which are placed in the image plane, can utilize various low-level features. The most popular and widely used are: shape, texture, colour, luminance, context of the information (background, geographical, meteorological etc.) and behaviour (mostly movement). The comparison can utilize one of similarity assessment methods, ranging from classical to those incorporating artificial intelligence. Moreover, it is possible to use more than one feature and similarity metric at the same time, but such an approach is rather rare. Usually, each recognition method is limited to only one feature. On the other hand, the literature survey shows that combining images coming from different sources (instead of different features of the same image) is gaining the popularity among researchers [5]. The image fusion has been widely accepted in diverse fields like medical imaging, aircraft navigation guidance, robotic vision, agricultural and satellite imaging. For these applications, image fusion is a necessary stage in order to achieve better understanding of the observed phenomena as well as improving decision making. The approach is motivated by weaknesses of individual imageries, which can be eliminated by joining their unique features. However, there are many situations when different sensors are not available and only one type of image is a source of features. Hence, we should use as much object information as it is possible. It is obvious that every type of object representation has its advantages and drawbacks, and the choice is not always easy and evident. It depends on many conditions, e.g. application and user requirements, situation during the image acquisition process (especially the hardware parameters, weather or lighting). Fig. 1 shows a typical scheme of CBIR system, where a query image is compared to the images stored in the database. The comparison is performed in several feature spaces independently. In this case, an image database (Image DB) collects all the images, yet is not utilized directly in the comparison stage. It is processed in the offline manner in order to extract visual descriptors (based on low-level features). The same descriptors are extracted from a query image. Comparing the descriptors can be performed according to a strategy, which simulates the way humans compare images. In the paper we focus on visual descriptors related to shape and colour, since they are the most popular in the literature and guarantee good efficiency when it comes to a single-feature type of recognition [1,2,9,13]. To improve the CBIR efficiency we join them into parallel, sequential or mixed structures. Visual Features Amongst many types of visual features describing single objects two seem to be especially attractive and popular in use. Those are shape and color. Both are strongly influenced by HVS and the way it works. In image analysis or recognition the shape as a description is regarded especially applicable when a particular object (or objects) is identified. The areas are numerous but regarding the importance and number of applications special attention is paid to: medical problems, optical character recognition, machine vision, ecology, forensic, military and visual data retrieval. The importance of shape in the discussed problem comes from its easy localisation and segmentation from the background (when compared to other features). The shape used for recognition can be considered as a binary object, which is represented by a whole, including its interior or as a boundary (contour). It is crucial to uniquely characterize the shape and stay invariant to translation, scale and rotation [8]. Shape descriptors can be classified in various ways [4,8,10,12,13]. The most popular classification is based on the mentioned distinction. The second (as described e.g. in [13]) is based on whether the shape is represented as a whole (global) or by a set of local primitives (structural methods). The third one distinguishes between spatial and transform domains [10]. Every shape descriptor should be robust to as many shape distortions as possible. They are considered as differences between the object under recognition and the reference object belonging to the same class, but stored in the database. Firstly, spatial transformations have to be described. Those are the most obvious problems and therefore for years the most explored and solved ones. One can take into consideration translation, rotation in the image plane, the Pobrane z czasopisma Annales AI-Informatica http://ai.annales.umcs.pl Data: 30/07/2023 17:02:12 U M C S change of its size and the influence of the projection of a three-dimensional object into a two-dimensional plane. On the other hand, the description algorithm has to take into account more challenging problems, e.g. varying amount of points, noise, discontinuity and occlusion, which is equivalent to a lack of some parts or added parts to a shape. If the contour representation is used, one has to solve also the problems of selecting the starting point and the direction of tracing the outline. Colour is the second mentioned feature. In good lighting conditions human-being pays attention: firstly to intensity and colour of objects, secondly to shape and movement, then to texture and other properties. Therefore, the colour seems to be the second very popular and promising feature and it is easy to point out many colour descriptors in the literature. Usually they are based on colour-subspace histograms and dominant values. Nowadays, when the MPEG-7 standard [1] is being introduced, the most promising are compact descriptors, which join colour information and its distribution: Scalable Color (SCD), Dominant Color (DCD), Color Layout (CLD). In our recent works we have been using also specific, simplified representations, like RGB colour histogram, 8x8 pixels intensity thumbnail of an image, three (R,G,B) thumbnails, mean RGB value together with mean intensity, dominant values H and V in the HSV colour model. The traditional scheme for a system applied in querying and indexing images is composed of four main elements. The feature extractor is the first one. The second is the block devoted to comparison and classification. Separately, the storage element has to be considered. Finally, the front-end has to be provided. The results achieved by the above scheme strongly depend on the two parameters: the efficiency of comparisons and accuracy of description. In order to improve the retrieval results we propose to combine queries using descriptors from the shape and colour domains, used jointly. That gives the possibility of increasing the influence of advantages of particular methods and reducing the influence of the drawbacks. Hence, in our work we concentrate on the combination of the two features briefly described in this section. It comes from the obvious observation that in the case of large visual databases the usage of single descriptors can not be enough. The idea is promising but not new. One can find examples of its successful implementation [5][6][7]. However, the existing approaches are usually based on joint descriptors from the same domain (e.g. colour or texture). For many real image data sets this is not suitable, because they contain significantly richer and heterogeneous information. Therefore our approach emphasizes the possibility of joining queries in different domains. As it was mentioned, the idea of combining a few methods in the field of pattern recognition is definitely not new. Fusion at the decision level is employed to increase classification accuracy of an image beyond the level accomplished by individual classifiers. Rank-based decisions provide more opportunities compared to other numerical score measurements. Fusion on the level of features, on the other hand, is much simpler to implement, but suffers from the incompatibility of individual scales and requires applying universal classifiers (instead of feature-specific, which is Pobrane z czasopisma Annales AI-Informatica http://ai.annales.umcs.pl Data: 30/07/2023 17:02:12 U M C S undoubtedly better). The elementary query processes can be joint in a sequential or parallel way. However, mixed strategy is also possible. The first approach consists of n iterative queries. Each single query limits the initial dataset and creates an image subset by means of seeking and sorting images according to a similarity measure (e.g. distance metrics or correlation). Next, the obtained subset is used as an initial dataset for the next query. The whole process is repeated n times giving the resulting images. In this approach it is important to create the sequence of descriptors in a way that each consecutive query produces successive approximation of the required result. In the second approach we assume parallel use of descriptors to get n independent results. After that we apply certain voting rules (i.e. two-out-of-three, three-out-of-five and similar) and select the resulting images. It should be noticed that in both approaches we can use different classifiers adequately to different features, which gives distinctly better and more trustworthy results. Each strategy has its own advantages and drawbacks. The first one (sequential) utilizes an intuitive flow, which is similar to the iterative way of seeking images by humans, while the second one (parallel) makes it possible to avoid a situation, when images correctly found in the first stage are eliminated during the process of reducing the dataset in further stages. In practice, each strategy has its own specific application. According to several experiments we have performed, the sequential order is better for the image retrieval based on examples, while the parallel one tends to be more appropriate, when only one resulting image is needed. However, in the experiments presented in this paper, some other properties are also explored (see section 3. for examples). Joining queries may significantly improve the retrieval accuracy. It can be observed using a simplified example of the CBIR system which consists of three descriptors and three comparators, respectively. We denote the mean retrieval rate of the pairs descriptor comparator as: P1, P2 and P3. In this case we take each single retrieval as an independent event and assume that each one works under its optimal conditions. It is a very basic approach, yet a more sophisticated decision taking solutions can be found in [6,7]. The total rate P of such a combined system can be calculated (on the interval <0,1>) according to the following formula: where: P i = 1 −P i ∀i ∈ {1, 2, 3}. For example, if the mean retrieval accuracy of a single descriptor-comparator pair is equal to 0.8 (80%), which is a typical rate for the state-of-the-art methods, then the combined accuracy will increase to 0.9. Experimental flags retrieval In order to explore the properties and possible applications of algorithms based on fusion of low-level image features an experiment with flags retrieval was performed. The idea here was to verify the influence of our strategies on retrieval results. The images used in experiments were collected from the "Flags of the World" database, which consists of over 74,000 of pictures (flag images of various kindsnational, regional, provincial, state, municipal, historical, civil, war, international, organisation, naval, political, sport, personal, positional, fictional, etc.). The experimental database was limited to flags only (24,000 images). Other symbols, coats of arms, etc. were eliminated. Every single experiment was conducted based on the same idea. The query image (taken from the database) was presented to the retrieval system that used combination of descriptors and a few closest base elements were presented as an output. The stress was put here on the results of joining methods of different kinds, mainly colour and shape descriptors. It was interesting for us what properties will be emphasized in that case. It is obvious that if only the colour descriptors are used, the result from the above 20 thousand flags will be rather homogeneous. For example providing the Polish flag to the system will result in several dozen of exactly the same white and red flags, because one can find many of them across the world (Austrian states: Tirol and Upper Austria, Czech cities: Bubanec, Holesovice-Bubny, Karlin, German city Bad Durrenberg, etc.). Therefore the results of that kind are not even presented here. The special emphasis is put on co-operation of completely different features (descriptors). Two exemplary results from many performed ones are described in this section. In our opinion, they are the most interesting and confirm the relevance of combination of various descriptors'. The first case (see Fig. 2) was based on fusion of Colour Layout, Scalable Colour and Edge Histogram descriptors. As it can be seen in the figure, thanks to this, various characteristics were emphasized by the system. On one hand, the colours presented in the query image were important. On the other hand, the star was also influencing the results. This is an obvious result of the parallel strategy, where every element of the scheme has its contribution to the result (in this case equal for every component). The second presented test (see Fig. 3) used the United States flag, a very characteristic one, as a query. This time combination of RGB Histogram and Moments Invariants was applied. The appearance of flags other than the mentioned one is very interesting because, as it turned out, in the tested fusion scheme the most important property of the query image was the domination of red-white stripes in the image. Probably it was the result of the sequential strategy, where firstly the histogram is used and secondly, in the selected subset, the shape descriptor becomes more important. Conclusions In the article we showed some aspects of multi-tier content-based image retrieval, employing visual description provided by more or less standard low-level features. The Pobrane z czasopisma Annales AI-Informatica http://ai.annales.umcs.pl Data: 30/07/2023 17:02:12 U M C S ideas presented here can be applied in many different fields of digital image processing and pattern recognition. This approach is universal and, as it was proved, can be successfully implemented. Moreover, its main advantage over the existing methods is the possibility of joining descriptors from various domains and compare them using specific metrics to get better efficiency. In the article we showed some aspects of multi-tier content-based image retrieval, employing visual description provided by more or less standard low-level features. The ideas presented here can be applied in many different fields of digital image processing and pattern recognition. This approach is universal and, as it was proved, can be successfully implemented. Moreover, its main advantage over the existing methods is the possibility of joining descriptors from various domains and compare them using specific metrics to get better efficiency.	2016-09-01T08:36:37.373Z	2010-01-01T00:00:00.000	{ "year": 2010, "sha1": "efc50b97dcc587b0f3b407e2c89235140b314092", "oa_license": "CCBY", "oa_url": "https://journals.umcs.pl/ai/article/download/3266/2460", "oa_status": "GREEN", "pdf_src": "ScienceParsePlus", "pdf_hash": "bd304b1e64a88c251df37ee4ab56fff24c06754f", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [ "Computer Science" ] }
212415846	pes2o/s2orc	v3-fos-license	Alpha-lipoic acid attenuates cyclophosphamide-doxorubicin-induced hepatic perturbation in rats Background and Objectives: The clinical use of cyclophosphamide-doxorubicin (CP-DOX) in breast cancer treatment may cause hepatotoxicity. This study assessed the protective effect of alpha-lipoic acid (ALA) against hepatotoxicity induced by CP-DOX in albino rats. Materials and Methods: Thirty-six adult male albino rats were randomized into six groups (A-F) of n = 6. Group A (control) was treated intraperitoneally (ip) with 0.3 mL of normal saline 8 hourly for 48h. Group B was treated with 10 mg/kg of ALA 8 hourly ip for 48 h. Group C was treated with a dose of CP-DOX (150/20 mg/kg) for 24 h. Group D was pre-treated with ALA 8 hourly for 48 h before treatment with a dose of CP-DOX ip for 24h. Group E was co-treated with a dose of CP-DOX and ALA ip 8 hourly for 48 h. Group F was treated with a dose of CP-DOX for 24 h before treatment with ALA ip 8 hourly for 48 h. After treatment, rats were euthanized; blood samples were collected and evaluated for serum liver function markers. Liver samples were evaluated for biochemical markers and histology. Results: Liver catalase, superoxide dismutase, glutathione (GSH), and GSH peroxidase levels were significantly (P < 0.001) decreased in CP-DOX-treated rats. Aminotransferases, alkaline phosphatase, gamma glutamyl transferase, lactate dehydrogenase, total bilirubin, conjugated bilirubin, and malondialdehyde levels were significantly (P < 0.001) increased in CP-DOX-treated rats. The liver of CP-DOX-treated rats showed hepatocyte necrosis. However, CP-DOX-induced hepatotoxicity was significantly reversed in rats pre-treated (P < 0.001), co-treated (P < 0.01), and post-treated (P < 0.05) with ALA when compared to CP-DOX-treated rats. Conclusion: Pre-treatment with ALA produced the best protective effect against CP-DOX-induced hepatotoxicity. antioxidant nucleophiles such as glutathione (GSH) causing hepatic depletion of antioxidant defence thereby increasing vulnerability to free radical attack culminating in oxidative stress (OS). [10] Furthermore, the hapatotoxic effect of DOX is speculated to be associated with free radicals generated during its hepatic biotransformation [11] leading to hepatic mitochondria damage, OS and hepatocyte damage. [12] Alpha-lipoic acid (ALA) is an antioxidant that can decrease OS response by scavenging reactive oxygen species. [13] It is reduced to dihydrolipoic acid, which is generally regarded as the most bioactive form of ALA and the form responsible for most of the antioxidant effect. [14] Unlike other antioxidants, ALA is both fat and water-soluble; therefore, it can cross biological membranes easily and produce antioxidant action both in the cytosol and in the plasma membranes. [15] In addition to its antioxidant activity, it has been reported to act as a down-modulator of the activities of mediators of inflammation. Also, it has stimulatory effect on some endogenous antioxidants thereby facilitating their activities. [16] It can protect biomolecules such as DNA, lipids and proteins from assaults by free radicals and can inhibit cell apoptosis [17] This study evaluated the protective effect of ALA against hepatotoxicity induced by CP-DOX in albino rats. Animals Thirty-six adult male albino rats were purchased from the animal breeding unit of the Department of Pharmacology and Toxicology, Faculty of Pharmacy, Niger Delta University, Nigeria. The rats were kept in cages in a well-ventilated room under natural condition with free access to food and water. The rats were allowed to acclimatize for 1 week before the commencement of the experiment. Rats were handled according to the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Science. This research was approved by the Research Ethics Committee of the Department of Pharmacology and Toxicology, Faculty of Pharmacy, Niger Delta University, Nigeria. Animal treatment • Group A (control) was treated intraperitoneally (ip) with 0. Animal sacrifice After treatment, rats were fasted overnight and euthanized (ether anaesthesia). Blood samples were collected from the heart in plain sample containers. Blood samples were allowed to clot, centrifuged and serum samples were separated and analysed for liver function markers. Liver samples were excised and homogenized in ice-cold 0.1 M Tris-HCl buffer (pH 7.4). The resultant homogenates were centrifuged at 1200 g, at 20 min and the supernatants were obtained for biochemical analyses. Oxidative stress marker analyses Liver protein was determined according to Lowry et al. 1951. [21] GSH was measured as reported by Sedlak and Lindsay, 1968. [22] Superoxide dismutase (SOD) was assayed according to Sun and Zigma, 1978. [23] Catalase (CAT) was measured as described by Aebi, 1984. [24] Glutathione peroxidase (GPx) was determined using the method of Rotruck et al. 1973. [25] Malondialdehyde (MDA) was assayed as reported by Buege and Aust, 1978. [26] Histological study At the end of treatment, liver samples were excised and sections were fixed in 10% formal saline. Liver tissues were dehydrated in an ascending series of ethanol (70, 80, 96 and 100%). Liver tissues were processed and embedded in paraffin block, transverse sections (5 μm) were cut and stained with H and E, and examined for histological changes using a light microscope. Statistical analysis Data are expressed as mean ± standard error of mean. Values were analyzed using one-way analysis of variance followed by Tukey's test for post hoc comparison using GraphPad Prism 5.0 software (GraphPad Software Inc, La Jolla, CA, USA). P < 0.05; < 0.01 and < 0.001 were selected as the criteria for significance. Effect on biochemical parameters in liver tissues The liver levels of ALT, AST, ALP, LDH and GGT were normal (P > 0.05) in rats treated with ALA when compared to control [ Table 1]. On the other hand, liver ALT, AST, ALP, LDH and GGT levels were significantly (P < 0.001) increased in rats treated with CP-DOX when compared to control. The increases in liver ALT, AST, ALP, LDH and GGT levels were calculated to be 310.8%, 365.8%, 345.1%, 433.9% and 290.0% respectively [ Table 1]. However, liver ALT, AST, ALP, LDH and GGT levels were significantly decreased in rats pre-treated (P < 0.001), co-treated (P < 0.01) and posttreated (P < 0.05) with ALA when compared to rats treated with CP-DOX [ Table 1]. Effects on liver oxidative stress markers and histology Normal (P > 0.05) liver levels of SOD, CAT, GSH, GPx and MDA were obtained in rats treated with ALA when compared to control [ Table 2]. However, significant (P < 0.001) decreases in liver SOD, CAT, GSH, and GPx levels with increases in MDA levels were obtained in rats treated with CP-DOX when compared to control [ Table 2]. On the other hand, liver SOD, CAT, GSH, and GPx levels were increased whereas MDA levels were decreased significantly in rats pre-treated (P < 0.001), co-treated (P < 0.01) and posttreated (P < 0.05) with ALA when compared to rats treated with CP-DOX [ Table 2]. Furthermore, normal histology was observed in the liver of control rat [ Figure 8a] whereas hepatocyte necrosis was observed in the liver of rat treated with CP-DOX [ Figure 8b]. Also, hepatocyte necroses were dIscussIon Hepatotoxicity is one of the primary reasons for the withdrawal of drugs from the market. Studies have shown that 5% of all hospital admissions are associated with hepatotoxicity caused by drugs. [27] Experimental studies suggest that OS could be an essential pathologic factor in drug-induced hepatotoxicity. [28] ALA is an antioxidant that has gained considerable attention due to its free radical scavenging activity and the propensity to inhibit OS. [29] This study assessed the protective effect of pre-treatment, co-treatment and post-treatment with ALA against hepatotoxicity induced by CP-DOX in rats. In this study, CP-DOX induced hepatotoxicity was determined by microscopic and biochemical evaluations. Drugs stimulate the production of a variety of serum biochemical and histopathologic indicators of hepatotoxicity. Biochemical markers which include AST, ALP, CB, TB, LDH and GGT have been used to assess the functionally of the liver as a measure of its wellbeing. Also, the aforementioned parameters are usually elevated as a consequence of hepatotoxicity caused by chemical assault. [30] In this study, hepatic assault caused by CP-DOX was confirmed by remarkable increases in the serum levels of ALT, AST, ALP, CB, TB, LDH, and GGT. However, the hepatic assault induced by CP-DOX was reduced in rats pre-treated, co-treated, and post-treated with ALA with most reduction observed in rats pre-treated with ALA. This was evident by observed decreases in serum ALT, AST, ALP, CB, TB, LDH, and GGT levels. Also, the extent of hepatic damage caused by CP-DOX was assessed by measuring ALT, AST, ALP, LDH, and GGT contents of liver tissues. Experimental studies have shown that the hepatic activities of the aforementioned parameters can be up-regulated as a consequence of uncontrollable or untreated hepatic insults by drugs. [31] In this study, ALT, AST, ALP, LDH, and GGT levels were remarkably elevated in the liver tissues of rats treated with CP-DOX which are signs of hepatocyte degeneration and functional incapacitation of the liver. [32] Interestingly, hepatic activities of ALT, AST, ALP, LDH, and GGT were decreased in the liver of rats pre-treated, co-treated, and post-treated with ALA with most decreases observed in rats pre-treated with ALA. The ability of ALA to restore hepatic function in CP-DOX-treated rat is a vivid attestation to its inherent potential to prevent or abrogate hepatotoxicity that can arise from the clinical use of CP-DOX. Studies suggested that disturbance in oxidant-antioxidant system caused by OS culminating in antioxidant depletion is involved in the pathogenesis of drug-induced hepatotoxicity. [33] In this study, treatment with CP-DOX produced low hepatic antioxidant (SOD, CAT, GSH and GPx) levels. This is an evidence of an over whelming OS which might have surmounted the activities of antioxidants leading to their Several studies have demonstrated that LPO marked by higher levels of MDA caused by free radicals is frequently associated with hepatotoxicity induced by drugs. [34] In this study, treatment with CP-DOX caused a remarkable increase in the hepatic activity of MDA. This is an evidence of the breakdown of hepatic poly unsaturated fatty acid via OS caused by reactive oxygen species. Interestingly, the hepatic peroxidative activity of CP-DOX was ameliorated as evidenced by reductions in MDA levels in rats pre-treated, co-treated and post-treated with ALA with most amelioration observed in rats pre-treated with ALA. In addition to the evaluation of serum biochemical markers, the microscopic assessment of liver histology is also used as a confirmatory investigation for drug-induced hepatotoxicity. [35] In this study, histological examination of the liver section of rat treated with CP-DOX showed hepatocyte necrosis which supports observed changes in evaluated biochemical parameters. This observation can be attributed to the ability of CP-DOX to produce excess free radicals, and to suppress free radical scavenging capacity of the liver via antioxidant depletion thereby increasing the vulnerability of the liver to more free radical assault leading to OS. This might have resulted to hepatic biomolecular damage creating an enabling environment for necrosis. [36] Interestingly, hepatocyte necrosis decreased in rats pre-treated, co-treated and post-treated with ALA. The hepatotoxic effect of CP has been primarily attributed to its toxic metabolite (acrolein) which has been associated with the production of free radicals causing OS and biomolecular damage. [37] Also, studies have associated the hepatotoxic effect of DOX to free radical generation leading to OS, inflammation and cell apoptosis. [38] The protective effect of ALA observed in this study might be due to its antioxidant effect. The hepatic oxidative activity of CP-DOX might have been down-regulated by the antioxidant action of ALA. Studies have shown that ALA is a unique antioxidant that scavenges free radicals in fat and water-soluble environments in its oxidized and reduced dihydrolipoic acid form. [39] It is effective in recharging enzymes in the mitochondria "the energy centers" of cells [40] and can prevent DNA, lipids and proteins from damage caused by OS. [41] It can increase antioxidant gene expression thereby facilitating antioxidant production and activity. [42] conclusIon This study discovered that pre-treatment with ALA produced the best protective effect against CP-induced hepatotoxicity than co-treatment and post-treatment. Pre-treatment with ALA may be clinically used to prevent hepatotoxicity that may arise with the use of CP-DOX.	2020-03-05T10:22:34.185Z	2020-01-01T00:00:00.000	{ "year": 2020, "sha1": "7ab8b2ba182b234fc2fc7042449bd3bc8bb845ea", "oa_license": "CCBYNCSA", "oa_url": "https://doi.org/10.4103/jmms.jmms_64_19", "oa_status": "GOLD", "pdf_src": "WoltersKluwer", "pdf_hash": "c1d65f22392e676c5ddcef68fb415ce5a961f3d7", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
252458700	pes2o/s2orc	v3-fos-license	Characterization of CRH-Binding Protein (CRHBP) in Chickens: Molecular Cloning, Tissue Distribution and Investigation of Its Role as a Negative Feedback Regulator within the Hypothalamus–Pituitary–Adrenal Axis Corticotropin (ACTH) is a pituitary hormone playing important roles in stress response within the hypothalamus–pituitary–adrenal (HPA) axis. The biosynthesis and secretion of ACTH are controlled by multiple factors, including corticotropin-releasing hormone (CRH). As a key hypothalamus-derived regulator, CRH binds to corticotropin-releasing hormone receptor 1 (CRHR1) in the anterior pituitary gland to regulate ACTH synthesis and release. Thus, CRH-binding protein (CRHBP), which binds CRH with high affinity to inhibit CRH-induced ACTH secretion from pituitary cells, draws wide attention. In contrast to the extensive investigation of CRHBP in mammals and other lower vertebrates, the gene structure, tissue expression and physiological functions of CRHBP in birds remain largely unknown. In the present study, using chicken (c-) as our animal model, we examined the gene structure, tissue expression and functionality of CRHBP. Our results showed that: (1) cCRHBP cDNA encodes a 345 amino acid precursor, which shares high sequence identity with that of mammals, reptiles, frogs and fish; (2) cCRHBP is abundantly expressed in the brain (cerebrum and hypothalamus), pituitary and ovary; (3) cCRHBP inhibits the signaling of cCRHRs induced by cCRH, thus reducing the cCRH-induced ACTH secretion from cultured chick pituitary cells; (4) stress mediators (e.g., glucocorticoids) and stress significantly upregulate CRHBP mRNA expression in chickens, supporting its role as a negative feedback regulator in the HPA axis. The present study enriches our understanding of the conserved roles of CRHBP across vertebrates. In addition, chicken is an important poultry animal with multiple economic traits which are tightly controlled by the HPA axis. The characterization of the chicken CRHBP gene helps to reveal the molecular basis of the chicken HPA axis and is thus beneficial to the poultry industry. Introduction In vertebrates, corticotropin (ACTH), which is released from the anterior pituitary gland, plays important roles in stress response and other physiological activities, including steroid hormone biosynthesis and adrenal development [1][2][3]. As the critical part of the hypothalamus-pituitary-adrenal (HPA) axis, the biosynthesis and secretion of ACTH are controlled by both hypothalamic stimulatory factors and inhibitory factors (e.g., CRIF) [4][5][6]. For example, neurons in the paraventricular nucleus (PVN) of the hypothalamus synthesize and secrete corticotropin-releasing hormone (CRH), which binds corticotropin-releasing hormone receptor 1 (CRHR1), expressed in the anterior pituitary corticotroph to regulate the ACTH synthesis and release [7][8][9]. The neuropeptide W (NPW), recently identified in chickens, which binds to the neuropeptide B/W receptor 2 (NPBWR2), inhibits ACTH synthesis and release [10]. CRHBP, a 37 kDa secreted glycoprotein, is reported to be actively involved in ACTH release regulation [11,12]. Studies have shown that CRHBP can bind CRH with nanomolar (IC 50 = 0.54 nM) affinity [13,14]. Using luciferase assays, CRHBP inhibits CRH-mediated CRHR1 activation in HEK293T cells [15]. Moreover, CRHBP also attenuates CRH-induced ACTH release from cultured rat anterior pituitary cells [15][16][17] and mouse pituitary AtT-20 cell line [18]. Compared to the wildtype mouse, the mouse with CRHBP overexpression in the anterior pituitary shows the increase in hypothalamic CRH using in situ hybridization [19,20]. In the mouse with CRHBP deficiency, a significant loss in body weight and an increase in anxiety-like behavior have been detected, thus supporting that CRHBP is a negative regulator in CRH signaling [21]. The full-length cDNAs encoding CRHBP are originally cloned from the human liver and rat brain [16]. They all encode a 322 amino acid precursor, including an N-terminal signal peptide of 23 amino acids, an N-linked glycosylation site and ten conserved cysteines, which form five consecutive disulfide bonds [16]. Subsequently, the CRHBP gene is further identified in other vertebrate species, including mice [18], sheep [22] and Xenopus laevis [23]. Using in situ hybridization, CRHBP is found to be expressed in the central nervous system (CNS), including the prefrontal cortex [24], central and basolateral amygdala [25], the bed nucleus of the stria terminalis [26], ventral tegmental area [27] and various hypothalamic regions [28]. In addition to the CNS, CRHBP is also highly expressed in the pituitaries of rats [29] and mice [30]. In contrast to the detailed and extensive studies in mammals and lower vertebrates, the information of CRHBP gene remains unknown in birds. In chickens, the cDNA sequence and gene structure of CRHBP have been predicted in the Ensembl database; however, its sequence feature and tissue expression need further confirmation. In addition, as a negative regulator in the HPA axis reported in mammals, whether CRHBP plays similar role in chickens needs to be investigated. Therefore, using chicken as the animal model, our present study aims to: (1) clone and analyze the gene structure and sequence characteristics of chicken (c-) CRHBP; (2) investigate the tissue expression of cCRHBP; (3) examine the effect of cCRHBP on the signaling of cCRHRs induced by cCRH; (4) examine the effect of cCRHBP on ACTH secretion induced by cCRH in anterior pituitary cells; (5) examine the effect of glucocorticoids and stress on the cCRHBP mRNA expression in chickens. The present study, for the first time, characterizes the chicken CRHBP gene, thus enriching our understanding of its conserved roles across vertebrates. Ethics Statement All animal experiments were conducted in accordance with the Guidelines for experimental animals issued by the Ministry of Science and Technology of the People's Republic of China. The experimental protocol performed in this study was approved by the Animal Ethics Committee of the College of Life Sciences, Sichuan University, China, and the assurance number is 20210308008 (8 March 2021). Chemicals, Primers, Peptides, Antibodies and Animals All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), and restriction enzymes were obtained from TaKaRa (Dalian, China). Dexamethasone (A2324) was purchased from APExBIO (Houston, TX, USA). Dexamethasone 21-phosphate disodium salt (D807084) was purchased from Macklin (Shanghai, China). All primers used in this study were synthesized by Youkang Biotechnology (Chengdu, China) and are listed in Table 1. Chicken (c-) CRH peptide (41 amino acids, SEEPPISLDLTFHLLREVLEMARAE-QLAQQAHSNRKLMEII) with the amidated C-terminus was synthesized using solid-phase Fmoc chemistry (GL Biochem, Shanghai, China), dissolved to 100 µM in Dulbecco's Modified Eagle's Medium (DMEM, Hyclone, Logan, UT, USA) and stored at −80 • C. The purity of the synthesized peptide was greater than 95% (analyzed by HPLC) and its structure was verified by mass spectrometry. Rabbit anti-ACTH polyclonal antibody (ab74976) was purchased from Abcam (Cambridge, UK). Mouse anti-His monoclonal antibody (HT501) was purchased from TransGen Biotech (Beijing, China). Rabbit anti-β-actin monoclonal antibody (#4970) was purchased from Cell Signaling Technology (Danvers, MA, USA). All chickens (Lohmann layer) employed in the present study, including adult chickens (1 year old) and chicks (3 weeks old), were purchased from a local commercial company in Chengdu. Total RNA Extraction, Reverse Transcription and Quantitative Real-Time PCR Assay Six adult chickens (3 males and 3 females) were sacrificed, and various tissues were collected, including the cerebrum, midbrain, cerebellum, hindbrain, hypothalamus, spinal cord, anterior pituitary, heart, liver, cecum, rectum, testis and ovary. All tissue samples were stored at −80 • C for RNA extraction. According to the manufacturer's instructions, total RNA was extracted from chicken tissues using RNAzol reagent (Molecular Research Center, Cincinnati, OH, USA) and dissolved in diethylpyrocarbonate (DEPC)-treated water. Total RNA was reverse transcribed into cDNA using Moloney murine leukemia virus (MMLV) reverse transcriptase (Takara, Dalian, China) according to our previous study [31]. Firstly, in a volume of 5 µL, 2 µg of total RNA and 0.5 µg of oligo-deoxythymidine were mixed, heated for 10 min at 70 • C and chilled for 2 min at 4 • C. Secondly, in a reaction mixture with a total volume of 10 µL, the first strand buffer, 0.5 mM of each deoxynucleotide triphosphate and 100 U of MMLV reverse transcriptase were added. Reverse transcription (RT) was performed at 42 • C for 90 min. The quantitative real-time PCR (qPCR) experiment was used to investigate the mRNA expression of cCRHBP according to our previously established method [32]. The qPCR primers of the chicken CRHBP were designed based on the amplified sequence of Gallus gallus (OP259501) using the Primer-BLAST tool at NCBI, as listed in Table 1. The primers (10 µM), dNTP (10 mM), Easy Taq Buffer, Easy Taq DNA polymerase (TransGen Biotech), Eva Green (Biotium), MilliQ-H 2 O and templates were mixed in a total volume of 20 µL. Then, the reaction mix was conducted on the CFX96 Real-time PCR Detection System (Bio-Rad). The amplification conditions included an initial denaturation for 10 min at 94 • C followed by 20 s denaturation at 94 • C, 15 s annealing at 60 • C and 30 s extension at 72 • C for 40 cycles. To assess the specificity of qPCR amplification, a conventional PCR reaction and gel electrophoresis were performed before the qPCR, followed by melting curve analysis after the qPCR. The relative mRNA levels of cCRHBP in the chicken tissues were calculated using the comparative CT quantification (2 −∆∆CT ) method of qPCR. Moreover, the mRNA levels of cCRHBP were normalized by that of β-actin and then expressed as the fold difference compared with that of the midbrain. Cloning, Sequence Alignment and Synteny Analysis of Chicken CRHBP Based on the predicted sequences of the chicken CRHBP deposited in Ensembl Database (ENSGALT00010027397.1), gene-specific primers were designed to amplify the cDNA containing a complete open reading frame (ORF) of the cCRHBP from the chicken brain using RT-PCR. The amplified PCR product of cCRHBP was cloned into the pTA2 vector (TOYOBO, Osaka, Japan) and sequenced (Youkang, Chengdu). To determine whether the cloned chicken CRHBP is orthologous to CRHBP identified in other vertebrate species, the neighboring genes of CRHBP in genomic regions of chicken and other vertebrate species were examined, which was formatted using a genome browser Genomicus, available online at https://www.genomicus.bio.ens.psl.eu/genomicus-93.01/ cgi-bin/search.pl (accessed on 16 January 2022) [33]. Tissue Expression Analysis of Chicken CRHBP Using RNA-seq Data In addition to the qPCR, RNA-seq datasets were also employed for the investigation of the CRHBP tissue expression in chickens. One RNA-seq dataset from red jungle fowl (SRP016501) was established by Burge et al. in an effort to reveal the evolutionary dynamics of gene regulation across vertebrates [34]. The other RNA-seq dataset, also from the red jungle fowl (E-MTAB-6769), was obtained from seven organs (cerebrum, cerebellum, heart, kidney, liver, ovary and testis) throughout developmental stages from early organogenesis to adulthood [35]. In the present study, based on the RNA-seq databases, we quantified the gene expression level using the transcript quantitative analysis tool Salmon v0.10.2 and default parameters. The relative abundance of chicken CRHBP transcripts was expressed as the transcripts per million (TPM). Generation of Chicken CRHBP Conditioned Medium In the present study, human embryonic kidney (HEK) 293 cells were used to generate the chicken CRHBP-conditioned medium (cCRHBP CM ). The full-length coding sequence of the chicken CRHBP (including a His-tag fused at the C-terminus) was first cloned into a pcDNA3.1(+) expression vector and sequenced by Youkang Biotechnology (Chengdu, China). Then, HEK293 cells were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum (HyClone, Chengdu), 100 U/mL penicillin G and 100 mg/mL streptomycin in a 90 mm culture dish (Nunc, Rochester, NY, USA) and incubated at 37 • C with 5% CO 2 . A day before transfection, cells were seeded into 6-well plates at a density of 3 × 10 5 cells per well. The cells were then transfected with a mixture containing 1000 ng cCRHBP-His expression plasmid (or an empty pcDNA3.1 vector as a negative control) and 2 µL of jetPRIME (Polyplus transfection, Illkirch, France) in 200 µL transfection buffer. After 24 h of transfection, serum-free medium was substituted for the original medium. The conditional medium (cCRHBP CM and pcDNA CM ) was harvested after 24 h and subjected for concentration through ultrafiltration spin. Chicken CRHBP protein in the cCRHBP CM was detected by western blot. Briefly, the concentrated conditional media were separated on 15% SDS-PAGE gels (Yamei, Shanghai) and transferred to a polyvinylidene difluoride (PVDF) membrane (Sigma-Aldrich, USA). The membrane was incubated in 5% nonfat dry milk (TBST solution) for two hours at room temperature to lessen nonspecific binding. Next, the membrane was washed three times with TBST, followed by incubation with the anti-His primary antibody (1:2000) for the duration of the night at 4 • C. After washing three times with TBST, the membrane was incubated with HRP-conjugated goat anti-mouse secondary antibody (1:5000, A9044, Sigma) for two hours at room temperature. Finally, blots were detected using an ECL chemiluminescence detection kit (Thermo Fisher Scientific, Waltham, MA, USA). Effect of Chicken CRHBP on the Signaling of CRHRs Induced by cCRH in Chinese Hamster Ovary (CHO) Cells In the present study, using the pGL3-CRE-luciferase receptor system, which was capable of the monitoring receptor-stimulated cAMP-PKA signaling pathway, we determined the effect of CRHBP on the signaling of cCRHRs activated by cCRH in CHO cells. In chickens, two types of CRH receptors, cCRHR1 and cCRHR2, have been cloned [36]. Based on the methods described in our previous studies [31,37], the signaling assays of chicken CRHRs (cCRHR1, cCRHR2) were first performed in CHO cells. Briefly, CHO cells were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin G and 100 g/mL streptomycin in a 90 mm culture dish and incubated at 37 • C with 5% CO 2 . The CHO cells were seeded into 6-well plates one day before transfection. The cells were then transiently cotransfected with a receptor expression plasmid (cCRHR1 or cCRHR2) and a pGL3-CRE-luciferase reporter construct. The transfection mixture contained 250 ng of receptor expression plasmid, 750 ng of luciferase reporter construct, 2 µL of jetPRIME and 200 µL transfection buffer. After 24 h, the cells were subcultured on 96-well plates for an additional 24 h prior to treatment with CRH and CRHBP. The following are the processing steps for CRH and CRHBP. For the CRH treatment, the cells were treated with 100 µL DMEM containing the desired dosages of cCRH (10 −12 to 10 −6 M) for 6 h. Finally, CHO cells were lysed using cell culture lysis buffer (Promega, Madison, WI, USA). According to the manufacturer's instructions, the luciferase activity of the cell lysate was measured using a multimode microplate reader (TriStar LB941, Berthold Technologies, Bad Wildbad, Germany). For the CRHBP treatment, the cells cotransfected with cCRHR1 (or cCRHR2) and pGL3-CRE-luciferase reporter constructs were exposed to a fixed dose of cCRH (1 nM) in combination with increasing amounts of cCRHBP CM (0-20 µL added, total volume 60 µL/well). The pcDNA CM (0-20 µL added, total volume 60 µL/well) was added to be employed as a negative control in the present study. After 6 h, the luciferase activity of CHO cell lysates was measured. The luciferase activity was represented as a percentage of the group treated with 1 nM CRH. Effect of Chicken CRHBP on the ACTH Secretion Induced by CRH in Cultured Chick Pituitary Cells According to our previously established method [38,39], anterior pituitaries were isolated and subjected for the primary culture from 3-week-old chicks. Pituitaries were digested by 0.25% trypsin at 37 • C for 30 min under sterile conditions. Dispersed pituitary cells were cultured at a density of 5 × 10 5 cells/well in the 48-well plates (Corning, Tewksbury, MA, USA) with medium 199 (M199, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing 15% fetal bovine serum and incubated at 37 • C with 5% CO 2 . After 18 h of culture, the cells were treated with cCRHBP CM (20 µL added, total volume 120 µL/well) in the absence or presence of cCRH (5 nM) for 4 h. The conditional medium was then harvested for ACTH measurement through Western blot. Meanwhile, pituitary cells were lysed in RIPA buffer (100 µL/well) to examine the expression level of β-actin as an internal control. Effect of Dexamethasone (DEX) on CRHBP mRNA Expression in Chickens In the present study, in vitro and in vivo studies were further designed to examine whether glucocorticoids regulate the expression of CRHBP in the hypothalamus and pituitary in an effort to determine its role in the HPA axis. Firstly, for the in vitro studies, pituitary cells were treated with various concentrations of DEX (0 nM, 1 nM, 10 nM and 100 nM) for 4 or 24 h. The total RNA was extracted from cultured pituitary cells and prepared for CRHBP mRNA expression analyses using qPCR. For the in vivo studies, according to our earlier study [10], DEX was administered subcutaneously, and the hypothalamus and pituitary were collected for RNA extraction to determine the CRHBP mRNA expression using qPCR. In the experiment, three-week-old male chicks were reared at 22 • C with a 16L:8D (16 h light, 8 h dark) photoperiod and were fed with commercial diet regularly and water freely. Similar-weight chicks (~300 g/chick) were divided into two groups (n = 6): the control group (saline) and the experimental group (DEX). For adaptive training, a drug-free syringe needle was plunged subcutaneously into every chick's abdomen at 9 a.m. every day for seven consecutive days. For the experimental group, the chicks were treated by a single subcutaneous injection of DEX (4 mg/kg body weight) in a 500 µL volume. For the control group, the chicks were subcutaneously injected with an equal volume of saline. Chicks were sacrificed three hours after injection, and hypothalamus and pituitary tissues were collected for RNA extraction. Simultaneously, all plasma samples were collected and subjected for ACTH (RXJ600533CH) and corticosterone (RXJ600484CH) concentration based on ELISA kits purchased from Ruixin Biotechnology company (Quanzhou, China). Effect of Stress on CRHBP mRNA Expression in Chickens In vertebrates, the HPA axis is actively employed for stress responses [5]. In the present study, in an effort to identify the CRHBP as a regulator in the HPA axis involved in stress response, its mRNA expression in the hypothalamus and pituitary were examined in chicks with stress treatments. The similar-weight male chicks (3-week-old) were first divided into two groups (n = 6): the experimental group and the control group. Three stress treatments were employed. For stress treatment 1: fasting for 24 h (the chicks were fed water freely but deprived of food for 24 h versus chicks which were fed food and water freely). For stress treatment 2: fasting for 48 h (the chicks were fed water freely but deprived of food for 48 h versus chicks which were fed food and water freely). For stress treatment 3: restraint for 1 h (the chicks were placed in a homemade cage (15 × 15 × 20 cm) with restricted mobility for 1 h versus chicks which were unrestrained). After the stress treatment, the chicks were sacrificed, and their hypothalamus and pituitary tissues were collected for RNA extraction. Following that, the CRHBP mRNA expression were examined using qPCR assay. In the present study, ACTH (RXJ600533CH) and corticosterone (RXJ600484CH) concentrations in all plasma samples were measured based on ELISA kits purchased from Ruixin Biotechnology company (Quanzhou, China). Data Analysis The protein bands of Western blotting were quantitated using densitometric analysis (ImageJ 1.52a, National Institutes of Health, Bethesda, MD, USA). The relative ACTH protein levels normalized by intracellular β-actin were represented as a percentage compared with the control group. The luciferase activities were represented as a percentage of the group treated with 1 nM CRH. The relative mRNA levels of cCRHBP in pituitary cells were first calculated as ratios to β-actin and then expressed as a percentage compared to their respective controls. The data were analyzed by one-way ANOVA followed by the Dunnett's test in GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). To validate our results, all experiments were repeated twice or thrice. Cloning, Sequence Alignment and Synteny Analysis of Chicken CRHBP According to the predicted cDNA sequence of the chicken CRHBP deposited in the Ensembl Database (ENSGALT00010027397.1), we cloned the cDNA containing the complete open reading frame (ORF) of cCRHBP from the chicken brain tissue by RT-PCR. The cloned cCRHBP cDNA (accession no. OP259501) is 1038 bp in length and consists of 7 coding exons, which encodes a precursor protein with 345 amino acids ( Figure 1). Sequence alignment revealed that the chicken CRHBP shares a high amino acid sequence identity with the CRHBP precursors from the Japanese quail (98.55%), zebra finch (85.80%), human (68.99%), mouse (68.12%), sheep (65.71%), Chinese soft-shelled turtle (79.71%), Xenopus tropicalis (67.63%) and spotted gar (60.87%). Furthermore, the ten cysteine residues, which form five disulfide bonds, and the N-linked glycosylation site were found to be conserved between chickens and other vertebrates ( Figure 2). In addition, in the N-terminus of the chicken CRHBP, a putative signal peptide consisting of 46 amino acids was detected, which was longer than the signal peptides detected in mammals [16,18,22], reptiles [23] and fish [40,41]. Tissue Expression of CRHBP in Adult Chickens Using qPCR, we examined the mRNA expression of CRHBP in adult chicken tissues. As shown in Figure 4A, the chicken CRHBP is abundantly expressed in the cerebrum, hypothalamus, pituitary and ovary, and weakly in the hindbrain, spinal cord and testis. In addition to qPCR, the two RNA-seq datasets deposited into the public database were also employed to analyze the expression pattern of CRHBP. According to the RNA-seq dataset from red jungle fowl (SRP016501), the expression of CRHBP mRNA was abundant in the cerebrum, hypothalamus and ovary ( Figure 4B), consistent with our qPCR results. In another RNA-seq dataset from red jungle fowl (E-MTAB-6769), the expression of CRHBP mRNA showed an abundance in the brain ( Figure 4C) and ovary ( Figure 4D) during fetal (E10, E12, E14 and E17) and postnatal development stages (P0, P7, P35, P70 and P155). pituitary cells were first calculated as ratios to β-actin and then expressed as a percentage compared to their respective controls. The data were analyzed by one-way ANOVA followed by the Dunnett's test in GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). To validate our results, all experiments were repeated twice or thrice. Cloning, Sequence Alignment and Synteny Analysis of Chicken CRHBP According to the predicted cDNA sequence of the chicken CRHBP deposited in the Ensembl Database (ENSGALT00010027397.1), we cloned the cDNA containing the complete open reading frame (ORF) of cCRHBP from the chicken brain tissue by RT-PCR. The cloned cCRHBP cDNA (accession no. OP259501) is 1038 bp in length and consists of 7 coding exons, which encodes a precursor protein with 345 amino acids ( Figure 1). Sequence alignment revealed that the chicken CRHBP shares a high amino acid sequence identity with the CRHBP precursors from the Japanese quail (98.55%), zebra finch (85.80%), human (68.99%), mouse (68.12%), sheep (65.71%), Chinese soft-shelled turtle (79.71%), Xenopus tropicalis (67.63%) and spotted gar (60.87%). Furthermore, the ten cysteine residues, which form five disulfide bonds, and the N-linked glycosylation site were found to be conserved between chickens and other vertebrates (Figure 2). In addition, in the N-terminus of the chicken CRHBP, a putative signal peptide consisting of 46 amino acids was detected, which was longer than the signal peptides detected in mammals [16,18,22], reptiles [23] and fish [40,41]. Using synteny analysis, we found that CRHBP exists in chickens and other examined species, including humans (Homo sapiens), mice (Mus musculus), turtles (Pelodiscus sinensis), frogs (Xenopus tropicalis), quails (Coturnix japonica), finches (Poephila guttata), sheep (Ovis aries) and gars (Lepisosteus oculatus) ( Figure 3). Moreover, some genes adjacent to CRHBP were consistent with those species examined, indicating that the chicken CRHBP was orthologous to the CRHBP genes of humans and other species. also employed to analyze the expression pattern of CRHBP. According to the RNA-seq dataset from red jungle fowl (SRP016501), the expression of CRHBP mRNA was abundant in the cerebrum, hypothalamus and ovary ( Figure 4B), consistent with our qPCR results. In another RNA-seq dataset from red jungle fowl (E-MTAB-6769), the expression of CRHBP mRNA showed an abundance in the brain ( Figure 4C) and ovary ( Figure 4D) during fetal (E10, E12, E14 and E17) and postnatal development stages (P0, P7, P35, P70 and P155). Functional Analyses of Chicken CRHBP Studies have shown that CRHBP binds CRH with nanomolar affinity, despite the structural difference between CRHBP and CRHR1 [14]. In addition, CRHBP inhibits CRHmediated CRHR1 activation in HEK293T cells [15]. In the present study, the effect of cCRHBP on the signaling of CRHRs was examined in cultured CHO cells. Using the pGL3-CRE-luciferase reporter system which was capable of monitoring receptor-stimulated cAMP-PKA signaling pathway, the effect of cCRH for the two CRH receptor subtypes (cCRHR1 and cCRHR2) transiently expressed in CHO cells were set in present study. As shown in Figure 5A, cCRH activates cCRHR1 (EC 50 Effects of CRHBP on ACTH Secretion in Cultured Chick Pituitary Cells Given that cCRHBP was highly expressed in the chicken pituitary, and cCRHB inhibited the signaling of cCRHR1 activated by cCRH in CHO cells, the effect of cC on the pituitary ACTH secretion induced by cCRH was examined in the present As shown in Figure 6, cCRHBPCM (20 μL added, total volume 120 μL/well) inhibit ACTH secretion induced by cCRH (5 nM). Under similar conditions, the 20 μL cCRH (total volume 120 μL/well) showed no effect on the baseline ACTH secretion. In the present study, the full length of cCRHBP cDNA was ligated into the expression plasmid, which was further transfected into HEK293 cells. The conditional medium from HEK293 cells with the cCRHBP-His expression plasmid was collected and concentrated. Western blot detected the band size of 38 kDa of chicken His-tagged CRHBP, which is in line with the report in humans [42], supporting that cCRHBP is a secreted protein ( Figure 5B). To determine the effect of the chicken CRHBP on the signaling of cCRHRs activated by cCRH, increasing amounts of cCRHBP CM were added to the CHO cells that were transiently transfected with cCRHRs. As shown in Figure 5C,E, cCRHBP CM (2-20 µL added, total volume 60 µL/well) was able to inhibit the signaling of cCRHR1 or cCRHR2 activated by cCRH (1 nM) in a dose-dependent manner. In contrast, the maximal volume of cCRHBP CM (20 µL added, total volume 60 µL/well) showed no influence on the basal luciferase activities of cCRHR1 or cCRHR2 in CHO cells. As a negative control, as shown in Figure 5D,F, any amount of pcDNA CM (2-20 µL added, total volume 60 µL/well) showed no effect on the signaling of cCRHR1 and cCRHR2. The present study supported that cCRHBP was able to efficiently inhibit the signaling of cCRHRs activated by CRH in CHO cells. Effects of CRHBP on ACTH Secretion in Cultured Chick Pituitary Cells Given that cCRHBP was highly expressed in the chicken pituitary, and cCRHBP also inhibited the signaling of cCRHR1 activated by cCRH in CHO cells, the effect of cCRHBP on the pituitary ACTH secretion induced by cCRH was examined in the present study. As shown in Figure 6, cCRHBP CM (20 µL added, total volume 120 µL/well) inhibited the ACTH secretion induced by cCRH (5 nM). Under similar conditions, the 20 µL cCRHBP CM (total volume 120 µL/well) showed no effect on the baseline ACTH secretion. The Expression Regulation of CRHBP mRNA by DEX and Stress in Chickens In vertebrates, the ACTH secreted by the pituitary will further target the adrenal to promote glucocorticoid (GC) secretion, principally the corticosteroid (CORT) in chickens. To maintain the homeostasis of the HPA axis, GC could provide feedback on the hypothalamus and pituitary to inhibit CRH and ACTH synthesis/secretion. Whether cCRHBP participates in the feedback regulation was examined in the present study. In the cultured chick pituitary cells, the effect of DEX on CRHBP mRNA expression The Expression Regulation of CRHBP mRNA by DEX and Stress in Chickens In vertebrates, the ACTH secreted by the pituitary will further target the adrenal to promote glucocorticoid (GC) secretion, principally the corticosteroid (CORT) in chickens. To maintain the homeostasis of the HPA axis, GC could provide feedback on the hypothalamus and pituitary to inhibit CRH and ACTH synthesis/secretion. Whether cCRHBP participates in the feedback regulation was examined in the present study. In the cultured chick pituitary cells, the effect of DEX on CRHBP mRNA expression was first examined. As shown in Figure 7A,B, using qPCR, various doses of DEX (1, 10, 100 nM) significantly stimulated CRHBP mRNA expression after 4 h and 24 h treatment. Furthermore, the upregulation level of CRHBP mRNA expression in the 24 h after DEX treatment was significantly higher than that in the 4 h after DEX treatment. Genes 2022, 13, 1680 13 of 20 significantly increased after 24 and 48 h of fasting, but its expression was steady after 1 h of restraint. Compared to the hypothalamus, the expression of CRHBP in the pituitary was considerably upregulated in response to all stress treatments ( Figure 8B). Additionally, all three stress treatments significantly elevated the ACTH and corticosterone concentrations in the plasma ( Figure 8C,D), supporting that the HPA axis was activated in response to the stress treatments. In the present study, through in vivo studies, DEX was further injected subcutaneously to determine its effect on CRHBP mRNA expression in the chickens. As shown in Figure 8A,B, DEX (4 mg/kg body weight) substantially enhanced the expression of CRHBP in the hypothalamus and pituitary. In the present study, the concentrations of ACTH and corticosterone in plasma before or after the DEX injection were also detected. As shown in Figure 8C,D, DEX significantly decreased ACTH and corticosterone concentrations, supporting the feedback effect of DEX on the HPA axis. Glucocorticoids, as the end products of the HPA axis to be stress-mediators, will be rapidly synthesized and secreted in response to the varied stressors [43]. Therefore, in the present study, the impact of stress on the CRHBP mRNA expression was further investigated. As shown in Figure 8A, the expression of CRHBP in the hypothalamus was significantly increased after 24 and 48 h of fasting, but its expression was steady after 1 h of restraint. Compared to the hypothalamus, the expression of CRHBP in the pituitary was considerably upregulated in response to all stress treatments ( Figure 8B). Additionally, all three stress treatments significantly elevated the ACTH and corticosterone concentrations in the plasma ( Figure 8C,D), supporting that the HPA axis was activated in response to the stress treatments. 24 h treatment (B). The mRNA levels were first calculated as the ration to that of β-actin and then expressed as a percentage of the control group. Each data point represents the mean ± SEM of 4 replicates (n = 4). , p < 0.01 vs. control; *, p < 0.001 vs. control. Discussion The present study, for the first time, characterizes the chicken CRHBP gene. Sequence analyses reveal that it shares a high sequence similarity with its counterparts in vertebrates, thus indicating its conserved role across species. Tissue expression analyses reveal that the CRHBP mRNA is abundantly expressed in the cerebrum, hypothalamus, pituitary and ovary. Functional assays demonstrate that CRHBP inhibits the signaling of cCRHRs activated by cCRH in CHO cells, thus reducing the cCRH-induced ACTH release in cultured chick pituitary cells. Moreover, stress-mediators (e.g., glucocorticoids) have been found to increase CRHBP mRNA expression in in vitro and in vivo studies. Together with the observation that stress stimulates CRHBP mRNA expression, our study supports the conserved role of cCRHBP as a negative feedback regulator in the chicken HPA axis. As shown in Figure 2, ten cysteine residues and one glycosylation site are conserved among the species examined, including chickens. The cysteine residues, which form five successive disulfide bonds, are essential for sustaining the biological activity of CRHBP [11]. Meanwhile, the asparagine N-linked-type oligosaccharides are reported to be essential for CRHBP binding [44]. In the present study, the amino acids (Arg68, Arg82, Arg79 and Asp85) have been detected to be conserved among the species (Figure 2). Using photoaffinity labeling experiments, the amino acid residues Arg46 and Arg59 of rat CRHBP are identified as the interaction sites with human/rat CRH 6-33 [45]. Moreover, Huising et al. report that Arg56 and Asp62 of hCRHBP are required for binding to r/hCRH [15]. The essential amino acid conservation of chicken CRHBP with mammalian species suggests their similar binding features to their targets. Additionally, synteny analysis supports that chicken CRHBP is orthologous to its counterparts in other vertebrates (Figure 3), indicating its conserved role across species. CRHBP Is Widely Expressed in Chicken Tissues In this study, qPCR and RNA-seq datasets were employed to detect the expression pattern of CRHBP in chicken tissues. Both the qPCR assay from chicken and the RNA-seq dataset from red jungle fowl demonstrate that cCRHBP mRNA is abundantly expressed in the central nervous system (CNS), pituitary and ovary. Within the CNS, cCRHBP shows a high expression level in the cerebrum and hypothalamus, which is accordant with the previous studies in rodents [26,28,46]. In the present study, in line with the reports in Xenopus laevis [23] and zebrafish [47], the CRHBP mRNA is also detected to be expressed in the brain. In addition, in the RNA-seq dataset from red jungle fowl (E-MTAB-6769), CRHBP has been found to be highly expressed at all developmental stages from fetal to postnatal ( Figure 4C), suggesting that CBP-BP may play important roles in the chicken brain. In rodents, the CRHBP expressed in the brain, including the amygdala, bed nucleus of the stria terminalis (BNST), ventral tegmental area (VTA), prefrontal cortex (PFC) and various hypothalamic regions, is suggested to play key roles in stress-related disorders such as depression [27], anxiety [24], addiction [48] and Alzheimer's disease [49]. In addition to the CNS, chicken CRHBP is also found to be abundantly expressed in the pituitary gland ( Figure 4A). Our result is consistent with early reports that CRHBP is expressed in the rat and mouse pituitary [26,29,30]. Especially, the mouse CRHBP is detected to be highly expressed in the pituitary gland with a sexually dimorphic pattern, with females exhibiting much higher expression levels [30,50]. Single-cell sequencing from our research group further reveals that chicken CRHBP is abundantly expressed in pituitary folliculostellate cells (FS cells) [51], being different from the detection in female mice that CRHBP is expressed in pituitary cells, including corticotropes, lactotropes and gonadotropes [30]. The FS cells in the chicken pituitary are hypothesized to function as a paracrine/autocrine signaling hub, influencing neighboring cells [51]. Therefore, chicken CRHBP may function as a paracrine factor to regulate the wide range of pituitary functions, such as CRH-induced ACTH synthesis and secretion. Interestingly, chicken CRHBP is also detected to be highly expressed in the ovary (Figure 4), suggesting its potential role in female chickens. The present study differs from a previous report in humans, in which CRHBP mRNA is not expressed in human ovarian follicles [52]. However, in rhesus monkeys, CRHBP mRNA is detected in theca cells of preovulatory follicles [53]. By binding plasma CRH, CRHBP may attenuate the hyperstimulation of the HPA axis caused by elevated placental CRH during pregnancy in humans [54,55]. In chickens, the CRHBP in the ovary may play a similar role in order to alleviate the stress response during egg production. CRHBP Affects the Signaling of cCRHRs in CHO Cells In the present study, using the pGL3-CRE-luciferase system, we demonstrated that cCRHBP CM inhibited the signaling of cCRHRs activated by cCRH in a dose-dependent manner in CHO cells ( Figure 5C). Our result is consistent with the finding in mammals, in which rCRHBP inhibits the activation of CRHR1 by 50 pM h/rCRH with an IC 50 of 2.65 nM, as measured by a cAMP-luciferase reporter assay [15]. Present study is also consistent with the finding in common carp that recombinant CRHBP inhibits the r/hCRH-mediated CRHR1 activation [56]. In present study, cCRHBP CM also inhibit the signaling of cCRHR2 activated by CRH (1 nM) in a dose-dependent manner ( Figure 5E). Our result is partially similar to the finding in common carp, in which CRHBP inhibits the r/hCRH-mediated CRHR2s activation by cAMP-driven luciferase activity [56]. In nonmammalian species, including birds, CRH is also a potent thyrotropin (TSH)-releasing factor involving TSH expression and secretion through CRHR2 [57,58]. The present study supports the potential role of CRHBP involving chicken TSH synthesis and release. Until the present, multiple functional mechanisms of CRHBP have been proposed, including the inhibition of CRHR1 [16][17][18], the promotion of CRHR2 [59,60], the independent of CRHRs [46,61] or as an escort protein of CRHR2α [62,63], which vary from tissues to species. The conserved amino acids (Arg68, Arg82, Arg79 and Asp85) detected in the chicken CRHBP revealed in the present study, together with the conserved motif (ARAE motif, Ala-Arg-Ala-Glu) detected in cCRH, with the report from the mammals that CRHBP binds CRH with nanomolar affinity [13][14][15]17], supports that chicken CRHBP may play an inhibitory function by decreasing the amount of 'free CRH', hence reducing the signaling of cCRHRs. CRHBP Is a Negative Feedback Regulator in HPA Axis Corticotropin (ACTH), as the critical part of the HPA axis, its biosynthesis and secretion from the pituitary gland, are controlled by the corticotropin-releasing hormone (CRH). In the present study, in chick pituitary cells, we demonstrated that cCRHBP CM can inhibit CRH-induced ACTH secretion ( Figure 6). Our result is consistent with the reports in mammals, in which CRHBP attenuates the CRH-induced ACTH secretion from primary anterior pituitary cells and AtT-20 cells [15,16,18]. Being the inhibitor of pituitary ACTH synthesis and release, the present study directly supports CRHBP as the negative feedback regulator in the HPA axis. In the HPA axis, corticotropin (ACTH) will further target the adrenal to promote GC release to induce the serial cellular reactions [1,64]. As an essential negative feedback regulator of the HPA axis, glucocorticoids inhibit the expression of the pro-opiomelanocortin (POMC) gene in the pituitary gland, thus reducing ACTH synthesis [65,66]. In the present study, we found that DEX may significantly increase the expression of CRHBP mRNA in cultured chick pituitary cells ( Figure 7A, B). In addition, glucocorticoids also positively upregulated the pituitary CRHBP mRNA expression with a single subcutaneous injection of DEX (4 mg/kg body weight) ( Figure 8B). Consequently, in chickens, both in vitro and in vivo findings support that the expression of CRHBP mRNA is upregulated in the pituitary gland under GC treatment. In addition to the pituitary, hypothalamic CRHBP mRNA expression can also be upregulated by a subcutaneous injection of DEX ( Figure 8A). As the inhibitor for ACTH synthesis and secretion, the elevated CRHBP expression in the hypothalamus-pituitary axis in response to the DEX treatment will further inhibit ACTH release, thus supporting its role as a negative feedback inhibitor in the HPA axis. The present study is in line with the detection in mammals [11,12]. In addition, our result is also similar with the finding in rats, in which adrenalectomy reduces pituitary CRHBP mRNA expression to 8% of sham adrenalectomy rats [67]. In vertebrates, as the most important neuroendocrine system in response to stress, the HPA axis will be activated in response with many kinds of stressors, including temperature and starvation [43,68,69]. In the present study, the effect of different stress treatments on the expression of CRHBP was further investigated. Both fasting and restraint treatments could significantly increase the expression of CRHBP in the pituitary ( Figure 8B), thus being in line with the detection in rats that restraint stress significantly increases the pituitary CRHBP mRNA levels from 30 min to 2 h [67]. The present study is also in line with the observance in mice that restraint stress elevates the pituitary CRHBP mRNA levels, particularly in the female pituitary [50]. In addition to the pituitary, fasting treatment also positively upregulates the expression of CRHBP in the chicken hypothalamus ( Figure 8A). The hypothalamus, as the convergence center from various nuclei and nerve fibers, plays vital physiological functions, including stress, reproduction, energy metabolism and thermoregulation [70]. The present study is in accordance with the report from the fish and Xenopus laevis that the hypothalamic CRHBP expression is regulated by stress [23,40,41]. In present study, the ACTH and corticosterone concentrations in the plasma were detected to be upregulated, revealing the activation of the HPA axis in response to various stress treatments. The upregulation of CRHBP mRNA expression is thus most likely regulated via enhanced stress mediators (e.g., glucocorticoid) release. The stress-induced upregulation of CRHBP mRNA may further augment the glucocorticoid-mediated feedback inhibition of the HPA axis, thus supporting its role as a negative feedback regulator in the HPA axis. In summary, the gene information and tissue expression of CRHBP have been investigated in chickens for the first time. Functional assays prove that chicken CRHBP inhibits the signaling of cCRHR1 (or cCRHR2) activated by cCRH in CHO cells, which further reduces the ACTH secretion in chick pituitary cells. In combination with the observance that glucocorticoids and stress significantly stimulate CRHBP mRNA expression in chickens, the present study supports that CRHBP may function as a negative feedback regulator along the HPA axis, possibly by modulating the CRH-CRHR1 signaling pathway. As an important poultry animal, the growth, reproduction, disease resistance, etc., of chickens are severely impacted by the HPA axis [71][72][73][74]. The characterization of the chicken CRHBP gene helps to reveal the molecular basis of the chicken HPA axis and is thus beneficial to the poultry industry.	2022-09-23T15:16:42.123Z	2022-09-20T00:00:00.000	{ "year": 2022, "sha1": "21eadbf22b8aec0f8718e6fea026db7e51760111", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2073-4425/13/10/1680/pdf?version=1663907301", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "c583dbb148a157fa891a987934c9c87478d24848", "s2fieldsofstudy": [ "Biology", "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
245013418	pes2o/s2orc	v3-fos-license	Comparative meta-omics for identifying pathogens associated with prosthetic joint infection Prosthetic joint infections (PJI) are economically and personally costly, and their incidence has been increasing in the United States. Herein, we compared 16S rRNA amplicon sequencing (16S), shotgun metagenomics (MG) and metatranscriptomics (MT) in identifying pathogens causing PJI. Samples were collected from 30 patients, including 10 patients undergoing revision arthroplasty for infection, 10 patients receiving revision for aseptic failure, and 10 patients undergoing primary total joint arthroplasty. Synovial fluid and peripheral blood samples from the patients were obtained at time of surgery. Analysis revealed distinct microbial communities between primary, aseptic, and infected samples using MG, MT, (PERMANOVA p = 0.001), and 16S sequencing (PERMANOVA p < 0.01). MG and MT had higher concordance with culture (83%) compared to 0% concordance of 16S results. Supervised learning methods revealed MT datasets most clearly differentiated infected, primary, and aseptic sample groups. MT data also revealed more antibiotic resistance genes, with improved concordance results compared to MG. These data suggest that a differential and underlying microbial ecology exists within uninfected and infected joints. This study represents the first application of RNA-based sequencing (MT). Further work on larger cohorts will provide opportunities to employ deep learning approaches to improve accuracy, predictive power, and clinical utility. www.nature.com/scientificreports/ resolution [15][16][17] . Shotgun sequencing circumvents culture and PCR-based limitations by enabling a comprehensive view of the identity and functional gene content of microbial consortia populating a clinical specimen 18 . Metagenomics analysis has previously shown high concordance with culture results for diagnosing PJIs 19 ; however, to the best of our knowledge, no work has yet been done comparing metatranscriptomics results to culture results in this context. Our goal was to compare 3 NGS techniques (16S, MG, and MT) in their ability to distinguish microbial profiles in synovial fluid and blood from patients undergoing primary, aseptic revision, and revision for PJI. Additionally, we assessed concordance of these NGS techniques to culture-based methods. We hypothesized that untargeted techniques, including MG and MT would outperform 16S amplicon sequencing taxonomically resolving and differentiating microbial profiles associated with PJIs. Additionally, we wanted to evaluate the utility of shotgun MG and MT methods for detecting antimicrobial resistance genes in clinical specimens. Methods Statement of ethical approval. This study was approved by the internal review board of Thomas Jefferson University (IRB protocol 17D.059). All patients in this study provided informed consent prior to surgical procedures either during pre-operative office visits or in the pre-operative area. Study methods were performed in accordance with approved guidelines. Study cohorts and patient classification. The study cohort consisted of a total of 30 patients, with a mean age of 68.1 years (range 53-85) undergoing knee or hip surgery in a single institution between September 2018 to January 2019. The study samples were subdivided into 3 groups, patients undergoing primary arthroplasty (native joints), patients with aseptic failure, and patients with PJI. Individuals were excluded from the study if they were under the age of 18, had a body mass index (BMI) greater than 40, or had a diagnosis of diabetes with a current HbA1c > 8. Patients with primary joints had a diagnosis of osteoarthritis in either the knee or hip and were receiving a TKA or THA. Patients were excluded from the primary arthroplasty group if they had any previous surgical procedure on the operative joint, had received a corticosteroid or viscosupplement injection within 9 months of the procedure, or showed any signs of infection or osteoarthritis in the contralateral joint. Patients with infected joints had undergone a TKA or THA and presented with signs of infection and met the ICM criteria for a PJI 20 . Patients were assigned aseptic revision group if they were undergoing revision arthroplasty but did not meet the ICM criteria for PJI. Patient demographics, cohorts, and procedure information are in Table 1. Applicable physical exam, clinical laboratory results, and ICM PJI classification of patients are provided in Supplementary Table S1. Sample collection and preservation. Sample collection for this study was performed after obtaining Institutional Review Board approval. All patients consented to participate in the study. Peripheral blood samples were collected from patients in the preoperative area in Vacutainer Collection tubes with Sodium Heparin (Becton, Dickinson and Company, Franklin Lakes, NJ), with collection volumes ranging from 2 to 4 mL. A volume of 10-20 mL synovial fluid was aspirated from the hip or knee joint and collected in sterile, nuclease-free 50 mL conical copolymer polypropylene screw-cap centrifuge tubes. Upon collection, an equal volume of DNA/RNA Shield (Zymo Research, Irvine, CA) was added to each volume of synovial fluid for sample preservation. Samples were deidentified, stored on ice, and then shipped overnight with ice packs to Contamination Source Identification laboratories (Huntingdon, PA) for sample preparation. Further details regarding synovial fluid, blood, skin swab, and negative control skin and air samples can be found in Supplementary Materials. Analytical validation, sample preparation, library preparation, and illumina sequencing. All methods describing analytical validation of metatranscriptome (MT) sequencing using ERCC RNA controls are described in Supplementary Materials. Details regarding sample collection, preservation, preparation for each NGS method, and Illumina sequencing can also be found in Supplementary Materials. For synovial fluid, blood, and negative control saline solution samples, DNA and RNA were extracted for downstream 16S, MG, and MT sequencing. For the negative control skin and air swabs, only DNA was extracted for 16S and MG analysis. For a list of all patient samples processed see Table 1 and for a list of all samples including controls see Supplementary Table S2. Bioinformatic analysis. A description of all bioinformatics and statistical analyses for 16S, MG, and MT datasets are described in the Supplementary Materials. Briefly, MT and MG sequence data were subject to quality filtering and adaptor trimming, human sequence removal, and annotation in Kraken2 21 . 16S sequences were merged, de-noised, quality filtered and grouped into ASVs within the DADA2 software package 22 . Normalization and statistical analyses on 16S data were performed within QIIME2 and R 23 . Microbial annotation count data underwent cumulative sum scaling (CSS) normalization prior to unsupervised and supervised clustering of 16S, MG, and MT data. Statistical differences in clustering between groups were evaluated using the PERMANOVA test. To account for contaminant sequences, CPM normalized ratios of sample:controls counts of matched taxa were calculated prior to random forest modeling. To screen for the presence of antibiotic resistance genes, filtered microbial reads were aligned against the Comprehensive Antibiotic Resistance (CARD) Database via BLAST (min e-value = 1e−10) to detect antibiotic resistance genes in MG and MT data for each sample 24 Beta diversity of synovial and blood microbiota. Unsupervised principal coordinates analysis (PCoA) and supervised partial least squares discriminant analysis (PLS-DA) modeling were conducted to assess differences in microbial communities among primary, aseptic, and infected samples within synovial fluid and matched blood specimens (Fig. 3a Distinction between primary and non-primary samples was observed from MT synovial fluid PLS-DA model (Fig. 3a), as no overlap is observed between 95% confidence ellipses plotted around primary and non-primary Table 1. Patient characteristics and cohorts. Summary of the patient characteristics including cohort, age, and procedure received. TKA total knee arthroplasty, THA total hip arthroplasty, TKR total knee replacement, THR total hip replacement, L left, R right, B/L bilateral, M male, F female. www.nature.com/scientificreports/ clusters (error rate: 11-14%). Increased overlap between primary and non-primary confidence ellipses were noted for MG synovial fluid, resulting in a poorer model performance (error rate: 20-25%) compared to the MT PLS-DA model (Fig. 3b). PLS-DA models built using MT and MG data from blood samples revealed overlap between groups, indicating reduced model performance in differentiating between cohorts compared to models generated using synovial fluid samples. Random forest modeling. MT analysis yielded the most accurate predictive random forest models considering both synovial fluid and blood (respectively 79.0% and 75.2%) compared to MG (74.3%, 68.6%) and 16S (38.6%, 51.0%). Rhizobiales and Archromobacter were among the top predictors for classifying MT blood samples, while Bacteroidales and M.intenstinales were most predictive for MT synovial fluid samples. A comprehensive list of top predictors of synovial fluid and blood sample groups is in Supplementary Table S3. Detection of PJI associated pathogens and synovial fluid culture concordance. Differential clustering of infected MT samples appears to be driven by increased E. coli expression (LDA = 4.04, p = 0.00094) in infected samples, compared to primary and aseptic samples (Fig. 4a, Supplementary Table S4). S. epidermidis (LDA = 3.55, p = 0.004) exhibited significantly higher abundance within non-primary MT samples compared to primary joints. Of the 7 bacterial taxa identified by culture, 6 were detected using MT sequencing (Fig. 4a, Supplementary Table S4). Sample SF-23 yielded partial concordance with culture, as this sample was culture-positive for K. pneumoniae, which was detected, as well as vancomycin resistant Enterococcus sp., which was not detected (Supplementary Table S5). However, sequences for the SF-23 MT sample yielded 95% identity with Enterococcus but could not be differentiated from alternative genera within the Lactobacillales order. While the MG dataset yielded the same concordance with culture as MT, MG samples clustered indistinctly by cohort. Interestingly, the conserved signature of high abundance of E. coli within the infected synovial fluid samples of the MT dataset was not observed within the MG SF data (Fig. 4b, Supplementary Fig. S4). Notably, this was likely due to true biological signal and not contamination, as 2000 of the 2032 contigs (assembled using only sequences initially identified as E. coli) were classified by BLAST (with NCBI's nucleotide database) once more as E. coli. Antibiotic resistance gene screening of synovial fluid. MT Assessment and removal of environmental contamination. Due to the high potential of environmental contamination within untargeted microbial genomic investigations, rigorous environmental controls were collected to assess the microbiome of the respective environments encountered during the sample collection process. Considering the MG dataset, consistent contaminating microorganisms of prominent (top 10) abundance were observed across sampled skin swabs, air swabs, and NaCl ( Supplementary Fig. S5). An unclassified taxon within the Clostridiales were identified as the most prominent contaminant within both the air swab and NaCl samples (average relative abundance of 10% and 14%, respectively), whereas the Cutibacterium acnes were the prominent contaminant within the skin swab MG samples (average relative abundance of 19%). The Cutibacterium acnes were also observed as a prominent contaminant when considering the MT dataset, and www.nature.com/scientificreports/ comprised 13%, 9%, and 14% community abundance within skin swab, NaCl, and air swab samples, respectively. The fungi Malassezia restrictica, which was not observed as a prominent contaminant within the MG dataset, was identified at 9%, 5%, and 10% abundance within skin swab, NaCl, and air swab samples, respectively within the MT data. Blank samples were included on each sequencing run to account for potential contamination biases and to control for environmental organisms. After CPM-r normalizing our data with run-specific blank sample annotation data, a consistent signature of Ralstonia picketti was discovered within the primary joints of the MT dataset (Fig. 6). A total of 6 out of 10 primary joints yielded a consistent profile of elevated Ralstonia picketti abundance in comparison to the remaining samples. The trend of increased Ralstonia abundance was not observed within the MG samples, nor identified as a prominent environmental contaminant within either the MG or MT datasets. Consequently, this taxon was identified as the most significantly enriched species within the primary joints when considering the MT dataset (LDA score = 3.90, p = 0.008). www.nature.com/scientificreports/ Discussion PJIs are a complication for patients undergoing total hip (THA) and knee (TKA) arthroplasty and continue to pose a public health concern since current methods lack accuracy to identify causative pathogens. Pitfalls associated with current assays mandate a robust clinical test system that rapidly and precisely detects pathogens associated with infection. Our study assessed: (1) utility of NGS technologies, including, 16S rRNA gene (16S), metagenomics (MG), and metatranscriptomics (MT) to characterize microbial communities of synovial fluid and blood samples from patients classified under primary, aseptic revision, and revision for PJI, and (2) concordance of NGS to culture-based assays in identifying pathogens and ARGs from clinical specimens. Herein, we described the potential clinical utility of an analytically-validated MT method for identification of active microbial consortia in synovial fluid and blood specimens. In this study, MG and MT techniques outperformed 16S in distinguishing between sample groups (Supplementary Fig. S2). Moreover, our results revealed MT as a superior technique, yielding the most accurate represent pathogens identified by library preparation and culture-based methods while an X indicates that the pathogen was identified by culture but not sequencing. Infected synovial fluid samples from the MT dataset yielded differential clustering from remaining samples, with only one infected sample (SF-32), plotting distinctly from other infected samples (a). Heatmaps were generated using pheatmap version 1.0.12 (https:// cran.r-proje ct. org/ web/ packa ges/ pheat map/ pheat map. pdf) and ggplot2 version 3.3.5 (https:// cran.r-proje ct. org/ web/ packa ges/ ggplo t2/ ggplo t2. pdf) within R 3.6.1 (https:// www.R-proje ct. org/). Parts of the heatmap marked with stars (*) and X were added using Adobe Photoshop version 21 (https:// www. adobe. com/ produ cts/ photo shop). www.nature.com/scientificreports/ random forest (RF) models and differentiation between primary and non-primary groups. Top predictive taxa for classifying MT samples using our RF model, revealed several taxa associated with surgical infections 27,28 . These findings highlight how MT-based classification models can simultaneously identify several active biomarker taxa associated with PJIs. Unlike MG and 16S, MT captures the active microbial profile by sequencing and annotating RNAs from a specimen, circumventing issues associated with DNA-based methodologies 29 . Our results suggest that identifying active organisms can be a promising discriminator and relevant clinical tool, particularly with PJI where treatment can be focused on highly active microorganisms. MT profiling of clinical samples revealed superior taxonomic resolution of microbial consortia compared to MG and 16S methods. Moreover, MT exhibited greater differential quantitative signals (i.e., relative abundance) Figure 5. Antibiotic resistance gene (ARG) heatmap. Heatmap displaying log transformed counts of antibiotic resistance genes identified to be actively expressed within each synovial fluid sample when considering the MT dataset. The y axis displays the observed ABX resistance gene, which are stratified by the respective antibiotic to which resistance is conferred. Samples are stratified along the X-axis by their respective "status" and are ordered from left to right as primary joints, aseptic revisions, and infected revisions, respectively. This heatmap was generated using pheatmap version 1.0.12 (https:// cran.r-proje ct. org/ web/ packa ges/ pheat map/ pheat map. pdf) and ggplot2 version 3.3.5 (https:// cran.r-proje ct. org/ web/ packa ges/ ggplo t2/ ggplo t2. pdf) within R 3.6.1 (https:// www.R-proje ct. org/). www.nature.com/scientificreports/ compared to the other methods. Since MG generates sequence data from all DNA present in a sample, this method is prone to false positives for pathogen detection 29,30 . These false positives occur because DNA degrades over time, and inactive microbial populations, or naked DNA, remain detectable using MG. The greater discrimination from MT is likely because it generates sequence data from active transcripts, thus avoiding potentially latent, dead, or inactive microbial populations 29 . For instance, infected joints subject to MG sequencing exhibited an increased relative abundance of several PJI-associated taxa [31][32][33] . While S. epidermidis and C. acnes are both skin commensals, they are also associated with PJIs due to the invasiveness of prosthetic joint procedures. Notably, these taxa can exhibit elevated ARGs from patients with PJIs compared to similar strains on the skin of healthy patients 34 . Although no infected samples returned culture positive for E. coli, biomarker enrichment analysis of MT data revealed E. coli to be significantly enriched in infected samples and least expressive in primary joints. Infected samples may not have been able to return culture positive for E. coli due to sensitivity limitations associated www.nature.com/scientificreports/ with culture-based methods 35 . The possibility of cross-contamination is unlikely since the signal of E. coli in negative controls were minimal. One case study isolated variants of E. coli from a PJI patient with history of recurring UTIs involving E. coli and K. pneumoniae, implying UTIs could be a contributing factor for increased expression of E. coli among infected joints 36 . Contrary to current literature, our results imply that other factors may be contributing to E. coli expression in infected joints from this study, since there were no reports of UTIs among our cohort. Apart from known risk factors like UTIs and surgical area proximity to gastrointestinal tract, MT methods can serve a crucial role in addressing uncertainties surrounding identification of risk factors and treatment of E. coli-associated cases of PJI. Additionally, our study revealed a potential underlying "native" joint microbial community. MT data showed highest expression of Ralstonia spp. in primary joint specimens. Although the genus Ralstonia has been identified as a contaminant in other studies 37,38 , Ralstonia picketti was not observed to be among the top 10 most abundant taxa in any of our controls ( Supplementary Fig. S5), in contrast to its relatively high CPM-r abundance in the primary joints subject to MT (Fig. 6). Additionally, S. aureus and K. pneumoniae were also more abundant in primary joints, relative to non-primary joints. Native joint space was historically thought to be a sterile environment, void of any microorganisms, but recent literature has unveiled the existence of an endemic microbiome 39,40 . A study of 40 patients undergoing primary TKA, 12 were found to have at least one organism identified using NGS 40 . Results of our study support these findings and suggests that the joint microbiome differs based on the presence of a prosthesis. Results from this study revealed that MT yielded a high culture-positive concordance of 83%. The single case of partial concordance in our study could be due to multiple reasons, including underdeveloped databases, quality of sample, and inconsistencies with culture-based assays 41 . Of particular note, the microbe not identified by MT, a vancomycin resistant Enterococcus sp., was isolated via culture from broth only. Previous reviews of test characteristics have demonstrated the poor clinical utility and reliability of broth-only isolates which draws into question the reliability of the identification of this non-concordant microorganism 42 . Additionally, MT was also superior in ARG detection and concordance with culture antibiotic susceptibility testing, as it detected ARGs in 3 cases concordantly with culture where MG failed to identify ARGs overall. In particular, Aminoglycoside resistance genes were expressed across all groups, whereas Phenicol resistance was largely observed in aseptic joints. Interestingly, bBoth drug classes contain wide-spectrum antibiotics that have been used in treatment of PJIs and infections relating to eyes and urinary tract [43][44][45] . Regarding ARG, a broad range of resistance mechanisms were noted throughout the study. In particular, Aminoglycoside resistance genes were expressed across all groups, whereas Phenicol resistance was largely observed in aseptic joints. Interestingly, both drug classes contain wide-spectrum antibiotics with Aminoglycosides being used in treatment of PJIs and Phenicols being utilized for infections relating to eyes and urinary tract [43][44][45] . This finding can possibly be explained due to the absence of selective pressures on the different cohort's microbiome. Patients within the aseptic joint cohort were not previously treated with any courses of antibiotics or operative procedures with the goal of treating PJI. Phenicol resistance could have mainly been observed among aseptic joints due to the lack of selective pressure of a more common broad spectrum antibiotic. Several of the ARG genes noted in this group are protective against older and less commonly used antibiotics (i.e., chloramphenicol) therefore their expression may be found more commonly in the aseptic cohort when compared to the septic revision cohort. Additionally, there were some multidrug resistant mechanisms noted in this aseptic cohort. This study utilized the ICM criteria to define the presence or absence of PJI, however as newer diagnostic techniques emerge it is possible that some of the aseptic cases may have indeed been due to an underlying infection. With the advent of newer, more sensitive techniques, such as MT, and with further clinical research a future update to the diagnosis of PJI may rely more heavily on laboratory diagnostic data. This is of course out of scope of this study and further work with larger cohorts and longer clinical follow up are needed. Within the scope of this study, our results suggest MT can provide the unique ability to accurately detect microbes and ARGs to a greater extent than other existing techniques. As is the case in all infectious disease, accurate identification of resistant strains is crucial to ensure appropriate antibiotic treatment is administered. While these techniques can detect low levels of microbes, it is important to reduce the risk of sample contamination. Analysis of negative controls, air, and skin swabs demonstrated that contaminants are relatively common. Of the prominent contaminants identified, C. acnes is a known common PJI-associated pathogen, particularly in total shoulder arthroplasty, although to a much lesser extent than other common pathogens, S. aureus and S. epidermidis 33,46,47 . False positive results involving common pathogens can lead to incorrect PJI diagnoses, which may incur unnecessary procedures and treatment for patients. As demonstrated in this study, using blank samples enable controlled analyses by revealing contaminant organisms, thus reducing the effect of contamination on final results. This study is not without limitations. Since 16S sequencing requires selecting primers targeting hypervariable regions of the gene, primers for different regions than the ones used in this study may yield different results. Within this study, blood and synovial fluid were collected, but it is unknown if swabbing of infected tissue/ implants would impact results. Though other studies have depleted human DNA from clinical samples before extraction to improve microbial signal yield 48 , a human DNA depletion step did not occur during this study. Considering these limitations, we demonstrate the potential for MT to offer significant improvements over 16S sequencing and even MG in the detection of active microorganisms and ARGs. Future clinical validation studies are warranted to establish the utility of MT as a clinical diagnostic test. Preliminarily, this study suggests MT may be a valuable method for diagnosing suspected PJIs and able to improve detection of pathogens and ARG identification. Future work towards the development of a clinically valuable assay should investigate the efficacy of MT for diagnosing clinical disease states, including PJIs. Analytical validation experiments would be required to assess the limit of detection, precision, sensitivity, and specificity of this technology when applied to blood and synovial samples with known outcomes, similar to the analysis done in other NGS assay validation experiment 1,4 .	2021-12-11T06:16:22.747Z	2021-12-01T00:00:00.000	{ "year": 2021, "sha1": "adb8ab6ce10e1fe5df433752c88eda9021f3fc03", "oa_license": "CCBY", "oa_url": "https://www.nature.com/articles/s41598-021-02505-7.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "a3263b380f65b70905a97c6c32fa5daf38d269b8", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
27727218	pes2o/s2orc	v3-fos-license	Rhabdomyosarcomatous differentiation in a spermatocytic seminoma with review of literature The sarcomatous differentiation occurring in spermatocytic seminoma (SS) renders an aggressive behavior with metastatic potential to this relatively indolent neoplasm. Correct identification of this sarcomatous component is essential as further management differs. Herein, we report a case of young male with SS with rapid increase in size of the tumor. Histopathology revealed a rhabdomyosarcomatous component infiltrating the rete-testis and epididymis along with a well-circumscribed SS. INTRODUCTION Spermatocytic seminoma (SS) is an uncommon testicular germ cell neoplasm with distinct histopathological features. It usually occurs in older men with mean age of 53.6 years (range 19-72 years). [1] It bears an excellent prognosis but tends to behave aggressively and renders a poor prognosis if associated with sarcomatous transformation. Patients with sarcomatous transformation at the time of presentation already have metastatic disease and this is almost fatal. [2,3] The sarcoma component may present as rhabdomyosarcoma, angiosarcoma, leiomyosarcoma, myxoid liposarcoma or undifferentiated sarcoma. [4] The identification and diagnosis of the sarcomatous component is pertinent for planning further management. We describe a case of a young man with SS developing rhabdomyosarcomatous transformation. Clinical findings A 38-year-old man presented with long standing history of swelling in right hemiscrotum. He underwent right testicular biopsy in a local medical college, which was diagnosed as seminoma. The patient was referred to our institute for further evaluation. On examination, he gave a history of sudden increase in size of the mass. There was a right testicular mass measuring 10 × 10 cm, with scrotal violation and left testis was normal. His general physical examination was normal. The serum tumor markers i.e., alfa-fetoprotein (AFP) and beta HCG were within normal. Hematological and biochemical parameters were normal. Chest X-ray, CECT abdomen and pelvis were normal. He underwent right high inguinal orchidectomy and hemiscrotectomy in view of scrotal violation. Retroperitoneal lymph node dissection (RPLND) was not performed as radiologically there was no lymphadenopathy. In addition to local and abdominal radiotherapy, chemotherapy (with vincristine, actinomycin D and cyclophosphamide) was given. Two months postoperatively, the patient is on follow-up and doing well. Pathological Findings The right high inguinal orchidectomy specimen measured 7 × 6 × 4 cm with attached vas deferens and scrotal skin. The testicular shape was maintained. On cut surface, a tumor measuring 6 × 6 cm was seen replacing the entire testicular parenchyma. The tumor was yellow, soft, and homogenous with a lobulated appearance. Most of the nodules were gelatinous with some appearing darker in color and firm in consistency than the rest [ Figure 1a]. DISCUSSION SS is a relatively rare, well-defined pathological entity first described by Masson [5] comprising 3-7% of all seminomas. [3] It is an indolent neoplasm with long duration of symptoms, early stage at presentation, absence of metastasis and bears an excellent prognosis. [6] However, dedifferentiation or transformation to a sarcoma which occurs in approximately 6% of cases renders a poor prognosis with a metastatic potential. [7] Literature review cites small series [3] and a few case reports [8][9][10] of occurrence of sarcomatous differentiation in SS. The occurrence of rhabdomyosarcomatous differentiation is rare and has been described in the literature in eight previous case reports [ Table 1]. [6,8,9,11] In the present case, the sarcomatous transformation occurred at a much early age of 38 years compared to the previous cases. The sudden increase in size should always suggest emergence of a sarcomatous component. Light microscopic findings are usually diagnostic after one appreciates the two components distinctly. However, it is important to be aware of misinterpreting the focal areas of atypical stromal overgrowth within conventional germ cell tumor. True sarcomatous component can be distinguished by its expansile growth pattern, infiltration into the surrounding elements and characteristic morphology of the distinct sarcomatous components. [4] Immunohistochemistry helps in highlighting and further categorizing the sarcomatous element. The seminomatous component was well circumscribed but the rhabdomyosarcomatous component was seen infiltrating and destroying the rete testis and epididymis. Identification of epithelial structures of rete testis is important as these may be mistaken for epithelial component of a mixed nonseminomatous germ cell tumor. The origin of the sarcomatous elements of these tumors and the relation to the spermatocytic component is uncertain. Few have implied [12,13] this sarcoma component to be a teratomatous element of a mixed germ cell tumor as the occasional occurrence of sarcomatous elements (chiefly rhabdomyosarcoma, angiosarcoma, and leiomyosarcoma) in gonadal and extragonadal germ cell tumors is well known. The serum elevation of alpha-fetoprotein and beta-HCG favors this hypothesis. The serum tumor markers were not elevated in the present case. However, other authors view the sarcomatous component as an 'anaplastic transformation' or 'dedifferentiation' occurring within the well-differentiated neoplasm which seems most plausible. [3] The development of this tumor, first with slow enlargement of the testis followed by rapid increase in size tends to favor the explanation of sarcomatous differentiation occurring in SS. The treatment of choice in SS is orchidectomy, followed by surveillance to detect tumor in the contralateral testis. However, in the presence of sarcomatous differentiation, adjuvant chemotherapy or radiotherapy is beneficial. As the risk of metastases if remarkably increased with this transformation, a close follow-up is essential; however, prognosis is dismal despite aggressive multimodality approach. [3,6] In conclusion, this case describes the rare sarcomatous differentiation occurring in SS heralded by a rapid increase in size, which portends a poor prognosis to this indolent neoplasm. Accurate and timely diagnosis is pertinent for planning further management of the patient.	2018-04-03T01:01:12.867Z	2012-10-01T00:00:00.000	{ "year": 2012, "sha1": "a7f3bb857ebfb3b8bee9deed55ee6b5778e3fdf7", "oa_license": "CCBYNCSA", "oa_url": "https://doi.org/10.4103/0970-1591.105760", "oa_status": "GOLD", "pdf_src": "ScienceParsePlus", "pdf_hash": "71b40d1354ca2c58d04f6d7bccbef24ebd688807", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
233821746	pes2o/s2orc	v3-fos-license	Study of the Properties of Open Graded Asphalt Mixtures With the addition of SBS Porous asphalt (PA) is widely used in a growing number of countries where porous asphalt is applied for a variety of purposes, e.g. for the effective drainage of rainwater, traffic safety (high slip resistance), the control of noise pollution and lower temperatures surrounding the city. However, it has many other disadvantages, such as poor resistance to rutting, poor resistance to fatigue, and PA is susceptible to raveling (wastage of aggregates from the pavement surface), due to effects of climatic and traffic loading. In general, this type of mixtures is not as good as traditional mixtures. This research aims to study and improve the properties of porous mixtures using SBS. In this paper, laboratory tests were carried out to the materials involved in the composition of this mixture: binder, aggregate, and additive. SBS is used in the proportion of (2.0, 3.0, and 4.0) % of the weight of the binder. It was found that this additive leads to reduce the permeability and air void, but not as large as that without polymer modifier by (1.7 %, 3%, and 3.5%), while in the case of abrasion loss (aged and unaged) decrease by (4.1, 6.67 and 10.92) (4.7, 6.3and 2.6)% respectively. The drain down value is decreased by (16.5%, 38.25%, and 43.51%) respectively, from original asphalt cement. 1-Introduction Open-Graded Asphalt, also known by different names: Graded Friction Course (OGFC), Porous Friction Course (PFC), permeable European mix (PEM) and Porous Asphalt (PA), which in Europe was mightily used, for instance, Netherlands, France, and Germany, whereas in Asia e.g. China, Japan, and Korea [1]. The open-graded asphalt mix is defined as the thin wear surface of the "HMA" hot mix asphalt, pavement that is used worldwide due to its safety properties that have an affirmative influence on a driver and is orderly used as the last lane on the interstate and high-speed low-volume expressways. The open-graded asphalt mix is light compared to dense asphalt mix and can cover more road surfaces. Open-graded asphalt mixture layer that improves drainage when it rains. The rainwater flows vertically through the road surface to the base course and then horizontally for the end of the road. Open asphalt mixes consist of a high proportion of coarse aggregate, which creates a high percentage of air and thus leads to rainwater flowing vertically across the road surface to the base layer and laterally to the end of the road [3]. On the other side Kandhal and Mallick, (1999) [4], studied the percentage of aggregate passing through sieve number 4.75 mm. They found that the ratio does not exceed 20% to maintain contact with a stone-on-the stone in the skeleton of the coarse aggregate and to ensure and provide sufficient permeability high air spaces in the open asphalt mixture. The benefits of open asphalt IOP Publishing doi:10.1088/1757-899X/1090/1/012002 2 pavement include reducing splash and spray, decrease wet skidding, diminish the risk of water planning, and improve visibility of pavement; signs in wet weather [5]. And compared to dense hot asphalt (DGHMA), this type of mixture improves driving quality and noise reduction efficiency. Also, several studies have shown that the lowest concentrations of particles and total suspended soil pollutants in the proven runoff of asphalt mixes with open gradients compared to conventional DGHMA [6]. Table (1) exhibit specifications for porous Asphalt [7]. Styrene Butadiene Styrene (SBS) Modifier utilized in this research is (SBS) collected from the local markets. Table (6) shows the characteristics of the Modifier. Design of Marshall Molds Porous friction course (PFC)specimens were prepared by mixing the aggregates, cement filler, and bitumen in its mold of Marshall; (diameter 101.4mm and high 64mm) with a weight of around1200 grams [9]. 50 blows on each side (marshal hammer) are used to compress the samples as indicated (ASTM D7064.13) [8]. Figure (2) displays some of the prepared specimens. Mixing and Compaction Temperature In this paper was determined temperatures (mixing and compaction) from rotational viscometer test (Asphalt Institute 2003) [10]. The results display that the temperature of mixing approximately (155 °C), and temperature of compaction approximately (144°C) as shown in Figure (3). Cantabro Abrasion Loss The Cantabro test is used to determine the abrasion resistance of porous pavement, this examination was conducted according to American specifications [13]. One group was tested in the unaged condition using the (Los Angeles) machine test method and the other was aged for 7 days at 140˚F (60˚C) in the oven. To measure the abrasion resistance of the open-graded specimens, the initial mass of a sample was measured and then the sample was placed inside the cylinder without any steel charge at speed (30-33) rpm so, that the number of rotations does not exceed ,300 rotations, at 77 ° F (25 ° C), after that the sample is removed and then weighed again. Figure (4) illustrate specimens before and after the abrasion loss test (Before and After Test). Allowed limits of 20 % maximum for un-aged specimens and 30 %maximum for aged specimens. Abrasion loss was calculated using Equation. Where Ai and Af are the initial and final masses of the sample, respectively. (2) a. Before b-After According to (ASTMD6390 -11) [14], un-compacted samples were prepared (the mass of the sample is 1200 ± 200 g) so that the value of draindown should not exceed 0.3 %. Then, placed the assembly in the oven at the temperature would be the anticipated plant production temperature as well as 15°C above for one hour ± 5.0 min. The assembly is removed from the oven and cooled at 24°C, as shown in figure (5). The draindown percentage is calculated using the following equation: - where: A= mass of the empty wire basket gm, B= mass of the wire basket and sample gm, C= mass of the empty catch plate gm, and D= mass of the catch plate plus drained material gm. Hydraulic Conductivity Test (Permeability) Permeability is one of the most important features used in the evaluation of the open-graded asphalt mixtures. The minimum value of permeability of the porous asphalt is100m/day. To determine the rate of flow water was used the Falling-Head test apparatus as shown in the figure (6). According to Darcy law, the (K) value is calculated for compacted paving mixture, using the following equation: -(4) where: K = coefficient of water permeability, cm/s a = inside cross-sectional area of inlet standpipe, l = thickness of test specimen, cm A = cross-sectional area of test specimen, cm2 T = average elapsed time of water flow between timing marks, s h1 = hydraulic head on specimen at time t1, cm h2 = hydraulic head on specimen at time t2, cm = natural logarithmic function and tc = temperature correction for viscosity of water. Asphalt binder content selection Depending on test method (ASTM D 7064-13) [8], the content of asphalt for porous mixtures was determined using five ratios (4.0 to 6.0) % by weight of the mixture, with an increase of 0.5 %. Practical tests showed that the optimum asphalt ratio is (5.2) as shown in figure (7) and table (8). 6. The SBS polymer effect on Open-Graded Asphalt Mixtures Properties. The SBS polymer effect on Air Voids The results registered that the Va of the specimens containing (2, 3, 4) % modifier SBS decreased by 1.7 %,3%, 3.5% respectively, as shown in the figure (8). , Figure 9. Air voids results. (9) and (10), it is possible to notice a decrease in abrasion loss aging and un-aging for porous asphalt after adding the polymer SBS. Figure 10. Effect of SBS polymer on un-aged. Figure 11. The SBS polymer Effect on aged. Conclusions The following conclusions was determined based on the testing results: - Recommendations 1. It is recommended to use SBS polymer to obtain high-performance asphalt concrete and improve durability (aging and un-aging) for porous asphalt. 2. Adopting other types of polymers mixing with asphalt cement with different percentages such as Crumb Rubber (CR), High-Density Polyethylene (HDPE) and Styrene Butadiene Rubber (SBR), also using waste materials as modified such as (fly ash, slag, glasses, and plastic). Acknowledgments The researchers would like to thank the University of Technology, workers in the asphalt laboratory.	2021-05-07T00:04:10.233Z	2021-03-01T00:00:00.000	{ "year": 2021, "sha1": "441395e59d77cf85fdde3f82d4cae30140c5cf8b", "oa_license": "CCBY", "oa_url": "https://iopscience.iop.org/article/10.1088/1757-899X/1090/1/012002/pdf", "oa_status": "GOLD", "pdf_src": "IOP", "pdf_hash": "237ee33f2df61092817115a13e3ea62c8a15971d", "s2fieldsofstudy": [ "Engineering", "Materials Science" ], "extfieldsofstudy": [ "Physics", "Materials Science" ] }
53984941	pes2o/s2orc	v3-fos-license	Effect of Haematogenous Oxidation Therapy with Ul- traviolet-C Irridation in Alloxan-Induced Diabetes and Poloxamer 407-Induced Hyperlipidemia in Rabbits Background: Recently diabetes and hyperlipidemia (HL) considered a major source of mortality. Chemical treatments could minimize the symptoms but, still the disease exists. Previously Ultraviolet was used for treatment of ailments related to infection and metabolism. Methods: The study evaluates the effects of Haematogenous Oxidation Therapy (HOT) on the blood when a low dose of Ultraviolet-C (UV-C) is directly irradiated to the blood in a diabetic rabbit model and to evaluate the effects of treatment on diabetic rabbit. Type 1 Diabetes and hyperlipidemia were induced by intravenous injection of alloxan monohydrate and subcutaneous injection of poloxamer 407, respectively. A 10 ml blood was collected from diabetic rabbits, blood was being perfused with oxygen for 10 seconds and UV-C was irradiated to the blood, UV-irradiated blood was transfused back to the original rabbits. The HOT treatment was performed a total of 10 times. It was evaluated the effects of the HOT treatment on diabetes and HL through hematological and biochemical analyses before and after HOT treatment were performed. Results: The results indicated that the reduced body weight was increased and blood glucose levels were significantly reduced after the HOT treatment was performed when compared to those prior to the HOT treatment. In addition, CRE, BUN and UA levels indicating renal functions were significantly reduced when compared to those prior to the HOT treatment. When the HOT treatment was performed in a diabetic and HL rabbit model, our results indicate that blood glucose levels and lipids profile were improved. Conclusions: Biochemical and Hematological analyst was demonstrating that the HOT was effective to alleviate diabetes and HL. Background Ultra violet irradiation (UBI) of blood was hailed as a miracle therapy for treating serious infections in the mid of 19th century. However, this time period coincided with the widespread introduction of penicillin antibiotics, which were rapidly found to be an even bigger miracle therapy. Moreover, another major success of UBI, which was becoming used to treat polio, was also eclipsed by the introduction of the Salk vaccine. Starting in the 1960s UBI fell into disuse in the West and has now been called "the cure that time forgot" [1] . Recently, there has been a gradual increase in mortality due to coronary heart diseases (CHDs). Factors such as hypercholesterolemia, cigarette smoking, diabetes mellitus, and sedentary lifestyle are key contributors to the development of hyperlipidemia (HL) and atherosclerotic cardiovascular disease. Apart from these factors, there are evidences that suggest certain chemicals can also cause hyperlipidemia and may substantially lead to CHDs. Poloxamer 407 was one such example [2] . Poloxamer 407-induced hypertriglyceridemia and hypercholesterolemia are well known incident. Elevated triglycerides and inhibition due to lipoprotein lipase was the main feature of poloxamar 407-induced hyperlipidemia [3] . Diabetes was a metabolic disease with high blood glucose levels caused by the metabolic disorder resulting from defects of hormones such as insulin, glucagon or glucocorticoids involved in the metabolism of glucose or by abnormal reactions in the pathway [4] . Because the diabetes was characterized by high blood glucose levels, which leads to a wide range of malfunctions in the metabolic control over carbohydrates, proteins, fats, and electrolytes, it was closely associated with increases in various chronic degenerative diseases [5] . Diabetes emerges as the disease causing social problems because it has a very high prevalence as a typical chronic metabolic disease. Diabetes goes beyond endemic limits and was becoming an epidemic internationally [6,7] . The incidence of diabetes was very low in Korea when compared to that in Western countries. However, the incidence was gradually increasing in Korea due to recent changes in diet, environment, and lifestyle [8,9] . Oral hypoglycemic agents and insulin formulation for the treatment for the diabetes are continuously developed. However, because it was the chronic disease which was not fully cured once patients develop diabetes, it causes a serious problem [10] . Thus, in this study, it was evaluated the effects of Haematogenous Oxidation Therapy (HOT) blood irradiation on the diabetes and HL using physical methods with UV light rather than drug therapy such as insulin and statins injection in order to get over diabetes-and HL-causing serious problems with an emphasis on diabetes. Design of ultraviolet blood irradiation device In this study, HOT device was produced to identify effects of ultraviolet blood irradiation on the blood in a diabetic animal model. Figure 1 shows a simple drawing and photo of the HOT device. In brief, a fixing holder for quartz cuvette was installed in the center and ultraviolet (UV) lamps were installed on both sides of the holder. The UV lamps are designed to adjust the distance (5 mm ~ 120 mm) from the holder in the center. Ultraviolet blood irradiation was performed in the cuvette. Experimental animals Adult male eight New Zealand White rabbits of body weighing 2 -2.5 kg were used in the study. All the rabbits were kept in cages with wide square mesh at the bottom to avoid coprophagy and maintained under controlled conditions of humidity, temperature (22 ± 2°C) and 12 hours' light and dark cycle. Food and water were provided ad libitum. They were fasted for 18 hours prior to the experiment, allowing free access to water only. The experimental protocols were approved by the Institutional Animal Ethics Committee. All experimental protocols (CBU2013-0010) were approved by the Committee on the Care of Laboratory Animal Resources, Chonbuk National University and were conducted in accordance with the Guide for the Care and Use of Laboratory. Induction of experimental diabetes -Procedure for injecting alloxan monohydrate The 10 rabbits weighing between 2 to 2.5 kg were made diabetic by injecting intravenously 110 mg/kg body weight of alloxan monohydrate (A7413, Aldrich), a dose confirmed to induce diabetes by our previous study [11] . Before giving alloxan, the normal blood glucose levels of all rabbits were estimated. After 2 hours of alloxan injection, the 5% Dextrose injected to the all-diabetic rabbits intraperitoneally to prevent a hypoglycemic condition of rabbits with alloxan. After 72 hours of alloxan injection, the blood glucose levels of all surviving rabbits were determined by the glucose oxidase method. Induction of experimental hyperlipidemia -Procedure for injecting poloxamar 407 Poloxamar 407 (Sigma) was prepared at a concentration of 30% (w/w) in sterile distilled water. The 10 rabbits weighing between 2 to 2.5 kg were made hyperlipidemic by injecting 137.5 mg/kg (0.7 ml/kg) subcutaneously after shaving the dorsal surface of injection, dose repeated weekly for 10 weeks. Haematogenous Oxidation Therapy It was confirmed that the diabetes was induced by measuring blood glucose levels in rabbits at 72 hours (3 days) after alloxan was injected. The blood was collected from diabetic rabbits after 1 week. The blood was perfused with oxygen for 10 seconds. For the HOT, UV was irradiated to the blood collected through auto transfusion and the blood was transfused back to the original rabbit. Anticoagulation Sodium Citrate Solution (BOIN ACDA SOLN, SBD Co., Ltd.) was used to prevent coagulation of the blood when it was collected. 10 ml blood was collected from the vein by using 20 ml syringe (needle gauge 26). UV light with the intensity of 10.290 J/cm 2 was irradiated to the blood which passes at a constant flow rate using the syringe pump in the HOT device. Once UV was irradiated to the blood, the blood collected on the syringe in the other side of the cuvette. After the HOT was performed, the blood transfused back to the original rabbit. The HOT treatment was performed a total of 10 times. Rabbits were stably raised in a laboratory animal breeding facility. Food and water were sufficiently supplied. Biochemical analysis Blood was collected from the ear marginal vein. A Nova Stat Profile ® pHOx ® Ultraanalyzer (NOVA Biomedical Corp., Waltham, MA, USA) was used to measure blood gas, electrolytes, and anion gap. After clotting, blood serum was separated by centrifugation at 3000 rpm for 20 min. The levels of glucose, enzymes, lipids, and proteins were analyzed using a Model 7020 auto analyzer (Hitachi, Tokyo, Japan). Serum insulin and HbAlc measurements Fasting serum insulin levels were measured with a rat insulin enzyme-linked immune absorbent assay kit (Life Span Bio Sciences, Inc., LS-F21890) according to the manufacturer's protocol. HbA1c ELWASA kit (ALPCO Diagnostics, Windham, NH, USA) were quantities using commercially available kits according to the manufacturers' protocols. Statistical analysis Data were expressed as means ± standard errors of the mean (SEMs). Differences between groups were evaluated by oneway analysis of variance with the Bonferroni post hoc test or by calculation of Spearman's rank correlation coefficient, as appropriate, using Prism 5.03 (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was set at p < 0.05. Serum glucose, insulin, and HbA1c concentration In the diabetic rabbit model induced by alloxan, glucose was increased significantly compared to normal rabbits as shown in figure 2. However, in the diabetic rabbit model undergoing the HOT treatments, glucose levels were reduced over the course of the treatment. bits. Data were reported as means± SEMs (n = 10). : p < 0.05; : p < 0.01; and *: p < 0.001, Bonferroni post hoc test following oneway ANOVA versus the NC; normal control prior diabetes was induced and before HOT: p < 0.05; ##: p < 0.01; and ###: p < 0.001, Bonferroni post hoc test following one-way ANOVA versus Diab; diabetic (after diabetes induced and prior HOT),4th, 10th treatment, and A10W; fourth treatment, tenth treatment, and 10 weeks after last treatment respectively. Insulin concentration in the blood was significantly decreased in the diabetic group without HOT treatment compared to the normal group (NC) as shown in figure 3. However, the insulin concentration in the HOT group was almost similar to that of NC. Therefore, an increase in the insulin concentration of blood in diabetic groups receiving the HOT treatment reduced blood glucose. The HbA1c in the diabetic group without HOT treatment was significantly increased compared to the normal group. However, the HbA1c in the diabetic group that received the HOT treatment was significantly reduced compared to the group without the HOT treatment. Figure 3: Effects of HOT on the renal characteristics by serum analysis in alloxan-induced diabetes (a) and 407-induced hyperlipidemic(b) rabbits.CRE, creatinine; BUN, blood urea nitrogen; UA, uric acid; creatine kinase (CK). Data were reported as means± SEMs (n = 10). : p < 0.05; : p < 0.01; and : p < 0.001, Bonferroni post hoc test following oneway ANOVA versus the NC; normal control prior diabetes was induced and before HOT: p < 0.05; ##: p < 0.01; and ###: p < 0.001, Bonferroni post hoc test following one-way ANOVA versus Diab; diabetic (after diabetes induced and prior HOT), 4th, 10th treatment, and A10W; fourth treatment, tenth treatment, and 10 weeks after last treatment respectively. Kidney function test In diabetes and HL rabbit prior HOT treatment; CRE, BUN, UA and CK levels were elevated significantly when compared to normal group as shown in figure 4a and 4b. However, after HOT treatments sessions were started these values were significantly reduced when compared to those before HOT was applied. Figure 4: Effect of HOT on the liver characteristic by serum metabolic enzymes analysis in alloxan-induced diabetic (a) and 407-induced hyperlipidemic(b) rabbits. AST, aspartate aminotransferase; ALT, alanine aminotransaminase; LDH, lactate dehydrogenase; ALP, alkaline phosphatase. Data were reported as means± SEMs (n = 10). : p < 0.05; : p < 0.01; and : p < 0.001, Bonferroni post hoc test following oneway ANOVA versus the NC; normal control prior diabetes was induced and before HOT: p < 0.05; ##: p < 0.01; and ###: p < 0.001, Bonferroni post hoc test following one-way ANOVA versus Diab; diabetic (after diabetes induced and prior HOT), and ), and vs hyperlipidemic rabbits (after HL being induced and prior HOT), 4th, 10th treatment, and A10W; fourth treatment, tenth treatment, and 10 weeks after last treatment respectively. Liver function test In the diabetic and HL rabbit model induced by alloxan, after one week AST, ALT, LDH, and ALP levels were significantly increased compared to normal rabbits as shown in figure 5a and 5b. However, after HOT treatments sessions were started these values showed significantly time-dependent reduction when compared to those before HOT was applied. . T-CHO, total cholesterol; HDL, high density lipoprotein; LDL, low density lipoprotein; TG, triglyceride; T-PRO, total protein; Alb, Albumin. Data were reported as means± SEMs (n = 10). : p < 0.05; : p < 0.01; and : p < 0.001, Bonferroni post hoc test following oneway ANOVA versus the NC; normal control prior diabetes was induced and before HOT: p < 0.05; ##: p < 0.01; and ###: p < 0.001, Bonferroni post hoc test following one-way ANOVA versus Diab; diabetic (after diabetes induced and prior HOT), and vs hyperlipidemic rabbits (after HL being induced and prior HOT), 4th, 10th treatment, and A10W; fourth treatment, tenth treatment, and 10 weeks after last treatment respectively. Lipid profile In the diabetic and HL rabbit model, T-CHO, LDL, TG, T-PRO, and Alb levels were increased significantly compared to those of normal rabbits as shown in figure 6a and 6b. However, after HOT treatments, these levels significantly decreased when compared to those before HOT were applied. In addition, HDL level significantly decreased compared to normal group. However, in rabbit subjected HOT treatment, the level was elevated significantly. Blood gas, Hb, Hct, and lactate Blood concentration of PO 2 , SO 2 , Hb, and Hct were significantly decreased in diabetic rabbits compared to normal rabbits. However, in HOT-treated rabbits, these parameters significantly improved. Oppositely, PCO 2 and lactate were significantly elevated in diabetic rabbits compared to normal rabbits. Never the less, www.ommegaonline.org Vol 5:1 pp 52 in after HOT treatment, the concentration significantly reduced as shown in table 1. Blood ions Blood concentration of Mg 2+ and Ca 2+ were significantly decreased in diabetic rabbits compared to normal rabbits. However, in HOT-treated rabbits, these parameters significantly increased. On the other side, K + , Cl -, and AG were significantly elevated in diabetic rabbits compared to normal rabbits. Never the less, in after HOT treatment, the concentration significantly reduced. Na + concentration although significantly reducedin diabetic rabbits compared to normal group did not increased after HOT treatment as shown in table 2. Data were reported as means± SEMs (n = 10). : p < 0.05; : p < 0.01; and *: p < 0.001, Bonferroni post hoc test following one-way ANO-VA versus the NC; normal control prior diabetes was induced and before HOT: p< 0.05; ##: p< 0.01; and ###: p< 0.001, Bonferroni post hoc test following one-way ANOVA versus Diab; diabetic (after diabetes induced and prior HOT), 4th, 10th treatment, and A10W; fourth treatment, tenth treatment, and 10 weeks after last treatment respectively. Discussion To best of our knowledge, there was no published data concerning effect of HOT in HL and diabetic animal models. However, in a previous study, the result indicated that UBI was safe when used in diabetic rabbits induced by alloxan. The glucose level was significantly reduced and the body weight was significantly increased after UBI treatments compared to diabetic rabbit prior UBI sessions were started [11] . This experiment followed the same protocol, however, the difference was only that the blood was perfused with oxygen for 10 seconds using sterile oxygen cylinder. A report stated that HOT treatment improves insulin efficiency and blood glucose levels were reduced. In addition, vision was improved in some diabetic patients with different stages of retinitis undergone HOT treatments because RBC aggregation was suppressed [12] . Normally, protein metabolized and hence excreted by kidney as a urea which was considered a final product of protein and amino acid. High blood urea level indicates renal problem. BUN directly affected by protein intake and considered as an indicator of renal function [13] . The result in Figure 4 showed an increased level of Cre, BUN, UA,and CK, which in an agreement with other study which showed that an increased level of Cre, BUN, and UA in diabetes was well documented by [14] . In contrast, after treatment with HOT rabbits exhibited a significant reduction in the levels of these indicators, suggesting that HOT effectively enhanced renal function. As kidney was also responsible of regulating ions and electrolyte homeostasis, so an imbalance in electrolytes such as Mg 2+ , Ca 2+ , K + , Na + , Clconsidered a deterioration in kidney function of diabetic rats [15] . As shown in table 1 these indicators were significantly improved by HOT treatments. AST, ALT, LDH, and ALP are indicators of liver function and their elevations indicated liver stress as mentioned [15,16] . These indicators as shown in Figure 5 were significantly reduced by HOT treatments which supports that HOT has hepatoprotective effect. This was in a good agreement with our previous study [11] we reported that AST, ALT, LDH, and ALP were significantly improved after UBI treatment. Our result in Figure 6 as expected showed an elevation in T-CHO, TG, LDL, and decreased level in HDL in diabetic rabbits before HOT sessions were started which agreed with a previous study [7] who found that diabetic patients are under the risk of Hypertriglyceridemia and hypercholesterolemia with respect to heart and coronary disease. On other hand after HOT treatments, these indicators were significantly improved. Hence, HOT could be used to prevent cardiovascular disease. The result was in a good agreement with our previous study. T-PRO and Alb were significantly decreased in diabetic rabbits. However, after HOT treatment, these indicators were significantly increased, this result coincided with a conclusion made other investigators [11] . Table 1 indicated that pO 2 was significantly reduced in diabetic rabbits without HOT treatment whereas pCO 2 was significantly increased in diabetic rabbits with HOT. However, these values were significantly improved after HOT treatments which agreed [17,18] . Lowered blood pH, reduced HCO 3 -, and increased lactate levels were observed in the diabetic indicating acidic blood, which may be a sign of ketoacidosis [19] . [20] who found that UBI improved oxygen delivery, blood elements, and stimulation of mitochondrial oxidation may help quick recovery of many aliments. This result coincided with Mattman and Lida Alloxan causes a high reduction in insulin release by the destruction of the β-cells of the islets of Langerhans, results in hyperglycemia [21] . Impaired glucose tolerance was attained due to lack of insulin in alloxan-induced diabetic rats by destructing the β-cells which leads to type I diabetes [22] . However, insulin level in rabbits subjected to HOT were showed a significant increment in insulin level compared to diabetic rabbits. Fasting serum HbAlc level was an important indicator for patients with diabetes who have cardiovascular diseases. The HbAlc level has been found to be directly proportional to the blood glucose concentration [14,23] . As expected, the HbA1c levels were increased in the diabetic groups in our study, which were lowered by the 10 times treatment protocol of HOT. This outcome could be due to improved insulin secretion [14] . AST, ALT, LDH, and ALP which indicates liver injury caused by hyperglycemia andhyperlipidemia due to tissue insult [24,25] . However, HOT enhanced the liver condition of hyperlipidemic rabbits. This result come in a good agreement with our previous study concluded that AST, ALT, ALP, and LDH significantly reduced in diabetic rabbits undergoing UBI for eight times with blood oxygenation [11] . Dyslipidemia associated with chronic kidney diseases as concluded by Tsimihodimos V et al. [26] However, after HOT was applied, hyperlipidemic rabbits showed a significant enhancement in kidney status which can be clarified by a significant decrement in the measured levels of parameters. These results coordinated with a recent study [11] which found that in diabetic rabbits subjected to UBI treatment, a marked enhancement in kidney function namely CRE, BUN, and UA levels were lowered significantly. In our study the reason behind this could be attributed to UV implied in HOT which enhanced tissue blood perfusion and oxygenation. Conclusion The study evaluated the effectiveness of HOT in treatment of type 1 diabetes and HL by using auto-transfusion. Evaluation was performed through some biochemical and hematological parameters. As a result, HOT effectively lowered glucose level, improved lipid profile enhanced body weight in diabetic rabbits, showed a hepato-protective, cardio-protective and protects against renal damage. In addition to, blood gas and electrolytes imbalance were improved. However, further study to elucidate the adverse effect of HOT and to elaborate in mechanism of action using some molecular and biological techniques. This experiment was a part of a series of experiments in our lab using to remind the scientific community about an old, cheap and effective method of treatment that forgotten for long time due to an advancement in synthetic and chemical pharmaceutical agents.	2019-04-10T13:12:31.300Z	2018-11-19T00:00:00.000	{ "year": 2018, "sha1": "6aa03840e8b299f85ba97f74e20e210c81a89036", "oa_license": "CCBY", "oa_url": "https://www.ommegaonline.org/articles/publishimages/15238-JDO-18-RA-1930.pdf", "oa_status": "GOLD", "pdf_src": "MergedPDFExtraction", "pdf_hash": "9c743954c623151be0a539549a49ff9a0954994e", "s2fieldsofstudy": [ "Medicine", "Environmental Science" ], "extfieldsofstudy": [ "Chemistry" ] }
213094264	pes2o/s2orc	v3-fos-license	The effect of zircon particle size on the surface properties of sanitaryware glaze Zircon (zirconium silicate; ZrSiO4) is an essential compound of sanitaryware glaze due to contributions to the final properties of the surface. In this study, the effect of zircon particle size on the surface properties (microstructural development, opacity, roughness, hardness and bacterial activity) were studied. Initially, zircon powder was milled by planetary mill at different times (0–25 min) and added to an industrial sanitaryware glaze recipe. Four different glaze suspensions were prepared, applied to a sanitary ware body and sintered in an industrial tunnel kiln at 1220 °C for 16 h. The effect of milling is clearly observed via SEM investigations by uniform distribution of zircon grains with a size reduction to 0.38 μm. This uniform distribution and reduction in particle size provides improvements in opacity (L* value measured as 91.47) and hardness (Hv was 6.3 (±0.3) GPa) values. According to AFM studies, the average roughness decreased to 10.94 nm when a 25 min milling process was used. The antibacterial activities of two samples (milled for 0 and 25 min) were analyzed against Escherichia coli and Staphylococcus aureus. Although both samples did not show any antibacterial activity, the number of viable bacteria on the samples milled for 25 min decreased drastically compared to the one not milled. A smoother glaze surface facilitated less sites for adherence and accommodation of bacteria and hence a decrease in bacterial colonization was obtained. Introduction Several functions of sanitaryware, such as corrosion-wear resistance, dirt repellency, cleanability, optical properties and antibacterial activity are directly related to the surface characteristics of glazes. Since expectations on the quality of sanitaryware increases, improvements of these functions are requisite and, therefore, many recent studies have focused on the surface properties of these products. Studies vary from changing the properties of the starting mixture (raw material type, particle size), firing conditions [1][2][3][4][5], addition of antibacterial compounds or salts [6][7][8], creating hydrophobic and oleophobic surfaces [9,10]or a combination of these conditions [11]. Higher refractive index (1.92-1.97), wear-impact resistance, thermal shock and the chemical durability of zircon impart excellent properties to the surface of sanitaryware glaze [12,13]. Owing to the high cost of zircon, several studies have been conducted aiming to reduce its usage or completely replace it by more economical alternatives. Despite these studies, zircon is still one of the main raw materials used in the sanitaryware industry. The effect of zircon on the functions of sanitaryware has therefore been well investigated. A certain amount of zircon should be used to enhance chemical and wear resistance. As the addition of zircon becomes greater than 10% mass, a higher resistance against alkali and acidic solutions is obtained [14]. ZrO 2 is a highly durable oxide against ion exchange and alkaline attack. Since the soluble of the ionic compound, such as ZrO +2 , Zr +4 and HZrOO −3 , form only lower and higher pH values, ZrO 2 based raw materials are preferred in order to obtain glazes with high chemical resistance [15]. The particle or grain size of zircon is another parameter studied and it directly affects optical properties and wear resistance. By decreasing the particle size of zircon, the possibility of a Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. light-particle interaction increases and this will consequently increase light scattering [16]. In addition, the distribution of the zircon particles is important. A non-uniform distribution of zircon forms clusters that results in a glassy phase on the surface of glaze. A uniform distribution of zircon provides more sites for light scattering and increases L * value [17]. The homogenous distribution of these hard crystals throughout the surface leads to an enhancement on the hardness of glaze [18]. Several approaches have been taken in order to obtain antibacterial surfaces; antibacterial agent release (e.g. silver coatings), contact killing (e.g. TiO 2 based materials) and anti-adhesion/bacteria-repellent. The use of molecules provides anti-adhesion or tailoring surface topography [19]. One way to tailor surface topography is to decrease the surface roughness that inhibits the attachment of bacteria cells by reducing the contact area between the cells and the surface area. When the dimension of surface topography is less than the cells (in the range of submicron or nano), the attachment of the cells is inhibited. In addition, submicron surface topography creates repulsive forces between bacteria and surface that reduce the possibility of attachment [20]. In the current study, the effect of the particle size of zircon on surface properties (whiteness, microstructure, roughness and bacterial activity) of sanitaryware glaze is investigated. As the particle size of zircon decreases to submicron scale, improvements in surface properties are obtained owing to a uniform distribution of zircon and reduced roughness values. 2. Experimental procedure 2.1. Sample preparation Zircon (Zircobit MO, Bittosi, Italy) was milled four different times; for 0, 5, 15 and 25 min, to observe the effect of zircon particle size on surface properties, using ZrO 2 balls (3 mm in diameter) and ZrO 2 jar in a planetary ball mill (Retsch, PM4, Germany) at 450 rpm. Milled zircon powders were added to an industrial sanitaryware glaze and glaze suspensions were prepared using an eccentric jar mill, mixed with water (30 wt%) and binder (carboxy methyl cellulose) for 17 min. The oxide composition of industrial glaze is given in table 1, and glazes are designated as GZ-0, GZ-5, GZ-15, and GZ-25 regarding the zircon milling time. Green sanitaryware bodies of 10×10 cm were coated by prepared glaze suspension being sprayed in 25 g amounts. The glazed samples were kept at room temperature for 2 h, then dried at 110°C for 24 h. The samples were then sintered in an industrial tunnel kiln at 1220°C for 16 h. Characterization The particle size distribution values of the zircon powders were measured using the laser diffraction technique (Malvern Mastersizer E 2000, UK). L * , a * and b * parameters were measured with a colorimeter (Konica Minolta Chroma Meter CR-400, Japan ).The distribution of zircon grains and the overall surface appearance were also examined by scanning electron microscopy (SEM) (Zeiss Supra 50 VP, Germany). SEM images were segmented by thresholding and analyzed automatically using Image J 1.52a software. Since the grains were not perfectly round, a minimum feret diameter of 250 grains from each sample was measured and then the average grain size was calculated. Chemical investigations of vitreous phase were performed using SEM with energy dispersion x-ray spectrometry (SEM-EDX) (Hitachi SU5000, Japan). X-ray diffraction (Rigaku MiniFlex, Japan) was performed using monochromatic Cu-Kα radiation (λ=1.5406 Å). The surface roughness of the glazed samples was characterized by atomic force microscopy (AFM) (QScope-250, Canada) using a non-contact mode equipped with a Si 3 N 4 cantilevered scanner of 50×50 μm scan size. The surface roughness was evaluated by measured values of the root mean square roughness (Rq) and the average roughness (Ra). Rq is the standard deviation of the height values from the average height in the image. Ra is the mean height value as calculated from the entire surface plane. The hardness of the glaze was measured by the indentation method (Shimadzu HMV-G, Japan). The measurement was carried out using a diamond indenter with a 10 N load for ten seconds. Antibacterial-activity test The antibacterial activity of the GZ-0 and GZ-25 samples against Escherichia coli and Staphylococcus Aureus was examined according to ISO 22196 (measurement of antibacterial activity on plastics and other non-porous surfaces) [21]. Prior to antibacterial activity test, both samples were sterilized. Bacterial suspensions were pipetted on the surface of samples then the surfaces were covered with sterilized plastic film. Incubation was done at a temperature of (35±1)°C and a relative humidity of not less than 90% for (24±1) h. The test was conducted at the Saniter Laboratory (Istanbul, Turkey), which is accredited by the Turkish Accreditation Agency. The number of viable bacteria was quantified by elapsed time (t=0 h, t>24 h) with the following equation being used for the calculation of antibacterial activity: where R represents antibacterial activity, A is the average number of viable bacteria immediately after inoculation on the control specimen, B is the average number of viable bacteria on the control specimen after 24 h, and C is the average number of viable bacteria on the samples (GZ-0 and GZ-25) after 24 h. Results and discussion Planetary ball mills produce high energy density via rotation of the supporting disk and rotation of the jars in counter direction in the ratio 1:2. This difference in speeds forms an interaction between frictional and impact forces that causes high dynamic energies. Due to these forces high and very effective degree of size reduction of the planetary ball mill. Therefore, it is commonly used during the particle size reduction down to the nm scale [22][23][24]. Mia et al examined the grinding rate of a gibbsite powder by using a planetary ball mill. The median diameter of the powder was 51.1 μm and at the highest speed within 5 min the particle size reduced to one tenth of the initial size [25]. This demonstrates how efficiently planetary ball mill grinds ceramic powders in a short period of time. The effect of milling time on the particle size distribution of zircon can be seen in figure 1. The median particle size of the non-ground zircon was measured as 1.65 μm (average particle size: 1.74 μm, with it decreasing to 0.45 μm (average particle size 0.48 μm) after 5 min. Since the practical grinding limit was approached after 15 min, the median particle size became constant at 0.38 μm (average particle size: 0.32 μm) for 15 and 25 min According to L * a * b * parameters (table 2), the whiteness of the glazes showed a slight increase against a reduction in the particle size of the zircon. When zircon is added directly into a glaze formulation, fine zircon particles are always favorable for high opacity and whiteness. The measured L * values for these types of glaze vary between 89-93and the results given in table 2 are compatible with previous studies [14,17]. If zircon particles are ofa particle size inthe range of 0.60-0.75 μm and an aspect ratio of around 1, the number of possible sites that diffract an incident light beam increases with higher opacity and whiteness being approached [3,17]. Schabbach et al studied the relationship between zircon particle size and glaze color. They used three different particle sizes (200, 100 mesh and micronized) of zircon opacifier at a 5 wt% amount, and found that L * values rose with decreasing particle size [16]. GZ-0 is the industrial production glaze at Kaleseramik A Ş Thanks to the increased whiteness value of zircon grain size reduction, the same whiteness value can be obtained using less zircon. Therefore, glaze cost can be reduced. The significant effect of milling can be seen from the BSE images of samples that are presented in figures 2(a)-(h). Larger, bright zircon grains (indicated by arrow) were poorly distributed in GZ-0 from figures 2(a) and (b). When milling was applied for 5 min, micron-size zircon particles still existed in microstructure as given in figures 2(c) and (d). Cluster of zircon crystals was observed for these samples. Zircon clusters are the results of the combination effects of gas evolution and liquid phase formed during sintering. In the elimination step of bubbles, they make zircon grains to move and accumulate at the grains boundaries. As gas releasing is completed, zircon crystals are not able to return their initial position [17]. After milling for 15 and 25 min, these micron-size zircon grains were almost disappeared. Also, uniform distribution of zircon was obtained for both samples as shown in figures 2(e) and (g). The finer size of zircon can be beneficial to return its original location. This uniform distribution had important contribution to improve the surface properties of sanitarywares. Some dark regions (indicated by circles) were observed around the zircon particles for all samples. Two explanations were given for the formation of these zones. The presence of residual quartz may form such regions. Wang et al showed that formation of cracks was observed around the grain boundaries when residual quartz existed due to thermal expansion differences between the phases. The other explanation on the formation of these zones was generation of bubbles. These were the regions where bubbles have been eliminated from the surface. The remaining areas were refilled with the liquid phase that was formed at the sintering temperature [17]. The average grain size of zircon for all samples was measured from processed BSE images. Representative segmented and analyzed images of GZ-25 are shown in figures 3(a), (b). The measured grain size values from GZ-0 to GZ-25 are 1.49 μm, 1.53 μm, 0.57 μm and 0.33 μm, respectively. Castilone et al identified a critical zircon amount (>3wt%) for the recrystallization of zircon grains. Recrystallization occurs via dissolution of fine zircon particles and the subsequent coarsening of undissolved zircon particles [26]. Since the amount and size of the coarser zircon particles was lower for GZ-15 and GZ-25, the final zircon grainsize lowered for both samples. As the size of zircon grains reduced, possible dissolution of these grains in the liquid phase might be observed. Blonski et al showed the relation between size and dissolution behavior of zircon opacifier and pigments. The dissolution rate was slightly higher than 2.5% when the size was over 10 μm. As size decreased to 1 μm, the increase in dissolution was 2% [kongre kitabı]. To observe dissolution behavior of zircon, the chemical composition of vitreous phase for all samples was analyzed by EDX and the results are represented between figures 4(a) and (h). Spectrum 1-4 correspond to the vitreous phases in glazed surfaces. Obtained EDX spectra and chemical analysis revealed that chemical compositions of vitreous phase were similar. The wt% of Zr varied from 1.6 to 2.1, no relation can be established between grain size and Zr content. The x-ray diffraction (XRD) analyses of samples are presented in figure 5. According to XRD result, all samples contain zircon as a single phase. It must be pointed out that the intensity of the major peak of zircon from (200) increased against decreasing in zircon grain size. This proves the effect of milling on the distribution zircon grains. Since the finer zircon resulted uniform distribution on the surface, enhanced reflections from tetragonal zircon crystals from (200) was obtained. Homogeneous distribution of the zircon grains improved the Vickers hardness of GZ-25 to 6.3 GPa from 5. be concluded that the contribution of the crystal phase on the hardness of glaze is more pronounced than for the glassy phase. Since the hardness of zircon is lower compared to ZrO 2 , the values obtained in this study are acceptable [27]. Surface images of the glazed samples from the AFM analysis are shown in figures 6(a)-(d). The presence of large zircon grains can be seen in figures 6(a) and (b). In addition, it was detected that the accumulation of these grains increased the roughness of the glaze surfaces. As milling time rose to 15 and 25 min, even the elimination of coarser zircon grains resulted in a reduction in roughness (figures 6(c) and (d)), although submicron scale roughness still appeared. As shown in table 4, the Rq and Ra values, milling provided a 28% smoother surface than non-milled. The methods to impair the bacterial activity of a material can be done either active or passive ways. Active methods kill the bacteria while passive methods inhibit bacteria attachment. In this study, second method was examined by generating a smoother surface with less topographic features. It was anticipated that this type of surface can reduce the possible sites for bacterial attachment and subsequent biofilm formation. AFM measurement of GZ-25 showed that nanometric surface topography was generated and this inhibited the attachment by reducing the contact area between bacteria cells and the surface. Hence a great diminish was obtained in the number of viable bacteria for both types of the bacteria. Table 5 shows the antibacterial test results. The number of viable E. coli bacteria for GZ-0 was 2400, it dropped to 90 for GZ-25. A rapid decrease was also observed for S.Aures, smoother surface produced around a fortyfold decrease in the number of viable bacteria. Since all the R values are lower than 2, the samples did not show any antibacterial activity. However, a significant reduction was achieved due to the presence of submicron roughness as can be seen from the AFM image ( figure 6(d)). Conclusions (1) The surface properties of opaque sanitaryware glaze are directly influenced by the initial particle size of zircon. Lower particle sizes resulted in a uniform distribution of zircon crystals on the surface and increased the whiteness (L * ) value. (2) The hardness of the glaze was directly related to the distribution of zircon particles, and a lower initial size increased the hardness of the surface. (3) The effect of milling was observed by AFM studies of the surfaces. The average roughness of GZ-0 was 39.51 nm, which decreased to 10.94 nm for GZ-25. (4) A reduction in surface roughness provided an important contribution to the bacterial activity of the glazed samples. Even though the samples did not show any antibacterial activity, the amount of viable bacteria decreased by around twofold without the use of any antibacterial compound, such as silver or TiO 2 .	2020-01-02T21:47:02.085Z	2020-01-13T00:00:00.000	{ "year": 2020, "sha1": "eb67ac4ead8b8414281dda8da719c4a4b3a8a7b2", "oa_license": "CCBY", "oa_url": "https://doi.org/10.1088/2053-1591/ab657d", "oa_status": "GOLD", "pdf_src": "IOP", "pdf_hash": "5abdfb5f2f0e98fe6c38ab8381d12570f22ac030", "s2fieldsofstudy": [ "Materials Science" ], "extfieldsofstudy": [ "Materials Science" ] }
2555633	pes2o/s2orc	v3-fos-license	Variability in Abundance of Temperate Reef Fishes Estimated by Visual Census Identifying sources of sampling variation and quantifying their magnitude is critical to the interpretation of ecological field data. Yet, most monitoring programs of reef fish populations based on underwater visual censuses (UVC) consider only a few of the factors that may influence fish counts, such as the diver or census methodology. Recent studies, however, have drawn attention to a broader range of processes that introduce variability at different temporal scales. This study analyzes the magnitude of different sources of variation in UVCs of temperate reef fishes off Patagonia (Argentina). The variability associated with time-of-day, tidal state, and time elapsed between censuses (minutes, days, weeks and months) was quantified for censuses conducted on the five most conspicuous and common species: Pinguipes brasilianus, Pseudopercis semifasciata, Sebastes oculatus, Acanthistius patachonicus and Nemadactylus bergi. Variance components corresponding to spatial heterogeneity and to the different temporal scales were estimated using nested random models. The levels of variability estimated for the different species were related to their life history attributes and behavior. Neither time-of-day nor tidal state had a significant effect on counts, except for the influence of tide on P. brasilianus. Spatial heterogeneity was the dominant source of variance in all but one species. Among the temporal scales, the intra-annual variation was the highest component for most species due to marked seasonal fluctuations in abundance, followed by the weekly and the instantaneous variation; the daily component was not significant. The variability between censuses conducted at different tidal levels and time-of-day was similar in magnitude to the instantaneous variation, reinforcing the conclusion that stochastic variation at very short time scales is non-negligible and should be taken into account in the design of monitoring programs and experiments. The present study provides baseline information to design and interpret results from visual census programs in temperate reefs. Introduction Accounting for or minimizing ''unexplained'' sampling variability represents a challenge in all fields of ecology, given its importance for correctly interpreting field data [1][2][3][4][5]. Most monitoring programs of reef fish populations aim to assess spatial and/or temporal changes in their abundance; for example, to assess the effects of fishing by comparing protected to unprotected areas. Indices of abundance of reef fishes collected through such programs are affected by multiple sources of variability, whose magnitude depends on the characteristics of the habitats and species monitored. Despite the importance of understanding the sources of variability for experimental design and data analysis, surprisingly few studies have quantified the variability associated with different temporal scales and processes, which may introduce ''noise'' in the data and potentially confound the results. Different methods are used to collect field data for estimating reef-fish abundance (e.g., gillnets, trapping, video recording and visual censuses, see [6][7][8][9][10][11][12][13]). Among them, underwater visual census (UVC) is the most commonly used in shallow waters because it offers several advantages: censuses are non-destructive sampling techniques, therefore suitable for use in marine protected areas (MPAs) and on long-lived rare species; they work for a wide range of fish sizes and behaviors; and they are cost-effective in terms of money, time and logistics, compared to video recording. In addition, divers may register several variables and gather direct observations while they count fish (e.g., Reef Life Survey Program, URL: http:\\www.reeflifesurvey.com). Drawbacks of UVC techniques, however, are biases in the estimates of abundance of cryptic species and small fishes, problems with recounting mobile individuals, lack of a permanent record, and diver avoidance of some species [10,11,[14][15][16][17]. Sources of variation in visual census datasets were classified by Thompson & Mapstone [4] in three general categories: (1) real changes in abundances due to recruitment and/or loss of individuals to populations; (2) temporary local shifts in the distribution of individuals, without net change in population size; and (3) sampling error. Sampling error is composed of systematic error (due to low detectability of some species or fishes) and random factors (e.g., counting errors), both influenced by the diver and directly related to his/her experience [14][15][16]. Detectability is defined as the probability of observing a particular species during a given sampling occasion conditional on its presence at that location [18]. UVC-based studies routinely assume that detectability equals one for all species, although in reality it may vary substantially among reef fishes and habitats in relation to behavioral and life-history characteristics. For example, estimation bias may be substantial in the case of cryptic or small species [17,19,20]. The random portion of the sampling error is composed of simple counting error plus real variation in local abundance introduced by random displacements of fish across the boundaries of the sampling units, at very short time scales (e.g., seconds to minutes). Both contribute to what McClanahan et al. [14] called ''instantaneous variation'', which they found to be a large source of variation for the coral reef fishes they surveyed, often overlooked in previous works (i.e., which attributed it to larger temporal scales). Counts of fast swimming and schooling species are particularly affected by this source of variation. Aside from diver effects, which can be readily estimated [19], the remaining sources of instantaneous variability in fish counts are difficult to discriminate as they all contribute to base sampling error [14,16,21]. At larger temporal scales, displacements in response to tidal level or time-of-day (e.g., in response to light) may result in increased variation at the scale of hours, while seasonal migrations associated with changes in water temperature or reproductive cycles would increase variation at the scale of months. The assessment of actual changes in abundance over longer time periods (often the scale of interest) resulting from trends in recruitment and mortality, or from shifts in distribution (e.g., shifts in latitudinal range associated with climate change), needs to take into account the magnitude of all sources of variability. However, most studies based on UVC data typically consider only a few such sources (e.g., diver effect, time-of-day) [22][23][24], and methods used to partition variability amongst the different components, including not only temporal but also spatial variation, may be inappropriate if they fail to account for the different sources. The investigation of sources of error and variability in UVC is a rising and fast-developing field due to its relevance for a correct interpretation of the data and for the design of appropriate monitoring programs (e.g., [4,14,16,17,[21][22][23][24][25]). The aim of this study was to assess the magnitude of temporal sources of variation in UVCs conducted on a low-diversity reeffish assemblage, typical of temperate shallow reefs in the northern Atlantic Patagonian coast. Two types of variation were considered: (1) differences in time-of-day and tidal level, treated as deterministic factors, and (2) those associated with different temporal scales, treated as stochastic factors. A unified statistical framework based on nested random-effect models was used to analyze the data, while accounting for spatial heterogeneity between reefs. The paper presents an original case study from temperate waters, which provides information relevant for survey design and interpretation of past UVC data used for monitoring temperate reef systems. Northern Patagonia rocky reefs and fishes In the study area, reefs are formed by isolated small rocky outcrops that extend for a few hundred meters on an otherwise flat, soft bottom. These reefs are mainly linear structures, typically breaks or ledges (up to 4 m high and 6 m wide) located along the edge of submerged abrasion limestone platforms, where cavities are formed. Crumbled portions of the ledges determine width and structural complexity of the reefs. The northern Patagonia reef-fish assemblage has a low species diversity: it is composed of 29 species belonging to 21 families [26], but only five of species (four families) are conspicuous and commonly found in the reefs: Pinguipes brasilianus, Pseudopercis semifasciata, Sebastes oculatus, Acanthistius patachonicus (see [27] for a taxonomic update for this species) and Nemadactylus bergi [26]. This work focuses on these most common non-cryptic species, which are easily detectable by standard strip transect visual censuses. While N. bergi is a schooling species, the rest are sedentary demersal fishes, strongly associated to refuges [26]. Study site Fish censuses were conducted at 12 shallow rocky reefs near Punta Pardelas (42.7uS 64.3uW), Nuevo Gulf, from March 2007 to February 2008. This site was selected because of the large number of reefs and good visibility (between 5 m and 10 m). Because the abundance of P. semifasciata in Pardelas was too low to provide reliable estimates of variability, data from UVCs conducted in San José Gulf (42.3uS 64.4uW) were used (see [24] for details), corresponding to three shallow rocky reefs censused between July 2002 and May 2004 (reefs B, C and E in [24]). Those reefs were similar in shape, topography and depth to the reefs from Pardelas, and the visual census technique used was comparable (see below). The reefs selected at Nuevo Gulf were small ledges (24 m to 150 m in length by up to 5 m in width), separated from each other by 50 m to 1.2 km. Reefs from San José Gulf were much farther apart (from 3.7 km to 19.6 km), up to 3 m wide, and between 52 m and 315 m in length. Reefs depth ranged between 5 m and 16 m at low tide. The tidal regime is semidiurnal and the mean tidal amplitude is 3.8 m in Nuevo Gulf and 5.7 in San Jose Gulf, but spring tides could reach 5.7 m and 8.8 m, respectively. Underwater visual census methods Censuses of the 12 reefs selected in Nuevo Gulf were all conducted by the same diver, by swimming along a fixed 25 m65 m strip transect along the reef ledge. A single transect was randomly positioned in each reef at the beginning of the study, and delimited by iron pickets driven permanently into the bottom. Three sequential passes were made of each transect, swimming at a constant speed (24 m/min), to count individuals of A. patachonius, S. oculatus and P. brasilianus, in that order, using standard methodology [4,16,24,28]. To increase the probability of detection of the schooling species N. bergi, whose counts involved at most one single school, this species was counted on each pass and the maximum number encountered recorded. At San José Gulf, the entire reefs were censused by three divers, who counted all P. semifasciata observed while swimming along the ledges. Three replicated censuses were completed on each sampling occasion, one by each diver in random order, waiting 5-10 minutes between successive censuses. No significant effects of order of census (i.e., diver disturbance) nor diver identity were detected when those effects were evaluated as fixed factors using these data [24]. Because fish are only present on a narrow band (generally less than 3 m wide) along the reef ledges, and given the good visibility conditions in the area, divers were able to survey the whole width of the reefs. Divers swam continuously, only counting fishes larger than 10 cm of total length in order to avoid possible biases associated with small size categories (e.g., [4,17]). They also avoided counting fish that appeared from behind. Sampling design The reefs were censused at controlled tidal levels (low vs. high tide) and times of day (morning vs. afternoon), and at different time periods (from minutes to months), in order to quantify the variability in fish counts associated with the different factors and temporal scales. The censuses conducted in Nuevo Gulf to evaluate short-term effects were concentrated in the fall, when water temperature reaches a maximum of ,17uC (minimum temperature occurs in August and is approximately 9uC). In order to evaluate the effects of tidal state and time-of-day on fish counts, sampling dates and times were selected so that each factor was allowed to vary one at a time while keeping the other factor constant (for further details see Text S1). These deterministic factors were evaluated separately and prior to the onset of the remaining temporal censuses; this approach was similar to that used by Thompson & Mapstone [4]. Once the effect of time-ofday was found to be non-significant for all species (see Results), the effect of tidal state was evaluated by surveying all transects at low and high tides during the same day. Tidal amplitude on the sampling day (5 May 2007) was 4.3 m, whilst it ranged between 3 m and 4.5 m over the study period. To estimate instantaneous variation, three replicated counts were made for each species on each of the 12 transects, waiting 5 minutes between successive series (each series consisted of three sequential passes, one per species as explained earlier). These censuses were done on 4 April 2007 (transects 1 to 9) and on 4 May 2007 (transects 10 to 12) (Fig. 1). To estimate day-to-day (i.e., daily) variation in fish counts, all transects were surveyed once a day during three consecutive days (between 0900 hrs and 1300 hrs), on 19-21 March 2007. The initial date was selected opportunistically depending on logistics and weather conditions, and the initial census was delayed ,45 min per day in order for the tidal height to remain approximately constant. Variability at the scale of weeks was assessed based on censuses conducted on 19-21 March 2007, 29 March 2007, and 4 April 2007 (Fig. 1). Finally, to estimate intra-annual variation, all transects were surveyed on an approximately monthly basis (n = 9), between 0900 hrs and 1300 hrs, from March 2007 to February 2008, covering an annual cycle. Sampling dates did not follow a strictly regular schedule, but were dependent on weather; thus tidal height varied among censuses. Censuses of P. semifasciata in San José Gulf were conducted over a two-year period to investigate the species' temporal pattern of reef occupancy [24]; hence that data set only allowed assessment of instantaneous and intra-annual variability in counts, but not the effects of tidal height, time-of-day, and daily and weekly variation (Fig. 1). These latter sources of variability therefore contribute to the intra-annual variability estimated. Statistical analysis The variability of fish counts associated with different factors and temporal scales was estimated separately for each species using log-linear mixed and random-effect models. The models were fitted by restricted maximum likelihood (REML) using the lme4 and nlme libraries [29,30] implemented in the R software [31]. REML was preferred to maximum likelihood (ML) because it compensates for the downward bias of the ML estimates of variance components [29,32] and it is the recommended methodology for estimating variance components in unbalanced designs [32,33]. In all cases (mixed-and random-effect models), the transect identity was treated as a random effect in order to account for variation associated with spatial heterogeneity, i.e., heterogeneity among the experimental units given by reef depth, reef geomorphology, bottom type, availability of refuges, etc. Time-of-day and tidal state were evaluated as fixed factors, with levels: morning and afternoon, and low and high tide, respectively. Their significance was assessed by a t-test, conditional on the estimates of the random effects variance parameters [29]. In order to quantify the variability contributed by the different temporal scales, a nested random-effect model was fitted, which included day, week and month as random factors, all nested within transect (see Text S2). All random effects were assumed to be normally distributed with mean zero and variances s 2 x , where x represents a given random effect. The variance estimated for each of the random effects (spatial and temporal) measures the contribution of each source to the total variability. The residual error was interpreted as the base sampling error or instantaneous variability. The percent contribution of each source to the total variance was calculated from the ratio of each variance to the sum of all variance components. The significance of each random component was evaluated by likelihood ratio tests [29], comparing the full model with one in which the given component had been dropped, one at a time. Additionally, the nested model for P. semifasciata included year as a random factor given that the data were collected over a two-year period. Note that the standard deviation (s) estimated with log-transformed data is close to the coefficient of variation (CV) in the natural scale, as The CVs corresponding to variation due to spatial heterogeneity among reefs, CV SPAT , and temporal variation added at increasing temporal scales,CV TEMP (TEMP: instantaneous, day, week, month) were calculated from the respective estimated variances. Confidence bounds (95%) for each standard deviation were calculated using the function intervals of the nlme library. Seasonal patterns of abundance for each species were examined by plotting the monthly average (over reefs) best linear unbiased predictions (BLUPs) [29] corresponding to the intra-annual effects. In addition to the nested model above, separate models were fitted to subsets of the data corresponding to each temporal scale (e.g., only the instantaneous or the daily replicates), to evaluate the sensitivity of the estimates of spatial variation. Fish counts were log-transformed to obtain additive effects. Due to the absence of some species in a few sampling occasions, we added a constant ( = 1) to all raw fish counts [ln(y+1)], except in the case of P. semifasciata, whose counts were always positive. In the latter, counts were standardized as number of individuals per 25 m of transect length, because the three reefs surveyed had different lengths. We excluded a few reefs considered as marginal nonrepresentative habitat in the cases of S. oculatus and N. bergi, where the mean abundance over all censuses was less than one fish per transect. Coefficient of variation vs. mean counts The relationship between the empirical CVs and mean fish counts was examined for the instantaneous, daily, weekly and monthly scales. Percent CVs were calculated from the raw counts for each species and transect, by selecting the censuses replicated at each temporal scale. Diurnal and tidal variation Fish abundance did not vary significantly with time-of-day or tidal state; the inclusion of the fixed effect in the models did not reduce the residual variation significantly ( Table 1). The exception was P. brasilianus, for which counts were significantly higher at low tides (p = 0.034) ( Table 1). This effect, however, was not consistent across reefs: while in eight transects abundance was a 44% higher on average at low tide, in the remaining four it was stable or decreased slightly (mean = 28%). These differences were unrelated to the depth of the reefs. Temporal variation Daily and weekly components of variability in fish counts were in general of similar magnitude to the instantaneous variation. A remarkably low daily variation (with broad 95% confidence bounds) was estimated for N. bergi and P. brasilianus (Fig. 2). In all cases, the contribution of the daily component to the overall variability was not statistically significant, while that of the weekly scale was significant for A. patachonicus, S. oculatus and P. brasilianus ( Table 2). The intra-annual component showed the highest CV for most species, indicating strong seasonal patterns in fish abundance; the contribution of the intra-annual variation was statistically significant, except for S. oculatus and P. semifasciata, whose densities remained rather stable throughout the year ( The temporal variation in counts of A. patachonicus and P. brasilianus was relatively small at the instantaneous, daily and weekly scales, with respective CV TEMP of 0.11, 0.09 and 0.18 in A. patachonicus, and 0.19, 0.08 and 0.22 in P. brasilianus, much lower than those of the intra-annual component (CV month of 0.56 and 0.66 for the two species). The total contribution of the short time scales (i.e., instantaneous, daily and weekly) to the overall variability was 5.6% in A. patachonicus and 12.8% in P. brasilianus, compared to 34.1% and 60.3% due to the intra-annual component. The temporal variation in S. oculatus counts was relatively small at all time scales with the exception of the weekly scale, which had the largest contribution (CV week = 0.41, compared to CV instantaneous~0 .18, CV day~0 .13 and CV month~0 .15). The contribution of the monthly scale was not significant (Table 2), although shallower reefs presented a slight seasonal pattern, with a winter drop in abundance. Similar to S. oculatus, repeated and intra-annual counts of P. semifasciata had relatively low and similar CVs (CV instantaneous~0 .16 and CV month~0 .18) ( Temporal variation of N. bergi counts was higher than that observed for sedentary species, although the daily and weekly contributions were not statistically significant ( Table 2; Fig. 2). Fish counts were dominated by a strong seasonal component: this species was virtually absent in winter and spring (Table 2; Fig. 3). Spatial heterogeneity When separate models were fitted to subsets of the data corresponding to each temporal scale (e.g., only the instantaneous or the daily replicates), the estimated spatial variation in fish counts was similar across temporal scales and factors analyzed, for all the species studied. This supported the assumption made in the nested model that the spatial variability was time-invariant. In the case of N. bergi, however, theCV SPAT estimated from the mixed ( Table 1) and random models ( Table 2) are not directly comparable because different transects were used for model fitting. Sebastes oculatus showed the largest spatial variability (CV SPAT between 1.08 and 2.47) (Tables 1 and 2; Fig. 4), with fish counts ranging from 0 to up to ,200 individuals per transect. In both S. oculatus and A. patachonicus spatial variability was larger than temporal variability, amounting to 94% and 60% of the total variance, respectively. On the other extreme, P. semifasciata and P. brasilianus showed the smallest spatial variation; their counts per 25 m of linear reef ledge had the narrowest range: between 1 and 5 for P. semifasciata, and between 5 and 45 for P. brasilianus (CV SPAT equal to 0.10 and 0.44, respectively) (Fig. 4). Coefficient of variation vs. mean The CVs of the raw fish counts for sedentary species were inversely related to their mean abundance (Fig. 5). Although there were several cases in which both were low, high mean abundance always had low CVs. This relationship was consistent for all the temporal scales considered: all showed a similar pattern while the level of variability increased with time elapsed between censuses. Nemadactylus bergi was excluded because its much larger CVs and few records did not allow detecting any clear pattern. Discussion Our results showed that instantaneous variation represents a non-negligible (.10%) basal level of variability that should be taken into account in the design of monitoring programs, or when interpreting past visual census estimates of reef fish abundance in northern Patagonia. McClanahan et al. [14] drew attention to this important and often ignored source of variation, which can lead to difficulties in detecting differences over time and between sampling methods or sites (e.g., in response to management regulations). In their case, the estimates of instantaneous variation could be somewhat inflated by the inclusion of between-diver differences. The most relevant feature of our approach is that it allowed a direct comparison between the magnitude of the different sources of variability -both spatial and temporal-that affected fish counts, by incorporating them into random-effect models. An advantage of this method is that it can be applied to unbalanced designs such as the one used in this study. This is not the case of standard methods used for variance decomposition based on analysis of variance, which do require that the sampling design be balanced [3,32,33]. Diurnal and tidal variation In our comparisons, fish counts did not differ significantly between morning and afternoon, similarly to other studies [4,16,34,35]. Nor could we detect an effect of tidal state for most species, a result that is also consistent with previous works [34,36]. The only exception was P. brasilianus, which was more abundant at low tide in some of the reefs (Table 1). This species is the only among the species studied that occupies the intertidal zone during high tide (AJI, personal observation), suggesting that some fish could move between subtidal and intertidal rocky areas following the tidal cycle, and that fish abundance may increase in subtidal shallow reefs during the low tide. Most previous studies did not consider tidal state explicitly in their sampling designs, thus any potential effects of tide may add ''noise'' or be confounded with those of other factors. Although we did not find a strong influence of tide on our comparisons, its effects were evaluated based on censuses made on a single day; therefore possible interactions with other factors (e.g., season, tidal amplitude, spawning activity) could not be evaluated. This limitation is particularly relevant because tide is a complex factor, correlated with other variables such as current speed and direction, depth and availability of suitable intertidal habitats. Furthermore, as current speeds are highly influenced by bottom topography, the extrapolation of our results to other sites in relation to the effect of tide would not be appropriate. Fish movements in response to currents are well known by fishers, and they were demonstrated for other reef species [37]. Temporal variation in fish counts Compared to UVCs conducted in warmer regions, the variability in fish counts estimated in this study was low for sedentary species, but similar for schooling species [4,14,17,38]. Among time scales shorter than a year, the contribution of the Fixed-effect coefficients for levels afternoon and high-tide, and their significance (in parenthesis), and standard deviation (SD) of spatial and residual components of variability. The number of replicates (i.e., number of reefs censused) for each data set is indicated in the ''Transects'' column. Significance is indicated as: ns = not significant (p.0.05), * = significant (p,0.05), ** = highly significant (p,0.01). Spatial variation was highly significant in all cases. doi:10.1371/journal.pone.0061072.t001 instantaneous variation was in general similar to that of the other short temporal scales (i.e., daily and weekly). Variability in counts between low and high tides, and between morning and afternoon, was similar in magnitude to the instantaneous variation (i.e., the residual error in the nested random models), reinforcing the conclusion that differences in counts could be due to instantaneous stochastic variation, similar to the results reported by McClanahan et al. [14]. In the case of P. semifasciata, the repeated counts were conducted by different divers, which may have inflated the estimates of instantaneous variation, even though the diver effect was found to be non significant [24]. Similarly, as tidal state was not controlled in the sampling design of the temporal censuses, it may have contributed to the variability estimated for P. brasilianus. Except for P. semifasciata and S. oculatus, the other species analyzed (A. patachonicus, P. brasilianus and N. bergi) showed high intra-annual variation associated with a strong seasonal pattern (Fig. 3). Abundance trends for these species paralleled the annual cycle of water temperature in Nuevo Gulf: fish abundance increased slowly during spring and summer, peaked in autumn when water temperature is maximum (17uC in March-April), and diminished gradually towards late winter, when the water is coldest (9uC in August-September). This trend was much more marked for N. bergi, which was virtually absent during winter (for more details on the seasonal patterns of abundance see [24,39]). The levels of variability estimated for the different species were consistent with their life history attributes and behavior. The instantaneous variability was smallest for A. patachonicus, followed by P. semifasciata, S. oculatus, P. brasilianus and N. bergi, in that order. This ranking can be mostly explained in terms of degree of siteattachment and swimming speed. Acanthistius patachonicus, for example, is a sedentary species commonly observed in close proximity (within 5 m) to the refuge [40], which facilitates censusing. This makes A. patachonicus a reliable candidate for assessing the status of the reefs and the impact of angling and spear-fishing, or even for detecting small changes in abundance. Pinguipes brasilianus and P. semifasciata are more mobile than A. patachonicus. While these species can be observed resting on the bottom on their pelvic fins, in general they swim in and out of the refuges and of the area censused. For example, one individual of P. semifasciata was spotted in places distant up to 46 m along a reef ledge over a 1-hour period (although most, i.e., 30 of 35 fish, remained within 13 m) (LAV, unpublished data), and it is common to observe fish more than 20 m far from the reefs feeding on soft bottoms (AJI, personal observations). The schooling species N. bergi had the highest variability at all temporal and spatial scales due to high mobility and variation in group size. Similar patterns have been found for others schooling species (e.g., Lutjanidae, Acanthuridae and Scaridae families [4,14,25]). When counting these species, observation events regularly record fish schools as a unit, whilst in sedentary species they record individual fish. This explains the high variability observed, as the presence or absence of a school could easily result in counts changing from 0 to 100 individuals. The information provided by visual censuses on schooling species would be limited to detecting their presence and seasonal trends in the reefs. Standard deviations (SDs) of spatial and temporal components of variability, and their percent contribution (%VC) to total variance for each species. The number of replicates (i.e., number of reefs censused) for each species is indicated in the ''Transects'' column. The significance of the variance components is indicated in the column denoted with ''p'': ns = not significant (p.0.05), * = significant (p,0.05), = highly significant (p,0.01), * = very highly significant (p,0.001). The instantaneous variation was estimated as the residual error of the nested random models; therefore its significance was not evaluated. doi:10.1371/journal.pone.0061072.t002 Spatial variation The design of impact studies should address the spatial heterogeneity in order to avoid confounding its effects with the factors under investigation [3]. Our results show that patterns of spatial heterogeneity differed among species, a result of the complex interactions between morphological, physical and ecological characteristics of the reefs, and the particular microhabitat and food requirements of each of the species studied. Amongst the sedentary species, S. oculatus showed the highest spatial variation due to marked contrasts in abundance between shallow and deep reefs. Conversely, P. brasilianus, a pinguipedid with an apparently weak microhabitat association [39], had low spatial variation (Fig. 2). In the case of P. semifasciata, reefs with high fish abundance were intentionally selected to investigate seasonal patterns [24]; hence their spatial variation (the lowest estimated) is not representative of the variability corresponding to a random collection of reefs. Overall, spatial heterogeneity for each species was consistent across the different temporal scales and/or factors analyzed; thus environmental differences between reefs could be considered stable between seasons. Implications for design of monitoring programs The instantaneous variation, understood as baseline variation or error in fish counts, tends to obscure relevant patterns at larger temporal and/or spatial scales. Its magnitude sets the minimum variance to be used in power analyses to calculate minimum sample sizes required for detecting any given change in abundance. Given that instantaneous variation and detectability are strongly related to swimming behaviour, shape and size ( [4,9,[14][15][16] and our results), and are independent of spatial heterogeneity [16], it is possible to use published results for other systems and similar species to explore power and sample size requirements. In the case of schooling species, for example, only large effects could be detected given the high variability of UVC counts at all scales. In general, the power of UVC experiments may be increased by augmenting either the number of replicates or the length of the transects. The level of sampling effort would of course depend on a trade-off between the desired precision and logistics and monetary constrains. Increasing the number of replicates per transect would increase the statistical power by reducing the variance of the average in proportion to 1/n. Faster reductions may be possible by increasing the length of the transects in cases in which instantaneous variability is caused not so much by counting errors but by fish moving along a reef ledge, in and out of the sampled portion. This is the case of P. semifasciata and P. brasilianus. In such cases, the best alternative, for any given total distance covered, would be to use longer transects rather than to replicate short transects. Overall, the choice between increasing the number of replicates or the length of the transects should depend on the size and shape of the fishes' home ranges, and on their degree of association with specific portions of the reefs. Longer transects would be required in studies focused on scarce or rare species, given the inverse relationship between variability and mean fish counts found for all sedentary species at all temporal scales (Fig. 5). This relationship is consistent with previous studies [4,14]. Some studies have found that sampling sites and instantaneous variation in counts made by a single diver were more important sources of variation than diver identity [14][15][16]. On this basis, the authors proposed that a better choice to increase power would be to increase sampling effort using the same group of divers rather than spending valuable water time to undertake prolonged diver retraining [16]. Diver inter-calibration and training would become important for longer-term studies in which divers change over time [16]. When the focus is on temporal comparisons, our results, in concordance with those of Thompson & Mapstone [4], suggest that the use of fixed transects is a good strategy to increase power, and to remove the spatial heterogeneity by treating the sampling units as a random effect. In most cases, high between-site variability could obscure most temporal patterns if not accounted for. Additionally, for inter-annual comparisons or in short timescale experiments, UVCs should be done preferentially during periods of highest abundance, when CVs are lower. Regarding diurnal variation, our results agree with the recommendation of Thompson and Mapstone [4], that sampling during the middle of the day (between 2 to 3 h after sunrise and 2 to 3 h before sunset) is a good strategy to minimize possible timeof-day effects. It is noteworthy that time-of-day effects are generally associated to crepuscular periods [41,42], not investigated in this study. Finally, tidal state could not be discarded as a factor that may affect abundance estimates; fixing the tidal state on sampling designs or evaluating the tidal effects on experiments or monitoring programs appears to be necessary. Supporting Information Text S1 Details of sampling design. Dates and sampling hours for time-of-day and tide experiments. (DOC) Text S2 Statistical models. Hierarchical random-effect model used to estimate variance components for each species. (DOC)	2016-05-12T22:15:10.714Z	2013-04-04T00:00:00.000	{ "year": 2013, "sha1": "12ab25c35f2a7fe4f4e2cbe41fa3486efa3bd55a", "oa_license": "CCBY", "oa_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0061072&type=printable", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "12ab25c35f2a7fe4f4e2cbe41fa3486efa3bd55a", "s2fieldsofstudy": [ "Environmental Science" ], "extfieldsofstudy": [ "Geography", "Medicine" ] }
210164898	pes2o/s2orc	v3-fos-license	$R$ matrix for generalized quantum group of type $A$ The generalized quantum group $\mathcal{U}(\epsilon)$ of type $A$ is an affine analogue of quantum group associated to a general linear Lie superalgebra $\mathfrak{gl}_{M\|N}$. We prove that there exists a unique $R$ matrix on tensor product of fundamental type representations of $\mathcal{U}(\epsilon)$ for arbitrary parameter sequence $\epsilon$ corresponding to a non-conjugate Borel subalgebra of $\mathfrak{gl}_{M\|N}$. We give an explicit description of its spectral decomposition, and then as an application, construct a family of finite-dimensional irreducible $\mathcal{U}(\epsilon)$-modules which have subspaces isomorphic to the Kirillov-Reshetikhin modules of usual affine type $A_{M-1}^{(1)}$ or $A_{N-1}^{(1)}$. Introduction A generalized quantum group U(ǫ) associated to ǫ = (ǫ 1 , . . . , ǫ n ) with ǫ i ∈ {0, 1} is a Hopf algebra introduced in [17], which appears in the study of solutions to the tetrahedron equation or the three-dimensional Yang-Baxter equation. The generalized quantum group U(ǫ) of type A is equal to the usual quantum affine algebra of type A (1) n−1 , when ǫ is homogeneous, that is, ǫ i = ǫ j for all i = j. But it becomes a more interesting object when ǫ is non-homogeneous, which is closely related to the quantized enveloping algebra associated to an affine Lie superalgebra [22], or which can be viewed as an affine analogue of the quantized enveloping algebra of the general linear Lie superalgebra gl M\|N [21], where M and N are the numbers of 0 and 1 in ǫ, respectively. We remark that the subalgebraŮ(ǫ) of U(ǫ) associated to the Lie superalgebra gl M\|N was also introduced in [7] independently, as symmetries appearing in the study of wave functions of quantum mechanical systems [23]. When the parameter ǫ is standard, that is, ǫ M\|N = (0 M , 1 N ), it is shown in [17] that there exists a unique R matrix on the tensor product of finite-dimensional U(ǫ M\|N )-modules W s,ǫ (x), which correspond to fundamental representations of type A (1) N −1 with spectral parameter x when N ≥ 3. Indeed, the R matrix is obtained by reducing the solution of the tetrahedron equation, and the uniqueness follows from the irreducibility of tensor product W l,ǫ (x) ⊗ W m,ǫ (y) for generic x and y. An explicit spectral decomposition of the associated R matrix is obtained by analyzing the maximal vectors with respect toŮ(ǫ M\|N ). By applying fusion construction using the R matrix in [17], a family of irreducible U(ǫ M\|N )-modules is constructed in [16], which are parametrized by rectangular partitions inside an (M \|N )-hook. Moreover the existence of their crystal base is proved together with a combinatorial description of the associated crystal graphs. It can be viewed as a natural super-analogue of Kirillov-Reshetikhin modules (simply KR modules) of type A (1) ℓ , which is a most important family of finite-dimensional irreducible modules of quantum affine algebras (cf. [5,14]). The results in [17] and [16] suggests that there is a close connection between finitedimensional representations of U(ǫ M\|N ) and U q (A (1) n−1 ). The purpose of this paper is to extend the results in [17] and [16] to arbitrary parameter sequence ǫ, and find a more concrete connection between the finite-dimensional representations of U(ǫ) and U q (A (1) ℓ ). From a viewpoint of representations of gl M\|N , the sequence ǫ represents the type of Borel subalgebras of gl M\|N , which are not conjugate to each other. It is not obvious whether the representation theory of U(ǫ) is the same under a different choice of permutations of ǫ M\|N . For example, if we change the Borel in the generalized quantum group, then the defining relations and the crystal structure associated to U(ǫ)-modules become much different from the ones with respect to ǫ M\|N as ǫ gets far from ǫ M\|N (cf. [2,15]) . We first show that there exists a unique R matrix on the tensor product of finitedimensional U(ǫ)-modules W s,ǫ (x) of fundamental type (Theorem 4.10). Since the existence of R matrix for arbitrary ǫ was shown in [17], it suffices to prove the irreducibility of tensor product W l,ǫ (x) ⊗ W m,ǫ (y) for generic x and y. We use a method completely different from [17]. Indeed, motivated by the work [6], we introduce a functor called truncation, and show that it sends any U(ǫ)-module with polynomial weights to a U(ǫ ′ )-module, preserving the comultiplications in tensor product, where ǫ ′ is a subsequence of ǫ. This in particular enables us to define an oriented graph structure on W l,ǫ (x) ⊗ W m,ǫ (y) when x = y = 1 with additional arrows other than the ones associated to U(ǫ). With this structure, we prove the connectedness of the crystal (Theorem 4.8), and hence the irreducibility for generic x and y. Next, we prove that the truncation functor is compatible with the R matrix. This immediately implies that the spectral decomposition of the R matrix for U(ǫ) is the same as that of type A (1) ℓ (Theorem 5.2) and hence does not depend on the choice of ǫ. As an application, we construct a family of irreducible U(ǫ)-modules W (r) s,ǫ which yields the usual KR modules under truncation (Theorem 5.3). We conjecture that W (r) s,ǫ has a crystal base as in the case of ǫ = ǫ M\|N . We expect that the compatibility of truncation with the R matrix will also play a crucial role in understanding arbitrary finite-dimensional U(ǫ)-modules in connection with those of type A (1) ℓ . There are other recent works on the finite-dimensional representations of quantum affine superalgebra associated to gl M\|N [24,25,26]. It would be interesting to compare with these results. The paper is organized as follows. In Section 2, we review basic materials for a generalized quantum group and its crystal base. In Section 3, we present the classical Schur-Weyl duality forŮ(ǫ) and then realize the irreducible polynomial representation ofŮ (ǫ). In Section 4, we prove the main theorem on the existence of the R matrix. In Section 5, we construct KR type modules of U(ǫ) using the R matrix. Acknowledgement The authors would like to thank Euiyong Park and Masato Okado for helpful discussions and thank Shin-Myung Lee for careful reading of the manuscript and comments. Let P = i∈I Zδ i be the free abelian group generated by δ i with a symmetric bilinear form ( · \| · ) given by (δ i \|δ j ) = (−1) ǫi δ ij for i, j ∈ I. Let { δ ∨ i \| i ∈ I } ⊂ P ∨ := Hom Z (P, Z) be the dual basis such that δ i , δ ∨ j = δ ij for i, j ∈ I. Let I = { 0, 1, . . . , n − 1 } and Throughout the paper, we understand the subscript i ∈ I modulo n. When ǫ = ǫ M\|N , the Dynkin diagram associated to the Cartan matrix ( α j , α ∨ i ) 0≤i,j≤n is where denotes an isotropic simple root. Let q be an indeterminate. We put I even = { i ∈ I \| (α i \|α i ) = ±2 } and I odd = { i ∈ I \| (α i \|α i ) = 0 }, and set Definition 2.1. We define U(ǫ) to be the associative Q(q)-algebra with 1 generated by q h , e i , f i for h ∈ P ∨ and i ∈ I satisfying , and the Serre-type relations (i ∈ I even and \|i − j\| = 1), (2.5) and (2.6) e i e i−1 e i e i+1 − e i e i+1 e i e i−1 + e i+1 e i e i−1 e i −e i−1 e i e i+1 e i + (−1) ǫi [2]e i e i−1 e i+1 e i = 0, We call U(ǫ) the generalized quantum group of affine type A associated to ǫ (see [17]). . There is a Hopf algebra structure on U(ǫ), where the comultiplication ∆, the antipode S, and the couint ε are given by for h ∈ P ∨ and i ∈ I. Let η be the anti-automorphism on U(ǫ) defined by for h ∈ P ∨ and i ∈ I. It satisfies η 2 = id and We have an isomorphism between U(ǫ) and U( ǫ) where ǫ is obtained from ǫ by permutation of ǫ i 's, which is not an isomorphism of Hopf algebras [20, Theorem 2.7] (cf. [19, 37.1]). . . , ǫ n ) be the sequence given by exchanging ǫ i and ǫ i+1 in ǫ. Then there exists an isomorphism of algebras τ i : U(ǫ) −→ U( ǫ) given by where the inverse map is given by be the µ-weight space of V . For a non-zero vector u ∈ V µ , we denote by wt(u) = µ the weight of u. Let P ≥0 = i∈I Z ≥0 δ i and let O ≥0 be the category of U(ǫ)-modules with objects V such that which is closed under taking submodules, quotients and tensor products. There is another comultiplication on U(ǫ) given by (while ∆ op + is used in [16]). Let ⊗ and ⊗ + denote the tensor product with respect to ∆ and ∆ + , respectively. For U(ǫ)-modules M and N , we have a U(ǫ)-linear isomorphism ψ : M ⊗ N −→ M ⊗ + N given by for u ∈ M µ and v ∈ N ν with µ = i µ i δ i and ν = i ν i δ i . Let us recall the notion of crystal base for V ∈ O ≥0 [16] (cf. [2]). The Kashiwara operators e i andf i on V for i ∈ I are defined as follows. Suppose that u ∈ V µ is given. Case 1. Suppose that i ∈ I odd and (ǫ i , ǫ i+1 ) = (0, 1). We definẽ Suppose that i ∈ I odd and (ǫ i , ǫ i+1 ) = (1, 0). We definẽ So we defineẽ i andf i on V to be the usual Kashiwara operators on the lower crystal base of U q (sl 2 )-module induced from ζ. In other words, if u = k≥0 f ! and e i u k = 0 for k ≥ 0, then we definẽ Case 4. Suppose that i ∈ I even and (ǫ i , ǫ i+1 ) = (1, 1). Let ξ : U q (sl 2 ) → U(ǫ) i be the Q(q)-algebra homomorphism given by ξ(e) = −e i , ξ(f ) = f i and ξ(k) = k −1 i . Then the induced comultiplication ∆ ξ on U q (sl 2 ) is So we defineẽ i andf i on V to be the Kashiwara operators on the upper crystal base of Let A 0 be the subring of Q(q) consisting of f (q)/g(q) with f (q), g(q) ∈ Q[q] and g(0) = 0. (1) L is an A 0 -lattice of V and L = µ∈P ≥0 L µ , where L µ = L ∩ V µ , (2) B is a signed basis of L/qL, that is B = B ∪ −B where B is a Q-basis of L/qL, Let us call B/{±1} a crystal of V , which is an I-colored oriented graph. We have a tensor product rule for crystals (see [2] and [16,Proposition 3.4]). Proof. The proof is almost the same as in [16,Proposition 3.4], where the order of tensor product is reversed due to a different convention of comultiplication. The crystal structure on B ⊗ℓ /{±1} for ℓ ≥ 1 can be described explicitly by Proposition 2.5, which is the same as in [2] or [16] except that the tensor product order is reversed. 3. Schur-Weyl duality and polynomial representations ofŮ(ǫ) satisfying the Yang-Baxter equation; where R ij denotes the map acting as R on the i-th and the j-th component and the identity elsewhere on V ⊗3 (cf. [11]). Let H ℓ (q −2 ) be the Iwahori-Hecke algebra of type A over Q(q) generated by h i for i ∈ {1, . . . , ℓ − 1} subject to the relations; acts as R on the i-th and (i + 1)-th component and the identity elsewhere. Then we have an analogue of Schur-Weyl duality forŮ(ǫ) (cf. [11]) as follows. The proof is similar to the case when ǫ i = 0 for all i. Polynomial representations ofŮ(ǫ). Recall that M is the number of i's with ǫ i = 0 and N is the number of i's with ǫ i = 1 in ǫ. Let P be the set of all partitions. [4]). We denote the set of all (M \|N )-hook partitions by P M\|N . For a Young diagram λ, a tableau T obtained by filling λ with letters in I is called semistandard if (1) the letters in each row (resp. column) are weakly increasing from left to right (resp. from top to bottom), (2) the letters in I 0 (resp. I 1 ) are strictly increasing in each column (resp. row). Let SST ǫ (λ) be the set of all semistandard tableaux of shape λ. Then SST ǫ (λ) is non-empty if and only if λ ∈ P M\|N . For T ∈ SST ǫ (λ), let w(T ) be the word given by reading the entries in T column by column from left to right, and from bottom to top in each column. For We fix ℓ ≥ 2, and let W denote the symmetric group on {1, . . . , ℓ}. Suppose that λ ∈ P is given with i≥1 λ i = ℓ. Let T λ + be the standard tableau obtained by filling λ with {1, . . . , ℓ} row by row from top to bottom and from left to right in each row, and let T λ − be the tableau obtained by filling λ with {1, . . . , ℓ} column by column from left to right and from top to bottom in each column. Let is the tableau obtained by acting w λ on the letters in T λ − . Let W λ + (resp. W λ − ) be the Young subgroup of W stabilizing the rows (resp. columns) of T λ + (resp. T λ − ). Then the q-deformed Young symmetrizer is given by . Suppose that a is a letter in T λ − such that a + 1 is located in the same column. We put C a = 1 + h a . Then we have Next, suppose that a is a letter in T λ − , where there is another letter d to the right. Let b be the letter at the bottom of column where a is placed, and c = b + 1 the letter at the top of the column where d is placed. Let G λ a be the set of minimal length right coset representatives of W ab × W cd in W ad . We define the Garnir element at a to be The collection of boxes in the Young diagram λ corresponding to the letters from a to d in T λ − is called a Garnir belt at a. Then we have the following relations [3, (15)]; Let T be a tableau of shape λ with letters in I, and let T (i) be the letter in T at the position corresponding to i in For σ ∈ W , let T σ be the tableau given by replacing Let a be a letter in T λ − with d to the right in the same row and with b, c as above. Let w 0 be the longest element in W ab × W cd , and letG λ a = w 0 G λ a w 0 . Let u 1 , . . . , u s and u s+1 , . . . , u r+s be the letters in T corresponding to c, . . . , d and a, . . . , b in T λ − , respectively. Then we may identify σ ∈G λ a with a permutation on {1, . . . , r + s} satisfying σ(1) < · · · < σ(s) and σ(s + 1) < · · · < σ(s + r) so that T σ is the tableau obtained from T by replacing u i 's with u σ(i) 's for 1 ≤ i ≤ r + s. With this identification, we letl(σ) be the length of σ as a permutation on {1, . . . , r + s}, and put where σ is the permutation on {1, . . . , r + s} corresponding to w 0 ww 0 and Hence it follows from (3.5) and that (3.6) We have For σ ∈G λ a , let U σ be the subtableau of T σ corresponding to the Garnir belt at a, where U = U id . We define d a (T σ ) in the same way as in d(T ) only by using the letters in U σ . Let l p > · · · > l 1 ≥ r q > · · · > r 1 be the distinct letters appearing in U , where l i and r j are located in the left and right columns of U , respectively. Let m i (resp. n j ) be the number of occurrences of l i 's (resp. r j 's) in U , which remain in the same column after applying σ. Let m ′ i (resp. n ′ i ) be the number of l i 's (resp. r j 's) which are placed on the right (resp. left) column of U σ after applying σ to U . Note that one can check easily that By (3.8), (3.9), and (3.10), we have By (3.3), (3.7) and (3.11), we have This proves the identity in the lemma. Case 2. Suppose that Suppose that l 1 = r q . In this case, d a (T ) is the same as in Case 1, and Note that where the last summand is equal to m ′ p n ′ 1 . By similar arguments as in (3.10), we have This also proves the identity in the lemma as in (3.11). (2) Suppose that we have filled a subdiagram of λ from 1 to i. Then fill the first row (resp. column) of the remaining diagram with i + 1 if ǫ i+1 = 0 (resp. ǫ i+1 = 1). ( Proof. (1) It is clear that V ǫ (λ) is invariant under q h for h ∈ P ∨ . It suffices to check f i V ǫ (λ) ⊂ V ǫ (λ) for i ∈I since the proof for e i is the same. The proof is similar to the case when ǫ = (0, . . . , 0) (cf. [9]). For column-semistandard tableaux U and V of shape λ, where we may assume that T is column-semistandard by (3.3). If such T ′ is not semistandard, then we may apply Lemma 3.2 to T ′ so that v T ′ is a linear combination of T ′′ 's which is column-semistandard and T ′ < T ′′ . Repeating this process finitely many times, we conclude that f i T is a linear combination of v S 's for some S ∈ SST ǫ (λ). Therefore, (2) Since V ǫ (λ) = Y λ (q)V ⊗ℓ and Y λ (q) is a primitive idempotent up to scalar multiplication [10], it follows from Theorem 3.1 that V ǫ (λ) is an irreducibleŮ(ǫ)-module. Recall that the dimension of the irreducible H ℓ (q −2 )-module S λ generated by Y λ (q) is the number of standard tableaux of shape λ. We may have an analogue of the Robinson-Schensted type correspondence, which is a bijection from the set of words of length ℓ with letters in I to the set of pair of standard tableau and semistandard tableau of shape λ (cf. [4]). Comparing the dimensions of V ⊗ℓ and its decomposition into (3) The character of V ǫ (λ) is equal to that of polynomial representations of the general linear Lie superalgebra gl M\|N corresponding to λ ∈ P M\|N , and wt(v H λ ) is maximal [8,Theorem 2.55]. This implies that e i v H λ = 0 for all i ∈I and hence v H λ is a highest weight vector. Remark 3.5. The character of V ǫ (λ) for λ ∈ P M\|N is called a hook Schur polynomial [4], which depends only on ǫ up to permutations. The tensor product of two polynomial representations is completely reducible and the multiplicity of each irreducible component is given by usual Littlewood-Richardson coefficient. Proof. The proof is similar to that of [16,Proposition 3.3]. Let I µ ǫ be the subspace of V µ ǫ spanned by the vectors induced from the relation (3.5), which includes the relations in Lemma 3.2. Since I µ ǫ is aŮ(ǫ)-submodule, the quotient V µ ǫ /I µ ǫ is isomorphic to V ǫ (λ) by Proposition 3.4. So we have a well-definedŮ(ǫ)-linear map given by π µ (v T1 ⊗ . . . ⊗ v Tr ) = v T where T is the column semistandard tableau whose i-th column (from the left) is T i for 1 ≤ i ≤ r. Since the decomposition of V µ is equal to the usual Pieri rule of Schur functions, it has exactly one component isomorphic to V ǫ (λ). Hence π µ is equal to the projection onto V ǫ (λ) up to scalar multiplication. Let L µ ǫ = L ǫ ((1 µ1 )) ⊗ . . . ⊗ L ǫ ((1 µr )) be the crystal lattice of V µ ǫ . By [16, Theorem 4.14], π µ (L µ ǫ ) is a crystal lattice of V ǫ (λ) whose wt(H λ )-weight space is equal to A 0 v * H λ . Since the crystal of V ǫ (λ) is connected, we conclude that { v * T \| T ∈ SST ǫ (λ) } is an A 0 -basis of π µ (L µ ǫ ) which is equal to L ǫ (λ). For a parameter x ∈ Q(q), we denote by W s,ǫ (x) a U(ǫ)-module V , where V = W s,ǫ as a Q(q)-space and the actions of e i , f i , ω j are given by for i ∈ I, j ∈ I, and m = (m 1 , . . . , m n ) ∈ Z n + (ǫ). Here we understand e 0 = e n . Proof. It follows from the same arguments as in Lemma 3.6 that (L s,ǫ , B s,ǫ ) is a crystal base of W s,ǫ . The crystal SST ǫ ((s)) is connected with highest element H (s) . Since the crystal B s,ǫ /{±1} of W s,ǫ is equal to SST ǫ ((s)) as anI-colored graph, B s,ǫ /{±1} is connected as an I-colored oriented graph. Put Ω j = ω j for 1 ≤ j ≤ i − 1 and ω j+1 for i ≤ j ≤ n − 1, and let φ(ω ′ j ) = Ω j , φ(e ′ j ) = E j , and φ(f ′ j ) = F j for j = 1, · · · , n − 1. Let us check that Ω j , E j , F j satisfy the relations in Definition 2.1. Note that D i−1i = q −1 i . First, the relations (2.1) and (2.2) are trivial. Let us check that (2.3) holds. Let E j and F l be given for j, l ∈ I ′ . If j = l or j = l = i − 1, then it is clear. 3) holds. We can check the relation (2.4) by the same argument. Next, consider the relations (2.5). The first one is immediate. So it is enough to show the second one. We may only consider four non-trivial cases when the pair of relevant indices in I ′ are (i − 2, i − 1), (i − 1, i − 2), (i − 1, i), (i, i − 1) with the first index in the pair in I ′ even . In case of (i − 2, i − 1), we have which is zero, since e 2 i−2 e i−1 + e i−1 e 2 i−2 = (q i−1 + q −1 i−1 )e i−2 e i−1 e i−2 and hence The proof for (i, i − 1) is the same. In case of (i − 1, i − 2) and (i − 1, i), the proof reduces to the case of (i − 2, i − 1) or (i, i − 1) by applying τ i to E l 's for l = i − 2, i − 1, i. Finally let us check the relation (2.6). We may only consider the cases when the relevant triple of indices in In case of (i − 1, i, i + 1) and i ∈ I ′ odd , we have which is zero by (2.6) for U(ǫ) with respect to i + 1 ∈ I odd . The proof for (i − 3, i − 2, i − 1) is the same. The proof for (i − 2, i − 1, i) reduces to the previous cases by applying τ i to E l for l = i − 2, i − 1, i. We leave the proof for F j 's to the reader. 4.3. Truncation to U(ǫ ′ )-modules. Let ǫ ′ be as in Section 4.2. Suppose that M ′ is the number of j's with ǫ ′ j = 0 and N ′ is the number of j's with ǫ ′ j = 1 in ǫ ′ . For a submodule V of V ⊗ℓ (ℓ ≥ 1), we define where wt(V ) is the set of weights of V . For any submodules V, W of a tensor power of V, it is clear that as a vector space. Proof. (1) Let us assume that 2 ≤ i ≤ n − 2 since the proof for the other cases is similar. Let j ∈( I ′ ) and k ∈ I \ {i} given. It is clear from (4.3) that We have similar formulas for F j for j ∈( I ′ ). Hence V ′ is invariant under the action ofŮ(ǫ ′ ). In fact, V ′ is isomorphic to the natural representation ofŮ(ǫ ′ ) (2.15). (2) We see that the actions of E j , F j , K j (j ∈( I ′ )) on V ′ ⊗ V ′ are equal to those of (4.7) respectively. This implies that V ′ ⊗ V ′ and hence (V ′ ) ⊗ℓ are invariant under the action of U (ǫ ′ ). For example, in case of Then the action of ∆( Proposition 4.5. Let λ ∈ P M\|N be given. is non-zero if and only if λ ∈ P M ′ \|N ′ . In this case, we have as aŮ (ǫ ′ )-module. We may define tr ǫ ǫ ′ and have similar results for U(ǫ)-modules in O ≥0 . Proposition 4.7. 4.4. Irreducibility of W l,ǫ (x)⊗W m,ǫ (y). Let us show that W l,ǫ (x)⊗W m,ǫ (y) is irreducible for l, m ∈ Z + and generic x, y ∈ Q(q). When ǫ = ǫ M\|N , the irreducibility is shown in [17]. In this paper, we give a different proof of it, which is also available for arbitrary ǫ. Proof. Let us assume without loss of generality that M, N ≥ 1 with ǫ 1 = 0. We first claim that there exists a sequence i 1 , . . . , i r ∈ I such that (ǫ i k , ǫ i k +1 ) = (0, 0) for 1 ≤ k ≤ r and for some \|m ′ 1 ∈ W l,ǫ ′ and \|m ′ 2 ∈ W m,ǫ ′ , where x is = e is or f is for each 1 ≤ s ≤ r. Suppose that there exists k with ǫ k = 1 such that m 1k = 1 or m 2k = 1. Let i and j be the maximal and minimal indices respectively such that i < k < j and ǫ i = ǫ j = 0. If there is no such (i, j), then we have ǫ = ǫ M\|N and identify this case with the one of ǫ = (0 M −1 , 1 N , 0). Since we will choose i 1 , . . . , i r in {i, i + 1, . . . , j − 1}, we may assume for simplicity that m ab = 0 for a = 1, 2 and b ∈ {i, . . . , j}. Suppose that L > 1. We may assume that Then by tensor product rule in Proposition 2.5 we have for some i < x < y < z < j. Here we assume that z≤v≤j−1 e v in m 1 is empty if there is no such z. Now we take the following steps to construct b ′ in (4.9). Step 1. If there exists z such that y < z < j and m 1z = · · · = m 1j−1 = 1, then by applying f z f z−1 . . . f j−1 to b, m 1 in (4.10) is replaced by (4.11) m 1i e i + x≤u≤y e u + z+1≤v≤j−1 Repeating this step, (4.11) is replaced by Hence we may assume that m 1 in (4.10) is of the form m 1i e i + x≤u≤y+1 e u + m 1j e j . Step 2. If m 1j = 0, then we have Hence we may apply the induction hypothesis to conclude (4.9). Step e v + m 2j e j , respectively. We may keep this process until m 1j −d = 0, which belongs to the case in Step 2, or y+d+1≤v≤j−1 e v is empty. In the latter case, m 1 is replaced by m 1i e i + x≤u≤j−1 e u + (m 1j − d)e j so that we may apply f j−1 and use induction hypothesis to have b ′ . This proves the claim. By construction of b ′ and its weight, we have M−1 (cf. [1]). Finally, since dim Q(q) (W l,ǫ ⊗ W m,ǫ ) (l+m)δ1 = 1 and B l,ǫ ⊗ B m,ǫ /{±1} is connected, it follows from [2, Lemma 2.7] that W l,ǫ ⊗ W m,ǫ is irreducible. This completes the proof. 4.5. Existence of R matrix. For l, m ∈ Z + and x, y ∈ Q(q), consider a non-zero Q(q)linear map R on W l,ǫ (x) ⊗ W m,ǫ (y) such that ∆ op (g) • R = R • ∆(g), (4.12) for g ∈ U(ǫ), where ∆ op is the opposite coproduct of ∆ in (2.7), that is, ∆ op (g) = P •∆(g)•P and P (a ⊗ b) = b ⊗ a. We denote it by R(z), where z = x/y, since R depends only on z. We say that R(z) satisfies the Yang-Baxter equation if we have Here R ij (z) denotes the map which acts as R(z) on the i-th and the j-th component and the identity elsewhere. We call R(z) the (quantum) R matrix. We also define v ′ (m, l, t) in the same manner. For 0 ≤ t ′ ≤ min{l, m}, we may regard as a Q(q)-space, and let P l,m t : W l,ǫ ′′ (x) ⊗ W m,ǫ ′′ (y) −→ W m,ǫ ′′ (y) ⊗ W l,ǫ ′′ (x) be aŮ(ǫ ′′ )linear map given by P l,m t (v ′ (l, m, t ′ )) = δ tt ′ v ′ (m, l, t ′ ). Then we have the following spectral decomposition of P R ǫ ′′ (z) for some ρ t (z) ∈ Q(q). By Proposition 4.5 and Lemma 5.1, we also have the following spectral decomposition of P R ǫ (z) where we understand P l,m t as defined on W l,ǫ (x) ⊗ W m,ǫ (y). Then we have the following explicit description of P R ǫ (z), which is proved in case of ǫ = ǫ M\|N [17]. Then we define a U(ǫ)-module It is proved in [16] that W s,ǫ is irreducible, and it is isomorphic to V ǫ ((s r )) as aŮ(ǫ)-module. One may use a similar argument as in the proof of Theorem 5.3 to prove the irreducibility of a tensor product of W l,ǫ (x)'s and its image under R matrix in some special cases. Let l 1 , . . . , l r ∈ Z + and x 1 , . . . , x r ∈ Q(q) be given and let ǫ ′ = ǫ M\|0 . Proposition 5.6. If M is sufficiently large and W l1,ǫ ′ (x 1 ) ⊗ · · · ⊗ W lr,ǫ ′ (x r ) is irreducible, then W l1,ǫ (x 1 ) ⊗ · · · ⊗ W lr ,ǫ (x r ) is also irreducible. Proof. It follows from Lemma 5.1 and the same argument as in Proposition 5.6.	2020-01-13T09:35:18.000Z	2020-01-13T00:00:00.000	{ "year": 2021, "sha1": "8ad1f5fcfd7c18c6daa1d22908b1d5b4f15d5a11", "oa_license": null, "oa_url": "http://arxiv.org/pdf/2001.04119", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "8ad1f5fcfd7c18c6daa1d22908b1d5b4f15d5a11", "s2fieldsofstudy": [ "Mathematics" ], "extfieldsofstudy": [ "Mathematics" ] }
231184060	pes2o/s2orc	v3-fos-license	Comparative study of dexamethasone nebulisation with magnesium sulphate nebulisation in preventing post operative sore throat following endotracheal intubation Introduction: Post-operative sore throat is one of the most common complications following endotracheal intubation. Though considered minor complication, it may cause significant patient dis-satisfaction. Various non-pharmacological and pharmacological trials have been used with variable results. Objectives: To compare the efficacy of nebulised dexamethasone with that of nebulised magnesium sulphate in decreasing the incidence and severity of postoperative sore throat (POST). Materials and Methods: In this prospective double blind study 90 patients undergoing surgery under general anaesthesia with endotracheal intubation lasting <3hr were randomly assigned into two equal groups. Group D received dexamethasone 8mg (2ml) with 3ml saline nebulisation and group M received magnesium sulphate (50%W /V 2ml) with 3ml saline nebulisation 30 min before the induction of anaesthesia. Primary outcome assessed was incidence and severity of POST. Secondary outcome assessed were the incidence of post-operative hoarseness and cough. Results: Compared to group M, significantly lesser number of patients in group D had post-operative sore throat at 0hr (p= 0.0262), 4 hr (p=0.00022), 8 hr (p=0.00039) and 12hr (p=0.000657). None of the patients in group D had any hoarseness of voice at 0hr, 4 hr, 8 hr of assessment (p= <0.05). Except one patient in group M, none of our patients in either of the group had cough at any point of assessment. Conclusion: Preoperative dexamethasone nebulisation just before induction of anaesthesia is an effective method of reducing the incidence and severity of POST following endotracheal intubation. Dexamethasone nebulisation reduces the severity of sore throat more effectively than magnesium sulphate nebulisation. Introduction Sore throat is one of the common postoperative complaint that leads to morbidity and patient dissatisfaction. It is 5 th most frequent adverse clinical anaesthesia outcome. 1 The incidence of sore throat has been reported to be very high [21%-65%]. 2 Irritation and inflammation of the airway, mechanical injury during intubation, damage to the mucosa due to the pressure from the endotracheal tube cuff and dehydration of the mucosa were considered the cause of postoperative sore throat (POST). 3,4 Although symptoms subside without any treatment, the management for POST is still advised because it enhances patient satisfaction, acceptance of anaesthesia and improves the activities after discharge. 5 Prophylactic management of POST is recommended to improve the quality of anaesthesia care of duration of time a patient stays in the postanesthesia care unit because of POST increases the cost of care. Patients with POST had a 14min longer stay in the post anaesthesia care unit and 25 min longer stay in the ambulatory care unit and were discharged 51 min later from the facility compared with those who did not complain of POST. 5 Different agents like ketamine gargle 6 and nebulisation, 7 endotracheal tube spraying with Beclomethasone, 8 intravenous injection of dexamethasone, 9 magnesium sulphate gargle 10 and Lozenges, 11 nebulized lignocaine, 12 lignocaine jelly have been used with variable success for decreasing both the incidence and severity of POST. Corticosteroids are used in the perioperative period to enhance the effects of analgesics and antiemitics. Inhaled carticosteroids deliver the drug to the airways without systemic effects. Dexamethasone is a potent steroid that has 26.6 and 6.6 times stronger antiinflammatory and immune-suppressant effects than cortisol and prednisone, respectively. It has been supported that it is very useful for relieving POST. 13,14 Therefore, inhaling dexamethasone may be used as a method to reduce POST following general anesthesia [GA]. As proven by previous studies N-methyl Daspartate [NMDA] receptors are involved in nociception and inflammation. 15,16 NMDA receptors are present all over the nervous system. Magnesium is an antagonist of the NMDA receptors and prevents the central sensitization of peripherally inflicted nociceptive stimulus. Nebulisation will deliver the drug to the airways with no or minimal systemic effects. Both magnesium sulphate and dexamethasone are readily available in the operation theatre. Present study was done to compare the efficacy of dexamethasone nebulisation pre-operatively with that of magnesium sulphate nebulisation. In addition we also observed for other airway related complaints like hoarseness and cough, postoperatively Materials and Methods This was a prospective double blind randomised study conducted after obtaining approval from our hospital ethical committee (IEC NO: SIMS & RC/IECC/05/2017). Written informed consent was obtained from patients after proper explanation regarding the study. ASA1 & ASA2 physical status patients aged between 20 to 50yrs admitted for elective surgery under general anaesthesia with endotracheal intubation with mallampatti score 1-2, undergoing surgeries lasting less than 3hrs duration were selected for the study. 90 patients were chosen by simple chit picking method into two groups of 45 patients each. To show 50% reduction in the incidence of POST (which is reported to be 65%) at α =0.05, confidence interval of 95% and a power of 90%, we required 30 patients each group. On adding 10%-15% patients loss due to multiple attempts at laryngoscopy and due to bucking during extubation, each group needed 40 to 45 patients. Patients with history of sore throat, on long term anti-inflammatory analgesic medications, patients who have undergone insertion of devices which stimulate oral cavity, larynx, pharynx like nasogastric tube, endoscopic nasobiliary drainage tube, mallampatti grading >2, patients who are undergoing head and neck surgeries, patients who needed more than two attempts at endotracheal intubation and use of bougie and styllete, obese, diabetic, pregnant patients, surgeries lasting for more than 3hrs, surgeries in prone position were excluded from the study. Group D received dexamethasone 8mg [2ml] with 3ml of normal saline for nebulisation. Group M received MgSo4 [50% W/V 2ml] with 3ml of normal saline for nebulisation 30 min before the induction of anaesthesia in the preoperative receiving room. The patients were nebulised using mask connected to wall mounted oxygen driven source. (8lt, 50psi) A nurse blinded for the study nebulised the patient using the study drug prepared by an anaesthesiologist not involved in the study. All the intubations were done by an anaesthesiologist with >5yrs experience. All patients were kept nil oral overnight and were premedicated with table. Ranitidine150mg and table. Alprazolam 0.5mg 2hrs before the surgery. After the nebulisation for 30 min in the receiving area, patients were shifted into the OT. Patient premedicated with inj.glycopyrrolate 0.2mg and midazolam 1mg. Injection fentanyl l was given 2mcg/kg. Patient was induced with inj. propofol 2mg/kg. Intubation was done using non-depolarising muscle relaxant, inj.Vecuronium0.15mg/kg. Intubation was performed as smooth direct laryngoscopy after 4min of injecting vecuronium. Endotracheal tube size 7 was selected for females and 8 was selected for males. A single use portex high volume low pressure cuffed endotracheal tube was used for the intubation. Patient were excluded from the study if intubation failed in two attempts or O2 saturation dropped below 95% during intubation. Anaesthetic gas mixture was used to inflate the endotracheal tube cuff till no leak was heard around the tube. Cuff pressure was adjusted to 20-22CmH2O using cuff inflator/ pressure gauge PORTEX TM. Cuff pressure was monitored every half an hour to maintain the cuff pressure of 20 to 22CmH2O. Maintenance of anaesthesia was the same in both the groups using isoflurane 1MAC and vecuronium divided doses with 50%O2+50%N2O. 20 min before the completion of the procedure inj. ondansetron 4mg and inj.pethidine0.5mg/kg mg IV was given to prevent post operative nausea and vomiting and bucking on the tube respectively. Neuromuscular blockade was reversed using neostigmine 0.05mg/kg and glycopyrrolate 0.001mg/kg. Gentle suctioning of oropharynx was done. Extubation was performed using the same method in both the groups when the patient was fully awake. Patients who bucked or coughed during extubation were excluded from the study. In the recovery room patient received O2 by mask 5lt/min and inj. Paracetamol 1gm 8 th hr. Assessment: Patients were assessed in the postoperative ward by a nursing staff blinded for the study. Incidence of sore throat, discomfort, pain and other symptoms were assessed at the time of shifting to postoperative ward [0hr], 4hr, 8hr, 12hr and 24hrs according to the grading shown in Fig.1. Fig. 1 Total incidence of POST was calculated by dividing the number of patients who experienced postoperative throat pain at least once by the total number of patients. The patients were then examined to determine the occurrence of any other postoperative complications and the findings were recorded in detail. Patients who had POST grading more than 2 were instructed to gargle aspirin 75mg dispersible tablet. Statistical Analysis The collected data was analysed using statistical package for social sciences (SPSS, V-20) Descriptive and inferential statistical analysis has been carried out in the present study. Results on continuous measurements are presented on Mean ± SD and results for P value were calculated using t-test. Categorical measurements are presented in number. Chi square test is used to compare the difference in each group. Significance was assessed at 5% level of significance. If P <0.005 is considered statistically significant. Results From Aug 2017 to March 2018, 90 patients were enrolled for the study. Three patients in group M were excluded due to bucking during extubation. Two patients in group D were excluded due to insertion of nasogastric tube and two patients bucked during extubation. So for statistical analysis we considered 40 patients in each group. Both groups were comparable with respect to age, distribution of sex, ASA physical status, duration of surgery (Table 1). Nearly 85% of patients in group M were intubated in first attempt where as 15% of patients were intubated in second attempt. In group D 82.5% of patients were intubated in first attempt and 17.5% were intubated in second attempt. This was not statistically significant (P=0.338). POST (at rest and on swallowing), cough and hoarseness of voice were assessed at 0, 4, 8, 12 and 24 hrs according to the scales shown in the Fig. 1. Incidence of POST was 27.5% in group D and 57.5% in group M and the difference was statistically significant at all times of assessment. (Table 2). Compared to group M, the number of patients in group D who had POST were significantly low at rest at 0 hr (p= 0.0262), 4 th hr (p=0.00022), 8 th hr (p=0.00039) and 12hr (p=0.000657). (Table 2). Also duration of pain relief was significantly longer (12hrs) in group D compared to group M (P=0.000657). Only one patient in each group had POST at 24hr which was statistically not significant. (P= 1.00) ( Table 2) Incidence of pain on swallowing was also more in patients in group M at all points of assessment compared to group D. (Table 2). Though the highest incidence of sore throat occurred at 4 th hr postextubation in both the groups, it showed declining trend. This decline was significant in group D. (Table 2) None of the patients in group D had postoperative hoarseness which was statistically significant at 0hr, 4 th hr, 8 th hr of assessment. At 24hrs none of the patients in both the study groups had any hoarseness. (Table 2) None of the patients in group D had cough at any point of assessment where as one patient in group M had cough at 4 th hr and 8 th hr which was clinically and statistically not significant. (P=1.00) ( Table 2) Discussion Post-operative sore throat, cough, and hoarseness of voice are common, uncomfortable, distressing sequelae after tracheal intubation. Throat irritation in the presence of a large abdominal or thoracic incision can be very annoying especially in the presence of inadequate analgesia since any attempt to cough to clear the throat causes severe pain. The contributing factors for POST include young age, females, gynaecological surgery, use of succinylcholine, larger tracheal tubes, high cuff pressure. [3][4][5] POST can be multifactorial in origin, including mechanical injury during laryngoscopy and intubation causing aseptic inflammation, continuous pressure by the inflated tracheal tube cuff on tracheal mucosa causing damage and dehydration of the mucosa. 6,13,19,20,23 Although many drugs are used through different routes to relieve the POST, these could result in unwanted side effects. Ketamine gargle might cause adverse hemodynamic effects and taste may not be acceptable. Higher doses of drugs are required when used through intravenous route causing unwanted side effects. Gargling requires patient co -operation. So we selected method of nebulistion to deliver the study drug to throat. Dose of drug used is very minimal and acts topically avoiding adverse systemic effects. Present study shows that prophylactic dexamethasone nebulisation helps in significant reduction in incidence of sore throat at 0hr (P =0.0262), 4 th hr (P = 0.00022), 8 th hr (P = 0.00039) and 12 th hr (P= 0.000657) post extubation when compared to magnesium sulphate nebulisation. Severity of sore throat also was less compared to group M. (Fig. 2) There was no incidence of moderate sore throat (grade 2) in group D patients. None of the patients in group D had hoarseness of voice. Incidence of severe sore throat (grade 3) was not found among both the groups. This may be because we used well defined inclusion and exclusion criterias and intubations were done by an experienced anaesthesiologist, duration of surgery was less than 3hrs, cuff pressure was monitored through out the procedure and bucking on the endotracheal tube was avoided. Usually steroids and nebulised adrenaline are used for the treatment of POST. 18 Intravenous dexamethasone or hydrocortisone is used for this purpose. It has been shown that both, topical and intravenous dexamethasone had reduced the incidence of POST. 13,14,18 But glucose intolerance, fluid retention are concerns with the use of intravenous steroids. So we used nebulised dexamethasone at a dose of 8mg. Widespread recognition of under treatment of sore throat by clinicians has led to the development of preemptive strategies for its alleviation. So we used nebulisation just before the induction of anaesthesia. Our study showed significantly lower incidence POST in dexamethasone group at all points of assessment. This was similar to the results of Salama et al. 21 Tabari et al 22 studied the effectiveness of betamethasone gel applied to the tracheal tube and intravenous Dexamethasone on POST on 225 ASAI and ASAII patients undergoing elective abdominal surgery with tracheal intubation who were randomly allocated into three groups: the betamethasone gel group, the intravenous dexamethasone group and control group. They concluded that widespread application of betamethasone gel over tracheal tubes effectively reduced POST, compared to intravenous dexamethasone. These findings are consistant with the topical application of steroid on the upper airway before endotracheal intubation. Magnesium sulphate gargle 10 and lozenges 11 have been used for treating POST. We selected magnesium sulphate nebulistion to avoid large doses and for ease of administering the drug. Not much literature is available about the use of nebulized magnesium sulfate for attenuation of POST. Blitz et al. 24 used nebulised magnesium sulphate for the treatment of acute asthma without any systemic and local adverse outcomes. To the best of our knowledge, there is currently no study that compares the efficacy of nebulised dexamethasone with that of nebulised magnesium sulphate on the incidence and severity of POST. In Borzan et al 11 study, there was a significantly decreased incidence of POST when subjects sucked on a lozenge containing magnesium preoperatively at 2 nd hr and 4 th hr but not immediately or 24hrs postoperatively. Incidence of POST is more common at 4 th to 6 th hr is due to the gradually developing local Inflammation. But in Our study, more than 50% of patients (Table 2) in magnesium sulphate group had significant POST at 4 th hr and 6 th hr. This shows that magnesium sulphate is not very effective in controlling POST. Even though incidence of POST was not significantly reduced with the use of nebulised magnesium sulphate, there was significantly reduced incidence of cough. In our study, group M patients who complained of hoarseness had prolonged duration of surgery (>120 min). This may be the cause, because of prolonged cuff pressure causing irritation of mucosa and odema of vocal Cords. 25 So in our study magnesium sulphate nebulisation did not reduce the incidence of POST, but had a role in attenuating the severity of sore throat since none of the patients had grade 3 POST in group M also. In the present study, the overall incidence of POST was 27.5% in group D and 57.5% in group M which is the same as evaluated by Macario et al. 2 Even though few patients in magnesium sulphate group had relief from sore throat, it was not statistically significant. Limitations of the Study Firstly we did not use humidity moisture exchanger in the gas delivery circuit and dry gases are implicated in the development of postoperative sore throat 19. Secondly we don't know how efficient is dexamethasone nebulistion in relieving sore throat in case of multiple attempts at laryngoscopy, prolonged intubation due to long procedures, use of bougies. Repetation of the nebulistion can be considered postoperatively in these patients. Thirdly the scale used to assess POST was a subjective scale and may be associated with bias. Conclusion Preoperative dexamethasone nebulisation just before induction of anaesthesia is an effective method of reducing the incidence and severity of POST following endotracheal intubation. Magnesium sulphate has a role in reducing the severity of sore throat. So we conclude that preoperative dexamethasone nebulisation reduces the incidence and severity of sore throat more effectively than magnesium sulphate nebulisation.	2021-01-07T20:20:04.303Z	2020-12-15T00:00:00.000	{ "year": 2020, "sha1": "b75818ea05a31abb15032a7855c5124bcaf1e0d5", "oa_license": "CCBYNCSA", "oa_url": "https://www.ijca.in/journal-article-file/7424", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "b75818ea05a31abb15032a7855c5124bcaf1e0d5", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [] }
255066722	pes2o/s2orc	v3-fos-license	Attitudes, deliberation and decisions In this paper I discuss the challenges of several authors to the claims I make in Decision Theory with a Human Face regarding the relation between preference and choice, the nature of conditional desire, the semantics of conditionals, attitudes to chances and their role in individuating prospects, belief change under growing awareness and choice under ambiguity 1. Preferences are a type of judgement. 2. Qualitative attitudes have conceptual, methodological and explanatory priority over numerical ones. 3. Rational choice is constrained only by preference. Judgmentalism, in turn, she takes to be motivated by an internalism about the requirements of rationality: that they concern only the relationship between an agent's attitudes and not their relationship to features of the external world. Internalism is a view she is willing to grant for the purposes of discussion, but she rejects my argument for it and, more importantly, regards it as an insufficient basis for judgementalism. Her main claim however is that judgementalism is false because, contrary to (3), attitudes other than preference rationally constrain choice. This leads her to reject the Choice Principle. I agree with a great deal of what Thoma says: in particular that I am committed both to internalism and to the first and second of the above claims. But I don't make the third claim in my book and indeed consider it false. Thoma thinks that my statement of the Choice Principle commits me to it. But with this I disagree. I also disagree that the falsity of claim 3 means that the Choice Principle requires modification. I take up these issues below, but first let me respond to Thoma's criticism of my defence of internalism. Thoma argues that internalism cannot be defended by appeal to the 'ought implies can' principle because it overshoots its target. In particular, she argues, it would imply that those who had false beliefs about the requirements of rationality would not be rationally required to obey them, something she takes to be false. Instead, she suggests, internalism should be defended on the grounds that norms of rationality must be actionguiding. This latter point I agree with, but I see it as being in tune with the application of the 'ought implies can' principle to rationality constraints. And I don't agree that the requirement to conform to the requirements of rationality is itself a requirement of rationality. Let me spell this last point out a bit. It is true that you ought to conform to the requirements of rationality. But what kind of 'ought' is contained in this claim? It is not, I suggest, the ought of rationality, but some other kind of imperative (perhaps one rooted in the prudential advantages of being rational). We know that it can't be the rational ought precisely because someone who didn't respect it in virtue of not knowing what the requirements of rationality were would not be acting or judging in a manner that is inconsistent by their own lights. Consider someone with intransitive preferences who believed that it was rationally required that they be so. Then it would both be the case that they have irrational preferences and so ought to revise them to make them transitive and that they ought not to both retain their belief about the requirement to have intransitive preferences and adopt transitive preferences. The second ought is an ought of rationality because it derives from a consistency requirement. Suppose that the first is as well (as Thoma claims and I deny). Then it would follow (assuming that rationality does not impose contradictory requirements) that the person rationally ought not to retain their belief that rationality requires intransitive preferences. But while they undoubtedly ought not to retain this belief, it is equally undoubtedly not an ought of rationality (on an internalist view). It follows that the requirement to conform to the requirement to have transitive preferences cannot be a rationality requirement. Thoma's main claim is that judgementalism is untenable because there are norms governing the framing of decision problems that are not directed at preferences. Her argument for this is compelling. Some normative constraints on framing, such as the ideal of taking into account everything of relevance to the decision, are not internal norms of rationality. But some are: such as incorporating all contingencies into one's representation of a decision problem that one considers (more-than-marginally) relevant to the choice one will make. And one could conform to the Choice Principle even if one didn't respect these norms because the principle doesn't by itself constrain how alternatives are framed. So, she argues, the Choice Principle does not exhaust the rationality constraints on choice. All this is true. But I do not claim otherwise in my book. On the contrary, I castigate behaviourism (p. 60) for its failure to recognise that the choice an agent makes depends on how the set of alternatives is conceived and, as Thoma herself points out, I argue against the view that 'anything goes' when it comes to framings (p. 13). Nonetheless, Thoma thinks that my endorsement of the Choice Principle commits me to falsely claim (3), that only preferences constrain choices. But the Choice Principle says that preference determines which of the available options are/should be chosen. For an internalist, 'available options' must be read subjectively, as the alternatives the agent believes are real options for her. So interpreted, the Choice Principle does allow that an agent's choice depends on what options she considers available to her, not just because she cannot choose what is not available but also because her preferences over these alternatives may depend on how they are conceived by her. If this is correct, then the Choice Principle should not be abandoned, but supplemented with an account of how decision problems should be framed. 1 Thoma makes two interesting suggestions in this regard. Firstly, that formulating decision problems requires reference to non-preference attitudes such as beliefs and to attitudes to objects that are neither actions nor outcomes: in particular, to properties. And, secondly, that there are rationality conditions relating attitudes to properties to attitudes to prospects. I agree with both claims: in many cases it is our preferences for properties that explains our preferences for fully described outcomes. It is, for example, my preference for sweet fruits (over sour ones) and my belief that this pear before me is sweet that explains my desire for it. And this explanation implicitly depends on it being the case that my preferences for properties impose rationality constraints on my all-things-considered preferences (this point is familiar from Pettit (1991)). This being said, since an attitude to a property can be captured by an attitude to a proposition, it seems to me that all the ingredients for such an account of the role of property preferences are already in place in my book. When I explain my desire for a pear in terms of my attitude to the property of sweetness, it is the sweetness of pears (or, more broadly, of fruits, or even of edibles) that explains my desire, not an 'unattached' property of sweetness. Pears being sweet is something that is captured by a proposition: the set of worlds in which the pears are sweet. The property attitudes that explain my desire for the pear are attitudes to propositions like this one. And it will follow from the treatment of attitudes to propositions that I give that my attitudes to properties so-conceived will constrain my attitudes to alternatives that instantiate them. Note that it does not follow on this account that I will always desire the pear in front of me just because I prefer my pears sweet, even when I believe this pear to be a sweet one. The pear may also be discoloured and I may also dislike the property of discolouration in fruit. So the relevant attitudes to look to for explanations in this case are those directed at the proposition that is the intersection of the pears-being-sweet propositions and the fruits-being-discoloured propositions. In conclusion, I fully concur with the importance of exploring how all-thingsconsidered preferences over options depend on attitudes to the properties that they instantiate. But such an account is best viewed as an enrichment of the Rationality Hypothesis and so would supplement the Choice Principle, rather than replace it. 2 Once an agent has formed all-things-considered preferences over a set of options in the light of the properties of their outcomes that she considers relevant, she should choose the option that she most prefers (as required by the Choice Principle). If this option is not in fact best, given her beliefs and her attitudes to properties, then her preferences must fail to reflect these attitudes and so she must have violated one of the requirements of rationality. Conditional desire (Joyce) Over the years since the publication of his ground-breaking book The Foundations of Causal Decision Theory and my review of it, Joyce and I have exchanged views on numerous occasions on the nature of supposition and its role in reasoning and decision making. 3 There is, as he says, much that we agree on; perhaps most significantly he has persuaded me of the correctness of causal decision theory. But there is also some residual disagreement relating mainly to the role of suppositions in evaluating actions and about the kinds of judgements that can provide foundations for a decision theory. Since both issues draw on disagreement about the best expression for desirability under the supposition that some proposition is true, let me start by saying something about the motivation for my approach to this topic. To keep things simple, let's focus on the case of evidential supposition. When we suppose that, as a matter of fact, it will rain tomorrow, we put ourselves in a judgemental state in which we look at the various possibilities in the light of this fact. The supposed truth of it raining provides, as it were, the backdrop for judgements of both the credibility and the desirability of other propositions. For this reason, I use measures of conditional credibility and desirability that factor out the credibility/desirability of the condition assumed true. This feature is exemplified by the normalisation of them with respect to the tautology, with the conditional probability of the tautology always equalling one on my account and its conditional desirability always equalling zero. 4 Together with the usual consistency requirements this normalisation yields, in the case of evidential supposition, the following expressions for conditional desirability (V (·\|A)) and probability (P(·\|A)), given that A: Both expressions define what Joyce calls a relative measure of an attitude. The conditional probability of getting wet given that it will rain, for instance, doesn't express anything about the prior credibility of rain-only about the probability of getting wet from the rain relative to the probability of it raining. Analogously, my proposed expression for the conditional desirability of getting wet, given that it will rain, doesn't express anything about the desirability of rain. Rather it measures the desirability difference that getting wet makes when it rains. Joyce (2021) argues, and I agree, that the notions of suppositional belief and desire expressed by relative measures are not the only ones of potential interest. For one can also define absolute notions of suppositional belief and desire such that the probability or desirability of a proposition under the supposition of A measures not its probability or desirability in the light of, or relative to, the supposed truth of A, but simply the probability or desirability of the state of affairs that holds if both the proposition and A is true, or would hold if both were. Joyce opts for just such an absolute measure of suppositional desirability, but retains a relative measure of suppositional probability (conditional probability)-a combination that I find odd. What is at stake in the choice between absolute and relative measures of suppositional desirability? Joyce argues that adopting an absolute measure allows him to make sense of the kind of deliberation involved in choice and in particular the kind of crosssuppositional comparisons that he takes to be essential to it. Consider his example of the choice between going birding and going surfing in the light of one's uncertainty about whether one's friend will bring binoculars or a wetsuit. He and I are agreed that birding should be chosen over surfing just in case the expectation of desirability on the supposition of birding exceeds the expectation of desirability on the supposition of surfing, where these expectations are calculated relative to a suppositional probability P * A measuring the probability of propositions on the supposition that A is or were true. Where we disagree is over how to interpret this quantity and what sort of reasoning is required to determine its value. Joyce identifies the choice-worthiness of A with the absolute suppositional desirability of the status quo induced by A-ing, denoted in his paper in this volume by J * A (T * ). He claims moreover that cross-suppositional comparisons are essential to making choices. The choice between birding and surfing for example requires a comparison, he says, between the desirability of your friend bringing binoculars (or a wetsuit) on the supposition that you choose birding and on the supposition that you choose surfing. But this is demonstrably not the case because, on Joyce's theory, for any action A, J * where the w i are the different possible final outcomes. So we can express the relevant quantities in his example as follows: Notice that no cross-suppositional comparisons are involved in determining the values on the right-hand sides of these equations. So one can assess whether or not J * bird (T * ) > J * sur f (T * ) without them. This example brings out our broader disagreement about how to think of choices between acts. For Joyce when we compare act A to act A * , we calculate the desirability of the status quo on the supposition that we perform A and compare that to the desirability of the status quo on the supposition that we perform A * . So we make a comparison of one object (T * ) across two different suppositions. In contrast, I claim that we compare the desirability of two different objects: that of the situation obtained by A-ing to that of the situation obtained by A * -ing. Contrary to what Joyce claims, no cross-suppositional desirability comparisons are required for this because we simply compare the desirability, from our current perspective, of the two different situations that we (expect) would be achieved by A-ing or A * -ing. And so the quantity measured by J * A (T * ), as he defines it, is best thought of as the desirability of the state of affairs we expect to achieve by A-ing (or the expected gain in desirability in virtue of A-ing) and not as a suppositional desirability. The claim that evaluation of action requires no cross-suppositional comparisons of desirability does not depend on rejecting the possibility of making these kinds of comparisons. But in my book I do argue against seeking foundations for decision theory in cross-suppositional preference comparisons of the form B\|A D\|C (typically glossed as a preference for B given that A over D given that C). My objection was essentially that A and C serve in this expression to determine different standpoints from which the prospects B and D are to be evaluated, not as objects of preference, and that an evaluation cannot be made, at a single moment, from two different standpoints. My rejection of cross-suppositional comparisons of this sort puzzles Joyce for a couple of reasons. Firstly, he thinks we are in fact capable of making them. And, secondly, he thinks forbidding such comparisons makes it difficult to explain the use of suppositions in the assessment of actions. I have already shown that the second concern is misguided, but I grant his first point. I should clarify however. I did not (intend to) claim that we cannot make cross-suppositional comparisons of probability and desirability. We can. For instance, we can compare the evidential conditional desirability of B given that A to that of D given that C by computing V (B\|A) from V (AB) − V ( A) and V (D\|C) from V (C D) − V (C) and seeing which is greater. What I don't think we should do is take such comparisons as primitive and use them to derive numerical desirabilities, because I don't see that we can make the comparisons without already having at least rough numerical desirabilities to hand. So it is only in the context of the project of supplying foundations for cardinal measures of both desirability and choice-worthiness that I reject use of cross-suppositional comparisons. Conditionals (Huttegger & Rothfus; Hájek) Conditionals and suppositional reasoning of the kind discussed in the previous section are closely related. Suppose I want to be first in line for a new show but would rather not leave before finishing my dinner. In thinking about whether to leave right away (without dinner) I consider whether, were I not to, I would still arrive in time for the show. The opinions I form can be reported using conditionals like 'If I were to leave later, I would be late for the show' and 'If I were to leave now, I will be first in line'. Since it matters to me whether or not I can both eat my dinner and make the show, I want to get the right answer to the question about what would happen if I were to leave later. It is difficult to explain why I should be interested in settling the question if there is no truth to the matter. The semantics for conditionals developed in Bradley (2012) takes this truth-aptness of conditionals at face-value and embeds it within a broadly suppositional theory of conditionals in the tradition of Adams (1975), Stalnaker (1968) and McGee (1989). The crucial feature of this account is that the role of the truth makers for conditionals are played by counterfacts: facts about the worlds picked out by the antecedent of the conditional being evaluated and not (typically) facts about the actual world. Propositions are therefore modelled not as sets of possible worlds, but as sets of ordered sets of possible worlds, where the latter specify the facts under all relevant suppositions. At the most general level this semantics imposes no constraints on how the facts and the counterfacts may combine. But it is possible to distinguish the truth conditions for indicative and subjunctive/counterfactual conditionals by imposing constraints on the relationship between them that are characteristic of the different kinds of supposition associated with these two types of conditional. From this one can derive a number of their distinguishing properties; properties that explain the different roles that they play in our thinking and in a discourse. These include Adams' famous thesis that rational degrees of belief in conditionals are conditional degrees of belief, something widely reckoned to be true but which has proven very difficult to accommodate within standard semantic theories. It is baked into this account of conditionals that there is a close relationship between their semantics and the use to which they are put in the kind of suppositional reasoning that we employ in deliberating about what to do. So unsurprisingly they serve in my book to support an account of deliberation aimed at choice of action. But conditionals also play a number of other roles in DTHF. They are used to (re-)individuate the outcomes of actions to capture what would have happened if uncertainty had been resolved differently, something that Allais' paradox (Allais, 1953) suggests is a matter of concern for agents. And they provide for a Savage-style representation of actions as conjunctions of indicative conditionals (that specify the outcome of the performance of the action in each state of the world), thereby allowing for a derivation of Savage's theory within the extended Jeffrey-framework developed in my book. Huttegger and Rothfus' (2021) paper provides a fourth application, to the modelling of planning and sequential choice within this extended Jeffrey-framework. In their model of sequential choice, conditionals are used to express the content of plans at natural nodes in a decision tree, by specifying for each eventuality open at that node what will be chosen if that eventuality transpires. A plan is dynamically consistent only if it can permissibly be chosen at all nodes reached in the course of its implementation. Remarkably Rothfus and Huttegger are able to show that choice of plan in accordance with desirability maximisation is dynamically consistent if the conditionals that express plans have just the semantic properties that, in my theory, characterise indicative conditionals. Furthermore, although satisfaction of these properties is not necessary for base-line dynamic consistency, it is so for their stronger condition of preferential stability: that a preference for one plan over another at some node should not be reversed at any downstream node obtained under both plans. The question that naturally arises is: what properties of conditionals are necessary and sufficient for the dynamic consistency and preferential stability of choice in accordance with causal decision theory? Addressing it is not straightforward since we lack an account of the causal expected utility of a plan, qua conjunction of conditionals, at a natural node. My suggestion would be that it is the average of the causal expected utilities of the possible actions that could be performed at that node with the weight on an action being given by the probability of the eventuality upon which its performance is contingent. But clearly more work needs to be done here. These applications provide explanations of the role of conditionals in decision making and, more generally, in thought and talk, thereby giving indirect support for my account of conditionals. Hájek (2020) grants this, but thinks that the metaphysical cost of postulating the existence of counterfacts is too high a price to pay for these explanatory benefits. It isn't that Hájek believes that there are no counterfacts-just that there aren't that many of them. In particular, he denies that to every conditional there corresponds a counterfact that makes it true or false (a thesis he dubs Counterfactual Plenitude). Nor, it should be emphasised, does he deny that conditionals have truthvalues. Rather, he thinks most conditionals are false; something that follows in his view from the fact that there is typically no way of settling the question of whether the consequent of the conditional would have been true had its antecedent been. Now Hajek's main target in his paper is Stefánsson's (2018) version of this view and his use of my semantics to underwrite his rejection of the claim that most conditionals are false. And I think he incorrectly attributes some of Stefánsson's views to me. Although I take counterfacts to be the truth-makers for conditionals, I am not committed to what he calls Primitive Counterfacts Realism: neither to counterfacts being primitive nor to realism about them. In the first place, the semantic role that counterfacts play in my theory doesn't determine any particular view about their metaphysical status (and I largely stayed clear of expressing one). Secondly counterfacts are not even semantically primitive in my theory. The only primitives are possible worlds, from which the ordered sets of worlds are constructed for the purposes of defining truthconditions. Consequently, my theory has no more metaphysical commitments than standard possible world semantics. If there are other possible worlds than the actual one, then counterfacts exists. If they are real then so are the counterfacts, etc. I am of course committed to the existence of counterfacts and Hájek thinks that this is a mistake. I don't, on the whole, see much value in directly engaging in arguments for and against their existence however (any more, say, than arguing about whether or not imaginary numbers exist). The primary question surely is whether in postulating their existence we are able to explain and/or rationalise the role conditionals play in thought, talk and choice in a way that we cannot by denying their existence. Or whether, on balance, the explanation afforded by so-doing is sufficiently better (in terms of strength, simplicity and fit perhaps) than the alternatives on offer. DTHF shows what you can do with a theory which embraces counterfacts. What theory that does without them is even in the same ballpark in terms of strength, simplicity and fit? 5 Hajek's main reason for rejecting Counterfactual Plenitude is that we typically have no way of determining what the counterfacts are. He is surely right about this. If we had tossed a coin yesterday evening to settle what to have for dinner, would it have landed heads or tails? We usually cannot say. But questions about what exists cannot be settled by what we are able to discern. It is often difficult or even physically impossible to discern what is occurring very far away or what will occur far into the future, but this not in itself a decisive reason for doubting that there is some fact (some future or distant fact) of the matter as to what is or will occur. So why does Hájek think that these epistemic considerations are decisive in the case of conditionals? He asks whether it's true that 'If Sophie had gone to the parade, she would have seen Pedro dance', and concludes that it is not, because there are "so many possible relevant ways for Sophie to have gone to the parade and not seen Pedro dance: by standing at the wrong place, getting there at the wrong time, looking away at the crucial time, …" (Hájek, this volume). That there are many possible ways in which Sophie could fail to see Pedro dance is reason, Hájek thinks, for saying it must be false that she would have seen him dance. But equally there are many possible relevant ways for Sophie to have gone to the parade and seen him dance, so it must also be false that 'If Sophie had gone to the parade, she would not have seen Pedro dance'. We are led to conclude that most counterfactuals are false. It seems clear to me that this argument is mistaken. If Sophie had gone to the dance she would either have seen Pedro dance or she would not have. We don't know which is true-that she would have seen him dance or that she would not have-but we do know that one of them is. What I think is correct is that there is no fact about the actual world which determines which it is. From this I draw support for my claim that counterfactuals are not true at stand-alone possible worlds but at ordered sets of them. 'If Sophie had gone to the parade, she would have seen Pedro dance' is true at those ordered sets of possible worlds in which the possible counteractual world under the supposition that Sophie was at the parade is one in which she saw Pedro dance. Its false at those in which the counteractual world is one in which she failed to see him dance. No ordered set contains both or neither, since at every possible world in which Sophie goes to the parade she either sees Pedro dance or she does not, and not both. In summary, Hajek's mistake is to infer from the true claim that, for most ordinary counterfactuals, no set of facts about the actual world suffices to determine whether the counterfactual is true or not, to the false conclusion that there are no counterfacts. The choice we face, rather, is to infer either that there are no counterfacts and that most counterfactuals are not truth-apt or that the truth or falsity of counterfactuals is not determined by the facts but by the counterfacts. It is the latter path that I chose. Individuation and chance (Mongin & Baccelli; Goldschmidt & Nissan-Rozen) In their wide-ranging paper, Mongin and Baccelli (2020), (1) contest that the Bolker-Jeffrey theory of decision has the advantages I claim for it; (2) argue against certain uses of the 'redescription strategy' to defend expected utility (EU) theory, and (3) cast doubt on the version of this strategy that I use to defuse the Ellsberg paradox (Ellsberg, 1961). I will not say much about the first, as their discussion of the merits of using the Bolker-Jeffrey theory as the basis for reorganising normative decision theory is nuanced and insightful and I agree with many of their points. In particular, I agree that the use of an atomless algebra of propositions seems to be in tension with the project of developing a theory of rationality for bounded agents, especially when combined with the assumption of complete preferences. Indeed, this explains (in part) why I think that it is so important to develop a theory that allows for incomplete preferences, a project developed in later parts of the book. One point of disagreement however about the first issue. Mongin and Baccelli approve, I think, of the ambition of recovering Savage's expected utility theory from the Bolker-Jeffrey one. But they doubt that I have succeeded in doing so, because Bolker's uniqueness theorem is not strong enough for an SEU representation. In the book I rely primarily on Joyce's representation theorem to overcome this problem, but Mongin and Baccelli dislike the reliance on a second primitive in Joyce's framework, something which they point out is a departure from the main tradition in decision theory. This is all true. But I have proved a 'traditional' representation theorem for an enriched version of the Bolker-Jeffrey theory elsewhere (Bradley, 2007) and point out in the book that one could equally well work with it rather than Joyce's. So I think that the derivation of Savage's theory within the enriched Bolker-Jeffrey framework is solid. Let's turn to the second issue. Defendants of expected utility theory often deal with putative counterexamples to their theory by recourse to the 'redescription strategy', i.e., by arguing that if the elements of a decision problem are properly described, taking into account all that is relevant to the agent, then the counterexample is revealed to be no such thing. Mongin and Baccelli argue that such use of the redescription strategy should be neither indiscriminate nor lazy. Merely refining the outcomes in the Allais paradox to include feelings of regret or disappointment is an example, they claim, of such laziness since it serves merely to disarm the particular counterexample without offering any resources for predicting choices in similar circumstances. On these methodological grounds they prefer the route taken by non-EU theories, of proposing models which explain why we observe the patterns of choices that we do. As Mongin and Baccelli acknowledge, I don't actually endorse the instances of the redescription strategy that they dislike and indeed draw on their point (often ignored by philosophers) that the Allais preferences are primarily a problem for the von Neumann and Morgenstern theory and only indirectly (i.e., in combination with some other assumptions) for Bayesian versions of EU theory. More importantly, I think that they are entirely correct in arguing against indiscriminate uses of the redescription strategy. But under what conditions is such a strategy acceptable? Redescriptions should be based on generalisable claims about what factors matters to an agent's decisions that can be fruitfully employed to support predictions and explanations of their choices in a variety of situations. Redescriptions of states of the world will be useful, for instance, if they pick out features about which agents are uncertain and which they believe affect the outcomes of their choices, while redescriptions of consequences must pick out features that agents plausibly care about. These are not terribly precise criteria and it might take a while to determine whether a proposed redescription meets them or not. But the claim that individuals care not just about intrinsic properties of their circumstances (e.g. their income level) but also relational ones (e.g. how well off they are compared to others around them) is the sort of hypothesis that does meet the criteria, whereas I think the jury is still out as to whether the claim we experience feelings of regret does (the challenge being that of saying how such feelings depend on the formulation of the decision problem). Let me turn to the third issue raised by them: my approach to the Ellsberg paradox. In the book I show that the pattern of preferences exhibited in the Ellsberg paradox are justifiable within Bayesian decision theory, provided that we accept that agents are concerned not just about final monetary outcomes but also their chances of obtaining these outcomes. For if they are, then the outcomes of the different prospects in Ellsberg's set-up should be re-individuated in terms of the chances of monetary outcomes. This redescription suffices to show why no violation of the axioms of Bayesian decision theory (and in particular of the Sure-thing principle) is implied by the pattern of preferences identified by Ellsberg. Goldschmidt and Nissan-Rozen (2021) correctly interpret my project here of enriching Jeffrey's framework by taking chances of goods and bads themselves to be objects of agents' attitudes "as a way to pursue the re-individuation strategy while avoiding the threat of triviality" (this volume). Is this threat successfully avoided? I think that it quite clearly is. Firstly, the hypothesis that some agents do in fact value the chances of outcomes is empirically fruitful, as evidenced by the explanation it provides of a number of phenomena unconnected to those exhibited by Ellsberg's paradox. One is our concern for the fairness of procedures which, I argue, is in part a matter of sensitivity to the chances that these procedures confer. A second is the value we attribute to succeeding at tasks that are less-than-sure to succeed, something that is hard to explain if the relatively low chance of success at these tasks did not enhance their value. Secondly, the hypothesis is theoretically fruitful. This is amply illustrated by Goldschmidt and Nissan-Rozen's paper in which they show that it implies interesting constraints on value: in particular, that the desirability value of a risky prospect or lottery can be represented as the sum two of expectations, respectively of the prospect's intrinsic value and of its instrumental value. That these two kinds of value can be so neatly separated is both surprising and significant. As is a corollary of this, that both the expected intrinsic value and the expected instrumental value of a set of mutually exclusive and exhaustive prospects must equal zero. The non-triviality of the framework is thus clearly established by them. Mongin and Baccelli don't say otherwise directly. Instead, they claim that the redescription supported by my proposal is not the natural one and propose instead that outcomes be individuated in terms of the chances of drawing a ball of a given colour from an urn of a given composition. But why should agents care about this? Ball colours are (typically) of no concern to us in themselves; it is only because in the Ellsberg choice problem ball colours are associated with monetary amounts that we track them. So re-individuation of the states in terms of the chances of colour draws has some merit, but not re-individuation of the outcomes. Because they now omit from the description of the outcomes precisely what it is that agents might plausibly care about, they recover the violation of the Sure-thing principle. But this simply serves to confirm my original claim: that the paradox arises only if we don't take into account everything that agents care about. Unawareness (Mahtani) Decision Theory with a Human Face attempts to develop a theory of rational decision making tailored to agents that are limited in their cognitive resources. Such agents face decision problems without full awareness of all relevant considerations and without having a settled opinion on all those that they are aware of. And the sorts of attitude changes they can undergo are not restricted to updates in the light of newly acquired information: they also include suspension of opinion, formation of opinion through deliberation and inference (rather than information acquisition), and the gain in (and sometimes loss of) awareness of the possibilities they face. The standard Bayesian theory of conditionalization is not adequate as a model of such changes and so there is a need to supplement it with further principles. Mahtani's (2020) paper is concerned with the principles applying to situations in which an agent is initially unaware of one or more possibilities. In my book I define such a situation as one in which judgements about certain possibilities are not available to an agent when deliberating about what to think or do. Mahtani is critical of this characterisation on the grounds that one may be aware of, and have attitudes, to possibilities at some moment of time, even if these are not available to one's consciousness at that time. I don't deny this. On the contrary, 'availability' is something that comes in grades, spanning from cases in which a possibility is immediately available to judgement, through those in which they are accessible given enough time and effort, all the way to those in which possibilities are essentially inaccessible without some external intervention. Perhaps the word 'unawareness' should only apply to the latter cases and the others are better characterised as states of inattention. In any case the differences are important and Mahtani is right to ask for greater clarity. What I propose as a starting point is a four-tiered model. In the inner core are one's attitudes to the prospects to which one is paying attention. In one layer out from them are the prospects to which one has an attitude but to which one is not paying attention (the objects of implicit attitudes). In the next, those of which one is aware but to which one is not paying attention and towards which one has not formed an attitude. Finally in the outer layer are those prospects of which one is strictly unaware. Right now, for instance, I am paying attention to the question of what rationality requires of us, but not as to whether there is milk in the fridge. On this question however I do have a belief (now brought into my consciousness by the process of writing), namely that there is. On other questions, such as whether most residents in Kathmandu keep milk in their fridges, I have no opinion (though this entirely conceivable possibility, now having been brought to my attention, is one on which I could form an opinion with a little effort). Finally, there are no doubt all sorts of possibilities that I cannot at present conceive of, but of which I cannot of course provide an example! (Examples applying to others are easily found however: surely, for instance, ancient Egyptians had no opinion on which of the current smartphones has the longest battery life and could not have formed an opinion because they lacked the conceptual resources required to conceive of smartphones.) Mahtani focuses her discussion on the intermediate layers, consisting of possibilities outside of my immediate attention, but firmly within the realm of conceivability. These are just the sorts of things that one gives attention to if prompted by the circumstances and about which one has little difficulty in forming an opinion. I agree with her that cases like these are often discussed in the literature on unawareness. But that doesn't mean that there aren't cases in which new possibilities are brought to one's attention about which one has no information at all. Moreover, it seems to me that it is better to start with such cases (i.e. those involving no new information), so that one can better separate the effects of a change in the domain of one's awareness from changes that result from the gain of new information and from making new inferences. Suppose for instance that you are not aware that more than two horses can be entered for a race and that you have invested a good deal of effort in gathering information and forming a view as to how probable it is that either of the two entrants, Speedy and Steady, will win. Suppose that at the last minute you are told that there is another horse in the race about which you know nothing. How should this discovery affect your credences? In my book I apply three principles to address this question. 1. Success: Your new domain of awareness should contain the possibility of this 3 rd horse running (and winning). 2. Consistency: Your new credal state should be consistent. Conservatism: If what you learn does not give you reason to revise some aspect of your credal state, then you shouldn't revise it. It is the last of these conditions that does most of the work. For when conjoined with the thought that merely learning of new possibilities does not change the balance of reasons for and against old possibilities it implies that the relative probability of Speedy and Steady should not change just because another horse has been entered. 6 This is not to deny that the entertaining of new possibilities can trigger new enquiry or inference that ultimately leads to revisions of ratios of old probabilities (as the examples of Steele and Stefansson 2020 show). But this should be modelled separately, downstream from the initial response to the growth in awareness. The view that a rational agent will, in situations in which they are made aware of a new possibility but acquire no further information about it, adopt new credences that preserve the ratios between credences in all propositions in her old algebra is what Mahtani calls Reverse Bayesianism (hereafter RB). 7 In her paper she presents an objection to it based on a pair of contrasting examples, one involving an extension to the set of possibilities and one involving a refinement of it. She also argues that the problem of awareness is misconstrued by me and that a solution to it is available within a broadly Bayesian framework by adopting a dispositionalist account of credence. Mahtani's objection to RB is completely sound, but her examples raise complicated issues about how propositions are to be individuated when we shift from one algebra of possibilities to another that are not immediately apparent. So let's put her objection in more abstract form, which will also serve to show how general it is. Without loss of generality consider a 2-element partition {L, R} of the state space e.g., the partition {Landlord, Tenant}. Suppose that you become aware of a new possibility M (e.g., Other}, distinct from both, so that the {L, R} partition must be extended to the {L, R, M} one. Then RB requires that the relative probability of L to that of R stays the same. But an extension of the {L, R} partition by M is also a refinement of the {L, ¬L} partition by M and ¬M, since R ¬L ∧ ¬M, L L ∧ ¬M and M ¬L ∧ M. So RB also requires that the probabilities of L and ¬L stay the same. These two constraints are consistent only if we assign probability zero to M, which trivialises the whole thing. Mahtani argues that extending attitudes to possibilities of which one was previously unaware requires more information than Reverse Bayesianism draws on. This information can come, she contends, from our prior unconscious attitudes to the propositions that enter into our awareness. Indeed she suggests that the whole problem of belief change under growing awareness as I have posed it should be rejected because it presupposes that one cannot possess an attitude to propositions of which one is not conscious. In contrast, on the dispositionalist view of attitudes (which she recommends), attitudes are constituted by behavioural dispositions of one kind or another: to assent to an utterance, to accept a proposition or to bet on its truth, when prompted to do so. Since one may be in possession of the relevant disposition without being aware of its object, the dispositionalist doesn't think the question of how to extend one's attitudes to new objects ever arises. Mahtani is right in saying that the problem of awareness growth cannot be solved without more information than RB uses, but I don't think that what she proposes constitutes an adequate solution to it. I cannot survey all possible dispositionalist accounts that might support her account, but I think they will all fail for much the same reason. So let's just focus on the version that says that to believe X to degree x is to be disposed to bet on X at odds x when offered the opportunity to do so. Clearly X does not have to be available to consciousness for this criterion to be applied and so a dispositionalist can argue that the problem of what belief to adopt to a proposition when one first becomes aware of it doesn't arise: one already has a belief! Only if its coming into consciousness brings new relevant information in its wake is anything required of one. And in this case the appropriate response (says Mahtani) is to conditionalize one's degrees of belief on the new evidence. So no modification of the Bayesian model is required in order to handle cases of new awareness. But how can a disposition to bet at some odds serve as a justification for having the corresponding degree of belief? Imagine that I have never heard of a quetzal. Suppose also that I am disposed to accept bets at even odds on the truth of any proposition concerning objects of whose existence I had been previously unaware that have a name beginning with the letter 'q'. So I am disposed to bet at even odds, for instance, on the proposition that quetzals can fly. But what of it? It is neither correct to say that in virtue of this disposition I have a credence greater than 0.5 in quetzals being able to fly, nor that I should adopt this credence when I become aware of their existence. Such betting dispositions only serve as plausible markers for credences in circumstances in which someone is able to rationally assess the expected value of the bet-which in these cases they cannot. So Mahtani's solution is not satisfactory. I prefer the one proposed by Roussos (2020), which starts with the observation that refinement and extension of the set of possibilities one conceives of involve different embeddings of one's old algebra into the new one. To illustrate consider the two lattices respectively based on the two sets of basic possibilities {L, R} and {l, m, r }. There are several ways in which the first 4-element lattice can be mapped onto the second 8-element one, each of which determines a consistent application of RB. There is, for example, the 'extension' mapping E such that E(L) l, E(R) r , E(L ∨ R) l ∨r and E(L ∧ R) l ∧m ∧r . And there is the 'refinement' mapping R such that R(L) Given the extension mapping, RB says that relative probabilities of l and r should equal those of L and R. Given the refinement mapping, on the other hand, it says that relative probabilities of l and m ∨ r should equal those of L and R. It should be clear therefore why RB generates an inconsistency if we apply it simultaneously to multiple embeddings. In the counterexample to RB presented before for instance I applied both the extension and refinement mappings that have just been characterised. But this was a mistake: in adopting a new algebra of possibilities following the growth of awareness, I should not treat both r and m ∨ r as the counterparts to the element R of the old algebra. To apply RB one must first choose an embedding. Sometimes an extension embedding seems more sensible, sometimes the refinement one. In Mathani's first case, involving the landlord and tenant, new awareness of the possibility that some other person might be around is intuitively a case of extension. On the other hand, her second 'coin' case, is intuitively one in which we refine the tail-possibility. So the two situations are quite different and this fact should be represented in a satisfactory account of awareness growth. There is clearly still much work to be done in providing one. First, RB needs to be reformulated so that it takes as the input to the attitude revision procedure not just an old and new algebra, but also an embedding of the former into the latter. Second, RB needs a more substantial account of the grounds for embedding in one way rather than another. And finally, there is the task of showing how this account of pure awareness growth combines with other mechanisms for attitude change and in particular those based on acquisition of evidence and on inference. Ambiguity (Steele) In DTHF I argued that in circumstances of informational poverty agents may reasonably eschew adoption of precise degrees of belief for all relevant contingencies and yet nonetheless act as if they had precise beliefs when making a choice. The idea was that she could in effect temporarily adopt a probability for decision purposes but without long-term commitment to it, so that the adopted probability did not constrain subsequent belief revision. Steele (2020) argues that such 'as-if' Bayesianism is unsatisfactory because it doesn't protect the agent from the possibility of sure losses in diachronic decision problems. The dilemma Steele sets up is the following. Consider a decision problem, such as the diachronic version of the Ellsberg paradox, that requires an agent with imprecise credences to make a first decision at time t 0 and then a second one at later time t 1 when she has received some new evidence. Suppose that at t 0 the agent selects a precise 'prior' probability for decision purposes in accordance with her adopted rule of choice for proxy beliefs and maximises expected utility relative to this probability. Suppose that she does this again at t 1 , by first revising her initial imprecise set of credences by point-by-point conditionalization, selecting one from the updated set in accordance with her choice rule, and then maximising expected utility relative to the chosen probability. By standard arguments if the probability function chosen at t 1 is not equal to the prior chosen at t 0 , conditioned on the newly acquired evidence, then she will be vulnerable to sure loss. Steele shows that neither of the two rules I consider-MaxEnt and linear averaging-satisfy this requirement. In general, as she points out, only a rule of proxy choice that satisfies the External Bayesianity condition will ensure that it is respected. 8 So her argument leaves us with three options. 1. We can adopt a rule of proxy selection that satisfies External Bayesianity (geometric averaging being the most salient example). 2. We can deny that the possibility of sure losses counts decisively against a decision method. 3. We can deny that an agent should handle diachronic decision problems in the manner sketched above. All three routes seem open to me. Geometric averaging is a form of averaging with many advantages and perhaps the guarantee of immunity to sure loss is sufficient reason to use it for proxy selection. But I don't personally regard such immunity as decisive: it is just one consideration to be weighed against others. If one's preferences change over time, this can make one vulnerable to exploitation by someone who anticipated how they change (see DTHF,p. 286). But that doesn't make it rational to stick with one's preferences just in order to avoid such a possibility. Finally, even if avoiding sure loss is a decisive consideration, an agent can still assure it by being resolute in her choice of proxy. In particular, I think there is more to the second resolute strategy identified by Steele than she grants. When the less-thanfully opinionated agent adopts precise degrees of belief for the purposes of decision making she can reasonably do so for the entirety of the decision problem at hand, without needing to re-apply the selection rule at every node. For the idea is simply to adopt a set of beliefs to work with, recognising that although the choice of this set is not determined by the evidence she holds, it is not totally arbitrary either. That this entails that her precise credence at t 1 is different from the one that would be selected from the set of probabilities obtained by conditioning her initial imprecise credal state on any new evidence, is arguably neither here nor there.	2022-12-25T15:24:38.370Z	2022-02-01T00:00:00.000	{ "year": 2022, "sha1": "e8cb78a12cb8cf5f47b8cd649208aa2588b4fa01", "oa_license": "CCBY", "oa_url": "https://link.springer.com/content/pdf/10.1007/s11229-022-03468-6.pdf", "oa_status": "HYBRID", "pdf_src": "SpringerNature", "pdf_hash": "e8cb78a12cb8cf5f47b8cd649208aa2588b4fa01", "s2fieldsofstudy": [ "Philosophy" ], "extfieldsofstudy": [] }
1536394	pes2o/s2orc	v3-fos-license	Videoarthroscopic treatment of glenohumeral osteoarthritis* Objetive To evaluate possible benefits obtained through the use of surgical videoarthrosco- py in the management of glenohumeral osteoarthritis. Methods We evaluated 37 patients (38 shoulders) who underwent through surgical videoar- throscopy in the period between November 1999 and May 2009 (minimum follow-up of two years). Twenty five patients attend for revaluation and thirteen were interviewed by telephonic contact. Functional assessments were performed (UCLA, Constant, and measu- rement of range of motion –ROM-), as well as pre and post surgical radiographics. We eva- luated the influence of the following factors in the final results: the presence of chondral lesions, joint space narrowing, osteophyte presence, associated injuries (rotator cuff torn or instability), and follow-up. Among those patients interviewed by phone we evaluated the satisfaction level and if they would submit themselves again to the surgical procedure. Results It was observed significant gain towards to the function (UCLA) and the internal rotation, as well as the association between dissatisfaction and pre surgical joint space reduced. Among the operated patients, 84% were satisfied with the results and 86.6% would repeat the procedure. Conclusion Surgical videoarthroscopy presents a relevant role in management of the glenohumeral osteoarthritis, providing improvement of functional results and high levels of satisfaction. Introduction Osteoarthritis of the glenohumeral joint is not uncommon and may affect more than 20% of the elderly population. Its therapeutic management begins with conservative methods, with the aims of alleviating painful symptoms and improving range of motion. Lifestyle changes, analgesic and anti-inflammatory medication, physiotherapy, joint infiltrations with corticoids and viscosupplementation have been mentioned in the literature. [1][2][3][4][5] When conservative methods fail, total arthroplasty or hemiarthroplasty provide significant relief of painful symptoms and functional improvement, particularly in more elderly populations (over the age of 60 years). However, in younger populations (under the age of 50 years) that are active, these procedures do not present the same results, due mainly to the high functional demands made by this age group, their functional expectations and the length of survival of the implants, especially the glenoid component. 1,4,6,7 Among patients with this profile, arthroscopic management may provide relief for painful symptoms and functional improvements. However, it is incapable of restoring joint cartilage that presents lesions. 8,9 The arthroscopic procedures of lavage and debridement provide satisfactory short-term results for 70 to 88% of these patients. [8][9][10] The aim of the present study was to evaluate the results from videoarthroscopic treatment among patients with glenohumeral osteoarthritis. shoulders operated. Five patients (seven shoulders) were excluded from the study due to death; three patients were excluded because their cases evolved to arthroplasty; 18 patients (20 shoulders) were excluded because they could not be contacted; and two patients (two shoulders) were excluded because they refused to supply data for the investigation. The patients selected were evaluated before and after the operation. The preoperative evaluations were done by reviewing the medical files and the initial radiographs, and the following data were gathered: age, gender, dominance, side affected, ranges of motion (EAA, EAP, ER I, ER II and IR), radiographic evaluation of the joint space (in the true anteroposterior and simple axillary lateral views), 11 radiographic classification according to Samilson and Prieto 12 ( Fig. 1), function evaluation using the University of California at Los Angeles (UCLA) score and presence of associated lesions. Methodology We reviewed the operative records of each patient, thus obtaining the classification of the chondral lesion as described by Outerbridge 13 (Fig. 2), and the surgical procedures performed (debridement, resection or non-resection of osteophytes or microfractures and treatment of associated lesions). The results from capsulotomy and microfractures years of follow-up with those with less than five years of follow-up. We also evaluated the influence of the length of follow-up on the functional results, in comparing the groups of chondral lesions (mild to moderate versus advanced), size of osteophyte (Samilson 1 and 2 versus Samilson 3) and joint space (preserved versus reduced), since a difference in length of follow-up between the groups compared might directly influence the results. In relation to the associated lesions, we identified two groups of patients: one with rotator cuff injuries and the other with instability (Bankart or Slap). Two patients (two shoulders) who presented both instability and rotator cuff injury in association were taken to belong to the group of rotator cuff injuries. Among the patients who said that they were dissatisfied in the postoperative subjective UCLA evaluation, we analyzed which preoperative factors (degree of chondral degeneration, Samilson classification stage and joint space) contributed towards that level of satisfaction, along with the influence of the length of follow-up. The statistical analysis was done using the resources of the PASW statistical software, version 18. The results were described in terms of descriptive measurements for quantitative variables and frequency tables for the qualitative variables analyzed. The significance of the chondral degeneration, Samilson and Prieto stage, joint space, length of follow-up and resection of the osteophyte among the patients with Samilson and Prieto stage 3, in relation to the functional result, along with the influence of the length of follow-up on the patients level of satisfaction, was evaluated using nonparametric Mann-Whitney tests. The differences between the preoperative and postoperative ranges of motion and UCLA scores were assessed using the nonparametric Wilcoxon test. Contingency tables were used to correlate the patients' degree of satisfaction with the preoperative factors of chondral degeneration, Samilson and Prieto stage and joint space. Fisher's chi-square test was used to investigate the statistical significance of associations between these variables. To compare the means of the Constant and UCLA variables of the group of patients with associated pathological conditions present (rotator cuff injury or instability) with those of the patients in the full sample, the t test for one sample was used. In this study, the specific value to be tested was the calculation of the general mean of 24 patients for the Constant and UCLA variables. In all the statistical tests used, the significance level was taken to be 5%. Thus, associations were considered to be statistically significant if the p value was less than 0.05. Results Among the 24 patients (24 shoulders) on whom the functional results were analyzed, the preoperative mean UCLA score was 16 and the postoperative score was 28, with a significant difference between them (p = 0,000) (Fig. 3). The mean postoperative Constant score was 71.8. were not assessed separately, since these were performed on limited numbers of patients (four and three, respectively). The postoperative evaluations were made by two independent examiners who had not participated in the surgical procedures. Twenty-four patients came for the examination (24 shoulders), at which they underwent assessments of range of motion (EAA, EAP, ER I and ER II by means of a goniometer and IR by means of the difference in vertebral level achieved, between the operated and contralateral sides) and the length of postoperative follow-up, with radiographic evaluation (preservation or non-preservation of the joint space and the Samilson and Prieto classification) and functional evaluation by means of the Constant and UCLA scores. The patients were also asked whether they would go through the same surgical procedure again if necessary. The 13 patients (14 shoulders) who were unable to come for the physical examination were interviewed over the telephone and were evaluated regarding their current degree of satisfaction (satisfied or dissatisfied, in accordance with the UCLA score) and whether they would go through the same surgical procedure again. One patient in this group had undergone treatment in both shoulders: we considered evaluating each shoulder separately, since we judged that the subjective evaluation on one would not influence that of the other. The preoperative and postoperative ranges of motion and functional evaluations were compared. We investigated the influence of the degree of chondral degeneration, the size of the humeral or glenoid osteophyte (in accordance with the Samilson and Prieto classification), degree of preservation of the preoperative joint space, the length of postoperative follow-up and the presence of rotator cuff injuries or instability associated with glenohumeral osteoarthritis in postoperative functional assessments (Constant and UCLA). We divided the occurrences of chondral degeneration into two groups: one with Outerbridge grades 1, 2 and 3 (mild to moderate chondral degenerations) and the other with Outerbridge grade 4 (advanced chondral degenerations). In relation to the size of the osteophyte, one group included patients with osteophytes smaller than 8 mm (mild and moderate arthrosis; Samilson and Prieto stages 1 and 2) and the other group included patients with osteophytes larger than or equal to 8 mm (advanced arthrosis; Samilson and Prieto stage 3). Among the patients in the second group (osteophytes larger than 8 mm), the influence of resection of the osteophyte on the postoperative functional result was analyzed. The length of postoperative follow-up among the patients evaluated ranged from 2 to 11 years. To analyze its importance regarding the postoperative functional evaluations, we compared the results between the patients with up to five the patients with advanced degeneration, 4.53 years; this difference was not significant (p = 0.402) (Fig. 4). Thirteen of the 24 patients presented reduced joint space (less than 2 mm) and 11, preserved joint space in the preoperative radiographic evaluation. In the preoperative functional evaluation, the UCLA of the first group was 15 and of the second group, 21, without any significant difference between these values (p = 0.081). After the operation, the patients with reduced joint space presented UCLA of 26 These 24 patients presented the following mean preoperative ranges of motion: active anterior elevation = 160°, passive anterior elevation = 160°, external rotation with arm beside the body = 50°, external rotation with the arm abducted at 90° = 70° and medial rotation limited to six vertebral levels. The postoperative values were as follows: active anterior elevation =155°, passive anterior elevation = 160°, external rotation with arm beside body = 45°, external rotation with arm abducted at 90° = 78° and internal rotation limited to three vertebral levels. With the exception of the internal rotation (p = 0.043), there were no difference between the preoperative and postoperative range of motion measurements (Table 1). Among the 24 patients, 12 presented mild to moderate chondral degeneration (Outerbridge 1, 2 and 3) and the other 12 presented advanced chondral degeneration (Outerbridge 4). In the preoperative functional evaluation, the UCLA score of the first group was 18.5 and of the second group, 17, and there was no significant difference between these values (p = 0.706). After the operation, the patients with mild to moderate abnormalities presented UCLA of 29.5 and Constant of 75. The patients with advanced abnormalities presented UCLA of 27 and Constant of 78. The differences between the two groups were not significant (p = 0.367 and p = 0.862). The mean length of follow-up for the patients with mild to moderate degeneration was five years and for There was an association between unsatisfactory results in the subjective assessment and the presence of reduced preoperative joint space (p = 0.024) ( Table 6). Among the 37 patients included in the study, 33 (89%) would go through the surgical procedure again. (Fig. 7). Among the 24 patients, 13 presented lengths of follow-up less than or equal to five years and 11 presented follow-ups longer than five years. The preoperative UCLA scores (15 and 15), postoperative UCLA scores (28 and 28) and postoperative Constant scores (77 and 74) did not present any significant differences (p = 0.931, 0.907 and 0.642). For these 24 patients, the mean preoperative UCLA and postoperative Constant were respectively, 16.6, 25.6 and 71.9; the mean length of follow-up among this population was 5.3 years. Of these patients, 16 presented rotator cuff injuries associated with osteoarthritis and had preoperative UCLA = 17.5, postoperative UCLA = 24.3 and postoperative Constant = 70.6; the mean length of follow-up among these patients was 5.25 years. There was no significant difference between these values and those of the compete group (p = 0.503, 0.540, 0.740 and 0.929) ( Table 2). The five patients in whom the associated pathological condition was instability presented preoperative UCLA = 14.8, postoperative UCLA = 25 and postoperative Constant = 74.4. The mean length of follow-up among these patients was 4.84 years. There was no significant difference in these values in comparison with the complete group (p = 0.403, 0.860, 0.647 and 0.413) ( Table 3). The values in parentheses are the standard deviations of the means for each value. Discussion Glenohumeral osteoarthritis may result in significant functional incapacity. From the patient's perspective, the impact from this pathological condition is comparable to that of chronic comorbidities such as congestive heart failure, diabetes and coronary diseases. 1 Clinically, such patients present with pain, which may interfere with their nighttime rest; and also with overall loss of range of motion with occasional blockade, which may be due to free intra-articular bodies. 2 Pain at the extremities of movements may result from impact syndrome, whereas pain in the middle of the range, particularly below shoulder level is associated with mechanical symptoms. 14 In physical examinations, the symptoms of chondral lesions may resemble those of other intra-articular or extra-articular diseases, such as subacromial impact, tenosynovitis of the biceps and labral lesions. 3,14,15 On inspection, muscle hypotrophy and bone prominences are searched for, and the scapular-thoracic rhythm is assessed. The range of motion, both passive and active, generally presents limitations. 3,14, 15 Ellman described a compressionrotation test that helps to differentiate chondral lesions from impact syndrome: a internal and external rotation maneuver with the arm beside the body, at the same time as performing compression of the humeral head in the direction of the glenoid, which is done before and after bursal infiltration using lidocaine. The symptoms that are alleviated on the second occasion are related to impact syndrome. 14 In radiographic evaluations, glenohumeral osteoarthritis is classically characterized by asymmetrical reduction of the joint space, subchondral sclerosis, cyst formation and osteophyte formation (in the humeral head or glenoid). Free bodies can be seen inside the joint. 2 Samilson and Prieto developed a classification system that was originally described for degenerative joint alterations resulting from arthropathy due to instability, which today is applied to arthrosis of other etiologies. The classification takes into consideration the size of the osteophyte, whether it is located inferiorly on the humeral head or on the glenoid, and any presence of irregularities on the joint surface, observed in anteroposterior radiographic view of the glenohumeral joint. The arthrosis is mild if the osteophyte is smaller than 3 mm, moderate if between 3 and 7 mm, in association with mild irregularity of the joint surface, and severe if greater than 7 mm, in association with diminished joint space and bone sclerosis. 12 The size of the osteophyte is correlated negatively with range of motion. 16 In arthroscopic evaluations, chondral lesions are classified in accordance with the system proposed by Outerbridge. 13 Grade 1 represents softening of the cartilage. Grade 2 presents fragmentation and fissures covering an area less than or equal to 1.5 cm in diameter. Grade 3 presents fragmentation and fissures covering an area greater than 1.5 cm in diameter. Grade 4 presents erosion of the subchondral bone. These chondral lesions can be found in 5% to 17% of routine arthroscopic evaluations. 3,4 Total arthroplasty or hemiarthroplasty provides significant pain relief and functional improvement, especially in more elderly populations (over the age of 60 years). 1,4,6,7 On the other hand, Sperling et al. 17 observed that among patients under the age of 50 years who underwent hemiarthroplasty or total arthroplasty of the shoulder, the rate of unsatisfactory results was approximately 56%, thus suggesting that for this group of patients, another therapeutic approach should be used. Several studies in the literature have demonstrated good results from arthroscopic approaches for treating glenohumeral osteoarthritis. Ogilvie-Harris and Wiley 15 conducted a retrospective analysis on 439 patients who underwent arthroscopic shoulder surgery and found 54 cases of glenohumeral osteoarthritis. Of these, 29 presented associated diseases. These patients underwent removal of the arthroscopic debris and chondral fragments, and synovectomy. Satisfactory results were achieved in twothirds of the patients with slight degenerative alterations (superficial lesions in the joint cartilage) and on one-third of the patients with severe degenerative alterations (with exposure of the subchondral bone). Ellman et al. 14 reported on a group of 18 patients with degenerative joint diseases the clinically resembled impact syndrome. Among these 18, ten came to a diagnosis of glenohumeral osteoarthritis even before the operation. During the operation, no cases of complete rotator cuff injury were found, but partial joint lesions were found in five shoulders (three A1 and two A3). The arthroscopic procedures consisted of debridement of the unstable cartilaginous fragments, removal of free bodies and partial synovectomy, Subacromial decompression was performed in 15 patients. Among the 18 patients, ten presented a minimum duration of symptom relief greater than six months. Richards and Burkart 18 presented preliminary results from arthroscopic debridement associated with release of the rotator interval and capsulotomy, for treating glenohumeral osteoarthritis. In addition to pain reduction, there were increases in anterior elevation, external rotation and internal rotation. The alleviation of the painful symptoms was due to elimination of the joint debris and diminution of the joint contact pressure. Weinstein et al. 10 followed up 25 patients for 12 months, who had undergone arthroscopic debridement of glenohumeral osteoarthritis. Nine of these patients presented an associated disease. The procedures for treating the osteoarthritis consisted of arthroscopic lavage, debridement of labral and cartilaginous lesions, removal of free bodies, partial synovectomy and resection of the osteophyte, in addition to treatment for the associated diseases. At the end of the follow-up, it was observed that 8% of the results were excellent, 72% good and 20% unsatisfactory. There was no statistical correlation between good results and the degree of radiographic alterations and degenerative joint alterations. Pain was the most important factor in evaluating the patients. Cameron et al. 9 retrospectively analyzed 61 patients who underwent debridement, with or without associated capsulotomy, for treating grade IV chondral lesions. The patients were divided according to the location of the lesion (humeral, glenoid or bipolar) and the size of the osteochondral defect (greater than or less than 2 cm 2 ). The indication for capsulotomy was a restriction of more than 15° in any plane of the range of motion. Improvements in painful symptoms were observed in 88% of the patients, based on a visual analogue pain scale and on the increase in the score of the American Shoulder and Elbow Surgeons (ASES). Among the patients, 87% stated that they would undergo this surgical procedure again, if necessary. The location and size of the lesions did not have any influence on the improvements in pain and functional scores. Kerr and McCarty 19 analyzed 19 patients (20 shoulders) who underwent arthroscopic debridement to treat glenohumeral osteoarthritis. No difference in functional results was found between the patients with mild-tomoderate degenerative alterations (Outerbridge 2 and 3) and those with advances degenerative alterations (Outerbridge 4). However, patients with unipolar impairment presented better results than did those with bipolar impairment. Van Thiel et al. 20 followed up 71 patients with glenohumeral osteoarthritis who underwent arthroscopic debridement. Of these, 22% evolved to arthroplastic procedures after a mean of 10 months of follow-up, while 78% continued without arthroplasty over a follow-up of 27 months. The group of patients who did not evolve to arthroplasty presented larger joint spaces and lower stages in the Samilson classification from preoperative radiographs and, at the end of the follow-up, better functional results and fewer painful symptoms. In this group of patients, 87% said that they would undergo this procedure again. In our series of patients, we obtained a significant difference in UCLA scores from before to after the operation (p = 0.000), and this was concordant with previous studies in relation to functional improvement. The mean postoperative Constant score was 71.8, which was considered satisfactory. We did not find any relationship between the functional results and the degree of chondral degeneration (p = 0.367 and 0.862 for the postoperative UCLA and Constant scores), and this was concordant with what had previously been reported by Weinstein et al., 10 Kerr and McCarty 19 and Cameron et al. 9 The reduction in joint space also did not influence the functional results (p = 0.153 and 0.663 for the postoperative UCLA and Constant scores), thus resembling the findings of Van Thiel et al. 20 There was a tendency (p = 0.081) for the preoperative UCLA to be greater in the patients with preserved joint space. We did not find any correlation between the Samilson classification stages (osteophyte size) and the functional results (p = 0.727 for the postoperative UCLA and Constant scores), which was concordant with the reports of Weinstein et al. 10 Although the radiographic classification used in that study had been drawn up at their own clinic, it resembled the Samilson classification with regard to progression of the osteophyte. On the other hand, Van Thiel et al. 20 presented better functional results among patients with lower Samilson stages in the preoperative radiographic evaluation. Among our patients with osteophytes larger than 8 mm, there was no influence on the functional results caused by resecting the osteophyte (p = 0.730 and 0.864 for the postoperative UCLA and Constant scores). Neither Van Thiel et al. 20 nor Weinstein et al. 10 mentioned any influence from resecting the osteophytes on their results. Our sample presented a mean follow-up of 5.13 years, with a range from 2 to 11 years. There was no difference in the functional results between the group of patients with less than five years of follow-up and those with more than five years of follow-up (p = 0.907 and 0.642 for the postoperative UCLA and Constant scores), thus suggesting that the improvement in functional results could be long-lasting. The length of follow-up also did not interfere with the functional evaluation when we took into account the degree of chondral degeneration, Samilson classification stage or joint space. In relation to the length of follow-up, our study differs from the remainder of the literature, in which the length of follow-up was a maximum of two years. 20 We found high incidence of rotator cuff injuries and instability associated with glenohumeral arthrosis. An association between glenohumeral arthrosis and both intra and extra-articular disease had already been mentioned in the studies by Ogilvie-Harris and Wiley 15 and Ellman et al. 14 Although our sample was of limited size, we did not find any influence from these diseases on the functional result (postoperative UCLA and Constant, with p = 0.540 and 0,740 in patients with rotator cuff injuries, and p = 0.860 and 0.647 in patients with associated instability). In relation to the influence of rotator cuff lesions on treatments for glenohumeral osteoarthritis, Wirth et al. 21 observed that small lesions, independent of whether they had been repaired concomitantly with the arthroplastic procedure, did not interfere with the final result from hemiarthroplasty. Iannotti and Norris 22 analyzed the influence of preoperative factors on the results from shoulder arthroplasty for treating glenohumeral arthrosis, and found that small repairable rotator cuff injuries that were limited to the supraspinatus did not affect the postoperative score of the American Shoulder and Elbow Surgeons (ASES). In our series of patients, all the associated rotator cuff lesions were successfully repaired and, although the treatment type was different, the results were concordant with what had been proposed by Iannotti and Norris 22 and Wirth et al., 21 regarding the presence of repairable lesions of the rotator cuff associated with glenohumeral osteoarthritis. Millett and Gaskill 23 presented their preliminary results. They suggested that the lower osteophyte might compress the axillary nerve close to the lower capsule, thereby causing symptoms similar to those of quadrilateral space syndrome. In addition to extensive joint debridement, capsulotomy and resection of the lower osteophyte, decompression of the axillary nerve was performed. Among their 26 patients (27 shoulders) with a mean follow-up of 20 months, there was an increase in the satisfaction rate, diminution of pain, increase in mean range of motion and improvement of the ASES score. One of the patients in our sample (M.A.B.N) underwent arthroscopic debridement at the age of 29 years. Radiographically, he had a lower osteophyte in the humeral head that was larger than 8 mm (Fig. 8); and clinically, he presented painful limitation of the range of motion, along with pain on the posterior face of the shoulder, thus suggesting axillary nerve compression. After arthroscopic debridement and complete resection of the osteophyte (Fig. 9), this patient evolved with improvement of the range of motion and painful symptoms. Differently to what was proposed by Millet, we did not do any intraoperative controls using fluoroscopy. At the end of the surgical procedure, radiography was performed in true anteroposterior view, in order to verify the resection of the lower osteophyte. We also did not perform additional decompression of the axillary nerve, and resection of the osteophyte was sufficient for improving the compressive symptoms. After five years of follow-up, the patient is satisfied with the procedure that was performed, with few painful symptoms and the following range of motion: EAA = 170°, ER I = 30°, ER II = 70° and IR = 5 th lumbar vertebra (Fig. 10). In assessing the level of satisfaction, in addition to the 24 patients (24 shoulders) examined, we also analyzed the 13 patients (14 shoulders) who were contacted by telephone. Out of these 38 shoulders, 32 (84%) presented satisfactory results in the subjective UCLA evaluation. Among the unsatisfactory results, we did not find any correlations with the degree chondral degeneration (p = 0.645), preoperative Samilson classification stage (p = 1.000) or length of follow-up (p = 0.542). On the other hand, there was a significant association between shoulders with unsatisfactory results from the subjective assessment and reduced joint space in the preoperative radiographic evaluation (p = 0.024). Although this association was from a subjective assessment, it followed the trend of the results of Van Thiel et al., 20 in which the patients with preserved joint space before the operation presented better functional evaluations and fewer painful symptoms at the end of the follow-up. In the same way, these authors reported that 87% of the patients would go through the same surgical procedure again, which did not differ from our results, in which 89% would go through the procedure again. This is directly related to the patients' level of satisfaction. In our sample, we had a significant loss of patients from the follow-up. Out of the 65, five died for reasons unrelated to the surgical procedure, 18 could not be found because of changes of address and two refused both to come for the examination and to undergo subjective assessment over the telephone. Three patients evolved to total arthroplasty within two years after arthroplastic debridement and were therefore excluded from the data analysis. Out of the 37 patients (38 shoulders) that remained, 13 (14 shoulders) were unable to come for a physical examination (eight of them were living in other cities, which made it impossible to come for the examination). Among these patients, the assessment was made by means of telephone contact. Functional results from 24 patients were analyzed. We did not find any significant difference in the functional results when we took into account the degree of chondral degeneration, size of the osteophyte, preservation of the joint space, length of postoperative follow-up and presence of rotator cuff injuries. This may have been due to the limited number of patients, which might have interfered with the statistical analysis. Although the postoperative Constant score presented a mean of 71.8, which was considered satisfactory, we did not have a preoperative value for evaluating the functional gain and for adding value to the functional gain obtained through the UCLA score. Other limitations of our study included the retrospective study model and the lack of a control group. Future studies using a prospective model, with a control group and with fewer losses from the follow-up are needed in order to consolidate our results. Conclusion Arthroscopic management of the arthrotic shoulder provided improvement of the functional results and high satisfaction levels. The reduced joint space in the preoperative radiographic assessment negatively influenced the satisfaction level in the final evaluation. Conflicts of interest The authors declare that there was no conflict of interests in conducting this study.	2017-10-20T17:17:06.012Z	2013-06-11T00:00:00.000	{ "year": 2013, "sha1": "1ecc483ef705918e144be588acfec7380f1da3cd", "oa_license": "CCBYNCND", "oa_url": "https://doi.org/10.1016/j.rboe.2011.05.001", "oa_status": "HYBRID", "pdf_src": "PubMedCentral", "pdf_hash": "1ecc483ef705918e144be588acfec7380f1da3cd", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
245543507	pes2o/s2orc	v3-fos-license	A Simple Predictive Score to Distinguish between Disseminated Histoplasmosis and Tuberculosis in Patients with HIV Disseminated histoplasmosis is a common differential diagnosis of tuberculosis in disease-endemic areas. We aimed to find a predictive score to orient clinicians towards disseminated histoplasmosis or tuberculosis when facing a non-specific infectious syndrome in patients with advanced HIV disease. We reanalyzed data from a retrospective study in Cayenne Hospital between January 1997–December 2008 comparing disseminated histoplasmosis and tuberculosis: 100 confirmed disseminated histoplasmosis cases and 88 confirmed tuberculosis cases were included. A simple logit regression model was constructed to predict whether a case was tuberculosis or disseminated histoplasmosis. From this model, a score may be obtained, where the natural logarithm of the probability of disseminated histoplasmosis/tuberculosis = +3.917962 × WHO performance score (1 if >2, 0 if ≤2) −1.624642 × Pulmonary presentation (1 yes, 0 no) +2.245819 × Adenopathies > 2 cm (1 yes, 0 no) −0.015898 × CD4 count − 0.001851 × ASAT − 0.000871 × Neutrophil count − 0.000018 × Platelet count + 6.053793. The area under the curve was 98.55%. The sensitivity of the model to distinguish between disseminated histoplasmosis and tuberculosis was 95% (95% CI = 88.7–98.3%), and the specificity was 93% (95% CI = 85.7.3–97.4%). In conclusion, we here present a clinical-biological predictive score, using simple variables available on admission, that seemed to perform very well to discriminate disseminated histoplasmosis from tuberculosis in French Guiana in well characterized patients. Introduction With an HIV prevalence greater than 1% for over 3 decades, French Guiana is the French territory where the human immunodeficiency virus (HIV) epidemic is most preoccupying [1]. Disseminated histoplasmosis and tuberculosis have consistently remained among the top AIDS-defining illnesses, disseminated histoplasmosis being the first [2,3]. During HIV infection, both histoplasmosis and tuberculosis are often disseminated infections. In persons with advanced HIV, in the absence of treatment, the dissemination of the pathogen may cause a rapid and potentially fatal evolution, often in a context of hemophagocytic lymphohistiocytosis [4,5]. In the absence of rapid diagnostic tests, invasive diagnostic methods are often necessary and presumptive treatment is often given guided by both knowledge of the local epidemiology-the respective incidences of disseminated histoplasmosis and tuberculosis-and clinical judgement [6]. Although the clinical dilemma is often framed as a dichotomy between disseminated histoplasmosis or tuberculosis, in fact, coinfections are common [7]. The biological confirmation through pathogen identification by culture is long and may be difficult, although direct examination or pathology may yield rapid results [8]. Although progress is in the pipeline [9][10][11], in practice, rapid and sensitive antigenic detection techniques are still not available in most endemic countries [12,13]. The non-specific nature of the clinical and paraclinical findings for both diseases makes the differential diagnosis between disseminated histoplasmosis and tuberculosis difficult in disease-endemic areas [14]. Ever since the first publication by Samuel Darling [15], numerous publications have reported cases of histoplasmosis resembling tuberculosis. We have recently mapped estimates for histoplasmosis and tuberculosis incidence and case fatality for Latin America showing that, for a median scenario of 50% of symptomatic histoplasmosis cases and a historical level of 40% case fatality, 9/21 (43%) Latin American countries had an equivalent of greater incidence of disseminated histoplasmosis than tuberculosis, and that 14/21 (67%) countries had an equivalent or greater number of disseminated histoplasmosis deaths than tuberculosis [16]. In French Guiana, because of a good knowledge of the epidemiologic context, clinicians generally suspect histoplasmosis and tuberculosis in immunosuppressed patients at admission [4,17]. Although lengthy hospitalizations are often required before the pathogen is identified, despite proactive sampling of fluids and tissues, presumptive treatment is common in order to avoid potentially fatal therapeutic delays, usually antituberculosis therapy prevailing on antifungal therapy [18,19]. Although it has long been assumed that these two diseases are similar, this was not based on any direct comparison. In this context, we had performed a comparative study between tuberculosis and histoplasmosis, suggesting that, although there were many similarities, there were some differences that allowed experienced physicians to distinguish between the two diagnoses [14]. Hence, tuberculosis was more associated with pulmonary signs and an elevated CRP, whereas histoplasmosis was associated with cytopenia and/or a digestive presentation. Whether these striking features at the level of a study population had any value when caring for an individual patient was not clear. Since then, to our knowledge, no other direct comparisons have been made. We aimed to further the analyses to determine if we could find a predictive score to orient clinicians towards disseminated histoplasmosis or tuberculosis when facing a non-specific infectious syndrome in patients with advanced HIV disease. Study Design A retrospective study took place at Cayenne Hospital (Cayenne, French Guiana) between January 1997-December 2008 [14]. The study population consisted of patients from the HIV hospital cohort, which is part of the French Hospital Database on HIV, for which data have been systematically collected since 1992. Inclusion and Exclusion Criteria The inclusion criteria were an age ≥ 18 years, hospital admission or outpatient visit before admission, inclusion in the French Hospital Database, confirmed HIV infection, confirmed tuberculosis by culture and identification of Mycobacterium tuberculosis or confirmed disseminated histoplasmosis by direct examination and/or culture of Histoplasma capsulatum, and biological screening less than 7 days before treatment initiation. The inclusion date was that of treatment initiation for tuberculosis or disseminated histoplasmosis. The exclusion criteria were concomitant tuberculosis and histoplasmosis, a history of tuberculosis or histoplasmosis, and an immune reconstitution disease due to tuberculosis or disseminated histoplasmosis. If the diagnosis of tuberculosis or histoplasmosis was only done by polymerase chain reaction, we did not include the patient. Clinical evaluation of the patient's general condition upon admission used the Eastern World Health Organization performance status score. Statistical analysis of anonymized was performed with Stata 16 (Stata Corporation, College Station, TX, USA). A pulmonary presentation was defined as clinical symptoms or signs of the bronchopulmonary sphere (cough, dyspnea, abnormal auscultation) or abnormal chest X-ray. This was coded as a dichotomous variable. Clinically palpable lymphadenopathies >2 cm were also coded as a dichotomous variable. Based on our previous analysis and stepwise multivariate model [14], we selected variables to construct multiple logistic regression models. The dependent variable was tuberculosis or disseminated histoplasmosis, respectively, coded 0 or 1. Hence, if the OR was significantly < 1, the variable was associated with tuberculosis, and, if the OR was significantly > 1, then the variable was associated with histoplasmosis. Biological variables-CD4, Neutrophils, Aspartate-Amino-Transferase (ASAT), and Platelets-were included as continuous variables in order to make marginal predictions for different values of these variables. We did not include "local" variables, such as the place of residence or the duration of stay in French Guiana or ethnicity, because these variables would not be transposable in other contexts. We used Akaike's Information Criterion to select the most parsimonious model, and we used Hosmer-Lemeshow's goodness of fit test. We then used the logit coefficients to construct a predictive score. We performed postestimation analyses estimating sensitivity, specificity, and positive and negative predictive values. Finally, we plotted the full model's ROC curve. The alpha risk was set at 5%. For continuous variables, the reference laboratory threshold used 1 for those with platelets < 150,000/mm 3 , and 0 for those with higher platelet counts. For CD4 and for neutrophil counts, we used the median value as a cutoff, 1 being lower that the median, and 0 being higher that the median. To avoid overfitting, we also tried the model on a training random sample of the dataset and then used the coefficient from the training model to make predictions on the testing set with the remaining observations. To compute the probability of having histoplasmosis, we exponentiated the logit coefficient from the training model and then divided the odds by the odds + 1 − Probability = odds/(1 + odds). Ethical and Regulatory Aspects The study and database were approved by the Institut National de la Santé et de la Recherche Médicale (INSERM (IRB00000388, FWA00005831). Patients gave written informed consent for the study and the publication of results. Table 1 shows the most parsimonious Logit regression model obtained on 188 observations, 100 confirmed disseminated histoplasmosis cases, and 88 confirmed tuberculosis cases. From this model, a score may be obtained, where the natural logarithm of the probability of histoplasmosis compared to tuberculosis = +3.917962 × WHO performance score (1 if >2, 0 if ≤2) −1.624642 × pulmonary presentation (1 yes, 0 no) +2.245819 × adenopathies > 2 cm (1 yes, 0 no) −0.015898 × CD4 count − 0.001851*ASAT − 0.000871 × neutrophil count − 0.000018 × platelet count + 6.053793. Supplementary file 1 provides an Excel spreadsheet computing the probability for the specific values of an individual patient. Figure 1 shows the ROC curve of the predictive model and Figure 2 shows the evolution of sensitivity and specificity for different cutoffs. Figure 1 shows the ROC curve of the predictive model and Figure 2 shows the evolution of sensitivity and specificity for different cutoffs. Table 2 shows the performance of the multivariate model in classifying disseminated histoplasmosis and tuberculosis. Sensitivity of the model to distinguish between disseminated histoplasmosis and tuberculosis was 95% (95% CI = 88.7-98.3%), and the specificity was 93% (95% CI = 85.7.3-97.4%). The positive predictive value was 93.9%, and the negative predictive value was 93%, but the respective numbers did not correspond to real life proportions. Overall, 94.1% of patients were correctly classified. Table 2 shows the performance of the multivariate model in classifying disseminated histoplasmosis and tuberculosis. Sensitivity of the model to distinguish between disseminated histoplasmosis and tuberculosis was 95% (95% CI = 88.7-98.3%), and the specificity was 93% (95% CI = 85.7.3-97.4%). The positive predictive value was 93.9%, and the negative predictive value was 93%, but the respective numbers did not correspond to real life proportions. Overall, 94.1% of patients were correctly classified. We looked at the patients that were wrongly classified (data not shown). For missed disseminated histoplasmosis, there were too few misclassified observations to do robust statistics, but the missed disseminated histoplasmoses tended to have higher mean platelet counts than correct predictions, 27,0142 versus 165,032 per mm 3 , respectively, and higher mean neutrophil counts, 2838 versus 2276 per mm 3 , respectively. To eliminate overfitting artefacts, we randomly split the dataset into a training set and a testing set, where the coefficients obtained from the training set (n = 94) were used to compute the probability on the testing set (n = 94). With this, the sensitivity on the training set was 83% (70-91%), and specificity was 91.8% (80-97.7%). Figure 3 showed that, according to the multivariate model and the predictive score derived from it, as platelet counts and neutrophil counts decline, and as CD4 counts decline, the probability of disseminated histoplasmosis increases. This was not clear for Aspartate-Amino-Transferase concentrations. We looked at the patients that were wrongly classified (data not shown). For missed disseminated histoplasmosis, there were too few misclassified observations to do robust statistics, but the missed disseminated histoplasmoses tended to have higher mean platelet counts than correct predictions, 27,0142 versus 165,032 per mm 3 , respectively, and higher mean neutrophil counts, 2838 versus 2276 per mm 3 , respectively. To eliminate overfitting artefacts, we randomly split the dataset into a training set and a testing set, where the coefficients obtained from the training set (n = 94) were used to compute the probability on the testing set (n = 94). With this, the sensitivity on the training set was 83% (70-91%), and specificity was 91.8% (80-97.7%). Figure 3 showed that, according to the multivariate model and the predictive score derived from it, as platelet counts and neutrophil counts decline, and as CD4 counts decline, the probability of disseminated histoplasmosis increases. This was not clear for Aspartate-Amino-Transferase concentrations. Discussion Here, we show that, when comparing microbiologically confirmed cases of disseminated histoplasmosis and tuberculosis, a model using variables that are available in any hospital was able to correctly identify cases of tuberculosis or disseminated histoplasmosis with great accuracy, as shown by an area under the ROC curve of 98.55%. Although some authors have tried to discriminate patients [20], this is the first study to calculate sensitivity and specificity of variables to distinguish disseminated histoplasmosis from tuberculosis. The wide availability of variables from common clinical and paraclinical data could make it very useful for clinicians who wish to distinguish between the two in endemic contexts, a situation that corresponds to much of Latin America [16]. In French Guiana, disseminated histoplasmosis has been high on the research and clinical agendas, and microbiological facilities are well developed, but we have observed that patients on antituberculosis drugs with no confirmed diagnosis had a twofold increased risk of dying than those with a positive diagnosis [21]. Unfortunately, antigen detections tests or urine LAM for tuberculosis have still not been implemented as routine in patients with advanced HIV disease. Thus, the present score may be of future use to avoid misdiagnoses and treatment delays. The limitations of the present study are that, although this comparison models a common and emblematic differential diagnosis, the back-to-back comparison of disseminated histoplasmosis and tuberculosis is reductive and falsely dichotomizes real clinical situations with advanced HIV, where other differential diagnoses may be evoked. The diagnosis of disseminated histoplasmosis and tuberculosis relied on microbiology, the gold standard, but we now know that the sensitivity of these methods is lower than other methods of antigen testing for disseminated histoplasmosis or Gene Xpert for tuberculosis [22]. Therefore, perhaps there are a number of more difficult diagnoses that are not captured by this model. Furthermore, for the sake of clarity, we excluded coinfections with both tuberculosis and disseminated histoplasmosis, a situation that is not uncommon and may even be very frequent in some epidemiological contexts [7]. Superficial lymphadenopathies were clinically measured, which may have introduced some variability and imprecision. The relatively small sample size could be seen as a weakness, but the magnitude Discussion Here, we show that, when comparing microbiologically confirmed cases of disseminated histoplasmosis and tuberculosis, a model using variables that are available in any hospital was able to correctly identify cases of tuberculosis or disseminated histoplasmosis with great accuracy, as shown by an area under the ROC curve of 98.55%. Although some authors have tried to discriminate patients [20], this is the first study to calculate sensitivity and specificity of variables to distinguish disseminated histoplasmosis from tuberculosis. The wide availability of variables from common clinical and paraclinical data could make it very useful for clinicians who wish to distinguish between the two in endemic contexts, a situation that corresponds to much of Latin America [16]. In French Guiana, disseminated histoplasmosis has been high on the research and clinical agendas, and microbiological facilities are well developed, but we have observed that patients on antituberculosis drugs with no confirmed diagnosis had a twofold increased risk of dying than those with a positive diagnosis [21]. Unfortunately, antigen detections tests or urine LAM for tuberculosis have still not been implemented as routine in patients with advanced HIV disease. Thus, the present score may be of future use to avoid misdiagnoses and treatment delays. The limitations of the present study are that, although this comparison models a common and emblematic differential diagnosis, the back-to-back comparison of disseminated histoplasmosis and tuberculosis is reductive and falsely dichotomizes real clinical situations with advanced HIV, where other differential diagnoses may be evoked. The diagnosis of disseminated histoplasmosis and tuberculosis relied on microbiology, the gold standard, but we now know that the sensitivity of these methods is lower than other methods of antigen testing for disseminated histoplasmosis or Gene Xpert for tuberculosis [22]. Therefore, perhaps there are a number of more difficult diagnoses that are not captured by this model. Furthermore, for the sake of clarity, we excluded coinfections with both tuberculosis and disseminated histoplasmosis, a situation that is not uncommon and may even be very frequent in some epidemiological contexts [7]. Superficial lymphadenopathies were clinically measured, which may have introduced some variability and imprecision. The relatively small sample size could be seen as a weakness, but the magnitude of the area under the curve, and the fact that training and testing models on randomly split samples yielded similar results, suggests that overfitting was not a problem. As the context is specific to French Guiana, the score extracted should nevertheless not be taken at face value and replace sound clinical practice until it has been validated elsewhere in different contexts, a task that seems feasible given the near universal availability of the variables composing the score. Furthermore, as other diagnostic methods are scaled up across Latin American hospitals, and beyond, the score may require further validation and recalibration. Positive and negative predictive values are probably not valid because the relative proportion of disseminated histoplasmosis and tuberculosis in our sample did not necessarily reflect the real situation. Further prospective studies could help obtain these values for the site where the study is performed. Conclusions In conclusion, we here present a clinical-biological predictive score, using simple variables available on admission, that seemed to perform very well to discriminate disseminated histoplasmosis from tuberculosis in French Guiana in well characterized HIV patients. This should be confirmed by external validation in different epidemiological contexts, and beyond the dichotomy disseminated histoplasmosis-tuberculosis, before we see if it is useful for clinicians who most often still do not have access to rapid antigen detection tests, and for patients for whom therapeutic delays can lead to early death. Although Samuel Darling's differential diagnosis remains common at the bedside, this data suggests that, in fact, in patients with advanced HIV, single infections by M. tuberculosis or H. capsulatum are quite different. Data Availability Statement: Anonymized data can be made upon reasonable request at cicec@chcayenne.fr. Conflicts of Interest: The authors declare no conflict of interest.	2021-12-30T16:21:03.340Z	2021-12-27T00:00:00.000	{ "year": 2021, "sha1": "ffdf606048bb2066dc0e12f39fed7dd05bf84d65", "oa_license": null, "oa_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8777677", "oa_status": "GREEN", "pdf_src": "MergedPDFExtraction", "pdf_hash": "41202f964e39dd4e121aee3da399e381f365ef66", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
245375392	pes2o/s2orc	v3-fos-license	Removal of Organic Dyes from Water and Wastewater Using Magnetic Ferrite-Based Titanium Oxide and Zinc Oxide Nanocomposites: A Review Heterogeneous photocatalysis using titanium dioxide (TiO2) and zinc oxide (ZnO) has been widely studied in various applications, including organic pollutant remediation in aqueous systems. The popularity of these materials is based on their high photocatalytic activity, strong photosensitivity, and relatively low cost. However, their commercial application has been limited by their wide bandgaps, inability to absorb visible light, fast electron/hole recombination, and limited recyclability since the nanomaterial is difficult to recover. Researchers have developed several strategies to overcome these limitations. Chief amongst these is the coupling of different semi-conductor materials to produce heterojunction nanocomposite materials, which are both visible-light-active and easily recoverable. This review focuses on the advances made in the development of magnetic ferrite-based titanium oxide and zinc oxide nanocomposites. The physical and magnetic properties of the most widely used ferrite compounds are discussed. The spinel structured material had superior catalytic and magnetic performance when coupled to TiO2 and ZnO. An assessment of the range of synthesis methods is also presented. A comprehensive review of the photocatalytic degradation of various priority organic pollutants using the ferrite-based nanocomposites revealed that degradation efficiency and magnetic recovery potential are dependent on factors such as the chemical composition of the heterojunction material, synthesis method, irradiation source, and structure of pollutant. It should be noted that very few studies have gone beyond the degradation efficiency studies. Very little information is available on the extent of mineralization and the subsequent formation of intermediate compounds when these composite catalysts are used. Additionally, potential degradation mechanisms have not been adequately reported. Introduction Nowadays, concern around efficient management of water use has become topical, with interest not only limited to agricultural and industrial sectors but also attracting public health and sustainable economic development proponents. The wide range of anthropogenic activities that use water results in the generation of highly toxic effluents, which are rich in organic pollutants, rendering them unsuitable for reuse in agricultural activities and human consumption. As a result, water decontamination has become the focus of attention for several studies. [12]. Unit cell structure of (b) normal spinel ferrite, and (c) inverse spinel ferrite [25]. Republished with permission from Elsevier. Due to the small band gap energy of ferrites, which makes them effective under absorption of visible light irradiation, they are extremely suitable for the removal of organic pollutants in water and wastewater treatment processes [22,23]. Methods of Synthesis of Magnetic Spinel Ferrites Synthesis methods play an important role in the development of magnetic nanoparticles as this controls the electrical, optical, and magnetic properties of the material [15]. Co-precipitation requires careful monitoring of pH in order to obtain pure spinel ferrites [25]. Advantages associated with this method include low cost, short synthesis time, high product yield, and production of uniformly sized particles [45]. For example, El-Okr et al. [46] synthesized magnetic CoFe 2 O 4 using the co-precipitation method and obtained crystallites between 11 and 45 nm in size, with saturation magnetization ranging from 5 to 67 emu/g. The authors reported that the difference in crystallite size and saturation magnetization (Ms) values was associated with the variation of parameters such as pH and calcination temperature. The hydrothermal synthesis method enables particle size control and flexibility in terms of surface modification. It is based on the wet-chemical synthesis; typically, this occurs in sealed reactors or autoclaves at high vapor pressures (from 0.3 to 4 MPa) and elevated temperatures (130 to 250 • C) [47,48]. Some noteworthy advantages of this method include low temperature for synthesis, high purity, simple reactions, cost-effectiveness, and good dispersibility of the MNPs [49]. For instance, Zhao et al. [41] prepared cobalt ferrite using the hydrothermal method. The resultant material had 70 nm crystallites with a saturation magnetization (Ms) of 86 emu/g. The sol-gel method is extensively used for the synthesis of spinel ferrites [50][51][52][53][54][55]. The process involves the transition of a system from a liquid phase (sol) to a solid phase (gel), through chemical reactions such as hydrolysis and condensation polymerization of the metallic precursors [25]. Thus, its widespread acceptance is driven by the low cost associated with the method, better homogeneity, composition control, and narrow particle size distribution at relatively low temperatures [25,45]. The sol-gel method also allows for good control of the structural and magnetic properties of MNPs [45]. Sajjia et al. [56] prepared cobalt ferrite nanoparticles by a sol-gel method. Their results demonstrated that the saturation magnetization was 67.3 emu/g and the particle sizes were between 7 and 28 nm, according to the calcination temperature of nanoparticles. The combustion synthesis method of MNPs is based on the thermodynamics principles and chemistry of propellants and explosives [57][58][59]. The method requires a powdered mixture, typically consisting of an oxidizing agent containing the metal ions of interest such as oxidizing reagents, and a reducing agent such as urea or glycine [57,[60][61][62][63]. Thus, the combination of total valences of reducing agent (fuel, urea) and oxidizing agent achieved gives the following expression: −40 + 6n = 0, where n is the number of moles of urea (in this case, n = 6.67 mol) for the combustion reaction. Since it is complete combustion, the stoichiometric reaction (Φe = 1) follows the definition described for the oxygen balance and equals zero. To accomplish this, the content of oxygen from the nitrates is completely oxidized by a reducing agent (fuel) in the mixture [61]. A study conducted by Salunkhe et al. [65] evaluated the magnetic properties of cobalt ferrite nanoparticles prepared using the combustion method using glycine as fuel. The results showed that the crystallite size was 38 nm and saturation magnetization was 67.3 emu/g. Table 1 summarizes the structural and magnetic properties of CoFe 2 O 4 MNPs synthesized using various methods. Table 1. Effect of synthesis method in structural and magnetic properties of catalyst cobalt ferrite [65][66][67]. Characterization Methods Before the synthesized magnetic materials are used as catalysts, an investigation of the various properties, which influence their performance, is essential. Some of the key parameters are size, shape, and surface area. Therefore, this information can be elucidated using one or a combination of the following techniques: X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), BET-N 2 analysis, vibrating sample magnetometer (VSM). X-ray diffraction (XRD) gives information about the structural properties, crystallite size, and crystalline phases of magnetic nanoparticles as catalysts. The BET-N 2 adsorption-desorption isotherm is a technique commonly used to evaluate the porosity and specific surface area of MNPs. For example, smaller particles have a larger surface area, leading to higher photocatalytic activity due to a larger number of active sites [23]. More details are explained in Section 5. The magnetic behavior of the ferrites (saturation magnetization) is evaluated using a vibrating sample magnetometer (VSM). The information obtained from this technique gives an idea of the recovery potential of the photocatalyst. Figure 2 shows the magnetic behavior of various ferrites at room temperature. With the exception of zinc ferrite, which has a low magnetization of saturation, the other ferrites have a high magnetization of saturation values, which implies good magnetic and recyclability properties [67]. A study conducted by Salunkhe et al. [65] evaluated the magnetic properties of cobalt ferrite nanoparticles prepared using the combustion method using glycine as fuel. The results showed that the crystallite size was 38 nm and saturation magnetization was 67.3 emu/g. Table 1 summarizes the structural and magnetic properties of CoFe2O4 MNPs synthesized using various methods. Characterization Methods Before the synthesized magnetic materials are used as catalysts, an investigation of the various properties, which influence their performance, is essential. Some of the key parameters are size, shape, and surface area. Therefore, this information can be elucidated using one or a combination of the following techniques: X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), BET-N2 analysis, vibrating sample magnetometer (VSM). X-ray diffraction (XRD) gives information about the structural properties, crystallite size, and crystalline phases of magnetic nanoparticles as catalysts. The BET-N2 adsorption-desorption isotherm is a technique commonly used to evaluate the porosity and specific surface area of MNPs. For example, smaller particles have a larger surface area, leading to higher photocatalytic activity due to a larger number of active sites [23]. More details are explained in Section 5. The magnetic behavior of the ferrites (saturation magnetization) is evaluated using a vibrating sample magnetometer (VSM). The information obtained from this technique gives an idea of the recovery potential of the photocatalyst. Figure 2 shows the magnetic behavior of various ferrites at room temperature. With the exception of zinc ferrite, which has a low magnetization of saturation, the other ferrites have a high magnetization of saturation values, which implies good magnetic and recyclability properties [67]. This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, and the experimental conclusions that can be drawn. The morphology, including shape, and particle size of magnetic nanoparticles, can be determined using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Figure 3a,b demonstrates the difference in morphology and structure of cobalt ferrite affected by different methods of synthesis [68]. [67]. Republished with permission from Elsevier. The morphology, including shape, and particle size of magnetic nanoparticles, can be determined using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Figure 3a,b demonstrates the difference in morphology and structure of cobalt ferrite affected by different methods of synthesis [68]. Thermal decomposition studies are crucial for the development of nanocatalysts as most of them are synthesized at high temperatures. Thermogravimetric analysis (TGA) or differential scanning calorimetry (DSC) are used to determine the optimum temperature required for the synthesis of magnetic nanoparticles due to the possibility of a loss of activity during their preparation. Finally, these characterization techniques are also employed to investigate if any changes occur by decomposition or degradation, and whether they retain their magnetic properties after the photocatalytic process. Photocatalytic Application of Magnetic Ferrites and Their Nanocomposites Photocatalytic degradation is a sequence of chemical reactions promoted by light resulting in the breakdown of the target compound [69]. The photocatalytic activity is effectively dependent on the surface area and electronholes separation efficiency of the catalyst [69]. Figure 4 shows a schematic of the photocatalytic mechanism of magnetic ZnFe2O4/ZnO nanocomposite during methylene blue and methylene orange dye degradation [69]. Thermal decomposition studies are crucial for the development of nanocatalysts as most of them are synthesized at high temperatures. Thermogravimetric analysis (TGA) or differential scanning calorimetry (DSC) are used to determine the optimum temperature required for the synthesis of magnetic nanoparticles due to the possibility of a loss of activity during their preparation. Finally, these characterization techniques are also employed to investigate if any changes occur by decomposition or degradation, and whether they retain their magnetic properties after the photocatalytic process. Photocatalytic Application of Magnetic Ferrites and Their Nanocomposites Photocatalytic degradation is a sequence of chemical reactions promoted by light resulting in the breakdown of the target compound [69]. The photocatalytic activity is effectively dependent on the surface area and electronholes separation efficiency of the catalyst [69]. Figure 4 shows a schematic of the photocatalytic mechanism of magnetic ZnFe 2 O 4 /ZnO nanocomposite during methylene blue and methylene orange dye degradation [69]. The morphology, including shape, and particle size of magnetic nanoparticles, can be determined using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Figure 3a,b demonstrates the difference in morphology and structure of cobalt ferrite affected by different methods of synthesis [68]. Thermal decomposition studies are crucial for the development of nanocatalysts as most of them are synthesized at high temperatures. Thermogravimetric analysis (TGA) or differential scanning calorimetry (DSC) are used to determine the optimum temperature required for the synthesis of magnetic nanoparticles due to the possibility of a loss of activity during their preparation. Finally, these characterization techniques are also employed to investigate if any changes occur by decomposition or degradation, and whether they retain their magnetic properties after the photocatalytic process. Photocatalytic Application of Magnetic Ferrites and Their Nanocomposites Photocatalytic degradation is a sequence of chemical reactions promoted by light resulting in the breakdown of the target compound [69]. The photocatalytic activity is effectively dependent on the surface area and electronholes separation efficiency of the catalyst [69]. Figure 4 shows a schematic of the photocatalytic mechanism of magnetic ZnFe2O4/ZnO nanocomposite during methylene blue and methylene orange dye degradation [69]. It has been reported that nanocatalysts that have high surface areas exhibit higher photocatalytic activity compared to their larger counterparts with lower surface area. The smaller nanoparticles support the easy transition of electrons from the valence to conduction band, thereby generating electron-hole pairs when exposed to UV-visible radiation. The generated electrons and holes further interact with dissolved oxygen and water to produce highly reactive free radical species capable of degrading the methylene dyes [69,70]. The general photocatalytic degradation mechanism of magnetic nanocomposites towards organic pollutants is demonstrated by Equations (2)- (8). h + + H 2 O absorbed → H + + •OH (6) h + + OH absorbed − → •OH (7) •OH + MB dye → degraded products (8) Reduction and oxidation take place at the photo-excited surface of the photocatalyst. Recombination between e − and h + can occur for the use of redox reaction. The e − and h + that do not recombine are transferred to the surface of redox reaction and undergo reduction process and oxidation process to form superoxide ion (O 2 − ) and ·OH, respectively. OH − then leads to the production of strong oxidizing ·OH radicals. Meanwhile, the negative e − reacts with the oxygen (O) molecule to form a ·O 2 − . This ·O 2 − also produces ·OH radicals via the formation of HO 2 • radicals and H 2 O 2 . The radicals formed from the reaction are used to degrade the organic pollutant [71,72]. Nickel Ferrite and Nanocomposites NiFe 2 O 4 has generated a lot of interest because of its excellent features. These include being a soft ferrimagnetic or ferrite n-type semiconductor with low coercivity, chemical stability, and electrical resistivity. These make it an excellent material in different applications such as in magnetic resonance imaging enhancement, magnetic recording media, and electronic devices, as well as in catalysis [20]. Following the description in Section 2, NiFe 2 O 4 is completely composed of an inverse spinel structure comprising a face-centered cubic lattice. NiFe 2 O 4 consists of tetrahedral sites occupied by half of the Fe 3+ cations, while the rest of the Fe 3+ and Ni 2+ cations are distributed over the octahedral sites [73][74][75]. Figure 5 shows the XRD spectra and SEM images of neat zinc oxide, nickel ferrite, and their nanocomposites [76]. In the study conducted by Adeleke et al. [76], no secondary peaks or secondary phases of material were observed; this demonstrated the effectiveness of the synthesis method used in this study. Furthermore, SEM images demonstrated the effect of doping ferrite with zinc oxide. The high degree of agglomeration and different morphologies observed on the ZnO/Fe 2 O 4 catalyst were attributed to the magnetic attraction between nickel ferrite and zinc oxide layers [76]. Several studies have demonstrated that magnetic NiFe 2 O 4 and its nanocomposites are effective photocatalysts for the removal of dye from water and wastewater, due to their high adsorption capacity and strong photocatalytic properties [16,[77][78][79][80][81][82]. Khosravi and Eftekhar [83] synthesized magnetic NiFe 2 O 4 using a sol-gel method and evaluated its effectiveness as an adsorbent for the removal of Reactive Blue 5 (RB5) dye. Parameters such as pH, temperatures, and catalyst concentration were evaluated during RB5 degradation [83]. Maximum degradation (90%) was achieved under acidic conditions (pH = 1) at room temperature using an (adsorbent/catalyst loading of 0.03 g/L). Several studies have demonstrated that magnetic NiFe2O4 and its nanocomposites are effective photocatalysts for the removal of dye from water and wastewater, due to their high adsorption capacity and strong photocatalytic properties [16,[77][78][79][80][81][82]. Khosravi and Eftekhar [83] synthesized magnetic NiFe2O4 using a sol-gel method and evaluated its effectiveness as an adsorbent for the removal of Reactive Blue 5 (RB5) dye. Parameters such as pH, temperatures, and catalyst concentration were evaluated during RB5 degradation [83]. Maximum degradation (90%) was achieved under acidic conditions (pH = 1) at room temperature using an (adsorbent/catalyst loading of 0.03 g/L). These findings were corroborated by Zhu et al. [16] when they evaluated the photocatalytic degradation of Congo Red dye using NiFe2O4/ZnO as a catalyst. In their study, the NiFe2O4/ZnO nanocomposite resulted in a 94% removal of Congo red solution under simulated solar light irradiation in 10 min. Nickel ferrite was also shown to be effective when coupled with other metal oxides such as TiO2. In a study done by Hung and Thanh [84], a magnetic nanocomposite of NiFe2O4/TiO2 degraded 98% of methyl orange dye after 14 h of UV or visible light irradiation. Although the reaction time was rather long, the photocatalyst had a high saturation of magnetization (40 emu/g), which makes it easily recyclable for reuse. The results obtained in these studies demonstrate that nickel ferrite nanocomposites are potential candidates for wastewater treatment in large-scale applications. Additional studies that illustrate the efficacy of nickel ferrite, nickel ferrite-based titanium oxide, and zinc oxide catalysts in the degradation of variant organic pollutants are summarized in Table 2. These findings were corroborated by Zhu et al. [16] when they evaluated the photocatalytic degradation of Congo Red dye using NiFe 2 O 4 /ZnO as a catalyst. In their study, the NiFe 2 O 4 /ZnO nanocomposite resulted in a 94% removal of Congo red solution under simulated solar light irradiation in 10 min. Nickel ferrite was also shown to be effective when coupled with other metal oxides such as TiO 2 . In a study done by Hung and Thanh [84], a magnetic nanocomposite of NiFe 2 O 4 /TiO 2 degraded 98% of methyl orange dye after 14 h of UV or visible light irradiation. Although the reaction time was rather long, the photocatalyst had a high saturation of magnetization (40 emu/g), which makes it easily recyclable for reuse. The results obtained in these studies demonstrate that nickel ferrite nanocomposites are potential candidates for wastewater treatment in large-scale applications. Additional studies that illustrate the efficacy of nickel ferrite, nickel ferrite-based titanium oxide, and zinc oxide catalysts in the degradation of variant organic pollutants are summarized in Table 2. Zinc Ferrite and Nanocomposites Zin ferrite has a small bandgap of around 1.9 eV, which gives a good response to the visible light, as well as excellent photochemical stability, considerable magnetism, and cost-effectiveness. As a result, it has also attracted attention by researchers in the photocatalysis process [85]. The compound consists of a fully normal spinel structure, where its tetrahedral sites are occupied only by Zn 2+ cations and the Fe 3+ ions are distributed in the octahedral The compound consists of a fully normal spinel structure, where its tetrahedral sites are occupied only by Zn 2+ cations and the Fe 3+ ions are distributed in the octahedral sites [86]. Figure 6 shows the XRD patterns and SEM micrographs of the zinc oxide-, zinc ferrite-, and zinc ferrite-based zinc oxide nanocomposites [17]. The photocatalyst ZnFe2O4/ZnO had diffraction peaks similar to those of neat ZnFe2O4 and ZnO. The sharp peaks showed good crystallinity of the nanocomposite [17], which demonstrates the efficacy of the synthesis method used in this study. The SEM images revealed the effect of incorporating ZnFe2O4 nanoparticles into the pores of the ZnO matrix. The authors observed that a high content of ZnO nanoparticles was formed; these were better defined in the ZnFe2O4/ZnO nanocomposites [17]. Significant efforts have been devoted to investigating ZnFe2O4-based photocatalysts for water and wastewater treatment, with the aim of removing organic pollutants [87][88][89][90]. Yuan et al. [91] investigated the photocatalytic activity of a ZnFe2O4/TiO2 nanocomposite where the pure ZnFe2O4 and TiO2 were obtained via the co-precipitation method. The results showed that the ZnFe2O4/TiO2 and pure TiO2 resulted in 95% and 20% of degradation of phenol respectively during 180 min of irradiation under UV-Visible. This demonstrated that the ZnFe2O4/TiO2 nanocomposite catalyst was more effective than pure TiO2 in the degradation of phenol. In addition, Shao et al. [85] evaluated the application of ZnFe2O4/ZnO nanoparticles in the photodegradation of methylene blue dye. The findings demonstrated that even after three cycles, the photocatalytic activity of the magnetic nanocomposite ZnFe2O4/ZnO (65%) was better compared to that of pure ZnO (58%), indicating the significance of ZnFe2O4 in the suppression of ZnO photo-corrosion. This was attributed to the photostability of ZnFe2O4 nanoparticles [92]. The photocatalyst ZnFe 2 O 4 /ZnO had diffraction peaks similar to those of neat ZnFe 2 O 4 and ZnO. The sharp peaks showed good crystallinity of the nanocomposite [17], which demonstrates the efficacy of the synthesis method used in this study. The SEM images revealed the effect of incorporating ZnFe 2 O 4 nanoparticles into the pores of the ZnO matrix. The authors observed that a high content of ZnO nanoparticles was formed; these were better defined in the ZnFe 2 O 4 /ZnO nanocomposites [17]. Yuan et al. [91] investigated the photocatalytic activity of a ZnFe 2 O 4 /TiO 2 nanocomposite where the pure ZnFe 2 O 4 and TiO 2 were obtained via the co-precipitation method. The results showed that the ZnFe 2 O 4 /TiO 2 and pure TiO 2 resulted in 95% and 20% of degradation of phenol respectively during 180 min of irradiation under UV-Visible. This demonstrated that the ZnFe 2 O 4 /TiO 2 nanocomposite catalyst was more effective than pure TiO 2 in the degradation of phenol. In addition, Shao et al. [85] evaluated the application of ZnFe 2 O 4 /ZnO nanoparticles in the photodegradation of methylene blue dye. The findings demonstrated that even after three cycles, the photocatalytic activity of the magnetic nanocomposite ZnFe 2 O 4 /ZnO (65%) was better compared to that of pure ZnO (58%), indicating the significance of ZnFe 2 O 4 in the suppression of ZnO photo-corrosion. This was attributed to the photostability of ZnFe 2 O 4 nanoparticles [92]. Further study by Patil et al. [93] investigated the photocatalytic activity of ZnFe 2 O 4 nanoparticles synthesized using a combustion method. Their activity was tested on synthetic wastewater made up of the following dyes: Methylene Blue, Rose Bengal, Evans Blue, and Indigo Carmine. Degradation efficiencies of 98, 99, 82, and 87% were recorded for each dye, respectively [93]. The antibacterial activity of ZnFe 2 O 4 against diverse gram-negative bacterial strains such as Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Bacillus was also investigated. A variation in the antibacterial activity towards the different bacterial strains was observed [93]. Other work conducted by Sripriya et al. [94] reported on the excellent photocatalytic performance of ZnFe 2 O 4 in the degradation of 4-chlorophenol (4-CP). They further reported that factors such as particle size and surface area significantly affected the activity. Further studies on the use of magnetic ZnF 2 O 4 nanocomposites as photocatalysts are listed in Table 2. Cobalt Ferrite and Nanocomposites Cobalt ferrite is a hard ferrimagnetic material that has a face centered cubic structure [95]. It exists as a normal spinel structure or inverse spinel structure, depending on the synthesis method. In the normal spinel structure, the Co 2+ ions are distributed in the tetrahedral sites, while the octahedral sites are occupied by Fe 3+ ions. For the inverse spinel structure, the tetrahedral sites are occupied by half of the Fe 3+ ions and the rest of the octahedral sites are distributed by Fe 3+ and Co 2+ ions. It is important to note that the magnetic properties vary depending on the structures [95,96]. Figure 7 shows the XRD patterns and TEM micrographs of the cobalt ferrite and cobalt ferrite-based zinc oxide nanocomposite [97]. Pristine XRD patterns were obtained with no impurities; this demonstrated that the co-precipitation method used for synthesis was highly efficient [97]. The photocatalytic activity and stability of ZnO/CoFe2O4 were confirmed by showing 10 cycles for successive reuse. Since ZnO/CoFe2O4 is recyclable and easily recovered magnetically; it is a good candidate for use as a low-cost photocatalyst for water and wastewater treatment. Additional studies on the photocatalytic performance of cobalt ferrite and their nanocomposites in the degradation of organic pollutants are summarized in Table 2. Manganese Ferrite and Nanocomposites MnFe2O4 is a soft spinel ferrite with high magnetic permeability and moderate saturation magnetization, high chemical stability, high electrical resistance, and special optical properties [102]. A combination of these factors makes it attractive for use in different applications such as biomedical drug delivery and catalysis [103,104]. MnFe2O4 is considered a mixed spinel ferrite in which the tetrahedral and octahedral sites are both are occupied by Mn 2+ and Fe 3+ ions [104,105]. High reaction temperatures affect the synthesis of the magnetic ferrite, resulting in variation in particle sizes of the Several studies have demonstrated the potential of cobalt spinel ferrite doped with metals oxides (TiO 2 or ZnO) as photocatalysts for water and wastewater treatment for organic pollutants removal. This is due to the various attributes that include chemical stability, small band-gap energy that leads to activation by visible light [5,12,[98][99][100], magnetic properties, and higher surface area. A study conducted by de Oliveira et al. [4] demonstrated that the magnetic nanocatalyst of CoFe 2 O 4 coupled to TiO 2 resulted in 100% degradation of diuron degradation. This study also observed that CoFe 2 O 4 /TiO 2 nanoparticles displayed good saturation magnetization, demonstrating that they can be easily separated for reuse. Furthermore, Li et al. [101] reported good performance of magnetic TiO 2 /CoFe 2 O 4 nanocomposite for methylene blue (MB) degradation (98%) in 300 min. The good per-formance of magnetic TiO 2 /CoFe 2 O 4 nanocomposite was attributed to the presence of CoFe 2 O 4, which not only improved the UV light absorbance but also enhanced the response to the visible light region. A study done by Chandel et al. [102] reported that the ZnO/CoFe 2 O 4 nanocomposite displayed 94% degradation efficiency towards Methylene Orange (MO) and 92% removal for malachite green (MG) dye. The authors attributed the hydroxyl radicals (OH • ) and holes (h + VB ) as the main reactive species responsible for the degradation of MO and MG dyes. The photocatalytic activity and stability of ZnO/CoFe 2 O 4 were confirmed by showing 10 cycles for successive reuse. Since ZnO/CoFe 2 O 4 is recyclable and easily recovered magnetically; it is a good candidate for use as a low-cost photocatalyst for water and wastewater treatment. Additional studies on the photocatalytic performance of cobalt ferrite and their nanocomposites in the degradation of organic pollutants are summarized in Table 2. Manganese Ferrite and Nanocomposites MnFe 2 O 4 is a soft spinel ferrite with high magnetic permeability and moderate saturation magnetization, high chemical stability, high electrical resistance, and special optical properties [103]. A combination of these factors makes it attractive for use in different applications such as biomedical drug delivery and catalysis [103,104]. MnFe 2 O 4 is considered a mixed spinel ferrite in which the tetrahedral and octahedral sites are both are occupied by Mn 2+ and Fe 3+ ions [104,105]. High reaction temperatures affect the synthesis of the magnetic ferrite, resulting in variation in particle sizes of the material, which in turn affects other parameters such as saturation magnetization. A study conducted by Chang et al. [106] demonstrated through X-ray diffraction analysis that no other peaks attributed a second phase were observed for manganese ferrite and titanium oxide. Additionally, peaks attributed to pure materials (MnFe 2 O 4 and TiO 2 ) were observed in the XRD pattern of MnFe 2 O 4 /TiO 2 nanocomposite (Figure 8). The findings demonstrate that the magnetic nanoparticles were successfully synthesized via a hydrothermal followed by the sol-gel method [106]. The SEM micrograph of MnFe 2 O 4 /TiO 2 (Figure 9) showed that agglomerated spherical particles were produced. The results showed that 90% degradation of Congo red dye was achieved in 35 min under UV-vis irradiation. Additionally, Arief et al. [103] showed that the same nanocomposite was effective for Rhodamine B dye degradation (95%). This was attributed to the presence of a narrow band gap energy (1.95 eV) of MnFe2O4/ZnO. Silambarasu et al. [108] tested the performance of MnFe2O4 on the degradation of methylene blue dye. The manganese ferrite achieved 96% dye decolorization and exhibited saturation magnetization (Ms) of 39.7 emu/g. The magnetic properties indicated that the product could be easily recovered for potential reuse. The MnFe 2 O 4 nanoparticles display a well-defined morphology with particle size around 15-20 nm. Meanwhile, it is clearly visible that the particle size of MnFe 2 O 4 /TiO 2 is uneven and relatively large [106]. Numerous studies have demonstrated that manganese ferrite-based metal oxide nanocomposites are effective in the photocatalytic degradation of organic dyes. For example, Zamani et al. [107] evaluated the photocatalytic performance of a magnetic MnFe 2 O 4 /ZnO nanocomposite for Congo red dye (CR) removal. The results showed that 90% degradation of Congo red dye was achieved in 35 min under UV-vis irradiation. Additionally, Arief et al. [103] showed that the same nanocomposite was effective for Rhodamine B dye degradation (95%). This was attributed to the presence of a narrow band gap energy (1.95 eV) of MnFe 2 O 4 /ZnO. Silambarasu et al. [108] tested the performance of MnFe 2 O 4 on the degradation of methylene blue dye. The manganese ferrite achieved 96% dye decolorization and exhibited saturation magnetization (Ms) of 39.7 emu/g. The magnetic properties indicated that the product could be easily recovered for potential reuse. There are few studies reporting the application of manganese ferrite and manganese ferrite-based zinc oxide and titanium oxide nanocomposites in water and wastewater treatment for organic pollutants removal. Copper Ferrite and Nanocomposites CuFe 2 O 4 is one of the magnetic nanoparticles that has become a promising candidate in the catalysis field due to the presence of surface hydroxyl groups, good chemical, and thermal stabilities, a small band gap, and magnetic properties [109]. These characteristics make it attractive as a photocatalyst for work on water and wastewater treatment for organic pollutants degradation. Copper ferrite has an inverse spinel structure, where the tetrahedral sites are occupied by half of the Fe 3+ ions and the rest of the octahedral sites are occupied by Fe 3+ and Cu 2+ ions [110]. Therefore, besides the cubic crystal structure, the copper ferrite also presents a tetragonal crystal structure that depends on the synthesis method and annealing temperature [110]. In the XRD patterns of TiO 2 /CuFe 2 O 4 nanocomposites, the peaks associated with the neat TiO 2 and CuFe 2 O 4 were observed without any further secondary phase ( Figure 10) [109]. This demonstrated that the nanocomposite photocatalyst was successfully prepared using the Sol-Gel method and the pure titanium oxide and copper ferrite nanoparticles remained with their structure during the synthesis processes. CuFe2O4 is one of the magnetic nanoparticles that has become a promising candidate in the catalysis field due to the presence of surface hydroxyl groups, good chemical, and thermal stabilities, a small band gap, and magnetic properties [109]. These characteristics make it attractive as a photocatalyst for work on water and wastewater treatment for organic pollutants degradation. Copper ferrite has an inverse spinel structure, where the tetrahedral sites are occupied by half of the Fe 3+ ions and the rest of the octahedral sites are occupied by Fe 3+ and Cu 2+ ions [110]. Therefore, besides the cubic crystal structure, the copper ferrite also presents a tetragonal crystal structure that depends on the synthesis method and annealing temperature [110]. In the XRD patterns of TiO2/CuFe2O4 nanocomposites, the peaks associated with the neat TiO2 and CuFe2O4 were observed without any further secondary phase ( Figure 10) [109]. This demonstrated that the nanocomposite photocatalyst was successfully prepared using the Sol-Gel method and the pure titanium oxide and copper ferrite nanoparticles remained with their structure during the synthesis processes. Figure 11a,b shows the SEM micrographs of CuFe2O4 and TiO2/CuFe2O4, where the presence of agglomerated particles distributed randomly is apparent. The introduction of TiO2 in the CuFe2O4 influenced the morphology of the nanocomposite TiO2/CuFe2O4. The surface of the nanocomposite was much rougher than that of the CuFe2O4. The TiO2 agglomerated on the surface of CuFe2O4 can provide more active sites for the nanocomposite and improve its photocatalytic activity during organic pollutants degradation [109]. The TiO 2 agglomerated on the surface of CuFe 2 O 4 can provide more active sites for the nanocomposite and improve its photocatalytic activity during organic pollutants degradation [109]. Several studies have reported the photocatalytic activity of copper ferrite and its nanocomposites in water and wastewater treatment [109,111,112]. For instance, a study done by Anandan et al. [110] reported good performance of magnetic CuFe 2 O 4 as photocatalyst for the degradation of methylene blue (MB) dye in the presence of peroxydisulphate under UV-vis light. The activity of the copper ferrite was attributed to the effect of the peroxydisulphate in the photocatalyst, which improved the photocatalytic degradation of methylene blue (95%) 75 min. Before the addition of the oxidant peroxydisulphate to the cobalt ferrite, the nanoparticles showed 16% MB dye degradation in 75 min. A recent study conducted by Janani et al. [113] evaluated the magnetic nanocomposite ZnO/CuFe 2 O 4 as a catalyst for methylene blue dye degradation under visible light. In their study, they demonstrated that the ZnO/CuFe 2 O 4 photocatalyst was efficient in the degradation of methylene blue dye (86%) in 77 min. The authors associated the activity of the nanocomposites with the hydroxyl radicals and holes generated, which play a principal role in the degradation of the dye. Furthermore, the photocatalyst also remained stable after six cycles of reuse. More studies on the activity of copper ferrite nanocomposites for the degradation of organic pollutants are listed in Table 2. Several studies have reported the photocatalytic activity of copper ferrite and its nanocomposites in water and wastewater treatment [109,111,112]. For instance, a study done by Anandan et al. [110] reported good performance of magnetic CuFe2O4 as photocatalyst for the degradation of methylene blue (MB) dye in the presence of peroxydisulphate under UV-vis light. The activity of the copper ferrite was attributed to the effect of the peroxydisulphate in the photocatalyst, which improved the photocatalytic degradation of methylene blue (95%) 75 min. Before the addition of the oxidant peroxydisulphate to the cobalt ferrite, the nanoparticles showed 16% MB dye degradation in 75 min. A recent study conducted by Janani et al. [113] evaluated the magnetic nanocomposite ZnO/CuFe2O4 as a catalyst for methylene blue dye degradation under visible light. In their study, they demonstrated that the ZnO/CuFe2O4 photocatalyst was efficient in the degradation of methylene blue dye (86%) in 77 min. The authors associated the activity of the nanocomposites with the hydroxyl radicals and holes generated, which play a principal role in the degradation of the dye. Furthermore, the photocatalyst also remained stable after six cycles of reuse. More studies on the activity of copper ferrite nanocomposites for the degradation of organic pollutants are listed in Table 2. Mixed-Metal Ferrites and Nanocomposites The introduction of different cations in the spinel ferrite system is required to improve the physicochemical properties of spinel ferrites. For instance, the substitution of magnetic cations such as Mn 2+ , Ni 2+ , Co 2+ , and Cu 2+, and diamagnetic ions such as Zn 2+ and Cd 2+, in spinel ferrites systems, changes the structural, morphological, opto-magnetic, and catalytic properties [114,115]. In general, this is attributed to the distribution of metallic ions in the tetrahedral and octahedral sites [115,116]. Ciocarlan et al. [117] evaluated the structural, morphological, and photocatalytic properties of magnetic nanoparticles Co 0.5 Zn 0.25 M 0.25 Fe 2 O 4 /TiO 2, where M represent Ni 2+ , Cu 2+ , and Mn 2+ ions. XRD patterns for TiO 2 -based magnetic nanocomposites showed that the introduction of TiO 2 into the magnetic nanoparticles affected their structural properties ( Figure 12). catalytic properties [114,115]. In general, this is attributed to the distribution of metallic ions in the tetrahedral and octahedral sites [115,116]. Ciocarlan et al. [117] evaluated the structural, morphological, and photocatalytic properties of magnetic nanoparticles Co0.5Zn0.25M0.25Fe2O4/TiO2, where M represent Ni 2+ , Cu 2+ , and Mn 2+ ions. XRD patterns for TiO2-based magnetic nanocomposites showed that the introduction of TiO2 into the magnetic nanoparticles affected their structural properties ( Figure 12). Finally, in terms of photocatalytic activity, the results demonstrated that approximately 80% and 75% of methylene orange (MO) and methylene blue (MB) were effectively degraded by Co 0.5 Zn 0.25 Ni 0.25 Fe 2 O 4 /TiO 2 . The authors attributed the good photocatalytic activity to the Ni 2+ ions and synergistic effect in combination with Co 2+ ions [117]. Several other studies have also demonstrated the photocatalytic performance of complex-structured magnetic nanocomposites. For example, a Mn 1−x Ni x Fe 2 O 4 catalyst with varying concentrations of nickel (x = 0.1, 0.2, 0.3, 0.4, and 0.5) was evaluated for indigo carmine dye degradation by Jesudoss et al. [118]. Amongst the obtained photocatalysts, the Mn 0.5 Ni 0.5 Fe 2 O 4 catalyst exhibited higher photocatalytic performance in the degradation of indigo carmine dye, with 96% degradation within a 180 min period. The material exhibited excellent saturation magnetization of 35.0 emu/g, demonstrating that this can be recoverable after a catalytic reaction. The authors concluded that the concentration of Ni 2+ ions affected the structure of MnFe 2 O 4 , and that a high concentration of nickel ions reduced the crystallite size and increased the surface area, thereby affecting photocatalytic activity (Figure 14). Finally, in terms of photocatalytic activity, the results demonstrated that approximately 80% and 75% of methylene orange (MO) and methylene blue (MB) were effectively degraded by Co0.5Zn0.25Ni0.25Fe2O4/TiO2. The authors attributed the good photocatalytic activity to the Ni 2+ ions and synergistic effect in combination with Co 2+ ions [117]. This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, and the experimental conclusions that can be drawn. Several other studies have also demonstrated the photocatalytic performance of complex-structured magnetic nanocomposites. For example, a Mn1-xNixFe2O4 catalyst with varying concentrations of nickel (x = 0.1, 0.2, 0.3, 0.4, and 0.5) was evaluated for indigo carmine dye degradation by Jesudoss et al. [118]. Amongst the obtained photocatalysts, the Mn0.5Ni0.5Fe2O4 catalyst exhibited higher photocatalytic performance in the degradation of indigo carmine dye, with 96% degradation within a 180 min period. The material exhibited excellent saturation magnetization of 35.0 emu/g, demonstrating that this can be recoverable after a catalytic reaction. The authors concluded that the concentration of Ni 2+ ions affected the structure of MnFe2O4, and that a high concentration of nickel ions reduced the crystallite size and increased the surface area, thereby affecting photocatalytic activity ( Figure 14). A study by Naik et al. [119] evaluated the performance of nanostructured zinc-doped cobalt ferrites (Zn x Co 1−x Fe 2 O 4 with (x = 0.0 to 0.6 with the step of 0.2) in the photocatalytic degradation of Congo Red (CR) and Evans Blue (EB) dyes. They established that the photocatalytic performance of cobalt ferrite increased with an increase in Zn-doping up to x = 0.4 then decreased thereafter (Figure 15). Figure 14. Effect of Ni 2+ ions doped MnFe2O4 magnetic nanoparticles on the photocatalytic degradation (PCD) efficiency. The conditions used were IC = 150 mg/L, photocatalyst = 50 mg/100 mL, and λ = 365 nm) [118]. Republished with permission from Elsevier. A study by Naik et al. [119] evaluated the performance of nanostructured zinc-doped cobalt ferrites (ZnxCo1−xFe2O4 with (x = 0.0 to 0.6 with the step of 0.2) in the photocatalytic degradation of Congo Red (CR) and Evans Blue (EB) dyes. They established that the photocatalytic performance of cobalt ferrite increased with an increase in Zn-doping up to x = 0.4 then decreased thereafter (Figure 15). Additional studies revealed that higher Zn 2+ doping concentrations increased the bactericidal properties of the CoFe2O4 towards human pathogens. For both Congo red (CR) and Evans Blue (EB) dyes, Zn0.4Co0.6Fe2O4 nanoparticles showed good photocatalytic activity in 150 min of irradiation time. The study suggests that the synthesized nanoparticles are suitable for photocatalytic applications. More studies of photocatalytic activity of mixed metal ferrites nanocomposites are summarized in Table 2. The study suggests that the synthesized nanoparticles are suitable for photocatalytic applications. More studies of photocatalytic activity of mixed metal ferrites nanocomposites are summarized in Table 2. It is important to note that, besides the focus of the development of magnetic nanocomposites for water and wastewater treatment, new recent trend of development of new materials is emerging. For example, Mir et al. [138] studied how confining AuNPs in a porous Si template can significantly enhance the photocatalytic activity of MO. The pores prevent agglomeration of nanoparticles and eliminate the need for any functionalization. Confinement of the AuNPs in the Si nanocavities prevents electron-hole recombination and facilitates the transfer of hot carriers from the Si support to accelerate the photocatalytic efficiency. The results showed that the recyclable, low-band gap photocatalytic system has economic and environmental advantages that promote implementation of catalytic and separation processes in continuous flow mode, with the advantages associated with easier phase separation and product recovery, enhanced safety, and easier operation [138]. Another recent trend of study explored functional elastomeric copolymer membranes designed by nanoarchitectonics approach for Methylene Blue Removal. The results demonstrated specific adsorption abilities up to 18 mg/g of grafted cyclodextrins [139]. The findings obtained in these studies show that more studies can be explored in order to develop new nanomaterials that are sustainable and safer for water and wastewater applications. Factors Affecting the Photocatalytic Activity of Magnetic Nanocomposites The photocatalytic activity of magnetic nanocomposites is dependent on several factors such as surface area, concentrations of dopant metal ions, pH, and catalyst loading. Catalyst Surface Area Nanomaterials with a high specific surface area exhibit enhanced catalytic activity. A decrease in particle size takes one to the increase of the surface area, which improves the dispersion of the nanomaterials in solution. This results in an enhanced photon absorbance, leading to the retrieval of their photocatalytic performance [140][141][142][143]. A study done by Padmapriya et al. [144] evaluated the effect of surface areas of magnetic nanoparticles system Ni x Zn 1−x Fe 2 O 4 , with different concentrations of Ni 2+ ions (0.0 ≤ x ≤ 1.0) in photocatalytic degradation of methylene blue dye (Figure 16a). new materials is emerging. For example, Mir et al. [138] studied how confining AuNPs a porous Si template can significantly enhance the photocatalytic activity of MO. T pores prevent agglomeration of nanoparticles and eliminate the need for a functionalization. Confinement of the AuNPs in the Si nanocavities prevents electro hole recombination and facilitates the transfer of hot carriers from the Si support accelerate the photocatalytic efficiency. The results showed that the recyclable, low-band gap photocatalytic system h economic and environmental advantages that promote implementation of catalytic a separation processes in continuous flow mode, with the advantages associated with eas phase separation and product recovery, enhanced safety, and easier operation [138]. Another recent trend of study explored functional elastomeric copolym membranes designed by nanoarchitectonics approach for Methylene Blue Removal. T results demonstrated specific adsorption abilities up to 18 mg/g of grafted cyclodextr [139]. The findings obtained in these studies show that more studies can be explored order to develop new nanomaterials that are sustainable and safer for water a wastewater applications. Factors Affecting the Photocatalytic Activity of Magnetic Nanocomposites The photocatalytic activity of magnetic nanocomposites is dependent on seve factors such as surface area, concentrations of dopant metal ions, pH, and catalyst loadin Catalyst Surface Area Nanomaterials with a high specific surface area exhibit enhanced catalytic activity decrease in particle size takes one to the increase of the surface area, which improves t dispersion of the nanomaterials in solution. This results in an enhanced phot absorbance, leading to the retrieval of their photocatalytic performance [140][141][142][143]. A study done by Padmapriya et al. [144] evaluated the effect of surface areas magnetic nanoparticles system NixZn1−xFe2O4, with different concentrations of Ni 2+ io (0.0 ≤ x ≤ 1.0) in photocatalytic degradation of methylene blue dye (Figure 16a). The results demonstrated that better photocatalytic activity was found for t nanocatalyst Ni0.6Zn0.4Fe2O4, which had a high surface area (36.6 m 2 /g) compared to t other samples. It can be understood from this study that surface area is also related to active sites on the catalytic surface, which enhance the photocatalytic activity. Manikandan et al. [115] demonstrated that the photocatalytic degradation of Chlorophenol (4-CP) using the photocatalyst ZnFe2O4 was affected by the particle size a morphology of the catalyst. The results demonstrated that better photocatalytic activity was found for the nanocatalyst Ni 0.6 Zn 0.4 Fe 2 O 4, which had a high surface area (36.6 m 2 /g) compared to the other samples. It can be understood from this study that surface area is also related to the active sites on the catalytic surface, which enhance the photocatalytic activity. Manikandan et al. [115] demonstrated that the photocatalytic degradation of 4-Chlorophenol (4-CP) using the photocatalyst ZnFe 2 O 4 was affected by the particle size and morphology of the catalyst. Additionally, a study conducted by Jia et al. [145] reported that the photocatalytic activity of ZnFe 2 O 4 nanoparticles for methylene blue dye degradation was related to the surface properties and surface defects of the photocatalyst. Effect of Catalyst Amount The efficiency photocatalytic reactions can be influenced by the amount of the catalyst used. Padmapriya et al. [144] reported that the photocatalytic degradation of methylene blue dye was found to increase with an increase in the amount of the magnetic catalyst Ni 0.6 Zn 0.4 Fe 2 O 4 until a certain amount of nanocatalyst loading (Figure 16b). However, further increase in the catalyst loading demonstrated negative influence of the degradation plateaued. The authors also reported that adding an amount of the magnetic nanocatalyst increased the active sites on the catalyst surface, which in turn increased the amount of •OH (hydroxyl) and •O 2 − (superoxide) radicals and degraded the methylene blue (MB) dye. Furthermore, the excess of amount of nanocatalyst beyond the optimum may have resulted in the agglomeration of catalyst particles and generated turbidity, which resulted in the decrease of the photocatalytic degradation efficiency [140,142,146]. Effect of pH Generally, the solution pH is an important variable in water and wastewater treatment as it has a significant influence on the photocatalytic degradation process of organic compounds [10]. The variation of pH alters the surface charge of heterogeneous catalysts and, consequently, changes the photocatalytic activity of catalyst [147,148]. Figure 16c shows the influence of different pH (3, 4, 5, 6, 7, 8, and 9) on the photodegradation of methylene blue using the nanomagnetic catalyst Ni 0.6 Zn 0.4 Fe 2 O 4 in a study conducted by Padmapriya et al. [144]. The results from this study showed that high photocatalytic degradation efficiency was achieved at pH = 3, due to electrostatic attraction between the anionic dye (MB) and the positively charged surface of nanocatalyst. The authors demonstrated that at pH values above 7, the nanocatalyst surface became negatively charged, leading to electrostatic repulsion between the methylene blue dye and the catalyst, which reduced the photocatalytic degradation efficiency. More studies demonstrating the same behavior were reported by Mirkhani et al. [149] and Suwarnkar et al. [150]. Reusability of the Magnetic Nanocatalyst Heterogeneous photocatalysis technology is always looking for an ideal photocatalyst, one that is reusable and that possesses high photocatalytic efficiency, a large specific surface area, and ability to absorb visible light [138]. Thus, the recyclability of catalysts is one of the key steps towards the sustainable application of photocatalysts and development of heterogeneous photocatalysis technology for water and wastewater treatment. The recyclability of catalysts is also related to their actual operational costs. Krishna et al. [100] reported the reusability of the CoFe 2 O 4 /TiO 2 nanocatalysts for acid blue 113 (AB113) dye degradation through magnetic separation where its photocatalytic activity was found to be retained up to six consecutive cycles and without considerable loss of photocatalytic activity and stability ( Figure 17). Table 3 shows additional results of the activities of reused magnetic nanocomposites for organic photodegradation processes. Most of the magnetic nanocomposites are recyclable up to more than three runs, demonstrating their stability during their application for water and wastewater treatment for organic pollutants removal. Therefore, these studies are indicators for possible industrial or large-scale application. [100]; and (c) magnetic responsiveness of CoFe2O4/TiO2 with an external magnetic field [153]. Republished with permission from Elsevier. The Overlooked Social Dimension The focus of most water and wastewater-related research has been on the technical aspects of the problem and improvements in terms of water quality and in minimizing environmental and health impacts, with very limited attention to its basic social and cultural sustainability dimensions [158]. A study done by Wichelns et al. [159] demonstrated that there is a need for a paradigm shift from the 'treatment for disposal' to the 'treatment for reuse' since wastewater contains pollutants such as organic and inorganic compounds which may pose health risks if not well managed [159]. Additionally, even when wastewater is treated using advanced technologies and health risks are carefully addressed and controlled, irrespective of all scientific evidence, the social perception remains the driver of the success or failure of wastewater reuse schemes [158]. Depending on public perceptions, impressions, and attitudes, the development of a wastewater scheme can be supported or constrained. Negative public perception can prevent well-planned projects from moving forward. On the other hand, positive public perception, which leads to greater acceptance, is the key element for the successful implementation of wastewater treatment [159,160]. Saad et al. [158] reported that various local communities around the world have rejected several water and wastewater treatment projects by their governments due to inadequate community consultation which resulted in negative public perception. In summary, it can be said that recognizing the role of the social base for wastewater management from risk reduction to reuse can have major implications, for example, on the choice and effectiveness of the technologies employed. [100]; and (c) magnetic responsiveness of CoFe 2 O 4 /TiO 2 with an external magnetic field [153]. Republished with permission from Elsevier. The Overlooked Social Dimension The focus of most water and wastewater-related research has been on the technical aspects of the problem and improvements in terms of water quality and in minimizing environmental and health impacts, with very limited attention to its basic social and cultural sustainability dimensions [158]. A study done by Wichelns et al. [159] demonstrated that there is a need for a paradigm shift from the 'treatment for disposal' to the 'treatment for reuse' since wastewater contains pollutants such as organic and inorganic compounds which may pose health risks if not well managed [159]. Additionally, even when wastewater is treated using advanced technologies and health risks are carefully addressed and controlled, irrespective of all scientific evidence, the social perception remains the driver of the success or failure of wastewater reuse schemes [158]. Depending on public perceptions, impressions, and attitudes, the development of a wastewater scheme can be supported or constrained. Negative public perception can prevent well-planned projects from moving forward. On the other hand, positive public perception, which leads to greater acceptance, is the key element for the successful implementation of wastewater treatment [159,160]. Saad et al. [158] reported that various local communities around the world have rejected several water and wastewater treatment projects by their governments due to inadequate community consultation which resulted in negative public perception. In summary, it can be said that recognizing the role of the social base for wastewater management from risk reduction to reuse can have major implications, for example, on the choice and effectiveness of the technologies employed. Added to this is the creation of economic incentives for the public and private sector institutions to invest in sanitation and to generate income for private operators as well as secure their sustainability [161]. Conclusions and Recommendation The development and application of magnetic ferrite-based titanium oxide and zinc oxide nanocomposite as catalysts are extremely promising for the removal of organic pollutants from water and wastewater, as shown by various studies presented in this review. Studies demonstrated that these catalysts can be prepared by different methods such as sol-gel, co-precipitation, hydrothermal, and combustion. However, the methods of synthesis are chosen based on their advantages. The magnetic nanoparticles (MNPs) have several advantages, including that they are easily separated by an external magnetic field without loss of the nanocatalyst, which can be reused up to several runs of experiments. In most of studies, the magnetic based titanium oxide and zinc oxide nanocomposite exhibited an excellent catalytic activity for organic pollutants removal. Additionally, some studies showed that these catalysts were even effective after more three successive cycling runs. The catalytic activity of the MNPs as catalysts is a direct outcome of its intrinsic characteristics as well as of its synthesis method; nevertheless, the catalytic performance can be influenced by conditions that are imposed on these materials to prepare them for a given application. Additionally, the method of synthesis plays a principal role in the physicochemical properties of the catalyst obtained. However, the synthesis of magnetic nanoparticles and their relevance for organic dyes removal from water and wastewater still require more investigation in order to achieve the optimum optimization for large-scale for subsequent practical applications. Finally, studies of the application of MNPs-based oxides nanocomposites in water and wastewater treatment are still few; however, more studies are still required. Additionally, with this technology in progress, scientists have enough supporting theory to upscale and provide a cleaner environment and safe drinking water to human populations. Conflicts of Interest: The authors declare no conflict of interest.	2021-12-22T16:31:09.927Z	2021-12-18T00:00:00.000	{ "year": 2021, "sha1": "ffcde22005664cc112c25d357e9614a31eb21a23", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2073-4344/11/12/1543/pdf?version=1640076432", "oa_status": "GOLD", "pdf_src": "Adhoc", "pdf_hash": "03c8a772f6122dc5453c7fb21e276d0f200ae3c2", "s2fieldsofstudy": [ "Environmental Science", "Materials Science", "Chemistry" ], "extfieldsofstudy": [] }
201187744	pes2o/s2orc	v3-fos-license	Ingested sharp foreign body presented as chronic esophageal stricture and inflammatory mediastinal mass for 113 weeks: Case report Introduction Impacted foreign bodies in the esophagus have the potential to cause serious complications. Ingested sharp objects carry the risk of acute complications as: perforation, acute mediastinitis, and acute bleeding. Rarely, such foreign bodies might migrate through the esophageal wall and present as chronic esophageal foreign body. Case presentation We present a case of a 36-month-old girl presented with solid food dysphagia and regurgitation proved to be secondary to esophageal stricture after 26 months of accidental ingestion of aluminum can tab which has migrated through the wall of the upper esophagus into the mediastinum. After two trials of endoscopic treatment; she underwent thoracotomy and partial esophagectomy. Multiple trials of dilation and Mitomycin C injection were followed because of re-stricture. Conclusion Foreign body impaction or secondary stricture needs to be considered in the differential diagnosis of children presenting with new onset dysphagia and regurgitation. Metallic Foreign body might be even radiolucent. Practitioners should keep a high index of suspicion for a retained esophageal FB in the child with gastrointestinal or respiratory symptoms that do not respond to standard therapy. Introduction Foreign body (FB) ingestion is an everyday occurrence and a common emergency presentation in the pediatric population. Most of the FBs travel through the gastrointestinal tract without any complications. The natural narrowing areas through the gut serve as places for FB impaction [1]. Impacted FBs in the esophagus have the potential to cause serious complications, apart from significant distress to the patient and the family [1]. Esophagoscopy offers a safe and a widely available intervention for retrieval of FBs in the esophagus. Sometimes it is difficult to differentiate chronic esophageal foreign body (CEFB) ingestion from those patients with an upper respiratory tract infection or asthma or esophageal stenosis [2,3]. Ingested sharp objects carry the risk of perforating the esophagus leading to acute mediastinitis or acute bleeding, or rarely leading to abscess or fistula formation [4,5]. We present a case of a 36-month-old girl who presented with solid food dysphagia and regurgitation secondary to esophageal stricture after 26 months of accidental ingestion of an aluminum can tab that has migrated through the wall of the upper esophagus as a technical perforation. This case study was performed and is being reported in line with the SCARE criteria [6]. Case presentation Thirty-six-month-old girl; not known to have any medical or congenital illness; presented to our center complaining of persistent solid food dysphagia and regurgitation since the age of 10 months. However, her symptoms worsened over the last three months. The parents deny any history of recurrent choking or aspiration. She had no atopy, and her mother could not link her symptoms to any specific foods. Her drug, family and psychosocial history were irrelevant. Her examination revealed a normal looking child except for growth parameters which were on 5th percentile. Her basic laboratory investigations were normal. Chest X-Ray was normal. She underwent diagnostic upper endoscopy by consultant pediatrics gastroenterologist (EA) and revealed a stricture at the upper esophagus with dilated pre-stenotic pouch filled with food. A flexible infant scope (Olympus ® 4.9 mm) could not pass through the stricture. Barium swallow was performed and delineated the anatomy of the stricture at the upper esophagus. Fig. 1. Trial of antegrade esophageal dilatation (using flexible and rigid scopes) under general anesthesia failed at the first time, repeated four weeks later after gastrostomy and retrograde approach also failed by pediatrics surgeon (ZB) and (EA). Due to the fixed stricture and posterior outpouching, chest computed tomography (CT) scan with IV contrast was carried out. and revealed a circumferential soft tissue thickening involving the upper thoracic esophagus more pronounced at its right posterior lateral wall containing multiple rim-enhancing collections; the largest of which measures about 1.1 × 0.6 cm with linear hyperdense material measuring about 1.5 cm causing complete obstruction of the esophagus at this level and compression effect on the trachea which was displaced to the left side. Fig. 2. The patient underwent thoracotomy by the pediatrics surgeon (ZB)_. An aluminum can tab with reactionary inflammatory mass around it were obstructing the esophagus and found to erode the esophageal wall with intramural elements. Removal of the foreign body along with the mass, partial esophagectomy with primary anastomosis were performed due to relatively wide esophageal perforation with in the inflammatory mass. No postoperative complications were detected. Fig. 3 Video 1. Retrospectively, the mother recalled a history of chocking episode and vomiting at the age of 10 months, which was not investigated at that time. After recovery, she was able to tolerate oral intake. She presented two weeks later with dysphagia and regurgitation. Her upper endoscopy revealed re-stricture formation at the esophagectomy site. She underwent multiple sessions of esophageal dilatation using buggies up to 11mm by (EA). Due to the re-stricture the decision was to apply Mitomycin to reduce the need to re-dilatation. Mitomycin C was prepared immediately before application at a concentration of 0.4 mg/ml and applied at the site of dilatation. The last dilatation session was four months ago (6 months postoperatively). The parents were taught about the risk of FBs ingestion and about the importance of notice any episodes of chocking and they were adherent. The patient is doing well; she gained weight and is able to tolerate solid food. Her family were satisfied about the treatment and outcome. Discussion CEFB is defined as an impacted FB for at least one-week duration [4]. Miller et al. reported 8% of CEFB from a total 552 cases of FB ingestion [4]. They stated that the duration of impaction was less than 12 weeks in 83% of the patients and only 2 patients had a duration longer than two weeks [4]. Glover and colleagues presented a case of a child with a 2-year history of chronic cough after witnessed FB ingestion [3]. Also, in a case series of 5 children with FBs, Gilchrist et al. reported the retention duration from 2 to 24 months [5]. In addition, Cole et al. described 3 cases of prolonged FB retention from 3 to 12 months complicated by mediastinitis [7]. Moreover, Skaff et al. reported a case of 2-yeaar-old girl presented with intramural CEFB for 11 months [8]. To the best of our knowledge, we have presented a case for the longest duration of sharp FB impaction that presented after 26 months (approximately 113 weeks) as progressive dysphagia and solid food intolerance and revealed to have intramural "can tab" with technical perforation, esophageal stenosis, esophageal pouching and inflammatory mediastinal mass. According to Miller et al., a ''classic'' esophageal perforation was defined as erosion through the mucosal and muscular layers of the esophagus such that there was a direct connection between the esophageal lumen and the mediastinum [4]. This was evaluated either by: (1) direct visualization of an esophageal perforation in the operating room (endoscopically or through an open approach) or (2) radiographically through visualization of esophageal contrast spilling into the mediastinum. A ''technical'' esophageal perforation is defined by meeting any one of the following criteria: (1) foreign body erosion through the mucosal layer of the esophagus; (2) a foreign body that had been walled off in either the mucosal or muscular layer of the esophagus; or (3) radiologic evidence of contrast leaking from the esophageal lumen into a loculation or space in the wall of the esophagus, but not extending into the mediastinum [4]. In their review, a total of 17 patients were found to have technical perforation. One of the main theoretical pathophysiology for development of CEFB and its complications is mucosal ulceration with prominent underlying necrosis of the muscularis propria followed by muscular parenchyma replacement by granulation and fibrous tissue, and a marked mixed inflammatory infiltrate extending into the surrounding tissue [4,9]. Recognition of the presenting symptoms of this condition is critical for the diagnosis and treatment of retained FBs. Incidence is greatest in children aged 6 months to 4 years. This reflects the tendency of small children to use their mouths in the exploration of their world [1,10]. When pediatric patients do present with CEFB, their symptoms differ from those of patients with acute FBs. Vomiting, dysphagia, drooling of saliva, and respiratory symptoms were the most common presenting symptoms in several studies [4,10,11]. In our case, the 36 months-patient presented with symptoms related to the complications of the FB ingestion-stricture formation-as progressive dysphagia and regurgitation for solid foods. Most common FBs ingested in children are coins [4,11]. Being radiopaque hasten the process of diagnosis. Although the FB in our case was metallic, it was not visualized on neither her chest X-ray nor in the upper gastrointestinal series done for her. It is already reported that aluminum is a low molecular weight compound, relatively radiolucent, unlike most other common metals. Therefore, ingested aluminum objects are difficult to appreciate on radiographs [12,13]. The ingested FB in this case was Aluminum can tab with sharp edges. In one study by Singh et al., the main factor that is associated with FB ingestion complication is the late presentation [14]. CEFB has been associated with severe complications including bronchoesophageal fistula, aortoesophageal fistula, esophageal perforation with resulting mediastinitis/abscess, complete esophageal stricture/esophageal obstruction, pulmonary edema, esophageal diverticulum, esophageal stricture requiring re-dilatation and malnutrition [2][3][4][5]14]. The main treatment modality is rigid esophagoscopy under general anesthesia. However, in severe cases, thoracotomy may be needed [4]. In this article, the patient underwent thoracotomy with removal of the FB and partial esophagectomy. The case was further complicated by severe stenotic tissue with pouching and intramural location of the FB. Although the FB was removed, surgical site re-strictured again. Esophageal dilatation improved the condition of the patient temporarily. Unfortunately, she needed frequent dilatation. Mitomycin C is a popular choice for benign strictures [15]. Our case presents an example of how safe and effective Mitomycin C application in children with esophageal strictures requiring multiple frequent dilatations. Conclusion This report highlights and adds important learning points regarding esophageal FB ingestions in children. First, FB impaction or secondary stricture needs to be considered in the differential diagnosis of children presenting with new onset dysphagia and regurgitation. Second, metallic FBs might be even radiolucent, and X-ray might not be enough to exclude FB ingestion. Third, impacted FB in the esophagus may remain asymptomatic for some time and become symptomatic only when complications develop. Therefore, practitioners should keep a high index of suspicion for a retained esophageal FB in the child with gastrointestinal or respiratory symptoms that do not respond to standard therapy. Finally, CEFB can lead to significant esophageal injury and complications that may enforce the physicians to go for aggressive surgeries. As Esophageal dilatation would be considered a safe procedure in cases of esophageal strictures; Mitomycin C might be considered in resistant reoccurring strictures. Ethics approval The institutional review board is not required. Sources of funding No funding. Author contribution All authors contributed significantly and in agreement with the content of the article. Tashtush, Al Quran and Aleshawi collected all data and photographs to draft the manuscript. Altamimi was the gastroenterologist who perform the esophagoscopy. Yusef was the pediatric consultant who follow the patient. Bataineh performed the surgery. Rousan and Khasawneh were the radiologist. Tashtush, Al Quran and Aleshawi wrote the manuscript for submission. All authors presented substantial contributions to the article and participated of correction and final approval of the version to be submitted. Conflicts of interest The authors declare that they have no competing interests. Registration registration number None.	2019-08-23T02:03:46.422Z	2019-08-01T00:00:00.000	{ "year": 2019, "sha1": "e84374ce506151271e32cd653fb050d08de96cd4", "oa_license": "CCBY", "oa_url": "https://doi.org/10.1016/j.amsu.2019.07.028", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "ad3760963de1f759b86689dda5374d76bd546b32", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
267506492	pes2o/s2orc	v3-fos-license	Atypical Presentation of Pemphigus Vulgaris: Nail Involvement in a 20-Year-Old Male Pemphigus vulgaris (PV) mainly causes blistering of the skin and mucous membranes, with nail unit involvement being rare. Nail involvement may serve as an indicator of disease severity. We present a case of a 20-year-old male with PV who had both cutaneous and nail findings, with nail changes corresponding with disease severity. The patient with biopsy-confirmed PV, on prednisone and mycophenolate, presented to the emergency department with an acute flare of PV and severe mandibular pain and lymphadenopathy. At follow-up in our outpatient department, the physical examination was significant for onychomadesis and onycholysis of the fingernails. Prednisone and mycophenolate dosages were increased, and rituximab infusions were initiated. Bullae and mucosal lesions resolved on the follow-up, and nail changes improved. This case appends an unusual perspective to the limited literature on PV-associated nail changes, especially in younger patients. It advocates for meticulous history taking and physical examination and supports a correlation between nail symptoms and PV disease severity. Introduction Pemphigus vulgaris (PV) is an autoimmune vesiculobullous disease that causes blistering of the skin and mucous membranes [1].Nail involvement is uncommon and underreported [2].We describe a case of PV in a 20-year-old male who presented with onychodystrophy, corresponding with the severity of his oral and cutaneous lesions and improving with PV treatment. Case Presentation A 20-year-old male with a diagnosis of PV presented with painful oral and cutaneous lesions.Two months before his initial visit to our dermatology clinic, the patient underwent a punch biopsy, with histopathology showing suprabasal clefting and a dermal eosinophil-rich infiltrate.Direct immunofluorescence testing showed intracellular deposits of C3 and IgG in the epidermis, consistent with a diagnosis of PV. Prior to presenting to our clinic, the patient was initially managed with 20 mg of oral prednisone daily and 500 mg of mycophenolate twice daily.Despite initial improvement, after one month of treatment, he presented to the emergency department with mandibular pain and edema, submandibular lymphadenopathy, and bullae and erosions posterior, soft and hard palate, cheeks, and gums.He met the systemic inflammatory response syndrome criteria, indicating a heightened state of inflammation based on physiologic parameters, including heart rate, respiratory rate, leukocyte count, and body temperature.He was given a 10-day course of oral amoxicillin/clavulanate, and prednisone was increased to 40 mg. On presentation to our dermatology clinic, three weeks after his prednisone dosage was increased, his bullae were healing, and he did not develop any new ones.His clinical examination was notable for hyperpigmented patches on his chest, back, abdomen, axillae, and upper medial thighs, corresponding to ruptured vesicles and bullae.No intact bullae were present.However, superficial ulcerations were observed in the bilateral buccal mucosa.There was onychomadesis, separation of the proximal and distal parts of the nail plate, and brown discoloration involving his fingernails (Figure 1), without involvement of the toenails. FIGURE 1: Clinical presentation of bilateral index fingernails at initial visit showing onychomadesis and brown discoloration of the nail plate proximally The black arrows indicate the presence of onychomadesis, while the blue arrow indicates brown discoloration of the nail plate. The patient was started on sulfamethoxazole and trimethoprim 800 mg/160 mg three times per week, as well as omeprazole and a calcium vitamin D supplement.He was prescribed rituximab (1,000-mg IV infusion on Days 1 and 15), but the treatment was complicated by a grade 2 infusion reaction during the first infusion.He was continued on mycophenolate 500 mg twice daily and oral prednisone 40 mg daily, with a plan of decreasing his dose by 5 mg every two weeks.When he returned to the clinic for his two-week follow-up, he reported no new oral or cutaneous lesions and noted continued improvement in his existing lesions but not in his fingernail changes.At his seven-week follow-up, however, no developing onychomadesis was noted proximally, and the previously observed signs of onychomadesis had migrated distally, consistent with nail growth and clinical improvement (Figure 2).A comparison of clinical photographs at each visit helped evaluate nail improvement over time. Discussion We report the case of a 20-year-old patient with PV who exhibited nail changes affecting his fingernails, corresponding to a flare in his PV.Nail changes associated with PV are either uncommon or underreported [2].In a retrospective study conducted in the United States analyzing nail changes in patients with pemphigus (N = 141 patients), not limited to PV, 26% of patients had non-fungal nail changes that correlated with pemphigus disease severity, the most common being pachyonychia (25%), paronychia (25%), and onycholysis (21.5%) [2].In PV patients, specifically, nail changes were present in 47% of cases.In a single-center retrospective observational study in Poland with 67 ethnically Polish patients, nail involvement was present in 13% of cases, with paronychia (13%), nail discoloration (10%), and Beau's lines (7%) being the most common findings [3].In a retrospective study with 79 patients in Iran, nail involvement was present in 32% of cases, with paronychia (10%) and onychomadesis (8%) being the most common changes [4].In a case-control study in India involving 25 patients with PV, 80% exhibited nail changes, with paronychia (40%) and onychorrhexis (32%) being the most common findings [5].In a case series of 15 patients with PV and nail involvement, paronychia was the most frequent, present in 60% of cases, followed by onychomadesis (33%) [6].In a retrospective study conducted on 25 Egyptian patients with PV, the predominant nail changes were onycholysis and subungual hyperkeratosis, which were observed in 16 patients (64%) [7]. With PV-associated nail changes, fingernails are more often affected than toenails, particularly the first three, possibly because these are used more often and subject to local trauma [8].In a case study of a 55year-old female who experienced chronic, treatment-resistant paronychia, her onychodystrophy was the initial presentation of PV [9].The onset of nail changes may precede a flare [10], present concurrently with mucosal and cutaneous lesions, or be the only symptom [7].While nail changes often signify the progression of PV, they may also reflect treatment efficacy.For example, in a case report of a 34-year-old female, her PVassociated paronychia improved rapidly following treatment with oral tofacitinib and rituximab infusions [11].In our patient, nail involvement presented with the initial PV lesions worsened with flare and improved with systemic treatment. PV generally affects older individuals, most commonly in their fourth to sixth decade of life [12].In a crosssectional analysis of 1,795 adult pemphigus patients in the United States, not limited to PV, 78.5% of patients were at least 50 years old, and only 3.9% of adults with PV were under 30 years old [13].Our patient is 20 years old, which makes his presentation of early-onset PV, in addition to his having associated nail involvement, atypical. The cause of PV-associated nail changes and why they might be rare remains unclear, but it may be because the nail bed is an "immunologically privileged" site where the immune system is less active [9,14].In the nail immune system, the quantity and function of antigen-presenting cells are markedly diminished compared to those in the cutaneous or mucosal epidermis.Furthermore, major histocompatibility class II and CD209 expression by Langerhans cells in the proximal nail matrix is notably decreased, along with decreased functionality and/or quantity of natural killer and mast cells around the human nail apparatus [8].Additionally, it is hypothesized that the nail unit has a lower density, reduced expression, or relative sequestration of desmoglein 1 (DSG1) and DSG3 compared to the skin and mucosal epidermis.These are the proteins targeted by autoantibodies in PV.Furthermore, the nail matrix is devoid of a granular layer, and the nail bed epidermis is merely two to three cells thick [11].As a result, the nail unit exhibits low levels of DSG1 and DSG3 expression.This low expression could serve as a protective mechanism, as a high concentration of autoantibodies targeting these desmosomal glycoproteins would be necessary for the nails to be affected in PV, potentially shielding the nail unit from autoimmune attacks [8,14]. While PV may present with nail changes, onychomadesis may stem from a wide range of etiologies.In the absence of characteristic PV symptoms or a known PV diagnosis, onychomadesis should prompt consideration of a broad differential diagnosis, including bacterial infections, viral illness, trauma, medication side effects, systemic disease, and idiopathic nail changes [15].In this case, the patient's established diagnosis of PV provided context for the observed nail changes.A limitation of our study is the possibility of conflating PV-related nail changes with similar presentations caused by other etiologies, emphasizing the importance of a thorough history taking and physical examination. Conclusions This case of a 20-year-old patient with PV highlights the clinical significance of nail changes as an integral aspect of disease presentation and progression.Our findings underscore that nail involvement may be a clinical finding in patients with PV, although variably reported in the literature, and may correlate with disease severity.Recognizing nail changes as potential indicators of PV activity can lead to a more timely diagnosis and tailored management strategies.Our observations also contribute to the growing understanding of PV pathophysiology, particularly the role of the nail unit as an "immunologically privileged" site, potentially influencing the expression and impact of autoantibodies in PV. FIGURE 2 : FIGURE 2: Clinical presentation of fingernails showing more distal onychomadesis The black arrows indicate the presence of onychomadesis.	2024-02-07T16:06:59.723Z	2024-02-01T00:00:00.000	{ "year": 2024, "sha1": "2abc0f0b2f5ed536ff28e53d47ec2638d0e448ba", "oa_license": "CCBY", "oa_url": "https://assets.cureus.com/uploads/case_report/pdf/212735/20240205-23086-w18jw0.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "09135aa5bf47e853a9a9f8a98f5fc26f3b6a9be0", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [] }
64591020	pes2o/s2orc	v3-fos-license	Cyclone , Salinity Intrusion and Adaptation and Coping Measures in Coastal Bangladesh Although households in the coastal areas of Bangladesh undertake various adaptation and coping measures to minimise their vulnerability to cyclone hazards and salinity intrusion, these autonomous measures have received little attention in the past. However, the Government of Bangladesh has recently emphasised the importance of understanding these measures so that necessary interventions to make households more resilient to natural hazards and the adverse impacts of climate change can be introduced. This paper, based on secondary sources, explores adaptation and coping measures that households in the coastal areas of Bangladesh undertake to minimise their vulnerability to cyclone hazards and salinity intrusion. This paper shows that many of the adaptation and coping measures contribute to making households less vulnerable and more resilient to cyclone hazards and salinity intrusion, although some coping measures do the opposite as they reduce households’ adaptive capacities instead of improving them. This paper argues that the adaptation and coping measures that contribute to reducing households’ vulnerability to natural hazards need to be supported and guided by the government and NGOs to make them more effective. Additionally, measures that make households more vulnerable also need to be addressed by the government and NGOs, as most of these measures are related to and constrained by both poverty, and because the households have little or no access to economic opportunities. Introduction Bangladesh, which is one of the most disasterprone countries in the world, regularly experiences various types of natural hazards, including floods, cyclones and storm surges, tornados, river-bank erosion, droughts, landslides, and earthquakes (Ministry of Environment and Forests [MEF], 2005; Disaster Management Bureau [DMB], 2010).Cyclonic storm surges and floods are major killers and the cause of most direct and indirect damage in Bangladesh (MEF, 2005).Furthermore, climate change will exacerbate many of the existing natural hazards that the country faces (MEF, 2005; Ministry of Environment and Forests [MEF], 2009; Ministry of Disaster Management and Relief [MDMR], 2015) (see Table I).Climate change will lead to an increase in both the frequency and intensity of climatic events, although it will not necessarily create new hazards.Bangladesh's low level of social and economic development, inadequate infrastructure, lack of institutional capacity, and higher dependency on the natural resource base increases its susceptibility to existing natural hazards and the adverse effects of climate change (MEF, 2005), with the population living in the coastal areas being more exposed to these impacts than those living in other parts of Bangladesh (MEF, 2005;Parvin, Takahashi, & Shaw, 2008;Warner et al., 2012). Despite the high level of vulnerability to multiple natural hazards, coastal people of Bangladesh have a long tradition of employing various adaptation and coping measures to cope with these hazards (MEF, 2009;Ahmed, Haq, Nasreen, & Hassan, 2015;Paul & Routray, 2011).It has been argued that autonomous measures employed by people have largely been ignored and not integrated into a systematic approach, by either government or non-government organisations (Ahmed et al., 2015;Paul & Routray, 2011).However, in recent years, the Government of Bangladesh has focused on community-based disaster risk management (MEF, 2009;DMB, 2010).The three major government policy documents related to climate change and disaster management are the Bangladesh Climate Change Strategy and Action Plan (BCCSAP) (MEF, 2009), the National Plan for Disaster Management 2010-2015 (NPDM) (DMB, 2010), and the National Disaster Management Policy (NDMP) (MDMR, 2015).These documents focus on community-based disaster risk reduction and climate change adaptation measures to strengthen community and household capacity to cope with natural hazards and the adverse effects of climate change. 12 To strengthen community and household capacity, each of the three documents emphasises the need for interventions to be built on the understanding of the existing susceptibility of communities and households to natural hazards, and the adaptation and coping measures they currently use to deal with natural hazards and the adverse effects of climate change.Thus, a comprehensive understanding of what adaptation and coping measures households undertake to cope with natural hazards is essential in designing effective interventions that suit local needs and make households more resilient to natural hazards and the adverse impacts of climate change (DMB, 2010;MEF, 2009).However, adaptation and coping measures that households of the coastal areas of Bangladesh undertake to cope with natural hazards and the effectiveness of these measures have not been adequately investigated (Paul & Routray, 2011). To address this gap, this paper explores adaptation and coping measures that households of the coastal areas of Bangladesh undertake to minimise their vulnerability to two major natural hazards-cyclone and salinity intrusion.This paper is based on secondary sources.The first section of this paper describes the coastal area of Bangladesh; the second 1 Both NPDM and NDMP include natural, environment and human induced hazards. 2 Each of the three policy documents integrates disaster risk reduction measures and climate change adaptation measures as disaster risk reduction measures not only increase community and household level capacity to cope with natural hazards but also serve as adaptation measures to the adverse effects of climate change. presents the cyclone and salinity situation in coastal Bangladesh; and the third investigates adaptation and coping measures that households of the coastal areas of Bangladesh undertake in relation to these two hazards. Coastal Areas of Bangladesh The coastal areas of Bangladesh include 30 per cent of the total land area of the country and have a population of 39.41 million, which is 27 per cent of the country's total population (Paul & Rashid, 2017).Based on ecological and physiographic characteristics, the coastal area is divided into three zones: eastern, central, and western (Paul & Rashid, 2017).The landward boundary of the coastal area has been determined based on three indicators: influence of tidal waters, salinity intrusion, and cyclones/storm surges (Ministry of Water Resources [MWR], 2005).Of the country's 64 districts, 19 belong to the coastal area.These 19 districts have a total of 147 upazilas (subunit of a district), with 48 of these located on the exposed coast and 99 on the interior coast (see Figure 1).The exposed coast meets the sea directly and the interior coast is situated behind the exposed coast.The exposed coast is highly vulnerable to natural hazards and to the impacts of sea level rise.About 60 islands and 177 char lands (accreted land) are identified in the coastal area (MWR, 2005;Sarwar, 2005). The coastal areas of Bangladesh are prone to both natural and human-made hazards.These hazards have adversely affected lives and livelihoods and slowed down the pace of social and economic development in the coastal region (MWR, 2005).Furthermore, low income and poor housing and sanitation conditions have intensified the vulnerability of coastal communities (Parvin, Takahashi, & Shaw, 2008).The vulnerability of the coastal areas of Bangladesh is being aggravated further by climate change and its adverse impacts.Saline water intrusion, extreme events, drainage congestion, and changes in coastal morphology have been identified as key vulnerabilities in the coastal area due to climate change (MEF, 2005(MEF, , 2009)). Cyclones The entire coastal area of Bangladesh, especially the islands and exposed upazilas, is a geographical 'death trap' because of its exposure to cyclones and associated storm surges (Ahmed, 2008;Parvin, Takahashi, & Shaw, 2008;Saha & James, 2017) 2 outlines some of the major cyclones in Bangladesh. Salinity Intrusion Salinity is a major problem in some areas of coastal Bangladesh.About 6 million people are already exposed to high salinity (>5 parts per thousand) and the number is expected to rise to 13.6 million by 2050 and 14.8 million by 2080 due to climate change.The population most affected by this will be of that located in the Bagerhat, Khulna and Satkhira districts (Pender, 2010).The increased severity and frequency of cyclones and the associated storm surges have also exacerbated the gradual increase in salinity levels (Warner et al., 2012). Increased saline levels in the soil negatively affect rice production (Pender, 2010;Warner et al., 2012).It is argued that suitable areas for transplanted aman rice cultivation will decrease from 88 per cent to 60 per cent with a 32 cm rise in sea level in Bagerhat, Khulna and Satkhira districts (Pender, 2010).A study based in Satkhira shows that 81 per cent of the 360 surveyed households suffer from high salinity in their rice fields, compared to only two per cent a decade ago (Warner et al., 2012).Salinity also diminishes productions of vegetables, fruits, and other crops; it also negatively affects other means of livelihood, such as pottery, as saline clay is not suitable for making ceramics (Pender, 2010). Higher salinity levels in water sources also negatively affect people's health and wellbeing.Evidence suggests that high levels of salinity are associated with changes in menstruation of women and higher rates of miscarriage (Warner et al., 2012).Moreover, the consumption of salty water is partly responsible for an increased number of waterborne diseases, such as diarrhoea and dysentery, which primarily affect children (Warner et al., 2012;Paul & Rashid, 2017). 3 Increased salinity also causes stomach problems in cattle (Pender, 2010). Adaptation and Coping Measures Associated with Cyclones and Salinity Intrusion The terms 'adaptation' and 'coping' are sometimes used interchangeably, despite differences between them (van der Geest & Warner, 2015;Warner et al., 2012;Monnereau & Abraham, 2013;van der Geest & Dietz, 2004;CARE International [CI], 2009).Adaptation refers to 'longer-term adjustments to more permanent changes in the climate' while coping refers to 'short-term responses to the impacts of sudden or unusual events' (van der Geest & Warner, 2015, p. 126).Adaptation is associated with longer-term livelihoods security, and sustainable use of resources, while coping is associated with short-term and immediate survival measures, often motivated by a crisis and prompted by a lack of alternatives (CI, 2009).Hence, it can be argued that the measures of adaptation help people to moderate harm or exploit beneficial opportunities, whilst coping measures aid people to face and manage adverse conditions, emergencies, or disasters by using available skills and resources (United Nations Office for Disaster Risk Reduction [UNISDR], 2009). Coping measures can be categorised as either non-erosive or erosive coping measures (Warner et al., 2012;van der Geest & Warner, 2015).Non-erosive coping measures do not jeopardise long-term or future livelihood security of households while erosive coping measures, despite the possibility of having 3 Although the current recommended dietary intake of sodium is 2 g/day, the population of Dacope Upazila, Khulna District, consume 5 to 16 g/day sodium during the dry season in drinking water alone, depending on drinking water source (Khan et al., 2011). short-term benefits, jeopardise long-term or future livelihood security of households (Warner et al., 2012;van der Geest & Warner, 2015).Thus, coping measures are successful when households allocate resources to overcome a crisis without compromising the long-term livelihood security, though coping measures are unsuccessful when households employ measures to overcome a crisis that undermine their long-term livelihood security. Households in the coastal areas of Bangladesh undertake various adaptation and coping measures to manage the adverse impacts of cyclones and salinity intrusion.Despite this, access to and control over the resources (natural, human, social, physical, and financial resources) that are necessary for the adoption of these measures vary among communities (such as a village), and even among households within a community (CI, 2009;Blaikie, Cannon, Davis, & Wisner, 1994).Adaptation and coping measures adopted by households are dependent on many factors, such as the social, cultural, and economic background of the households; physical location; the nature of the hazards; and household's level of vulnerability and ability to absorb shock (Parvin, Takahashi, & Shaw, 2008;Paul & Routray, 2011;Saha, 2016).Although this paper does not deal with variations in adopting adaptation and coping measures based on the aforementioned factors, it is worth mentioning that-based on these factors-measures undertaken by households vary within a community, between communities within a coastal region, as well as among the three coastal regions of the coastal Bangladesh (Paul & Routray, 2011;van der Geest & Dietz, 2004;van der Geest & Warner, 2015). Adaptation and Coping Measures Associated with Cyclones Adaptation and coping measures associated with cyclone hazards can be categorised into three stages (although some measures may not be exclusive to a particular stage): pre-cyclone (long-term adaptation and when a cyclone is imminent), just prior to and during cyclone, and post-cyclone. Households undertake various long-term adaptation measures to reduce their vulnerability to cyclones.They build houses on a raised earth platform to protect family members and belongings during cyclones, avoid housing materials that are susceptible to surge water, and construct houses in a way so that houses face less wind forces (see Figure 2 and Figure 3) (Alam & Collins, 2010;Paul & Routray, 2011).Moreover, some people plant bigbranched trees around the homestead to protect life, houses and properties from wind and storm surges (Alam & Collins, 2010;Paul & Routray, 2011;Garai, 2017).Households also undertake some measures to reduce their vulnerability to cyclones when a cyclone is imminent.Such measures include: collecting crops from fields; hiding valuables and food in the earth; tying houses to adjacent big trees using strong ropes; inserting new poles around the houses if houses are not made of brick; sending important things to relatives living in safer areas; increasing religious activities; and sending domestic animals to highland areas or untying domestic animals so that they can survive surge water if the warning signal is at or close to the highest danger (Alam & Collins, 2010;Parvin, Takahashi, & Shaw, 2008;Paul & Routray, 2011;Garai, 2017). Just prior to and during the cyclone and induced storm surge, people try to save themselves by taking refuge in safe places, such as cyclone shelters, neighbouring houses, or in the ceilings of their houses (Alam & Collins, 2010;Paul & Routray, 2011;Garai, 2017). There are multiple tasks that people do during the post-cyclone period, such as: searching for friends and family; looking for materials needed to rebuild houses; taking dried food that had been stored as precaution, or whatever food is available to them, such as wetted rice, bread and sweet potato, before outside help arrives; arranging community cooking for several days; travelling a long distance to collect water; using tablets to purify water; searching for means of livelihoods; reducing the number of meals per day and relying on inexpensive foods, such as flattened rice and gur; seeking assistance from relatives, neighbours and unaffected local people; relying on relief; using savings; going to cities for work; taking out loans, including conditional loans, from various sources such as relatives, friends, money lenders and local NGOs; and selling assets, such as land, cattle, fishing and agricultural equipment (Alam & Collins, 2010;Parvin, Takahashi, & Shaw, 2008;Paul & Routray, 2011;Saha, 2016, Garai, 2017). It is worth noting that some of the measures that households undertake during the postcyclone period-such as reducing the number of meals per day, taking out loans (particularly conditional loans from money lenders), and selling assets-are erosive coping measures as these measures risk households' food and livelihood security and make them more vulnerable.These erosive coping measures are very common in the coastal areas of Bangladesh.For instance, a study conducted in the three villages of Barguna and Patuakhali districts of Bangladesh shows that more than 90 per cent of the surveyed households reduced the number of meals per day to cope with the crisis following a cyclone (Paul & Routray, 2011).These erosive coping measures are mainly linked to poverty and a lack of an alternative livelihood, or livelihood stress resulting from the failure to secure income after the cyclone, as the cyclone adversely affects or destroys livelihoods of households, particularly households that are dependent on agriculture or on selling agricultural labour (Saha, 2016, Rabbani, Rahman, & Mainuddin, 2013). Adaptation and Coping Measures Associated with Salinity Intrusion Households in the coastal areas of Bangladesh undertake several measures to adapt and cope with the changing conditions associated with increased salinity intrusion (see Figure 4).These measures can be categorised as field or agricultural-based measures and non-field or non-agricultural-based measures.Households employ agricultural-based measures to reduce the negative effect of salinity intrusion on agricultural production, while they employ nonagricultural measures to reduce livelihood dependency on agriculture (Rabbani, Rahman, & Mainuddin, 2013).The two main agriculturalbased measures that households undertake are using new saline-tolerant rice cultivars and reducing the salinity content of soil by washing rice fields with fresh water (see Table 3) (Mainuddin, Rahman, Islam, and Quasem, 2011;Warner et al., 2012;Rabbani, Rahman, & Mainuddin, 2013).To flush freshwater through agricultural fields to reduce salinity, farmers dig canals or purchase fresh water from neighbouring canals and ditches (Mainuddin et al., 2011;Warner et al., 2012).Other agricultural-based measures include raising seedbeds with fresh soil (Warner et al., 2012;Rabbani, Rahman, & Mainuddin, 2013), using fertilisers to prevent soil salinity (Khanom, 2016;Pouliotte, Islam, Smit, & Islam, 2006), and applying sugar to the rice fields to reduce salinity (Warner et al., 2012). Another measure that is related to agriculture is converting croplands into shrimp farms, which in turn contributes to the increased salinity intrusion (Pouliotte et al., 2006;Khanom, 2016;Swapan & Gavin, 2011).For instance, a study reveals that almost 90 per cent of the land previously used for rice and vegetable production has been converted into shrimp ponds in the study area located in Koyra Upazila of Khulna District of Bangladesh (Swapan & Gavin, 2011).However, in addition to having severe negative ecological impacts, shrimp farming as a response to saltwater intrusion does not equally benefit all households that convert agricultural lands into shrimp ponds.Although shrimp farming offers significant benefits for large landowning households, small landowners and other poor and landless households do not usually benefit from this new livelihood opportunity (Pouliotte et al., 2006;Swapan & Gavin, 2011). Non-agricultural measures that households employ include taking out loans from nongovernmental organisations, neighbours, and relatives; increasing the number of hours worked to earn more; increasing the number of household members (generally women or children) working for a wage income; reducing household expenditure; migrating elsewhere to find work; selling household assets, including land and livestock; leasing out the land to others; changing eating habits; raising goats as an alternative to cattle; and switching to nonfarm activities such as rickshaw/van pulling (Rabbani, Rahman, & Mainuddin, 2013;Pouliotte et al., 2006).A study conducted in four villages of Shyamnagar Upazila of the Satkhira District shows that more than 70 per cent households took out loans, reduced household expenditure and changed eating habits to cope with the impact of salinity intrusion on rice production.Additionally, 55 per cent of households tried to earn more by increasing working hours; about 30 per cent employed temporary migration of a household member/(s); 25 per cent switched to non-farm activities; and about 23 per cent sold household assets to cope with the impact of salinity intrusion on rice production (Rabbani, Rahman, & Mainuddin, 2013). Salinity also has a gendered dimension, as women are traditionally responsible for collecting water for drinking and cooking.Women are trying to adapt to the scarcity of fresh water, due to salinity, by collecting rainwater or developing a jar filter system and a pond sand filter system to get fresh water from saline water.Other than these three practices, women collect water from very deep tube wells by walking a long distance, which may take three to four hours a day.This naturally results in health hazards (Ahmed, 2008;Rahman, 2009). (2012) Conclusion Households in the coastal areas of Bangladesh are relentlessly trying to reduce their vulnerability to cyclone hazards and salinity intrusion by undertaking various adaptation and coping measures.However, while many of these adaptation and coping measures reduce their vulnerability, and enhance resilience, some of the undertaken coping measures, such as reducing the number of meals per day and selling households' assets-including land and livestock-are erosive coping measures as they jeopardise their food and livelihood security.Despite helping households to address immediate needs and overcome a crisis, these erosive coping measures make households more vulnerable instead of making them resilient.These erosive coping measures may, at worst, result in the complete destitution of households. Adaptation and coping measures that contribute to reduce households' vulnerability should be supported and guided by the government and NGOs to make them more effective.Erosive coping measures that make households further vulnerable also need to be addressed by the government and NGOs since most of these coping measures are related to poverty and households' limited-if any-access to economic opportunities.One of the most important actions that need to be undertaken by the government and NGOs is the creation of alternative livelihood opportunities for coastal communities, since livelihood stress is one of the major factors that increase their vulnerability to natural hazards (Saha, 2016;Pouliotte et al., 2006;MEF, 2009).Finally, supporting measures that make a positive contribution, and addressing those measures that are making households more vulnerable to natural hazards, is only possible when disaster risk reduction activities instigated by the government and NGOs can be integrated with the activities undertaken by households and communities to reduce vulnerability to natural hazards. Figure Figure 1: Coastal Area of Bangladesh Source: Sarwar (2005) Cyclones and Salinity Intrusion in the Coastal Areas of Bangladesh . Tropical cyclones with high winds and storm surges strike Bangladesh, on average, every three years (MEF, 2009; International Organisation for Migration [IOM], 2010), with the majority of cyclones striking Bangladesh during the April to early June and late September to November periods (United Nations Development Programme [UNDP], 2007).Cyclones have killed more than 450,000 people in Bangladesh since 1970 (United Nations [UN], 2010), with the majority of these deaths caused by two cyclones: the Bhola Cyclone (1970), which caused the deaths of 300,000 people, and Cyclone Gorky (1991), which killed 140,000 people (Department of Disaster Management [DDM], 2013; Saha & James, 2017).Table Figure 2 : Figure 2: A Temple-shaped House on a Raised Earth Platform Figure 4 : Figure 4: Measures Undertaken at the Household Level to Cope with Salinity Intrusion in Four Villages of Satkhira District, Source: Warner et al. (2012) Conclusion	2018-12-11T06:51:21.271Z	2017-06-30T00:00:00.000	{ "year": 2017, "sha1": "4716f59d93c20415a206de26cfeae81af23c52d2", "oa_license": "CCBY", "oa_url": "http://spaceandculture.in/index.php/spaceandculture/article/download/234/pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "4716f59d93c20415a206de26cfeae81af23c52d2", "s2fieldsofstudy": [ "Economics" ], "extfieldsofstudy": [ "Business" ] }
247315476	pes2o/s2orc	v3-fos-license	Comparison of CYGNSS and Jason-3 Wind Speed Measurements via Gaussian Processes Wind is a critical component of the Earth system and has unmistakable impacts on everyday life. The CYGNSS satellite mission improves observational coverage of ocean winds via a fleet of eight micro-satellites that use reflected GNSS signals to infer surface wind speed. We present analyses characterizing variability in wind speed measurements among the eight CYGNSS satellites and between antennas. In particular, we use a carefully constructed Gaussian process model that leverages comparisons between CYGNSS and Jason-3 during a one-year period from September 2019 to September 2020. The CYGNSS sensors exhibit a range of biases, most of them between -1.0 m/s and +0.2 m/s with respect to Jason-3, indicating that some CYGNSS sensors are biased with respect to one another and with respect to Jason-3. The biases between the starboard and port antennas within a CYGNSS satellite are smaller. Our results are consistent with, yet sharper than, a more traditional paired comparison analysis. We also explore the possibility that the bias depends on wind speed, finding some evidence that CYGNSS satellites have positive biases with respect to Jason-3 at low wind speeds. However, we argue that there are subtle issues associated with estimating wind speed-dependent biases, so additional careful statistical modeling and analysis is warranted. I. INTRODUCTION W IND is a crucial component of the atmosphere and climate, having significant implications in numerous areas of daily life, from safety and transportation to industry and science. Recording accurate wind measurements provides critical information for precisely defining weather hazards [1], building skyscrapers, and landing aircraft [2]. Reliable wind speed observations are also consequential in allowing us to efficiently conduct crop spraying [3], monitor the global climate [4], and avoid the obstruction of essential global shipping routes [5]. Scientists and engineers have developed a diverse suite of tools for measuring wind speeds. Weather stations and buoys are often equipped with anemometers to measure wind speed directly. Many earth-observing satellites carry sensors capable of inferring wind speeds. A thorough review of satellite-based methods for measuring wind speeds is provided in [6], which includes radiometers, scatterometers, and altimeters, which are usually attached to low-earth-orbiting satellites. In addition, geostationary satellites are capable of inferring upper-air wind speeds by detecting movements in clouds via derived motion winds algorithms [7]. The Cyclone Global Navigation Satellite System (CYGNSS) is a fleet of eight micro-satellites that use the scattered signals from existing GNSS satellites to infer wind speeds at the ocean surface [8], [9]. CYGNSS is a relatively new and low-cost system that, due to its ability to distribute its sensing effort over eight satellites, has the advantage of greater spatial-temporal coverage of the oceans relative to a single-satellite system, an important feature for its mission of monitoring tropical cyclones. Our primary goal is to study the internal variability in wind speed measurements among the eight CYGNSS satellites and across each satellite's starboard and port antennas. As noted in [10], this variability is still under study, and further calibrations are a possibility: "it is more likely the differences in bias, both between antennas and between spacecraft, are caused by residual errors in the engineering calibration, which is performed individually for each spacecraft and antenna. This is an ongoing area of investigation by the CYGNSS project team . . ." Our secondary goal is to study differences between CYGNSS and Jason-3 wind speed measurements. Several recent articles study statistical properties of CYGNSS wind speed measurements. CYGNSS was compared with weather model forecast winds and found to be positively biased at low wind speeds and negatively biased at high wind speeds, but no comparisons were made among individual CYGNSS satellites [11]. In the tropics, a similar pattern of positive bias for low wind speeds and negative bias for high wind speeds, relative to hourly-averaged buoy data, was detected [10]. CYGNSS biases with respect to modeled and sensed wind speeds were assessed in the context of building a complex bias correction algorithm [12]. The spatial-temporal patterns of low-earth-orbiting satellite observations, coupled with the inherent spatial-temporal variability in wind speeds, present a data-analytic challenge for conducting the desired comparisons in our study. By design, the eight CYGNSS satellites do not measure winds concurrently at the same locations, so it is difficult to draw direct comparisons between observations from most pairs of these satellites. Thus, we rely on repeated approximate crossings between CYGNSS and Jason-3 for indirect comparisons. Jason-3 uses reflected signals from its radar altimeter to infer wind speed and other ocean surface parameters. We believe that Jason-3 is a suitable comparison because, like CYGNSS, it retrieves a snapshot of surface wind speed, as opposed to an hourly average or a model forecast, both of which can be overly smooth. To maximize statistical power, it is important to make judicious use of the observations arising from the limited number of nearby CYGNSS-Jason-3 passes, while avoiding a subjective determination of which passes constitute a close-enough match. To address these issues, we analyze the data using carefully constructed Gaussian process models. Gaussian process models have become an indispensable tool for analyzing and interpolating scattered remote sensing data. Recent examples include the analysis of Argo float data [13], Orbiting Carbon Observatory-2 data [14], [15], surface temperatures [16], Microwave Atmospheric Satellite data [17], and Jason-3 wind speeds [18]. Our models for CYGNSS and Jason-3 wind speeds contain bias and variance parameters that are directly related to our study goals. In particular, each model has parameters that are interpreted as the expected difference between CYGNSS starboard-and port-measurements and Jason-3 measurements if they had measured wind speed at the exact same location and time. These parameters are estimated via maximum likelihood and generalized least squares, which uses a variance-minimizing linear combination of observations to make efficient use of the available data. Computational challenges often associated with Gaussian process models are overcome by downsampling across time and using a state-of-the-art Gaussian process approximation implemented in the publicly available GpGp R package [19]. The Gaussian process model and associated computational techniques are the main methodological novelties of this work. We find that there are significant and persistent differences among some pairs of the CYGNSS sensors of a magnitude up to 1.11 m/s. There are smaller differences between the starboard and port sensors from the same satellite. Five of the eight CYGNSS satellites have a negative bias with respect to Jason-3 measurements. No substantial differences in variances among the eight satellites were detected. These results are successfully validated against a traditional empirical analysis, which shows similar trends but higher uncertainty. In addition, we investigate the possibility that bias depends on wind speed, finding some evidence that CYGNSS measurements are larger than Jason-3 at low wind speeds, though we argue that more careful analysis is needed. We conclude the paper with a discussion of the results and suggestions for how to modify the models to study variation in the biases. All of the code necessary for reproducing our results is available in a GitHub repository at https://github.com/WillBekerman/satellite-wind-speeds. II. DATASETS AND DATA PROCESSING We compile one year of measurements recorded by CYGNSS and Jason-3 between September 28, 2019 and September 25, 2020, specifically, CYGNSS Level 2 Science Data Record Version 3.0 and Jason-3 Level-2 X-GDR Data. Due to missing records in the Jason-3 data during the weeks of February 1, 2020 to February 14, 2020 and June 13, 2020 to June 19, 2020, we omit these periods from our data collection, yielding 49 total weeks of satellite measurements for our analysis. After acquiring the data in NetCDF format, we process the data in R, retaining information about spatial location, time of measurement, wind speed, CYGNSS satellite number, and CYGNSS sensor (port vs. starboard). We omit any observations with missing data and standardize the times to seconds since In Figure 1, we compare the wind speed measurements taken over the same latitudes by CYGNSS 1, CYGNSS 4, and Jason-3 during the week of November 30 to December 6, 2019. The value in each pixel is the average of all measurements taken within the pixel over the week. While the spatial patterns of wind speeds are similar among the three satellites, there are subtle differences. CYGNSS 4 appears to record larger wind speeds than CYGNSS 1, and Jason-3 tends to have less-smooth wind fields with more of the largest values. The pixel-wise comparisons in Figure 1 can be misleading because even though the satellites have complete coverage in a one-week period, the timing of the measurements will differ among satellites, so differences could be attributable to the interaction between locally changing wind conditions and the observational times, rather than bias. In seeking to quantify these differences between satellite measurements, the simplest approach is to directly compare measurements recorded by CYGNSS and Jason-3 taken within small space-time windows. To explore the feasibility of the space-time matching analysis, we plot the time-varying distances between the eight CYGNSS satellites and Jason-3 over the course of one full day in Figure 2. All of the CYGNSS satellites come reasonably close to Jason-3 at one or more points during the day, with some variability in the number and proximity of such occurrences. By contrast, while some pairs of CYGNSS satellites nearly always measure winds at nearby locations, some pairs never do, like CYGNSS 1 and CYGNSS 4. In the next section, we propose a model designed to address the challenge of making efficient use of the CYGNSS and Jason-3 comparisons. III. ANALYSIS A. Model Description We first describe the statistical model used in our analysis in mathematical notation, and then provide interpretations for the model and its statistical parameters. Our analysis strategy is to fit separate models to datasets consisting of Jason-3 data and one CYGNSS satellite, and repeat the analysis for each week and each CYGNSS satellite, which means we will have many separate fits of the same general model formulation. This allows us to study whether biases are consistent over time. In the interest of keeping the number of symbols manageable, we do not provide notation for each individual model fit; the notation below is a model for an arbitrary CYGNSS satellite in an arbitrary week. Our results will contain a re-estimation of the parameters for each dataset. We label the n observations from both CYGNSS and Jason-3 with i = 1, . . . , n. We use Y i for the wind speed associated with observation i and model it as follows: The term µ i is intended to capture broad-scale variation in wind speed over time t i and latitude, independent of satellite. The mapping k(i) indicates which sensor produced observation i, with k = 1 indicating Jason-3, k = 2 indicating CYGNSS starboard, and k = 3 indicating CYGNSS port. We model space-time variation in wind speeds with the Gaussian process (GP) Z, with inputs spatial location x i and time t i , assumed to have mean zero and space-time Matérn covariance function where θ 1 controls the variance of Z, θ 2 is the Matérn smoothness parameter, θ 3 controls the spatial-decay of the covariances, and θ 4 the temporal decay. Since Z is a mean-zero process that depends only on spacetime location, and not the satellite or random independent error, we interpret it as a wind speed anomaly, with the caveat that the anomaly is relative to a specific linear-in-time, cubic-in-latitude mean field. The mean field contains an intercept b 0 , which means that a 1 , a 2 , and a 3 are not separately identifiable, but differences such as a 2 − a 1 and a 3 -a 1 are. As a consequence, without additional outside information, our analysis is not able to determine whether CYGNSS or Jason-3 is biased with respect to the true wind field, but it is capable of assessing whether CYGNSS and Jason-3 are biased with respect to one another via estimation of differences such as a 2 − a 1 . In terms of the model, we are principally interested in how CYGNSS starboard, CYGNSS port, and Jason-3 measurements would differ if they had measured wind speed at the same location at the same time. To see how this quantity relates to our model parameters, suppose that measurement i was taken by CYGNSS starboard and measurement j was from Jason-3, and those two measurements were recorded at the same time and location. Then meaning that a 2 − a 1 is the bias and 2σ 2 is the variance of the difference. These are the parameters of interest in our study. By comparing the estimates of a 2 − a 1 and a 3 − a 1 across CYGNSS satellites, we can achieve our primary goal of understanding variability among the CYGNSS measurements. B. Model Estimation We fit our model separately to each of the 49 weeks and each of the eight CYGNSS satellites, producing a total of 392 model fits. To reduce computational burden, we fit each model using 20,000 randomly selected observations from CYGNSS and 20,000 from Jason-3, and we employ a popular computationally efficient Gaussian process approximation proposed by [20], implemented in the R package GpGp [19]. Each model fit delivers estimates of the model parameters via maximization of the approximate likelihood function, as well as standard errors for the mean parameters. C. Model-Based Results In Figure 3, for each week and each CYGNSS satellite, we plot the CYGNSS vs. Jason-3 bias estimates a 2 −a 1 (starboard) and a 3 − a 1 (port). Estimates for starboard and port from the same week and CYGNSS satellite are connected by a black line. The bias estimates vary by week, satellite, and antenna. Five of the eight CYGNSS satellites (1, 2, 3, 5, and 6) produce negative biases with respect to Jason-3 for every week and for both antennas. The three other satellites (4, 7, and 8) have a mix of negative and positive biases across weeks. CYGNSS 1 has the largest negative biases, on average approximately -0.83 m/s for starboard and -0.94 m/s for the port antenna. Nearly all of the CYGNSS 1 biases are more negative than the most negative CYGNSS 4 bias, suggesting that CYGNSS 1 and CYGNSS 4 were persistently biased with respect to one another during our study period. Within each satellite and antenna, the bias estimates vary by roughly 0.5 to 1.0 m/s from week to week. The differences across weeks in the biases could be due to uncertainty in the parameter estimates, rather than a bias that truly varies over time. By inspecting the black lines in Figure 3, we observe differences between the estimates of the starboard and port biases from the same satellite within the same week. Figure 4 explores these differences in more detail by directly plotting estimates of a 3 − a 2 , which measure the bias between the starboard and port antennas. Most of the starboard vs. port bias estimates are smaller than the CYGNSS vs. Jason-3 biases, with most magnitudes less than 0.25 m/s, and show a mix of both negative and positive biases, though there are notably more negative than positive biases. CYGNSS 5 is the most lopsided with 46 of the 49 biases being negative. Figure 5 displays estimates of √ 2σ, which we recall from Equation (7) is the model's standard deviation of the difference between two observations taken by different sensors at the same location and time. The estimates generally fall between 0.6 and 0.9 m/s, meaning that the size of the noise is roughly equal to the largest CYGNSS vs. Jason-3 biases. There is some variation of the estimates of √ 2σ across weeks but no substantial differences among the eight CYGNSS satellites. D. Empirical Explorations To validate our model-based results, we conduct additional analyses based on simple averages of differences between CYGNSS and Jason-3 wind speeds that fall within small spacetime windows. For each of the eight CYGNSS satellites, each antenna, and each week between September 28, 2019 and September 25, 2020, we divide the week into two-hour windows and find the pair of CYGNSS and Jason-3 observations that are closest in distance within the two-hour window, ignoring any windows that do not have a pair that fall within 25km. We then take the average of the differences between the selected pairs of CYGNSS and Jason-3 wind speeds. We refer to the averages of these differences as our empirical bias estimates. Analogously to Figure 3, we plot in Figure 6 the empirical bias estimates over all CYGNSS satellites, antennas, and weeks. The general patterns of the empirical and model-based bias estimates are quite similar. The same five CYGNSS satellites have largely negative biases with respect to Jason-3, while the other three have a mix of negative and positive biases. The average size of the biases are similar as well, ranging roughly from -1.0 to 0.05 m/s. As in the model-based analysis, the port biases in CYGNSS 5 are, on average, more negative than the starboard biases. The empirical biases differ in that the variation across weeks is larger than in the model-based biases, which produces more overlap among the eight CYGNSS satellites and two antennas. For instance, whereas there was essentially no overlap between the CYGNSS 1 and CYGNSS 4 model-based biases, the empirical biases show more substantial overlap. In addition, the difference between the CYGNSS 5 starboard and port model-based biases is more clear than the difference between the empirical biases. To further compare the model-based and empirical bias estimates, we average the estimates over the 49 weeks and plot them in Figure 7. The estimates generally follow the 45 degree line, with the model-based estimates being slightly more positive than the empirical estimates. The points tend to cluster by satellite, suggesting that the difference between starboard and port within a CYGNSS satellite is generally smaller than the differences among the eight CYGNSS satellites. Interestingly, every starboard point is northeast of its corresponding port point, indicating that the average empirical and model-based starboard biases are more positive than the corresponding port biases for every CYGNSS satellite. Previous studies have explored whether bias depends on the magnitude of the wind speed. These analyses are complicated by the fact that we never have access to the "true" wind speed. One could take the more accurate measurement as the "true" wind speed and estimate bias as a function of the more accurate measurement. This approach is not without its drawbacks; when one measurement is high, the other is likely to be lower, due to standard regression-to-the-mean. To partially circumvent this issue, we plot in Figure 8 the difference between CYGNSS and Jason-3 (CYGNSS minus Jason-3) against their average. As before, we break the year into two-hour intervals and within each interval, we extract the closest pair of observations, provided that the closest distance is less than 25km. We see that among the port antennas, CYGNSS measurements are usually larger than Jason-3 for small average wind speed, but for larger average wind speeds, Jason-3 records tend to be larger. The pattern is similar for all satellites. The overall negative bias for satellites 1, 2, 3, 5, and 6 is also evident from the plots. The patterns for the starboard antennas are similar (not shown). IV. CONCLUSIONS Our main finding is that during our study period of September 2019 to September 2020, persistent biases existed among the wind speed measurements recorded by the eight CYGNSS satellites and between some CYGNSS satellites and Jason-3. Considering the averages of the model-based parameter estimates over the study period, the largest bias between pairs of CYGNSS sensors was 1.11 m/s (CYGNSS 4 starboard minus CYGNSS 1 port), and the largest CYGNSS vs. Jason-3 bias was -0.94 m/s (CYGNSS 1 port minus Jason-3). We discovered smaller biases between the starboard and port antennas within a satellite, with the largest average bias being 0.25 m/s (CYGNSS 5 starboard -CYGNSS 5 port). It is not surprising to us that the two sensors on the same CYGNSS satellite would be reasonably well-calibrated with respect to one another. Since they tend to measure wind speeds at fairly close locations, direct comparison across antennas is easier. However, direct comparisons between CYGNSS satellites are more difficult due to the fact that some pairs nearly always measure wind speeds at disparate locations. Similar to other studies that make indirect comparisons with forecast winds or buoy data, we use Jason-3 as an intermediary to achieve indirect comparisons among every pair of CYGNSS satellites. We believe that Jason-3 data is appropriate because both CYGNSS and Jason-3 attempt to measure snapshots of wind speed within small space-time windows and they pass each other somewhat regularly. These findings were facilitated by the use of a Gaussian process model that contained parameters directly related to the expected difference between measurements from different instruments taken at the same time and location. In addition to the bias parameters, the models contained a parameter related to the variance of the difference between two observations taken by different sensors at the same time and location. We did not find substantial differences in the estimates of noise parameters among the models for the eight CYGNSS satellites. The size of the noise averaged about 0.75 m/s, meaning that the noise was of roughly equal magnitude to the largest biases, implying that about half of the mean square errors between measurements from different sensors was due to bias, half due to noise. In other words, eliminating the bias could potentially reduce the mean squared errors by a factor of two. We validated the model-based findings with a more traditional empirical analysis that searched for matching pairs of observations from different sensors within small spacetime windows. The general pattern of the estimates in the empirical analysis was similar to that in the model-based analysis. The analyses differed in that the week-to-week variation in the empirical estimates of the bias was larger. This is expected because the model-based estimates use generalized least squares, which provides variance-minimizing parameter estimates under the assumed model. Some studies have suggested that the size and direction of the bias may depend on the magnitude of the true wind speed. We caution against over-interpreting these results because we do not have access to the true wind speed. If a particular sensor is chosen as the reference, we expect to see positive bias at low wind speeds and negative bias at high wind speeds due to regression-to-the-mean effects, even if the bias does not vary with wind speed. We attempted to mitigate this effect by comparing paired differences against paired averages, finding that generally the Jason-3 wind speeds increase relative to Pair Average (m/s) Fig. 8. CYGNSS minus Jason-3 vs. the average of CYGNSS and Jason-3 for pairs of observations. Each pair represents the two closest observations from each two hour window over the entire year, provided that the distance is less than 25 km. CYGNSS as the average of their two measurements increases. This aspect is certainly worthy of more exploration, with consideration of the aforementioned statistical issues. Due to the differing accuracies of CYGNSS and Jason-3, the average may not be the best estimate of the wind speed. We could also seek out a third wind speed measurement to serve as the baseline. One could also pursue model-based estimates of biases that depend on true wind speed. To this end, consider the following extension of our model: which contains a sensor-dependent slope multiplying the wind speed anomaly Z(x i , t i ). Inferences about biases that depend on wind speed could be obtained via estimation of a k(i) and b k(i) . This is still a Gaussian process model, so we could use the same methodology to fit the model, though one would have to be careful about non-identifiability of parameters; for example, the variance of Z(x i , t i ) is not identifiable separately from the b k(i) parameters. One could imagine that the bias depends on various other factors, such as latitude, time, or GNSS satellite. This sort of variation could be handled within our model framework by adding interactions between the bias and the desired factor. To capture biases that vary in space, we could extend our model as where W 1 , W 2 , and W 3 are independent spatial Gaussian processes. Then we can interpret a 2 −a 1 +W 2 (x i )−W 1 (x i ) to be the spatially-varying starboard bias, and a 3 − a 1 + W 3 (x i ) − W 1 (x i ) as the spatially-varying port bias. We suspect that we would need to use more than one week of data at a time to accurately estimate a spatially-varying bias. We maintain a Github repository at https://github.com/ WillBekerman/satellite-wind-speeds which contains all data, as well as R scripts to replicate our analysis.	2022-03-09T04:43:13.368Z	2022-03-07T00:00:00.000	{ "year": 2022, "sha1": "140aa2552257699480aa0d130333665cd1e195c9", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "140aa2552257699480aa0d130333665cd1e195c9", "s2fieldsofstudy": [ "Environmental Science" ], "extfieldsofstudy": [ "Mathematics" ] }
155567035	pes2o/s2orc	v3-fos-license	Emergence of Corporate Philanthropy: Chapter Bangladesh The study objective is to focus on emergence of corporate philanthropy on Bangladesh. Corporate philanthropy is a phenomenon which associates the business sector with the social sector. Social historians and researchers alike as a subset of a larger corporate social responsibility (CSR) subject, philanthropy provides an opportunity for corporations to establish an ethical and moral mantra within the organization. The study also aims to find out, understand the perspective of attributes, legal context, importance, advantages and disadvantages, competitive advantage, Sectors contribution analysis of emergence of corporate philanthropy on Bangladesh. The study used published secondary source of data from various relevant sources reports, websites, journals, publications and the researcher have used own personal experience regarding the country’s corporate behavior and following background and their impact on this research paper. After conceptualizing the various situations and literatures review the study find out that corporate philanthropy is a new concept in the corporate world and increasing clients/ customers demand on the products and services towards the corporate in the meantime cooperate responsibilities towards their clients/ customers also increasing day by day, so emergence of the corporate philanthropy in Bangladesh is a vital part of the corporate sector and Bangladeshi business culture for centuries and it seems that an emphasis on charitable contributions from philanthropic programs has enhanced the visibility of this practice in Bangladesh. INTRODUCTION According to Philip L. Fioravante (2008) research paper, corporate philanthropy is a phenomenon which associates the business sector with the social sector. Social historians and researchers alike as a subset of a larger corporate social responsibility (CSR) subject, philanthropy provides an opportunity for corporations to establish an ethical and moral mantra within the organization (Gan, 2006;Madr igal & Boush, 2008). An organization is comprised of people who assume the responsibility of cultivating and maintaining a culture supportive of philanthropy and its range of objectives. Successful philanthropyachieving the goal is as vita l to an organization as the "core business" (Bruch & Walter, 2005). Philanthropic initiatives are complex and thus need to be developed, communicated, implemented, monitored, and lastly sustained, in order to guarantee its viability as a strategic tool. Corporate philanthropy, where a corporation donates a portion of its resources to a societal cause, has been an important part modern business corporation. Today it is examined that most of the famous and non famous corporation of the world that some amount of funds donated underscores the importance today's corporations place on philanthropic activity. Philanthropy is defined as a means by which public organizations externally exhibit corporate social responsibility -widely defined by a myriad of scholarly authors (Carroll, 1979;Gan, 2006;Halme & Laurila, 2009). Corporate philanthropy, where a corporation donates a portion of its resources to a societal cause, has been an important part modern business corporation. Today it is examined that most of the famous and non famous corporation of the world that some amount of funds donated underscores the importance today's corporations place on philanthropic activity. Corporate Philanthropy mirrors individual philanthropy except for the fact that a corporation, not an individual, is donating funds, time, or talent. Although done on a larger scale, corporate philanthropy is still done without any expectation of direct corporate gain such as increases in revenue, but usually involves indirect gains such as enhancing a company's brand, engaging employees, recognition, etc. Corporate philanthropy is a phenomenon which associates the business sector with the social sector. Social historians and researchers alike as a subset of a larger corporate social responsibility (CSR) subject, philanthropy provides an opportunity for corporations to establish an ethical and moral mantra within the organization. An organization is comprised of people who assume the responsibility of cultivating and maintaining a culture supportive of philanthropy and its range of objectives. Successful philanthropyachieving the goal is as vital to an organization as the core business. Philanthropic initiatives are complex and thus need to be developed, communicated, implemented, monitored, and lastly sustained, in order to guarantee its viability as a strategic tool. There can be internal institutional benefits as well. Increased interaction between staff of corporate giving units and business divisions can help build understanding within the company of how it can address the often complex social issues that exist in various places within their global market. The emergence of a cadre of personnel with experience in both fields is likely to further encourage cross-fertilization of ideas and skills. While many companies have long-standing philanthropy programs, many have begun to apply business thinking and models to their philanthropic objectives. For a growing number of firms, philanthropy is no longer developed separately from corporate strategy. The issues that companies target for financial support are increasingly aligned with corporate risk management and bottom-line objectives. Purpose Corporate philanthropy, where a corporation donates a portion of its resources to a societal cause, has been an important part modern business corporation. Today it is examined that most of the famous and non famous corporation of the world that some amount of funds donated underscores the importance today's corporations place on philanthropic activity. The study focuses on emergence of corporate philanthropy on Bangladesh. Corporate philanthropy is a phenomenon which associates the business sector with the social sector. Social historians and researchers alike as a subset of a larger corporate social responsibility (CSR) subject, philanthropy provides an opportunity for corporations to establish an ethical and moral mantra within the organization. This paper also aims at understanding the perspective of attributes, legal context, importance, advantages and disadvantages, competitive advantage, Sectors contribution analysis of emergence of corporate philanthropy on Bangladesh. Methodology The study uses secondary data collected mainly from the various published documents, office circular, official web folders other related websites, journals, publications etc. all information and reference is provided in the reference section of this research paper. In addition to this the researcher has used personal observation and experience regarding the country's socio economic and corporate sectors and their impact and standpoint of emergence corporate philanthropy on Bangladesh. Literature Review Many types of research articles, based on analytical, empirical, qualitative and mixed methods approaches inquisitive about CSR (corporate social responsibility) now authors innovate the new phenomenon of "Philanthropy" as a subset of CSR (corporate social responsibility). According to Friedman (1970) showed his self-professed communalist propensity and his theories concerning market mechanisms, capita l structure, and the conception of social responsibility. His hypothetical spot centered on the word "social" and concluded, "There are no 'social' values, no 'social' responsibilities in any sense other than the public values and responsibilities of individuals" (p.126). This perspective may initially appear to have a positivist paradigm. However, Friedman (1970) later asserted there is a association between the company and the consumer in an economic sense that drives the rules the rendezvous in an open, free of fraud, and responsible manner. Corporate social responsibility as evidenced continues to play a role in the strategic direction and financial performance of a company. The economic aspects have reasonable correlation to positive market presence and therefore consumer perception. In addition, these perceptions and the extent of CSR exhibited by a company affect buying behaviors (Bird, Hall, Momentè & Reggiani, 2007;Lockett, Moon & Visser, 2006). Continuing, Lockett et al. (2006) stated, "A though CSR [philanthropy] is addressed by many disciplines [it] has become an increasingly salient feature of business and the environment, to which managers are expected to respond" (p. 115). Agreeing with this postulation creates the further opportunity to study cause and effect variables such as brand strategy and how it is affected by philanthropic initiatives employing a qualitative (phenomenological) design study. According to Philip L. Fioravante (2008) social sciences such as sociology and psychology contribute to ethical decision-making, consumer perceptions and brand loyalty. Data analysis within case studies and interviews have provided clear correlations between CSR [philanthropy] and "marketing" objectives such as revenue generation, market share, brand reputation, and market differentiation. Understanding the potential impact of philanthropy in all of its forms enables a corporation to alter its value proposition and ultimately shape the manner in which it employs this phenomenon in the business strategy. Strategic marketing has a myriad of meanings and applications across industries. Philanthropy can add altruistic and capitalistic contribution to an organization. By analyzing how corporations use philanthropy for strategic marketing purposes, conclusions are possible that are drawn on the deep value beyond the "feel good" and towards a business growth driver. Attributes of Corporate philanthropy  Like standard philanthropy, corporate philanthropy focuses on the treating the cause of a problem or issue instead of the symptom.  Unlike standard philanthropy, corporate philanthropy must be done through a corporation directly or a corporation's own non-profit entity.  Funding for corporate philanthropy mainly comes from the company's contributions and is usually treated as a business expense. o Funding can also consist of individual donations if, for example, someone wanted to donate to a corporation's non-profit. o Companies are allowed to deduct up to ten percent of pre-tax income for direct charitable contributions -this includes giving to the company's foundation. Most companies deduct closer to one percent.  Some of the common forms of corporate philanthropy are: o Cash donations: including grants, donations, sponsorships -whenever money exchanges hands. o In-kind donations: such as donating products, access to employee volunteer groups, the use of a company's facilities, property, or services as exampleswhenever non-monetary support is given. These are some basic attributes discussed in above. It is not framed within these small characteristics. The days are passing and new corporation build up. So, new concept of philanthropy is introduced in corporate sector. Because in the era of modern evolution of Corporate Social Responsibility (CSR). The legal context of corporate philanthropy and law in Bangladesh The concept of philanthropy in Bangladesh is rooted in custom, tradition and religion. Philanthropy in Bangladesh has transcended generations and spanned communities. Influenced by an amalgam of cultures derived from Hinduism, Buddhism, Christianity and Islam, Bangladesh has a rich and traditional heritage manifest in its diverse customs, art, literature, music and people's way of life. Throughout Bangladesh's history, the practice of giving to others without expecting any return and helping the distressed often began at home and formed a crucial part of the socialization process in which family values and traditions were instilled in young people. These activities had both voluntary and religious dimensions. Over time, individuals and groups in Bangladesh undertaking philanthropic initiatives have worked to incorporate professionalism and expertise through organizational and management structures to make their work more sustainable. Therefore, what started out as a purely humanitarian effort has evolved into concrete mechanisms for providing services to the public. To these end societies, trusts, clubs, associations, foundations and other entities were established with the primary objective of rendering social services. More recently, the Bangladeshi business community and corporations has also been engaged in social welfare activities. Well-established corporate bodies have engaged in nonprofit welfare activities ranging from job creation to providing education and medical care. The Ispahani Group for example, a prominent business house, is well known for its charitable activities and specifically an eye hospital. Corporate donors and employers associations have also established schools to train child laborers following U.S. pressure on the child labor industries. Yet while there is a great deal of potential for corporate philanthropy activities, contributions from business enterprises to charitable activities remain negligible. Why corporate philanthropy in Bangladesh? The corporate philanthropy has a long history in Bangladesh. These philanthropic activities included donations to different charitable organizations, poor people and religious institutions. Till now, most of the businesses in Bangladesh are family owned and first generation businesses. They are involved in community development work in the form of charity without having any definite policy regarding the expenses or any concrete motive regarding financial gains in many instances. The discussions on corporate philanthropy practices in Bangladesh in its modern global terms, are relatively new, but not so for the concept itself. Because, being a part of the global market, it is difficult to ignore philanthropy standard specifically in the export sector. In general, it is true that in Bangladesh, the status of labor rights practices, environmental management and transparency in corporate governance are not satisfactory, largely due to poor enforcement of existing laws and inadequate pressure from civil society and interest groups like Consumer Forums. Globally, as philanthropy practices are gradually being integrated into international business practices and hence is becoming one of the determining factors for market accesses, it is becoming equally instrumental for local acceptability. A focus on CSR in Bangladesh would be useful, not only for improving corporate governance, labor rights, work place safety, fair treatment of workers, community development and environment management, but also for industrialization and ensuring global market access. The Importance of corporate philanthropy in Bangladesh Corporate philanthropy in Bangladesh ranges from financial contributions to employees volunteering time on the clock. While philanthropy costs the company, it also provides benefits for the company, community and employees. Understanding the significance of corporate philanthropy helps each company justify the expense to upper management and investors. Community Benefits: No matter what the motivation for corporate philanthropy, the community and specific organizations helped through the program benefit. When a company offers financial support, local organizations are able to afford supplies and programs that might otherwise go unfunded. These benefits for the recipients of the philanthropic efforts strengthen the community as a whole. Depending on the specific help offered by a corporation, the efforts may result in a cleaner community, more opportunities for residents and a boost to the local economy. Morale: Companies who emphasize public service and volunteerism may notice a boost in morale, particularly if the employees value the idea of giving back to the community. Employees who work together on a charity project gets to know one another beyond the typical scope of work. The work may even improve teamwork on work projects. When companies of Bangladesh allow employees to volunteer during the work day, you show them that the company cares about the community and emphasizes a sense of giving. Recruitment: In Bangladesh most employees want to do philanthropic work. Establishing each company in Bangladesh as a philanthropic company gives the company a potential edge when recruiting new employees. Job seekers who see that financial giving is part of the company culture are likely to see that as a positive for the company. Individuals who already volunteer, work with charities and do other philanthropic work on their own are particularly likely to be drawn to a company with the same values. The company's philanthropic efforts give you one more bullet point to add to recruitment information. Company Image: As news of the company's philanthropy spreads, the community develops an impression of the company. People tend to view philanthropic businesses favorably because they are supporting the community that provides them with business. In some cases, this positive image encourages community members to utilize company's services over a competitor who fails to become involved with the community. To maintain a positive company image, the philanthropic efforts need to be helpful and done primarily for the benefit of the recipients. By arranging some philanthropic works companies of Bangladesh can build their company's image throughout the world. Advantages and Disadvantages of Corporate philanthropy in Bangladesh Corporate philanthropy is a general term for the actions that businesses take to improve their communities and society in general. Corporate philanthropy can include donations of money or of time and labor at community centers or for improvement projects, or for fundraising for a cause. There are both clear advantages to corporate philanthropy, as well as a disadvantage. Community Support and Market Creation: A major advantage that companies gain from their philanthropic practices is the support of communities and the surrounding markets. Essentially, by using profits derived from the community to benefit that same community businesses can greatly increase their prospects of future revenue flows. Market Development through Reputation: Market development can also occur through the improved reputation of the business. The goodwill that a company can generate through corporate philanthropy can increase customer's interest and favorable opinions of the company. This may lead to increased sales, especially when the philanthropy is combined with effective advertising and co-branding. Direct Giving can hamper Goals: On the other side direct philanthropy created through pure donations of money can make it difficult for a business to actually change what it wants to change. A donation to a nonprofit agency may put control of the funds beyond the reach of the business. There is no guarantee that the agency will improve the community or offer any of the benefits that the business can gain from philanthropy. If we think this in the perspective of Bangladesh than the situation is very dangerous. There are many community agencies are formed in Bangladesh whose main purpose is to undertake some community wealth and uprooted the business in secret. So, this makes a bad impression about philanthropic work by the business community. Time, Cost, and Decisions: If a corporation decides and invest money in the community itself, then there are other disadvantages. The business must form a team and decide how much money to give and where it should be given to produce the most impact. Decisions affect not only the length of the project, but also any marketing that goes along with it. In the end, the company must be prepared to spend time, money and decision-making power on a project that has no direct benefits. The competitive advantage of Corporate philanthropy When it comes to philanthropy, executives increasingly see themselves as caught between critics demanding ever higher levels of corporate social responsibility and investors applying pressure to maximize short-term profits. Increasingly, philanthropy is used as a form of public relations or advertising, promoting a company's image through high-profile sponsorships. But there is a more truly strategic way to think about philanthropy. Corporations can use their charitable efforts to improve their competitive context--the quality of the business environment in the locations where they operate. Using philanthropy to enhance competitive context aligns social and economic goals and improves a company's long-term business prospects. Addressing context enables a company not only to give money but also leverage its capabilities and relationships in support of charitable causes. Taking this new direction requires fundamental changes in the way companies approach their contribution programs. Adopting a context-focused approach requires a far more disciplined approach than is prevalent today. But it can make a company's philanthropic activities far more effective. Corporate philanthropy is on the top of CRS hierarchy Source: Google Image. Sectors contributing in philanthropy in Bangladesh Many corporations of Bangladesh have come forward to arrange philanthropic work. Bangladesh is a developing country and it is properly understood by different companies in Bangladesh. So, they are come together in arranging a campaign of philanthropy. Donation of different charitable funds, helping poor and affected people, providing scholarship for students, make different initiatives funds etc. The Banking Sector continues to make the largest contribution to philanthropy in Bangladesh as their corporate philanthropy. The Commercial Banks directed and encouraged by the Bank of Bangladesh have made cash donations to individuals and projects in a wide variety of sectors. Trade Associations such as BGMEA and BKMEA and the International Brands sourcing from Bangladesh garment factories have set up training centers, work place day nurseries and have welfare funds, worker insurance schemes and education and cultural activities as part of their corporate philanthropy. Conclusion Corporate philanthropy involves members of corporations taking responsibility for the society but it also expects people who have more resources and more influence to use their privileged position to help those who are less well off. While the phenomenon of philanthropy has been a necessary part of Bangladeshi business culture for centuries, it seems that an emphasis on charitable contributions from philanthropic programs has enhanced the visibility of this practice. By understanding and the importance of corporate philanthropy one of the Government Ministries should devise a 'world-class' Bangladesh specific definition of corporate philanthropy and a Bangladesh philanthropic Policy. However, the principal motivation of Bangladeshi firms to engage in philanthropic activities has stemmed from a sense of their moral obligation to give back to society as businesses control a bulk of society's resources and the financial inability of the government to fix social problems.	2018-12-16T06:17:36.782Z	2012-01-01T00:00:00.000	{ "year": 2012, "sha1": "9c2b0bb36dd00d7cd1fe86bdc2ec19fba05d9d74", "oa_license": "CCBYNC", "oa_url": "https://i-proclaim.my/journals/index.php/gdeb/article/download/204/194", "oa_status": "HYBRID", "pdf_src": "Anansi", "pdf_hash": "b13ccc0a020208486eab7c349f47488b45957db9", "s2fieldsofstudy": [ "Business" ], "extfieldsofstudy": [ "Political Science" ] }
26714909	pes2o/s2orc	v3-fos-license	Is it only the regulatory status? Broadening the debate on cisgenic plants In current debates on emerging technologies for plant breeding in Europe, much attention has been given to the regulatory status of these techniques and their public acceptance. At present, both genetically modified plants with cisgenic approaches—using genes from crossable species—as well as transgenic approaches—using genes from different species—fall under GMO regulation in the EU and both are mandatorily labelled as GMOs. Researchers involved in the early development of cisgenic GM plants convey the message that the potential use and acceptance of cisgenic approaches will be seriously hindered if GMO regulations are not adjusted. Although the similar treatment and labelling of transgenic and cisgenic plants may be a legitimate concern for the marketability of a cisgenic GM plant, there are concerns around their commercialization that reach beyond the current focus on (de)regulation. In this paper, we will use the development of the cisgenic GM potato that aims to overcome ‘late blight’—the most devastating potato disease worldwide—as a case to argue that it is important to recognize, reflect and respond to broader concerns than the dominant focus on the regulatory ‘burden’ and consumer acceptance. Based on insights we gained from discussing this case with diverse stakeholders within the agricultural sector and potato production in Norway during a series of workshops, we elaborate on additional issues such as the (technical) solution offered; different understandings of the late blight problem; the durability of the potato plant resistance; and patenting and ownership. Hence, this paper contributes to empirical knowledge on stakeholder perspectives on emerging plant breeding technologies, underscoring the importance to broaden the scope of the debate on the opportunities and challenges of agricultural biotechnologies, such as cisgenic GM plants. The paper offers policy-relevant input to ongoing efforts to broaden the scope of risk assessments of agricultural biotechnologies. We aim to contribute to the recognition of the complex socio-ecological, legal and political dimensions in which these technological developments are entangled as a means to acknowledge, discuss and respond to these concerns and thereby contribute to more comprehensive and responsible developments within agricultural biotechnology. Background The regulatory status and public acceptance of emerging technologies for plant breeding have been considerably debated among scholars, policymakers, innovators and non-profit organizations, though there has been little public debate on the topic [1,2]. Recent developments within biotechnology, such as cisgenic approaches-the genetic modification (GM) of a recipient organism with genes from a crossable, sexually compatible, variety of the same or closely related species [3]-bring to the fore the debate on the applicability of current genetically modified organisms (GMO) regulation and whether exemptions should be made for new plant breeding techniques [4][5][6][7][8][9]. Cisgenic GM potato that is resistant to 'late blight' is one example of a product developed using emerging plant breeding technologies, and serves as a good illustration of the complex socio-ecological, legal and political dimensions in which these agricultural biotechnologies are entangled. As to date, potato production is significantly plagued by late blight which is the most devastating potato disease worldwide. A funguslike organism called Phytophthora infestans causes the disease. It is estimated to result in an annual global loss of 7 billion Euros [10] and present control measures in conventional potato production involve excessive use of fungicides. Improving host plant resistance, and thereby reducing the need for fungicides, is considered to be the most sustainable way to control late blight [11,12]. Traditional introgression breeding for late blight resistance is challenging, mainly because the potatoes are genetically complex and reproduce vegetatively. New plant breeding techniques such as cisgenic approaches hold promises. It is possible to use the cisgenic approach to improve late blight resistance in potato cultivars because the genes that make potatoes resistant to late blight-so-called resistance (R) genes-originate from wild potato species found in South and Central America [13]. Therefore, in recent years, the use of genetic modification to develop a late blight resistant potato has shifted from a transgenic approach-a genetic modification of a recipient organism with genes from an unrelated organism-to a cisgenic approach. This shift is driven by the potential that if successful, cisgenic plants will be more accepted by public as the inserted gene is from crossable species and the product is thereby more similar to traditionally bred plants. In the debate about GM regulation in Europe and specifically the difference between 'cisgenic' and 'transgenic' approaches, it is argued that exempting cisgenic plants from GMO regulations may herald a new future for agricultural biotechnology [14]. Currently, no distinction between cisgenic and transgenic approaches is made within European GMO regulation and this has become a highly controversial issue. Proponents of deregulation argue that a cisgenic plant can have the exact same properties and is as safe as a plants obtained by traditional introgression breeding [7,[15][16][17]. It is therefore considered inconsistent that plants with the same properties are differently regulated. Furthermore, it is questioned whether cisgenic plants fall under the definition of a GMO, as this definition states that a "GMO means an organism […] in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination" [18]. As cisgenic potatoes do not have a novel combination of genetic material compared to conventionally bred plants, it is argued that they should therefore not fall under the GMO legislation [15]. In most jurisdictions, for example in the Cartagena Protocol on Biosafety and the European GMO legislation, there are both process-and product-related criteria, though the focus in the EU has been on process criteria (e.g. [9]). Those in favour of a strict regulation indeed point to the process criteria to argue that the same technique is applied with cisgenic and transgenic approaches and that therefore cisgenic should fall under the GMO regulation. Another argument stresses that one of the reasons for current regulation of GMOs is the traceability and labelling of the GMO products so as to guarantee consumers' right to know which products contain GMO. If cisgenics is exempted from GMO regulation, consumers would not be able to actively choose products that they know are not genetically modified [14]. The outcome of the re-evaluation of the EU GMO legislation was expected by the end of 2015, but the process keeps delayed and currently no decision has been made. Indeed, the re-evaluation of the GMO legislations and the request for exempting cisgenic approaches from this regulation arguably open up sensitive issues about GMO regulation. For example, whether all GMOs should be regulated in the same way, whether new biotechnologies in plant breeding techniques should fall under the GMO definition, or whether the regulation of GMOs should be process or product based [7]. Researchers involved in the development of cisgenic GM 'late blight resistant' (LBR) potato actively engage in these policy discussions, conveying a message that burdensome GMO regulations prevent the commercialization of cisgenic plants and that their future use and acceptance will be seriously hindered if GMO regulations fail to differentiate between cisgenic and transgenic organisms [8,10,15,17,[19][20][21]. Indeed, the need for a re-evaluation of the current GMO regulation is also wanted because of new genome editing techniques, such as CRISPR/Cas9 and synthetic biology that were not existing when the current regulation was established. The rise of these techniques, such as CRISPR/Cas9-which is currently considered as the most precise and site-specific genome editing technique and is used to delete, insert or replace gene sequences [22,23]-are described as promising developments for improving crop traits and increasing the use of plant genetic resources in pre-breeding and breeding work [24,25]. Combining cisgenic approaches with new genome editing techniques could enable more efficient use of genetic resources and possibly more precise crop breeding. For instance, using CRISPR/cas9 to introduce cisgenes into a plant could potentially build trust in the precision and safety of cisgenic approaches and thereby "tackle some objections to the application of biotechnologies in agriculture" [24]. Furthermore, synthetic biology approaches have the potential for designing disease resistance in crops and could enable the construction of novel, resilient immune response networks in plants [26]. A synthetic biology approach could potentially be used to construct synthetic R genes or multi-R-gene constructs to improve late blight resistance. This could complement current potato breeding approaches that exploit naturally occurring R genes found in wild potato species. Irrespective of the potential opportunities that new biotechnological approaches such as CRISPR/Cas9, cisgenic or synthetic biology could bring about, GMOs currently remain controversial. When it comes to cisgenic plants, there are other concerns that are hindering a successful commercialization than the current focus on the (de)regulation. These concerns include issues such as corporate control over seed markets and the food chain, consumers' and farmers' right to know and choose, the coexistence of different agricultural production systems, intellectual property rights, power relations, scientific uncertainties, ambiguities and values underlying the knowledge development and concerns about the human domination of nature [27][28][29]. The persistent controversies in views and debates on GMOs in general contain polarized arguments that sustain permanent different viewpoints and disagreement about the facts and values and their interwoven nature [30]. These controversies that surround agricultural biotechnologies and the aim to develop truly "responsible governance of agricultural biotechnology" [29] give little reason to assume that clarifying the regulatory status of cisgenic plants and exempting them from GMO regulations are sufficient to assure their marketability and/or public acceptance towards this technology. Moreover, new European legislation on GMOs allows Member States to restrict the cultivation of GMOs on their own territory based on socio-economic impacts, environmental or agricultural policy objectives, or with the aim to avoid the unintended presence of GMOs in other products (Directive 2015/412). This reflects the increased international recognition and significance to consider a broader range of issues beyond the environmental and human safety-related aspects-such as sustainability, social utility and ethical justifiability-when assessing the use of agricultural biotechnologies [31,32]. Furthermore, the recognition to consider a broader assessment frame has been accompanied with a shift in the relationship between science and society, going from 'science and society' to 'science in and with society' (e.g. [33,34]), or 'science with, and for society' (European funding program Horizon 2020). This has resulted in an increase of attention on addressing societal needs, anticipating impacts, assessing alternatives, and conducting public outreach of scientific work. These approaches aim for more transparency in research processes and outcomes, and rely on a participatory and inclusive process (e.g. [35][36][37]). This shift has changed how research, research-based policies, technological developments and technology assessments are developed and performed. Motivated by these thoughts, and particularly the call for broadening risk assessments of GMOs through participatory processes, we organized a series of three workshops with the aim to identify stakeholder perspectives on biotechnological developments within agriculturesuch as cisgenic LBR GM potato. In these workshops, stakeholders involved in the potato production and the agriculture sector in Norway discussed concerns and questions related to sustainability, ethical and social considerations raised by the potential cultivation of cisgenic LBR GM potato in Norway. Two workshops focused on sustainability aspects and used the Problem Formulation Option Assessment method as a way to stimulate discussion (see [38] for more details on the use of this method and specific outcomes). The third workshop focused on social and ethical aspects and used the Ethical Matrix as a starting point for discussion (see [39] for more details on this workshop and the use of the Ethical Matrix during their workshop). An interesting data set was produced from the three workshops which when analysed thematically highlight a number of important issues raised by the stakeholders, namely: the (technical) solution offered; the problem phrasing of the potato disease; the durability of the resistance of the GM potato; and patenting and ownership. By drawing on questions, opinions, concerns and uncertainties that this diverse group of stakeholders shared or raised on these issues, we argue for a broader recognition of, and reflection on the implications arising from the cultivation of cisgenic plants, and cisgenic LBR potato within Europe in particular. The stakeholders participating included potato breeders, seed potato retailers, seed potato and potato producers, representatives from the potato processing industry, agricultural advisors, regulators and representatives of environmental, agricultural and consumer interests and researchers within the fields of crop plants and plant pathogens, and social, economic and ethical issues related to GMOs. The main intention with these stakeholder discussions was to explore the diversity of views among the participants in the workshops, rather than reaching any form of consensus or arrive at representative conclusions. Consequently, the participants did not always agree with each other about arguments, questions, concerns and claims made during the discussions and we recognize that many more concerns and questions than we address can be identified and would benefit from further research and reflection. In this paper, we highlight the issues that were given particular attention during the workshop discussions by stakeholders. The paper is structured in four sections in which each of the issues (i) the (technical) solution offered; (ii) the problem phrasing of the potato disease; (iii) the durability of the resistance of the GM potato; (iv) and patenting and ownership are described in more detail. We describe how the issues were addressed by workshop participants, present relevant scientific literature in the field and make suggestions for future research needs. A key contribution of this paper is the empirical insight on stakeholder perspectives on emerging plant-breeding techniques, thereby underscoring the importance of broadening the scope of the debate on the opportunities and challenges of agricultural biotechnologies, such as cisgenic GM plants. In terms of its policy relevance, the paper offers a valuable contribution to ongoing efforts and a growing need to broaden the scope of risk assessments of agricultural biotechnologies, as for instant reflected in the new European legislation on GMOs (Directive 2015/412). By addressing these concerns, we aim to broaden the debate so as to acknowledge, discuss and contribute to respond to them. A technical fix to consumer concerns The shift from transgenic to cisgenic approaches in the development of LBR GM potato was based on the availability of resistance (R) genes in wild potato species. The assumption was that this approach would be ethically more acceptable to the public as these genes are from crossable species instead of different species [21]. However, past and present debates on this topic, and the difficulty to decide on regulation for it, have shown that there are additional concerns that reach beyond the regulatory questions. Indeed, when the development of cisgenic technologies is considered to be "a result of taking the opinions and concerns of consumers seriously" [40] and is aimed to respond to consumer concerns, it fails to recognize the broader issues that are persistently addressed within society about GM approaches in general. During the workshops, it became clear that most of the Norwegian stakeholders in the potato and agriculture sector consider it as a reasonable assumption that cisgenic plants will decrease consumer concerns to some degree. Still, they highlighted that it is too simplistic to think that this technical fix is sufficient to alleviate all concerns associated with GM crops. Moreover, there is no scientific consensus on the safety of cisgenic plants. For example, the GMO panel of the European Food and Safety Authority [41] concluded in 2012 that the risks associated with cisgenic and traditionally bred plants are the same, while transgenic plants may result in novel risks. This conclusion was gene based, meaning that because the genes are known to be safe, the cisgenic approach was also considered safe. However, even if the inserted gene is known, this does not mean that uncertainties associated with the transformation process are known. Hence, in contrast to the conclusion of the EFSA, it is argued that, in terms of food and environmental safety, cisgenic approaches cannot be regarded as equivalent to conventional breeding. Neither can the safety of the products resulting from traditional breeding and cisgenic technologies be considered as similar, based on the unknown behaviour and effects of the genes in the transformation process and their functioning within the mechanisms of the host plant [42][43][44]. Furthermore, the issues reaching beyond the safety of the cisgenic plant seem to get little attention from researchers in the field of these emerging breeding techniques. For them, it seems that the ability of cisgenic plants to get more public acceptance is a common ground to pursue cisgenic approaches and advocate for a less strict regulation [2]. Although the 'more natural' approach of cisgenic compared to transgenic may affect the view of consumers to this technique to some degree, cisgenic breeding remains a genetic modification, which remains controversial, and consumers still favour, for example, labelling of such a technique [14]. The assumption that cisgenic plants will alleviate the reluctant attitude of consumers raised concerns among some of the Norwegian stakeholders about the effect this may have on the consumers' trust in their farming practice, their products, and the agriculture sector in general. This prevented these stakeholders to fully embrace this technological shift as the solution for late blight. Their concerns go beyond ambiguities on the regulatory status of cisgenic plants or safety issues, to include their relationship with consumers and the potential damage that a lack of transparency, communication and research of alternatives may cause. Moreover, some stakeholders were concerned that a loss of trust from the public could result in a decrease of subsidies for the agriculture sector. For years, the Norwegian agriculture sector receives one of the highest governmental support levels among the OECD countries [45,46]. Some of the stakeholders emphasized that the current financial support from the government stems from the trust in quality of domestic agricultural products. These subsidies constitute a large share of the income of Norwegian farmers and thus an important reason for them to carefully assess the potential implications that different methods for plant breeding may have on their trustworthiness. The limited amount of empirical research on consumer's acceptance of GM food indicates different perceptions of consumers towards cisgenic and transgenic plants; this arguably demonstrates that the acceptance of cisgenic approaches is not straightforward. A study in 2010 demonstrates a more reluctant than tolerant attitude towards GM food in Europe [47], as does a study in 2017 under Norwegian consumers [48]. Research by Lusk and Rozan [49] indicates a higher acceptance of cisgenic than transgenic approaches for GM food, based on nationwide surveys in the US and France. Their research demonstrates a significant difference between these nations, with a twice as high acceptance level in the US. Moreover, European citizens perceive a lack of knowledge on GM approaches, hindering them to make a well-informed decision about their willingness to consume GM products [50]. Although Delwaide et al. claim that the different perceptions on transgenic and cisgenic plants confirm the hypothesis that public will be more tolerant to the latter than the former, it is not yet clear what the conditions for acceptance of a GM application are. Indeed, a research performed by Mielby et al. [51] indicates that more knowledge of different GM approaches does not lead to consumers developing an attitude based on differences between methods (i.e. transgenic or cisgenic). Rather, consumers consider the purposes of GM applications as important, such as a medical application or food application. Furthermore, consumers choose to depend on people they consider experts and trustworthy to make informed decisions. This gives trust an important role in the view people develop of GM food [52][53][54]. Overall, there is relatively limited information available about current perception of European public on cisgenic and transgenic plants to get a comprehensive and accurate representation of consumers' preference and public acceptance. More empirical studies and surveys are required to reveal and document the support base from public, and assess whether a further development of cisgenic plants is safer, desirable, acceptable and can be marketable. Additionally, the debates on cisgenic approaches would benefit from including broader issues mentioned by different stakeholders within the potato and agricultural industry-both on the level of cisgenic approaches as well as the more general level of GMOs. Failing to acknowledge these broader issues and relying on a technical fix to overcome the safety and consumer concerns with cisgenic GM approaches potentially sustains a scattered and under-communicated discussion and severely complicates constructive and responsible developments within agricultural biotechnology. The importance to recognize alternatives Different understandings of the late blight problem, approaches to breed for more resistant potato varieties and "models for agriculture" will lead to a different set of farm practices to try to fight late blight. In this particular case, characterizing the problem, approaches and solutions is mostly framed within the currently dominant approach of conventional potato production that involves excessive use of fungicides to control the disease [3,10]. The message conveyed is that despite considerable efforts during the last 150 years, traditional introgression breeding for durable late blight resistance has largely been unsuccessful [13]. Genetic modification is expected to contribute in solving these challenges as it allow researchers to introduce several R genes into a potato variety in which the qualities appreciated by farmers, processors and consumers remain the same. Reflection on the dominant approach in research and governance of agriculture reveals the different values on which the approach is based, and may lead to different practices, evaluations and perspectives on cisgenic plants [55]. Risk assessment of GM crops is however, typically narrowly framed, where technological benefits and risks are compared to current dominating production systems (most commonly conventional agriculture). This may cause a tunnel vision, where only particular problems, tactics and solutions are discussed. Contrary to this approach, we argue that it is crucial to critically reflect on whether the solution that is offered is responding to the societal need that it is supposed to address. Moreover, the true origins of the problem should be identified, different future visions explored and alternative approaches and solutions investigated. 1 Exploring alternative approaches includes considerations of their benefits, adverse effects, uncertainties, ambiguity, existing knowledge gaps and the changes required for large-scale implementation of this alternative [56]. During our workshops, some stakeholders emphasized that the message conceived seems to be that, based on the current (dominant) agricultural model, improving host plant resistance is the best strategy to fight late blight, and that GM is the only means to develop potato varieties with durable resistance to late blight that also satisfy consumer demands. They stressed that there is limited attention and investigation of alternative approaches to fight late blight. Stimulating critical reflection is useful to reveal the understanding of a particular societal need, problem, and proposed solution by different stakeholders or different agricultural models. It is also a useful means to demonstrate the different interests and future visions of stakeholders (e.g. what kind of society do we want?) and opens up opportunities to direct technological developments in ways that are perceived to be socially desirable [34]. A way to manifest critical reflection on the underlying values of a technological development or an agricultural practice is to open up the assessment processes to a more participatory model, with deliberation among different stakeholders. Indeed, the current trend to emphasize the importance of recognizing underlying values, assumption and beliefs to achieve "responsible governance of agricultural biotechnology" [29] underscores the significance to critically reflect on these hidden values, norms, assumption and visions within agriculture to understand the different perspectives and solutions offered. The aspiration for more critical reflection was also shared by some of the stakeholders, who highlighted that there is little consideration of more overarching questions related to the late blight problem and challenged the "narrow scope" in the frames of conventional largescale potato production. For example with regard to whether the severity of the late blight problem is partly a result of industrial agriculture dominated by largescale monoculture production. These stakeholders called for a broader problem formulation frame, pointing to questions related to the role of agricultural policies and regulations in promoting large-scale industrial potato production and "roads not taken" when searching for alternative approaches to breed for late blight resistance. Current agricultural policy, incentives, structures and regulation are implicitly focussed and structured to support and sustain conventional agriculture as the dominant approach, for example by defining 'organic agriculture' as a separate category whereas conventional agriculture is meant with agriculture (e.g. [57,58]). Critically reflecting on the effect of this dominant approach, acknowledging the complex socio-ecological systems and networks of which agricultural biotechnologies are part of is important as well as being open to learn from other agriculture systems [e.g. 28]. Learning from other agricultural systems could also facilitate responding to broader concerns, such as the contribution to sustainable development, the societal needs and the ethical justifiability of a technological development within agriculture (As an example, see http.//www.agriculturesproject.org). An assessment of a biotechnological application that takes these aspects into account benefits from input and deliberation with for example practitioners of agriculture and those working with the governance of agriculture, rather than solely expert-driven or academic actors focusing on agriculture. This may lead to create more understanding between different stakeholder groups, enables responsible approaches to generate more support for decisions and responds to the opinions and concerns of stakeholders in a desirable way. The quest for durable late blight resistance A host plant and its pathogen (e.g. virus, bacteria or fungus) are typically in a constant evolutionary arms race. In our case, the potato plant is continuously trying to develop ways to defend itself from late blight, while P. infestans is continuously searching for ways to break these defence mechanisms. P. infestans has a remarkable capacity to rapidly adapt to resistant host plants [59], and R genes introduced to commercial potato varieties through traditional introgression breeding have consequently been defeated shortly after the potato variety are put in commercial production [13]. Traditional breeding for potatoes with late blight resistance that lasts over time has therefore been a vexing challenge. One of the main aims for using genetic modification when developing LBR potato varieties is to achieve resistance that is lasting over time [10]. Novel molecular technologies have advanced researchers' ability to identify and characterize genes that provide late blight resistance and transfer these genes from wild potato species to commercial ones [13]. Still, several questions remain about the molecular mechanisms underlying the observed resistance of potato plants [60][61][62]. Similarly, more research is needed to advance the understanding of the molecular and genetic mechanisms of virulence and adaptability in the P. infestans population [63]. Further research of these knowledge gaps is crucial in the search for ways to achieve durable LBR potato varieties. Still, these uncertainties are seldom acknowledged and openly discussed in public, as for instance illustrated with the lack of attention that these issue are given in a recent report written by leading European researchers involved in the development of cisgenic LBR potato [64]. Rather, the message conveyed by researcher working in this field is that GM potato will significantly contribute to a more sustainable potato production by providing the potato plant with a defence system that lasts over time [10,20,[64][65][66][67]. They argue that this approach makes it easier for researchers to introduce several R genes into the same potato variety, which is expected to tip the 'evolutionary' balance in favour of the potato plant and against P. infestans, as it will be more difficult and time consuming for P. infestans to overcome multiple defence mechanisms at once. When this approach is presented to public, it is however largely under communicated that the durability of the cisgenic LBR GM potato can only be assessed in retrospect, meaning that it is problematic to predict how long the resistance will last until the potato is put in large-scale production and cultivated over time. It is also important to be aware that successful-i.e. durable-application of the cisgenic LBR GM potato hinges on careful monitoring and development of resistant management strategies. This implies that the P. infestans population is monitored to detect if any of the R genes of a certain GM potato variety are broken by the pathogen, and that strategies to halt virulence development in the P. infestans population are established prior to large-scale cultivation [68]. These strategies must be adapted to the area where the GM potato will be grown. In Norway, for instance, the genetic diversity of the P. infestans population is particularly high [69], which may strengthen the adaptive potential of the pathogen, and possibly influences its ability to overcome resistance. Finally, it is important to clarify questions about the distribution of the responsibilities and the costs for carrying out monitoring and resistance management. Farmers and agricultural advisors must be sufficiently trained to carry out these tasks, which might be challenging, particularly in developing countries. For instance, one of the first outbreaks of resistance development among target organisms on GM Bt maize were reported in South Africa, partly explained by farmers' failure to comply with resistant management strategies such as refuge requirements [70]. Some of the stakeholders questioned the durability of the GM approach, and emphasized that farmers and consumers may be left with the misconception that this approach is the solution to the late blight problem. As phrased by Mullins [71]: "Demonstrating durability of generated resistance is key to underpinning grower confidence". Hence, a lack of transparency about the uncertainties associated with such a statement, as well as the conditions that this approach hinges on might result in a backlash against trust that farmers and public place on the researchers driving this technology. It may also limit the ability to open up for a broader and more inclusive discussion about how to address the uncertainties and limitations of this proposed solution, as these uncertainties and limitations are to a large extent unknown. Therefore, candidness and transparency about the uncertainties, knowledge gaps and limitations of using GM approaches to develop durable LBR potato varieties should be stimulated throughout the different levels of research and development of cisgenic plants. Patent rights and ownership A major issue in the debate about GM technology is the intellectual property rights for plant varieties. The genetic modification of organisms is currently a patentable practice. However, patenting plant traits has given reasons for concerns by a broad range of the Norwegian stakeholders. Within the development of cisgenic crops, proponents emphasize that because the inserted gene stems from a crossable and/or sexually compatible variety; the obtained crop is in theory not different from a potentially traditionally bred crop. Thus for them, the similarity between cisgenic and conventional crops makes the product of cisgenic approaches closer to traditionally bred plant varieties, thereby arguing for easing the regulation of cisgenic crops. Despite this argued similarity, cisgenic crops are considered to be sufficiently different to be patentable. Although patent laws and requirements differ among nations, in general patents are granted if inventions are "new, involve an inventive step and are susceptible of industrial application" (Article 52, EPC 2016). What can be patented when it concerns biological inventions is the 'biological material'-in this case the plant-possessing specific trait(s) as a result of the invention, or a process that enables a plant to be produced possessing specific trait(s) as a result of the invention (Article 8, DIRECTIVE 98/44/EC), for example the trait to be resistant to late blight. Nevertheless, if a biotechnological product fulfils these criteria and can obtain a patent, one could argue that it is reasonable to have an elaborated regulatory (safety) assessment in place to assess the risks, need and desire of this novel trait in the crop before it is released on the market. Therefore, a paradox emerges in the arguments in favour of deregulation of cisgenic crops, namely that the claim for novelty that is required to obtain a patent is the same reason why deregulation or easing the regulation of this method seems questionable, and arguably irresponsible. Indeed, if it is questioned whether cisgenic plants should fall under the (strict) GMO regulation, it simultaneously raises questions whether it still fulfils the criteria to be patentable. This illustrates that this discussion is not only about whether an elaborated safety assessment should be in place. It also touches upon the perceived ambiguity whether cisgenic approaches should be a patentable practice, thereby questioning the criteria of the 'inventive step' . The idea of a form of intellectual property right in agriculture is not new. In fact, developers of a new variety can get a "plant breeders' rights" (PBR), giving the breeder the exclusive control over the breeding of a particular variety. 2 The most concerning difference for stakeholders between PBR and patents however concerns the reuse of farm-saved seeds/potatoes to be used as seeds the next year. Under PBR, a potato farmer can save a part of its harvest to reuse the next year, which is significant in the Norwegian potato industry, as farmers take around 70-75% of their seed potatoes from their own crop. 3 In contrast to a PBR, a patent on a plant trait does not allow the use of farm-saved seeds/potatoes and so farmers need to buy new seed potatoes every year, thereby increasing their costs significantly. Moreover, the difference between patents and PBR also affects breeding programmes. In general, the access to technology as well as genetic material is essential for the development of new plant varieties. Within PBR, this is regulated in the 'breeder's exemption' that allows breeders to use each other's varieties in breeding programmes without necessarily obtaining a licence or agreeing on financial compensation [72]. The importance of facilitating the development of new plant varieties is reflected in international treaties such as the International Union for the Protection of New Varieties of Plants (UPOV 1961(UPOV /1978(UPOV /1991, the Agreement on Trade-Related Aspects of Intellectual Property Rights (WTO-TRIPS-1994), and the International Treaty on Plant Genetic Resources for Food and Agriculture (IT PGRFA-2001). In contrast to PBR, patents always demand permission from the patent holder in order to be used in breeding programmes, leading to a longer procedure and potentially more costs. Another concern related to the patentability of cisgenic crops concerns the traceability of cisgenic plants. This becomes extra complicated when traditional breeding becomes capable of developing and commercializing a variety with for example resistance to late blight, and both LBR potato via traditional breeding as well as GM co-exist. It then becomes difficult, if not impossible, to check whether a plant is cisgenic in retrospect and to keep control of where the patentable plant ends up, whether it crosses with other species and the assessment thereof. Moreover, there is uncertainty about the legal and economic consequences for farmers for unintended contamination through gene flow, as well as for the patent holder to potentially argue that his patent was breached. For some of the Norwegian stakeholders, the concern with patenting is predominantly about the power and potential monopoly of biotech companies that obtain patents over plant varieties and the influence this can have on the seed and food prices, the power relations within the sector and the independent research on the safety of cisgenic plants and further uses, such as breeding by other research institutions (see also [73]). Entry of the patent system into for example the potato sector, contributes to the concentration and reduction of the diversity of companies and the possibilities for strategic use, which may lead to lack of clarity in the market and to monopolistic behaviour [74]. Patenting has the potential to increase the control of a few corporations over seeds and crops, thereby increasing farmer dependency and a reduction of biodiversity [27,[75][76][77]. The potential effects and implications for stakeholders and the uncertainty that patenting involves creates uneasiness among Norwegian stakeholders in the sector. Even though experimenting with patent regimes is ongoing, it seems that the fundamental principle of patenting is receiving significant resistance. As cisgenic crops are considered GM products, they have to get through regulatory processes to get approval. Currently the costs for complying with regulatory processes can cost millions of euros for each application and typically takes years to complete [78,79]. This burden of high costs to comply with regulatory processes is used as an argument in favour of exempting cisgenic plants from GM regulation, so that small and medium-sized enterprises can compete with the bigger, usually multinational corporations that are currently dominating the market (e.g. [8,71,80]). Therefore, in theory, easing regulation could benefit small enterprises. In practice however, it is questionable whether this will actually benefit small-and medium-sized enterprises, as they may easily be overruled by larger companies due to the larger capacity of these companies to develop new GM varieties. As patenting generates revenues for more research and enables seeking for the costly approvals of other inventions, it can cause a "Matthew effect" [81] in which the larger firms and institutes will get larger, while the small firms and research institutes are unable to keep up with this development and grow ever smaller. Thus, the potential to patent cisgenic crops raises significant concerns about the implications for different actors in the sector and demands reflection on what kind of consequences patenting entails and calls for further investigation on the role of (different) patent policies within agriculture. Transparency about who will benefit, how power relations may shift or increase, and deliberation on these implications is warranted. Conclusion In this paper, we have discussed important concerns in the debate about the development of LBR GM potato in Europe, which go beyond the regulatory status of cisgenic LBR potato and consumer acceptance of cisgenic plants. Although we recognize the importance of clarifying the regulation of cisgenic plants, we argue that this debate is currently too narrowly focused and fails to recognize the broader issues that are persistently addressed within society about GM approaches in general and that, therefore, it is important to broaden the scope of the debate on cisgenic plants. Although the importance to proactively engage in 'post-research' issues by researchers involved in the development of LBR GM potato is emphasized and arguably contributes to the recognition of broader concerns, these are often phrased in the sense of 'educating' or 'informing' public, rather than listening and acting upon their concerns (e.g. [71]). By discussing concerns around the (technical) solution offered, the problem phrasing of the late blight potato disease, the durability of this solution, and patenting and ownership, we hope to contribute to a recognition of the complex socio-ecological, legal and political dimensions in which this technological development is entangled and stimulate discussion that takes this broader view into account. Importantly, we recognize that more concerns can be identified related to the development of cisgenic plants and that we touch upon questions and concerns that would benefit from deeper reflection and further investigation. While concerns mentioned in this paper are specifically formulated around the case of LBR GM potato, we do not consider them to be exclusive to this particular technological development. Rather, we believe that these issues can be found across different agricultural biotechnological developments and approaches-such as CRISPR/Cas9 and synthetic biology-and are therefore important concerns to reflect upon and respond to. Thus, in order to develop truly responsible governance for agricultural biotechnology, the scope of the debate on cisgenic approaches in Europe should be broadened to include other significant concerns raised by stakeholders within the agricultural sector, and the public in general.	2017-08-08T05:15:32.422Z	2017-06-26T00:00:00.000	{ "year": 2017, "sha1": "ec3ba70a61b0d03896e835e5008c78a7ac98c6cb", "oa_license": "CCBY", "oa_url": "https://enveurope.springeropen.com/track/pdf/10.1186/s12302-017-0120-2", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "ec3ba70a61b0d03896e835e5008c78a7ac98c6cb", "s2fieldsofstudy": [ "Environmental Science" ], "extfieldsofstudy": [ "Medicine", "Biology" ] }
3420908	pes2o/s2orc	v3-fos-license	Shikonin exerts antitumor activity in Burkitt’s lymphoma by inhibiting C-MYC and PI3K/AKT/mTOR pathway and acts synergistically with doxorubicin Burkitt’s lymphoma (BL) is a highly aggressive malignancy molecularly characterized by deregulation of the C-MYC proto-oncogene. Recently, it has been confirmed that phosphatidylinositol-3-kinase (PI3K) pathway activation is a crucial element in the malignant transformation of the B cells in BL. Despite the better outcome of adults with BL treated with high-intensity chemotherapy regimens, the overall survival rate for patients older than 60 years remains dismal. Shikonin, a natural naphthoquinone derived from Chinese herbal medicine plant, has the potential to induce cell death in a series of human cancer. In the present study, we investigated the effect and molecular mechanisms of Shikonin in treatment with BL. Shikonin suppressed cellular proliferation and induced caspase-dependent apoptosis in BL cells. Inhibition of C-MYC and suppression of PI3K/AKT/mTOR pathway played critical roles in SHK-induced apoptosis in BL both in vitro and in vivo. Besides, Shikonin potentiated doxorubicin-induced growth inhibition and apoptosis in vitro. Furthermore, the growth of a subcutaneous xenograft tumor model of BL was significantly inhibited by shikonin. Importantly, we did not find the effect of shikonin on liver function in mice. In summary, these data suggest that shikonin may be an encouraging chemotherapeutic agent in the clinical treatment of BL. constitutively activated transcription factor also involves the activation of PI3K through augmenting the BCR signaling. Additionally, C-MYC contributes to the activation of the PI3K pathway by promoting expression of mir-17-92 cluster, one of which decreases expression of phosphatase and tensin homolog deleted on chromosome ten (PTEN) 9 . Overall, these results demonstrate that C-MYC and PI3K pathway have emerged as promising therapeutic targets in BL. Natural herbs play a vital role in cancer treatment owing to their pharmacological effects and low toxicity 10,11 . Shikonin (SHK) is an active naphthoquinone derivative compound extracted from root tissues of the traditional Chinese medical herb Lithospermum erythrorhizon which had been broadly applied by ancient Chinese doctors for thousands of years to treat burns and to promote wound healing. Recent emerging researches confirmed that SHK had the potential to induce apoptosis in a variety of human tumor cell lines including leukemia cell lines in vitro and in vivo with minimal or no toxicity to healthy human cells [12][13][14][15] . The anti-tumor activity of SHK may involve in its ability to generate reactive oxygen species 12 , activate pro-apoptotic caspase family members, inhibit the expression of C-MYC 13 and suppress PI3K phosphorylation 14 . Thus much attention has been focused on the potential value of SHK in the therapy of kinds of cancer. However, the effect of SHK on the growth of BL and the possible mechanism had never been reported. In this study, we investigated whether SHK alone could have the anti-tumor effect on BL cells both in vitro and in a xenograft mouse model, and whether SHK could have the potential to act as a chemosensitizing agent to improve the therapeutic index of doxorubicin (DOX) in vitro. The results showed that SHK suppressed cellular proliferation and induced caspase-dependent apoptosis through inhibition of the expression of C-MYC and the modulation of PI3K signaling in BL cells. Besides, SHK strongly potentiated DOX-induced growth inhibition and apoptosis. The present data suggest that SHK may serve as a novel agent for the treatment of BL because of its likely targets. Shikonin suppresses cellular proliferation and induces caspase-dependent apoptosis in Burkitt's lymphoma cells. For investigating the effect of SHK on the proliferation of human BL cells, Raji and Namalwa cells were treated with different concentration of SHK for various durations. Cell viability measured by a MTT assay showed that SHK suppresses cellular proliferation in dose-dependent and time-dependent manners ( Fig. 1A-C). The two cell lines responded differently to the SHK treatment and Namalwa cells were apparently more sensitive to SHK than Raji cells. The differences noted between two cell lines were in agreement Previous researches demonstrated that induction of apoptosis is the main way for SHK-induced cell death in vitro 12,15 . So we detected whether apoptosis also took part in this process in BL after SHK treatment, and the apoptosis rate of Namalwa cells was examined using Annexin V and 7-AAD staining assay followed by flow cytometry analysis. As shown in Fig. 1D, the percentage of apoptotic cells dramatically increased with growing concentration of SHK from 250 to 1000 nM after incubation for 24 h. Furthermore, the pan-caspase inhibitor ZVAD-FMK (20 μM) was administered in advance for 2 h to determine whether the apoptosis induced by SHK was caspase-dependent. And we found that the apoptosis rate was significantly decreased, especially for the late apoptosis compared to the early apoptosis (Fig. 1D,E). To observe the nuclear condensation or fragmentation which signifies apoptosis, we used Hoechst 33342 and PI staining in Namalwa cells after 6 h incubation with SHK. As shown in Fig. 2A, on the contrast with untreated cells presenting round nuclei with dark blue color, part of the cells treated by SHK had shrunken nuclei in light blue color without or with red color indicating they were early apoptotic cells or late apoptotic cells, respectively. Furthermore, the numbers of apoptotic cells were also positively related to the concentration of SHK treatment. On western blot analysis (Fig. 2B), the same doses of SHK dose-dependently activated caspase pathway as evidenced by splicing events of caspase-9, -8, -3 and PARP, which indicate the initiation of apoptosis. All these data suggest that SHK activates caspase cascade and induces caspase-dependent apoptosis in BL cells. Shikonin inhibits both C-MYC and PI3K/AKT/mTOR activity in vitro. Numerous studies have established that C-MYC deregulation is one of the most important events for BL malignant transformation 16,17 . The previous study showed that C-MYC is a potential target of SHK in U937 cells 13 . Therefore, we evaluated the effect of SHK on C-MYC in Namalwa and Raji cells. As showed in Fig. 3A, SHK significantly inhibited the protein expression of C-MYC in a dose-dependent manner in both BL cells. C-MYC regulates a large number of micro-RNAs (miRs) that function as oncogenes apart from inducing cell proliferation and growth 18 . MiR-19a is one of the oncogenic miR-17-92-cluster members which is up-regulated by C-MYC and can cause the activation of the PI3K/AKT pathway through down-regulation of PTEN 9 . In this study, we found that SHK also induced a dose-dependent reduction of miR-19a for Namalwa cells (Fig. 3B). Similar results were obtained with Raji cells (Fig. 3C). A known C-MYC inhibitor 10058-F4 was used as a positive control drug. As shown in Fig. 3E,F, 10058-F4 inhibited the expression of both C-MYC and miR-19a. It is known that PI3K activity plays a central role in the development and ongoing maintenance of BL 19,20 . And several studies found that SHK could inhibit PI3K activity in different tumor cells 14,21 . So we first investigated the role of SHK on PI3K and its downstream AKT expression by western blot analysis (Fig. 3D), SHK decreased p110α which is one of the PI3K subunits and phosphorylation of AKT at Ser473 in a dose-dependent fashion in Namalwa and Raji cells. As expected, SHK also strongly inhibited phosphorylation of mTOR at Ser2448, a marker for mTORC1 activity 22 , as well as phosphorylation of p70S6K, the best-characterized target of mTORC1. Moreover, SHK successfully inhibited phosphorylation of mTOR at Ser2481, a marker for the presence of mTORC2 complexes 22 , which was consistent with the decreased level of phosphorylation of AKT at Ser473. The activity of mTORC1 and mTORC2 in Namalwa and Raji cells was completely inhibited by the treatment with 1000 nM SHK accompanied by slight degradation of protein expression of mTOR. The synergistic anti-proliferation activity of shikonin and doxorubicin is that Shikonin potentiates doxorubicin-induced apoptosis in Burkitt's lymphoma cells. Doxorubicin is one of the most commonly used chemotherapeutic drugs in patients with BL. Given the fact that the acquisition of doxorubicin resistance is a primary cause of chemotherapy failure and high doses of doxorubicin easily lead to toxic side effects, we tested the effect of combination therapy of doxorubicin and SHK by MTT assay. Therefore, Namalwa and Raji were treated with a series of doses of SHK or/and doxorubicin. As shown in Fig. 4A, SHK (200 nM) or doxorubicin (400 nM) decreased the cell viability of Namalwa cells to 49.33% or 73.63%, respectively, whereas the viability of Namalwa cells treated with the combination therapy decreased to 38.93% and the similar result was also observed in Raji cells. The combination index analysis showed that CI range values in Namalwa cells were 1.73 to 0.68 for fractional effect corresponding to 0.06 to 0.61 (Fig. 4B), indicating that SHK in combination with doxorubicin has synergistic anti-proliferation activity. Next, we examined whether this synergy effect of combination treatment of SHK and doxorubicin was due to induction of apoptosis. As shown in Fig. 4C-E, the results of the flow cytometric analysis revealed a marked increase in the proportion of apoptotic cells after combined treatment with SHK and doxorubicin for 24 h compared with that of SHK or doxorubicin alone. And SHK distinctly enhanced doxorubicin-induced activation of caspase-3 and PARP indicating the activation of apoptosis pathway (Fig. 4F). These data are consistent with MTT assay, suggesting that synergistic cell growth inhibition resulting from combination treatment with SHK and doxorubicin may owe to, at least partly, the induction of increased apoptosis in BL cells. Shikonin inhibits xenograft tumor growth. Given the potent inhibitory activity of SHK on BL growth in vitro, it is believed that SHK has potent anti-tumor effects in treating BL in vivo. Thus, we established NOD/SCID mice xenografts bearing Namalwa BL cells. As shown in Fig. 5A, there was a significant decrease in mean tumor volume for mice that received the intraperitoneal injection of SHK compared to which were treated with fat emulsion. The last average tumor volume of vehicle group and SHK group reached to 3356.61 mm 3 and 1842.63 mm 3 , respectively (P < 0.01). At the end of the experiment, tumors were isolated from mice and weighted. The mean tumor weight was significantly more substantial in control group compared with SHK-treated mice (P < 0.01) (Fig. 5B). Importantly, there was no significant difference between the vehicle group and SHK group in mice mean body weight (Fig. 5C). Moreover, there was also no significant difference in serum aspartate transaminase and alanine transaminase levels between SHK-treated mice and control mice (Fig. 5D). Additionally, consistent with our previous findings in vitro, the miR-19a expression of the SHK group was remarkably lower than the control group (Fig. 5E). To evaluate the histopathology changes in the tumor tissues, we performed hematoxylin and eosin (HE) and immunohistochemical staining after the tissues being extracted. As presented in Fig. 5F, HE staining demonstrated that SHK caused severe pathological changes including cell death, tumor necrosis and angiogenesis inhibition compared with control tissues. Furthermore, treatment with SHK obviously decreased the number of Ki-67-positive cells in tumors compared with controls. Similar results ). The volume of tumor was measured at the indicated times. Data are presented as mean ± SD (n = 5 per group). * and represents P < 0.05 and P < 0.01, respectively. (B) The scatter plot represents mean of the tumor weight from SHK-treated and control mice. Data are presented as mean ± SD (n = 5 per group). represents P < 0.01. (C) The body weight of mice was measured at the indicated times. Data are presented as mean ± SD (n = 5 per group). (D) Serum activity of alanine transaminase (ALT) and aspartate transaminase (AST) was measured in control and SHK group by spectrophotometric methods. Data are presented as mean ± SD (n = 5 per group). (E) RT-PCR was performed to evaluate the relative expression of miR-19a in Namalwa xenografts. Data are presented as mean ± SD (n = 3 per group). (F) Hematoxylin and eosin hematoxylin and eosin (HE) staining was performed to observe pathological changes in tumor tissues. Immunohistochemistry was performed to measure cell proliferation by Ki-67 staining, and the expression of C-MYC, P-AKT (Ser473) and P-mTOR (Ser2448). Original magnification: 400x. Samples from three animals in each group were analyzed, and representative data are shown. (G) Western blot analysis monitors the expression of C-MYC, PI3K and P-mTOR (Ser2448). Samples from two animals in each group were analyzed. GAPDH was used as a loading control. Cropped blots/gels were used in the figure and the gels had been run under the same experimental conditions; the fulllength blots/gels are presented in Supplementary Figure S4. Discussion With the development of intensive chemotherapy regimens, pediatric BL treatment is very successful, most of which can be cured 23 . However, the elderly patients often can not tolerate these rigorous BL regimens. Moreover, the bone marrow and the immune responses are consequently suppressed by these regimens, placing a premium on supportive care, which is to detect and treat infections promptly and efficiently. Hence, it is urgent to exploit new treatment regimens which are less immunosuppressive and better tolerated for BL. SHK is a small molecule natural compound derived from Chinese herbal medicine plant. Since its long history used in Chinese traditional medicine, it is considered to be much safe for human beings. Recent experiments found out that SHK had anti-tumor effects on glioma 24 , gastric cancer 25 , hepatocellular carcinoma 26 and leukemia 12,15 . In this study, we confirmed that SHK suppressed cellular proliferation and induced death in Raji and Namalwa BL cells in a dose and time-dependent manner. Apoptosis induced by chemotherapy, is one of the essential patterns participating in eliminating cancer cells. It is a consequence of a series of precisely controlled events involving cell death 27 . Two related pathways are resulting in apoptosis: the extrinsic pathway and the intrinsic pathway 28 . Generally speaking, the former involves activating caspase-8, and the latter involves activating caspase-9. Then activated caspase-8 or caspase-9 could trigger downstream proteins, such as caspase-3 and poly ADP-ribose polymerase (PARP). Our data presented that SHK induced apoptosis in BL cells via caspase-dependent pathways, which was validated by nuclear fragmentation, apoptotic cell morphology and pan-caspase inhibitor ZVAD-FMK. C-MYC deregulation plays a critical role in the pathogenesis of BL. Indeed, BET Bromodomain inhibitors that can inhibit C-MYC indirectly have been examined in mouse models of BL and been undergoing clinical trials in patients with hematologic malignancies [29][30][31] . Besides chromosome translocation, some other mechanisms are accounting for C-MYC deregulation in malignancy, for instance, gene amplification and insertional viral mutagenesis 18 . The C-MYC protein usually acts as a transcription factor that binds to DNA and regulates transcription. It can make the cell phase transition from the G0/1 phase to the S phase, which ultimately induces DNA replication, protein biosynthesis and cell proliferation and growth 32 . In our study, we found that SHK could significantly decrease the expression of C-MYC in protein level even at a low dose as well as its specific inhibi-tor10058-F4 did at a relatively high dose in BL cells. Apart from association with cell proliferation and growth, C-MYC ties up with a great number of micro-RNAs that function as oncogenes such as miR-19a 9 or tumor suppressor genes such as miR-34a 33 . Similar to the previous results, SHK also down-regulated the expression of miR-19a in a dose-manner in BL cells, which negatively regulated the expression of PTEN. It will be interesting to investigate the relationship between SHK and PTEN in future. It has proved that C-MYC dysregulation alone does not lead to lymphoma 34 and the t(8;14) is also detected in blood cells and bone marrow of healthy individuals 35 . Until recently an engineered mouse model expressing deregulated C-MYC and constitutively-active PI3K specifically in germinal center B cells identified that PI3K signaling could cooperate with C-MYC in the development of BL 7 . In the meantime, by comprehensive high-throughput RNA sequencing, Schmitz et al. further revealed that mutations of TCF3/ID3 genes resulted in continuous PI3K activation in a large panel of BL cases 8 . Therefore, more and more attention is paid to PI3K inhibitors and several pan-PI3K or isoform-selective PI3K inhibitors have been undergoing clinical trials in non-Hodgkin lymphoma 36,37 . In this study, we found that SHK inhibited protein expression of p110α, which is one of the catalytic subunits of Class IA PI3Ks. Moreover, SHK decreased the activity of mTORC1 and mTORC2, as evidenced by dephosphorylation of mTOR on Ser2448 and Ser2481. Paralleled with these results, it elicited potent suppression of phosphorylation of p70S6K and phosphorylation of AKT at Ser473 in BL cells, standing for one of the downstream targets of mTORC1 and a substrate of mTORC2, respectively. The data that both dephosphorylation of mTOR on Ser2448 and Ser2481 by SHK is exciting and inspiring because there is a negative feedback loop between mTORC1 and mTORC2 38 . Once mTORC1 was inhibited, mTORC2 could be further activated through PI3K signaling. It is the reason that single-agent rapamycin analogues have limited efficacy in cancer as a result of incomplete mTOR inhibition 39 . Although this dual inhibition of mTORC by SHK still needs more investigation, these data at least partly suggest SHK might directly target mTORC1/mTORC2. Long-term exposure of the tumor to certain drugs can lead to drug resistance. Multi-drug combination is a promising approach to overcome drug resistance 40,41 . The effect of SHK combined with DOX was evaluated and we found that low dose SHK potentiated anti-proliferation activity of DOX in BL cells. The molecular mechanisms of this synergistic effect may be involved with enhancement of apoptosis. Furthermore, our findings in vitro have been recapitulated in vivo in a subcutaneous BL model, indicating that the modulation of C-MYC and PI3K/AKT/mTOR is, at least, one of the molecular mechanisms by which SHK may manifest its effect against BL. In summary, our results indicate that cytotoxic effect mediated by SHK against BL attributes to caspase-dependent apoptosis. Inhibition of C-MYC and suppression of PI3K/AKT/mTOR activity play critical roles in SHK-induced apoptosis in BL both in vitro and in vivo. We also demonstrated that SHK has property to enhance chemotherapeutic sensitivity in BL cells. Altogether, these data suggest that SHK may be an encouraging chemotherapeutic agent in the clinical treatment of BL. MTT. Cell viability was monitored by 3-(4,5-dimethylthiazol-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) assay following the manufacturer's instructions. Briefly, cells (2 × 10 4 cells/well) were seeded in 96-well plates and treated with drugs for the indicated times. After incubation, 20 μl of MTT solution (5 mg/ ml) was added to each well and then the plates were incubated at 4 °C for 3 h. The absorbance of the reaction was measured by a 96-well plate reader (Bio-Rad, Hercules, CA, USA) at 570 nm. IC50 values (half maximal inhibitory concentration) were calculated. . Aliquots containing 30 μg proteins were separated on sodium dodecyl sulfate (SDS) -polyacrylamide gels containing 6-12% acrylamide gradients and then transferred to PVDF membranes (Millipore, Billerica, USA). The membranes were blocked for 2 h in Tris-buffered saline containing 0.1% Tween and 5% skim milk and then incubated with primary antibodies overnight at 4 °C. After washing three times, the membranes were incubated with anti-rabbit/mouse IgG antibody conjugated to horseradish peroxidase (Multi Sciences) for 2 h at room temperature. The results were visualized with the ECL detecting kit (Biological Industries, Cromwell, CT, USA). All primary antibodies were purchased from Cell Signaling Technology except C-MYC and p-mTOR (Ser2481) (Abcam, Cambridge, UK). Materials and Methods Hoechst 33342 and propidium iodide (PI) staining. Hoechst 33342 (Beyotime Biotechnologies, Shanghai, China) and PI (Multi Sciences) staining were used to analyze the nuclear morphology. Cells (2 × 10 5 cells) were treated with either DMSO (control) or shikonin at the indicated concentration for 6 h at 37 °C. After washed twice with PBS, the cells were incubated with Hoechst 33342 (1 mg/mL) and PI (5 mg/mL) at room temperature for 15 min and observed under fluorescence microscope (DFC450, Leica, Germany). Apoptotic cells were identified by morphologic changes in their nuclear assembly by observing chromatin condensation and fragment staining by Hoechst 33342 or both Hoechst 33342 and PI. Real-time PCR. Total RNA of BL cells were extracted using the TRIZOL Reagent (Invitrogen, CA, Carlsbad, USA) according to manufacturer's instructions. Reverse-transcribed complementary DNA was synthesized with the Prime-Script ® RT Reagent Kit (Takara, Tokyo, Japan). Amplification reactions were performed using SYBRP remix Ex Taq (Takara) in a 20 μL volume on a 96-well optical reaction plate in CFX96 Real-Time PCR Detection System (Bio-Rad). RT-PCR was performed with an initial denaturation at 95 °C for 5 min followed by 40 cycles including denaturing at 95 °C for 10 s, annealing at 60 °C for 20 s and extension at 70 °C for 10 min. Melting curves were computed after PCR product amplification. MiR-19a was normalized to U6. Comparative RT-PCR was conducted in triplicate, including no-template controls. The comparative cross-threshold (Ct) method was used to calculate relative expression. The primers for miR-19a and U6 were synthesized by RiboBio (Guangzhou, Guangdong, China). Combination index analysis. For evaluating the synergistic efficacies of shikonin and doxorubicin, the combination index (CI) isobologram method of Chou and Talalay was used. This commonly used analysis involves plotting concentration-effect curves for each agent and multiple diluted fixed-ratio combinations by using the median effect equation and the combination index equation. A value of CI less than, equal to or greater than 1 indicates synergism, additivity and antagonism, respectively. The synergy of Doxorubicin with shikonin was analyzed with the use of CalcuSyn software (Biosoft, Cambridge, UK). Establishment and treatment of Namalwa xenografts. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee. All experiments in vivo were performed in accordance with relevant guidelines and regulations. Male non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice (4 weeks old) were obtained from Shanghai Experimental Animal Center of the Chinese Academy of Sciences (Shanghai, China). For establishment of the xenograft, a mixture of 1 × 10 7 Namalwa cells and PBS was injected subcutaneously in the right oxter of each mouse. The tumor volumes were determined by measuring length (L) and width (W), and calculating volume (V = 0.5 × L × W 2 ) at the indicated time points. When each tumor volume reached about 100 mm 3 , the mice were randomly assigned to two groups (five mice per group): Vehicle, SHK (4 mg/kg). Mice were treated by intraperitoneal (i.p.) injection of 4 mg/kg SHK in fat emulsion every other day, or by i.p. injection of the same volume of the fat emulsion according to the same schedule. At the end of treatment, the mice were sacrificed, and the tumors were removed and weighed. Scientific RepoRtS \| (2018) 8:3317 \| DOI:10.1038/s41598-018-21570-z Immunohistochemistry. The extracted tumor tissues were fixed in 10% formalin at room temperature, processed and embedded in paraffin. Paraffin-embedded tissues were sectioned (5 mm thick). For histopathology analysis, the paraffin sections of tumors were stained with HE. Tissue sections were primarily stained with indicated antibodies for immunohistochemical analysis. Then biotinylated secondary antibodies detected the signal with DAB. Images were acquired using a Nikon E300 fluorescence microscope equipped with a Nikon digital camera (400x). Statistical analysis. All data are presented as the mean ± SD for at least three independent experiments. Comparisons between two groups were performed using Student's t-test and P < 0.05 was considered statistically significant. Data availability. Original data files are available upon a reasonable request.	2018-04-03T03:26:03.264Z	2018-02-20T00:00:00.000	{ "year": 2018, "sha1": "dba2d5dd96f582b57d042e7b6fe473a560ed33b5", "oa_license": "CCBY", "oa_url": "https://www.nature.com/articles/s41598-018-21570-z.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "dba2d5dd96f582b57d042e7b6fe473a560ed33b5", "s2fieldsofstudy": [ "Biology", "Medicine", "Chemistry" ], "extfieldsofstudy": [ "Chemistry", "Medicine" ] }
267066876	pes2o/s2orc	v3-fos-license	Serum IGFBP-1 as a promising diagnostic and prognostic biomarker for colorectal cancer Our previous study showed that levels of circulating insulin-like growth factor binding protein-1 (IGFBP-1) has potential diagnostic value for early-stage upper gastrointestinal cancers. This study aimed to assess whether serum IGFBP-1 is a potential diagnostic and prognostic biomarker for CRC patients. IGFBP-1 mRNA expression profile data of peripheral blood in colorectal cancer (CRC) patients were downloaded and analyzed from Gene Expression Omnibus database. We detected serum IGFBP-1 in 138 CRC patients and 190 normal controls using enzyme-linked immunosorbent assay. Blood IGFBP-1 mRNA levels were higher in CRC patients than those in normal controls (P = 0.027). In addition, serum IGFBP-1 protein levels in the CRC group were significantly higher than those in normal control group (P < 0.0001). Serum IGFBP-1 demonstrated better diagnostic accuracy for all CRC and early-stage CRC, respectively, when compared with carcinoembryonic antigen (CEA), carbohydrate antigen19-9 (CA 19-9) or the combination of CEA and CA19-9. Furthermore, Cox multivariate analysis revealed that serum IGFBP-1 was an independent prognostic factor for OS (HR = 2.043, P = 0.045). Our study demonstrated that serum IGFBP-1 might be a potential biomarker for the diagnosis and prognosis of CRC. In addition, the nomogram might be helpful to predict the prognosis of CRC. ELISA for IGFBP-1 The expression levels of serum IGFBP-1 were measured by a commercial ELISA kit (CUSABIO, Wuhan, China), and the procedure was carried out according to the manufacturer's recommendations in the ELISA kit.Briefly, 100 μl of serum sample at 20 fold-dilution and standard were added into antibody-coated 96-microwell plate, which were covered with the adhesive strip and then incubated for 2 h at 37 °C.Then, the liquid of each well was poured out, followed by the addition of 100 μl biotin-antibody (1X) in each well for 1 h at 37 °C.After removing the liquid and washing with wash buffer, 100ul horseradish peroxidase (HRP)-avidin (1X) was added to each well and incubated for 1 h at 37 °C.After washing the plate, TMB substrate was added into the microplates and incubated for 15-30 min in a dark environment for color development, and stop solution was used to stop the reaction.The optical density (OD) values were read at 450 and referenced to 570 nm wavelengths within 5 min after adding stop solution by microplate reader (Thermo Fisher Scientific, Boston, USA). The concentrations of serum IGFBP-1 were obtained by plotting a standard curve with a four-parameter logistic curve manner, and actual serum IGFBP-1 concentration must be multiplied by the dilution factor.Serum samples of patients and normal controls were measured simultaneously in the same batch.All measurements were done in duplicate. Measurement of tumor markers in clinical use The concentrations of serum CEA and CA19-9 were measured by an automatic electrochemical luminescence analyzer (Cobas e601, Roche, Germany).All tests of the two tumor markers were performed at the Department of Clinical Laboratory Medicine, the Cancer Hospital of Shantou University Medical College, and operated according to the instrument operating manual.In this study, the recommended clinical cutoff values of CEA, CA19-9 were 5.0 ng/mL and 27 U/mL, respectively. The level of serum IGFBP-1 in CRC patients and normal controls Subsequently, we performed ELISA to further evaluate serum levels of IGFBP-1 in CRC patients and normal controls.In total, 328 participants were recruited, 138 patients with CRC and 190 normal controls (Fig. 1).As shown in Supplementary Table S1, the mean age of 138 eligible patients was 58 years (range 26-82 years), of which 78 (56.5%) were males and 60 (43.5%) were females.The control group consisted of 147 (77.37%) males and 43 (22.63%)females aged between 40 and 80 years (mean, 56 years).The mean concentration of serum IGFBP-1 was 1569.455 ± 770.209 ng/mL, 1512.222± 818.971 ng/mL and 719.991 ± 379.340 ng/mL in CRC group (n = 138), early-stage CRC group (n = 68) and normal group (n = 190), respectively (Supplementary Table S2).To get a better view of its distribution and degree of dispersion, the levels of serum IGFBP-1 in three groups were shown in scatter plot (Fig. 2A) and box plot (Fig. 2B).The levels of serum IGFBP-1 in CRC group were higher when compared with normal group, which was confirmed statistically (P < 0.0001).In addition, the difference between early-stage CRC and normal controls is also significant (P < 0.0001).It can be observed that the distribution of CRC and normal controls is different in Fig. 2C.CRC group accounts for more histogram volume on higher concentration while normal group for more lower concentration.In a word, our data demonstrated that serum levels of IGFBP-1 significantly increase in CRC patients. The diagnostic value of serum IGFBP-1 in CRC and early-stage CRC To assess the diagnostic value of serum IGFBP-1 for CRC, we performed the ROC analysis to assess the ability of IGFBP-1 to distinguish CRC patients from normal controls.With an optimum diagnostic cutoff of 1258.387ng/ ml, ROC analysis displayed that serum IGFBP-1 achieved an AUC of 0.874 (95% CI 0.835-0.932)for distinguishing CRC from controls CRC (Fig. 3).The specificity and the sensitivity were 90.53% and 63.04%, respectively (Table 1).In addition, with the same cutoff value, IGFBP-1 could discriminate early-stage CRC from normal controls with a slightly lower AUC value of 0.812 (95% CI 0.795-0.908),a specificity of 90.53% and a sensitivity of 58.82% (Fig. 3 and Table 1).For better interpretation on clinical value of serum IGFBP-1, we also analyzed PPV, NPV, PLR and NLR, and the detail results were shown in Table 1.Interestingly, we evaluated the diagnostic performance of IGFBP-1 in CEA-negative, CA 19-9-negative CRC/early CRC.The results showed that IGFBP-1 also showed high diagnostic efficacy in CEA/CA19-9 negative CRC, and similar results were also obtained in CEA/CA19-9 negative early CRC, as shown in Table 2. Correlation between serum levels of IGFBP-1 and clinical data in CRC We evaluated the relationship between serum IGFBP-1 and clinicopathological characteristics of CRC patients by comparing serum IGFBP-1 positive rate in all 138 CRC patients.We defined that the positive level of serum IGFBP-1 in CRC patients was higher than 1258.387ng/mL.As shown in Supplementary Tables S3, there were no statistically significant associations between the positive rates of serum IGFBP-1 and depth of tumor invasion, Comparation of the diagnostic capacity of serum IGFBP-1 with CEA and CA19-9 We found that serum CEA and CA19-9 levels were detected in 126 CRC patients and 45 normal controls, according to the medical records and physical examination data, of which the diagnostic values were used to compare with serum IGFBP-1.The two/three-biomarker panel was established employing a logistical regression model with the predicted probability.As shown in Supplementary Fig. S2A,B and Table 3, the diagnostic efficiency of serum IGFBP-1 was significantly higher than those for CEA, CA19-9 or CEA+CA19-9 for both all-stage CRC and early-stage CRC.More importantly, when compared with IGFBP-1 alone, ROC analysis showed that the combination of IGFBP-1 and CEA, IGFBP-1 and CA19-9 or the three-biomarker panel (IGFBP-1+CEA+CA19-9) had a minute improvement of AUC to distinguish all stage CRC patients from controls (Supplementary Fig. S2A, Table 3).However, compared with detection of IGFBP-1 alone, IGFBP-1+CEA, IGFBP1+CA19-9 or the threebiomarker panel would reduce the diagnostic sensitivity (56.45% vs 40.32%, 48.40% or 43.55%, respectively, Supplementary Fig. S2B, Table 3). Prognostic value of serum IGFBP-1 in CRC We investigated whether serum IGFBP-1 could be used to predict prognosis of CRC.The 138 CRC patients recruited in this study were included for prognostic analysis.Using X-tile software, we set 1781.120 ng/mL as the optimal cut-off value to classify high and low expression of IGFBP-1.Univariate Cox analysis showed that age, gender, N stage, M stage, TNM stage and serum IGFBP-1 expression level were associated with prognosis of CRC.Multivariate Cox analysis further demonstrated that M stage (P = 0.010, HR = 3.811, 95%CI 1.373-10.580),TNM stage (P = 0.007, HR = 3.106, 95%CI 1.363-7.077)and serum IGFBP-1 (P = 0.045, HR = 2.043, 95%CI 1.014-4.115)were independent factors to predict the prognosis of CRC (Table 4).According to multivariate Cox proportional hazards regression analysis, the forest map was used to visualize the significant correlation between M stage, TNM stage, IGFBP-1 expression and OS of CRC patients (Supplementary Fig. S3).Furthermore, Kaplan-Meier revealed that the 5-year OS of CRC patients with high serum IGFBP-level was shorter than those with low serum IGFBP-1 level (16% vs 32%), and the difference was statistically significant (P = 0.019) (Fig. 4A). Nomogram for OS of CRC In order to better evaluate the prognosis of patients with CRC, the independent prognostic factors of M stage, TNM stage, and serum IGFBP-1 were used to establish a nomogram to forecast 1-, 3-and 5-years OS probability prediction (Fig. 5).From the nomogram, each prognostic factor was assigned a number of risk points, which www.nature.com/scientificreports/ was obtained by drawing a straight line directly upward to the "points" axis from the corresponding value of the prognostic factor.These points were added together to obtain "total points".The relationship between M stage, TNM stage, serum IGFBP-1 and OS can be visually shown in the nomogram (Fig. 5).The calibration plots were used to evaluate predictive capacity of the nomogram for 1-, 3-, and 5-year OS (Fig. 6).The results showed that the nomogram had a good prediction accuracy for OS.Akaike information criterion (AIC), bayesian information criterion (BIC) and C-index were used to evaluate the goodness-of-fit and discriminative ability of the nomogram.As shown in Table 5, the AIC and BIC of the nomogram were lower than those of TNM stage (291.994vs 305.737; 296.483 vs 307.234, respectively), suggesting that the nomogram had a higher goodness-of-fit for predicting OS.The C-index for the nomogram was 0.714 (95%CI 0.623-0.804),which was higher than that of TNM staging (0.651, 95%CI 0.575-0.727,P = 0.043).Time-dependent C-index analysis also showed that the nomogram showed a good prognostic accuracy for OS when compared with other single prognostic factors (Supplementary Fig. S4).Moreover, the DCA (Fig. 7) showed that the nomogram has higher overall net benefit than the traditional TNM stage systems alone in the majority of the range of threshold probabilities.Taken together, these results demonstrated that the nomogram had a better performance to predict OS of CRC patients when compared to the traditional TNM stage systems. Risk stratification based on the nomogram In order to determine whether the CRC patients could be effectively divided into two proposed risk groups based on the nomogram and OS, we calculated each patient's total point, and used the X-tile program to obtain the optimal cut-off value (1781.16ng/ml) to subdivide patients into low-and high-risk subgroups.Kaplan-Meier analysis and log-rank test showed that the high-risk group had shorter OS than those in the group of low-risk (39% vs 66%, P < 0.0001), and the median OS of CRC patients was less than 5 years (Fig. 4B).This stratification demonstrated that the nomogram could effectively divide those patients into the 2 risk subgroups with significant differences in OS. Discussion It is well known that endoscopy examination could help identify early-stage CRC 8,34 , but the invasiveness of colonoscopy limits its widely apply as a screening tool in a large number of asymptomatic populations 35 .Besides, the effectiveness of colonoscopy as a screening tool is closely related to the adequate detection and removal of colonic polyps, and the detection rate of colonoscopy is operator dependent in some extent 9 .In addition, some other noninvasive screening tests of CRC including stool blood tests, serum CEA, CA19-9 and blood septin9 gene tests were used to reduce the incidence and mortality of CRC 22,[36][37][38] .Nevertheless, because of the low sensitivity or high cost, none of these methods has been established as a recognized early screening tool 23,[39][40][41] .Therefore, it is urgent to find new methods with high sensitivity and specificity for the early diagnosis of CRC.In recent years, serum protein biomarkers are considered to be the most promising detection methods for population studies and routine clinical work 17,42,43 , because cancer phenotypic features seem most likely to be a direct reflection of changes in protein metabolism and function, which are also the targets of most anticancer drugs in clinical practice 44 , and these serum samples are noninvasive and easily accessible.Furthermore, a growing interest in the clinically potential use of circulating IGFBPs as diagnostic or prognostic biomarkers in cancers 45 . Our previous studies demonstrated that serum IGFBPs showed good diagnostic or prognostic performance in upper gastrointestinal cancer, including IGFBP-1 31,[46][47][48] .In this study, we found that blood IGFBP-1 mRNA is increased in GEO CRC patient dataset.In addition, in consideration of IGFBP-1 is a secreted protein, we carried out ELISA experiment to detect the levels of serum IGFBP-1 protein, and found that serum IGFBP-1 www.nature.com/scientificreports/expression was significantly increased in CRC, compared to normal controls (P < 0.0001).Meanwhile, serum IGFBP-1 showed a good diagnostic value in all stages of CRC with an AUC of 0.874, a specificity of 90.53% and a sensitivity of 63.04%.In terms of cancer screening, one of the most important attributes of a biomarker should be able to identify early cancers.For early-stage CRC, a certain diagnostic accuracy of serum IGFBP-1 could be observed (AUC 0.812, specificity 90.53% and sensitivity 58.82%).It is reported that CEA or CA-199 did not provide sufficient sensitivity and reliability for the early detection of CRC 21,23,49 .Similarly, our results also suggested that CEA and CA199 showed low diagnostic performance for early-stage CRC.Compared with CEA, CA19-9 or the twobiomarker combined panel (CEA+CA19-9), serum IGFBP-1 has higher sensitivity and AUC value to identify early-stage CRC (Table 3).Therefore, we believe that serum IGFBP-1 protein detection might be helpful for early diagnosis of CRC.Additionally, the combination of IGFBP-1 and CEA, IGFBP-1 and CA19-9, or the threebiomarker combined panel may further increase the diagnostic efficacy, especially with increased sensitivity, but there is no such effect for early-stage patients (Table 3). Although genetics plays an important role in risk stratification, the risk of developing CRC is also influenced by acquired factors, including age, race, male gender, and dietary habits 40 .Age is one of the risk factors that has been taken into account in most screening recommendations 50 .CRC death rates rose 1.2% per year in people younger than 50 years and 0.6% per year in those aged 50 to 54 years from 2005 to 2019 51 .Whereas a recent study showed similar prevalence at ages 45 to 49 years and 50 to 54 years, which provide clinically useful evidence for optimizing the age in CRC prevention and screening 52 .In our study, we found that the positive rate of serum IGFBP-1 was closely related to patient age. On one hand, IGFBP-1 is expressed in fetal liver and postpartum tissue, mainly in secretory endometrium, decidua gravidarum and liver.Pathologically, the expression of IGFBP-1 is elevated type 2 diabetes and some cancer patients 25 .Moreover, increasing studies indicate the expression and prognostic value of IGFBP-1 in serum/ tissue of patients with cancer remains equivocal and even controversial [53][54][55][56][57][58] .These results indicated that IGFBP-1 might have different expression pattern and prognostic value in different carcinomas, and the prognostic potential of serum IGFBP-1 in cancers should be further evaluated.So far, there is little literature involving the expression and the diagnostic or prognostic value of serum IGFBP-1 in CRC.Previously, most studies have focused on evaluating the relationship between circulating levels of IGFBP-1 and the risk of CRC, and the results seem to be contradictory.It is reported that higher plasma/serum IGFBP-1 levels were associated with a decreased risk of CRC 59,60 .Analogously, Vidal et al. revealed that lower concentrations of the plasma IGFBP-1 were associated with an increased risk of CRC, whereas higher levels of IGFBP-1 were related with a decreased risk of CRC in men only 61 .On the contrary, Wei et al. found that low blood IGFBP-1 expression was not obviously related with the increased risk of CRC 62 .Palmqvist et al. also reported similar results 63 .On the other hand, many in vitro and in vivo studies support the dual role of IGFBP-1 in tumor proliferation, migration, invasion, and adhesion through both IGF-dependent and IGF-independent molecular mechanisms, suggesting that the effects of IGFBP-1 are cell-specific and dependent on the type of target cells 25,30,56 .Moreover, as described in the literature, IGFBP-1 not only inhibited the invasion and migration of CRC cells SW480 and SW620, but also promoted CRC liver metastasis in mice transplanted with SW480 cells 64 .These findings suggest that IGFBP-1 may have a dual function, playing both positive and negative roles in the progression and metastasis of CRC.Liver metastasis (CLM) occurred only in mice injected with IGFBP-1 overexpressed SW480 cells, but CLM was not found in igfbp1 overexpressed SW620 cells, which may be related to the persistent expression of β-catenin, vimentin, and ZO-1 in IGFBP-1 overexpressed SW480 cells 64 .These results suggest that the role of IGFBP-1 in CRC is complex, but the specific mechanisms have not been clearly elucidated.Because there have been few studies on the role of IGFBP-1 in the development of colorectal cancer and the molecular mechanisms of action.Therefore, in future study, it is important to further explore its biological function in CRC.In our study, we identified that patients with higher serum IGFBP-1 expression had a worse OS.In addition, risk stratification demonstrated that the nomogram could effectively divide those patients into the high-and low-risk subgroups, and patients in the high-risk group had shorter OS.On the other hand, prognostic assessment of CRC patients remains a great clinical challenge.TNM stage system is the primary basis in estimating the prognosis of CRC 65 .However, TNM stage is based fully on the anatomical range of the disease, and may be affected by pathological assessment and tumor heterogeneity, which has limitations for survival analysis of CRC patients 66 .Here, we identified that serum IGFBP-1 was an independent prognostic factor.The nomogram based on serum IGFBP-1, M stage and TNM stage was established, of which the C-index was better than that of the TNM stage alone.A similar result was also observed in time-dependent C-index curve analysis.In addition, the decision curve analysis for 1-, 3-, and 5-year OS showed that the nomogram seemed to have higher overall benefit than TNM stage systems alone.These findings indicated that the nomogram seems to be more accurate in predicting OS than the traditional TNM stage system.In the study, age and lymph node metastasis were excluded after multivariate analysis, possibly due to sample size and clinical characteristics of patients.Therefore, it is necessary to verify whether age and lymph node metastasis are independent prognostic predictors with further large samples in the future. Several limitations of this study should be also taken into account: in our study, all patients were recruited from individuals with known cancers, and most were diagnosed based on the clinical symptoms of the disease.Nevertheless, the diagnostic sensitivity of IGFBP-1 may be different in individuals with asymptomatic disease or precancerous lesions, which needs to be further verified.Furthermore, these results were assessed in a singleinstitution study, which may lead to bias.Moreover, this study did not analyze the levels of serum IGFBP1 in patients of benign intestinal disease, which should be used as disease controls to assess the efficacy of serum IGFBP-1.Future studies need to address the role of IGFBP-1 in benign intestinal disease.The number of subjects in the control and CRC groups to compare the diagnostic value between IGFBP-1 and the classic clinical tumor markers (CEA and CA19-9) was not balanced, which may reduce statistical power.Whether serum IGFBP-1 had improved performance in the early diagnosis of CRC still needs further validation.Finally, there is a selection bias in the male-to-female ratio within the CRC group and control group.Future studies also need to verify whether the male-to-female ratio difference may influence the results.Therefore, in future study, a study with multicenter and large-scale samples should be completed to further verify the diagnostic and prognostic value of IGFBP-1 in CRC, and need to further evaluate the role of IGFBP1 in benign bowel disease. In conclusion, our findings offer a convenient and non-invasive for early diagnosis and prediction of outcomes for patients with CRC, as the blood-based IGFBP-1 are easy to obtain from routine admission laboratory tests.Meanwhile, we believe that serum IGFBP-1 detection is not meant to replace endoscopy or traditional prognostic assessment strategy, but contributes to identifying patients who may have CRC at an early stage.Furthermore, our study demonstrated that serum IGFBP-1 is an independent prognostic risk factor for CRC patients, and the construction of nomogram model containing serum IGFBP-1 might improve the accuracy of prognosis prediction. Figure 3 . Figure 3. ROC curve analysis in the diagnosis of CRC and early-stage CRC.(A) ROC curve for IGFBP-1 in all stage CRC versus normal control.(B) ROC curve for IGFBP-1 in early stage of CRC versus normal control.(C) Two groups versus normal controls group are in different colors.The area under the red line is 0.5, for reference. Figure 4 .Figure 5 . Figure 4. Kaplan-Meier curves for OS of patients with CRC.(A) Survival curve for serum IGFBP-1 with CRC patients, Log-rank test was used to evaluate the significant difference.(B) Survival curve of risk stratification for OS according to prediction of nomogram.The low risk: Total points ≤ 228.75 for OS, the high risk: Total points > 228.75 for OS.Log-rank test was applied to assess the significant difference.The number of people alive at each time point in the high and low IGFBP-1 groups was showed in "number at risk." Figure 6 . Figure 6.The calibration plots (A-C) are used to estimate predictive capacity of the nomogram for 1-, 3-, and 5-year OS probability.X-axis was the probability of 1-, 3-or 5-year OS predicted by nomogram.Y-axis was the actual OS of the patients included in the study. Figure 7 . Figure 7. Decision curve analysis the predictive accuracy of M stage, TNM stage, IGFBP-1 and nomogram in CRC patients.The decision curve of 1-(A), 3-(B), and 5-(C) year OS.The y-axis represents the net benefit, which is calculated by summing the benefits (true positive results) and subtracting the harms (false positive results).The horizontal line represents the assumption that no deaths happen. Table 1 . Evaluation of the detection value of IGFBP-1 in the diagnosis of CRC. Table 2 . Evaluation of the detection value of IGFBP-1 in the diagnosis of CEA/CA19-9-negative early CRC and CRC. Table 3 . Diagnostic performance of different biomarkers in 126 CRC patients and 45 normal controls. Table 4 . Cox analysis of OS for CRC. Table 5 . The C-index of IGFBP-1, M stage, TNM stage and nomogram for prediction of OS in CRC.AIC Akaike information criterion, BIC Bayesian information criterion.	2024-01-23T06:17:27.658Z	2024-01-22T00:00:00.000	{ "year": 2024, "sha1": "a6dcb853e7a5970d29ba15b6dff06b614ac3d28f", "oa_license": "CCBY", "oa_url": null, "oa_status": null, "pdf_src": "PubMedCentral", "pdf_hash": "323ed99f20f87a36dce2e047adc5684f9682378c", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
235972547	pes2o/s2orc	v3-fos-license	Effects of Black Cumin (Nigella sativa L.) on patients with cancer and tumor: A systematic Review Experimental studies demonstrated a positive effect of administration of Nigella sativa L. (Black cumin) and its active chemical components on cancer and tumor through the antioxidant and anticancer activity. This study aimed to determine the beneficial effect of the use of black cumin in patients with cancer and tumor. This systematic review includes 4 randomized controlled trials that investigated the effect of the administration of black cumin in human cancer. Articles were searched in PubMed, Cochrane library, Semantic scholar and directory of open access journal (DOAJ), open grey and grey literature report databases for studies from 1983 to 2020 before May using the following keywords, Nigella sativa, black cumin, cancer, tumor, etc. The results examined that N. sativa is much effective in treating breast cancer, gastrointestinal cancer, brain tumor, and acute myeloid leukemia. According to the limited evidence from the study, black cumin may have favorable effects on cancer and tumor. However, more research is needed on different types of cancer to confirm and establish the above findings. Introduction Nigella sativa L. or black cumin has become one of those popular medicinal plants that have been used for centuries for health benefits. Nigella sativa L. belongs to the Ranunculaceae family and is an annual herb with 8-12-inch-high pinnate, stratified leaves [1]. New products are also advocated from natural sources, as it is estimated that more than 300,000 herbal species exist globally among them only 15% have been explored for their pharmacological potential [2]. N. sativa, along with many medicinal plants (Ranunculaceae) has been considered as one of the most appreciated nutrient-rich herbs in the world's history, and numerous scientific studies are under its way to justify the typically claimed use of this species' Received: November, 24, 2020 Accepted: December, 04, 2020 small seed [3,4]. N. sativa is an herbaceous plant with wonderful therapeutic effects, such as antihypertensive, gastro-protective, nephroprotective, antioxidant, antimicrobial, Geno protective, neuroprotective, immunomodulatory, anti-inflammatory, hypoglycemic, hypolipidemic, anti-carcinogenic and hepato-protective [5,6]. N. sativa seeds with some active components such as essential oil, thymoquinone, p-cymene, and thymol are very rich in the fixed oil, essential fatty acids, alkaloids, phytosterols, glycolipids and phospholipids, saponins, and essential oil components. Thymoquinone (TQ) is a cytotoxic agent in many human tumor cell lines that seem to be immune to particular multi drugs [7]. There is evidence that most of N. sativa's therapeutic effects are due to the influence of TQ which has been the sativa's greatest bioactive component. Crude and TQ against noncommunicable diseases (cardiovascular disease, obesity, hypertension, diabetes, cancer, etc.) are beneficial from their seeds and oil, and communicable diseases (malaria, AIDS, hepatitis C virus, fungal, viral, parasitic infections, etc.) [8]. TQ had already observed pleiotropic anti-cancer effects, including chemo potentiation, anti-inflammation, immunomodulation, as well as radiosensitization [9]. In addition to inducing apoptosis across several different cancers, TQ has been shown to inhibit numerous tumorigenic signaling nodes, such as those involved in cell proliferation, epithelial to mesenchymal shift, invasive cell migration, and metastasis [10]. In this study, it was aimed to show the positive effects of N. sativa against human cancer. Literature search The reporting of this review follows the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. We searched PubMed, Cochrane library, Semantic scholar, and directory of open access journal (DOAJ) databases for studies from 1983 to 2020 before May. For gray articles, we searched open grey (www.opengrey.eu) and grey literature reports (www.greylit.org). The keywords used for searching (Nigella sativa OR Black cumin OR Black seed OR Kalonji) AND (Cancer OR Tumor OR Malignancy) to identify intervention studies investigating the effect of black cumin on cancer. Study selection and data extraction In this systematic review, we included published intervention studies (these include randomized controlled trials, clinical trials, and placebo-controlled trials), reporting the effect of black cumin on cancer and published in English). Trials were excluded if they did not meet the criteria above and were involved in animal studies or in vitro studies, did not use the interests of intervention or outcome. Two independent reviewers (MAH and AIC) screened the titles and abstracts of the initially identified studies to determine whether they would satisfy the selection criteria. Any disagreements about selection were resolved through consensus or consultation with a third author (TR). The data collection form included questions on the year of publication, geographic origin, types of intervention, doses, patients' type and characteristics (sample size, age, etc.). Figure 1 summarizes the study selection process. Quality assessment The quality of each study was assessed by following the Cochrane format [11] reported in Table 1. The quality assessed for each study are (1) quality of randomization, (2) quality of blinding, and (3) quality of the description of withdrawals and dropouts. Results We selected four human studies ( Table 2) that described the effect of N. sativa on human cancer [12][13][14][15]. In three days, Behnamfar et al. analyzed the incidence and magnitude of phlebitis, applying N. Sativa by a catheter upon the peripheral vein. In their research, they reported that the prevalence of phlebitis in the control group was optimum and that the occurrence of phlebitis in the intervention group was lowest during the 72 hours of the test, assuming that N. sativa oil seemed to have a massive effect on the intervention group [12]. Hussain et al. demonstrated the effect of applying N. sativa oil on acute myeloid leukemia. The primary result of this research concluded that at the end of 28 days, the severity of Literature review (11) Full-text articles assessed for eligibility (n = 16) Full-text articles excluded, with reasons (n = 12) Not reported the effect of black cumin on cancer (9) Not use randomized controlled trial (3) Studies included in qualitative synthesis (n = 4) oral mucositis was significantly lower in the N. sativa oil group compared with the control group. Indeed, the incidence of erythematic and ulceration among the N. sativa oil community was lower. At the end of 12 days, pain severity among the N. sativa oil group dropped considerably compared with the control group. The secondary outcome of this study mentioned that salivary TNF-a was substantially lowered by the use of N. sativa oil mouth rinse on day 18 and day 28 compared to the control group but salivary IL-6 was not considerably different in the N. sativa oil control samples compared to the corresponding group (p>0.05) [13]. Mousa et al. in their study found that maximum children took N. sativa for 3-9 months (average of 6 months) and loss of body weight was less severe in the intervention group than the control group (p=0.048). The intervention group of children experienced less febrile neutropenia (2.2%) than the control group (19.3%). The percentage of febrile neutropenia among medulloblastoma (MB) and children with primitive neuroectodermal tumors were higher among the control group than the intervention group. In the case of hematological toxic effects, there have been statistically significant disparities in the occurrence of neutropenia (p=0.001) and febrile neutropenia (p=0.001) between both the intervention group and the control group, but the incidence of anemia, thrombocytopenia, and leucopenia was not significantly different in both groups [14]. Rafati et al. established how acute radiation dermatitis might have been eliminated in breast cancer by applying N. sativa gel. The incidence of skin toxicity in the N. sativa gel group was significantly lower at weeks 3, 4, and 5 compared to the control group even though there were significant differences between the two groups in the size and severity of moist desquamation. The worst perception of pain among the control group was substantially higher than that of the N. sativa gel group (p<0.05). There was no dramatic difference in the skin-related quality of life between the two classes [15]. Discussion The present review showed the effects of black cumin on different types of cancer. Anticancer activity of N. sativa was described in a study where certain TQ (a chemical component of black cumin) had been used to manipulate phosphatase and tensin homolog (PTEN) expression and induce apoptosis in human breast cancer cells. In this study, it was found that TQ significantly inhibits the proliferation of DOX cells. It, therefore, triggered apoptosis and protein p53, and even some up-regulated PTEN by inhibiting the Akt pathway [16]. Another mouse study showed that TQ induced cytotoxicity and apoptosis, and also inhibited tumor growth [17]. TQ induces apoptosis and disrupts mitochondrial membrane potentials. It also activates the caspases 3, 8, and 9 in HL-60 cells to treat myeloblastic leukemia, thus showing its effect on myeloblastic leukemia [18]. TQ indicates significant cytotoxicity for cancer of such bladder. It prevents the rapid multiplication of cancer cells, and by activating caspases induces apoptosis. TQ's anticancer effect resulted in endoplasmic reticulum stress pathway activation and mitochondrial dysfunctions. It also boosted the Bcl-2 anti-apoptotic protein and blocked cytochrome C entrance [19]. TQ inhibited renal cell cancer (RCC) via cell migration, invasion, and epithelial-mesenchymal shift. This also increased the expression of E-cadherin and the level of hepatic kinase B1 phosphorylation was considerably upregulated following thymoquinone therapy [20,21]. TQ protects against the development of prostate cancer. It was found to inhibit the growth of DU145 and PC3 cells which caused prostate cancer by treating with TQ. TQ dramatically inhibits the DU145 and PC3 cell migration (p<0.05). The expression of epithelialmesenchymal transition (EMT) markers in prostate cancer cells have also been repressed, and TGF-ß, Smad2, and Smad3 expression have also been minimized with TQ [22]. The article has some strengths and limitations. Using proper guidelines of the systematic review and quality assessment of the selected studies are the strengths of the articles. Some limitations of the article are only used article published in English and had fulltext availability. N. sativa component potential anticancer activities have been identified thousands of years ago but appropriate scientific research with this essential traditional medicine is a very recent story. According to the limited evidence from the study, black cumin may have favorable effects on human cancer and tumor. It is worth emphasizing further research work behind this because it is a safe and effective anticancer agent. In particular, researchers should evaluate the active compounds more broadly, as very few authentic reports are available on the chemical composition of seeds or N. sativa exists. Also, more importance should be placed to investigate the appropriate molecular pathways of TQ and other components on different cancers as the current understandings are largely unclear. Regarding the potential benefits of N. sativa on cancer, the anti-carcinogenic effects of N. sativa remained assessed through restricted clinical trials lacking a placebo community. For ongoing studies, it was recommended that further double-blind, placebocontrolled randomized clinical trials test the impact of N. sativa on cancer diagnosis and prevention. Author Contributions MAH: Designed the study and final manuscript preparation; AIC: Conceived, and final manuscript preparation; MA: Designed the final data, tables, and graphs, conducted the experiment; TR: Conceived and final approval of the manuscript. Conflict of Interest The author declares no competing interest. Ethical declarations The study was approved by the ethics board of Noakhali Science and Technology University. Financial Support None.	2021-07-18T00:12:09.223Z	2020-01-01T00:00:00.000	{ "year": 2020, "sha1": "f083134da4d47f6e13792d5d991edfb183577bfe", "oa_license": "CCBYNC", "oa_url": "https://jcbior.com/index.php/JCBioR/article/download/51/PDF", "oa_status": "HYBRID", "pdf_src": "Anansi", "pdf_hash": "9719e21c1fe9fb50fdc3365368e36b183d13c6be", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
258187421	pes2o/s2orc	v3-fos-license	Assessing Video Game Balance using Autonomous Agents As the complexity and scope of games increase, game testing, also called playtesting, becomes an essential activity to ensure the quality of video games. Yet, the manual, ad-hoc nature of game testing leaves space for automation. In this paper, we research, design, and implement an approach to supplement game testing to balance video games with autonomous agents. We evaluate our approach with two platform games. We bring a systematic way to assess if a game is balanced by (1) comparing the difficulty levels between game versions and issues with the game design, and (2) the game demands for skill or luck. I. INTRODUCTION Game development is an iterative process [1]. Game developers start with a core mechanic, limited in scope, and then iterate, adding new features until they deem the game complete. For every new change in the game, game testers (playtesters) interact with the game and provide feedback to the game developers who then change the game. They use experimentation and trial-and-error to tweak the game mechanics and make it engaging for the players. Keeping the game challenging while avoiding boredom requires balancing it [2], which is hard to translate to the actual game specification and relies on empirical knowledge. This constant experimentation implies there are no clear requirements but a "vision" for games. In this paper, we propose an approach to assess video game balance (semi) automatically. Instead of manually testing games, we propose an automated approach with autonomous agents to aid game developers assess the game's balance. Schell [2] lists 12 common types of balance in video games. In this paper, we focus on two balance types: Challenge vs. Success about keeping the player engaged considering the game difficulty and the player's skills and Skill vs. Chance about needing luck instead of skills to succeed. Games of skills are more like athletic contests (which player is the best?) while games of chance are more random. For example, dealing out a hand of cards is a pure chance but choosing how to play them is pure skill. Similar to unit tests in continuous integration pipelines in traditional software development, with which a system warns developers when a test fails, we want to provide developers with an automated process that would warn them when a new version of their game is too far from a given balance. Our approach brings a systematic and autonomous way to identify if the game is balanced by (1) checking the difficulty spikes and (2) if the game demands more skill or luck. To do so, we train autonomous agents using Deep Reinforcement Learning (DRL). Training agents to play games with DRL is an ever-increasing research area [3] and machine learning models allow autonomous agents to master games. Video games offer an environment with reduced scope (compared to real life) that suits the training of autonomous agents. These approaches show great success in mastering simple and complex games [4]. In our approach, we incorporate the agents into the game development process and provide a solution for testing the game balance. Most game studios, especially the small ones, do not have the time or the budget to adopt complex and costly solutions. Yet, they can benefit from a more feasible approach. In the following, we describe our approach, how we implemented it, and how we validated our testing approach with two platform games. Section II discusses the related works and how our approach differs from them. Section III presents the approach. Section IV shows how we technically implemented the approach. Section V and Section VI are the case studies that use our approach. Section VII presents the discussion and Section VIII the Threats to Validity. Finally, Section IX shows the conclusion and future works. II. RELATED WORK We read 199 papers about video game testing 1 but, for the sake of space, here we will show the ones more close to the our testing objective: video game balancing. In the following we summarize, to the best of our knowledge, all the papers that relate to video game balancing. Isaksen, Gopstein, Togelius, et al. [5] used A-Star, MCTS and visualization to determine the game difficulty by verifing different attributes of the same game without changing its rules. The authors use metrics to predict the score of a platformer called Flappy bird. Gudmundsson, Eisen, Poromaa, et al. [6] tested the difficulty of a game level using autonomous agents, powered by a CNN, to simulate gameplay and the "success rate" against human players. They validated using a Match-3 game. They wanted to predict the difficulty of a new game level automatically. They claimed that the difficulty of new levels could be tested automatically. Roohi, Relas, Takatalo, et al. [7] predicted the pass rate (win the game) and churn (abandon the game) in new levels of a Match-3 game. They used gameplay data from autonomous agents and playtesters. The agents were trained using PPO within the Unity-ML engine. They claimed that the pass rate and churn of new levels could be tested automatically. DeLaurentis, Panchal, Raz, et al. [8] defined a framework that predicted the game balance using data from autonomous agents, trained with CNN and MCTS, to play against each other in a RTS multiplayer game. They assessed the playsession with a visualization tool together with the actions performed by the agents. Pfau, Liapis, Volkmar, et al. [9] and Pfau, Liapis, Yannakakis, et al. [10] used Deep Player Behavior Modeling (DPBM) and data from real players to model autonomous agents and play a MMO game. The idea was to replicate the human behaviour and then assess the playsession manually. de Mesentier Silva, Lee, Togelius, et al. [11] and Mesentier Silva, Lee, Togelius, et al. [12] created agents using A-Star and MCTS to explore a board game called Ticket to Ride. The focus was more exploring than dealing with the game balance. Liu, Chaoran, Yue, et al. [13] used procedural content generation to create new tower defense game levels to then playtest it autonomously using flat MCTS. Morosan and Poli [14], [15] used evolutionary algorithm to balance different games, like Pacman and StarCraft, using the win-rate as oracle. Preuss, Pfeiffer, Volz, et al. [16] used the feature of the opensource game OpenRA, an RTS, to balance the strategies the player can adopt. III. APPROACH We describe, now, our approach. Figure 1 shows the process of the feedback loop of manual game testing and how our approach automates part of it. There are three roles involved in this process: The Game Developer (GD), which refers to Game Designers and Programmers. It is the game developer who describes and implements how things should behave in 1 https://doi.org/10.5281/zenodo.7768876 the game. The Game Tester (GT) (Gameplay Tester, Playtesters or QA) find bugs and any other abnormality in the game. Game testers should test game quality by verifying gameplay, logical consistency, observability, progressive thinking, reasoning ability, and exhaustively testing features, game strategy, and functionality [17]. Therefore, game testers should understand the principles and the characteristics behind games and especially understand the game development context [18]. The Testing Agent (TA) is the autonomous agent that interacts with the game and reports the findings according to the test objectives. A. [GD] Modifies and Generates the Game The modifications in the game vary according to the game tester's feedback. For example, the game developer can simply tweak a parameter, like character speed, or introduce new gameplay mechanics, like the ability to jump. The bigger the change, the bigger the impact on the player's experience. B. [GD] Sets the Test Objectives The test objective depends on the modification made to the game. It is defined by the game developer and varies in scope. For example: find bugs, explore the levels, check the character collision, verify if the level can be completed, among many others. As video games have a large scope , developers isolate parts of the game to test. This strategy is similar to what Rare does with the game Sea of Thieves [19] where they use single scripted actions to verify the object's behaviour, like opening a door, for example. C. [GD] Trains the Agents Playing the game is a sequential decision-making process, where the players continuously make decisions and take actions based on received observations. Therefore, this problem can be modelled as a Markov Decision Process (MDP) [20]. The Agent (Player) interacts with the Game System. The Game State is the observation (representation) of the Game System. What the Agent can "see" of the game. The Game Action is a set of possible decisions (move, attack, jump, etc). Finally, Agent's Reward is the feedback used to measure the success or failure of the agent's actions in achieving some goal (winning, surviving, etc). The autonomous agent (or model) is the output of the training process. It is the autonomous agent who interacts with the game. D. [GT & TA] Interacts with the Game Testing a video game means playing it [19]. For every new change in the game, game testers interact with the game and provide feedback to the game developers who then change the game. This trial-and-error process relies on the empirical knowledge of the team and could be faster, more scalable, and more efficient. Indeed, as developers perform multiple changes or permutations thereof, keeping track of what works best for the game quickly becomes overwhelming. Figure 1. The testing process (in BPMN notation). The activities and artifacts show a typical manual process of game testing and how our approach complements it. E. [GT & TA] Reports the Feedback While the autonomous agent interacts with the game, we collect the metrics related to the testing objective. Game testers and testing agents have different testing objectives. For example, humans can assess subjective details of the game, like engagement heuristics, while autonomous agents can handle trivial details that are a burden for the human tester, like repetitive checks in the game versions. F. [GD] Assess the Feedback The game developer uses the feedback report to decide if the game is good enough to be released. Otherwise, they restart the process by making new modifications to the game. After receiving the feedback containing the data about the agent's performance, they can compare it with the previous versions of the game. IV. IMPLEMENTATION We now describe how we implemented our approach. One of the issues was how to separate the concerns between the game code, the libraries and frameworks (game engine), the DRL training code, and the testing detail (number of runs, logs, etc). Figure 2 shows the UML-like diagram of our architecture. We separated each class and made them responsible for only one job. We started with the first element, the game logic which is the game source code and its related assets. The game uses "Pygame" as framework (game engine). Then, we used two other libraries so we can use DLR methods to create the autonomous agents: Gym and StableBaselines. The training logic is the element that handles the training parameters like the reward function. To link the game and training logic, we used some instrumentation code based on the design patterns Observer and Command. Finally, we use another element to manage the testing code. A. [GD] Modifies and Generates the Game We simulate the development process by considering different versions of a game. The changes in version#1 are carried to the further versions. To have different versions of a game, we modify the game according to its game mechanics. We modify the game difficulty using the Doubling and Halving balancing methodology [2]. It says the developer should change the game attributes by high amounts: doubling or halving them. The objective of this method is to change something so that you can actually feel the difference right away. B. [GD] Sets the Test Objectives We focused our testing feedback on two game balance types: Challenge vs. Success is about keeping the player engaged considering the game difficulty and the player's skills; Skill vs. Chance is about games that depend, more or less, on luck instead of the players' skills to succeed. To better assess the game we use game testers (humans and agents) with different skill levels: novice and professional. Also, we added one random agent that performs actions without any reasoning. C. [GD] Trains the Agents Our approach uses DRL to train the models and create the Autonomous Agents. We use the game system to define (a) how to represent the game state and (b) what will be the Agent's Reward. These two details are specific to the game being tested and can be done in many ways. To train the agents, we use a Python library called Gym. It provides a number of already defined environments, which are games with defined action space and reward functions. This library is usually used to assess new machine-learning models. Using Gym, we defined a custom environment that includes an action space with the commands that are possible within the game, and a reward function. We also used Stable Baselines library, providing a selection of machine learning models, like PPO and A2C, to train the agents to play the game. We divided the skill level of the agents by training time, that is, the novice is trained with 100K steps while the professional with 1M steps. The machine used to run the training has a CPU Core i7 2.6 GHz, A GPU NVIDIA GeForce RTX3070, 32 GB DDR4 or RAM, and an SSD hard drive. As for the software, we use the Python language with the Stable Baseline library running on Windows with NVIDIA CUDA. To train the autonomous agents we used Training Parameters (Table I). For our experiments we chose to use the modelfree DRL models because (1) in our game environment, we cannot predict state transitions and rewards, and (2) the training cost (computation time) is lower. Thus, we chose two different models to train the agents: PPO and A2C. The action space, reward function, and observation space vary from game to game. Depending on the modification of the game, it is necessary to re-train the models (agents) for each new version. For example, when a new game mechanics heavily modifies the gameplay. We used human players to validate the performance of the agents. The agent must have a similar performance compared to the human players. Also, the performance across different versions of the game should follow the same pattern/trend. D. [TA] Interacts with the Game We defined a play session of 180 seconds where the autonomous agent plays the game two times. We use the median of the two runs to calculate each performance. Table II shows the Testing Parameters used in the game test scenarios. The player plays the game for N seconds, M times. Either a human or an agent plays the game. Each one is assigned a skill level, with the exception of the random agent. The human professional is someone with gaming experience while the novice is not a regular game player. As we are tackling the issue of balancing the game, we created metrics related to the score of the agent playing the game. These metrics are variables that work as a proxy for the agent's performance and, therefore, the game balance. They vary for each game but, in general, are related to the score of the game. F. [GD] Assess the Feedback We compare the balance metrics across the different versions of the game: • To balance the Challenge vs. Success we check spikes on the balance metrics when the agent plays in each game version. Also, we compare the performance of novice and professional skill levels. • To balance the Skill vs. Chance we compare the performance of the random agent with the other trained agents. If the random agent performs better, the game has a balance problem. V. CASE STUDY A. BATKILL For Case Study A, we modified a 2D action platformer open-source game called Batkill 2 . We did not touch the rules of the game. It consists of a single screen, where the character tries to stay alive while bats fly toward him. The goal is to kill as many bats as possible without being hit. For each bat killed, the player gets one point (+1 score). The player loses one life for each hit (-1 life). The bats spawn faster as the players kill them. The bats spawn in random locations. The actions are LEFT, RIGHT, ATTACK, JUMP. A. [GD] Modifies and Generates the Game We created five different versions of the game Batkill (Table III) by changing a set of variables in the game: bats is the number of enemies on screen; bat_speed is the enemies' movement speed; attack_cooldown is the time between the character's attacks; jump if the character can jump or not. We expect an increase in the difficulty on versions #2, #3, and #4. On version #5, with the addition of the jump mechanic, the difficulty should decrease as the player has one more resource to avoid enemies. B. [GD] Sets the Test Objectives To assess the balance of the game Batkill, we measure two variables. bats_killed, which is the number of enemies killed by the character; and hits_taken, which is the number of times an enemy hits the character. To have a balance between kills and hits, the balance metric (score) for the game Batkill is given by: score = bats_killed − hits_taken. C. [GD] Trains the Agents To train the agents to play the game Batkill, we started with giving rewards when an enemy is killed (BAT_KILLED +5) or a hit was taken (HIT_TAKEN -5). After that, we used a small negative reward (ATTACK -0.1) to avoid making the agent use the attack for no reason. After seeing humans playing the game, we verified that they did not jump often. That is why we penalized the agent if he jumped (JUMP -0.2). The idea is to keep the character on the ground for more time. We also gave a small reward when the player moved toward the enemy (MOVING_TOWARDS +0.1). Finally, we gave a small reward if the agent faced the nearest enemy (FACING_NEAREST_BAT +0.2). That is a movement humans do naturally, and we try to hint here. The PPO model had better performance (bigger reward) in all versions when the steps were more than 100K. For the PPO model, training a "novice" agent took around six minutes, while the "professional" took around one hour. The A2C model took about 10% more time to do the same training process. D. [TA] Interacts with the Game The trained agent interacts (plays) with the game autonomously. The behaviour of the agent resembles a human playing the game. However, the agent performs movements that are uncanny for humans. That is, a human player would never play like that. For example, even after putting a penalty for it in the reward functions, the agent used jump and attack constantly whether the enemy was near or not. Nevertheless, there were moments the agent stayed still, like a human player. E. [TA] Reports the Feedback The Table IV shows the results of the feedback report for the PPO and A2C agents, as well as the Random agent and the Human player. The score of each agent is also separated by skill level. The negative values mean that the agent/player got hit more times than kills. Figure 3 shows the results of the humans and agents playing the game. The score is the average between novice and professional players/agents. Version#1 is the easiest because the agents and human players had the best score among all other versions. Version#4 is the hardest, followed by versions #3, #5, and #2. F. [GD] Assess the Feedback In all game versions, the human outperforms any other autonomous agent, except on version#3. The PPO agent is the one that plays similarly to the human player, as the score across the game versions follows a similar pattern. In general, the A2C agent results are the lowest across all versions, except in the first version. 1) Challenge vs. Success: We can identify three big spikes in difficulty. The first two spikes made the game harder while the last one made the game easier: • Difficulty Spike: on game version#2, when we increase the number of enemies, the game got harder. • Difficulty Spike: on version#3, when we increase the speed of the enemies, the game got harder. • Difficulty Spike: on version#5, when we added the "jump" mechanic, the game got easier. 2) Skill vs. Chance: We identified two game versions where luck was more important than the player's skill. In those cases the random agent had similar (or better) performance to the other agents and human players: • Chance: on version#3, when we increase the number of enemies, the game depends more on luck than skill. • Chance: on version#4, when we decreased the time between the character attacks, the game depends more on luck than skill. Summary -Case Study A. Batkill • The PPO agent is the one that follows a similar pattern as the human players. • Across the game versions, we found two spikes of difficulty that made the game harder and one spike that made it easier. • The performance of the random agent on versions #3 and #4, compared to the other agents, shows that these two versions do not favour the player's skill. However, the introduction of jumping on version#5 helped making the game more skillbased. VI. CASE STUDY B. JUNGLE CLIMB We modified an open-source, 2D "infinite-runner" platform game 3 called Jungle Climb 4 . It consists of a single screen, where several platforms are drawn. Each platform has gaps randomly generated. The player character is initially placed below the platforms and has to climb them by jumping between gaps. After passing through the second platform, the screen will start scrolling upward. The objective of the player is to keep the character below the bottom line of the screen as long as possible, not letting the player be "scrolled down" for too long with the screen. For each step, the player is able to survive while above the second platform, it gets one point (+1 point). The scrolling speed of the platform increases as time passes. The actions are LEFT, RIGHT and JUMP. A. [GD] Modifies and Generates the Game We created three different versions of the game Jungle Climb (Table V) by changing two variables in the game: shift_speed is the rate the screen scrolls up; and max_gaps, which is the maximum number of gaps in each platform. We expect version#2 to increase the difficulty of the game and version#3 to make the game easier. B. [GD] Sets the Test Objectives To assess the balance of the game Jungle Climb, we measure two variables: max_points, which is the maximum number of points that shows how long the character was alive; and max_correct_jumps, which is the maximum of correct jumps, that is, when the jump connects to the next platform. The score is given by the rate between points and correct jumps: score = max_points + (max_correct_jumps * 100). C. [GD] Trains the Agents To train the game Jungle Climb we use the following set of rewards. At the beginning of every step, we compute the Below Threshold Reward (BTR). If the character has not passed the threshold of the screen where it starts to shift, the score will always be zero and the BTR will be greater than zero. We use this value so we can give the agent different motivations so it can exit this initial state more quickly: BTR = time_elapsed * 5 if score == 0 else 0. D. [TA] Interacts with the Game The trained model (agent) interacts (plays) with the game autonomously. However, aside from our efforts to train the model, the behaviour of the agent does not resemble a human player. The agent jumps continuously and, most of the time, without purpose. E. [TA] Reports the Feedback The Table VI shows the results of the feedback report for the PPO and A2C agents, as well as the Random agent and the Human player. The score is separated by skill level. Figure 4 shows the results (score) of all the agents playing the game versions. Version #1 is the easiest to play. The difficulty across the versions increases on version#2 and maintains on version#3. Even by adding one more gap to jump into each platform. The curve for the PPO agent and Human players are very similar, which shows that this agent is a good proxy to mimic human players. The PPO agent outperforms, in all three versions, all the other agents. On the other hand, the A2C agent performs poorly, even when compared to the random agent. 1) Challenge vs. Success: We could identify one big spike in difficulty: • Difficulty Spike: on game version#2, when we increase the speed the screen shifts, the game got harder. 2) Skill vs. Chance: The random agent has a similar pattern to the human player but with a very low score across all game versions. This indicates that the game does reward the player's skill in all versions. Therefore: • Skill: on versions #1, #2, and #3, the game depends more on skill than luck. Summary -Case Study B. Jungle Climb • The performance pattern of the PPO agent is similar to the human players. • There is a spike in difficulty on version#2 that is not balanced on version#3 by adding more gaps to jump. • The random agent does not outperform the human or the PPO, showing that the game is skill-based. A. Balancing Challenge vs. Success To deliver a good experience to the players, keeping them away from boredom or anxiety, game development requires trial and error and much experimentation. Our results show that it is possible to systematically automate part of the balance testing process. In our case studies, we showed that the agents could mimic the player's achievements and struggles, even without re-training the models. Thus we showed that it is possible to rely on autonomous agents to proxy the human's skill levels. These agents identified the spikes in difficulties among the versions. This setup provided quick feedback that game developers can use to promote changes in the game design. B. Balancing Skill vs. Chance Games are complex systems that mix mechanics that demand both skill and luck (chance). Finding the "sweet spot" requires a subjective aspect that only experienced developers have. Using the random agents proved to be useful in spotting when the game is rewarding, or not, the player's skills. When compared to the trained agents and humans, we noticed that, in certain versions, the random agents got the best results. With a closer look at the metrics, these exact same versions were the most difficult to play. The game developers can use the information to tweak the game accordingly, that is, make the game more skill-based or chance-based. C. Expectation vs. Reality With Case Study A we confirmed our design choices (Section V-A) that adding the jump ability would reduce the game difficulty, as it would add a new way to avoid getting hit. However, in Case Study B, we assumed that adding a new gap in the platforms would facilitate the climbing, which was not the case. This shows that the game developer's assumptions must be properly tested in practice. Our approach helps this process. D. Training the Agents and Balance Issues Training the agents demands an initial effort that pays off later in development. As each game has different mechanics, it takes time to discover which rewards will result in an agent playing the game well, or close to the human performance. While trying different rewards, the game shows some of its balance issues. For example, even a simple game like in Case Study B. Jungle Climb requires an effort to make the agents play reasonably well. E. Game Testers and Effort In our case studies, we asked two people to play the game for three minutes for each version, twice. Even with these two simple game scenarios and a few minutes of gameplay, both of the players reported tiredness after playing each game. This adds to the fact that manual testing on video games is tiresome and demands full concentration. In the game industry, game testers work for hours every day on the same game checking the same issues multiple times. The automation in the game testing aid these testers as well, so they can focus on subjective details of the game. F. Deterministic vs. Stochastic The two games used in our experiments are stochastic, they contain random gameplay elements that are not possible to predict. e.g., the bats in Case Study A and the gaps in Case Study B. Games like these pose difficulties when training the agents to play the game. As randomness is a feature in the games, automating the testing is even more valuable for these cases. G. Cost of Creating the Reward Function To make the DRL agents play the game, we must give rewards (or penalties). However, finding a combination of rewards that makes the agents play the game properly requires trial and error. Ultimately, the cost to produce "heuristics" to reward agents should be lower than creating a simple script that performs actions to the game. Thus, the process of creating cost-effective reward functions is an open challenge yet. VIII. THREATS TO VALIDITY We faced issues during the implementation of our approach that can become obstacles to its adoption. For example, creating the testing architecture ( Figure 2) demands an initial effort. Although this investment pays in the long run, we understand that spending engineering time with tooling instead of the game itself might not be welcomed by developers. However, many libraries allow a rapid configuration of the environment. In our cases, we used Python libraries (Gym and Stable Baselines) to speed up the process of training. Another issue is to define reward functions so that the agents can play the game properly. Games have different mechanics and even games within the same genre, e.g. platformers, do not have common rewards. Yet, scenarios within the same game can share the same rewards. For example, in larger games, we could split the chunks of the scenarios the developer wants to test and reuse the rewards with fewer modifications. IX. CONCLUSION In this paper, we proposed and implemented our approach to automate game testing to balance video games using autonomous agents. We described the process of training the agents, playing the game, and assessing the game balance. We focused on two game balance types: Challenge vs. Success and Skill vs. Chance. We validated our testing process with two platform games. We found difficulties spikes in the game versions and identified the versions which demanded more (or less) player skill. Game development often relies more on the developer's "feeling" rather than on any game specification. The result is an empirical manual cycle of development and testing. Although replacing manual testing is not possible, game developers can adopt a development pipeline with automated testing that provides quick feedback about the game balance. Our approach brings a systematic and autonomous way to identify if the game is balanced by (1) checking the difficulty spikes and (2) if the game demands more skill or not. We believe our approach is a step toward providing steady game development and games with better quality. For future work we want to expand the scope of the experiments and find easier ways to train the agents. We also want to add more skill levels for the agents aside from different ways to play the game, that is, a player profile/persona.	2023-04-19T01:16:10.296Z	2023-04-18T00:00:00.000	{ "year": 2023, "sha1": "1e5305a6171842571f234007d95da3342e640908", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "1e5305a6171842571f234007d95da3342e640908", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [ "Computer Science" ] }
234539773	pes2o/s2orc	v3-fos-license	Nanoscopic Quantication of Sub-mitochondrial Morphology, Mitophagy and Mitochondrial Dynamics in Patients With Mitochondrial Disease SLC25A46 mutations have been found to lead to mitochondrial hyper-fusion and reduced mitochondrial respiratory function, which results in optic atrophy, cerebellar atrophy, and other clinical symptoms of mitochondrial disease. However, it is generally believed that mitochondrial fusion is attributable to increased mitochondrial oxidative phosphorylation (OXPHOS)[1], which is inconsistent with the decreased OXPHOS of highly-fused mitochondria observed in previous studies. In this paper, we have used the live-cell nanoscope to observe and quantify the structure of mitochondrial cristae, and the behavior of mitochondria and lysosomes in patient-derived SLC25A46 mutant broblasts. The results show that the crista have been markedly damaged in the mutant broblasts, but that there is no corresponding increase in mitophagy. This study suggests that severely damaged mitochondrial cristae might be the predominant cause of reduced OXPHOS in SLC25A46 mutant broblasts. This study demonstrates the utility of nanoscope-based imaging for realizing the sub-mitochondrial morphology, mitophagy and mitochondrial dynamics in live cells, which may be particularly helpful for the quick assessment and diagnosis of mitochondrial abnormalities. Background The mitochondron is the cellular organelle which is most intimately associated with energy metabolism in mammals and most other eukaryotes. Mitochondrial dysfunction caused by nuclear DNA (nDNA) or mitochondrial DNA (mtDNA) defects lead to cellular respiratory chain and energy metabolism disorders, resulting in a group of multi-system diseases [1,2]. A number of mitochondrial diseases present aberrant mitochondrial morphology, including mitochondrial fragmentation or excessive mitochondrial fusion, which have an effect on mitochondrial function, leading to dysfunction of vital organs and tissues and accordingly threatening patients' health and survival [3][4][5]. The confocal microscope and transmission electron microscope are commonly used to observe mitochondrial morphology. However, a major shortcoming of the confocal microscope is that its spatial resolution is not high enough to visualize and quantitatively calculate the structure of sub-mitochondria, which is vital for examining the sub-mitochondrial damage that may affect mitochondrial function [6]. Transmission electron microscopy, however, is time-consuming, expensive, and unable to observe the variations of live-cell mitochondrial morphology in a dynamic manner. The spatial resolution of 100-120 nm achieved by the structured illumination microscopy (SIM) is su cient to observe the submitochondrial structure in living cells [7]. In this paper, we have set out to take advantage of the live-cell nanoscope − 3D-SIM to dynamically observe the sub-mitochondrial morphology originating from the broblasts of a patient carrying biallelic mutations in SLC25A46. Combined with the sub-mitochondrial structure identi cation/quanti cation and mitochondria-lysosome interaction quanti cation methods developed by our group [8][9][10][11], we have used this approach to identify and quantify the mitochondrial internal structure (cristae) and mitochondrial-lysosomal interaction of live cells. We consequently provide a new method for the identi cation of the mechanisms of mitochondrial oxidative phosphorylation (OXPHOS) dysfunction. Results The reduced metabolic ability of patient-derived SLC24A46 mutant broblasts The mitochondrial respiration function was investigated by examining OCR under both basal conditions and drug-induced mitochondrial stress using the Seahorse assay. The OCR was found to be signi cantly decreased in patient-derived SLC25A46 mutant broblasts compared to normal broblasts (Fig. 1A). After a detailed analysis, the basal respiration, oxygen consumption for ATP production, maximum oxygen consumption capacity of mitochondria, proton-leaked oxygen consumption, non-mitochondrial respiration, and the spare respiratory capacity in patient-derived SLC25A46 mutant broblasts were all lower than that of normal broblasts (Fig. 1B). The MTT assay re ects the metabolic ability of living cells by measuring the proliferation rates of cells. The results of this assay for the mutant and normal broblasts showed no apparent difference in the number of living cells between these two types of broblasts on Day 2 after cell seeding. However, subsequent to this time point, the normal broblasts showed vigorous metabolism and rapid proliferation rate on Day 4, Day 6, and Day 8 (Fig. 1D). Thus, the metabolic ability and cell proliferation rate of SLC25A46 mutant broblasts were signi cantly lower than that of normal broblasts (Fig. 1D). The imaging results showed that the cell density of normal broblasts was close to 80-90% on Day 8, while it only reached 40-50% in SLC25A46 mutant broblasts (Fig. 1C). Mitochondrial hyper-fusion in patient-derived SLC24A46 mutant broblasts with the live-cell nanoscope-3D-SIM imaging system The decreased metabolic ability of mutant broblasts suggested that the mitochondrial function in SLC25A46 mutant broblasts has been disturbed. Sanger sequencing results showed a homozygous, missense point mutation (c.1005A > T; p. Glu335Asp) in SLC25A46 mutant broblasts (Fig. 2D). To examine whether this mutation causes any changes in mitochondrial morphology, we used a nanoscope − 3D-SIM imaging approach to observe mitochondrial morphology in those two human cells. The images showed that the normal broblasts had round or medium length mitochondria ( Fig. 2A), while the SLC25A46 mutant broblasts showed slender, hyper-fused mitochondria (Fig. 2B). Imaris software (Nikon, Tokyo, Japan) was used to identify and analyze the mitochondria morphology (Fig. 2C). The results showed that the number of mitochondria in SLC25A46 mutant broblasts was signi cantly lower than what was observed in normal broblasts. In contrast, the average area and volume of mitochondria in mutant cells were signi cantly greater than those in normal broblasts (Fig. 2E). The comparative analysis of mitochondrial morphology showed aberrant hyper fusion of mitochondria in the patientderived SLC24A46 mutant broblasts. Severe damage of mitochondrial cristae in patient-derived SLC24A46 mutant broblasts Previously, mitochondrial fusion was considered to facilitate OXPHOS, and an increase of mitochondrial fusion will improve the mitochondrial OXPHOS level [12,13]. Mediated mitochondrial fusion was therefore regarded as a new therapeutic target for mitochondrial diseases [14,15]. However, our group found that the highly-fused mitochondria from SLC25A46 mutant broblasts resulted in reduced OXPHOS [16]. What is the underlying cause of this rare condition? One possibility is alterations in the cristae, a most important structures of the inner mitochondrial membrane (IMM), which are deemed as the core of ATP production and mitochondrial respiratory function [17,18]. Therefore, we decided to investigate whether structural defects of mitochondrial cristae lead to decreased OXPHOS. Using algorithm-based SIM imaging technology previously developed by our team [8], we identi ed and extracted cristae rst, then quantitatively analyzed the mitochondrial cristae for human-derived normal and patient-derived SLC25A46 mutant broblasts. The images showed that the mitochondrial cristae structure was visible and abundant in normal broblasts (Fig. 3A). In contrast, the cristae structure was damaged or even vanished in SLC25A46 mutant broblasts (Fig. 3B). After quanti cation analysis, the mean cristae number ( A similar tendency of mitophagy was observed in normal and SLC25A46 mutant broblasts Mitophagy is the general process by which the cell removes severely damaged mitochondria, consequently achieving the purpose of "quality control" of mitochondria within living cells [19,20]. We observed highly-fused mitochondria with severely damaged cristae structures in SLC25A46 mutant broblasts. This raised the obvious questions of whether or not these abnormal mitochondria induce mitophagy? Using the SIM image-based mitochondria-lysosome co-location analysis method in living cells [9], we can observe and quantify mitophagy in normal and SLC25A46 mutant broblasts (Fig. 4C). Our results demonstrate that only slight levels of mitophagy are occurring in both of these cell lines (Fig. 4A, Fig. 4B). After quantitative analysis, there was no statistically signi cant difference in the value of mitochondrial -lysosome co-location between the two cell lines. Western Blot also con rmed that the values of the LC3-II/LC3-I ratio were comparable between normal and SLC25A46 mutant broblasts (Fig. 4d), which was consistent with the results of the SIM image-based analysis method. In addition, with this nanoscope, we can straightforwardly monitor the mitochondrial dynamics and the mitochondrialysosome interaction dynamics (Fig. 4e). This novel nanoscope combined with a quanti cation analysis strategy can not only be used to observe mitochondrial morphology, but also to detect and quantify the damage of structures in sub-mitochondria, assess the extent of mitophagy, and monitor the dynamics of mitochondria and lysosome (Fig. 5). Therefore, this novel approach is a great approach for the observation and etiological diagnosis of mitochondrial damage in patients with mitochondrial disease. Discussion SLC25A46 is responsible for encoding a mitochondrial solute carrier protein [21]. We identi ed SLC25A46 is the human homolog of Ugo1, a protein of Saccharomyces cerevisiae and located in the mitochondrial outer membrane and involved in mitochondrial fusion [16,22,23]. So far, SLC25A46 has been found to be associated with various human diseases. Homozygous or compound heterozygous mutations of SLC25A46 led to a range of clinical syndromes, with the clinical feature of optic atrophy, cerebellar atrophy, progressive myoclonic ataxia, axonal peripheral neuropathy, autosomal recessive cerebellar ataxias (ARCA), lethal congenital pontocerebellar hypoplasia, and even Parkinson's disease [16,21,[24][25][26][27][28][29]. Mice with Slc25a46 dysfunction developed severe motor impairment, optic atrophy, and developmental defects of the nervous system, as well as premature death [30][31][32]. Currently, SLC25A46 is believed to affect mitochondrial dynamics due to the interaction with OPA1 and MFN2 [33,34]. The hyper-fused mitochondria and reduced mitochondrial respiratory function presented in patient-derived SLC25A46 mutant broblasts have also been con rmed by this study, as well as previous studies, which was supposed to be the pathogenic mechanism of a series of neurological diseases [35]. The MTT assay results from this study also strengthened the idea that the metabolic capacity of SLC25A46 mutant broblasts is signi cantly lower than that of control cells. However, there exists a contradiction between the morphology of highly-fused mitochondria and the decline of mitochondrial function. Traditionally, mitochondrial fusion has been veri ed to be vital for maintaining mtDNA stability and improving the tolerance of cells to high mtDNA mutations [36,37]. At the same time, mitochondrial fusion is also a protective factor for maintaining normal mitochondrial respiration function. The absence of mitochondrial fusion in the cerebellum has also been shown to result in a malformed mitochondrial distribution and function [38]. Moreover, mitochondrial fusion is required to support the normal development of embryos [3]. Why then do the SLC25A46 mutant cells examined in our study show mitochondrial hyper-fusion, but a decrease in mitochondrial respiratory function? The respiratory function of mitochondria is a series of oxidation-reduction reactions mediated by multiple complexes located on the mitochondrial inner cristae, which eventually produce ATP and provide energy for the tissues and cells in living organisms [39,40]. From this viewpoint, we hypothesized that SLC25A46 mutation causes structural abnormalities of cristae in highly fused mitochondria, consequently affecting mitochondrial respiratory function. Based on the identi cation and quanti cation method of mitochondrial cristae invented by our group, we analyzed the mitochondrial internal cristae of patientderived SCL25A46 mutant broblasts and human-derived normal broblasts. Our results showed that, compared with normal mitochondria, the number of mitochondrial cristae decreased, the length of cristae shortened, and the area of cristae was reduced in the SLC25A46 mutant broblasts. We even observed the disappearance of cristae in some mitochondria. Therefore, we posited that the structure of mitochondrial cristae in the mutant cells was damaged, which accordingly affected the mitochondrial respiratory function, as re ected by decreased aerobic respiration, reduced ATP generation and decreased metabolic capacity. Researchers have suggested that SLC25A46 plays a vital role in the interaction between the major structural proteins of the mitochondrial outer membrane and the mitochondrial cristae, and it is crucial for maintaining the structure and stability of the mitochondrial cristae. Immunoblot analysis revealed that MIC60 and MIC19 -two critical proteins of mitochondrial contact site and cristae organizing system (MICOS) complex obviously decreased in patient-derived SLC25A46 mutant broblast. And immunoprecipitation experiments showed SLC25A46 co-immunoprecipitated with MIC60, MIC19, OPA1 (located on IMM), MFN1 and MFN2 (located on OMM) [33]. MICOS complex, especially MIC60 and MIC19, is a crucial factor in cristae biogenesis [41,42]. Therefore, SLC25A46 is believed to be not only involved in maintaining the stability of OMM, but also an essential protein in the interaction and communication between the OMM and IMM, as well as the formation and maintenance of mitochondrial cristae [33]. They have used transmission electron microscopy and observed the signi cantly decreased cristae number and length from the patient-derived mitochondria [33]. This result from transmission electron microscopy is consistent with our nanoscope-based results. Mitophagy is a crucial step in mitochondrial quality control, which is used to remove damaged mitochondria [43,44]. Severe injury of mitochondrial cristae can induce mitophagy as well [45,46]. Therefore, we also hypothesized that the damaged mitochondrial cristae would increase the rate of mitophagy in SLC25A46 mutant broblasts. We monitored the dynamic changes of mitochondrial and lysosomal behavior in SLC25A46 mutant broblasts in real-time. We observed a contact and colocalization phenomenon between lysosome and mitochondria after mitochondrial fragmentation in SLC25A46 mutant broblasts. However, using the SIM image-based mitophagy quanti cation method we invented before, we determined that the overall tendency of mitophagy in the SLC25A46 mutant broblasts was not statistically different from that in normal broblasts, although mitophagy did occur in some mitochondria in the SLC25A46 mutant broblasts. Consequently, although the mitochondrial cristae were severely damaged in the SLC25A46 mutant broblasts, the damaged cristae alone did not appear to induce the occurrence of mitophagy. Currently, no studies have reported the mitophagy status of SLC25A46 mutant cells. Conclusions Overall, this study suggests that severely damaged mitochondrial cristae may be the predominant cause of reduced mitochondrial respiratory dysfunction in SLC25A46 mutant broblasts, but that the damaged mitochondrial cristae do not induce a signi cant increase in mitophagy. Through the usage of the SIM-based live-cell nanoscope and the quanti cation methods we developed, we can examine the morphology of the outer mitochondrial membrane (OMM), inner mitochondrial membrane (IMM), the rate of mitophagy, and also perform quantitative calculations of all these phenomena. Simultaneously, we can dynamically observe the behavior of mitochondria and lysosomes. We have thus achieved a comprehensive observation of mitochondrial morphology, internal structure, and mitophagy using a single technique. Therefore, this nanoscope is exceptionally suited for the observation and calculation of the mitochondria and sub-mitochondrial structures in live cells from patients with various mitochondrial diseases. The operation of the device is simple, rapid and accurate, which is helpful for the quick assessment and diagnosis of mitochondrial abnormalities. Cell culture The human-derived normal broblasts and patient-derived SLC25A46 mutant (c.1005A > T, p.Glu335Asp) broblasts cell lines were acquired after informed consent was obtained from the patients. The cells were cultured in Dulbecco's modi ed Eagle's medium (DMEM) medium (Gibco, Thermo Fisher Scienti c, USA) with 10% FBS (Gibco, Thermo Fisher Scienti c, USA) and 100 units/ml Anti-Anti (containing streptomycin and penicillin) (Gibco, Thermo Fisher Scienti c, USA) and incubated in a 5% CO2, 37℃ and 100% humidity incubator. Nanoscope -3D-SIM imaging The cells were seeded in a glass-bottom culture dish (MatTek Life Sciences, USA) and cultured for 24 hours in 2 ml DMEM containing 10%FBS and 100 units/ml Anti-Anti. Before imaging, cells were rst washed three times with a pre-warmed DMEM medium and then were incubated in a DMEM medium containing 100 nM Mito-Tracker Green (Invitrogen, USA) for half an hour. Cells for mitophagy analysis were co-incubated in DMEM medium containing 100 nM Mito-Tracker Green (Invitrogen, USA) and Lyso-Tracker Red (Invitrogen, USA) for half an hour. Cells were then washed three times with DMEM. The stained cells were photographed using the 3D-structure illumination microscope (Nikon, Tokyo, Japan). Western blot Protease inhibitor cocktail (Sigma, USA) and 2 × RIPA lysis and extraction buffer (Thermo sher Scienti c, USA) were added to the centrifuged cell pellets, and then were sonicated for 5 minutes each time, three times in total. The protein concentration was measured by using the Pierce BCA Protein Assay Kit (Thermo sher Scienti c, USA). 30ug protein for each sample and 4X NuPAGE LDS Sample Buffer (Thermo sher Scienti c, USA) were mixed at 4:1 ratio and denatured at 95℃ for 5 minutes, and then separated in 4-12% Bis-Tris gel (Invitrogen, USA). The gel was transferred onto a PVDF membrane (Invitrogen, USA) through the iBlot 2 gel transfer device (Life Technologies, USA). The transferred PVDF membrane was placed in the Intercept Blocking Buffer (LI-COR Biosciences, USA) for 45 minutes, and then incubated overnight in the primary antibody, rabbit anti-LC3B (cell signaling technology, USA) diluted in the blocking buffer at a ratio of 1:200 with Tween 20 diluted in the blocking buffer at a ratio of 1:1000. Rabbit anti-GAPDH (cell signaling technology, USA) was also diluted in the blocking buffer at a ratio of 1:2000 and set as the loading control. The next day, the PVDF membrane was washed for 10 minutes each time, three times in total. Then, the membrane was incubated in the secondary antibody, IRDye 800CW Goat anti-Rabbit IgG (LI-COR Biosciences, USA), for 120 minutes. The bands were detected by the LI-COR Odyssey Clx Imaging System (LI-COR Biosciences, Lincoln, NE). Sanger sequencing for mutation detection To detect the point mutation of SLC25A46 in human-derived normal and patient-derived broblasts, genomic DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen, USA). PCR products of 186 bp in length were ampli ed using GoTag mater mixes (Promega, USA). The following primer set was used for the ampli cation: Forward: TGCCAGTCTTTGTTCTGACG and Reverse: CCAAACACTCCTTCCTCCTG. The reactions were performed following the thermal cycling program: 95 °C for 2 minutes, followed by 30 cycles of 95 °C for 30 seconds, 56 °C for 30 seconds, and 72 °C for 30 seconds. A nal extension step was then performed at 72 °C for 4 minutes. Oxygen consumption rate (OCR) measurement Human-derived normal and patient-derived SLC25A46 mutant cells were seeded at a density of 1.0 × 10 4 cells/well with DMEM supplemented with 10% FBS in XFe96 cell culture plates (Agilent Technologies, USA). After incubation for 24 hours, the DMEM medium was removed and changed with the warmed XF DMEM Medium supplemented with 1 mM sodium pyruvate, 10 mM glucose and 2 mM L-glutamine at pH 7.4. All cells were treated with 1 µM oligomycin A, 1 µM FCCP, and 500 nM rotenone/antimycin A. The OCRs of the cells was assessed by using the XF Cell Mito Stress Test Kit (Agilent Technologies, USA). The Seahorse XF96 analyzer (Agilent Technologies, USA) was used for OCR measurement. Cell proliferation rate measurement (MTT assay) Human-derived normal and patient-derived SLC25A46 mutant broblasts were seeded in 96 well plates (Corning, USA) at a density of 3.0 × 10 3 cells/well with DMEM supplemented with 10% FBS and incubated at 37˚C, 5% CO2. 10 µl of MTT solution (Roche, USA) was added to 100 ul culture medium in each well at a nal concentration of 0.5 mg/ml. The following process was implemented according to the manual provided by the kit. The absorbance was detected at 570 nm by the microplate reader (BioTek, USA). Statistical analysis Graphpad Prism 7 software was used to display data. Independent-Samples T-test was used for statistical analysis. * was de ned as P < 0.05, as P < 0.01, * as P < 0.001, and **** as P < 0.0001. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests.	2020-11-19T09:14:33.910Z	2020-11-17T00:00:00.000	{ "year": 2020, "sha1": "e642550017cccb1594de01fd03cf553474c9f25f", "oa_license": "CCBY", "oa_url": "https://doi.org/10.21203/rs.3.rs-105738/v1", "oa_status": "GREEN", "pdf_src": "ScienceParsePlus", "pdf_hash": "44371787c7a1a538c75fd8ea34a1b3402b787b6b", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Chemistry" ] }
3930778	pes2o/s2orc	v3-fos-license	Extremely Low-Frequency Electromagnetic Fields Affect Myogenic Processes in C2C12 Myoblasts: Role of Gap-Junction-Mediated Intercellular Communication Extremely low-frequency electromagnetic fields (ELF-EMFs) can interact with biological systems. Although they are successfully used as therapeutic agents in physiatrics and rehabilitative practice, they might represent environmental pollutants and pose a risk to human health. Due to the lack of evidence of their mechanism of action, the effects of ELF-EMFs on differentiation processes in skeletal muscle were investigated. C2C12 myoblasts were exposed to ELF-EMFs generated by a solenoid. The effects of ELF-EMFs on cell viability and on growth and differentiation rates were studied using colorimetric and vital dye assays, cytomorphology, and molecular analysis of MyoD and myogenin expression, respectively. The establishment of functional gap junctions was investigated analyzing connexin 43 expression levels and measuring cell permeability, using microinjection/dye-transfer assays. The ELF-EMFs did not affect C2C12 myoblast viability or proliferation rate. Conversely, at ELF-EMF intensity in the mT range, the myogenic process was accelerated, through increased expression of MyoD, myogenin, and connexin 43. The increase in gap-junction function suggests promoting cell fusion and myotube differentiation. These data provide the first evidence of the mechanism through which ELF-EMFs may provide therapeutic benefits and can resolve, at least in part, some conditions of muscle dysfunction. Introduction Modern industrial society produces considerable levels of electromagnetic fields (EMFs), caused by widespread use of electric-power-generating facilities that deliver energy to domestic and industrial appliances. These EMFs are characterized by frequencies of 50 Hz to 60 Hz and are part of the extremely low-frequency (ELF) electromagnetic spectrum [1]. Even if ELF-EMFs are nonionizing radiation, they can interact with biological matter [2]. Since the 1980s, chronic exposure to ELF-EMFs has been implicated in carcinogenesis [3,4], even if epidemiological studies have not provided conclusive or statistically adequate data. This has mainly been due to confounding by variable exposure times and misclassification selection bias [5,6]. On the other hand, ELF-EMF-based therapies are commonly used in different medical fields, such as physiatrics and patient rehabilitation [7][8][9], with apparent beneficial effects. However, at present, the cellular mechanism(s) behind these effects remains unclear. Consequently, experimental investigations addressing how, and to what extent, ELF-EMFs influence living matter become urgent. Our studies have been specifically designed to provide new insight into the mechanism(s) responsible for the biological effects of ELF-EMFs on skeletal muscle. We previously described the effects of ELF-EMFs on the C2C12 myoblast cell line [10], a well-known in vitro model of skeletal muscle 2 BioMed Research International phenotype [11]. We demonstrated that ELF-EMFs affect intracellular reactive oxygen species production, mitochondrial membrane potential, and Ca 2+ handling, which have been shown to be all patterns strictly dependent on the cell differentiation stage in muscle tissue [12]. Such ELF-EMF-induced changes in mitochondrial activity and Ca 2+ homeostasis support the hypothesis that exposure to ELF-EMFs can effectively modulate regulation of the myogenesis in C2C12 myoblasts. Skeletal myogenesis is required for growth, maintenance, and repair of injured muscle [13]. It is a multistep developmental process determined by a complex cascade of events involving development and differentiation of myoblasts, their fusion to form primary and secondary myotubes, and their subsequent maturation into fully developed adult muscle fibers [14,15]. In recent years, several studies have led to significant improvements in the definition and understanding of the phases of the commitment and differentiation processes of skeletal muscle cells [15][16][17][18][19]. Many in vitro studies have focused on the role of gap-junction-mediated intercellular communication (GJIC) in specific and critical stages of myogenesis [20][21][22][23]. It has been hypothesized that GJIC might be involved in the onset of the differentiation process, to coordinate the myoblasts that are committed to differentiate, thus promoting their alignment and fusion, and, consequently, the intercellular diffusion of critical signaling molecules (i.e., Ca 2+ , inositol 1,4,5-trisphosphate, and adenosine 5triphosphate) [21,24,25]. The evidence that short exposure to ELF-EMFs has effects on both C2C12 myoblasts and myotubes through modulation of their redox status and Ca 2+ handling [10] also suggests a possible involvement of GJIC and, as a consequence, of long-term biological processes, like myogenesis [26]. To further clarify the mechanism(s) responsible for ELF-EMF-induced effects on myogenesis, in the present study we exposed differentiating C2C12 myoblasts to ELF-EMFs, and, in particular, we monitored their gap-junction permeability, which is considered to be a crucial function for myotube formation during progression through myogenesis. Equipment. A horizontal solenoid (Oersted Technology Corporation) was designed and built to deliver variable, homogeneous, sine-wave alternating-current magnetic fields at 50 Hz frequency and with intensities from 0.1 mT to 1.0 mT (±2%). This was used to expose large numbers of cells to ELF-EMFs simultaneously [10,27,28]. The horizontal cylindrical solenoid presents the following features: length, 340 mm; diameter, 113 mm; number of wire turns, 144; resistance at 20 ∘ C (Ω), 0.3; this device was mounted on a nonmagnetic supporting base and powered using a power supply (CW-801P; Elgar Electronics). In particular, the power supply was operated in its "closed-loop current mode": the output current and the generated magnetic field were consequently regulated against thermal and physical variations/drift of the coil. The final measured value of the solenoid coil constant was 4.830 Gauss/Amp. The solenoid was used in a cell culture incubator (5% CO 2 , 37 ∘ C; HERAcell, Kendro Laboratory Products GmbH, Hanau, Germany) for long periods, providing continuous exposure of the cells (1-7 days). During these treatments, any temperature increase in the incubator due to the solenoid was negligible. The tested value of background electromagnetic fields was less than 1 T (50 Hz). In particular it was in the order of 0.7 T (50 Hz) in the incubator and outside the switched on solenoid and of 100 nT (50 Hz) outside or inside the switched off solenoid placed in the incubator. Moreover, in laboratory areas between incubators, worktops, and cell cultures hood, electromagnetic fields measured 40-140 nT (50 Hz). The EMF meters used to measure the values of electromagnetic fields are F.W.BELL Tesla meters mod. 4190 (measuring range: 0.01-200 T, resolution: 0.01 T) and mod. 6010 equipped with an axial probe mod. HAD61-2508-05T (measuring range: 0.3-300 mT, minimum resolution: 0.01 mT) both from Sypris Test & Measurement (Orlando, FL). The cells were cultured in plastic dishes that are transparent to the ELF magnetic field. Chemicals and Materials. Unless otherwise indicated, the cell culture media, sera, and antibiotics were from Thermofisher (Monza, Italy), the cell culture plasticware was from Becton Dickinson Falcon (Sacco Srl, Cadorago, Italy), and the reagents and standards were from Sigma-Aldrich (Milan, Italy). Viability and Proliferation Assays. The analysis of cell viability was performed using trypan blue exclusion tests and cell growth using colorimetric assays based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Briefly, C2C12 myoblasts (4.0 × 10 3 cells/cm 2 ) were plated into 96-well plates (Corning-Costar, Milan, Italy) in growth medium. At selected times with the absence and presence of the ELF-EMF treatments, MTT was added to each well to a final concentration of 0.5 mg/mL. After 3 h at 37 ∘ C, the plates were centrifuged at 500 ×g for 5 min. The supernatants were removed, and 200 L dimethyl sulfoxide was added to each well. After 30 min at 37 ∘ C, the absorbance of each well was determined using a microplate reader (Spec-traMAX 190), at 560 nm. The trypan blue exclusion assay was performed by staining C2C12 myoblasts with a trypan blue dye solution (0.5% in phosphate-buffered saline), and the stained cells were counted using a hemocytometer (Bürker chamber). Blue stained cells were considered nonviable. Fusion Index. The extent of successful differentiation of C2C12 myoblasts into myotubes was determined by morphological analysis and Hoechst staining of the nuclei [29]. C2C12 myoblasts (4.0 × 10 3 cells/cm 2 ) were seeded in growth medium onto sterile coverslips (Ø, 12 mm) in 24-well plates. After 3 days, the growth medium was shifted to differentiation medium, and the C2C12 myoblasts were induced to differentiate for up to 7 days in the absence and presence of the ELF-EMF treatments. At each time point, the cells were washed twice with phosphate-buffered saline, fixed in 90% ethanol, and stained with Hoechst solution. Images were acquired using an inverted microscope (Olympus IX-70) equipped with a digital camera (Camedia C-5050; Olympus). At least 20 fields were analyzed for each experimental condition. The Fusion Index was quantified as the percentage of Hoechststained nuclei located within multinucleated cells (with at least two nuclei, as a result of myoblast fusion), based on the total analyzed nuclei. Western Blotting. C2C12 myoblasts (4.0 × 10 3 cells/cm 2 ) were seeded in growth medium into 100 mm diameter Petri dishes. After 3 days, the growth medium was shifted to differentiation medium, and the cells were maintained in the absence and presence of the ELF-EMF treatments. At each experimental point, the cells were scraped, lysed, and collected in sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.1 M dithiothreitol, and 0.002% bromophenol blue). Protein concentrations were determined using the protein assay kits (Bio-Rad DC; Bio-Rad, Segrate, Italy). The whole cell extracts were separated using SDS-PAGE on 7.5% or 12.5% (w/v) homogeneous slab gels (40 g protein/lane) and then transferred onto polyvinylidene fluoride membranes (Immobilon-P; Merck-Millipore, Vimodrone, Italy). Equal loading of protein samples was monitored using Coomassie blue staining of identical gels run in parallel (0.25% Coomassie blue solution; R 250/G 250 1 : 1; Bio-Rad). Red Ponceau S (Sigma-Aldrich) staining of each membrane was used to monitor the transfer efficiency. Microinjection/Dye-Transfer Assay. The differentiating C2C12 myoblasts were tested for their establishment of functional gap junctions using a microinjection/dye-transfer assay, following the protocol proposed by Mazzoleni et al. [30]. Briefly, C2C12 myoblasts (4.0 × 10 3 cells/cm 2 ) were seeded in growth medium into 60 mm diameter Petri dishes. After 3 days, the growth medium was shifted to differentiation medium, and the cell cultures were maintained in the absence and presence of the ELF-EMF treatments. For the dye-transfer assay, single cells within a monolayer were microinjected with a 10% (w/v) solution of the gap-junction-permeant fluorescent tracer lucifer yellow CH in 0.33 M lithium chloride. Microinjections were performed using glass capillary needles (Clark Electromedical Instruments, Edenbridge, UK) prepared in an automatic puller (Narishige, Tokyo, Japan). The needles were driven using a micromanipulator (Narishige 5240) linked to a fluorescence microscope (Olympus IMT2-SYFII). The fluorescent dye was injected into single cells under nitrogen pressure, using a microinjector (Eppendorf, Hamburg, Germany). Five minutes after the last injection, the cells were fixed with 4% paraformaldehyde in phosphatebuffered saline, and their dye-transfer was evaluated. The extent of functional GJIC was quantified by counting the number of fluorescent cells surrounding the microinjected cells (i.e., the number of dye-coupled cells/injection). For the precise quantification of functional GJIC in the cultures, at least 25 independent microinjection trials/dish were carried out in three separate dishes for each experimental point. Functional GJIC is expressed as the means ± SEM. Fluorescence and phase-contrast images of untreated and ELF-EMF-treated monolayers were obtained using the inverted fluorescence microscope (Olympus IMT2-SYFII) equipped with a digital camera (Olympus OM-4 Ti reflex). Statistical Analyses. Statistical analysis was performed using the Prism 4 software for Windows (GraphPad Software Inc., San Diego, CA, USA). Unless otherwise indicated, all of the data are expressed as means ± SD or ± SEM, as specified in the figure legends. Comparisons were made using -tests. Effects of ELF-EMF Exposure on C2C12 Myoblast Proliferation and Differentiation. The effects of ELF-EMF exposure on the C2C12 myoblast growth rate and viability were studied using a colorimetric assay and vital dye staining, respectively. These analyses were performed on undifferentiated C2C12 myoblast cultures exposed for up to 7 days to ELF-EMF treatments at different field intensities (0.1, 0.5, and 1.0 mT). As shown in Figure 1, exposure of the C2C12 myoblasts to these ELF-EMFs did not significantly affect either cell proliferation rates or viability; indeed, the numbers of nonviable cells did not differ between the ELF-EMF-treated C2C12 myoblast cultures and their corresponding untreated controls and remained from 10% to 15% of the total cells. The effect of ELF-EMF exposure on the myogenesis process of the C2C12 myoblasts was explored using morphological analysis and quantification of the expression of MyoD and myogenin, two early markers of myogenesis. The morphological analysis was quantified according to the Fusion Index, which was calculated as the percentage of C2C12 myoblasts (of the total) that underwent the differentiation process. The exposure to ELF-EMFs at the 0.5 mT and 1.0 mT intensities induced significant acceleration of the first phases of the myogenic process. Indeed, the C2C12 myoblasts showed significantly higher Fusion Index when exposed to ELF-EMF treatments with 0.5 mT after 2 days to 5 days and with 1.0 mT after 2 days to 3 days (Figures 2(b) and 2(c)). No significant differences were observed for the C2C12 myoblast cultures exposed to ELF-EMFs at 0.1 mT, in comparison with the untreated controls (Figure 2(a)). MyoD and myogenin expression levels showed some increases for the C2C12 myoblasts exposed to ELF-EMF treatments, with respect to the corresponding control cells (Figure 3). For the ELF-EMF treatment with 0.1 mT, this increase was evident only for myogenin expression after 72 hours of exposure. The exposure to 0.5 mT intensity triggered an increase of MyoD expression at 48 hours and of myogenin expression at 24 and 48 hours. Differently, 1.0 mT induced increased MyoD expression at 24 hours and increased myogenin expression at 72 hours of exposure. Influence of ELF-EMFs Treatments on C2C12 Myoblast cx43 Expression and GJIC. The effects of ELF-EMF treatments on differentiating C2C12 myoblasts were also evaluated by an analysis of the efficiency of their gap-junction coupling (dye-transfer efficiency) and by determining their expression levels of cx43, the major gap-junction component in skeletal myoblasts [20,31,32]. As illustrated by the representative Western blot and densitometric analyses in Figure 4, during the ELF-EMF treatment with 0.1 mT, a transient, but not significant, increase in cx43 expression was seen only at 24 hours, with respect to the corresponding control cells. Conversely, when the cells were exposed to ELF-EMF treatment with 0.5 mT, or 1.0 mT, these showed an increase in cx43 expression that became significant at 72 hours, with respect to the corresponding control cells (Figure 4). The influence of the ELF-EMF treatment on C2C12 myoblast gap-junction function was tested using the microinjection/dye-transfer assay at the early stages of C2C12 myoblast differentiation (i.e., 24, 48 h). These times were selected on the basis that it is the earlier times that are crucial for the onset of the differentiation program in these C2C12 myoblasts. In addition, after 48 h, the high cell density that was reached by the C2C12 myoblast monolayers made the single-cell approach for the assay difficult. The quantitative analysis of the extent of dye-coupling during the first 48 h of differentiation of the C2C12 myoblasts (e.g., see the representative phase contrast and corresponding fluorescence images in panel (a) and the graph in panel (b) of Figure 5) revealed an increased number of coupled cells in the cultures exposed to ELF-EMF treatment that became significant after 48 h at 1.0 mT, in comparison to the corresponding controls. The treatment of the differentiating C2C12 myoblasts with ELF-EMFs at 0.5 or 1.0 mT increased not only the expression of the major junction component, cx43, but also its assembly into functional membrane channels, which allowed direct cell-to-cell communication among the cells that were committed to differentiate. Discussion Epidemiological studies on the potential hazards of EMFs for human health have generated many controversies and attracted the attention of the media, the general public, Data are means ± SEM from three independent experiments. * < 0.05 and * * < 0.01 versus corresponding Ctr. and biomedical researchers [33,34]. On the other hand, there is evidence of beneficial effects of magnetic fields and EMFs in the treatment of various injuries and diseases [9]. Indeed, clinical benefits for EMFs have been claimed for 20 centuries, and even if there has been perplexity and medical skepticism concerning their use, the application of EMFs with a therapeutic aim is probably one of the most ancient treatments used by Man [35]. Today, both static and time-varying magnetic fields are applied therapeutically with success in the treatment of musculoskeletal injuries or dysfunction [36]. There are also a large number of experimental and clinical studies that have demonstrated that various exogenous EMFs at surprisingly low levels can have effects on a variety of biological systems and processes, most of which are of critical importance for diagnostics and therapy. Along with the evidence from in vitro cellular models, clinical and preclinical studies have suggested that magnetic field and EMF stimulation can accelerate healing processes and influence nerve repair and regeneration [7]. Skeletal muscle also represents a potential target for the biological effect(s) of ELF-EMFs, and this property might be of great importance, due in particular to their diffuse medical applications in physical and rehabilitation medicine. To better clarify this point, the objective of the present study was to investigate the effects of ELF-EMFs on the myogenic process. To achieve this, the C2C12 myoblastic cell line was chosen as a suitable in vitro model of skeletal muscle differentiation. The use of a well-characterized experimental model, the strict control of the experimental procedures, and the optimization of the protocol for this C2C12 myoblast exposure to ELF-EMFs have provided us with improved understanding of the cellular mechanism responsible for ELF-EMF-induced cell modifications, which have remained largely unknown to date. The data presented here demonstrate that under these experimental conditions ELF-EMFs at 50 Hz and with electromagnetic field strengths from 0.1 to 1.0 mT do not affect either C2C12 myoblast viability or proliferation rate. This thus initially demonstrates that there are no direct toxic effects of ELF-EMFs on this cell model. Even if this finding is in apparent contrast with what has been observed with other cell models, it is important to bear in mind that, in the many studies that have reported that ELF-EMFs can act on cell proliferation and cell-cycle progression, these effects have depended on the cell phenotype, as well as on the intensity, frequency, and wave form of ELF-EMFs applied, and length of the exposure [37][38][39][40][41][42]. In recent decades, a large amount of evidence has demonstrated prodifferentiating effects of ELF-EMFs on different cell phenotypes, and interesting data have also been obtained using adult stem cells, with the suggestion that ELF-EMFs represent an efficacious therapeutic approach [28,39,[43][44][45]. On the other hand, the mechanism(s) of action of ELF-EMFs on physiological processes and their intracellular molecular targets are far from being defined. In the cell model in the present study, the presence of ELF-EMFs enhanced the myogenic process. This process progresses through a highly ordered sequence of events, including morphological changes (i.e., cell alignment, fusion) and specific time-courses of gene expression patterns [46]. In addition, published data have strongly supported a key role for GJIC in the first phase of myogenic differentiation, when myoblasts that are committed to differentiate then adhere and fuse, to form gap junctions. These gap junctions then allow the intercellular diffusion of critical signals, such as Ca 2+ , inositol 1,4,5-trisphosphate, and adenosine 5 -triphosphate [24]. Indeed, when blockers of the permeability of these junction channels are used, or in the presence of inducible deletion of cx43, downregulation of myogenic factors is seen [21][22][23]47]. Furthermore, transfection of rhabdomyosarcoma cells with cx43 cDNA was shown to induce cell differentiation [48]. In particular, in myoblasts, cx43 expression is predominant, and it is a prerequisite for their fusion [32]. Indeed, cx43 gap-junction channels have been shown to provide the intercellular signaling pathways that are required for normal timing of skeletal muscle ontogeny and regeneration [20]. In our cell model, ELF-EMFs at 0.1 mT only transiently affected cx43 expression but at 0.5 mT and 1.0 mT modified GJIC activity through increases in both the expression of the cx43 protein and cell coupling in cultures committed to differentiate. These data are complementary to our previous report regarding the evidence that ELF-EMFs can affect the Ca 2+ handling and redox status of C2C12 myoblasts [10]. These processes are responsible for the phenotypic cell reactions to environmental stimuli, and they might provide the means through which ELF-EMFs can accelerate the myogenesis process through increased extent of the GJIC, as the regulation of Ca 2+ fluxes by gap junctions and their response to oxidative stress have been well demonstrated [49]. Conclusions In conclusion, although our data do not show clear dosedependence or threshold-induced effects, ELF-EMFs in the mT intensity range accelerated the myogenic process in these C2C12 myoblasts. In the same cell model, ELF-EMFs can affect the cellular redox state and intracellular Ca 2+ dynamics [10] and, consequently, cell metabolic activity. All of these events can be proposed as key triggers involved in increases in specific myogenic markers (e.g., MyoD and myogenin) and the promotion of cell fusion, which is made more efficient by the increased GJIC activity, all of which are steps that can be enhanced by ELF-EMFs. Taken together, all these evidences indicate for the first time the mechanism of action through which ELF-EMFs can promote skeletal muscle differentiation. This thus provides a scientific basis to what is considered an efficacious therapeutic means to resolve, at least in part, some conditions of muscle dysfunction. Disclosure Giovanna Mazzoleni and Maria A. Mariggiò are co-senior authors. Conflicts of Interest The authors report no conflicts of interest.	2018-04-03T05:16:43.493Z	2017-05-21T00:00:00.000	{ "year": 2017, "sha1": "cfb849d6af77432bad61ff248e86a47d4a1a25f7", "oa_license": "CCBY", "oa_url": "http://downloads.hindawi.com/journals/bmri/2017/2460215.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "cfb849d6af77432bad61ff248e86a47d4a1a25f7", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Medicine", "Biology" ] }
9477399	pes2o/s2orc	v3-fos-license	ZNF750 is a lineage-specific tumour suppressor in squamous cell carcinoma ZNF750 controls epithelial homeostasis by regulating epidermal-differentiation genes, a role underscored by its pathogenic mutations in esophageal squamous cell cancers (SCCs). However, the precise role of ZNF750 in SCC cell biology remains unclear. In this study, we report that ZNF750 is exclusively deleted, mutated and underexpressed in human SCCs, and low ZNF750 expression is associated with poor survival. Restoration of wildtype, but not mutant ZNF750 protein uniquely inhibited the malignant phenotypes of SCC cells both in vitro and in vivo. Notably, ZNF750 promoted the expression of a long non-coding RNA (TINCR), which mediated both cancer-inhibition and differentiation-induction effects of ZNF750. In addition, ZNF750 potently suppressed cell migration by directly inhibiting the transactivation of LAMC2. Together, our findings characterize ZNF750 as a crucial SCC-specific suppressor and uncover its novel anticancer-associated functions. INTRODUCTION Squamous cell carcinoma (SCC) is the most common human cancer. It arises from stratified epithelia, including the oral epithelium, epidermis and the epithelia covering the oesophagus, bladder, vulva and cervix. Approximately 300 000 new SCC cases per year occur in the United States alone, resulting annually in~100 000deaths. 1,2 Recently, we and others identified missense and truncating mutations as well as genomic deletions targeting ZNF750 in esophageal SCC (ESCC). 3,4 Functionally, we have shown that loss of ZNF750 was associated with impaired differentiation as well as a failure to repress tumour growth in ESCC, 3 suggesting that ZNF750 may act as a tumour suppressor. ZNF750 was reported to function as a transcriptional regulator of epidermal cell differentiation by inducing differentiation genes while inhibiting progenitor factors. 5 However, the precise role of ZNF750 in SCC cells largely remain unexplored. Particularly, the molecular events and signalling pathways associated with ZNF750 in SCCs await further characterization. Long non-coding RNAs (LncRNAs) have recently been reported to participate in the regulation of epidermal cell differentiation. For example, the LncRNA TINCR promotes differentiation of keratinocytes through a mechanism involving direct RNA:RNA interactions and recruitment of STAU1 protein to stabilize differentiation-specific mRNAs. 6 Another recent report demonstrated lncRNA/transcription factor network, which regulated epidermal differentiation. 7 However, whether and how these differentiation-associated LncRNAs are involved in the biology of SCC cells have not been fully addressed. In this study, we demonstrate that ZNF750 is frequently and exclusively targeted by genetic lesions in major types of human SCCs, including those cancers of the cervix (CSCC), head and neck (HNSCC) and lung (LSCC). Low expression level of ZNF750 is associated with poor prognosis of SCC patients. Furthermore, the tumour-suppressive role of ZNF750 is mediated through regulating key cancer genes such as TINCR and LAMC2. ZNF750 is exclusively mutated, deleted and under-expressed in different types of human SCCs To determine comprehensively the genetic abnormalities affecting ZNF750, multiple public datasets were re-analysed, including The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and Human Protein Atlas (see Materials and Methods). In TCGA whole-exome sequencing results, similar to our earlier findings in ESCC, 3 a number of mutations throughout ZNF750 were identified in various SCC types, including CSCC, HNSCC and LSCC ( Figure 1a). Of note, most of these somatic variants occurred at the beginning of Exon 2, which encodes for the evolutionally conserved C2H2 DNA-binding domain, highlighting the biologic relevance of this domain in SCC cells (Figure 1a). In addition, many of the mutations caused damaging effects to the ZNF750 protein (for example, Stopgain, Frameshift and Splicing mutations). We next compared ZNF750 gene dosage between tumour and normal samples using SNP 6.0 array data from TCGA, and found significant genomic deletions of ZNF750 in primary SCC samples from cervical, head and neck, and lung tissues (Figure 1c). Importantly, these genetic abnormalities were exclusively observed in squamous-type tumours (Figure 1c), underscoring the lineage-specific role of ZNF750 in squamous cancer biology. Next, in order to examine ZNF750 expression across different types of normal and cancer tissues at both mRNA and protein levels, we queried GEO cDNA microarray data (series GSE7307), TCGA RNA-seq data, as well as immunohistochemistry (IHC) results from Human Protein Atlas (see Materials and Methods). Notably, expression of both ZNF750 mRNA and protein was markedly higher in a variety of healthy squamous epithelium than nonsquamous tissues, again signifying its lineage-specific expression pattern and function (Figures 2a and b). Moreover, results from GEO (series GSE9750 and GSE25099) and Human Protein Atlas showed significantly decreased ZNF750 expression compared to its normal counterpart at both mRNA (Supplementary Figure 1) and protein levels ( Figure 2c) in CSCC and HNSCC. Congruent with these publically available data, we performed IHC analysis to stain samples from CSCC, HNSCC and LSCC (commercial tissue array, see Materials and Methods), and confirmed either lower or undetectable ZNF750 expression in tumour tissues ( Figure 2d). Notably, Kaplan-Meier analysis on the TCGA cohorts revealed that the downregulation of ZNF750 was significantly correlated with poorer outcome of patients with HNSCC and LSCC (Log-rank test, P = 0.04 and 0.01, respectively, Figure 2e). Collectively, these findings suggest that ZNF750 is uniquely compromised by multiple forms of genomic defects in human SCCs. As a p63-dependent transcription factor, ZNF750 regulates KLF4 expression in SCC cells ZNF750 was recently identified as a p63-downstream transcription factor, and activated KLF4 transcription in normal keratinocyte; 5 however, these regulations in the context of cancer cells are unknown. In order to investigate these transcriptional regulations in SCCs, we first depleted p63 using shRNA and confirmed that ZNF750 expression was decreased at both the mRNA and protein levels as addressed by qRT-PCR and western blot analysis (Figures 3a and b). We next analysed p63 and ZNF750 protein levels in a panel of different types of SCC cell lines (Figure 3c). The abundance of p63 protein tended to have positive correlation with that of ZNF750 in 10 SCC cell lines tested (Figure 3d). Concordantly, analysis of 86 squamous cancer cell lines in Cancer Cell Line Encyclopedia (CCLE) datasets showed a significant linear correlation between the mRNA levels of ZNF750 and p63 (Supplementary Table 1 and Figure 3e). We further observed a positive correlation between the levels of ZNF750 and p63 transcripts exclusively in squamous-type primary tumours based on TCGA RNA-seq datasets ( Figure 3f). Next, we sought to determine whether ZNF750 controls KLF4 expression in SCC cells. Upregulation of KLF4 transcript level was observed in ZNF750 over-expressing SCC cells ( Figure 3g); in contrast, KLF4 mRNA expression decreased in ZNF750 depleted cells (Figure 3h). Notably, the level of KLF4 was positively correlated with ZNF750 expression in SCC cell lines (Figure 3i). Consistently, we found a linear correlation between the mRNA levels of ZNF750 and KLF4 in CSCC, HNSCC and LSCC patient samples from TCGA RNA-seq datasets (Figure 3j). Collectively, these data suggested that ZNF750 is p63-dependent factor and regulates KLF4 expression in SCCs. Tumour-suppressive property of ZNF750 in SCC cells To explore the biological relevance of ZNF750 in SCCs, cell line models with ectopic ZNF750 expression were established. Previous studies found that the evolutionarily conserved atypical C2H2 zinc finger motif was required for its transactivation in normal keratinocyte; 5,8 therefore, we performed functional analysis with wildtype as well as C2H2 mutant ZNF750 in a number of In contrast, C2H2 mutant partially or fully lost its inhibitory effect on cell proliferation ( Figure 4a). Similarly, wildtype ZNF750, but not C2H2 mutant inhibited foci formation in a variety of SCC cells (Figures 4b and c). The tumour-suppressive potential of ZNF750 was further evaluated by xenograft tumour formation. Importantly, wildtype ZNF750 robustly suppressed tumour growth compared with the control group, whereas the C2H2 mutant had significantly less suppressive ability in vivo (Figure 4d). In addition, Ki-67 staining by IHC of xenograft tumours supported our findings that ZNF750 protein decreased the proliferative cell population ( Figure 4e). Moreover, the well-defined differentiation marker Involucrin (IVL) was highly expressed in tumours expressing wildtype ZNF750 (Figure 4e). Not surprisingly, neither ZNF750 wildtype nor C2H2 mutant affected p63 expression both in vivo and in vitro ( Supplementary Figures 2c and d). Unaltered p63 transcript level was also observed in ZNF750 depleted cells ( Supplementary Figure 2e), in agreement with earlier results 5 showing that ZNF750 was downstream of p63 (Figures 3a and b). We next addressed whether ZNF750 regulates other important malignant phenotypes. Notably, wildtype ZNF750 potently inhibited the migration of different types of SCC cells. In sharp contrast, the C2H2 mutant expressing cells showed minimal effect on migration (Figure 4f). We next determined cell adhesion by measuring the efficiency of SCC cells to adhere to collagen type I, and found that this cellular phenotype was significantly impaired by wildtype ZNF750, but was unaffected by the C2H2 mutant ( Figure 4g). As cellular signalling during cell adhesion is associated with anoikis resistance, 9 we examined the effect of ZNF750 on resistance to anoikis of SCC cells (Figures 4h and i). Concordantly, we attenuated anoikis-resistance only in wildtype ZNF750 but not C2H2 mutant cells. We confirmed that impaired tumour-suppressive functions of the C2H2 mutant was not due to the lack of expression or mis-localization (Supplementary Figures 2f and g). Importantly, ZNF750 hardly affected proliferation in non-SCC cells such as glioblastoma cells ( Supplementary Figures 2h and i). Taken together, these data strongly suggest that ZNF750 is a bona fide lineage-specific tumour suppressor, and the C2H2 domain is important for its biological functions in SCCs. Transcriptome analysis of ZNF750-regulated genes and processes To explore how ZNF750 suppressed SCC cell malignancy, we performed both cDNA microarray and whole transcriptome sequencing (RNA-seq) to measure global alterations in mRNA levels upon ZNF750 restoration. Gene ontology (GO) analysis revealed that ZNF750 activated a subset of genes highly enriched in processes regulating terminal epidermal differentiation similar to the function of ZNF750 in normal keratinocytes ( Figure 5a). Notably, genes repressed by ZNF750 were significantly enriched in processes highly relevant to cancer biology, including cell migration, proliferation, adhesion and cell death, all of which were shown to be regulated by ZNF750 in our experiments ( Figure 4). Gene set enrichment analysis (GSEA) further confirmed that ZNF750-upregulated genes were associated with an 'epidermal late differentiation signature' 7 ( Figure 5b) while ZNF750- LncRNAs have been shown to participate in the regulation of epidermal cell differentiation. To determine whether ZNF750 could regulate the expression of epidermis differentiation-related LncRNAs, we cross-compared LncRNAs whose levels were changed upon ZNF750 activation to those with differential expression during epidermal differentiation 6 ( Figure 5c). Interestingly, we identified TINCR as a candidate ZNF750-promoted LncRNA. Many epidermal progenitor genes contribute to the malignant phenotype of cancer cells, such as uncontrolled proliferation, enhanced migration, EMT and so on. 10 Thus, we hypothesized that ZNF750-mediated repression of epidermal progenitor genes might account for its tumour-suppressive functions in SCC cells, which we observed ( Figure 4). We performed similar crosscomparisons, and nominated a total of seven genes as possible ZNF750-repressed progenitor factors, namely THBS1, CTNNAL1, FST, LAMC2, NT5E, NRG1 and COL12A1 (Figure 5d). ZNF750 drives epidermal differentiation in SCCs Our transcriptome analysis showed that epidermal differentiation was the most significantly enriched process activated by ZNF750 in SCC cells. Therefore, the role of ZNF750 in differentiation regulation in SCCs was further characterized. We first validated that wildtype ZNF750 induced the epidermal differentiation complex 11 including the small proline-rich proteins 1B (SPRR1B), the calcium-binding protein S100A8 and IVL (Figure 6a and Supplementary Figure 3a), while suppressing the expression of the basal cell marker K14 in a variety of SCC cells (Figure 6b). Confirmation of the pro-differentiation function of ZNF750 was its ability to enhance the level of IVL protein both under organotypic and 2D culture conditions (Figure 6c, and Supplementary Figures 3b and c). Importantly, these changes were only triggered by wildtype but not C2H2 mutant ZNF750 (Figures 6a-c). Concordantly, ablation of endogenous ZNF750 either by siRNAmediated knockdown or CRISPR/Cas9-mediated genomic disruption decreased the expression of these differentiation factors (Figures 6d and e, and Supplementary Figures 3d and e). Furthermore, interrogation of TCGA RNA-seq datasets showed a linear correlation between the mRNA levels of ZNF750 and epidermal differentiation-related genes in CSCC, HNSCC and LSCC patient samples (Figure 6f). These positive correlations were exclusively observed in squamous-type tumours in TCGA RNA-seq datasets (Figure 6g), again underscoring the lineage-specific role of ZNF750 in cancer biology. We next asked whether differentiation stimuli lead to changes of the level of ZNF750. Consistently, stimuli of keratinocyte differentiation, including high confluence condition 12 or Ca 2+ concentration, 8 caused marked induction of ZNF750 expression (Figure 6h and Supplementary Figure 3f). Collectively, these results suggest that ZNF750 strongly promotes To determine further whether ZNF750 regulates the differentiation of non-malignant cells, such as keratinocytes, the expression levels of epidermal differentiation factors were measured in HaCaT (non-malignant keratinocytes 13 ) by qRT-PCR after siRNA-mediated ZNF750 depletion (Figure 6i). Indeed, differentiation genes were suppressed in ZNF750 silenced HaCaT cells (Figure 6i), highlighting the role of ZNF750 in enabling epidermal differentiation. ZNF750 regulation of TINCR LncRNA mediates differentiation, cell growth and cell migration in SCCs TINCR was identified as a ZNF750-regulated LncRNA; however, its roles in the settings of SCC are unknown. We next sought to understand whether and how TINCR is involved in the biology of SCCs and its relationship with ZNF750. In order to define the regulation between TINCR and ZNF750, we first confirmed the upregulation of TINCR transcripts in ZNF750 over-expressing SCC cells (Figure 7a), and the downregulation of TINCR in ZNF750 depleted cells (Figure 7b). Of note, TINCR transcript levels were significantly correlated with ZNF750 protein levels in SCC cell lines (Figure 7c). Moreover, transcripts of both TNCR and ZNF750 are activated during epidermal lineage induction (unpublished data) ( Figure 7d). Furthermore, we observed a positive correlation between the levels of ZNF750 and TINCR transcripts exclusively in squamous-type tumours on TCGA RNA-seq datasets (Figures 7e and f), suggesting that ZNF750 can activate both LncRNAs (such as TINCR) and coding genes in SCCs. Next, to explore the role of TINCR in SCC biology, we asked whether TINCR regulates SCC cell malignancy. Depletion of TINCR by siRNA resulted in an increased cell growth as well as migration activity (Supplementary Figures 4a-c). Moreover, silencing of TINCR resulted in the decrease of differentiation-related gene (Supplementary Figure 4d). We next probed whether TINCR was involved in ZNF750-mediated functions. Importantly, TINCR depletion partially rescued ZNF750-repressed malignancies, including cell growth (Figures 7g and h) and migration (Figure 7i). Silencing of TINCR also impaired the activation of differentiation-related gene by ZNF750 (Figure 7j). In addition, Kaplan-Meier analysis revealed that low levels of TINCR were significantly correlated with poor outcome of patients with HNSCC (Log-rank test, P = 0.03) in the TCGA cohort (Figure 7k). Collectively, these results strongly suggest that as one of the downstream targets of ZNF750, TINCR mediates the tumour-suppressive function of ZNF750 in SCC cells. Transcriptional repression of LAMC2 by ZNF750 in SCC cells LAMC2 is one of the seven candidate progenitor genes repressed by ZNF750 (Figure 5d). We and others previously have shown that LAMC2 promoted the proliferation and migration of several types of cancer cells. [14][15][16][17][18][19] Thus, we hypothesized that transcriptional repression of LAMC2 by ZNF750 might partially account for its tumour-suppressive activities. To determine whether ZNF750 regulates LAMC2 at the transcriptional level, we measured the levels of LAMC2 transcript and protein upon forced expression of either wildtype or C2H2 mutant ZNF750 (Figures 8a and b). LAMC2 expression was robustly decreased by wildtype but not C2H2 mutant ZNF750. A recent study found that ZNF750 interacted with target genes through recognizing the 'CCNNAGGC' DNA motif. 8 Thus, we searched for this motif near the transcription starting site of LAMC2, and identified it within regions of R2, R3, R4 (Figure 8c). R1 region that does not contain the motif was prepared as a negative control. ChIP-PCR results showed a prominent binding of ZNF750 to the R2 region (Figure 8d). To examine if this DNA region is responsible for ZNF750-mediated transcriptional regulation of LAMC2, dual-reporter luciferase assay was performed ( Figure 8e). Importantly, wildtype ZNF750 strongly suppressed the luciferase activity of R2 in 293 T cells (Figure 8e). These data indicate that ZNF750 directly inhibits the transcription of LAMC2 in SCC cells. We next asked whether LAMC2 was involved in ZNF750regulated cell migration. Depletion of LAMC2 inhibited cell migration ( Supplementary Figures 5a and b), whereas overexpression of LAMC2 enhanced this process ( Supplementary Figures 5c and d). Rescue experiments showed that reintroduction of LAMC2 in cells over-expressing ZNF750 significantly elevated the impaired cells' migration (Figures 8f and g). Taken together, these results strongly suggest that ZNF750 suppresses SCC cell migration by directly inhibiting LAMC2 transcription. DISCUSSION In this study, we demonstrated genomic and expression abnormalities and biological relevance of ZNF750, which is exclusive in human SCCs. Interestingly, TINCR LncRNA participated in control of cellular proliferation, migration and differentiation downstream of ZNF750. Furthermore, ZNF750 suppressed migration of SCC cells by directly inhibiting transactivation of LAMC2. Genome instability is one of the hallmarks of cancer, and somatic mutation and copy number loss are major causes of the inactivation of tumour suppressors. Based on our genomic analysis in three major types of human SCCs (CSCC, HNSCC and LSCC), 4.5% of ZNF750 mutations lead to detrimental frameshifts or premature truncations of ZNF750 protein. In addition, many ZNF750 as an SCC-specific tumour suppressor M Hazawa et al mutations were identified within the first 100 amino acids, which encodes for the evolutionally conserved C2H2 DNA-binding domain. We functionally validated that mutation of this domain resulted in its loss of function. A recent report studying normal keratinocytes also demonstrated that mutation of the C2H2 destroyed the ability of ZNF750 to activate differentiation genes. 5,8 Moreover, a cancer-derived mutant, ZNF750 S70X, caused mislocalization and abrogated its tumour suppressing function in ESCC. 4 Collectively, these data suggest that somatic mutations compromise ZNF750 function during tumorigenesis. ZNF750 as an SCC-specific tumour suppressor M Hazawa et al We further confirmed the downregulation of ZNF750 expression in these SCCs, which is consistent with its expression profiles in ESCC. 3,4 We also report, for the first time, the prognostic value of ZNF750 expression in all major SCCs, highlighting the biological relevance of ZNF750 in these malignancies. Tumour differentiation is an important clinical-pathological parameter affecting malignant potential. 10,20 Here, we showed that restoration of ZNF750 in SCCs cells activated a number of epidermal differentiation genes such as IVL, SPRR1B and S100A8. Importantly, these differentiation-related genes have been shown to be functionally relevant in SCCs. For example, S100A8/S100A9 were recently reported to inhibit G2/M progression and cell growth through enhancing PP2A phosphatase activity in HNSCC cells. 21 In addition, S100A8/S100A14 inhibited the oncogenic function of HPV16 E7 protein through regulating Casein Kinase IIdependent phosphorylation of E7. 22 Thus, ZNF750-regulated differentiation genes not only serve as markers of cellular differentiation but also modulate SCC cellular functions. We found that TINCR LncRNA mediates biological functions downstream of ZNF750. Recently, Kretz et al reported decreased TINCR expression in human SCC samples, 6 implying that TINCR has a tumour-suppressive role. However, oncogenic functions of TINCR through inhibiting KLF2 in gastric cancer was also observed recently. 23 Endogenous levels of KLF2 are undetectable in SCCs (unpublished data); therefore, the cancer-related function of TINCR might be context-dependent requiring further investigation. In summary, we show that somatic inactivation and decrease expression of ZNF750 contributes to the pathogenesis of SCC, and that ZNF750 expression is a novel biomarker for SCC prognosis. Through a variety of detailed mechanistic studies, both TINCR and LAMC2 were identified as functional downstream factors of ZNF750. These data will shed light on the understanding of the molecular basis of SCC biology. MATERIALS AND METHODS Cell culture HEK293T (kindly provided by Dr Yoshiaki Ito, Cancer Science Institute of Singapore), CSCC cell lines (C33A, CaSki, HT-3, ME180 and SiHa), skin SCC (SCC13), HNSCC cell lines (UMSCC1 and 93UV147) (kindly provided by Dr Timothy Chan, Memorial Sloan Kettering Cancer Center), and a spontaneously immortalized human keratinocyte cell line (HaCaT, kindly provided by Dr Norbert Fusenig, German Cancer Research Centre) were maintained in Dulbecco's Modified Eagle Medium supplemented with 10% (vol/vol) fetal bovine serum and 1% (vol/vol) penicillin/streptomycin (P/S), and LSCC cell lines (Calu-1, ChagoK1, H2170 and H226, kindly provided by Dr GOH Boon Cher, Cancer Science Institute of Singapore) were maintained in RPMI1640 supplemented with 10% fetal bovine serum and 1% P/S, at 37 ºC, 5% CO 2 in a humidified atmosphere. Cell lines were recently tested for the absence of mycoplasma. Cell lines were authenticated by short tandem repeat analysis with the Geneprint 10 System Kit (B9510, Promega, Madison, WI, USA) in 2014. Cell proliferation assay SCC cells were seeded into a 96-well plate at 3000 cells/well and cultured for the indicated time courses. Cell viability was assessed using the MTT (3-(4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) method. In brief, 10 μL of 12 mM MTT solution was added into each well followed by 3 h incubation, which was stopped by adding 100 μL of STOP solution (2% acetic acid, 16% SDS, 42% DMF). Samples were mixed thoroughly and measured at 570 nm for absorbance. Foci formation assay SCC cells were seeded into six-well plates at 1000 cells/well and cultured for 10 days, followed by fixation, staining with crystal violet and photographing the cells. For quantification, staining was dissolved in MTT stop solution, and measured at 570 nm for absorbance. Migration assay SCC cells were seeded onto transwell inserts (3422, Corning, Kennebunk, ME, USA) in 24-well plates and incubated for 48 h. The inserts were washed with PBS, and non-migrating cells were wiped off the top side. Migrated cells were fixed with 4% paraformaldehyde, stained with 4′,6-diamidino-2phenylindole (DAPI), and nuclei were counted. Adhesion assay Ninety-six-well tissue culture plates were coated with COL-I at 37°C for 1 h and rinsed with PBS. SCC cells were added to each well, and cells were allowed to incubate at 37°C for 30 min. After washing, adherent cells were estimated by MTT method described earlier. Western blotting Cells were lysed with lysis buffer (20 mM HEPES (pH 7.4), 350 mM sodium chloride, 1.5 mM magnesium chloride, 1 mM EGTA, 10% (v/v) glycerol, 1% Triton X-100, a mixture of protease inhibitors (Roche, Indianapolis, IN, USA), 0.2 mmol/L sodium orthovanadate and 1 mmol/L phenylmethylsulfonyl fluoride). BCA assay (Santa Cruz, Dallas, TX, USA) was used for protein quantification. Cell lysates or immunoprecipitation elutes were subjected to SDS-PAGE followed by conventional wet transfer. Membranes were incubated with antibodies as indicated and exposed to secondary horseradish peroxidase-conjugated antibodies (Millipore, Billerica, MA, USA). Antibodies used in this study were listed in Supplementary Table S2 cDNA vectors, shRNA vectors and siRNAs Expression vectors pLEX-ZNF750 (wildtype) and pLEX-C2H2 mutant ZNF750 (C2H2 mutant) were generously provided by Dr Paul A. Khavari (Stanford University School of Medicine). We also used another ZNF750 expression vector, SHC003-ZNF750 (which we reported previously 3 ), in some of the experiments to confirm that the results were not affected by different vector systems (Supplementary Figure 6). The shRNA construct for p63 was made with PLKO.1 backbone (Sigma-Aldrich) using Age I and EcoR I sites (Supplementary Table S3). LAMC2 over-expression and shRNA vectors were described previously. 24 Pooled siRNAs targeting human ZNF750 (M-014417-01) and TINCR (R-015725-00-0005) and scramble siRNA (D-001210-01) were purchased from Thermo Scientific (Rockford, IL, USA) (Supplementary Table S3). Transfections, viral particle production and infection Supplementary Table S4. Xenograft growth assay Female, 5-6 weeks, non-obese diabetic severe combined immunodeficient (NOD-SCID) mice were randomly allocated to control and experimental cohorts. Eight mice were used for control cohort; six and five mice were used for wildtype and C2H2 mutant groups, respectively. Three million UMSCC1 cells transduced with GFP (Control), wildtype or C2H2 mutant were mixed with 100 μl of Matrigel (BD Biosciences, San Jose, CA, USA) and injected subcutaneously in the upper flanks of the NOD-SCID mice. After 5 weeks, mice were euthanized; tumours were dissected and weighted. No blinding of investigators was performed. No statistical methods were used to determine the sample size of mice. The experiment was performed in compliance with ethical regulations of the Institutional Animal Care and Use Committee of the National University of Singapore. Bioinformatics and data analysis	2017-11-08T01:47:00.091Z	2016-11-07T00:00:00.000	{ "year": 2016, "sha1": "5f6753275329e75dbf0eeeefc11602f229ad4f64", "oa_license": "CCBYNCSA", "oa_url": "https://www.nature.com/articles/onc2016377.pdf", "oa_status": "HYBRID", "pdf_src": "PubMedCentral", "pdf_hash": "5f6753275329e75dbf0eeeefc11602f229ad4f64", "s2fieldsofstudy": [ "Biology", "Medicine" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
7887720	pes2o/s2orc	v3-fos-license	Research Article Isolation and Characterization of a Novel Facultative Anaerobic Filamentous Fungus from Japanese Rice Field Soil A novel filamentous fungus strain designated RB-1 was isolated into pure culture from Japanese rice field soil through an anaerobic role tube technique. The strain is a mitosporic fungus that grows in both aerobic and strict anaerobic conditions using various mono-, di-, tri-, and polysaccharides with acetate and ethanol productions. The amount of acetate produced was higher than that of ethanol in both aerobic and anaerobic cultures. The characteristic verrucose or punctuate conidia of RB-1 closely resembled those of some strains of the genus Thermomyces, a thermophilic or mesophilic anamorphic ascomycete. However, based on phylogenetic analysis with the small subunit (SSU) and large subunit (LSU) rDNA sequences, RB-1 was characterized as a member of the class Lecanoromycetes of the phylum Ascomycota. Currently, RB-1 is designated as an anamorphic ascomycete and is phylogenetically considered an incertae sedis within the class Lecanoromycetes. Introduction Rice fields are well known to be a major source of biological methane emission [1]. Many recent microbial analyses in rice fields have, therefore, focused on anaerobic microorganisms such as fermentative, acidogenic, and methanogenic microorganisms that are directly or indirectly involved in methane production [2]. Rice fields are usually flooded during the cropping season and drained after harvesting, and therefore, rice soils cycle through aerobic and anaerobic conditions throughout the year. During the drainage period, the major final decomposers of organic matter are aerobic or facultative anaerobic bacteria and filamentous fungi. Of these, many filamentous fungi are well known to secrete hydrolytic enzymes involved in biopolymer degradation, and therefore, they are considered to play significant roles in the decomposition of organic matter, especially in woods and upland soils. Filamentous fungi may also serve as decomposers of organic matter, such as plant debris and plant root exudation during the drainage period, in rice field soils. Kimura and Asakawa [3] showed the predominance of fungi in the microbial community of rice straw in a drained rice field using phospholipid fatty acids (PFLPs) as biomarkers. Hatamoto et al. [4] indicated by PCR-DGGE analysis that eukaryotes including fungi were associated with the decomposition of rice straw compost incorporated into rice soil especially at the midseason drainage. Many saprobic fungi have been isolated from Ukrainian rice soil before submerging and after draining [5]. However little is known about the ecological roles of such filamentous fungi during the water-logged phase because fungal species capable of anaerobic growth has not ever been isolated from rice soils. Although most filamentous fungi are obligate aerobes, some obligate anaerobic species, collectively called rumen fungi and classified as Chytridiomycota [6], do exist in spite of the fact that their habitat is limited to the gastrointestinal tract of herbivorous mammals. Tabak and Cooke [7] reported anaerobiosis of 13 fungal strains isolated from oxygen-limited environments, such as sewage sludges, polluted waters, and organically enriched soils. Previously, Wada [8] reported that an unidentified spore-forming filamentous fungus colonized rice straws in submerged rice field soil where the anaerobic condition was sufficient for sulfate reducing. Saito et al. [9] showed that cellulose filter papers or cellophane films incorporated into submerged rice field soil were transiently colonized by fungal hyphae ten days after flooding. However, further studies on those fungal-like organisms were not conducted. Recently, it has 2 International Journal of Microbiology been suggested that Fusarium oxysporum likely acquires energy for growth by denitrification [10] and by ammonia fermentation [11]. Fungal denitrification occurs in an O 2 limited environment in which N 2 O is generated as a final denitrified product because of the lack of N 2 O reductase to generate N 2 from N 2 O [10]. On the other hand, fungal ammonia fermentation occurs in an anaerobic environment in which NO 3 − is reduced to NH 4 + and ethanol is oxidized to acetate [11]. The fungal denitrification and ammonia fermentation in response to hypoxic conditions are considered to extensively distribute among many fungi [12]. These findings show the possibility that filamentous fungi capable of growth in the absence of oxygen are likely distributed in anaerobic environments in greater numbers than expected. These results suggest that some fungi capable of anaerobiosis certainly exist in rice soils. The author, therefore, attempted to isolate anaerobic filamentous fungi living in rice soils by applying the method of Hungate's roll-tube anaerobic technique [13] generally used in colonization of anaerobic prokaryotic cells. Here, the author reports the isolation and characterization of a novel facultative anaerobic fungus from Japanese rice field soil. Fungal Strain. Thermomyces lanuginosus NBRC 9738 purchased from the National Institute of Technology and Evaluation was used for comparison with RB-1. Media. Martin's medium [14] reduced with 0.03% cysteine hydrochloride was used for anaerobic fungal isolation from rice field soil. For usual cultivation, basal medium (0.1% KH 2 PO 4 , 0.05% MgSO 4 7H 2 O, 0.5% peptone, 0.1% yeast extract, and 1% glucose, (pH 7.0)) was used. For testing utilization of substrates, 1% glucose in the basal medium was substituted by the indicated substrates. For examining cultural and morphological characteristics, potato dextrose agar (PDA: NISSUI) was used. For anaerobic cultivation, media containing 0.0001% resazurine were prepared under an oxygen-free N 2 atmosphere for visual check of reducing conditions. Before inoculation, the media were reduced by aseptically adding cysteine hydrochloride (adjusted to pH 7.0) to a final concentration of 0.03%. The tubes, vessels, and flasks used were closed with cotton plugs for aerobic cultivation and butyl rubber stoppers for anaerobic cultivation. If needed, suitable amounts of CaCO 3 were added to the medium as a pH stabilizer to maintain the culture pH at 5.6-5.8. Media were solidified by adding 2% agar when required. Isolation Procedure. Isolation of the fungus used in this study was essentially carried out in accordance with the Hungate roll tube technique [13]. All procedures except soil sampling and aerobic incubation on PDA during the final step of isolation were carried out anaerobically under a stream of oxygen-free N 2 . Soil was collected from 0-10 cm depth from the rice field at Kanagi farm of the Teaching and Research Center for Biocoexistence at Hirosaki University, Aomori, Japan, in April 2004. Some of the soil properties were as follows: pH (H 2 O), 6.0; water content, 38.9%. The soil sample was serially diluted in a 0.03% cysteine hydrochloride solution (pH 7.0) and transferred to agar-solidified medium (reduced with 0.03% cysteine hydrochloride) role tubes, which were then incubated at 25 • C in a vertical position. After two months of incubation, the mycelium of a fungal colony formed on the agar was transferred to the same anaerobic fresh slant medium and incubated at 25 • C. After two repetitions of the procedure, the mycelium developed on the anaerobic slant was transferred to a PDA plate and aerobically incubated at 25 • C. A single spore isolate from the PDA plate culture was designated RB-1 and was used for further study. The cultures of RB-1 were maintained on PDA slants prepared in 25 mL tubes under both aerobic and anaerobic conditions. The RB-1 strain has been deposited in the Japan Collection of Microorganisms (JCM) as JCM13780. Morphological Observations and Cultural Characteristics. For morphological observations and determination of cultural characteristics, the isolate was cultured aerobically or anaerobically on PDA plates at 25 • C or on PDA thinly spread on a glass slide at 25 • C (slide culture). AnaeroPack (Mitsubishi Gas Chemicals Co., Inc.) was used for generating anaerobic atmosphere. After staining with lactophenol cotton blue, the morphology of the isolate was observed under differential interference contrast with the use of an Olympus microscope (BX50F4) or under phase contrast with an Olympus microscope (MX50). For macroscopic observations, a stereomicroscope (Olympus SZH10) was used. Colony colors were evaluated based on the Methuen Handbook of Color [15]. Phylogenetic Analysis. Genomic DNA of the isolate was prepared as described previously [16]. Nearly full-length small subunit (SSU) rDNA (1,760 bp) was PCR-amplified using the primer pair E21f (5 -ATCTGGTTGATCCTGCCAGT-3 ) and E1778r (5 -AATGATCCTTCCGCAGGTTC-3 ) [17]. For amplification of the internal transcribed spacer (ITS) region and the 5 end of large subunit (LSU) rDNA including the D1-D2 domains (ITS-LSU rDNA) (1,259 bp), a primer set ITS5 (5 -GGAAGTAAAAGTCGTAACAAGG-3 ) [18] and NL4 (5 -GGTCCGTGTTTCAAGACGG-3 ) [19] was used. The PCR products were directly sequenced using the Thermo Sequenase CY5.5 dye terminator cycle sequencing kit and the SEQ 4.4 personal sequencing system (Amersham). The sequences of SSU rDNA and ITS-LSU rDNA of the isolate are deposited in the DDBJ/GenBank/EMBL databases under the accession numbers AB246879 and AB517730, respectively. The determined sequences and those of relatively closely related taxa to the isolate retrieved from the DDBJ/GenBank/EMBL databases by searching with BLAST program were aligned using the CLUSTAL W program [20]. The evolutionary distances between the sequences were calculated using Kimura's two-parameter model [21], and the phylogenetic trees were constructed by the neighbor-joining method [22] using the MEGA 4.0 [23] computer program. Bootstrap analyses were conducted to assess the confidence limits of the branching by 1000 heuristic replicates. Biophysical and Biochemical Characteristics. All anaerobic incubations were carried out under an oxygen-free N 2 atmosphere. The temperature and pH profiles of the isolate were determined by measuring the diameters of the colonies developed under aerobic conditions. The temperature profile was tested on the PDA plate by incubating at 4-50 • C. The pH profile was tested on the basal medium plate at a pH range from 2.0 to 9.5. Incubations were carried out at 30 • C. For testing available substrates for fermentation, autoclaved or filter-sterilized substrates were added to 10 mL of basal medium instead of glucose in culture tubes, and the mycelium from the PDA slant was inoculated into the medium. Incubations were carried out at 30 • C for up to 3 months under aerobic or anaerobic conditions without shaking. The substrates were considered positive when acetate and ethanol were detected in the medium. Ethanol and acetate were measured as mentioned in Analytical Procedures. Time course of growth by the isolate under aerobic and anaerobic conditions was determined using 500 mL Erlenmeyer flasks with 100 mL of basal medium inoculated with the equivalent of 1.5 mg (dry weight) mycelium previously grown aerobically in 10 mL basal medium at 30 • C for 10 days. After inoculation, the flasks were incubated at 30 • C in a reciprocal shaker operating at 100 rpm. Samples were collected at various intervals by filtration through nitrocellulose filters and were measured after drying to a constant weight at 105 • C; the resulting filtrates were used to determine the residual glucose and fermentation products. Growth yields of the isolate on aerobic and anaerobic medium were determined using 125 mL serum bottles with 100 mL of basal medium inoculated with a trace amount (<0.1 mg) of mycelium from the PDA slant picked with a needle tip. After static incubation at 30 • C for 10 days, cultures underwent the same analytical procedures for growth analysis as described above. Growth ability of the isolate under anaerobic conditions was confirmed by a successive transfer experiment as described by Gleason and Gordon [24]. The mycelium of the isolate grown on the PDA slant was inoculated into 100 mL anaerobic basal medium prepared in a 125 mL serum bottle. The culture bottle was incubated at 30 • C and a small part of the mycelium grown in the medium was transferred every five days into the same medium. Exposure of mycelium to an atmospheric condition was avoided as possible with an immediate transfer to the next fresh medium. All experiments were repeated more than three times to confirm reproducibility. Analytical Procedures. Glucose was quantified using the anthrone-sulfuric acid method [25]. Ethanol was analyzed in a gas chromatograph (Shimadzu GC8A) equipped with an FID detector and a Porapaq N column (Waters). Organic acids were measured by using HPLC (Organic Acid Analysis System, Shimadzu) equipped with an ion exclusion column (Shim-pack SCR-101H) and detected by a post-column pH-buffered electroconductivity detection method [26] according to the manufacturer's instructions. Glycerol was enzymatically assayed with glycerokinase using commercial kits (Boehringer Mannheim). Gas samples were determined by gas chromatography (Shimadzu GC8A) equipped with a TCD detector and a WG-100 column (GL Sciences). Morphological and Cultural Characteristics. The strain designated RB-1 was a filamentous fungus isolated from Japanese rice field soil that could grow under both aerobic and anaerobic conditions. After six weeks of aerobic incubation at 25 • C on a PDA plate, the colonies were olive to olive-green in color, with a floccose texture, and few aerial mycelia. The color of the colonies was white initially and then turned from light green to dark green and eventually from olive to olive-green. Exudates and soluble pigment were not produced. In the vegetative stage, septate hyphae, which were 2.0-3.0 μm in diameter, changed in color from hyaline to bright brown with aging and were formed in the agar and on the agar surface. The morphological features of RB-1 after two months of aerobic incubation are shown in Figures 1(a)-1(e). Distinct conidiophore-like structures were absent (Figure 1(a)). Conidiogenous cells were cylindrical or indistinguishable from the vegetative hyphae (Figure 1(a)). Single or occasionally chained aleurioconidia, which were hyaline when young and brown to dark brown when mature, were borne on the tips of the slightly swollen conidiogenous cells or directly on the sides of the vegetative hyphae (Figures 1(a) and 1(b)). Mature conidia were globose-shaped or sometimes slightly subglobose-shaped single cells, 7.5-11.3 μm in diameter, and their surfaces were verrucose or punctuate (Figure 1(c)). Chlamydospores, which were smooth or verrucose, globose to oval, light brown to dark brown, and 9.0-11.0 μm in diameter, occurred frequently in the vegetative hyphae (Figure 1(d)). Sexual reproductive organ development was not observed. Instead, primordiumlike structures constructed with subglobose and globose cells were formed after two months of incubation (Figure 1(e)). Colonies on the anaerobic PDA plate were always white, and aerial mycelia were rarely observed, at least during the incubation period (up to 3 months). In the presence of CaCO 3 , colonies on the anaerobic PDA plate developed at a rate similar to that under aerobic conditions with dissolving insoluble particles of CaCO 3 . However, in the absence of CaCO 3 , colony development was slower than that under aerobic conditions and ceased when the colonies reached approximately 60 mm in diameter, which indicates that the lowered pH caused by acid production inhibited colony development. Although normal conidium development did not occur under any anaerobic conditions, some swollen cells looking little similar to conidium and chlamydospore structures were observed after three months of incubation (Figure 1(f)). Thermomyces lanuginosus [14], a fungal species apparently related morphologically to RB-1, did not grow under any anaerobic conditions. Moreover, acid production in its aerobic cultures was not observed different from those of RB-1. Hence further physiological and biochemical studies on T. lanuginosus were not carried out. Phylogenetic Analysis. The SSU rDNA sequence of RB-1 showed less than 96% similarity with those of other known fungal species and was most closely related to Bellemerea alpina (95.3% identity), which belongs to the order Lecanorales of the class Lecanoromycetes. A phylogenetic tree based on the sequences of SSU rDNA retrieved from the DDBJ/GenBank/EMBL databases is shown in Figure 2. Totals of 30 taxa (1,659 to 1,669 bp after alignment) were used. In this tree, RB-1 is placed within the class Lecanoromycetes of the phylum Ascomycota. T. lanuginosus showed a relatively low SSU rDNA sequence similarity (92.0% identity) with RB-1 and the presence in the class Eurotiomycetes ( Figure 2). In order to gain further insight into the classification of RB-1, a phylogenetic analysis using the LSU rDNA D1-D2 sequences was conducted. A DDBJ/GenBank/EMBL search revealed that the D1-D2 sequence of RB-1 had low similarity with those of related fungal species (less than 89% identity). The D1-D2 sequence of Caloplaca regalis, which is classified as Teloschistales of the class Lecanoromycetes, showed the highest similarity (88.6% identity) with that of RB-1. A phylogenetic tree constructed based on the D1-D2 sequences, which were retrieved from the DDBJ/GenBank/EMBL databases, is shown in Figure 3. Totals of 29 taxa (496 to 500 bp after alignment) were used to construct a phylogenetic tree. In this tree, RB-1 is related to Lecanoromycetes rather than to Eurotiomycetes. T. lanuginosus is placed in the class Eurotiomycetes as same as the phylogenetic tree constructed with the SSU rDNA sequences (Figure 2). The identity of the D1-D2 sequences between RB-1 and T. lanuginosus was 82.1%. Phylogenetic analyses using the ITS1 and ITS2 sequences were not carried out because ITS sequences related to those of RB-1 which are suitable for construction of phylogenetic trees with high confidence could not be retrieved from the DDBJ/GenBank/EMBL databases. Table 1. RB-1 grew by consuming glucose and producing acetate and ethanol as fermentation products under both aerobic (Figure 4(a)) and anaerobic (Figure 4(b)) conditions. Growth rate was almost similar between the aerobic and anaerobic cultures. Other possible products including secondary organic compounds, such as hydrogen, succinate, pyruvate, formate, citrate, lactate, glycerol, isopropanol, acetone, propionate, butyrate, glyoxalate, and malonate, were not detected. Growth and substrate consumption terminated immediately after the pH dropped below ∼4.7. Glucose was completely consumed in both aerobic and anaerobic cultures and growth was not terminated if the pH of the cultures was stabilized with CaCO 3 ; acetate and ethanol produced in the pH-stabilized aerobic culture were consumed immediately after the exhaustion of glucose (data not shown). Physiological and Biochemical Characteristics. Physiological and biochemical properties are summarized in The available initial pH for the growth of RB-1 ranged between 2. RB-1 could use D-glucose, D-galactose, D-mannose, Dfructose, D-xylose, L-arabinose, sucrose, maltose, cellobiose, trehalose, raffinose, starch, inulin, xylan, and pectin as fermentation and growth substrates. Fermentation on trehalose, inulin, and xylan was much weaker than that on other available substrates. All these substrates supported growth and were fermented to acetate, ethanol, and carbon dioxide. In the aerobic cultures, all substrates that were fermentable under anaerobic conditions were also converted to ethanol and acetate. On the other hand, RB-1 did not use L-rhamnose, lactose, melibiose, D-mannitol, Dsorbitol, glycerol, Avicell, carboxymethylcellulose, or chitin as fermentation and growth substrates. Growth yield and quantitative determinations of ethanol and acetate production on D-glucose are given in Table 2. More acetate than ethanol was produced under both anaerobic (1.8 fold) and aerobic (1.9 fold) conditions. Growth of RB-1 under anaerobic conditions (in a cysteine-reduced liquid medium) was sustained constantly from the initial culture after eleven successive transfers in the same medium. Discussion Because filamentous fungi are commonly recognized as obligate aerobic organisms, little interest has been paid to their ecological roles and functions in anaerobic environments, including flooded rice field soils. Accordingly, filamentous fungi have been considered to be less important constituents of natural anaerobic ecosystems; few studies on anaerobic filamentous fungi in natural habitats have been reported [7,[27][28][29]. In the present study, the author reports on the isolation of a novel facultative anaerobic filamentous fungus from Japanese rice field soil. Since no sexual reproductive organs were observed, from a morphological standpoint, RB-1 is currently considered a mitosporic fungus. The morphological characteristics of RB-1 are closely related to those of Thermomyces lanuginosus and Thermomyces verrucose [14]; the former species has also been isolated from Japanese field soil [30]. However, these species differ from RB-1 in forming single conidia from the conidiophores and their temperature profiles [31]. Moreover, T. lanuginosus showed no anaerobic growth as reported previously [32]. The phylogenetic statuses of T. lanuginosus and T. verrucose are indistinct, so they are currently classified as mitosporic ascomycetes (anamorphic ascomycetes) [33]. However, recently it has been shown that T. lanuginosus is placed in the class Eurotiomycetes and in the order Eurotiales based on the phylogenetic analysis using SSU rDNA and ITS sequences [34] as shown here (Figures 2 and 3). Usually, in mitosporic fungi, similarities in morphological characteristics do not necessarily indicate phylogenetic relations. Certainly, there are relatively low similarities of the SSU rDNA (92.0% identity) and the D1-D2 sequences (82.1% identity) between RB-1 and T. lanuginosus, which indicates that they are phylogenetically distant. According to the phylogenetic analysis based on the SSU rDNA and LSU rDNA sequences, RB-1 was probably placed within the class Lecanoromycetes of Ascomycota (Figures 2 and 3) although the precise phylogenetic position is currently unclear. Further phylogenetic studies using other markers such as elongation factors and mitochondrial rDNA should be required. Thus, although RB-1 is probably an anamorph of an unknown ascomycete considered an incertae sedis within the class Lecanoromycetes, the classification is still inadequate to define its precise taxonomic status; therefore, further detailed taxonomic analyses will be needed for nomenclature. RB-1 can grow both aerobically and anaerobically, unlike most other ethanol-and acetate-producing fungi; almost all of which can ferment but not grow under anaerobic conditions [35,36]. In successive transfer experiments for confirming the anaerobiosis, the growth ability of RB-1 in the strict anaerobic liquid medium reduced with cysteine was steadily maintained even after eleven successive transfers. A study of anaerobic growth of zygomycetes showed that strains that are apparently capable of anaerobic growth, but require extremely small amounts of oxygen for any growth, are diluted out during successive transfers in anaerobic medium, whereas a strain truly capable of anaerobic proliferation continues growth after ten successive transfers [24]. Consequently, it is concluded that RB-1 is a facultative anaerobic fungus. As far as known to the author, few facultative anaerobic filamentous "higher fungi" that can produce metabolic energy by simple fermentation, such as the yeast Saccharomyces cerevisiae, have been identified [28,29]. As shown in Figure 4 and Table 2, the amount of acetate was higher than that of ethanol, unlike other fermentable ethanol-producing fungi; almost all of which produce smaller amounts of acetate than ethanol [36]. A simple comparison cannot be made because of differences in the culture conditions and media between those previous studies and this study; hence the reason for this remains unclear, and metabolic analyses of RB-1 are ongoing. RB-1 produced significant amounts of ethanol and acetate under 8 International Journal of Microbiology complete aerobic conditions. This phenomenon is similar to that of the Crabtree-positive yeast cultures in which ethanol production occurs under full aerobic conditions when sugar is present in excess [37,38]. Hence, RB-1 is probably a Crabtree-positive organism. Productions of possible compounds other than ethanol, acetate, and carbon dioxide in the culture of RB-1 were not detected. If produced, their amounts may be smaller than the detection limits (<ca. 0.1-0.5 mM) of measuring systems (HPLC and GC) used in this study. Interestingly, RB-1 shows a significant acetate kinase (ACK) activity, thought to be found strictly in prokaryotic cells, during the growth under both aerobic and anaerobic conditions (unpublished data). Recently the presence of the ACK genes in several eukaryotes inducing fungi was identified [39]. However, the activity of ACK from those eukaryotes has not been confirmed. Thus, RB-1 may be an interesting organism for study of acetate metabolism. The author and coworkers are now attempting to purify ACK from RB-1. The phylogenetic, biophysical, and biochemical features of RB-1 indicated here can be distinguished from those of other known fungal species. Especially, RB-1 is phylogenetically far from known fungal genera. Hence, the author concludes that RB-1 is a novel fungus. As rice fields that are usually exposed to rotating periods of irrigation and drainage during cropping, plowed soils of the fields also rotate through anaerobic and aerobic conditions. Accordingly, microbial colonies will go through changes responsive to irrigation (anaerobic conditions) and drainage (aerobic conditions). Moreover, plowed soils of flooded rice fields are heterogeneous environments in which oxygen concentrations differ from those of other anaerobic sites. For example, bulk soils below the soil-water interface are strictly anaerobic; because oxygen is supplied through floodwaters and the aerenchyma of rice plants, the soil surface, the rhizosphere, and the rhizoplane are aerobic, respectively [40]. Facultative anaerobes are probably more adaptable microorganisms in such atypical sites compared to obligatory aerobic or anaerobic microorganisms. Hence, although the ecological role and function of RB-1 in rice soil ecosystem are currently unknown, the abilities of RB-1 to acquire metabolic energy and to proliferate in both aerobic and anaerobic environments may be advantageous in such environments. Previously, Wada [8] reported that an unidentified spore-forming filamentous fungus colonized rice straws in submerged rice soil where the anaerobic condition was sufficient for sulfate reducing. Recently, several researchers reported that when nitrate is supplied, many filamentous fungi can acquire metabolic energy in oxygenlimited and anaerobic environments by nitrate respiration and ammonia fermentation, respectively [10,11,41]. A recent research indicated that up to 89% of N 2 O emitted from soils to atmospheres could be attributed to fungal activity [42]. All these findings suggest that filamentous fungi capable of growth in the absence of oxygen are likely distributed in anaerobic environments in greater numbers than expected.	2014-10-01T00:00:00.000Z	0001-01-01T00:00:00.000	{ "year": 2009, "sha1": "1f79c6157bda7aa3088d3d0f88931a9ff132fa3b", "oa_license": "CCBY", "oa_url": "http://downloads.hindawi.com/journals/ijmicro/2009/571383.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "73db3675a108b3a7d336741666f575a1f82dda39", "s2fieldsofstudy": [ "Agricultural and Food Sciences", "Biology", "Environmental Science" ], "extfieldsofstudy": [] }
16681879	pes2o/s2orc	v3-fos-license	Novel regulatory role of neuropilin-1 in endothelial-to-mesenchymal transition and fibrosis in pancreatic ductal adenocarcinoma Pancreatic ductal adenocarcinoma (PDAC) is characterized by an intense fibrotic reaction termed tumor desmoplasia, which is in part responsible for its aggressiveness. Endothelial cells have been shown to display cellular plasticity in the form of endothelial-to-mesenchymal transition (EndMT) that serves as an important source of fibroblasts in pathological disorders, including cancer. Angiogenic co-receptor, neuropilin-1 (NRP-1) actively binds TGFβ1, the primary mediator of EndMT and is involved in oncogenic processes like epithelial-to-mesenchymal transition (EMT). NRP-1 and TGFβ1 signaling have been shown to be aberrantly up-regulated in PDAC. We report herein a positive correlation between NRP-1 levels, EndMT and fibrosis in human PDAC xenografts. Loss of NRP-1 in HUVECs limited TGFβ1-induced EndMT as demonstrated by gain of endothelial and loss of mesenchymal markers, while maintaining endothelial cell architecture. Knockdown of NRP-1 down-regulated TGFβ canonical signaling (pSMAD2) and associated pro-fibrotic genes. Overexpression of NRP-1 exacerbated TGFβ1-induced EndMT and up-regulated TGFβ signaling and expression of pro-fibrotic genes. In vivo, loss of NRP-1 attenuated tumor perfusion and size, accompanied by reduction in EndMT and fibrosis. This study defines a previously unrecognized role of NRP-1 in regulating TGFβ1-induced EndMT and fibrosis, and advocates NRP-1 as a therapeutic target to reduce tumor fibrosis and PDAC progression. Although the origin of fibroblasts is complex and dependent on several factors, the importance of endothelial-to-mesenchymal transition (EndMT) as an important source of activated fibroblasts is an emerging concept [29][30][31]. Besides the crucial function carried out as a part of the endothelium, endothelial cells have displayed the ability of acquiring an extreme form of cell plasticity, whereby they acquire a mesenchymal cell phenotype. Primarily, EndMT is characterized by the acquisition of mesenchymal cell surface markers such as N-cadherin, αSMA and types I/III collagen with the corresponding loss of endothelial cell surface markers like VE-cadherin, CD31, etc. [29,31]. While tightly regulated, EndMT is critical in the development of the primitive heart [32] and wound healing [33], and maladaptive EndMT has been linked to a variety of fibrotic pathologies including cancer [31,34]. Zeisberg et al. demonstrated convincing genetic evidence for EndMT-derived CAFs in the tumor microenvironment, whereby up to 40% of CAFs were derived via EndMT [35]. Mechanistically, several studies have established the involvement of TGFβ signaling in modulating EndMT [36][37][38][39]. However, mechanistic work involving EndMT in the context of diseased states like cancer remains obscure and warrants further investigation. In the current study, we hypothesized that NRP-1 is crucial in regulating TGFβ1-induced EndMT and associated fibrosis in PDAC. To test our hypothesis, we utilized translational loss of function and over-expression study approaches. Firstly, we demonstrate a robust positive correlation between NRP-1 expression, EndMT and associated fibrosis in human PDAC xenografts. Using loss of function and overexpression studies in vitro, we demonstrate a novel regulatory role of NRP-1 in TGFβ1-induced EndMT and fibrosis. Knockdown of NRP-1 limited while NRP-1 overexpression exacerbated TGFβ1-induced EndMT respectively. Overall, these data reveal a previously undetermined role of NRP-1 in regulating TGFβ1-induced EndMT as a potential therapeutic target to limit EndMT-associated fibrosis in PDAC. RESULTS Human PDAC tissue shows positive correlation between NRP-1 expression, EndMT and fibrosis H&E staining of human tumor tissues displayed a typical ductal morphology of PDAC ( Figure 1A). Further characterization of the xenografts is summarized in Tables 1 and 2. NRP-1 was aberrantly expressed in all the tissue samples at varying levels as assessed by immunohistochemistry studies ( Figure 1A). Furthermore, collagen content as detected by Masson's trichrome staining, demonstrated a significant positive correlation with NRP-1 expression ( Figure 1A, 1B). We observed a consistent increase in the expression of EndMT markers and pro-fibrotic markers with increasing NRP-1 expression at the transcript ( Figure 1C) and protein level ( Figure 1D, 1E). Contrarily, low levels of EndMT and pro-fibrotic markers were associated with reduced NRP-1 expression at the transcript and protein level ( Figure 1C, 1D, 1E). Overall, we observed a robust positive correlation between NRP-1 expression, EndMT markers and pro-fibrotic genes in human PDAC tissue ( Figure 1F), suggesting a previously undetermined role of NRP-1 in regulating EndMT and associated fibrosis in pancreatic tumors. NRP-1 siRNA leads to successful knockdown at the transcript, protein and functional levels in HUVECs We performed siRNA-mediated knockdown of NRP-1 in HUVECs. In vitro knockdown studies were performed in three groups: 1. non-transfected control, 2. transfected scramble control and 3. transfected siNRP-1. Data from second and third groups are shown in the manuscript since data from the non-transfected control and scramble control transfected HUVECs were similar (data not shown). NRP-1 knockdown was confirmed by a significant reduction in NRP-1 expression by qPCR and immunoblotting analysis at different time points (Figure 2A, 2B). NRP-1 is primarily involved in angiogenesis through its co-receptor function with VEGFR-2 by binding to VEGF-A [44]. We therefore performed the capillary-like tube formation assay in HUVECs to assess NRP-1 knockdown at functional level and observed that siNRP-1 demonstrated a significant reduction of number of nodes and tubes as compared to scramble control ( Figure 2C, 2D). Loss of NRP-1 inhibits TGFβ1-induced phenotypic switching typical of endothelial-tomesenchymal transition in HUVECs Light microscopy studies showed that TGFβ1stimulated scramble-transfected HUVECs demonstrated a distinctive morphological change from typical 'cobblestone-like endothelial cell morphology' to an enlarged spindle shaped manifestation, characteristic of 'mesenchymal-like cell morphology' ( Figure 2E). Additionally, we observed that these morphological changes were associated with cytoskeletal protein reorganization. In accordance, we observed an increase in α-actinin expression and a representative mesenchymal cell-like cytoskeletal protein re-organization ( Figure 2F). Interestingly, these distinctive morphological and ultrastructural protein re-organization changes were inhibited upon loss of NRP-1 ( Figure 2E, 2F). Phenotypic EndMT characteristics are accompanied by corresponding changes in the expression of cell surface markers [24,26]. In keeping with characteristics of reversal of EndMT, upon TGFβ1 stimulation, loss of NRP-1 significantly induced the expression of endothelial markers; VE-cadherin and CD31, and inhibited the expression of mesenchymal markers; αSMA, N-cadherin and transcription factor Slug as assessed by qPCR ( Figure 2G), immunoblotting ( Figure 2H) and immunofluorescence studies ( Figure 2I) when compared to scramble control transfected cells. Mechanistically, TGFβ signaling is crucial in regulating EndMT [36][37][38]. Knockdown of NRP-1 in TGFβ1-stimulated HUVECs resulted in significantly reduced expression of TGFβ1 ligand at the transcript ( Figure 2J) and protein ( Figure 2K) level when compared to the scramble control. The TGFβ1 isoform uses the same set of receptors as other isoforms consisting of TGFBR1 and TGFBR2 [47]. TGFβ1 binds to TGFBR2 initially, which further bind to TGFBR1 to form a complex with serine/threonine kinase activity, where TGFBR2 phosphorylates TGFBR1. This complex is further capable of phosphorylating downstream molecules belonging to the SMAD family of proteins that possess the transcription regulatory activity upon translocation to the nucleus. Knockdown of NRP-1 down-regulated TGFBR1 and TGFBR2 at transcript and protein levels as assessed by qPCR and immunoblotting, respectively, in TGFβ1stimulated HUVECs ( Figure 2J, 2K). Loss of NRP-1 was further accompanied by notably reduced SMAD2 phosphorylation as demonstrated by immunoblotting ( Figure 2K, Supplementary Figure 1A, 1B). Upon investigating the expression of TGFβ1-dependent profibrotic genes, like CTGF and Collagen 1, we observed that loss of NRP-1 in TGFβ1-stimulated HUVECs significantly decreased their expression at the protein level as detected by immunoblotting ( Figure 2H). As anticipated, reduction in SMAD2 phosphorylation and expression of pro-fibrotic genes was not observed in the scramble control HUVECs group ( Figure 2H, 2K). Intriguingly, in the absence of TGFβ1 stimulation, loss of NRP-1 significantly increased the expression of endothelial markers; VE-cadherin and CD31, however, the decrease in expression of mesenchymal markers; N-cadherin and αSMA, although moderately reduced, was not significant as assessed by qPCR (Supplementary Figure 1F). the morphology; for NRP-1 levels (indicated by brown color) by immunohistochemistry and for extent of fibrosis by Masson's trichrome staining (indicated by blue color). Representative images reveal varying levels of tissue differentiation (Scale bar = 100 μm), NRP-1 expression (Scale bar = 50 μm) and extent of fibrosis in the xenografts (Scale bar = 100 μm). (B) Quantification of NRP-1 levels and collagen content using the ImageJ software (expressed in AU, arbitrary units). NRP-1 levels positively correlate with EndMT markers and pro-fibrotic genes at the transcript (C) and protein level (D) as determined by qPCR and western blotting respectively. Correlation was determined by Spearman's Rho test (positive correlation: 0.8 < R ≤ 1, R = correlation coefficient). For C, data has been normalized to tissue sample having lowest expression of the respective genes that are plotted. (E) Representative immunohistochemistry images showing correlation between NRP-1 level and EndMT markers in tumor tissues having the lowest and highest NRP-1 expression as determined previously (Scale bar = 100 μm). (F) Summary of NRP-1-EndMT correlation. Lentivirus-mediated NRP-1 transduction in HUVECs leads to successful overexpression at the transcript, protein and functional levels We performed lentiviral-mediated overexpression of NRP-1 in HUVECs. In vitro overexpression studies were performed in three groups; 1. non-transduced control, 2. transduced lentiControl and 3. transduced lentiNRP-1. Data from empty control (LentiControl) and lentiNRP-1 are shown in the manuscript since data from the non-transduced control and empty control transduced HUVECs were indistinguishable (data not shown). NRP-1 overexpression was confirmed by a striking increase in NRP-1 expression by qPCR ( Figure 3A) and immunoblotting ( Figure 3B) analysis at different time points. The capillary-like tube formation assay exhibited that lentiNRP-1 demonstrated a significant increment in number of nodes and tubes as compared to lentiControl owing to the co-receptor function of NRP-1 during VEGF-induced angiogenesis ( Figure 3C, 3D). Overexpression of NRP-1 in TGFβ1-stimlated HUVECs induces distinct morphological and molecular changes consistent with EndMT Light microscopy studies showed that overexpression of NRP-1 in TGFβ1-stimlated HUVECs lead to a distinctive morphological change, in accordance with phenotypic switching associated with EndMT ( Figure 3E). Furthermore, we observed an increase in α-actinin expression and a mesenchymal cell-like cytoskeletal protein Yes Distant 681 Abbreviations: M-male; F-female. Data has been adapted from Lohse et al. [85]. 1 TNM classification of tumors of the exocrine pancreas: T2, tumor limited to the pancreas, more than 2 cm in greatest dimension; T3, tumor extends directly into any of the following: duodenum, bile duct, peripancreatic tissues; T4, tumor extends directly into any of the following: stomach, spleen, colon, adjacent large vessels; N0, no regional lymph node metastasis; N1a, metastasis in a single regional lymph node; N1b, metastasis in multiple regional lymph nodes. Patient alive when the study was published. No Gemcitabine No indication of disease 5FU-irinotecanoxaliplatin-leucovorin + Initial therapeutic response but progressed after 6 months Veliparib + Progression of disease Abbreviations: 5FU-5-fluorouracil. Data has been adapted from Lohse et al. [85]. Adjuvant, # Neoadjuvant, + Palliative. www.impactjournals.com/oncotarget re-organization upon NRP-1 overexpression as compared to lentiControl ( Figure 3F). In agreement with TGFβ1induced EndMT characteristics, NRP-1 overexpression significantly down-regulated the expression of endothelial markers; VE-cadherin and CD31, and induced the expression of the mesenchymal markers; N-cadherin, αSMA and Slug as assessed by qPCR ( Figure 3G), immunoblotting ( Figure 3H) and immunofluorescence ( Figure 3J). Interestingly, NRP-1 overexpression in the absence of TGFβ1 stimulation resulted in an increase in mesenchymal markers' expression but no significant decrease in the expression of all the assessed endothelial cell markers (Supplementary Figure 1G). Examining at the mechanistic level, we observed that overexpression of NRP-1 in TGFβ1-stimulated HUVECs resulted in significantly elevated expression of TGFβ1 at the transcript and protein level when compared to lentiControl ( Figure 3J, 3K). Additionally, overexpression of NRP-1 up-regulated TGFBR1 and TGFBR2 at transcript and protein level as assessed by qPCR ( Figure 3J) and immunoblotting ( Figure 3K) respectively, and led to increased SMAD2 phosphorylation as demonstrated by immunoblotting ( Figure 3K). Overexpression of NRP-1 along with TGFβ1-stimulation significantly augmented the expression of TGFβ1-dependent pro-fibrotic genes, like CTGF and Collagen 1 at transcript and protein level as compared to lentiControl ( Figure 3G, 3H). Thus our NRP-1 overexpression study was antagonistic with our loss of NRP-1 function study, and in accordance with our hypothesis. This confirms the novel in vitro regulatory role of NRP-1 in TGFβ1-induced EndMT. Loss of NRP-1 inhibits EndMT and fibrosis in vivo and results in reduced tumor growth Prior to in vivo studies, the knockdown efficiency of the shNRP-1 minicircle was tested in vitro in BxPC-3 cells. NRP-1 knockdown was confirmed by a significant reduction in NRP-1 expression by qPCR and immunoblotting analysis at different time points (Supplementary Figure 1C, 1D). Moreover, NRP-1 knockdown significantly affected the growth kinetics of BxPC-3 cancer cells in vitro as observed by the MTT assay for cell viability (Supplementary Figure 1E). Orthotopic pancreatic tumors were grown in athymic rats and followed till day 56 after implantation surgery. Ultrasound-targeted microbubble destruction (UTMD) of shNRP-1 minicircle was performed for NRP-1 silencing at day 28 post-implantation in a subset of animals. Total RNA and protein were extracted from the tumor tissues. qPCR data ( Figure 4A) and immunoblotting data ( Figure 4B) demonstrate successful silencing of NRP-1 in shNRP-1 minicircle delivered nude rats. Tumor tissues obtained from gene delivered animals were stained with H&E to characterize tissue type and morphology ( Figure 4C) and extent of fibrosis by Masson's trichrome staining. We observed a typical ductal morphology and increased collagen content, features consistent with the characteristics of PDAC. Histological assessments further revealed reduced NRP-1 expression and reduced extent of fibrosis in shNRP-1 minicircle delivered animals ( Figure 4C). Investigating the role of EndMT in tumor fibrosis, our qPCR analysis demonstrated significant changes in the EndMT markers at transcript level ( Figure 4A) and protein level ( Figure 4B) in shNRP-1 minicircle delivered nude rats when compared to scramble minicircle delivered rats. These data were further confirmed by immunohistochemistry for NRP-1 and EndMT markers ( Figure 4C). At day 56, we observed significant reductions in tumor volumes in shNRP-1 delivered animals compared to scramble treated group, as measured by volumetric assessment of explanted tumors ( Figure 4D). Representative CEU perfusion images of orthotopic pancreatic tumors on day 28 (before gene delivery) and day 56 (28 days after gene delivery) are shown in Figure 4E. CEU assessments at day 28 providing a pre-delivery baseline assessment, demonstrated no significant differences in normalized microvascular blood volume (MBV) and microvascular blood flow (MBF) between different tumors ( Figure 4G, 4H). For perfusion imaging, the blood pool signal measured from a region of interest placed in the left ventricular (LV) cavity of Matrigel TM was quantified as number of nodes and number of tubes, showing that siNRP-1 caused a significant anti-angiogenic effect as compared to scramble control. p < 0.05 and **p < 0.001 vs. scramble control. n = 5 in triplicate. (E) Confluent monolayer of HUVECs exhibits typical cobblestone morphology in scramble control under light microscope (20X, scale = 50 µm). TGFβ1 stimulation (10 ng/ml) caused marked morphological changes with the cells becoming enlarged and spindle shaped in scramble control transfected cells. These morphological changes were inhibited to some extent in siNRP-1 transfected TGFβ1-stimulated HUVECs. (F) Fluorescent microscopy images (scale = 10 µm) stained with alpha-actinin (α-actinin; green color), demonstrating cytoskeletal protein re-organization in scramble control transfected TGFβ1-stimulated HUVECs. Nuclei were stained with DAPI (blue color). HUVECs were transfected with scramble control and siNRP-1, and total RNA and protein was extracted at 24 h and 48 h, respectively. qPCR analysis demonstrates significant changes in the EndMT markers at transcript level (G) and protein level (H) by immunoblotting. These data were further confirmed by immunofluorescence for NRP-1 and EndMT markers (I) (scale = 20 µm; scale for magnified image = 10 µm) 48 h post-transfection. Nuclei were stained by DAPI (blue). (J) qPCR data analysis demonstrates significant down-regulation of TGFβ1, TGFBR1 and TGFBR2 and TGFβ1-responsive genes; Slug, Collagen I and CTGF at transcript level and (K) TGFβ1, TGFBR1, TGFBR2, pSMAD2, SMAD2 and (H) pro-fibrotic genes CTGF, Collagen I and Slug at protein level by immunoblotting upon NRP-1 silencing. p < 0.05, p < 0.01, p < 0.0001 vs. scramble control. n = 3-4 in triplicate. www.impactjournals.com/oncotarget the heart was used to normalize tumor plateau acoustic signal for systemic microbubble concentration [69]. Tumor area ( Figure 4F) and perfusion ( Figure 4G, 4H) was found to be greater and more diffuse throughout the tumor in scramble minicircle delivered rats, while reduced perfusion was observed in tumors treated with shNRP-1 minicircle. At day 56, the plateau signal intensity from tumors, representing MBV, was markedly reduced in the group treated with shNRP-1 minicircle as compared with scramble minicircle ( Figure 4G). Moreover, tumor MBF was moderately reduced in the group treated with shNRP-1 minicircle as compared with scramble minicircle ( Figure 4H). Given the transfection efficacy of ultrasoundmediated gene delivery coupled with delivery of high in vivo transfection efficiency of minicircle vectors, we would expect the shRNA knockdown to last up to 4-6 weeks in our animals. Day 56 in our study is 28 days after gene delivery, thus our significant knockdown of NRP-1 at the mRNA and protein level as consistent with prior studies using minicircle DNA vectors [70,71]. We cannot exclude the possibility that NRP-1 expression would have been re-gained by certain cell populations by this time, as transfection is not equal in each and every cell type. However, we observed significant knockdown of NRP-1 till day 28 presumably due to high transfection efficiency of shRNA-minicircle and ultrasound mediated delivery. DISCUSSION Pancreatic ductal adenocarcinoma remains the most malignant and common type of pancreatic cancer. Although considered rare in terms of incidence, it remains the 5th cause of cancer related deaths, with the lowest five-year survival rate [1,5]. Some of the important characteristics include; lack of early clinical symptoms and poor prognosis, multifaceted and complex genetic alterations and high incidences of distant metastases that contribute to the low median survival. Morphologically, PDAC is characterized by a dense fibrotic reaction termed as desmoplasia [14,17,18]. While previously considered to be benign, this unique compartment within the tumor has been shown to contribute to several events that initiate and promote carcinogenesis. In fact about 80-90% of PDAC tumor volume consists of this cancer-supportive compartment. Treating these patients becomes challenging owing to this dense stromal barrier, primarily composed of fibroblasts, making anti-fibrotic therapy a potential treatment modality. The origin of CAFs, contributing to the formation of dense stroma, is a subject gaining importance lately. These fibroblasts are responsible for releasing potentially oncogenic signals, such as TGFβ, VEGF and other growth factors. Among several potential mechanisms for their origin, EndMT has gathered attention recently [34,35]. Although first described during embryonic heart development, recent data suggest that maladaptive EndMT plays a major role in several fibrotic pathological disorders including pulmonary fibrosis, renal fibrosis and cancer [29][30][31][32][33]39]. Loss of endothelial cell surface markers and gain of mesenchymal cell markers represent the hallmark of this phenomenon. Notably, EndMT is a multifactorial process driven by molecular mediators like TGFβ1. Therefore, investigating the role of novel biological clues and targets regulating EndMT can provide mechanistic insights into the underlying molecular processes involved in PDAC progression. More importantly, validation of such targets can lead to potential treatment options for fibrosisrelated disorders, including PDAC. Here, we highlight EndMT as a potential source of CAFs in PDAC, with a discussion of proposed role of an angiogenic co-receptor, putative mechanisms and therapeutic implications. The role of neuropilins in carcinogenesis, particularly the NRP-1 isoform, has been well documented [43]. Originally discovered for its role during embryonic nervous system development, the discovery of its function as a co-receptor for VEGFR-2 during angiogenesis [49] prompted investigators to study its function in tumor angiogenesis and carcinogenesis. Subsequently, several was confirmed at protein level by immunoblotting. (C) Representative images from capillary-like tube formation assay demonstrating increased number of tubes in lentiNRP-1 compared to lentiControl at 6 h time point. (D) Tube formation ability of HUVECs on Matrigel TM was quantified as number of nodes and number of tubes and showed that lentiNRP-1 caused a significant pro-angiogenic effect as compared to lentiControl. p < 0.05 and *p < 0.01 vs. lentiControl. n = 5. (E) Confluent monolayer of HUVECs exhibits typical cobblestone morphology in scramble control under light microscope (scale = 50 µm). TGFβ1 stimulation caused marked morphological changes with the cells becoming enlarged and spindle shaped in cells transduced with lentiControl. Similar morphological changes were observed in lentiNRP-1 transduced HUVECs upon TGFβ1 stimulation. (F) Fluorescent microscopic images (scale = 10 µm) stained with alpha-actinin (α-actinin; green color), demonstrating cytoskeletal protein re-organization in lentiNRP-1 transduced HUVECs. Nuclei were stained with DAPI (blue color). HUVECs were transduced with lentiControl and lentiNRP-1, and total RNA and protein was extracted at 48 h. (G) qPCR analysis demonstrates significant changes in the EndMT markers at transcript level and protein level by immunoblotting (H). (I) These data were further confirmed by immunofluorescence for NRP-1 and EndMT markers (Scramble: scale = 20 µm, scale for magnified image = 10 µm; LentiNRP-1: scale = 10 µm, scale for magnified image = 10 µm) 48 h post-transduction in the photomicrographs. Nuclei were stained by DAPI (blue). (J) qPCR data analysis demonstrates significant down-regulation of TGFβ1, TGFBR1, TGFBR2 and (G) TGFβ1-responsive genes Slug, Collagen 1 and CTGF at transcript level and (K) TGFβ1, TGFBR1, TGFBR2, pSMAD2, SMAD2 and (H) pro-fibrotic genes Slug, Collagen I and CTGF at protein level by immunoblotting upon NRP-1 overexpression. Nuclei were stained by DAPI (blue) p < 0.05, p < 0.01 and p < 0.001 vs. lentiControl. n = 3-4 in triplicate. www.impactjournals.com/oncotarget Representative contrast-enhanced ultrasound perfusion images of orthotopic pancreatic tumors on day 28 (before gene delivery) and day 56 (28 days after gene delivery). Images are color coded, with bright yellow/orange signifying the highest acoustic signal and greatest perfusion. Tumor area (F) and perfusion (G, H) is greater and more complete throughout the tumor in scramble minicircle group, while reduced perfusion seen in tumors treated with shNRP-1 minicircle. (G,H) At day 28 there were no significant differences in microvascular blood volume (MBV) and microvascular blood flow (MBF) between groups. Normalized tumor blood volume (G) and blood flow (H) at day 56. At day 56, tumor blood volume and flow was significantly reduced in shNRP-1 minicircle group compared with scramble control group. p < 0.05, **p < 0.01 vs. scramble. n = 4 for each group. Tumor area and volume data are expressed as mean ± SEM. www.impactjournals.com/oncotarget studies highlighted that NRP-1 was aberrantly expressed in several cancer types, including PDAC, and was involved in a plethora of cancer initiating and promoting pathways due to its distinctive ability to bind numerous growth factors and ligands [44, 45, 50-53, 60, 61]. Moreover, the ability of NRP-1 to exert co-receptor function to TGFβ1 opened new realms for investigation into oncogenic processes like EMT [51,[56][57][58]. Together, these NRP-1 findings in conjunction with studies highlighting the role of TGFβ signaling in EndMT, helped us postulate that NRP-1 could be playing a previously unrecognized regulatory role in TGFβ1-induced EndMT and potentially contributes towards tumor fibrosis. Our correlation data obtained from human PDAC xenografts, in addition to in vitro knockdown and overexpression studies, led to an important observation in this study; NRP-1 plays a previously undetermined regulatory role in TGFβ-induced EndMT and associated fibrosis. Indeed, knockdown of NRP-1 inhibited, while overexpression of NRP-1 promoted EndMT and associated fibrosis. Our studies on human PDAC tissues in this report demonstrated a positive correlation between NRP-1 levels, EndMT markers and pro-fibrotic genes' expression ( Figure 1A-1E). We observed that NRP-1 was expressed in all the tissue samples, however, with varying degrees. Moreover, the fibrosis and immunohistochemistry quantification results confirmed the significant correlation between NRP-1 levels and the extent of fibrosis ( Figure 1A, 1B). These promising human data prompted us to specifically focus further on the role of NRP-1 in EndMTinduced tumor fibrosis. To our knowledge, this is the first study to report a novel function of NRP-1 in modulating TGFβ1-induced EndMT and associated tumor fibrosis. By measuring the NRP-1 transcript and protein expression along with the assessment of its in vitro angiogenic role, we confirmed success of our NRP-1 knockdown (Figure 2A-2D) and overexpression ( Figure 3A-3D) studies in HUVECs. Furthermore, we observed that loss of endothelial NRP-1 repressed EndMT characteristics. TGFβ1-mediated distinct morphological changes from cobblestone-like endothelial cell morphology to an enlarged spindle shaped, fibroblast like morphology ( Figure 2E) and cytoskeletal protein organizational changes were inhibited upon NRP-1 silencing ( Figure 2F). These microscopic modifications were in complete accordance with the changes in transcript ( Figure 2G) and protein ( Figure 2H, 2I) expression of endothelial (VE-cadherin and CD31) and mesenchymal (N-cadherin and αSMA) cell markers. Several evidences have highlighted the role of TGFβ1 signaling as a primary mediator in EndMT [36,37,39]. Therefore, we assessed this signaling pathway following NRP-1 silencing and overexpression. HUVEC-specific loss of NRP-1 downregulates and deactivates TGFβ signaling, culminating in reduced phosphorylated SMAD-mediated Slug, CTGF, and Collagen 1 transcription ( Figure 2H, 2K). Reduction in Slug expression in turn up-regulates the expression of the endothelial markers VE-cadherin and CD31, which inhibits the EndMT process. Furthermore, decreased levels of NRP-1 led to reduced TGFβ signaling and associated expression of TGFβ-responsive profibrotic genes ( Figure 2H, 2J, 2K). As anticipated, the overexpression studies demonstrated contrasting results when compared to the knockdown studies. NRP-1 overexpression promoted EndMT associated phenotypic switching in addition to the corresponding changes in expression of cell surface markers ( Figure 3E-3I). Additionally, increased levels of NRP-1 led to an elevated TGFβ signaling and associated expression of TGFβresponsive pro-fibrotic genes ( Figure 3H, 3J, 3K). In contrast to TGFβ1 stimulation combined with NRP-1 overexpression, NRP-1 when overexpressed in the absence of TGFβ1 was unable to facilitate the complete transitioning of endothelial cell into mesenchymal cell. The reason behind this phenomenon could be that the regulatory role of NRP-1 in EndMT is not independent of TGFβ1. Thus our knockdown and overexpression studies provide, for the first time, solid evidence of the involvement of NRP-1 in regulating TGFβ1-induced EndMT as a potential source of CAFs. Recently, in a study of sprouting angiogenesis Aspalter et al. reported, using loss-and gain-of-function experiments that NRP-1 negatively regulated SMAD2/3 activation downstream of TGFβ1 and Bmp9 signaling [72]. In another study, Hirota et al. reported that NRP-1 and β8 integrin cooperatively balanced TGFβ signaling in brain endothelial cells. Particularly, shNRP-1 produced enhanced pSMAD3 levels upon TGFβ stimulation [73]. These studies contrast with our current findings showing that NRP-1 enhances canonical TGFβ signaling in the process of EndMT, and suggest that the function of NRP-1 differs between EndMT and sprouting angiogenesis. The response of endothelial cells to TGFβ is extremely complex and can occur through two antagonistic pathways: ALK5/ SMAD2,3 or ALK1/SMAD1,5. In the case of TGFβ's failure to induce canonical SMAD2/3 signaling, TGFβ may interact with ALK1 (type I receptor) and activate SMAD1 and SMAD5. In endothelial cells, this leads to an increase in cell proliferation and migration [74]. Additionally, in the absence of canonical signaling, non-SMAD (non-canonical) pathways are also activated by TGFβ through either phosphorylation or direct interaction of its receptors. These non-SMAD pathways include various branches of MAP kinase (MAPK) pathways, Rho-like GTPase signaling pathways, and phosphatidylinositol-3-kinase (PI3K)/AKT pathways [75]. Although the activation of these other pathways cannot be ruled out, our observations regarding reduced TGFβ ligand levels upon NRP-1 silencing prompted us to mainly focus on the EndMT relevant canonical SMAD2/3 pathway. Furthermore, neuropilins can also promote non-canonical TGFβ signaling [76] and the activation of these non-canonical pathways can suppress canonical signaling [43]. These pathway interactions might be of importance in the angiogenesis studies outlined above. We hypothesize that the endothelial cell response in the NRP-1/TGFβ interaction is highly context dependent, and these cells respond differently under different physiological/pathological conditions. To directly link the preliminary in vitro knockdown studies with the pathobiology of tumor fibrosis, we further tested the effects of NRP-1 silencing in a clinically relevant orthotopic model of PDAC in athymic rats. This cell line can also be directly injected into the pancreas to establish orthotopic tumors [77][78][79]. However, we opted for the surgical implantation technique to establish our orthotopic tumors, as implanted tumors closely mimic the clinical course of the disease observed in human pancreatic cancer patients. We also preferred the implantation technique because direct injection of cells into the pancreas is sometimes associated with possible leakage, failure of primary tumor establishment and inconsistent metastatic patterns. The rats were delivered with shRNA-minicircle DNA vector targeted against NRP-1 using UTMD 28 days after tumor implantation, a validated noninvasive technique for gene delivery [80][81][82]. In our study we observed that at day 28 post-implantation, the tumor attained a size that was easily detectable by contrast enhanced ultrasound. We recorded baseline tumor perfusion at day 28 that was subsequently used for normalization to the perfusion recorded at day 56. This was important in our study as normalization ruled out the size/perfusion variations observed between different animals at the time of gene delivery and gave us an accurate estimate of size and perfusion reductions after gene delivery. Twenty-eight days after shNRP-1 minicircle delivery, rats demonstrated modest but significant NRP-1 knockdown along with basal increase in the endothelial markers, down-regulation of mesenchymal markers and TGFβ1-responsive Slug and CTGF expression ( Figure 4A, 4B, 4C) and reduced tumor fibrosis ( Figure 4C). Furthermore, the shNRP-1 delivered animals demonstrated a modest reduction in tumor volume, tumor area, microvascular blood volume and blood flow, compared to the scramble group ( Figure 4D-4H). These in vivo results suggest that loss of NRP-1 could potentially lead to reduced tumor growth, angiogenesis and fibrosis possibly through limiting EndMT as well as reducing NRP-1-VEGFR-2 signaling. We did not examine longer time points for longevity or survival advantage of NRP-1 knockdown. Survival analysis by Kaplan-Meier estimates will be a focus of our future studies along this line of investigation. A putative mechanistic schematic representation is shown to summarize the effect of endothelial cell loss and gain of NRP-1 on EndMT and tumor fibrosis ( Figure 5). Our in vitro and in vivo studies have further validated that TGFβ1-induced EndMT can be modulated in response to manipulations of NRP-1 levels. In our experiments, we also observed a critical role of the transcription factor Slug that exacerbates the process of EndMT as previously published [39]. The exact role of other signaling pathways and transcription factors is not entirely clear at this point and warrants further mechanistic work. The recent discoveries of EndMT in various pathologies, including cancer, suggest that modulating EndMT may represent a promising novel treatment option. Studies have demonstrated that NRP-1 knockdown increases chemosensitivity towards chemotherapeutic drugs in a variety of cancer cells, including pancreatic cancer cells [61,83]. Of interest, recently Zheng et al. highlighted the role of EMT in drug resistance and the potential of combining EMT inhibition with chemotherapy for the treatment of pancreatic cancer [84]. However, the exact contribution of EndMT towards drug resistance and the mechanistic role of NRP-1 in EndMT-dependent chemosensitivity are unknown. Moreover, the therapeutic benefit of inhibiting NRP-1-mediated EndMT combined with chemotherapy necessitates further investigation. In conclusion, we provide herein for the first time, solid evidence of a novel anti-NRP-1 therapy that could lead to a reduction in pancreatic tumor fibrosis and growth via a regulatory effect on TGFβ1-induced EndMT. We believe that therapies directed at targets like NRP-1 to inhibit EndMT have tremendous translational implications as they could delay PDAC tumor progression, possibly owing to impaired angiogenesis as well as limited CAF recruitment. Lentivirus-mediated NRP-1 overexpression studies HUVECs were transduced with lentiNRP-1 or Empty (referred to as lentiControl) vectors (Applied Biological Materials) according to manufacturer's protocols and cells were cultured as described above. Capillary-like tube formation assay 60 μl of Matrigel TM (Becton Dickinson) was added to each well of the 96-well tissue culture plate (Corning) and allowed to polymerize at 37°C, 5% CO 2 for 45 minutes under sterile conditions. 0.5 × 10 4 transfected/transduced HUVECs were plated onto the surface of the Matrigel TM and cultured as described earlier. All of the wells were stimulated with VEGF (50 ng/ml) to induce in vitro tube formation. The capillary-like network formation was observed at regular intervals until 24 h and photomicrographs were recorded at different time intervals for quantification. Cell viability assay MTT assay of cell viability (Vybrant, Life technologies) was performed to assess growth kinetics according to the manufacturer's protocol. 10,000 BxPC-3 cells transfected with shRNA or scramble minicircle were seeded in each well of 96 well plates (n = 6). 10 µl of the 12 mM MTT stock solution was added to each well and incubated at 37°C, 5% CO 2 for 4 hours. 50 µl of DMSO (Sigma) was added to each well and mixed thoroughly to solubilize the formazan. The pate was again incubated at 37°C, 5% CO 2 for 10 minutes. Each sample was mixed again and absorbance was read at 540 nm using an automated plate reader (Molecular Devices). Human xenograft studies Formalin-fixed paraffin-embedded and flash frozen human PDAC tissues were obtained from Dr. David Hedley (OICR, Toronto, Canada). qPCR and immunoblotting for NRP-1, EndMT and fibrosis markers was performed on these samples. Immunohistochemistry, hematoxylin-eosin (H&E) staining and Masson's trichrome staining (Polybiosciences Inc.) was performed on the human PDAC tissue following the manufacturer's guidelines. NRP-1 levels and fibrosis were quantified with light microscopy using the ImageJ software (NIH). Generation of orthotopic tumor model in athymic rats The in vivo study protocol was approved by the Animal Care and Use Committee at the Keenan Research Centre for Biomedical Science, Li Ka Shing Knowledge Institute, St Michael's Hospital, University of Toronto. Rowett Nude (RNU, strain 316) rats were purchased from Charles River. BxPC-3 human pancreatic cancer cells were purchased from ATCC and cultured in RPMI 1640 medium (ATCC) supplemented with 10% fetal bovine serum and antibiotics. (5 × 10 6 ) cells were injected (200 μl cells in RPMI medium + 200 μl Matrigel TM ) in the flank of athymic rats to establish heterotopic tumors. Pancreatic tumors grown subcutaneously were then harvested at exponential growth phase under aseptic conditions to establish the orthotopic model. Viable tissues were cut and minced into 3-mm 3 pieces. In recipient RNU rats, the tail of the pancreas, located in the splenorenal ligament within the spleen, was gently exteriorized via laparotomy. Five tumor fragments were sutured with 7-0 surgical sutures onto the pancreas so that tumor tissue is completely surrounded by pancreatic parenchyma. The pancreas was placed back in the original position and the muscle and skin were sutured. Post-mortem, tumor tissues were stained with H&E staining and Masson's trichrome staining for characterization. Contrast enhanced ultrasound perfusion imaging and ultrasound-mediated loss of NRP-1 in vivo The contrast-enhanced ultrasound (CEU) perfusion and gene delivery studies were performed as previously described [80,81]. CEU perfusion imaging of tumors was performed at 4 and 8 weeks after implantation surgery. At 4 weeks post tumor implantation surgery, ultrasoundtargeted microbubble destruction (UTMD) [80][81][82] was performed for NRP-1 silencing. For UTMD, shNRP-1 minicircle (SBI Biosciences) DNA vector (200 μg) was charge coupled to cationic microbubbles (1 × 10 9 ) and the suspension was administered intravenously during the external application of high power ultrasound over the pancreas (n = 4). Scramble minicircle (SBI Biosciences) DNA vector (200 μg) was delivered by UTMD as an appropriate control in a separate group of animals (n = 4). Animals were sacrificed 4 weeks after gene delivery (8 weeks post implantation). Tumor tissue and remote organs were collected for evaluation of tumor volume and other downstream analyses as previously described. Statistical analysis All data were expressed as mean ± SD unless otherwise indicated. The Student's t-test was applied when the means of two groups were being compared. Differences between multiple means were evaluated by One-way ANOVA (GraphPad Prism5) followed by Tukey's posthoc test to compare individual means. P value of < 0.05 was considered to be statistically significant. Spearman's Rho test was applied to perform bivariate correlation analysis between two variables, where values of R closer to 1 indicated positive correlation.	2018-04-03T00:37:18.550Z	2016-08-11T00:00:00.000	{ "year": 2016, "sha1": "16e16106d05333f91ba841a2b7432190ea598a40", "oa_license": "CCBY", "oa_url": "http://www.oncotarget.com/index.php?journal=oncotarget&op=download&page=article&path[]=11060&path[]=35025", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "16e16106d05333f91ba841a2b7432190ea598a40", "s2fieldsofstudy": [ "Biology", "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
15421070	pes2o/s2orc	v3-fos-license	Fatigue Reliability Analysis of Wind Turbine Cast Components : The fatigue life of wind turbine cast components, such as the main shaft in a drivetrain, is generally determined by defects from the casting process. These defects may reduce the fatigue life and they are generally distributed randomly in components. The foundries, cutting facilities and test facilities can affect the veriﬁcation of properties by testing. Hence, it is important to have a tool to identify which foundry, cutting and/or test facility produces components which, based on the relevant uncertainties, have the largest expected fatigue life or, alternatively, have the largest reliability to be used for decision-making if additional cost considerations are added. In this paper, a statistical approach is presented based on statistical hypothesis testing and analysis of covariance (ANCOVA) which can be applied to compare different groups (manufacturers, suppliers, test facilities, etc.) and to quantify the relevant uncertainties using available fatigue tests. Illustrative results are presented as obtained by statistical analysis of a large set of fatigue data for casted test components typically used for wind turbines. Furthermore, the SN curves (fatigue life curves based on applied stress) for fatigue assessment are estimated based on the statistical analyses and by introduction of physical, model and statistical uncertainties used for the illustration of reliability assessment. Introduction Wind energy has become an attractive source of renewable energy, and its installed capacity worldwide has grown significantly in recent years [1]. Offshore wind turbines have been installed many places, especially in the North Sea, and many new, larger wind farms are planned. Due to the harsh environmental conditions for offshore wind turbines, and the access difficulties for maintenance and repairs, it is very important to minimize failures of wind turbine components including fatigue failures of casted components [2] in the wind turbine drivetrain. Wind turbines are large structures exposed to wave excitations, highly dynamic wind loads and wakes from other wind turbines [3]. Thus, wind turbine components are exposed to stochastic loads that are varying randomly during the design working life. Due to highly variable loads, the components may fail due to fatigue, wear and other deterioration processes [3]. Casting defects are of high importance for the lifetime of structural casting. The fatigue life of cast iron components is often controlled by the growth of cracks initiated from defects such as shrinkage cavities and gas pores [4]. Further, different manufacturers apply different manufacturing processes of cast components, resulting in different fatigue lives of the produced components. This paper focuses on using analysis of covariance (ANCOVA) for comparing different groups/manufacturing steps of specimens from the casted components. An advantage of ANCOVA is that this method is able to handle different numbers of tests for various groups [5]. The result of 2 of 14 ANCOVA is useful in decision-making processes for companies and manufacturers to choose the better manufacturing process. This will lead to a higher quality of the manufactured components and increase the reliability of the produced components. Further, the results of the ANCOVA analysis are used for the reliability assessment of wind turbine cast components. For this reason, the uncertainties related to model evaluation and the effect of each uncertainty on the reliability level of the components is evaluated. Moreover, a case study is presented to illustrate the application of ANCOVA to compare different manufacturing steps of casted components according to fatigue life results and also the reliability assessment of chosen cast components in wind turbine components based on the ANCOVA results. Reliability Analysis Wind turbine component's reliability, usually explained as the probability of proper performance of the component throughout its lifetime. Structural reliability methods, such as first order reliability method and second order reliability method, can be used to evaluate the probability of failure/reliability [2]. Based on statistical analyses of statistically homogeneous datasets of fatigue lives, an appropriate stochastic model for the fatigue life can be established as described shortly below. Further, it is shown how a fatigue load model can be formulated based on simulations of the load effects for a specific wind turbine and represented by Markov matrices. Together with stochastic variables representing the uncertainties associated with the load assessment, and following the principles in, e.g., [6,7], the reliability can be estimated by First-Order Reliability Methods (FORM) or by simulation, see, e.g., [8]. Finally, the stochastic model established on the basis of the above statistical analysis in combination with the reliability model for the fatigue failure of wind turbine cast components can be applied for the calibration of partial safety factors to a specified target reliability level, e.g., 5 × 10 −4 per year as recommended in [7]. In Section 4, the reliability assessment and methodology to evaluate the probability of failure is briefly explained, see [2] for more details. Statistical Analysis of Fatigue Data Sets A major challenge in statistical analysis of fatigue data is to establish a statistically homogeneous dataset to be applied as the basis for the statistical analyses. Analysis of variance (ANOVA) is a statistical method that can be used to analyze the differences between mean values of different groups of, e.g., suppliers. ANOVA could be used as a statistical test to investigate equality of the means of several groups, and hence generalizes the classical statistical t-test to more than two groups [9]. Based on hypothesis testing, the null hypothesis by the ANOVA method is that the mean values of several groups are equal, and on the other hand, the alternative hypothesis is that the mean values are not equal [10]. The level of significance represents the probability of making a type I error and is denoted by α. The level of significance should not be made too small, because the probability of making a type II error will then be increased. In this paper the value of α = 0.05 is chosen. ANOVAs are useful in comparing (testing) three or more groups. A decision of whether or not to accept the null hypothesis depends on a comparison of the computed values of the test statistics and the critical values. The ANOVA analysis can be performed computationally as follows; for further details see [10]. Step 1: Formulation of hypothesis If a problem involves k groups, the following hypotheses are appropriate for comparing k group means [10]: H 0 : µ 1 = µ 2 = . . . = µ k H A : at least one pair o f group means are not equal (1) where µ is the mean value in the above equation. Step 2: Define the test statistic and its distribution The hypotheses of Step 1 can be tested using the following test statistic: in which MS b and MS w are the mean squares quantifying between and within variations, respectively, and F is a random variable following an F distribution with degrees of freedom of (k − 1, M − k); for more details, see [10]. The following table shows the calculation for the ANOVA. In Table 1, k is the number of groups, m j is the number of data in j-th group, X is the mean value of all data, X j is the mean value of data in the j-th group and M is the total number of data [10]. Between Step 3: The level of significance The level of significance is chosen to α = 5% in this paper. Step 4: Collect data and compute test statistic The data should be collected and used to compute the value of the test statistics (F) in Equation (2). Each data value is categorized according to the different groups to be compared statistically to each other. Step 5: Determine the critical value of the test statistic The critical value is a function of the level of significance and the degrees of freedom. If the computed value of Step 4 is greater than the critical value, the null hypothesis of Equation (1) should be rejected and the alternative hypothesis of Equation (1) accepted. In addition, the ANCOVA always involves at least three variables to be introduced: an independent variable, a dependent variable, and a covariate. The covariate is the variable likely to be correlated with the dependent variable. For application for fatigue data, these variables are chosen as: • The independent variable is the group types that we consider to compare to each other (for example, test facilities or suppliers); • The dependent variable is the fatigue life N and it is dependent on the test stress amplitude and the type of group; • The covariate is the test stress amplitude, σ [5]. The major distinction between the two analyses (ANOVA and ANCOVA) is that in ANOVA, the error term is related to the variation of logN around individual group means, whereas the ANCOVA error term is based on variations of logN scores around regression lines. The effect of the smaller within-group variation associated with ANCOVA is an increase of the power of the analysis. Note that the ANOVA distributions have a larger overlap than the ANCOVA distributions [5]. The analysis of covariance is a combination of the linear models employed in the analysis of variance and regression [11]. If it is assumed that data from k groups is available, then the starting point for the ANCOVA is exactly the same as for the ANOVA; the total sum of squares is computed. It is noted that ANCOVA can be used for linear regression methods and therefore the analysis is carried out using the "logarithm of fatigue life (logN)" and the "logarithm of test stress amplitude (logσ)". Hence, the covariate variable (x) is logσ and the dependent variable (y) is logN. Assuming that there is a linear relationship between the dependent variable and the covariate, we find that an appropriate statistical model is: where logN ij is the i-th observation on the response variable in the j-th group, logσ ij is the measurement made on the covariate variable corresponding to logN ij , log σ .. is the mean of the logσ ij values, µ is an overall mean, τ j is the effect/influence of the j-th group, β is a linear regression coefficient indicating the dependency of logN ij on logσ ij , and ε ij is a random error component. It is assumed that the errors ε ij are normally distributed with a mean value = 0 and a standard deviation of σ ε . The null hypothesis is "the group effect is zero ( k ∑ j=1 τ j = 0)" and if the null hypothesis is accepted, then logσ ij is not affected by the groups. Adjusted Mean An adjusted mean is the mean dependent variable that would be expected or is predicted for each group, if the covariate variable mean is equal to the grand covariate mean. The grand covariate mean is a unique logσ u and the adjusted fatigue life in each group is estimated according to this stress amplitude. In this way, for each group there is a unique point (logσ u , logN j,adj ). This value would be different in different groups, and these values can be used to compare the state of the regression lines to each other. According to adjusted means and slopes of regression lines, there are four types of regression line configurations, as shown in Figure 1 below. Energies 2017, 10, 466 4 of 13 "logarithm of fatigue life (logN)" and the "logarithm of test stress amplitude (logσ)". Hence, the covariate variable (x) is logσ and the dependent variable (y) is logN. Assuming that there is a linear relationship between the dependent variable and the covariate, we find that an appropriate statistical model is: where logNij is the i-th observation on the response variable in the j-th group, logσij is the measurement made on the covariate variable corresponding to logNij, logσ .. is the mean of the logσij values, μ is an overall mean, τj is the effect/influence of the j-th group, β is a linear regression coefficient indicating the dependency of logNij on logσij, and εij is a random error component. It is assumed that the errors εij are normally distributed with a mean value = 0 and a standard deviation of σε. The null hypothesis is "the group effect is zero (∑ = 0)" and if the null hypothesis is accepted, then logσij is not affected by the groups. Adjusted Mean An adjusted mean is the mean dependent variable that would be expected or is predicted for each group, if the covariate variable mean is equal to the grand covariate mean. The grand covariate mean is a unique logσu and the adjusted fatigue life in each group is estimated according to this stress amplitude. In this way, for each group there is a unique point (logσu, logNj,adj). This value would be different in different groups, and these values can be used to compare the state of the regression lines to each other. According to adjusted means and slopes of regression lines, there are four types of regression line configurations, as shown below. (c) parallel (same slope but without intercept); and (d) coincidence (exactly same lines) [12]. By adjustment of the point and slope for each group, comparisons can be performed. As mentioned above, the purpose of the ANCOVA is to test the null hypothesis that two or more adjusted population means are equal. Alternatively, the purpose could be formulated as to test the equality of two or more regression intercepts. Under the assumption of parallel regression lines, the difference between intercepts must be equal to the difference between adjusted means. The formula for the computation of adjusted means is: (c) parallel (same slope but without intercept); and (d) coincidence (exactly same lines) [12]. By adjustment of the point and slope for each group, comparisons can be performed. As mentioned above, the purpose of the ANCOVA is to test the null hypothesis that two or more adjusted population means are equal. Alternatively, the purpose could be formulated as to test the equality of two or more regression intercepts. Under the assumption of parallel regression lines, the difference between intercepts must be equal to the difference between adjusted means. The formula for the computation of adjusted means is: where log N j,adj is the adjusted mean for the j-th group; log N .j is the unadjusted mean (mean of fatigue life) for the j-th group; b w is the pooled within-group regression coefficient (for details see [5,11]); log σ .j is the mean of the stress amplitude for the j-th group; log σ .. is the mean of the logσ ij values. Testing the Homogeneity of Regression Slopes An assumption underlying the use of ANCOVA is that the population regression slopes are equal. If the slopes are not equal, the "Type of groups" effects differ at different levels of the stress amplitude; consequently, the adjusted stress amplitudes can be misleading because they do not convey this important information. When the slopes are the same, the adjusted means are adequate descriptive measures because the differences of the fatigue life are the same at different levels of the stress amplitude. If the slopes for the populations in an experiment are equal, that is, β , a reasonable way of estimating the value of this common slope from the samples is by computing an average of the sample b 1 values: where from Equation (5) we have: The slope b w is the best estimate of the population slope β 1 , which is the slope assumed to be common to all groups. As long as β , the estimate b w is a useful statistical value to use. Now the problem is to decide whether all groups have the same slope. The homogeneity of the regression F-test is designed to answer the question of the equality of the population slopes. The null hypothesis associated with this test is: The steps involved in the computation of the test are described next: Steps 1 and 2: Computation of within-group sum of squares and within-group residual sum of squares (SSres w ) This parameter can be evaluated by the following equation (for further details, see [5,11]): Step 3: Computation of individual sum of squares residual (SSres i ) The third step involves the computation of the sum of squares residual for each group separately, and then adding these residuals to obtain the sum of the individual residual sum of squares (SSres j ). The difference in the computations of SSres w and SSres j is that SSres w involves computing the residual sum of squares around the single b w value whereas SSres j involves the computation of the residual sum of squares around the b j values fitted to each group separately (Equation (13)). Step 4: Computation of heterogeneity of slopes sum of squares The discrepancy between SSres w and SSres i reflects the extent to which the individual regression slopes are different from the within-group slope b w ; hence, the heterogeneity of slopes SS is "SS het = SSres w − SSres i ", see [5]. Step 5. Computation of F-ratio The summary table for the F-test is as follows ( Table 2). If the obtained F is equal to or greater than F [α,j−1,N−2j] , then the null hypothesis H 0 : β In this method, diagnostic checking of the covariance model is based on residual analysis. Furthermore, the measure of uncertainty is not directly related to the uncertainty of the SN curve. The uncertainty of SN curves used in the next section has been evaluated by the maximum likelihood method (MLM) [2]. Reliability Assessment The fatigue strength of metals is often assumed to follow the Basquin equation (the equation is based on fully reversed fatigue (R = −1), i.e., the mean value is zero) and is written [12]: where N is the number of stress cycles to failure with constant stress ranges ∆σ; K and m are material parameters dependent on the fatigue critical detail. The fatigue strength ∆σ F may, e.g., be defined as the value of S for, e.g., N D = 2 × 10 6 [7]. If one fatigue critical detail is considered, then the annual probability of failure is obtained from: where P(fatigue failure in year t) is the probability of failure in year t and P COL\|FAT is the probability of collapse of the structure given fatigue failure-modeling the importance of the details/consequences of failure. The probability of failure in year t given survival up to year t is estimated by: where the limit state equation is based on the application of SN curves and Miner's rule for linear accumulation of fatigue damage, and by introducing stochastic variables accounting for uncertainties in fatigue loading and strength. Note that the probability of failure also depends on the repair and maintenance methods' accuracy and protective methods that have been applied on the component. The design equation can be written as follows, if used in a deterministic code-based verification: where n i,S represents the number of cycles per year at a specific stress level and T L is the design lifetime. It is assumed that for a wind turbine component, for a given fatigue life, the number of cycle according to the stress can be grouped as n σ , where the number of excitation at the specific stress range i is n i,S per year. In this paper, the Level II reliability method is used to estimate the reliability of the components [7]. The design parameter z is obtained from Equation (17), assuming that the fatigue partial safety factors are given. Consequently, the reliability equation is normalized and it became a function of the partial safety factors and presumed that the component is designed to the limit according to the design parameter z in the design equation. For a deterministic design, the following partial safety factors are introduced [13]: γ f a fatigue load partial safety factor multiplied by the fatigue stress ranges obtained by, e.g., rainflow counting. γ m a fatigue strength partial safety factor. The design value of the fatigue strength is obtained by dividing the characteristic fatigue strength by γ m . The characteristic fatigue strength can be defined in various ways, namely based on: • the mean minus two standard deviations of log K. • the 5% quantile of log K, i.e., the mean minus 1.65 times the standard deviation of log K. • the mean of log K. The corresponding limit state equation to be used in the reliability analysis is written: where X W is a stochastic variable modeling model uncertainty related to the determination of fatigue loads and X SCF is a stochastic variable modeling model uncertainty related to the determination of stresses given fatigue loads. In addition, ∆ models model uncertainty related to Miner's rule for linear damage accumulation [2]. In Equation (18), ∆, X W and X SCF are assumed to be log-normal distributed with mean values equal to 1 and coefficients of variation COV ∆ , COV W and COV SCF , respectively, and N(X W , X SCF , ∆σ, ∆σ F , ∆K, z) can be written as following equation: The coefficients of variation are estimated partly subjectively, but generally following the recommendations used as a basis for the material partial safety factors in IEC 61400-1, and also considering information from, e.g., [14]. The importance of the choices of the coefficients of variation is investigated by sensitivity analyses. It is noted that the reliability level obtained is in accordance with the target reliability corresponding to an annual probability failure of the order 5 × 10 −4 (annual reliability index 3.3) [15]. In Table 3, the stochastic model is shown. It is noted that m and ∆σ f are correlated with statistical parameters extracted from [2]. The stochastic model is considered as representative for the fatigue strength represented by SN curves. It is assumed that the design lifetime is T L = 25 year [13]. * Log-Normal Distribution; ** Normal Distribution. If the SN curves are obtained by a limited number of tests, then statistical uncertainty has to be accounted for. Table 4 shows indicative values of γ f γ m for the target reliability index equal to 3.3 as a function of the total coefficient of variation of the fatigue load: COV load = COV 2 W + COV 2 SCF . It is noted that more fatigue test data should be investigated to validate the indicative values in Table 4. Results and Discussion This section presents results obtained using representative fatigue test data from test specimens of cast components. The casted specimens are manufactured by two different manufacturing processes. The manufacturing processes are "sand casting" and "chill casting". In this section, the ANCOVA method is used to compare the different test laboratories and different extractions of samples for the tests and obtaining the fatigue test data. Comparison of Different Test Laboratories The fatigue tests were carried out by four different test laboratories. Table 5 shows the number of tests in each group. The comparison between groups (Tests labs) was performed using sand casting results because there was only one group with chill casting. In this configuration, the run-out samples were considered as broken samples with very high cycles in order not to exclude them from the analysis. A summary of the ANCOVA calculations is shown in Table 6. The value of b w shows that the stress amplitude and fatigue life were negatively correlated. First it was tested if the choice of test laboratory does not affect the fatigue test results. The null hypothesis is: H 0 , the choice of test laboratory does not affect the fatigue test results. The F-ratio is calculated according to Section 3. The study involves one covariate, four groups and 827 test results. The F-ratio calculation is summarized in Table 7. The obtained F was then compared with the critical value of F with three and 822 degrees of freedom and a level of significance equal to 5%; F (0.05, 3,822) equals 2.62, and the null hypothesis of "The choice of test laboratory does not affect the fatigue test results" was rejected. Next, the homogeneity of the slopes of regression lines was considered, i.e., it was tested if the slopes of the different regression lines were equal. The null hypothesis is written: The summary of the calculation is shown in Table 8. The critical value is F (0.05, 3, 821) = 2.62; hence, the null hypothesis was accepted. It was concluded that the slopes of the regression lines are similar. Figure 2 shows the results for the regression lines (note the results are normalized). It was seen that "Group 2" had the highest fatigue life and "Group 1" had the lowest fatigue life compared to the other testing places and the groups had the same slope. The critical value is F(0.05, 3, 821) = 2.62; hence, the null hypothesis was accepted. It was concluded that the slopes of the regression lines are similar. Figure 2 shows the results for the regression lines (note the results are normalized). It was seen that "Group 2" had the highest fatigue life and "Group 1" had the lowest fatigue life compared to the other testing places and the groups had the same slope. Comparison of Different Cutting Facilities The components from where samples were taken were cut up at different facilities. The cutting facilities were used to cut the sand casting samples after casting processes, and therefore they could be considered as a part of the manufacturing process for the casting samples. Table 9 shows the number of tests in each group. It is noted that the groups of cutting facilities are not the same as the groups of testing laboratories. Comparison of Different Cutting Facilities The components from where samples were taken were cut up at different facilities. The cutting facilities were used to cut the sand casting samples after casting processes, and therefore they could be considered as a part of the manufacturing process for the casting samples. Table 9 shows the number of tests in each group. It is noted that the groups of cutting facilities are not the same as the groups of testing laboratories. Group 1 107 8 96 3 214 Group 2 18 3 --21 Group 3 95 14 --109 Group 4 148 26 107 53 334 Group 5 345 63 99 51 558 Summation 713 114 302 107 1236 In this case, the comparison between groups was performed using sand casting and chill casting. The results of the ANCOVA for sand casting and chill casting are shown in Tables 10 and 11, respectively. The obtained F statistic was then compared with the critical value. In both casting methods, the null hypothesis of "The choice of cutting facilities does not affect the fatigue test results" was rejected. Next, the homogeneity of the regression test (slope of regression lines) was considered. The summaries of the calculation are shown in Tables 12 and 13. Based on Tables 12 and 13, the null hypothesis is rejected and slopes of regression lines are not the same. Figures 3 and 4 show the results for the regression lines of sand casting and chill casting, respectively (note the results are normalized). According to Figures 3 and 4, Group 2 has a different slope compared to the other groups, but the other groups are similar to each other. For sand casting, quite small differences between fatigue lives were obtained for cutting facilities 1, 3, 4 and 5. For chill casting, relatively larger differences in fatigue lives were obtained for cutting facilities 1, 4 and 5. According to Figures 3 and 4, Group 2 has a different slope compared to the other groups, but the other groups are similar to each other. For sand casting, quite small differences between fatigue lives were obtained for cutting facilities 1, 3, 4 and 5. For chill casting, relatively larger differences in fatigue lives were obtained for cutting facilities 1, 4 and 5. Reliability Analysis: Examples In the following, the SN curves that were derived in the above sections are used for reliability analysis in a case study. The parameters used for analysis are listed in Table 14. By using the design Equation (17), the design values (z) are determined for each group, as shown in Tables 15 and 16. Table 15. Design values (z) for testing lab groups (obtained from Equation (17)). Reliability Analysis: Examples In the following, the SN curves that were derived in the above sections are used for reliability analysis in a case study. The parameters used for analysis are listed in Table 14. By using the design Equation (17), the design values (z) are determined for each group, as shown in Tables 15 and 16. Table 15. Design values (z) for testing lab groups (obtained from Equation (17)). The reliability indices as a function of time for the groups of testing laboratories (Section 5.1) were estimated and are shown in Figure 5. Note that in this figure the annual reliability index is shown. The data from Group 2 result in the largest reliability level compared to the other groups. Moreover, the annual reliability index for cutting samples groups (Section 5.2) is shown in Figure 6. It is seen that the annual reliability indices at the end of the lifetime are of the order of 3.3, corresponding to the target annual reliability index = 3.3. Groups The reliability indices as a function of time for the groups of testing laboratories (Section 5.1) were estimated and are shown in Figure 5. Note that in this figure the annual reliability index is shown. The data from Group 2 result in the largest reliability level compared to the other groups. Moreover, the annual reliability index for cutting samples groups (Section 5.2) is shown in Figure 6. It is seen that the annual reliability indices at the end of the lifetime are of the order of 3.3, corresponding to the target annual reliability index = 3.3. Conclusions In this paper, the ANCOVA method is used to compare different groups related to the manufacturing process and associated quality control of cast components. The ANCOVA method is applied for fatigue failure data. Test data is used to illustrate the implementation of the ANCOVA method. The different test laboratories and cutting facilities are compared to each other. The results obtained from the ANCOVA analysis can be used for decision-making on which test laboratories and cutting facilities should be included in a statistical analysis in order to make sure that the statistical analyses are performed on data from a statistically homogeneous population. More parameters can be included, if relevant. Further, it is presented how to apply the statistical results from the ANCOVA analysis to estimate the reliability for generic cases, and to assess the required safety factors for deterministic The reliability indices as a function of time for the groups of testing laboratories (Section 5.1) were estimated and are shown in Figure 5. Note that in this figure the annual reliability index is shown. The data from Group 2 result in the largest reliability level compared to the other groups. Moreover, the annual reliability index for cutting samples groups (Section 5.2) is shown in Figure 6. It is seen that the annual reliability indices at the end of the lifetime are of the order of 3.3, corresponding to the target annual reliability index = 3.3. Conclusions In this paper, the ANCOVA method is used to compare different groups related to the manufacturing process and associated quality control of cast components. The ANCOVA method is applied for fatigue failure data. Test data is used to illustrate the implementation of the ANCOVA method. The different test laboratories and cutting facilities are compared to each other. The results obtained from the ANCOVA analysis can be used for decision-making on which test laboratories and cutting facilities should be included in a statistical analysis in order to make sure that the statistical analyses are performed on data from a statistically homogeneous population. More parameters can be included, if relevant. Further, it is presented how to apply the statistical results from the ANCOVA analysis to estimate the reliability for generic cases, and to assess the required safety factors for deterministic Conclusions In this paper, the ANCOVA method is used to compare different groups related to the manufacturing process and associated quality control of cast components. The ANCOVA method is applied for fatigue failure data. Test data is used to illustrate the implementation of the ANCOVA method. The different test laboratories and cutting facilities are compared to each other. The results obtained from the ANCOVA analysis can be used for decision-making on which test laboratories and cutting facilities should be included in a statistical analysis in order to make sure that the statistical analyses are performed on data from a statistically homogeneous population. More parameters can be included, if relevant. Further, it is presented how to apply the statistical results from the ANCOVA analysis to estimate the reliability for generic cases, and to assess the required safety factors for deterministic design such that a given target reliability level is obtained. It is noted that more data and more example structures are needed to perform a more general reliability-based calibration of safety factors with the proposed approach.	2017-04-19T08:35:22.255Z	2017-04-02T00:00:00.000	{ "year": 2017, "sha1": "a36ff78f09464baa421eedc2f3a52dd0e8d1c228", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/1996-1073/10/4/466/pdf", "oa_status": "GOLD", "pdf_src": "ScienceParsePlus", "pdf_hash": "adb2a6a98129a487545b7014013b5974bee08b8d", "s2fieldsofstudy": [ "Engineering" ], "extfieldsofstudy": [ "Engineering" ] }
40758490	pes2o/s2orc	v3-fos-license	No Laplacian Perfect State Transfer in Trees We consider a system of qubits coupled via nearest-neighbour interaction governed by the Heisenberg Hamiltonian. We further suppose that all coupling constants are equal to $1$. We are interested in determining which graphs allow for a transfer of quantum state with fidelity equal to $1$. To answer this question, it is enough to consider the action of the Laplacian matrix of the graph in a vector space of suitable dimension. Our main result is that if the underlying graph is a tree with more than two vertices, then perfect state transfer does not happen. We also explore related questions, such as what happens in bipartite graphs and graphs with an odd number of spanning trees. Finally, we consider the model based on the $XY$-Hamiltonian, whose action is equivalent to the action of the adjacency matrix of the graph. In this case, we conjecture that perfect state transfer does not happen in trees with more than three vertices. Introduction Let X be a simple undirected graph on n vertices, and let L = L(X) denote its Laplacian matrix. For any u ∈ V (X), we denote its characteristic vector with respect to the ordering of the rows of L by e u . The matrix operator U L (t) = exp(itL), defined for every real t ≥ 0, represents a continuous-time quantum walk on X. We say that X admits Laplacian perfect state transfer from a vertex u to a vertex v if there is a time t ≥ 0 such that U L (t)e u = γe v for some γ ∈ C. Here we work under the assumption that the graph X is the underlying network of a system of qubits coupled via nearest-neighbour interaction governed by the Heisenberg Hamiltonian with coupling constants equal to 1. If such a system is initialized in such a way that the state of the qubit located at the vertex u is orthogonal to the states of each of the other qubits, then Laplacian perfect state transfer in the graph between u and v is equivalent to a transfer of state from the qubit at u to the qubit at v with fidelity 1 (see Kay [16]). If we choose the XY -Hamiltonian instead, perfect state transfer is defined in terms of U A (t) = exp(itA), where A is the adjacency matrix of the graph. There are many similarities between both cases, mostly because both A and L are symmetric integer matrices with a positive eigenvector. If X is k-regular, then L = kI − A, thus exp(itA) and exp(itL) differ only by a constant. The problem of determining which graphs admit adjacency perfect state transfer has received a considerable amount of attention lately. For example, it was solved for paths and hypercubes (see Christandl et al. [8]), circulant graphs (see Bašić [3]), cubelike graphs (see Cheung and Godsil [7]) and distance-regular graphs (see Coutinho et al. [9]). The effect of certain graph operations was considered in Angeles-Canul et al. [1], Bachman et al. [2] and Ge et al. [11]. Recent surveys are found in Kendon and Tamon [17], and Godsil [15]. Bose et al. [5] constructed an infinite family of graphs admitting Laplacian perfect state transfer. Kay [16] observed that some Laplacian eigenvalues must be integers in order for Laplacian perfect state transfer to happen. Here we build upon this observation to solve the problem of determining which trees admit Laplacian perfect state transfer. Our main result is that, except for the path on two vertices, no tree admits Laplacian perfect state transfer. To achieve that, we first show that, in any graph, Laplacian perfect state transfer does not happen between twin vertices sharing one or two common neighbours. Then we apply the Matrix-Tree Theorem coupled with a precise understanding of Laplacian perfect state transfer to show that such phenomenon could only happen in trees between twin vertices. We also show how our methods can also be applied to other classes of graphs. In the last sections, we study the adjacency perfect state transfer and we will show that no tree with an invertible adjacency matrix admits perfect state transfer. The importance of our results is twofold. First, we rule out trees as candidates for graphs in which Laplacian state transfer can be achieved at large distances with relatively few edges. To this date, the best known trade off is obtained with iterated cartesian powers of the path on two vertices. We also progress in determining which trees admit (adjacency) perfect state transfer. Secondly, we succeed in exhibiting interesting connections between classical results in algebraic graph theory and relatively modern applications. Some results of this paper and an extensive elementary treatment of perfect state transfer can be found in Coutinho [10, PhD Thesis]. Laplacian Perfect State Transfer Throughout this paper, J denotes the all-ones matrix of appropriate size except for the all-ones column vector, which we denote by j. Consider a graph X with adjacency matrix A. Let D be the diagonal matrix whose entries are the degrees of the vertices of X. Then the Laplacian matrix of X is defined as L = D − A. The Laplacian matrix of a graph is positive semidefinite, and the multiplicity of 0 as an eigenvalue is equal to the number of connected components of the graph. The all-ones vector j is always an eigenvector for 0. Because L is symmetric, it admits a spectral decomposition into orthogonal projections onto its eigenspaces, which we will typically denote by with the understanding that 0 = λ 0 < λ 1 < ... < λ d . Suppose u and v are two vertices of X with corresponding characteristic vectors e u and e v . We say that an eigenvalue λ r is in the Laplacian eigenvalue support of u if F r e u = 0. We will denote the eigenvalue support of u by Λ u . We also define Λ + uv to be such that λ r ∈ Λ + uv if and only if F r e u = F r e v , and correspondingly Λ − uv to be such that λ r ∈ Λ − uv if and only if F r e u = −F r e v . Note that Λ + uv and Λ − uv are disjoint subsets of Λ u . The following result is a compilation of many well known facts about perfect state transfer, written in the context of the Laplacian matrix. It explicitly states which conditions on the spectral structure of the Laplacian and on the parity of its eigenvalues are equivalent to perfect state transfer. 2.1 Theorem. Let X be a graph, u, v ∈ V (X). Let L be the Laplacian matrix of X admitting spectral decomposition L = d r=0 λ r F r with 0 = λ 0 < λ 1 < ... < λ d . There exist a positive t ∈ R and a γ ∈ C satisfying exp(itL)e u = γe v if and only if all of the following conditions hold. (ii) Elements in Λ u are integers. Size of the Eigenvalue Support and Twin Vertices Let N (u) denote the set of neighbours of u in X. We say that u and v are twin vertices if N (u)\v = N (v)\u. Note that twin vertices may or may not be adjacent. A pair of twin vertices. Our first original contribution to the study of Laplacian perfect state transfer is the following lemma. 3.1 Lemma. Let X be a connected graph on n > 2 vertices admitting Laplacian perfect state transfer between u and v. Then \|Λ + uv \| ≥ 2 and \|Λ − uv \| ≥ 1. If \|Λ − uv \| = 1, then u and v are twins. Proof. Define (1) The eigenvalue 0 is in Λ + uv . Suppose it is the only eigenvalue in Λ + uv . Its corresponding eigenspace is spanned by j, thus z + = (1/n)j, which is only possible if n = 2. Now suppose that there is only one eigenvalue λ in Λ − uv . Then z − is an eigenvector for λ. As a consequence, any vertex w = v that is a neighbour of u must also be a neighbour of v and vice versa. Thus u and v are twins. We now proceed to show that some twins cannot be involved in Laplacian perfect state transfer. 3.2 Theorem. Suppose X is neither the cycle on four vertices nor the complete graph K 4 minus one edge. If u and v are twins sharing exactly either one or two neighbours, then Laplacian perfect state transfer does not happen between u and v. No Laplacian perfect state transfer between u and v. Proof. Let σ = 0 if u and v are not neighbours, and σ = 1 if they are neighbours. Let S be the set of common neighbours of u and v, and k = \|S\|. By hypothesis, note that k = 1 or k = 2. Suppose Laplacian perfect state transfer happens between u and v. Because u and v are twins, the vector that assigns +1 to u, −1 to v and 0 to all other vertices of the graph is an eigenvector of L with eigenvalue (k + 2σ). All other eigenvectors are orthogonal to this one, thus F r e v = −F r e u if and only if λ r = k + 2σ. Let e S be the characteristic vector of S. For all λ r ∈ Λ + uv , it follows from (1) and (2), we have uv with λ = 0, then λ is an integer and the power of two in the factorization of λ is larger than the power of two in the factorization of (k + 2σ). In particular, it turns out that λ ≥ k + 1. So we have If k = 1, then 3 ≤ n ≤ 4. It is easy to manually check that Laplacian perfect state transfer does not happen in K 3 , P 3 , K 1,3 and K 1,3 plus one edge. If k = 2, then 4 ≤ n ≤ 6. Computations carried out in SAGE show that the only graphs in this case in which Laplacian perfect state transfer occurs are the cycle on four vertices and K 4 minus one edge. If X is a tree, twin vertices in X must have a unique neighbour. As a consequence of our work in this section, we have the following corollary. 3.3 Corollary. If Laplacian perfect state transfer happens in a tree between u and v, then \|Λ − uv \| ≥ 2. No Laplacian Perfect State Transfer in Trees Recall a classical result in graph theory. We start showing a simple yet surprising application of Lemma 3.1 below. 4.2 Corollary. If a graph X has an odd number of vertices and an odd number of spanning trees, then Laplacian perfect state transfer does not happen in X. Proof. Let u and v be any two vertices of X. By Theorem 2.1.(iii), the elements in Λ + uv must be even in order for Laplacian perfect state transfer to happen between u and v. Note that the choice of u in Theorem 4.1 is irrelevant, so by using the Laplace expansion of a determinant, it is easy to observe that the number of spanning trees in a graph with n vertices is equal to the product of its non-zero Laplacian eigenvalues (with repetition) scaled by (1/n). Therefore all non-zero integral Laplacian eigenvalues of X are going to be odd, thus Λ + uv contains only the eigenvalue λ 0 = 0. By Lemma 3.1, Laplacian perfect state transfer cannot happen. Computations carried out in SAGE show that among the 853 connected graphs on seven vertices, 339 have an odd number of spanning trees. Graphs with an odd number of spanning trees are precisely those graphs that contain no non-empty bicycle and are also known as pedestrian graphs. See Berman [4] for more details. An example of a construction of pedestrian graphs consists of taking the successive 1-sums of odd cycles and edges. No Laplacian perfect state transfer in this graph. Corollary 4.2 rules out Laplacian perfect state transfer in trees with an odd number of vertices. We will now work on extending this result to all trees on more than two vertices. We will accomplish this by showing that integer eigenvalues are very bad candidates to belong to Λ − uv . 4.3 Theorem. Suppose X is a graph on n vertices whose number of spanning trees is a power of two. Let λ = 0 be an integer eigenvalue of X. If λ ∈ Λ − uv , then λ is a power of two. Proof. One of the alternative definitions for the rank of a matrix is that it is the largest order of any non-zero minor of the matrix. From Theorem 4.1, it follows that the rank of L is equal to (n − 1) over any field Z p with p an odd prime. Now let y be an integer eigenvector for λ, chosen with the property that the greatest common divisor of its entries is equal to 1. Suppose that there is an odd prime p dividing λ. It follows that Ly ≡ 0 (mod p). Because the rank of L over Z p is (n − 1), the vector y must be a scalar multiple of j over Z p , say y ≡ kj (mod p). If λ ∈ Λ − uv , the projection of e u onto any subspace of the λ-eigenspace must be the negative of the projection of e v . Thus y u = −y v . Therefore k ≡ −k (mod p), and because p is odd, the only possible solution is k ≡ 0 (mod p). This contradicts the fact that the greatest common divisor of the entries of y is equal to 1. Therefore if λ ∈ Λ − uv , no odd prime divides λ. 4.4 Corollary. No tree on more than two vertices admits Laplacian perfect state transfer. Proof. Let u and v be arbitrary vertices of the tree. Suppose that there are two distinct Laplacian eigenvalues λ and µ belonging to Λ − uv . If g is the greatest common divisor of all non-zero elements in Λ u , then Theorem 2.1.(iii) implies that both (λ/g) and (µ/g) must be odd integers in order for Laplacian perfect state transfer to happen. However Theorem 4.3 implies that λ and µ are two distinct powers of two. Thus if Laplacian state transfer occurs, there can only be one eigenvalue in Λ − uv . But that is not possible according to Corollary 3.3. No Laplacian Perfect State Transfer in Other Cases We remark that our technology can be applied to other situations. The following result is an example. Corollary. Suppose X is a graph on n vertices, n > 4. Suppose that the number of spanning trees of X is a power of two, and that X admits Laplacian perfect state transfer between u and v. Then u and v are twins with at least three common neighbours. Moreover, let k be the number of common neighbours of u and v. If u v, then k is a power of two. If u ∼ v, then k + 2 is a power of two. Proof. By Theorem 4.3, all eigenvalues in Λ − uv are powers of two. By Theorem 2.1.(iii), it follows that \|Λ − uv \| = 1. From Lemma 3.1, it follows that u and v are twins, and by Theorem 3.2, they have at least three common neighbours. Finally, let λ be the eigenvalue in Λ − uv . By Theorem 4.3, λ is a power of two. If u v, then λ = k. If u ∼ v, then λ = k + 2. Computations carried out in SAGE show that among the 853 connected graphs on seven vertices, the number of spanning trees of 83 of them is a power of two. Among these, 58 contain twin vertices with one or two neighbours in common. There are 11117 connected graphs on eight vertices. The number of spanning trees of 360 of them is a power of two. The corollary above rules Laplacian perfect state transfer in at least one pair of vertices on 247 graphs among them. No Laplacian perfect state transfer in this graph. We also make an observation that might be useful to rule out Laplacian perfect state transfer in bipartite graphs. Theorem. If X is a bipartite graph in which Laplacian perfect state transfer occurs, then the largest Laplacian eigenvalue λ of X must be an integer. Moreover, u and v are in the same colour class if and only if λ ∈ Λ + uv . Proof. First note that the Laplacian L = D − A is similar to the signless Laplacian Q = D + A. More specifically, if Σ is the diagonal matrix where Σ u,u = ±1 depending on the colour class of vertex u, then ΣLΣ −1 = Q. The signless Laplacian is a non-negative matrix, and it is irreducible. Hence we can apply the Perron-Frobenius theory ([6, Section 2.2]) to argue that the λ-eigenspace of L is one-dimensional and spanned by an everywhere non-zero vector which is positive on one colour class and negative on the other. From Theorem 2.1, it follows that λ must be an integer, and belongs to Λ + uv if and only if the entries corresponding to u and v in its eigenvector have the same sign. We checked in SAGE that among the 182 bipartite graphs on eight vertices, the largest Laplacian eigenvalue is an integer in only 10 of them. Adjacency Perfect State Transfer in Bipartite Graphs We denote the spectral decomposition of the adjacency matrix of a graph X by with the understanding that θ 0 > ... > θ d . Given two vertices u and v, we say that an eigenvalue θ r is in the eigenvalue support of u if E r e u = 0. We will denote the eigenvalue support of u by Φ u . We also define Φ + uv ⊂ Φ u to be such that θ r ∈ Φ + uv if and only if E r e u = E r e v , and correspondingly Φ − uv to be such that θ r ∈ Φ − uv if and only if E r e u = −E r e v . Similarly to what happened in Theorem 2.1, a necessary condition for perfect state transfer between u and v here is that E r e u = ±E r e v for all r, or equivalently, Φ u = Φ + uv ∪ Φ − uv . Vertices u and v satisfying this condition with respect to the adjacency matrix are called strongly cospectral. The following result characterizes the nature of eigenvalues in the support of vertices involved in perfect state transfer. 6.1 Theorem (Godsil [13], Theorem 6.1). If X admits perfect state transfer between u and v, then the non-zero elements in Φ u are either all integers or all quadratic integers. Moreover, there is a square-free integer ∆, an integer a, and integers b r such that Here we allow ∆ = 1 for the case where all eigenvalues are integers, and a = 0 for the case where they are all integer multiples of √ ∆. If the eigenvalues are integers, the following result is the adjacency matrix version of Theorem 2.1. 6.2 Theorem. Let X be a graph and u and v two of its vertices. Suppose the elements of Φ u are integers. Then there is perfect state transfer between u and v at time t ∈ R + and phase γ ∈ C if and only if the following conditions hold. (i) Vertices u and v are strongly cospectral. Suppose X is a bipartite graph, in which case the adjacency matrix of X can be written as If z is an eigenvector for A(X), we split it according to the blocks of A as z = (z 1 , z 2 ). This immediately implies that if θ is an eigenvalue for a bipartite graph X with corresponding eigenvector (z 1 , z 2 ), then −θ is an eigenvalue with eigenvector (z 1 , −z 2 ). As a consequence, we have the following result. 6.3 Lemma. If X is a bipartite graph and u ∈ V (X) is involved in perfect state transfer, then no eigenvalue in the support of u is of the form a+b √ ∆ 2 for non-zero integers a and b with ∆ a square-free larger than 1. is in the support of u. Then its algebraic conjugate is also in the support, and by the observation above, the values −θ and −θ are also eigenvalues in the support of u. The ratio condition (Godsil [12,Theorem 2.2]) states that a contradiction. No Perfect State Transfer in Certain Bipartite Graphs For more details about the next result, see Godsil [14]. 7.1 Theorem. If X is a bipartite graph with a unique perfect matching, then A(X) is invertible and its inverse is an integer matrix. If X is a tree, then A(X) is invertible if and only if X has a (unique) perfect matching. As a consequence of the theorem above, we have the following. 7.2 Theorem. Except for the path on two vertices, no connected bipartite graph with a unique perfect matching admits perfect state transfer. Proof. Suppose X is a bipartite graph with a unique perfect matching, and that u is involved in perfect state transfer. Let θ be an eigenvalue in the support of u, and recall from Theorem 6.1 that θ is a quadratic integer. By Theorem 7.1, (1/θ) must be an algebraic integer, and so θ is either +1, −1, or of the form (a + b √ ∆)/2 with a and b non-zero. Lemma 6.3 excludes the latter case, and hence the only eigenvalues in the support of u are +1 and −1. It is easy to see in this case that the connected component containing u is equal to P 2 , and so the result follows. The result above allows us to rule out perfect state transfer for a large class of trees. We can work a bit more in the case where perfect state transfer happens between vertices in different classes of the bipartition. Proof. From Lemma 6.3, we need only to consider the case where b √ ∆ is in the support of u. Because −b √ ∆ is the algebraic conjugate of b √ ∆, it follows that any eigenvector for b √ ∆ can be partitioned as (z 1 , √ ∆z 2 ), where z 1 and z 2 are rational vectors. As a consequence, the absolute value of the entries in the uth and vth position are different, and so these vertices cannot be strongly cospectral, a necessary condition for perfect state transfer. Now recall Equation 3, and observe that it implies that As a consequence, if perfect state transfer happens in a bipartite graph between vertices in different classes, it must happen with phase ±i. We use that to prove the following result. 7.4 Theorem. Suppose X is bipartite, perfect state transfer happens between u and v at time t, and u and v belong to different classes. Then the powers of two in the factorizations of the eigenvalues in the support of u are all equal. In particular, 0 cannot be in the support of u. From that it follows that θ r is also congruent to 2 α (mod 2 α+1 ), and that 0 cannot be in the support of u. All the results in this paper suggest that very restrictive conditions must hold for perfect state transfer to happen in bipartite graphs. In the context of trees, we could successfully rule out Laplacian perfect state transfer. For the adjacency case, we showed that all trees with a perfect matching do not admit perfect state transfer. We carried out computations in SAGE to check all trees up to ten vertices and we did not find any examples of perfect state transfer except if the tree is P 2 or P 3 . We therefore propose the following conjecture. Conjecture 1. No tree except for P 2 and P 3 admits (adjacency) perfect state transfer.	2014-08-13T08:01:12.000Z	2014-08-13T00:00:00.000	{ "year": 2015, "sha1": "312772bec62f8f054c327da25eea5d8383dd840d", "oa_license": null, "oa_url": "http://arxiv.org/pdf/1408.2935", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "312772bec62f8f054c327da25eea5d8383dd840d", "s2fieldsofstudy": [ "Mathematics" ], "extfieldsofstudy": [ "Mathematics", "Physics", "Computer Science" ] }
264309388	pes2o/s2orc	v3-fos-license	Psychosocial determinants associated with healthcare workers’ self-reported compliance with infection prevention and control during the COVID-19 pandemic: a cross-sectional study in Dutch residential care facilities for people with intellectual and developmental disabilities Background Healthcare workers’ (HCWs) compliance with infection prevention and control (IPC) is crucial to reduce the infection transmission risk. However, HCWs’ compliance with IPC in residential care facilities (RCFs) for people with intellectual and developmental disabilities (IDDs) is known to be suboptimal. Therefore, this study examined sociodemographic and psychosocial determinants associated with IPC non-compliance in this setting, to inform IPC policy and promotion programmes for adequate IPC behaviour. Methods An online questionnaire was administered to 285 HCWs from 16 RCFs between March 2021 and March 2022. Determinants associated with IPC non-compliance were assessed using logistic regression analyses. Results Being a woman (OR: 3.57; 1.73–7.37), and being a non-medical professional were associated with increased odds of non-compliance (social workers, OR: 2.83; 1.65–4.85; behavioural specialists, OR: 6.09; 1.98–18.72). Perceived inadequate education/training (aOR: 1.62; 1.15–2.27) and perceived time constraints/competing priorities (aOR: 1.43; 1.03–1.98) were also associated with increased odds of non-compliance, independent of sociodemographic variables. In contrast, the belief that the supervisor complies with IPC (descriptive norm supervisor) was associated with decreased odds of non-compliance (aOR: 0.60; 0.41–0.88). Conclusions To improve IPC in disability care settings, the implementation of tailored and structural IPC education and training programmes (e.g., on-the-job training) is recommended to increase HCWs’ capabilities and bridge the IPC compliance gap between medical and non-medical professionals. In addition, role models, particularly supervisors, are crucial for promoting IPC behaviour. Facilities should create a culture of IPC compliance by norm setting, acting on, and modelling IPC behaviours at all levels of the organisation (management, medical, and non-medical staff). Background Healthcare workers (HCWs) play a key role in controlling outbreaks and preventing infections in care institutions [1].HCWs should follow appropriate infection prevention and control (IPC) practices (e.g., hand hygiene and the use of personal protective equipment) to prevent the spread of infectious diseases, including Coronavirus Disease 2019 (COVID- 19).Especially in institutional care environments such as residential care facilities (RCFs) for people with intellectual and developmental disabilities (IDDs), the risk of emergence and spread of infections is high [2,3].In addition to the high-risk setting, HCWs' compliance with IPC is important because of the susceptibility of the patient population.Individuals with IDDs are more susceptible to infections due to their underlying medical and physical conditions [4][5][6][7]. Despite the importance of HCWs complying with IPC, this compliance has been reported to be suboptimal in RCFs for people with IDDs [8].Therefore, appropriate behavioural changes among HCWs are necessary.The application of behaviour change theories can be an effective way to improve the compliance of HCWs with IPC practices [9].By understanding the underlying factors that influence behaviour, it is possible to develop strategies that can encourage HCWs to adopt and maintain IPC practices. According to behaviour change theories, an individual's ability and willingness to adopt and maintain a behaviour are affected by a number of psychosocial determinants.At the individual level, cognitive factors, including individuals' beliefs about the risks and benefits of a behaviour, their attitudes towards a behaviour, and their perceived ability to engage in the behaviour -self-efficacy -can play a role [10][11][12][13].Next to individual determinants, the social environment can influence an individual's willingness and capacity to adopt a certain behaviour [10,14].For instance, social norms can influence behaviour by shaping an individual's beliefs and expectations about acceptable behaviour in a particular situation.In the context of IPC compliance, previous studies have shown that psychosocial determinants such as attitudes, self-efficacy, and social norms are established factors that influence IPC behaviour among HCWs [15][16][17][18][19][20][21]. Besides psychosocial determinants, IPC compliance may differ across sociodemographic factors as age, sex, and occupation.Previous studies in other healthcare settings have demonstrated that adequate IPC practices were more prevalent among younger HCWs [22], whereas other studies have argued that older HCWs reported higher levels of IPC compliance [23].Furthermore, prior studies have indicated that female HCWs were more likely to comply with IPC [23,24], whereas other studies did not demonstrate a relationship between age or sex and IPC compliance [25].Previous studies have also suggested an association between occupation and compliance with IPC, in which non-medical professionals reported lower compliance levels than medical professionals [8,[26][27][28]. Despite previous efforts in other healthcare settings, little is known about the determinants of IPC compliance among HCWs in disability care.Therefore, this study examined sociodemographic and psychosocial determinants associated with IPC non-compliance among HCWs in RCFs for people with intellectual and developmental disabilities (IDDs), to inform IPC policy and promotion programmes for adequate IPC behaviour. Study design and setting A cross-sectional questionnaire study was performed.This study was part of the larger mixed methods study NIEZT (Needs assessment for infection prevention among healthcare professionals outside the hospital) (grant number: 331618).The objective of the NIEZT study was to assess IPC compliance and its determinants among HCWs in Dutch RCFs for people with IDDs. In the Netherlands, approximately 110,000 individuals with IDDs reside in RCFs [29].The care setting is characterised by providing care to a range of different clients, including individuals with mild, moderate, severe, and profound intellectual disabilities [30].Due to the diversity in different client groups, and therefore different care needs, the disability care sector is also characterised by a broad range of different HCWs, including medical professionals (e.g., nurses and physicians), social workers (e.g., personal attendants), and behavioural specialists (e.g., psychologists and therapists). Participants Participants included HCWs from 16 Dutch RCFs for people with IDDs in the southern and western regions of The Netherlands.HCWs who had direct patient contact were considered eligible for the study.Exclusion criteria encompassed managerial or policy-related professionals, compliance gap between medical and non-medical professionals.In addition, role models, particularly supervisors, are crucial for promoting IPC behaviour.Facilities should create a culture of IPC compliance by norm setting, acting on, and modelling IPC behaviours at all levels of the organisation (management, medical, and non-medical staff ).Keywords COVID-19, Infection control, Long-term care, Intellectual disability, Developmental disability, Crosssectional studies such as managers and policy officers, as well as administrative personnel without direct patient contact.We aimed to include HCWs from a variety of occupations and educational backgrounds, in order to identify all relevant determinants of IPC compliance among the broad set of different HCWs in the disability care setting. Materials and procedure We developed an online questionnaire.The first part included questions regarding sociodemographic factors, such as age, sex, and occupation.The second part of the questionnaire assessed the levels of self-reported IPC compliance, which were based on national IPC guidelines of the National Institute for Public Health and the Environment (RIVM) [31,32].Additionally, we included questions on psychosocial determinants of IPC compliance. Prior to distributing the questionnaire to our participants, it was piloted among disability care physicians and reviewed by an infection control professional to ensure its applicability.The experts assessed the questionnaire to be applicable, and only minor modifications (i.e., shortening of the length) were applied. Convenience sampling was used to recruit participants.A contact person at each umbrella organisation in the disability care sector was contacted by email or telephone, inviting them to voluntarily participate and distribute the questionnaire among their staff members within their organisations in exchange for receiving a facility-specific report on HCWs' compliance with IPC and its determinants.Information on the study and its aims was provided.After two weeks, a reminder was sent to the contact person in case no responses were received.Responses were collected between March 2021 and March 2022 (during the COVID-19 pandemic: mainly Alpha and Delta variant period). Informed consent (digitally provided) to participate in this study was obtained from every participant before starting the data collection. Measures Outcome Self-reported IPC compliance was the outcome of interest.IPC compliance was measured by 16 items [31,32], covering multiple domains of IPC, including hand hygiene, personal hygiene, clothing requirements, use of personal protective equipment, laundry regulations, medical safety procedures, isolation procedures, and antibiotic prescription behaviour.Participants were requested to estimate how frequently they performed the IPC practices in their day-to-day activities using a 5-point Likert scale, with response options ranging from 1 ("never") to 5 ("always").HCWs' compliance was scored '1' (sufficient compliance) if the HCW responded either "always" or "most of the time" (score of 4 or higher on the 5-point Likert scale), otherwise the healthcare worker was scored '0' (inadequate compliance) [33,34], giving a total possible score range of 0-16.Finally, a total compliance score for all IPC practices was calculated by dividing the number of IPC practices marked as compliant by the total number of required practices (for the specific occupation) [1,23].The total score was expressed in terms of percentages.Subsequently, the total scores were dichotomised.A total compliance score of ≥ 80% was categorised as sufficient compliance, and a compliance score of < 80% as inadequate compliance.This cut-off point was chosen in accordance with previous studies [35,36].Detailed information on IPC compliance among HCWs in RCFs for people with IDDs has been described elsewhere [8]. In this study, inadequate compliance was operationalised as non-compliance to improve readability.However, it is important to note that compliance is a gradient that ranges from fully sufficient to complete non-compliance. Determinants Sociodemographic variables (non-modifiable determinants) Age, sex, and occupation were included as sociodemographic variables.Professional group was computed based on the occupation and associated job descriptions, tasks, and responsibilities [8]. Psychosocial determinants (modifiable determinants) The included items measuring psychosocial determinants comprised a range of items related to attitudes, self-efficacy, and social norms.The questionnaire included 16 individual 1-item statements.Individual statements were used to gain a more detailed understanding of participants' beliefs, which allows for more targeted interventions [37,38].The included psychosocial determinants were selected based on findings from our qualitative study [39] and concepts derived from the Health Belief Model, Theory of Planned Behaviour (including the attitude, social influence, and self-efficacy [ASE]model), Theory of Interpersonal Behaviour, and the Social Cognitive Theory [10][11][12][13], and adapted from previously developed instruments [20,33].Participants were asked to rate their level of agreement with each statement on a 5-point Likert scale ranging from 1 ("strongly disagree") to 5 ("strongly agree").Table 1 provides the measurement of psychosocial determinants. Statistical analyses For this study, only complete questionnaires were included.Participants who did not provide direct patient care (e.g., managers, policy-related professionals, and administrative personnel) were excluded (n = 38).Secondly, descriptive statistics were used to represent participant characteristics.In addition, descriptive statistics were provided for the distribution of answers for the psychosocial determinants.To assess the associations between determinants and IPC non-compliance, we used binary logistic regression.Firstly, associations between IPC non-compliance and all sociodemographic variables were assessed using univariable logistic regression analyses.Secondly, associations between IPC noncompliance and psychosocial determinants were assessed using multivariable logistic regression analyses, adjusted for sociodemographic variables.As we aimed to examine all relevant psychosocial determinants of IPC non-compliance, separate multivariable models were computed for each individual psychosocial determinant.To identify factors that could be targeted in interventions, it is important to focus on factors that have potential room for improvement [40].Therefore, items with high (dis) agreement (i.e., little contrast) -more than 90% of HCWs scored strongly (dis)agree or (dis)agree -were excluded from our models, as they are less likely to influence behavioural change.The resulting associations were reported using (adjusted) odds ratios (ORs) with 95% confidence intervals (CIs).For the psychosocial determinants, the odds of non-compliance were expressed for each point increase in psychosocial determinant scores.Moreover, interaction terms between each psychosocial determinant and professional group were added to the regression models to assess whether associations between psychosocial determinants and IPC non-compliance varied between different professional groups.A p-value < 0.1 was considered relevant to indicate effect modification.In case of effect modification, the associations between psychosocial determinants and IPC non-compliance were examined per professional group.Lastly, sensitivity analyses were conducted to assess the cut-off point for sufficient compliance by repeating the analysis with different operationalisations (cut-off points: 65%, 70%, and 75%).All analyses were performed using IBM SPSS Statistics version 27, and a p-value < 0.05 was considered as statistically significant. Study population In total, 323 complete questionnaire responses were received, of which 285 (88.2%) met the inclusion criteria.Out of the 20 facilities approached, responses were obtained from participants from 16 facilities (80%).Nonparticipation reasons were time constraints and staff shortages arising from the significant burden of COVID-19 cases and outbreaks in the facilities.Table 2 presents the participant characteristics.Most participants were women (87.4%), with a mean age of 43 (± 12.5 years).Half of the participants were social workers (55.1%). Determinants associated with IPC non-compliance To assess the determinants associated with IPC noncompliance, both sociodemographic variables (nonmodifiable determinants) and psychosocial determinants (modifiable determinants) were examined.Table 3 presents the results of the logistic regression analyses.Figure 2 provides a visual presentation of the results. Psychosocial determinants associated with IPC noncompliance After adjusting for sociodemographic variables (age, sex, professional group), perceived inadequate education/training was associated with increased odds of noncompliance (aOR: 1.62; 1.15-2.27,p = 0.006).In addition, perceived time constraints/competing priorities was associated with higher odds of non-compliance (aOR: 1.43; 1.03-1.98,p = 0.032).The association between Fig. 1 Distribution of responses to the items regarding psychosocial determinants perceived knowledge and IPC non-compliance varied for different professional groups (p = 0.1, relevant effect modification).Within the group behavioural specialists, perceived knowledge was associated with decreased odds of non-compliance (aOR: 0.07; 0.01-0.92,p = 0.043). Furthermore, the association between perceived skills and IPC non-compliance varied for different professional groups (p = 0.1, relevant effect modification).Within the group behavioural specialists, perceived skills was significantly associated with decreased odds of non-compliance Abbreviations.IPC = infection prevention and control, OR = odds ratio, CI = confidence interval, Ref = reference category, NA = not applicable a Analyses for the psychosocial determinants were adjusted for sociodemographic variables b Associations for each point increase in psychosocial determinant scores c Items were not applicable since they were not included in the analyses due to high (dis)agreement (i.e., little contrast), > 90% of HCWs scored strongly (dis)agree or (dis)agree d The results of the regression models including interaction terms between 'perceived knowledge' and 'professional group', and 'perceived skills' and 'professional group' revealed relevant effect modification (perceived knowledge, p = 0.1; perceived skills, p = 0.1).Therefore, the associations between these determinants and IPC non-compliance were examined per professional group (aOR:0.07;0.01-0.90,p = 0.042).For all participants, perceived skills was borderline significantly associated with decreased odds of non-compliance (aOR: 0.67; 0.45-1.01,p = 0.057).Descriptive norm supervisor was associated with decreased odds of non-compliance (aOR: 0.60; 0.41-0.88,p = 0.009). Sensitivity analyses for the cut-off point for sufficient compliance Sensitivity analyses using different cut-off points (i.e., 65%, 70%, and 75%) for sufficient compliance revealed highly similar results regarding the determinants associated with IPC non-compliance (data not shown). Discussion The examination of determinants associated with HCWs' compliance with IPC increases the understanding of factors affecting IPC behaviour, which informs the development of intervention programmes for improving compliance.This study assessed sociodemographic and psychosocial determinants of HCWs' self-reported compliance in residential care facilities (RCFs) for people with intellectual and developmental disabilities (IDDs).This study showed that the vast majority of included HCWs considered complying with IPC to be important and generally believed they were able to comply with IPC.In addition, fewer than half of the participants perceived clients to be at a high risk of infection, which reveals a The results of the regression models including interaction terms between 'perceived knowledge' and 'professional group' , and 'perceived skills' and 'professional group' revealed relevant effect modification (perceived knowledge, p = 0.1; perceived skills, p = 0.1).Therefore, the associations between these determinants and IPC non-compliance were examined per professional group potential underestimation of the risk of infection that clients face within RCFs. Regarding the associations between sociodemographic variables and IPC compliance, our findings demonstrated that non-medical professionals (social workers and behavioural specialists) had increased odds of noncompliance than medical professionals.This finding is consistent with previous studies [8,[26][27][28], which have indicated that health attendants, personal care workers, and non-clinical staff are more likely to be non-compliant compared to nurses and medical professionals.A potential reason for this may be the difference in professional background [39].In general, medical professionals are more acquainted with IPC practices, as this is often a part of their training, especially in light of performing medical and nursing procedures.Moreover, the results of this study revealed increased odds of non-compliance in female HCWs compared to their male counterparts.However, careful interpretation of this finding is advised, as previous studies have shown conflicting results.Some studies have suggested that women are more likely to comply with infection control guidelines than men [23,24], while others have found no significant differences between male and female HCWs [25].One should note that these previous studies were exclusively conducted among medical professionals.Therefore, caution is needed when comparing these findings with the findings of our study, which included both medical and non-medical professionals.Previous studies conducted in other healthcare settings also suggested an association between years of experience and IPC compliance levels during the COVID-19 pandemic [21,26].Overall, these studies indicated that HCWs with more (years of ) experience were more likely to comply with IPC practices.In our study, we did not include years of experience as a variable, because of the risk of multicollinearity.Nevertheless, we included age and occupation as covariates, thereby indirectly accounting for the years of experience of HCWs. Regarding psychosocial determinants, our findings suggested that the self-efficacy items perceived time constraints/competing priorities and perceived inadequate education/training were independently associated with IPC non-compliance.Previous studies have identified perceived time constraints and competing priorities as important determinants that negatively influence IPC compliance [41,42].HCWs often have multiple tasks and responsibilities, and compliance with IPC may sometimes conflict with other tasks or priorities, such as attending to patients' needs or administrative duties.Furthermore, inadequate education or training is commonly reported to be negatively associated with IPC compliance [42,43].Previous studies have indicated that HCWs who report receiving adequate IPC training have a higher likelihood of complying with IPC than those who report inadequate or no training [44].Our findings revealed that only among behavioural specialists, perceived knowledge and perceived skills were associated with IPC non-compliance.This can be attributed to differences in training and professional background.Knowledge and skills enhancing efforts such as education and training programmes may be less frequently aimed at non-medical professionals (e.g., behavioural specialists).In addition, non-medical professionals are often less aware of IPC procedures and tasks, and their role in preventing healthcare-associated infections [45]. This study's finding that HCWs' compliance with IPC is positively affected by the descriptive norm of the supervisor suggests that supervisors play an important role as role models for their subordinates.This is supported by previous studies in the nursing and hospital care setting, which have suggested that the presence of IPC role models is associated with increased IPC compliance among HCWs [17][18][19]46].The significance of the descriptive norm of the supervisor in our study, while the injunctive norm of the supervisor is not significant, may be explained by the fact that supervisors potentially have a strong social influence on their employees' behaviour through their actions (descriptive norm), rather than through explicit directives or expectations (injunctive norm) [47].This may reflect the influence of role modelling, whereby HCWs are more likely to adopt the behaviour of their supervisor as a positive role model, rather than as a result of feeling pressure to comply with their expectations. Besides sociodemographic and psychosocial determinants of IPC compliance, IPC behaviour may be influenced by other factors, including environmental and logistical barriers, such as lack of necessary equipment as well as organisational culture, leadership, and policies [25,27,28,39].These barriers can make it difficult for HCWs to practice effective IPC, even if they have motivation, knowledge, and skills to do so.Although sociodemographic and psychosocial determinants are important factors for IPC compliance, the influence of other contextual factors, such as environmental and logistical barriers, should also be considered. Strengths and limitations A strength of this study is the theoretical underpinning, as it uses concepts of multiple behaviour change theories.Previous studies have suggested the insufficiency of studies that used only one theory or a few theoretical models, and have highlighted that a broad theoretical underpinning enhances the quality of a study [46,48].Furthermore, the items included in our study were based on qualitative findings, thereby triangulating previous findings and providing additional insights into the topic [49]. This study has some limitations.Firstly, participants were selected by means of convenience sampling.This indicates that we cannot rule out some kind of selection bias, as perhaps, more IPC-minded HCWs were selected.Due to the recruitment method of participants (convenience sampling), it is challenging to accurately report the response rate, as the total number of professionals reached in the facilities is unknown.Nonetheless, we assume that the sample was rather representative of the study population since HCWs from different occupations (and therefore also different educational backgrounds) were reached.Secondly, the cross-sectional nature of the study precludes any causal inferences between compliance and the studied determinants [50].One should note that there are also other potential psychosocial determinants of IPC compliance, besides the psychosocial determinants included in our study.An important determinant is risk perception, for which the perceived risk of infection (i.e., perceived susceptibility) for HCWs and their perceived severity are important components [51,52].Nevertheless, due to the length of the questionnaire and the required time investment of HCWs, we had to make choices about which determinants to include.Thirdly, one should take note of the cut-off point of 80% for sufficient compliance, as this may be too strict.Nevertheless, sensitivity analyses demonstrated that different operalisations of the cut-off point did not reveal other significant relevant results, therefore, indicating the robustness of our findings.In addition, due to the relatively small sample size in our study, it is likely that some associations were missed due to limited power, for example in the different categories of professional groups.For future studies, an increased sample size per professional group can provide an additional understanding of specific psychosocial determinants associated with IPC noncompliance, providing further insights for the design of improvement strategies and its targeting. Implications for practice Our findings indicate the importance of increasing HCWs' capabilities to comply with IPC.From the background of behaviour change theories, an initial recommendation for facilities is to implement structural education and training programmes.These educational efforts should not only include technical knowledge, but also aim to increase awareness, practical skills, and attitudes needed for proper IPC compliance, which can potentially increase HCWs' capabilities.As the selfefficacy items perceived inadequate education/training and perceived time constraints/competing priorities were independently (of sociodemographic variables including professional group) associated with IPC non-compliance, there is a universal need among different HCWs for education and training programmes.Nevertheless, since professional group is a determinant of HCWs' IPC compliance, educational efforts should be tailored to the educational needs of different professionals.Targeted educational strategies are especially important since our results indicated that the associations between perceived knowledge and skills and IPC non-compliance vary across different professional groups, with only behavioural specialists' perceived knowledge and skills being associated with non-compliance.Therefore, we recommend directing education and training programmes towards the enhancement of knowledge and skills among behavioural specialists specifically. The implementation of tailored education and training programmes can bridge the gap in IPC compliance between different HCWs.For example, non-medical professionals (e.g., behavioural specialists) may need basic IPC training and education to increase their awareness of IPC, whereas medical professionals may need more advanced IPC training to cover specialised areas of IPC.These educational efforts should be ongoing and continuous, as the effectiveness of these strategies may diminish over time [53].Furthermore, educational programmes should incorporate an element that addresses perceived competing demands or time constraints.A potential strategy in this is professional-led education in which professionals come up with real-time examples and experiences in which they experienced competing demands or time constraints and discuss ways to overcome these challenges. In addition to the importance of enhancing HCWs' capabilities, the results of our study demonstrate the significant role of social norms − especially the descriptive norm of supervisors − on HCWs' IPC compliance.Therefore, an additional strategy to promote IPC compliance includes role modelling and norm setting.We recommend that facilities raise awareness among supervisors about the impact of their role model function, and actively encourage them and other key individuals to model good IPC practices.This sets a positive example for HCWs, helps to increase the social norm for IPC compliance, and reinforces the importance of IPC, which helps to create a culture in the workplace and organisation in which IPC is emphasised and valued [17,46].Role models can be individuals who occupy formal leadership positions (i.e., supervisors), but they can also be informal leaders who influence the behaviour of others through their actions and attitudes. Interventions regarding the use of role models have been implemented in the disability care sector in the Netherlands.On the initiative of the Limburg infection prevention and antibiotic resistance care network, some facilities started with the appointment of IPC contact persons.These individuals function as a first point of contact for their co-workers regarding IPC, act as a source of information, and play a stimulating role in raising awareness regarding the importance of IPC in the workplace. Previous studies have suggested that behaviour change methods should be used in combination [54,55].For instance, norm setting should be used in conjunction with other intervention functions, such as education and training and role modelling, to maximise the effectiveness of the intervention.A strategy that may enhance both capabilities and facilitate IPC behaviour as a norm within the organisation is on-the-job training.On-thejob training could be a comprehensive approach, as it incorporates different elements, including the opportunity for HCWs to gain new skills and knowledge relevant to their job and IPC tasks, immediate feedback (also further enhancing capabilities), reinforcement of IPC norms, and a personalised (and tailored) approach to learning.On-the-job training makes the learning process more relevant and meaningful [56]; therefore, it potentially results in increased motivation among HCWs to comply with IPC.Evidence from previous studies has shown that IPC education and training involving HCWs in a practical, hands-on approach and incorporating individual experiences is associated with decreased healthcare-associated infections and increased IPC compliance [57,58]. Conclusions Efforts to improve IPC compliance in the disability care setting should focus on strategies to enhance HCWs' capabilities and decrease the gap in compliance between medical and non-medical professionals.A recommendation for facilities is to implement tailored and structural IPC education and training programmes, for which onthe-job training can be a relevant educational approach.In addition, the presence of role models − especially supervisors − is important to promote IPC behaviour.Facilities should create a culture of IPC compliance by norm setting, acting on, and modelling IPC behaviours at all levels of the organisation, including management, medical, and non-medical staff. Figure 1 Figure1presents the distribution of response scores (1 to 5) for each psychosocial determinant.Regarding attitudes, 95.4% (n = 272) considered complying with IPC to be important.In addition, only 8.8% (n = 25) of participants reported that working according to protocols/guidelines evokes resistance.Regarding perceived interference with the professional-client relationship, 18.9% (n = 54) reported to strongly agree or agree.Furthermore, 19.3% (n = 55) reported that complying with IPC requires a lot of effort, and 34.4% (n = 98) reported that complying with IPC takes a lot of time.Moreover, 48.4% (n = 138) of participants expected the client to have a high risk of infection.Regarding outcome expectation, 78.6% (n = 224) expected that infections can be prevented when complying with IPC.Regarding general self-efficacy, 93.0% (n = 265) reported feeling confident about their ability to comply with IPC.In addition, 9.5% (n = 27) of participants reported that they were not educated/trained to comply with IPC.Moreover, 76.5% (n = 218) perceived sufficient knowledge, and 81.4% (n = 232) perceived sufficient skills to comply with IPC.Of the included HCWs, 17.5% (n = 50) reported forgetting to comply with IPC due to busyness with other tasks.Regarding social norms, 78.2% (n = 223) believed that colleagues comply with IPC, and 64.2% (n = 183) believed that the supervisor complies with IPC (descriptive norm).Moreover, 64.6% (n = 184) believed that colleagues think they should comply with IPC, and 70.9% (n = 202) Fig. 2 Fig. 2 Forest plot showing odds ratios (ORs) and 95% confidence intervals (CIs) for the logistic regression analyses between a sociodemographic variables and IPC non-compliance and b psychosocial determinants and IPC non-compliance.The circles correspond to the ORs.The error bars (horizontal lines) represent 95% CIs.The vertical line indicates the point of no effect (OR = 1) Abbreviations.IPC = infection prevention and control.Ref = reference category a Analyses for the psychosocial determinants were adjusted for sociodemographic variables bAssociations for each point increase in psychosocial determinant scores c Items were not included in the analyses due to high (dis)agreement (i.e., little contrast), > 90% of HCWs scored strongly (dis)agree or (dis)agree d Table 1 Measurement of psychosocial determinants Abbreviations.IPC = infection prevention and control Table 3 Logistic regression analysis examining the associations between both sociodemographic and psychosocial determinants and IPC non-compliance	2023-10-20T14:00:16.175Z	2023-10-19T00:00:00.000	{ "year": 2023, "sha1": "7e7a09170c293c944738f1844dd235ad8a4d24d1", "oa_license": "CCBY", "oa_url": "https://bmcpublichealth.biomedcentral.com/counter/pdf/10.1186/s12889-023-16912-0", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "be8f5f8d74950ffe15f8dc9a8a2384a124d5d98c", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
91939255	pes2o/s2orc	v3-fos-license	Relative Contribution of Framework and CDR Regions in Antibody Variable Domains to Multimerisation of Fv- and scFv-Containing Bispecific Antibodies Bispecific antibodies represent an emerging class of antibody drugs that are commonly generated by fusion of Fv or scFv antigen binding domains to IgG or Fab scaffolds. Fv- or scFv-mediated multimerisation of bispecific antibodies via promiscuous vH-vL pairing can result in sub-optimal monomer levels during expression, and hence, undesirable therapeutic protein yields. We investigate the contribution of disulphide stabilised Fv and scFv to Fab-Fv and Fab-scFv multimerisation. We show that monomer levels of isolated Fv/scFv cannot always be used to predict monomer levels of Fab-linked Fv/scFv, and that Fab-scFv monomer levels are greater than the equivalent Fab-Fv. Through grafting bispecifics with framework/CDR-‘swapped’ Fv and scFv, we show that monomer levels of disulphide stabilised Fab-Fv and Fab-scFv can be improved by Fv framework ‘swapping’. The Fab-Fv and Fab-scFv can be considered representative of the significant number of bispecific antibody formats containing appended Fv/scFv, as we also used Fv framework ‘swapping’ to increase the monomer level of an IgG-scFv bispecific antibody. This research may, therefore, be useful for maximising the monomeric yield of numerous pharmaceutically-relevant bispecific formats in pre-clinical development. Introduction Through simultaneous binding to two antigens, bispecific antibodies can invoke synergistic or novel biology and may offer enhanced clinical efficacy via improved drug targeting.Fv and scFv are discrete antigen binding domains that have been fused to Fab or IgG scaffolds to confer multispecificity in a wide variety of formats (reviewed in [1]).Conversion of Fv to scFv through introduction of a polypeptide linker between the vH and vL was originally carried out to stabilise the relatively weak vH-vL interface, but dynamic domain exchange ('breathing') between proximal scFv monomers can result in variable levels of monomer, dimer and higher-order multimers [2].The introduction of a disulphide (ds) bond in isolated scFv between the vH and vL prevents variable domain 'breathing' and thus fixes monomer:multimer ratios [2].The result of disulphide stabilised vH and vL 'mispairing' between proximal scFv monomers (in isolated scFv or scFv-containing bispecific antibodies), is a spectrum of dimer, trimer and higher order species.Formation of a vH-vL disulphide bond irreversibly locks all multimeric forms during expression/purification, and therefore enables the separation of desirable (monomeric) from undesirable (multimeric) species by purification.In isolated disulphide stabilised scFv (dsscFv), the presence of a linker enables stable dimer/multimer formation (Figure 1B).Conversely, isolated disulphide stabilised Fv (dsFv) might be expected to form 100% monomer, as there is no linker available to connect monomers together (Figure 1A).Fab or IgG fusion proteins appended with a dsFv may however have additional complexities.Figure 1C,D illustrates potential dimerisation mechanisms of formats appended with dsFv or dsscFv, demonstrating how inappropriate multimerisation can occur during cellular expression.The term 'mispairing' in the context of this paper is defined as that resulting from the intermolecular pairing of a dsvH in one intended dsFv/dsscFv on a carrier molecule, with a dsvL appended to a different carrier molecule of the same type.Antigen binding is retained in the resulting multimers owing to the fact that the vH-vL pairs are all 'appropriate'.However, multimers may have unwanted biological properties (e.g., receptor crosslinking, avidity effects) as well as concentration/storage issues, and must therefore be removed during subsequent bioprocessing steps.High levels of multimer formation during protein expression will thus ultimately result in low overall therapeutic protein yields. We compare Fab-dsFv [3,4] and Fab-dsscFv as exemplar Fv-and scFv-containing bispecific antibodies to investigate the influence of dsFv versus dsscFv on bispecific antibody monomer level, owing to the molecular simplicity compared to IgG appended formats.The Fab-dsFv can be considered to be a model for bispecific formats where vH and vL are appended off neighbouring C-terminii such as IgG(H)-Fv [5,6], whilst the Fab-dsscFv may be considered to be representative of formats containing appended scFv such as IgG(H)-scFv and IgG(L)-scFv [7][8][9][10][11][12].We further investigate the relative influence on multimerisation of dsFv and dsscFv framework (FW) and CDR residues through grafting a range of Fv FW/CDR 'swapped' Fab-dsFv and Fab-dsscFv.We also analyse the effect of the framework and CDR residues on Fv thermal stability to assess whether there is a relationship between thermal stability and monomer level.Lastly, we analyse the effect of Fv framework 'swapping' in an IgG-dsscFv bispecific antibody. Reagents All materials, reagents and human embryonic kidney (HEK) 293F cell lines were sourced from Life Technologies (Paisley, UK) unless otherwise stated. Plasmid Construction The Fab-dsFv light chain is constructed as signal peptide-vL1-cK-Ser(Gly 4 Ser) 3 -dsvL2, where vL1 is the humanised variable light domain 1, cK is the human kappa light chain constant domain and vL2 is the humanised variable light domain 2. The Fab-dsFv heavy chain is constructed as signal peptide-vH1-CH1-Ser(Gly 4 Ser/Thr) 3 -dsvH2, where vH1 is the humanised variable heavy domain 1, CH1 is the human gamma-1 heavy chain CH1 constant domain and vH2 is the humanised variable heavy domain 2. The Fab-dsscFv light chain is constructed as signal peptide-vL1-cK-Ser(Gly 4 Ser) 2 -dsvH2-(Gly 4 Ser) 4 -dsvL2.The Fab-dsscFv heavy chain is constructed as signal peptide-vH1-CH1.Isolated dsscFv were expressed in HL orientation and constructed as signal peptide-dsvH-(Gly 4 Ser) 4 -dsvL-10xHis, where 10xHis is a C-terminal epitope tag.Isolated dsFv were expressed as signal peptide-dsvH and signal peptide-dsvL-10xHis from separate plasmids.Linker sequences are listed here for clarification: Fab-dsFv light chain linker (SGGGGSGGGGSGGGGS), Fab-dsFv heavy chain linker (SGGGGSGGGGTGGGGS), Fab-dsscFv light chain linker (SGGGGSGGGGS), scFv linkers connecting vH2 and vL2 (GGGGSGGGGSGGGGSGGGGS).All isolated and Fab-linked Fv and scFv were disulphide stabilised, containing cysteines at vH44-vL100 (Kabat numbering).Proteins were expressed from one of two closely related CMV-containing UCB-modified mammalian expression plasmids; pNAFL was used for cloning and expression of light chain constructs while pNAFH was used for cloning and expression of heavy chain constructs and scFv.The two plasmids possess largely identical sequences and have been observed to have very similar functionalities. Antibody Expression Expression plasmids were co-transfected (1:1 ratio of heavy:light chain) into 293F cells using 293fectin™ transfection reagent according to the manufacturer's instructions.The cells were cultured in FreeStyle™ media with shaking at 37 • C. Cell culture supernatants were harvested 10 days post-transfection by centrifugation at 2000 rpm for 10 min.Supernatants were clarified by passage through a 0.22 µm filter. Octet Quantification Assay dsFv and dsscFv protein concentration in the cell culture supernatant was determined using an Octet (ForteBio) and software version 4.0.7.Penta-His biosensors were prepared according to the manufacturer's instructions and supernatants analysed using the following parameters: assay time 120 s, shake speed 200 rpm, dip and read time 120 s.A standard curve was generated using a purified His-tagged dsscFv (UCB) at a concentration of 1-100 µg/mL using a dose-response (4PL) equation.All standards and samples were read in duplicate.Octet limit of detection = 5 µg/mL. Size Exclusion Chromatography (SEC) 20 µg purified protein sample (100 µL of 0.2 mg/mL stock diluted in PBS, pH7.4) was injected onto either a Superdex 200 10/300 GL Tricorn column (GE Healthcare, Little Chalfont, UK) or a TSK Gel G3000SWXL, 7.8 × 300 mm, column (Tosoh Bioscience, Reading, UK) 3 days post-purification and developed respectively with an isocratic gradient of PBS, pH 7.4 at 1 mL/min or 200 mM phosphate, pH 7.0 at 1 mL/min.Signal detection was by absorbance at 280 nm.Gel filtration protein standards (BioRad, Watford, UK) were loaded for molecular weight estimation (chromatograms are shown in Supplementary Figure S1).Since all purified fusion proteins contain disulphide stabilised Fv/scFv, the observed monomer level is independent of protein concentration and unaffected by on-column dilution effects. Differential Scanning Fluorimetry Thermal stability analysis was carried out as described previously [2,13].Briefly, samples contained 3× SYPRO ® Orange dye and 0.1 mg/mL purified protein in PBS, pH 7.4.The mixture was dispensed in quadruplicate into a 384 PCR optical well plate, which was run on a 7900 HT fast real-time PCR System (Agilent Technologies, Stockport, UK).The PCR system heating device was set at 20 • C to 99 • C with a ramp rate of 1.1 • C/min; a charge coupled device monitored fluorescence changes in the wells.Intensity increase was plotted, and the inflection point of the slope(s) was used to generate the thermal stability transition midpoint (Tm).A higher Tm denotes a more stable protein domain.Thermograms are shown in Supplementary Figure S2. Comparison of dsFv and dsscFv A range of human vK1 vH3 variable region pairs with vH44:vL100 disulphide stabilisation was expressed transiently as C-terminally His-tagged dsFv or dsscFv in 293F cells.We used the vK1 vH3 sub-group, which is routinely used for humanisation and is thus considered to be a model v-region framework pairing.The scFv used in this study are in the HL orientation, whereby the C-terminus of the vH is connected to the N-terminal of the vL via a flexible peptide linker.We expressed the scFv in the HL orientation as we have previously observed higher monomer levels for these variable domain sequences in the HL orientation compared to the LH orientation.We used a 20 amino acid 4×G 4 S linker to connect the vH and vL as these long linkers have been shown to be effective at minimising non-covalent multimerisation [9,14].This is an important consideration in this study since we wanted to witness the importance of the variable domain sequences in the multimerisation process rather than those driven by any linker constraints.We used the vH44:vL100 disulphide bond position as this has previously been shown to be a preferred disulphide bond location that is well tolerated amongst different scFv and Fv [2,9,15]. Four dsFv and four dsscFv proteins were expressed transiently in 293F cells.The variable domain primary sequences in dsFv#1, dsFv#2, dsFv#3 and dsFv#4 are the same as those used respectively in dsscFv#1, dsscFv#2, dsscFv#3 and dsscFv#4.Fv#1-4 bind to three distinct target antigens: Fv#2 is specific for a serum protein, whilst the target antigens for Fv#1, Fv#3 and Fv#4 are all soluble cytokines.Fv#3 and Fv#4 bind to the same target antigen, but possess unique sequences and were discovered independently.Following expression, the cell supernatants were analysed by Octet using Penta-His tips.It has been suggested that expression of dsFv without a linker is unattainable or extremely inefficient in mammalian cells and can only be achieved in bacteria through periplasmic expression or refolding of cytoplasmic inclusion bodies [5].This is thought to be due to the weak association between vL and vH [16] resulting in inherently poor hetero-pairing.We successfully expressed four different dsFv in 293F cells, as judged by SDS-PAGE post-purification (Figure 2A), although the expression levels of dsFv#1 and dsFv#4 in the cell supernatant were below the limit of detection by Octet measurement.The expression levels of the dsFv were comparatively lower than the corresponding dsscFv, with dsFv#2 and dsFv#3 expression levels (29.4 µg/mL and 37.6 µg/mL) being respectively ~50% and 80% that of the equivalent dsscFv (Table 1).To put these yields in context, Fab and IgG expressed in 293F cells using the same plasmids and signal peptide, gave expression levels of 32.6 µg/mL and 39.8 µg/mL, respectively.The dsFv and dsscFv proteins were purified from the cell culture supernatant by a batch Ni 2+ -NTA method and the purified proteins were analysed by non-reducing and reducing SDS-PAGE (Figure 2A) and SEC (Figure 2B-E).SDS-PAGE analysis revealed prominent high molecular weight species in dsFv#1 and dsFv#4 samples (Figure 2A, lanes 1 and 4 respectively), which were also the two poorest expressing dsFv.These bands remained under reducing conditions and may represent host cell proteins that have co-purified with these dsFv.These bands are also visible in other samples, albeit to a much lower extent and were not detected by anti-His immune-blotting (data not shown).SEC analysis showed that dsFv#2 and dsFv#3 were 99-100% monomeric, whereas dsFv#1 and dsFv#4 appeared to display lower monomer levels of 40-44% (Table 1).Again, in the absence of a linker, it is improbable that this low monomer level represents multimeric dsFv and more likely reflects contamination by host cell proteins.Thus, dsFv#1 and dsFv#4 are also likely to be 100% monomeric.All dsscFv were highly monomeric, with monomer levels of 97.0%, 100.0%, 99.5% and 93.4% respectively for dsscFv#1, dsscFv#2, dsscFv#3 and dsscFv#4. The thermal stabilities (Tm's) of the purified dsFv and dsscFv proteins, determined by differential scanning fluorimetry, are displayed in Table 1.The Tm's of the dsscFv were towards the upper end of published scFv stabilities and were broadly comparable with their dsFv counterparts.The presence of the scFv linker appears to have marginally destabilised scFv#3, as the Tm of dsscFv#3 (61.2 • C) was several degrees lower than dsFv#3 (66.8 • C).Perhaps this represents the kind of steric constraint which could be alleviated with even longer linkers [11] or expression in the LH orientation.Similarly, the Tm of scFv#1 (57.8 • C) was slightly lower than dsFv#1 (59.5 • C).Conversely, the scFv linker appears to have stabilised dsFv#2 as the Tm of scFv#2 (76.9 • C) was slightly higher than dsFv#2 (75.2 • C).All dsFv and dsscFv displayed a single Tm, except dsFv#1 and dsFv#4, which displayed an additional minor unfolding transition at ~52 • C, which likely represents host cell protein contaminants. Comparison of Fab-dsFv and Fab-dsscFv The same dsFv or dsscFv (minus His-tag) was then fused to the C-terminal end of HER2 (4D5) Fab, which has been well characterised in the literature and has been shown to be 100% monomeric [17].We made Fab(LC)-dsscFv proteins, as opposed to Fab(HC)-dsscFv, because the Fab(LC)-dsscFv constructs were readily available.Following transient expression in 293F cells, the concentration of Fab-dsFv and Fab-dsscFv proteins in the culture supernatant was determined by a Protein G HPLC assay.The expression level of the Fab-dsFv ranged from 15.1-28.1 µg/mL, whereas the expression level of the Fab-dsscFv in the cell supernatant was somewhat lower at 15.8-21.8µg/mL (Table 2), although there was one exception where the expression level of Fab-dsscFv#4 (21.8 µg/mL) was higher than Fab-dsFv#4 (15.1 µg/mL). Table 2. Comparison of Fab-dsFv vs. Fab-dsscFv formats.Following the 10-day transient expression in 293F cells, the expression level of Fab-dsFv#1-4 and Fab-dsscFv#1-4 proteins in the culture supernatant was measured by a Protein G HPLC assay.Proteins were purified from the supernatant using Protein G HPLC, and the purified proteins analysed by G3000 SEC and differential scanning fluorimetry.Data shows mean ± SD from three independent transfections. Proteins were purified from the cell culture supernatant by Protein G HPLC and the purified proteins were analysed by SEC (Figure 3).Since Protein G binds to the CH1 domain, any light chain dimers that may have formed during expression will be absent from the purified material.Differential monomer levels were observed for Fab-dsFv and Fab-dsscFv proteins comprising equivalent sequences in the Fv position, with monomer levels of the Fab-dsscFv being higher in all four examples than the equivalent Fab-dsFv. Proteins were purified from the cell culture supernatant by Protein G HPLC and the purified proteins were analysed by SEC (Figure 3).Since Protein G binds to the CH1 domain, any light chain dimers that may have formed during expression will be absent from the purified material.Differential monomer levels were observed for Fab-dsFv and Fab-dsscFv proteins comprising equivalent sequences in the Fv position, with monomer levels of the Fab-dsscFv being higher in all four examples than the equivalent Fab-dsFv.In the three examples where the Fab-dsFv were inherently highly monomeric (Fab-dsFv#1: 87.5% monomer, Fab-dsFv#3: 83.7% monomer, Fab-dsFv#4: 76.0% monomer), conversion of dsFv to dsscFv resulted in only a minor increase in monomer level (Fab-dsscFv#1: 91.2% monomer, Fab-dsscFv#3: 91.8% monomer, Fab-dsscFv#4: 79.9% monomer) (Table 2, Figure 3A,C,D).However, in the single case where the Fab-dsFv had a very low monomer level (Fab-dsFv#2), the monomer level was almost doubled from 37.8% to 70.9% by conversion of the Fab-dsFv to a Fab-dsscFv (Table 2, Figure 3B).Isolated dsFv#2/dsscFv#2 appeared to be 100% monomeric, so the inherent tendency of Fv#2 to multimerise only becomes apparent when the dsFv or dsscFv is fused to a Fab.Thus, there appears to be no correlation of monomer level in free dsFv/dsscFv formats to that of the related Fab-dsFv/Fab-dsscFv formats, indicating that 'free intermolecular association' properties are not the same as 'tethered intermolecular association' properties. Thermal stabilities of the Fab-dsFv and Fab-dsscFv proteins were determined by differential scanning fluorimetry (Table 2).The isolated HER2 Fab has a Tm of 80.7 • C, and this was reduced by ~2 • C by fusion of dsFv/dsscFv.There was close correlation between the Fv Tm from dsFv/dsscFv and Fab-dsFv/Fab-dsscFv.For example, the dsFv Tm's of dsFv#2 and Fab-dsFv#2 respectively were 75.2 • C and 72.4 • C and the dsscFv Tm's of dsscFv#2 and Fab-dsscFv#2 respectively were 76.9 • C and 73.1 • C. The Fab appended dsFv/dsscFv generally displayed a Tm that was typically a few degrees lower than the isolated dsFv/dsscFv, except in the case of dsscFv#1, where the dsscFv Tm in Fab-dsscFv#1 (59.0 • C) was slightly higher than the isolated dsscFv#1 (57.8 • C), and in dsscFv#3, where the isolated and Fab appended dsscFv had equal Tm's (61.2 • C and 61.4 • C respectively).Thus, attachment to a Fab domain via a linker does not substantially affect thermal stability of dsFv/dsscFv. Table 3.Comparison of Fab-dsFv and Fab-dsscFv with wild type and FW/CDR-'swapped' Fv.Following the 10-day transient expression in 293F cells, the expression level of wild type and framework 'swapped' Fab-dsFv and Fab-dsscFv proteins in the culture supernatant was measured by a Protein G HPLC assay.Proteins were purified from the supernatant using Protein G HPLC, and the purified proteins analysed by G3000 SEC and differential scanning fluorimetry.Data shows mean ± SD from three independent transfections.* Same data as in Table 2, shown here for ease of comparison with FW/CDR 'swap' data. The reducing SDS-PAGE gels (Figure 4A,B) showed banding patterns which indicated that the constructs were being expressed correctly with bands at ~50 kDa and ~25 kDa (Fab-dsscFv) or a doublet at ~37 kDa (Fab-dsFv).Fab-dsFv with dsFv#2 or dsFv(FW#2/CDR#1) in the Fv position seemingly ran as a single band as the heavy and light chains are almost identical in size (Figure 4A, reduced gel, lanes 2 and 4).There was a small proportion of unreduced protein in all four Fab-dsFv samples under reducing conditions (Figure 4A, reduced gel).There was a small proportion of 'free' Fab in all Fab-dsscFv samples, which was most prominent in Fab-dsscFv(FW#2/CDR#1) (Figure 4B, reduced and non-reduced gel, lane 4). Fv framework 'swapping' influenced monomer levels more in the Fab-dsscFv format compared to Fab-dsFv.Percentage monomer data for wild type and 'swapped' Fab-dsscFv proteins is illustrated in the bar graphs (Figure 4B) and in Table 3. Grafting dsscFv#2 CDRs onto FW#1 resulted in a high monomer level (Fab-dsscFv(FW#1/CDR#2): 90.1% monomer, compared to Fab-dsscFv#2: 70.9% monomer).The monomer levels of Fab-dsscFv can thus be improved through the grafting of dsscFv CDRs onto a different framework, providing that the antigen affinity is retained within the FW-'swapped' dsscFv.In this particular case, essential antigen binding was retained when Fv#2 CDRs were grafted onto FW#1, as shown by antigen-based ELISA (Figure 4C).We have also shown analogous improvements in monomer level in a high density Expi293F cell line.Fab-dsscFv with wild type and framework 'swapped' dsscFv showed almost identical monomer levels to those seen in 293F cells, despite significantly higher (4-7 fold) expression levels in Expi293F cells (data not shown).This is an important observation as it shows that monomer level is unaffected by transient expression level, therefore a high-density cell line can be used to maximise expression with no negative influence on monomer level.Fab-dsscFv#1 and Fab-dsscFv(FW#2/CDR#1) showed similarly high monomer levels, although the lower expression and presence of more 'free' Fab observed in Fab-dsscFv(FW#2/CDR#1) suggests some instability upon CDR#1 grafting onto FW#2. To analyse the influence of dsFv framework and CDR sequences on Fab-dsFv thermal stability, the thermograms of wild type and 'swapped' Fab-dsFv were compared.Whilst the Tm profiles of Fab-dsFv with a common dsFv framework were quite different, the Tms of Fab-dsFv with common dsFv CDRs were very similar (Table 3, Supplementary Figure S2).This suggests that dsFv thermal stability is driven mainly by CDR residues and not framework residues.Whereas differences were seen between Fab-dsFv and Fab-dsscFv in terms of the contribution of Fv framework and CDR residues to monomer level, the contribution of the Fv CDRs to Tm was the same regardless of whether the Fv was a dsFv or dsscFv.The Fv Tm in 'swapped' Fab-dsscFv resembled those of the wild type Fv containing the same CDRs e.g., the Fv Tm in Fab-dsscFv(FW#1/CDR#2) (72.4 • C) was far more similar to that in Fab-dsscFv#2 (73.1 • C) than in Fab-dsscFv#1 (59.0 • C). Discussion Multimerisation of bispecific antibody formats during cellular protein expression is undesirable and necessitates removal of contaminating multimer.This can result in overall poor yields if the protein is particularly prone to multimerisation.In dsFv/dsscFv-containing bispecifics, the ability of some heavy and light variable domains in separate dsFv/dsscFv monomers to 'mispair' during cellular expression may be a reason for <100% monomeric proteins.In isolated scFv, the engineering of a disulphide bond between the vH and vL fixes any multimer formed during cellular expression.Nonetheless, we have previously shown that the disulphide bond is necessary in the final molecule to prevent multimerisation of monomers during and post-purification [2].The disulphide bond does not on the whole contribute any additional thermal stability nor does it affect the inherent propensity to form monomer/multimer in isolated scFv, at least when placed at the vH44:vL100 position [2]. SEC analysis showed that the isolated dsFv and dsscFv in this study were all highly monomeric, although two dsFv appeared to have a low monomer level.In the absence of a linker, it is likely that the lower apparent monomer level is a result of host cell protein co-purification, as opposed to dsFv multimerisation.We show that the monomer level of the isolated dsFv/dsscFv is not predictive of its true multimerisation potential in all protein fusion contexts.There was no correlation between the monomer levels seen in the isolated dsFv with the corresponding Fab-dsFv, and the isolated dsscFv with the corresponding Fab-dsscFv.For example, dsFv#2 and dsFv#3 were ~100% monomeric, but showed vastly different monomer levels when the dsFv was linked to a Fab (Fab-dsFv#3: 83.7% monomer, Fab-dsFv#2: 37.8% monomer).Hence our data suggest that dsFv and dsscFv monomer level screening may be more informative in the context of a relevant scaffold protein. We have observed differences in the monomer level between Fab-dsFv and Fab-dsscFv with equivalent sequences in the dsFv/dsscFv position.For sequences that are particularly prone to multimerisation, we have shown that conversion of the dsFv to dsscFv is one potential way to improve the monomer level.In three of the molecules we tested, the monomer level of the Fab-dsFv was high, but was modestly improved by conversion of the dsFv to dsscFv.One of the molecules we tested had a low monomer level as a Fab-dsFv, which was almost doubled upon conversion of the dsFv to dsscFv. Differences between Fab-dsFv and Fab-dsscFv monomer levels indicate that linker positioning may be a driving factor for multimerisation.The Fab-dsFv and Fab-dsscFv both contain two linkers.In Fab-dsFv, both variable domains are connected via either a S(G 4 S) 3 linker or a S(G 4 S/T) 3 linker to the Fab, whereas in Fab-dsscFv, only one variable domain is connected (via a S(G 4 S) 2 linker) to the Fab.As both Fv variable domains are coupled to the Fab in Fab-dsFv, longer S(G 4 S) 3 or S(G 4 S/T) 3 linkers are necessary in Fab-dsFv to prevent multimerisation induced by linker sterical constraints [4].In Fab-dsscFv, the dsscFv is coupled to the Fab by only a single linker, therefore there appears to be enough freedom of movement in the dsscFv even with a shorter S(G 4 S) 2 linker to prevent linker-induced multimerisation.It should be noted that we have previously observed identical monomer levels in Fab-dsscFv regardless of whether the linker between the Fab and dsscFv is 11 amino acids (S(G 4 S) 2 ) or 16 amino acids (S(G 4 S) 3 ).We have also previously observed identical Fab-dsFv monomer levels, whether the 16 amino acid linker between the Fab and dsFv is (S(G 4 S) 3 ) or (S(G 4 S/T) 3 ).The Serine to Threonine substitution has been used historically to facilitate cloning of HC/LC double gene vectors, but has no effect on expression or biophysical properties of Fab-dsFv (data not shown). Promiscuous v-region pairing appears to be less problematic in Fab-dsscFv, which is likely owing to more efficient intra-molecular dsvH-dsvL pairing as a result of the polypeptide linker connecting the two v-regions.In Fab-dsFv, the dsvH and dsvL are located on separate heavy and light chain polypeptides that must come together within the monomer without the aid of a linker connecting the two domains, whilst evading competition from Fab heavy and light chain interactions or light chain dimer interactions in the ER. Conversion of Fab-dsFv to Fab-dsscFv does not always result in efficient monomer formation.Engineering the vH-vL interface in dsscFv so as to alter the specificity, strength or speed of dsvH-dsvL assembly may thus serve to affect correct vH-vL pairing within the dsscFv monomer and thus monomer levels in Fab-dsscFv.Perhaps one way of inadvertently achieving this is through framework 'swapping', as we have shown that grafting the dsscFv CDRs from a multimerisation-prone Fab-dsscFv onto a Figure 1 . Figure 1.Potential dimerisation mechanisms of isolated and Fab-linked dsFv and dsscFv during cellular expression.(A) dsFv; (B) dsscFv; (C) Fab-dsFv; (D) Fab-dsscFv.Disulphide bonds are shown in red.During cellular expression, dsvH and dsvL can pair correctly to form disulphide stabilised monomers.'Mispairing' of dsvH and dsvL can result in unwanted disulphide stabilised dimers (and higher order multimers).Monomers and multimers do not interchange, as the disulphide bond within the dsFv/dsscFv fixes the monomer:multimer ratio, allowing purification of the desired monomeric species.Although Fab appended formats are shown, similar vH-vL 'mispairing' can occur in IgG formats appended with dsFv/dsscFv. Table 1 . Comparison of isolated dsFv and dsscFv.Following the 10-day transient expression in 293F cells, the expression level of dsFv#1-4 and dsscFv#1-4 proteins in the culture supernatant was measured by Octet.Proteins were purified from the supernatant using Ni 2+ -NTA resin, and the purified proteins analysed by S200 SEC and differential scanning fluorimetry.Data shows mean ± SD from three independent transfections.# Actual monomer level is likely to be ~100%; lower apparent monomer level indicates contamination by host cell proteins.* An additional minor Tm at ~52 • C was observed, which likely represents contaminating host cell proteins.LOD = Limit of detection. Table 4 . Comparison of IgG(H)-dsscFv with wild type and FW/CDR-'swapped' Fv.Following the 10-day transient expression in 293F cells, the expression level of wild type and framework 'swapped' IgG(H)-dsscFv proteins in the culture supernatant was measured by a Protein G HPLC assay.Proteins were purified from the supernatant using Protein A HPLC, and the purified proteins analysed by G3000 SEC. Table 4 . Comparison of IgG(H)-dsscFv with wild type and FW/CDR-'swapped' Fv.Following the 10day transient expression in 293F cells, the expression level of wild type and framework 'swapped' IgG(H)-dsscFv proteins in the culture supernatant was measured by a Protein G HPLC assay.Proteins were purified from the supernatant using Protein A HPLC, and the purified proteins analysed by G3000 SEC.	2019-01-30T17:13:25.369Z	2018-08-31T00:00:00.000	{ "year": 2018, "sha1": "3ea67bf7d63887210f5d42a7b088bdbb90cb3895", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2073-4468/7/3/35/pdf?version=1535959531", "oa_status": "GOLD", "pdf_src": "ScienceParseMerged", "pdf_hash": "3ea67bf7d63887210f5d42a7b088bdbb90cb3895", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Medicine", "Chemistry" ] }
25196285	pes2o/s2orc	v3-fos-license	Reactive Nitrogen Species Is Required for the Activation of the AMP-activated Protein Kinase by Statin in Vivo* The AMP-activated protein kinase (AMPK) is reported to mediate the beneficial effects of statin on the vascular functions, but the biochemical mechanisms are incompletely understood. The aim of the study was to determine how statin activates AMPK. Exposure of confluent bovine aortic endothelial cells to simvastatin (statin) dose-dependently increased phosphorylation of AMPK at Thr172 and activities of AMPK, which was in parallel with increased detection of both LKB1 phosphorylation at Ser428 and LKB1 nuclear export. Furthermore, statin treatment was shown to increase protein kinase C (PKC)-ζ activity and PKC-ζ phosphorylation at Thr410/Thr403. Consistently, inhibition of PKC-ζ either by pharmacological or genetic manipulations abolished statin-enhanced LKB1 phosphorylation at Ser428, blocked LKB1 nucleus export, and prevented the subsequent activation of AMPK. Similarly, in vivo transfection of PKC-ζ-specific small interfering RNA in C57BL/6J mice significantly attenuated statin-enhanced phosphorylation of AMPK-Thr172, acetyl-CoA carboxylase (ACC)-Ser79, and LKB1-Ser428. In addition, statin significantly increased reactive oxygen species, whereas preincubation of mito-TEMPOL, a superoxide dismutase mimetic, abolished statin-enhanced phosphorylation of both AMPK-Thr172 and ACC-Ser79. Finally, in vivo administration of statin increased 3-nitrotyrosine and the phosphorylation of AMPK and ACC in C57BL/6J mice but not in mice deficient in endothelial nitric-oxide synthase. Taken together, our data suggest that AMPK activation by statin is peroxynitrite-mediated but PKC-ζ-dependent. This article has been withdrawn by the authors. The LKB1 immunoblot from catabolic pathways and inhibits ATP-consuming anabolic pathways (17). Although the AMPK pathway is traditionally thought of as a regulator of metabolism, recent studies have demonstrated that AMPK may also act to maintain normal endothelial functions (18). AMPK exerts pleiotropic effects believed to be beneficial to endothelial functions and antiatherogenesis. These effects include, among others, induction of the eNOS/nitric oxide (NO) pathway to increase NO bioavailability; suppression of endothelial ROS production when stimulated by hyperglycemia or high FFA to improve endothelial FFA oxidation and limit lipid accumulation; inhibition of apoptosis and inflammation; and modulation of vascular tone (19 -21). Since many of the metabolic benefits and the endothelial protection conferred by statin are similar to those elicited by up-regulation of AMPK, we hypothesized that AMPK activation may mediate many of the pleiotropic and salutary activities exerted by statins on the cardiovascular system. Consistent with our hypothesis, several recent studies (22,23) demonstrated that statin can rapidly activate AMPK via increased Thr 172 phosphorylation in vitro, resulting in eNOS activation in cultured cells. Although statin is known to activate AMPK, the mechanism(s) by which statins activate AMPK was not yet defined. To this end, we examined the effects of statin on the kinases upstream of AMPK, specifically evaluating the actions on LKB1 and PKC-. Here we report that statin treatment results in LKB1-dependent AMPK activation through reactive nitrogen species, including peroxynitrite (ONOO Ϫ )-mediated but PKC--dependent mechanism. AMPK activation is required for the antiapoptosis effect of statin in endothelial cells exposed to high glucose. EXPERIMENTAL PROCEDURES Animals-Male eNOS knock-out (eNOS Ϫ/Ϫ ) and their littermates, C57BL6 mice, 10 weeks of age, were obtained from the Jackson Laboratory (Bar Harbor, ME). Mice were housed in temperature-controlled cages with a 12-h light-dark cycle and given free access to water and normal chows. These mice were randomly divided into control and statin-treated groups. Mice were abdominally injected with statin (5 mg/kg) for 4 h, and the control mice received a 0.9% physiological saline injection. The mice were euthanized with inhaled isoflurane. Mouse hearts, kidneys, livers, and aorta were removed and immediately frozen in liquid nitrogen. The animal protocol was reviewed and approved by the University of Oklahoma Health Science Center Institutional Animal Care and Use Committee. Cell Culture-All culture media were supplemented with penicillin (100 units/ml) and streptomycin (100 g/ml). A549 and HeLa S3 cells were grown in F-12K medium supplemented with 10% serum. BAEC and HUVEC were maintained in endothelial basal medium with 2% serum and growth factors prior to use. BAEC were serum-deprived overnight prior to experiments. For adenoviral infection experiments, infected BAEC were treated with statin in the presence of normal or high glucose concentrations and for the indicated length of time. Western Blot and Preparation of Subcellular Fractions-Western blots and preparation of nuclear, cytosol, and membrane fractions were performed as described previously (24). Western blot bands were qualified using the National Institutes of Health ImageJ program (version 1.37v). Assay of Protein Kinase C-Activity-PKC-was immunoprecipitated from untreated (control) or treated cells with an antibody against PKCovernight at 4°C in the presence of protein A/G-agarose. PKCactivity present in the immunoprecipitates was determined by its ability to activate pseudosubstrate derivative (50 M; ERMRPRKRQGSVRRRV) as described previously (24,25). AMPK Activity Assay-Total AMPK was immunoprecipitated from 500 g of protein using an antibody against AMPK␣, and AMPK activity was assessed by determining the incorporation of 32 P into the synthetic SAMS peptide as described (24,25). The difference between the presence and absence of AMPK is calculated as the AMPK activity. siRNA Gene Silencing of PKC-(25)-Small interfering RNA (siRNA) duplex oligonucleotides used in this study are based on the human cDNAs encoding PKC. PKC siRNA as well as a nonsilencing control siRNA were obtained from Santa Cruz Biotechnology. The working concentration of siRNA duplexes applied was 100 nM. HUVEC were transfected with PKC siRNA or nonspecific control siRNA by using Lipofectamine TM 2000 (Invitrogen) according to the manufacturer's instructions. Transfected cells were starved in serum-free medium for 6 h and then exposed to the indicated concentrations of statin. In Vivo Gene Silencing of PKC-siRNA (27)-C57BL/6J mice were injected retroorbitally with either mouse-specific PKC-siRNA or control siRNA (1 mg/kg, diluted in 200 l) every other 3 days for 6 days using in vivo-jetPEI TM from Polyplus Transfection (Illkirch, France) according to the manufacturer's recommendations. TUNEL Staining (27)-Apoptosis was assessed by TUNEL staining (TMR red) using a kit from Roche Applied Science and following the provided instruction manual. Percentage apoptosis was calculated from the number of TUNEL-positive cells divided by the total number of cells counted. (24,25). Mutations were confirmed by DNA sequencing, and plasmid DNA was extracted on a large scale using Qiagen EndoFree plasmid maxikit (catalogue number 12362) and transfected into HeLa-S3 using the Lipofectamine 2000 kit from Invitrogen (catalogue number 11668-019), according to the instructions provided by the supplier. 24 h after transfection, the cells were treated with statin or vehicle for 1 h. Both LacZ expression vector and untreated cells were used as control. Adenoviral Infection-BAEC were infected with adenovirus expressing a D194A mutant LKB1 (Ad-D194A), a S428A LKB1 (Ad-S428A), an AMPK dominant negative mutant (AMPK-DN), a PKCdominant negative mutant, or a constitutively active PKCmutant (PKC--WT), as described previously (25). A replication-defective adenoviral vector expressing GFP was used as control. Cells were infected with the adenovirus at a multiplicity of infection of 50 overnight and then washed and incubated in fresh medium for an additional 18 -24 h prior to the experiment. Under these conditions, ϳ85% of the cells showed positive for GFP. Statistical Analysis-Results are expressed as mean Ϯ S.E. of at least three independent experiments. Comparisons of the means were performed by the one-way or two-way analysis of variance. A value of p Ͻ 0.05 was considered statistically significant. RESULTS Statin Increases Phosphorylation of AMPK at Thr 172 and ACC at Ser 79 in BAEC-Statin has been shown to activate AMPK in cultured endothelial cells (22,23). To define the mechanism, we first examined whether the Ser 428 phosphorylation of LKB1, a well characterized AMPK kinase in endothelial cells (24), was altered by statin. Consistent with previous studies, statin (1-50 M) caused a dose-dependent increase in AMPK activity in cultured BEAC, as determined by examining phosphorylation of both AMPK (Thr 172 ) and ACC (Ser 79 ), the downstream AMPK substrate, by immunoblot analysis (Fig. 1) (25). Exposure of BAEC to statin (50 M) resulted in a 2.6-fold increase in AMPK-Thr 172 phosphorylation (Fig. 1A). AMPK activation by statin was further confirmed by enhanced phosphorylation of ACC at Ser 79 . No change in the expression of endogenous AMPK ␣ protein was observed by immunoblot analysis (Fig. 1A). To corroborate statin-induced AMPK activation, statin-enhanced AMPK activity was examined by Confluent BAEC were exposed to statin (1 to 50 M) for 2 h. The blot is a representative of three blots from three independent experiments. n ϭ 3. ࡔ, p Ͻ 0.05 compared with control. B, time course of statininduced AMPK phosphorylation at Thr 172 in BAEC. BAEC were exposed to statin (50 M) at the indicated time. The blot is a representative of five blots from five independent experiments. n ϭ 5. ࡔ, p Ͻ 0.05 as compared with the control conditions. C, dose-dependent effects of statin on LKB1 Ser 428 phosphorylation in BAEC. Confluent BAEC were exposed to statin (1-50 M) for 2 h. The blot is a representative of three blots from three independent experiments. n ϭ 3. ࡔ, p Ͻ 0.05 compared with control. D, time course of statin-enhanced LKB1 phosphorylation at Ser 428 in BAEC. BAEC were exposed to statin (50 M) at the indicated time. The blot is a representative of five blots from five independent experiments. n ϭ 5. ࡔ, p Ͻ 0.05 as compared with the control conditions. increased AMPK activity (6.5 Ϯ 0.7 versus 4.1 Ϯ 0.4 pmol/ min⅐mg protein, statin versus control, n ϭ 6, p Ͻ 0.05). AMPK activation by statin was also concentration-dependent. Although the maximal phosphorylation of AMPK by statin occurred at 50 M (Fig. 1A), the level of AMPK activation by statin at 50 M was comparable with that of 5-aminoimidazole-4-carboxamide-1-D-ribo-furanoside at 1 mM (data not shown). Our initial studies suggest that AMPK activation by statin is biphasic. Acute exposure for 45 min was required for statin (50 M) to activate AMPK. Increased phosphorylation of AMPK and ACC occurred 45 min following the initiation of statin treatment, with peak levels of activated protein occurring at 120 min (Fig. 1B). Chronic exposure of BAEC (Ͼ24 h) lowered the effective concentrations of statin to 5-10 M (data not shown). Compared with control, statin (5 M, 72 h) caused a drastic increase in phosphorylation of both AMPK (Thr 172 ) and ACC (Ser 79 ) (data not shown). Collectively, these data confirm that AMPK is activated by statin treatment. Statin Increases LKB1 Phosphorylation at Ser 428 in a Dose-dependent Manner-Previous studies have identified LKB1 as a kinase upstream of AMPK and determined that LKB1 phosphorylation at Ser 428 is important for peroxynitrite (ONOO Ϫ )-enhanced (24) or metformin-enhanced (25) AMPK activation in cultured endothelial cells. Since statin treatment activates AMPK, we next examined whether statin would affect LKB1 phosphorylation at Ser 428 . Statin (10 -50 M) for 2 h of treatment did not alter overall LKB1 levels but did result in significantly increased phosphorylation of LKB1-Ser 428 compared with untreated cells (Fig. 1C). Consistent with statin-mediated AMPK phosphorylation, there was a dosedependent increase in LKB1 phosphorylation at Ser 428 in response to statin (Fig. 1C). (28,29) have suggested that CaMKK-␤ functions as AMPK kinase under conditions in which intracellular Ca 2ϩ increases. We first determined if STO-609, a selective CaMKK-␤ inhibitor, or BAPTA-AM (20 M), an intracellular Ca 2ϩ chelator, altered calciumdependent AMPK activation in BAEC. As expected, exposure of BAEC to A23187 (1 M) significantly enhanced the phosphorylation of AMPK-Thr 172 in BAEC. Preincubation of BAEC with either STO-609 (1 M) or BAPTA-AM (20 M), significantly suppressed calcium inophore A23187-enhanced phosphorylation in BAEC ( Fig. 2A). Inhibition of Ca 2ϩ /Calmodulindependent Kinase Kinase (CaMKK) Does Not Alter Statin-induced AMPK Activation-Recent studies To determine if Ca 2ϩ or CaMKK-␤ is required for statinenhanced AMPK activation, STO-609 (1 M) or BAPTA were preincubated with BAEC prior to statin treatment. Neither STO-609 nor BAPTA-AM at the stated concentrations altered the basal or statin-enhanced phosphorylation levels of AMPK at Thr 172 or ACC at Ser 79 in BAEC (Fig. 2, B-E). These results suggest that CaMKK-␤ was not required for statin-induced AMPK activation in BAEC. Transfection of LKB1-expressing Plasmid Is Required for Statin-induced AMPK Activation in A549 Cells, Which Are Otherwise Deficient in LKB1-Since statin results in a parallel increase in both AMPK and LKB1 (Ser 428 ) phosphorylation, we next evaluated whether LKB1 was required for statin-induced AMPK activation. We first examined if statin activated AMPK in A549 or HeLa-S3, two tumor cell lines that have been reported to be deficient in endogenous LKB1 (30,31). Consistent with previous studies, we were unable to detect LKB1 protein in A549 cells, thereby confirming that A549 cells are deficient in endogenous LKB1 (Fig. 3A). Transfection of LKB1 wild type plasmids into A549 dramatically increased the detection of LKB1 protein in A549 cells (Fig. 3A), thus demonstrating the effective reconstitution of LKB1. In these LKB1-deficient A549 cells, we tested if statin would stimulate AMPK phosphorylation. Empirically, we found that statin did not alter the levels of AMPK-Thr 172 or ACC-Ser 79 in A549 cells. In contrast, after these A549 cells were transfected with LKB1-expressing plasmid, there was a significant increase in both AMPK (Thr 172 ) and ACC (Ser 79 ) phosphorylation (Fig. 3A). Similar results were also obtained in LKB1-deficient HeLa-S3 cells (data not shown). Collectively, these data suggest that LKB1 is required for statin-dependent AMPK activation. Genetic Inhibition of LKB1 Ablates Statin-induced AMPK Activation in Endothelial Cells-To further establish that LKB1 is required for statin-activated AMPK in BAEC, we suppressed LKB1 expression by applying siRNA in BAEC. LKB1 siRNA, but not control siRNA, suppressed the expression of LKB1 by 50% (Fig. 3B). Corroborating our previous results suggesting a requirement of LKB1 for statin-induced AMPK phosphorylation, we found that LKB1 siRNA, but not control siRNA, inhibited statin-dependent phosphorylation of both AMPK at Thr 172 and ACC at Ser 79 (Fig. 3B). These experiments offer additional support for the notion that LKB1 is required for stain-induced AMPK activation in endothelial cells. Phosphorylation of LKB1 at Serine 428 Is Required for Statin-induced AMPK Activation in Endothelial Cells-Since statin increased levels of LKB1 phosphorylation at Ser 428 (Fig. 1, C and D), we next evaluated whether LKB1 phosphorylation at Ser 428 was required for statin-induced AMPK activation. Using site-directed mutagenesis, we developed two LKB1 mutants in which an amino acid essential for LKB1 activation, aspartic acid 194 or serine 428, was mutated to alanine (LKB1-D194A or LKB1-S428A) (24,25). Adenoviral overexpression of LKB1 dramatically increased LKB1 expression in BAEC (25). Since statin treatment resulted in increased phosphorylation of both AMPK and ACC in BAEC infected with adenovirus encoding GFP, we next investigated whether adenoviral overexpression of the kinase-defective LKB1 mutant (LKB1-D194A) would prevent statin-dependent phosphorylation of AMPK. Although overexpression of the phosphorylation-defective LKB1 mutant (LKB1-S428A) did not affect the basal level of AMPK-Thr 172 in BAEC, it did abolish statin-enhanced phosphorylation of both AMPK-Thr 172 and ACC-Ser 79 (Fig. 3C). These data suggest that Ser 428 phosphorylation of LKB1 was essential for statin-induced AMPK activation in BAEC. Statin Increases PKC-Phosphorylation at Thr 410/403 in Both a Time-and Dose-dependent Manner-Previously we had determined that PKC-regulates AMPK activity by increasing Ser 428 phosphorylation of LKB1, which then stimulates AMPK Thr 172 phosphorylation (24). Therefore, we next determined whether PKC-activation was involved in statin-enhanced LKB1-dependent AMPK activation. Incubation of BAEC with statin (5-50 M) did not detectably increase total levels of PKCprotein, whereas it significantly increased PKC-phosphorylation at Thr 410/403 within 15 min of treatment (Fig. 4B). The statin-induced phosphorylation of PKC--Thr 410/403 occurred in a dose-dependent manner (Fig. 4A). In addition, statin significantly (p Ͻ 0.01) increased PKCactivity 2.1-fold relative to control groups (Fig. 4C), as determined by examining a PKC--specific substrate. Collectively, these results indicate that statin activated PKCprior to activating either LKB1 or AMPK in BAEC and suggest that PKCmay be required for statin-induced activation of LKB1 and/or AMPK. Inhibition of PKC-Abolishes Statin-enhanced AMPK Activation-To define the role of PKCin statin-induced LKB1 and AMPK activation, PKCactivity was suppressed by either pharmacological or genetic means. In order to suppress PKCactivity, a dominant negative PKC-mutant (Ad-PKC- (Fig. 5A). In parallel, overexpression of PKC--DN, which did not alter basal levels of LKB1 Ser 428 phosphorylation, abolished statin-enhanced LKB1 phosphorylation at Ser 428 (Fig. 5B). Since overexpression of the dominant negative PKC-mutant inhibited the statin-induced activation of both LKB1 and AMPK (Fig. 5, A and B) and since PKCactivation occurred prior to LKB1 and AMPK (Fig. 4B), as we did in BAEC in response to metformin (25), these results collectively support the prevalent concept that PKCmay function as an upstream kinase for both LKB1 and AMPK. In order to further confirm the essential role of PKC-, endogenous PKCin endothelial cells was suppressed by transfection of PKC--specific siRNA. As expected, BAEC transfected with control siRNA showed no significant change in PKClevels, whereas PKC-was undetectable in BAEC transfected with PKC--specific siRNA (Fig. 5C), confirming the reduction of PKCby siRNA transfection. Confirming the results using cells overexpressing the dominant negative form of PKC-, we found that expression of PKC--specific siRNA, but not control siRNA, abolished statin-induced phosphorylation of both AMPK at Thr 172 and ACC at Ser 79 , without altering the expression of AMPK␣ (Fig. 5D). We next determined if PKCinhibition alters statin-enhanced AMPK activity. To investigate statin-induced AMPK activity, GFP, PKC--WT, or PKC--DN was transiently overexpressed in BAEC. Following mock or statin treatment, AMPK was immunoprecipitated with antibodies raised against AMPK␣, which was then assayed for activity by the incorpora-tion of [ 32 P]ATP into the SAMS peptides. Statin was found to significantly increase AMPK activity in BAEC infected with either GFP or PKC--WT (Fig. 6A). However, overexpression of PKC--DN, which did not alter the basal levels of AMPK activity, abolished statinenhanced AMPK activity (Fig. 6A). These data provide further proof that PKCis essential for statin-induced AMPK activation in endothelial cells. Statin-induced LKB1 Translocation Is PKC--dependent-Previous studies have shown that LKB1 is predominantly localized in the nucleus, whereas AMPK is mainly localized in the cytoplasm (32,33). LKB1 must be exported from the nucleus to the cytosol to associate with STRAD and MO25 prior to becoming fully active (34,35). Since commercially available antibodies could not be used for LKB1 immunohistochemical stainings in BAEC, therefore, we examined whether statin altered the subcellular localization of LKB1 in HUVEC by immunohistochemical staining. As expected, LKB1 was found to reside predominantly in the nucleus of nonstimulated HUVEC (Fig. 6B). Statin increased the export of LKB1 from the nucleus to the cytosol in stimulated HUVEC (Fig. 6B). We further found that the PKCpseudosubstrate, a selective PKCinhibitor, abolished statin-induced LKB1 translocation to cytosol (Fig. 6B). In order to corroborate the immunofluorescent microscopy data, statin-enhanced LKB1 nuclear export was further examined in subcellular fractions. Immunoblot analysis of cellular fractions confirmed that statin treatment significantly increased cytoplasmic LKB1 protein levels and concurrently and reciprocally decreased nuclear LKB1 protein levels (Fig. 6C). Together, these data suggest that statin triggers LKB1 translocation from nucleus into cytosol by a pathway dependent on PKCactivity. PKC--dependent AMPK Activation in Vivo-We next determined if PKCwas required for AMPK activation in mice. C57BL/6J mice were injected retroorbitally with either mousespecific PKC-siRNA or control siRNA (1 mg/kg, diluted in 200 l) every other 3 days for 6 days. As shown in Fig. 7A, in vivo transfection of PKC-siRNA but not control siRNA significantly lowered the levels of PKCin isolated mouse aortas. In addition, statin significantly increased the phosphorylation of both AMPK-Thr 172 and ACC-Ser 79 in C57BL/6J mice or C57BL/6J mice treated with control siRNA (Fig. 7, B and C). Compared with the levels of phosphorylated AMPK and ACC in mice treated with control siRNA, transfection of PKC-siRNA significantly reduced statin-enhanced phosphorylation of AMPK-Thr 172 (Fig. 7, B and C) and LKB1-Ser 428 (Fig. 7D). These results suggest that PKC-was required for statin-en- (Fig. 8A). To investigate if statin activated AMPK via ONOO Ϫ , we monitored AMPK-Thr 172 phosphorylation under conditions where ONOO Ϫ was inhibited. Mito-TEMPOL (10 M), which did not alter basal O 2 . release in BAEC, markedly attenuated statin-enhanced phosphorylation of both AMPK-Thr 172 and ACC-Ser 79 (Fig. 8B). Conversely, overexpression of catalase, which increased catalase activity by 2.8-fold, as measured by the reduction of 1% H 2 O 2 absorption at 240 nm in BAEC overexpressing catalase, did not alter statin-enhanced phosphoryla-tion of AMPK-Thr 172 and ACC-Ser 79 (data not shown), indicating that hydrogen peroxide was not involved in statin-enhanced AMPK activation in BAEC. ONOO Ϫ -dependent Activation of AMPK-To further establish if ONOO Ϫ was involved in AMPK activation by statin in vivo, statin was given to eNOS Ϫ/Ϫ mice (attenuate ONOO Ϫ by lacking eNOSderived NO) or to the wild type C57BL/6J mice. Mouse aortas were isolated for assaying 3-nitrotyrosine (3-NT), a footprint for reactive nitrogen species, including ONOO Ϫ . The specificity of 3-NT staining was confirmed by the absence of staining when the antibody was omitted or was diluted in 10 mM 3-NT (data not shown). As shown in Fig. 8C, the proteins positive with the 3-NT antibody were only weakly visible in the aortic homogenates from sham-treated C57BL/6J mice. The levels of 3-NTpositive proteins in aortas of C57BL/6J were markedly increased by statin treatment (Fig. 8C). Compared with weak stainings of 3-NT in the aortas isolated from shamtreated eNOS Ϫ/Ϫ , statin did not increase 3-NT-positive proteins in the aortas from eNOS Ϫ/Ϫ mice (Fig. 8C), suggesting that NO from eNOS was required for statin-increased ONOO Ϫ in vivo. We next determined the effects of statin in C57BL/6J and eNOS Ϫ/Ϫ mice. As shown in Fig. 8D, administration of statin significantly increased the phosphorylations of both AMPK-Thr 172 and ACC-Ser 79 in C57BL/6J mice but not in eNOS Ϫ/Ϫ mice (Fig. 8D). Consistently, statin increased the phosphorylation of PKCin the aortas of C57BL/6J wild type mice but not in eNOS Ϫ/Ϫ mice (Fig. 8D). Taken together, these results suggested that the ONOO Ϫ -PKC-AMPK axis operates in statinenhanced AMPK activation in vivo. ONOO Ϫ -dependent and ONOO Ϫ -independent AMPK Activation in BAEC Exposed to High Glucose-We next determined if NO or ONOO Ϫ derived from eNOS caused a feedback activation of AMPK in BAEC. Since our earlier studies (1,2) had demonstrated that prolonged exposure of human aortic endothelial cells to high glucose (HG) resulted in ONOO Ϫ formation, we used this model to dissect the contribution of ONOO Ϫ in high glucose-induced AMPK activation. As depicted in Fig. 9A, exposure of BAEC to 30 mM D-glucose (HG) caused a biphasic increase of AMPK phosphorylation, which peaked at 2 and 48 h, respectively. In parallel, 3-nitrotyrosine, a footprint of ONOO Ϫ , was increased in BAEC treated with HG for 48 h. Uric acid (50 M), a potent scavenger for ONOO Ϫ , significantly suppressed HG-enhanced 3-nitrotyrosine at 48 h (Fig. 9B) not alter 3-nitrotyrosine in cells treated with normal glucose or HG for 2 h. These results suggest that HG increased ONOO Ϫ in BAEC at 48 h but not in BAEC exposed to normal glucose or HG for 2 h. We further determined if uric acid altered high glucose-enhanced AMPK activity. As shown in Fig. 9C, uric acid did not alter AMPK phosphorylation in BAEC exposed to normal glucose but markedly attenuated HG-enhanced AMPK activity at 48 h of exposure. Since the suppression of AMPK activity by uric acid was co-related with its inhibition on ONOO Ϫ formation in BAEC, these results strongly suggest that ONOO Ϫ generated by HG exposure activated AMPK in BAEC at 48 h. AMPK Activation by Statin Suppresses the Basal and Angiotensin-II-enhanced Superoxide Anions in BAEC-Previous studies have suggested that statin might exert its therapeutic effects by suppressing oxidant stress (5). Therefore, we next evaluated whether AMPK activation is required for the antioxidant effects of statin. Statin treatment was found to significantly lower the basal levels of O 2 . , assayed by the oxidation of dihydroethidium in BAEC (Fig. 10A). Furthermore, we determined that compound C, a potent AMPK inhibitor, abolished the effect of statin on O 2 . levels ( Fig. 10A) AMPK Activation Inhibits Endothelial Apoptosis in Cultured Cells-Previous studies demonstrated that AMPK inhibits apoptosis (18). Since statin is also known to inhibit endothelial apoptosis, we evaluated the role of AMPK in statin-mediated inhibition of apoptosis (37,38). As determined by TUNEL staining, exposure of BAEC to high glucose (30 mM), but not osmotic controls, significantly increased endothelial cell apoptosis (Fig. 10D). Exposure of BAEC to statin (5 M for 72 h) resulted in AMPK activation and significantly attentuated high glucose-induced apoptosis (Fig. 10D). Finally, inhibition of AMPK by adenoviral overexpression of AMPK-DN abolished the inhibitory effects of statin on apoptosis (Fig. 10D). These data indicate that AMPK activation by statin prevented high glucose-dependent endothelial cell apoptosis. DISCUSSION Recent studies have suggested that AMPK is a therapeutic target for treating diabetes. Oral hypoglycemic agents, such as metformin and rosiglitazone, have been reported to exert their therapeutic effects by activating AMPK (for a review, see Ref. 18). In the present study, we observe that LKB1 Ser 428 phosphorylation by atypical PKCis required for statin-stimulated AMPK activation. The key evidence can be summarized as follows. First, inhibition of LKB1 with D194A and S428A mutants or LKB1 siRNA effectively blocked AMPK activation induced by statin (Fig. 3, B and C). In addition, statin could not activate AMPK in either A549 or HeLa S3 cells that lack LKB1, but wild type LKB1 expression via adenoviral transfection restored the stimulatory effects of statin on AMPK in these cells (Fig. 3A). Second, STO-609, a potent CaMKK␤ inhibitor, failed to inhibit statin-induced AMPK activation in BAEC (Fig. 2). Third, statin increased PKCactivity prior to the phosphorylation of both LKB1 and AMPK in BAEC (Fig. 4C). Genetic inhibition of PKCeffectively blocked statin-induced AMPK phosphorylation (Fig. 5A) and AMPK activity (Fig. 6A). Fourth, statin increased export of LKB1 from the nucleus to the cytosol, previously reported to be an important and essential step for LKB1 to activate AMPK (25), and PKC-pseudosubstrate abolished statin-induced LKB1 translocation (Fig. 6, B and C). It is noteworthy that statinenhanced LKB1 phosphorylation in BAEC occurred within 30 min of treatment, which preceded the statin-induced increase in AMPK phosphorylation. These results suggest that LKB1 is an upstream activator of AMPK or a regulator of cellular signaling in concert with AMPK. Furthermore, the pleiotropic effects of statin thought to be beneficial to endothelial functions and antiatherogenesis might be mediated by PKC--dependent AMPK activation in endothelial cells. We previously observed that inhibition of c-Src or phosphatidylinositol 3-kinase activity by pharmacologic agents (PKC-siRNA) or genetic suppression (PKC--DN) of PKC-abolishes AMPK stimulation by metformin or by ONOO Ϫ in endothelial cells (25). In this study, we have extended these observation by showing that PKCis also required for statin-enhanced AMPK activation. Furthermore, we have for the first time demonstrated that in vivo inhibition of PKCwas required for statin-enhanced . enhanced by statin (n ϭ 6; †, p Ͻ 0.05, statin versus statin plus TEMPOL). B, ONOO Ϫ -dependent activation of AMPK in BAEC exposed to statin. Phosphorylation of AMPK (Thr 172 ) was attenuated by mito-TEMPOL. The blot is representative of four blots obtained from four independent experiments. Exposure of BAEC to 10 M statin significantly increased the phosphorylation of both AMPK and ACC (n ϭ 6; ࡔ, p Ͻ 0.01, control versus statin), whereas TEMPOL suppressed the effects of statins on both AMPK-P and ACC-P (n ϭ 6; †, p Ͻ 0.05, statin versus statin plus TEMPOL). C, statin increases 3-NT in the aortas of C57BL/6J mice but not in eNOS Ϫ/Ϫ mice. Mice were abdominally injected with statin (5 mg/kg) for 4 h, and the control mice received a 0.9% physiological saline injection. 3-NT-positive proteins were detected in the aortic homogenates in Western blots by using the specific antibodies. n ϭ 5; ࡔ, p Ͻ 0.05 (WT control versus WT statin); †, p Ͻ 0.05 (WT statin versus eNOS Ϫ/Ϫ statin). D, statin activates AMPK in C57BL/6J but not in eNOS Ϫ/Ϫ in vivo. Mice aortas were isolated and assayed for AMPK and ACC as described under "Experimental Procedures." Of note is that meformin increased AMPK-P and ACC-P in C57BL/6J mice but not in eNOS Ϫ/Ϫ mice. The blot is a representative blot from at least three blots from three independent experiments. n ϭ 5; ࡔ, p Ͻ 0.05. phosphorylation of AMPK and LKB1. These important findings imply that PKC--LKB1-AMPK is a common pathway for AMPK activation in vivo. The Finally, metformin significantly increased AMPK activity in the aortas and hearts of C57BL/6J mice but not those of eNOS Ϫ/Ϫ , although eNOS Ϫ/Ϫ mice expressed AMPK. Since eNOS Ϫ/Ϫ mice did not generate ONOO Ϫ in response to statin, the data strongly suggest that ONOO Ϫ is required for AMPK activation by statin. These results strongly suggest that NO-derived oxidants, such as ONOO Ϫ , might be required for AMPK activation by statin. Numerous studies from us and others (1,2,5) have demonstrated antioxidant effects of statin and AMPK in vivo. This apparently contradictory observation might be similar to ROS in ischemic preconditioning, in which low levels of ROS precondition the tissues to prevent massive production of reactive species in index hypoxia. Our results suggest that statin, like ischemic preconditioning, via the generation of low levels of oxidative stress by statin, "preconditions" the cells or tissues to alleviate oxidant production by activating AMPK. In line with this conclusion, we found that statin suppressed ROS triggered by Ang-II and high glucose. Further, we found that AMPK activation by statin is required P-AMPK Statin via PKC-Activates AMPK for the reduction of reactive nitrogen species (ONOO Ϫ ). Our earlier work (39,40) has also demonstrated that metformin, one of the most used antidiabetic drugs, activates AMPK by increasing ONOO Ϫ , and AMPK activation suppresses oxidant production. Thus, we consider that AMPK might function as a redox sensor, and AMPK activation might reduce oxidant stress by attenuating oxidant stress by other sources or by enhancing antioxidant potentials. Overwhelming evidence suggests that in humans, improved endothelium functions are one of the earliest observed clinical effects, following the initiation of statin treatment (7,8). Most importantly, statin therapy improves endothelial function by virtue of its antioxidant (8,9) and anti-inflammatory (8,9) effects as well as its ability to up-regulate eNOS (10,11). In the present study, we have for the first time shown that AMPK might be implicated in the antiapoptosis effects of statin in diabetes. This finding is in line with the clinical studies. Several clinical trials, including the Heart Protection Study (3) and the Collaborative Atorvastatin Diabetes Study (4), have shown significant benefits with low to moderate dose statin therapy in diabetic patients with and/or without overt cardiovascular diseases. In addition, overwhelming evidence suggests a beneficial effect of AMPK in both endothelial functions and the cardiovascular system. Activation of AMPK could therefore explain the beneficial effects of statin on these systems independent of lipid reduction (for a review, see Ref. 18). In an earlier study from us (39), we demonstrated that activation of AMPK by metformin increases NO formation and NO bioactivity in BAEC when stimulated by high glucose. Indeed, exposure of cultured human aortic endothelial cells to 5-aminoimidazole-4-carboxamide-1-D-ribo-furanoside dramatically reduced the markers of oxidant stress and prostacyclin synthase nitration caused by 30 mM glucose. 3 Thus, the beneficial effects of AMPK activation are mediated by two mechanisms: 1) increased NO; 2) decreased O 2 . release promoted by diabetes. In support of our findings, a recent study by Cohen et al. (41) found that S17834, a polyphenol compound that strongly and persistently stimulates AMPK phosphorylation and activity in HepG2 cells, prevented the accelerated atherogenesis typically observed in streptozotocin-induced type 1 diabetic LDLR Ϫ/Ϫ mice. In summary, the results reported here constitute the first direct evidence showing that PKC-is a critical regulator of AMPK activity. Statin stimulates PKC-, and PKC-can regulate AMPK activity by increasing ROS-dependent PKCactivation, which results in LKB1 Ser 428 phosphorylation, LKB1 nuclear export, and subsequent AMPK phosphorylation at Thr 172 by LKB1.	2018-04-03T01:57:15.785Z	2008-07-18T00:00:00.000	{ "year": 2008, "sha1": "12502b6f7d18f77fbf861b2180f526af010115fd", "oa_license": "CCBY", "oa_url": "http://www.jbc.org/content/283/29/20186.full.pdf", "oa_status": "HYBRID", "pdf_src": "Highwire", "pdf_hash": "ea51ecf01be884345df8290e052fc71699e0f33f", "s2fieldsofstudy": [ "Biology", "Chemistry" ], "extfieldsofstudy": [ "Chemistry", "Medicine" ] }
268674681	pes2o/s2orc	v3-fos-license	Bizarre Parosteal Osteochondromatous Proliferation (Nora’s Lesion) Affecting Carpal Bones of the Hand in a Middle-Aged Female: A Case Report A 45-year-old woman complained of left wrist pain and swelling for two years accompanied by limited dorsiflexion. Plain X-rays revealed an abnormal bony mass in the carpal bones, further evaluated using computed tomography and magnetic resonance imaging. Upon confirmation of the benign nature surgical excisional biopsy of the lesion, the histopathology confirmed the diagnosis of Bizarre parosteal osteochondromatous proliferation (BPOP). The patient has remained pain-free and actively involved in her routine for the past two years. BPOP, affecting the carpal bones of the hand, are exceptionally rare occurrence. Attentive preoperative evaluation helps in diagnosis and to initiate measures to avoid recurrence. Introduction Bizarre parosteal osteochondromatous proliferation (BPOP), also known as "Nora's lesion" described by Nora et al. in 1983, is relatively uncommon [1].It typically manifests as an abnormal growth on the outer surface of a short bone in the hands or feet.BPOP is a non-cancerous condition and does not display any tendency for malignancy, but it does have a propensity to recurrence after surgical excision [2].The proliferation of this lesion takes place on the outermost layer of the bone, although it does not have any connection to the normal part of the bone.Histopathological BPOP is characterized by an abnormal outgrowth from the outer bone surface, a combination consisting of bone, cartilage, and fibrous tissue [1,3,4]. BPOP progresses rapidly and has a high propensity for recurrence after surgical removal accounting for 50% [5].Due to its aggressive characteristics and confusing presentation on histopathology, it is essential to differentiate it from malignant conditions such as chondrosarcoma, parosteal osteosarcoma, and conventional osteosarcoma, as well as benign conditions like florid reactive periostitis, myositis ossificans, periosteal chondroma, and osteochondroma [5].Since its initial discovery, only a few cases documented in the literature, in this report, we present our experience with a single case of Nora's lesion. Case Presentation A 45-year-old woman came with a chief complaint of swelling on her left wrist over the dorsum that had been gradually progressive over the past two years with no history of antecedent trauma.Associated with pain, which aggravates on mobilization of the wrist and lifting heavy objects.On examination, the solitary mass was bony and hard in consistency, measuring about 3 cm by 3 cm (Figure 1), associated with tenderness.No skin adherence; wrist dorsiflexion is restricted terminally by twenty degrees due to a mechanical block of mass.Probable differentials were ectopic calcification or a tumor; as the tumor laboratory profile was within normal limits, an excisional biopsy was planned.Dorsal wrist arthrotomy was performed using standard incision, the multi-lobular mass with pseudo-capsule excised along with periosteum (Figures 4, 5).Bizarre parosteal osteochondromas of the bone, primarily occur on the proximal and middle phalanges, of metacarpal and metatarsal [6].On rare occasions, they can affect long bones, often in the upper limbs, as well as the skull and jaw [7].However, the presentation of carpal BPOP lesions is not described in the literature as far as our knowledge. Nora's lesions typically occur in adults during their second and third decades of life, with an average age of 30 to 33 years with no gender discrimination [4].Patients present with painful masses that gradually increase in size over several weeks or months.Although some patients may report joint stiffness or other mechanical symptoms.The exact cause of Nora's lesions remains unknown.Some theories correlate to the reparative process following trauma to the periosteum (the outer layer of bone), as observed in about 30% of cases in a study by Meneses et al [2].However, our patient had no precedent history of trauma. On plain X-rays, these lesions typically appear to arise from the bone's outer cortex but without any penetration [5].They are often localized at the metaphysis with spiculated or irregular surfaces [5,8]. Computed tomography scans provide better bony morphology. As a gross specimen, the lesion appears as a lump with a bumpy surface, covered in shiny cartilage and containing a hard inner core of bone [1].Under a microscope, it displays unusual cartilage growth with a lot of cell activity, which gives a picture like Grade I or II chondrosarcoma [1].There is also disorganized bone formation within the cartilage, along with irregularly calcified material and harmless bone cells, and there may be active growth of benign fibrous tissue [3,9].Cancerous growths like chondrosarcoma, parosteal osteosarcoma, and conventional osteosarcoma, as well as non-cancerous conditions like reactive periostitis, myositis ossificans, periosteal chondroma, and osteochondroma, are possible differentials here.Therefore, it is crucial to approach with attention [3,5]. The recommended treatment is an en-bloc surgical excision along with scraping the surface of the underlying cortical bone to reduce the likelihood of recurrence [3,4].However, there is a high chance of recurrence.Meneses et al. reported a 51% recurrence rate after the initial removal and a 22% recurrence rate after a second removal [2].Most recurrences happened within two years of the index surgery [1,4,10]. Conclusions It is worth noting that BPOP involving the carpal bones is extremely rare.However, after surgical excision, there is a high chance of recurrence.A thorough evaluation is essential to isolate from its close differentials.Thorough excision followed by histopathological confirmation is vital.However, constant surveillance is crucial for recurrence. 3 FIGURE 1 : FIGURE 1: Pre-operative clinical image lateral view (A) and anteroposterior view (B) depicting swelling of 3x3 cm over the dorsum of the left hand FIGURE 2 : FIGURE 2: Pre-operative sagittal (A), 3D image (B), and axial cut sections (C) of computed tomography images depicting a left wrist bony lesion arising from lunate bone FIGURE 3 : FIGURE 3: Pre-operative sagittal (A) and axial (B) magnetic resonance images depicting a left wrist bony lesion arising at the level of the lunate and triquetral proximally and capitate distally with increased signal intensity on the T2-weighted image (T2WI) image sequence FIGURE 4 :FIGURE 5 : FIGURE 4: Intra-operative image depicting (A) removal of lobulated bony mass with stalk attached to lunate (arrowhead) and (B) bed of the lesion showing complete en-bloc excision of the mass FIGURE 7 :FIGURE 8 : FIGURE 7: Two-year post-operative X-ray images (A) anteroposterior and (B) lateral view of hand showing no recurrence	2024-03-25T15:07:34.120Z	2024-03-01T00:00:00.000	{ "year": 2024, "sha1": "e39ff45cf7903f6e3312fd96e588742ee4f5c617", "oa_license": "CCBY", "oa_url": "https://assets.cureus.com/uploads/case_report/pdf/239815/20240323-702-1e3ajp6.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "9c108c4ea1755eb712abb68ff55372edd4cc2280", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [] }
259408303	pes2o/s2orc	v3-fos-license	The Effect of X-irradiation on the Shelf Life and Proximate Composition of Some Varieties of Tomatoes Commonly Grown in Benue State, Nigeria : Tomato production is a source of income to most rural producers in developing countries like Nigeria. Despite the numerous benefits from this crop, challenges of postharvest losses occasioned by lack of preservation techniques and storage facilities are making its production unprofitable in most developing countries in Africa. This research investigated the effect of X-Irradiation on the shelf life and proximate composition of some varieties of tomatoes commonly grown in Benue State. Five samples each of fully ripe Plum (Lycopersicon esculentum L.-Oval-shaped tomato of Italian origin), Juliet (Lycopersicon esculentum L.-1999 All American Selection Winner), Better Boy (Lycopersicon esculentum L. of USA origin), Giulietta F1 (Lycopersicon esculentum L. – a hybrid from France) and Cherry (Lycopersicon esculentum var. cerasiforme) tomatoes were collected from an experimental farm in Wannune, kilometer 54, Makurdi-Gboko road and exposed to X-irradiation doses of 0.10 mGy, 0.30 mGy, 0.61 mGy, 1.06 mGy and 1.67 mGy using the X-ray machine (Model: 1.2UG13GN) located at Musafaha Imaging Centre Makurdi, Benue State. Results of the investigation revealed that 0.30 mGy and 0.61 mGy are adequate for extension of shelf life of Plum tomatoes by 7 days; 0.30 mGy was effective in extension of shelf life of Juliet and Better Boy tomatoes by 5 and 6 days respectively while 0.61 mGy also proved adequate for extension of shelf life of Giulietta F1 and Cherry tomatoes by 6 and 7 days respectively. Proximate analysis of X-irradiated tomatoes showed no significant changes in the ash, moisture, fat, fibre and carbohydrate contents of all varieties of tomatoes considered (P˃0.05) except the protein contents of Juliet, Better Boy and Giulietta F1 that were significantly affected (P˂0.05). X-irradiation doses in the range of 0.30 mGy – 0.61 mGy are effective for extension of shelf life of tomatoes commonly grown in Benue State. Introduction Tomato (Lycoperscon esculentum L.) is one of the most widely cultivated and consumed fresh vegetable crops in the world today [1][2][3][4].Its consumption is second only to potato as a vegetable [5].The cultivated tomato belongs to the Solanum genus within the Solanaceae family of flowering plants.This family also known as the nightshade family includes other notable cultivated plants such as tobacco, pepper, potato and eggplant [6].Several cultivars of tomatoes which exhibit genetic diversity in terms of shape colour and size as well as taste are found in different parts of the world [7].Tomato is known to be excellent source of many nutrients and secondary metabolites that are very vital to human health, namely, mineral matter, vitamins, antioxidants, phenolics and organic acids [8].Thus, consumption of tomatoes has several health benefits such as reducing the risk of certain types of cancers and chronic degenerative diseases [9][10][11]. Tomato production is also a source of income to most rural producers in developing countries like Nigeria [12].Despite the numerous benefits from this crop, many challenges such as postharvest losses occasioned by the short shelf life of tomatoes are making its production unprofitable in most developing countries especially those in Africa [13].In Benue State for instance, tomato is produced in abundance Tomatoes Commonly Grown in Benue State, Nigeria but no serious efforts have been made in providing storage facilities and effective preservation techniques to enable farmers maximize profit on their tomatoes. Irradiation has proved to be a useful tool for the extension of shelf life of certain fruits and vegetables [14].Food irradiation is the only technique that can maintain food quality and ensure the safety of food without significantly affecting food sensory or nutritional attributes [15].Thus, the technology of food irradiation has been extensively employed for decontamination, disinfestation and shelf life improvement of food and agricultural products prone to rapid deterioration [16].It is a non-thermal, non-chemical and energy-efficient food preservation technique that involves precise exposure of food and agricultural commodities to ionizing radiations such as gamma rays (cobalt-60 and caesium-137) or machine generated X-rays and high energy electrons so that a prescribed quantity of radiation is absorbed [17,18].These types of radiation are chosen because they produce the desired food preservative effects and do not induce radioactivity to foods or packaging materials [19,20].The foods exposed to ionizing radiations is either prepackaged or in bulk to reduce the risk of foodborne illness, delay or eliminate sprouting or ripening [21,22]. Irradiation technique inactivates food spoilage organisms, including bacteria, moulds, and yeasts and is effective in lengthening the shelf-life of fresh fruits and vegetables by controlling the normal biological changes associated with ripening, maturation, sprouting, and finally aging.It also destroys disease-causing organisms, including parasitic worms and insect pests that damage food during storage [21]. This paper seeks to investigate the effect of x-irradiation on the shelf life and proximate composition of some varieties of tomatoes commonly grown in Benue State, Nigeria. Sample Collection Five varieties of fully ripe tomatoes were harvested from an experimental farm in Wannune, kilometer 54, Tarka Local Government Area of Benue State and packed in five baskets according to the varieties.The fruits were of the same maturity stage (red stage) and without blemishes, bruises or signs of infection.The fully ripe tomato fruits were used to test for extension of shelf life. Sample Preparation The fresh tomatoes were rinsed with water to remove dirt from the farm.The rinsed tomato fruits were allowed to dry for few minutes and after which each variety was subdivided into six (6) samples of thirteen fruits (13) fruits each and were labelled A, B, C, D, E and F with sample A serving as control.All the samples were packed in thirty (30) small baskets and taken to the X-ray machine for irradiation. Irradiation Procedure The X-irradiation of tomatoes was carried out using the single phase X-ray machine at Musafaha Imaging Centre, Makurdi, Benue State.Each tomato variety was subdivided into six samples and irradiated with X-rays of peak kilo voltages of 40 kvp, 50 kVp, 60 kVp, 70 kVp, 80 kVp and 5.00 mAs, 10.00 mAs, 15.00mAs, 20.00mAs, 25.00 mAs respectively.In order to convert these Kvp s and mAs to doses, Edmond's Formula for a single phase X-ray machine was used [23,24]. Where kVp = kilo voltage peak mAs = miliampere second SSD = Source to Skin Distance T = Total Filtration of the X-ray machine The total filtration (T) of the X-ray machine used in this work was 1.5 mmAl and a source to skin distance (SSD) of 100 cm was maintained throughout the exposure.The conversion arrived at the following doses: 0.00 mGy (control), 0.10 mGy, 0.30 mGy, 0.61 mGy, 1.06 mGy and 1.67 mGy. Proximate Analysis Proximate analysis was carried out to determine the percentages of ash, moisture, crude fibre, crude protein, fat and carbohydrate using methods of analysis described by Association of Official Analytical Chemists [25]. Determination of Ash Content Five grams (5 g) of sample were weighed in incinerated crucibles and then ashed in a muffle furnace at 600 °C for 4 hours.The ash content was then calculated using equation 2 [26]. Where w1 is the weight of empty crucible W 2 is the weight of crucible + food before drying W 3 is the weight of crucible + ash Determination of Moisture Content Five grams (5 g) of sample were weighed in petri dish of known weight.It was then dried in the oven at 104 ±1°C for 4 hours and later cooled in a desiccator and weighed.The moisture content was calculated using equation 3 [26]. Where W 1 =Weight of empty crucible W 2 =Weight of crucible + food before drying W 3 =Weight of crucible + sample after drying Determination of Crude Protein Protein content was determined using kjeldahl method, according to the procedure of Association of Official Analytical Chemists (AOAC).Concentrated H 2 SO 4 (12 ml) and two tablets of selenium catalyst were dropped into a kjeldahl digestion flask containing one 1 g of the sample.The flask was placed in a digester fume cupboard, switched on and allowed to digest for 45 minutes to obtain a clear colourless solution.The digest was distilled with 4 % of boric acid, and 20 % sodium hydroxide solution until distillation was completed.The distillate was then titrated with 0.1 mol/l of HCl until a violet colour was formed, indicating the end point.A blank was run under the same condition as with the sample.Total protein was then calculated using equation 4 [26]./ 100 (4) Determination of Crude Fibre Five grams (5 g) of sample were weighed into a 500 mL Erlenmeyer flask and 100 mL trichloroacetic acid digestion reagent was added.It was brought to boiling and refluxed for exactly 40 minutes.The flask was removed from the heater, cooled and filtered through a 15.0 cm whatman paper.The residue was washed with hot water, stirred once with a spatula and transferred to a desiccator and weighed (W 1 ) when cool.It was then ashed in a muffle furnace at 500 °C for 6 h ours, allowed to cool, and reweighed (W 2 ).The percentage crude fibre was calculated by applying equation 5 [25]. Where W 1 =Weight of crucible + fiber + ash W 2 =Weight of crucible + ash W 0 = Dry weight of food sample Determination of Fat Content Two grams (2 g) of sample were weighed on a chemical balance and wrapped in a filter paper and placed in an extraction thimble.25 mL of N-hexane was measured into the round bottom flask for fat extraction.After extraction, the flask and its contents were cooled in a desiccator and weighed for fat content.The percentage fat content was calculated using equation 6 [25]. Determination of Carbohydrate Content Carbohydrate content was determined by difference using equation 7 [27]. Statistical Analysis The data obtained in this research was subjected to Analysis of Variance (ANOVA) using Standard Package for Social Sciences (SPSS) version 25.0 at 5 % (α = 0.05) level of significant difference.Duncan Multiple Test was used to separate the mean values where significant differences existed.The statistical analysis was done on the basis of the null hypothesis that X-irradiation has no significant effect on the nutrient content of tomatoes and the alternative hypothesis that X-irradiation has significant effect on the nutrient content of tomatoes commonly grown in Benue State. Results and Discussion The results of the effect X-irradiation on the shelf life of Plum (Lycopersicon esculentum L.-Oval-shaped tomato of Italian origin), Juliet (Lycopersicon esculentum L.-1999 All American Selection Winner), Better Boy (Lycopersicon esculentum L. of USA origin), Giulietta F1 (Lycopersicon esculentum L. -a hybrid from France) and Cherry (Lycopersicon esculentum var.cerasiforme) tomatoes are presented in Figures 1 -5 respectively. Figure 6 presents the comparative analysis of the effect of X-irradiation on the shelf life of some varieties of tomatoes.The effect of X-irradiation on the proximate compositions of Plum, Juliet, Better Boy, Giulitta F1 and Cherry tomatoes are presented in Tables 1, 2, 3, 4 and 5 respectively. The Effect of X-irradiation on the Shelf Life of Tomatoes The result presented in Figure 1 shows that the control and other samples of Plum (Lycopersicon esculentum L. -Ovalshaped tomato of Italian origin) tomatoes X-irradiated with 0.10 mGy, 0.30 mGy, 0.61 mGy, 1.06 mGy and 1.67 mGy took 5, 7, 12, 12, 8 and 7 days to rotten respectively.This implies that X-irradiation doses of 0.30 mGy and 0.61 mGy were most effective in extending the shelf life Plum (Lycopersicon esculentum L. -Oval-shaped tomato of Italian origin) tomatoes by 7 days.In the same vein, Figure 2 shows that it took 6 days for the control (non-irradiated) sample of Juliet (Lycopersicon esculentum L. -1999 All American Selection Winner) while samples X-irradiated with 0.10 mGy, 0.30 mGy, 0.61 mGy, 1.06 mGy and 1.67 mGy took 7, 11, 10, 8 and 8 days to rotten respectively.This implies that doses of 0.30 mGy and 0.61 mGy were adequate for extension of shelf life of Juliet (Lycopersicon esculentum L. -1999 All American Selection Winner) tomatoes.More so, Figure 3 shows that the control and samples of Better Boy (Lycopersicon esculentum L. of USA origin) tomatoes X-irradiated with 0.10 mGy, 0.30 mGy, 0.61 mGy, 1.06 mGy and 1.67 mGy took 4, 6, 10, 8, 5 and 6 days respectively to rotten completely.This also implies that X-irradiation doses of 0.10 mGy, 0.30 mGy, 0.61 mGy, 1.06 mGy and 1.67 mGy extended the shelf life of Better Boy (Lycopersicon esculentum L. of USA origin) tomatoes by 2, 6, 4, 1 and 2 days respectively when compared to the control sample.A dose of 0.3 mGy which enhanced the shelf life by 6 days is therefore the most effective dose for extension of shelf life of Better Boy tomatoes.Figure 4 shows the effect of Xirradiation on the shelf life of Giulietta F1 (Lycopersicon esculentum L. -a hybrid from France) tomatoes.It was observed that the control sample and samples X-irradiated with 0.10 mGy, 0.30 mGy, 0.61 mGy, 1.06 mGy and 1.67 mGy took 5, 8, 9, 11, 7 and 7 days respectively to rotten completely.A dose of 0.61 mGy which extended the shelf life of Giulietta F1 (Lycopersicon esculentum L. -a hybrid from France) tomatoes by 6 days is thus the most effective dose.Nevertheless, the effect of X-irradiation on the shelf life Cherry (Lycopersicon esculentum var.cerasiforme) tomatoes as presented in Figure 5 shows that the control (non-irradiated) and samples exposed to X-irradiation doses of 0.10 mGy, 0.30 mGy, 0.61 mGy, 1.06 mGy and 1.67 mGy respectively took 6, 8, 10, 13, 12 and 9 days respectively to rotten completely.This implies that X-irradiation dose of 0.61 mGy extended the shelf life of cherry (Lycopersicon esculentum var.cerasiforme) tomatoes by 7 days and is the most effective dose.Figure 6 shows a comparative analysis of the effect of X-irradiation on the shelf life of Plum, Juliet, Better Boy, Giulietta F1 and Cherry tomatoes respectively.According to this Figure, Xirradiation doses in the range of 0.30 -0.61 mGy are adequate for extension of shelf life of these varieties of tomatoes considered. This research is in accordance with the work of Sombo et al. [28] who carried out a preliminary investigation of the effect of X-rays on the ripening and shelf life of locally grown Caynne, Roccoto and Annahein pepper in Benue State found that peak tube voltage in the range of 50 -55 kvp was effective in extending the shelf life of pepper.Yissah et al. [29] also found that the shelf life of Okra was greatly enhanced when X-irradiated with 0.05 Gy.Similarly, Ricciardi et al. [30] in their work on X-ray irradiation as a valid technique to prolong the shelf life of Ricotta cheese revealed that the artisanal Ricotta irradiated with 2 kGy and 3 kGy remained acceptable for more than 20 days whereas the control became unacceptable after 3 days while the industrial Ricotta x-irradiated at 0.5 kGy, 2 kGy and 3 kGy showed significant shelf life extension up to 84 days compared to the control which only lasted for 40 days.Other researchers such as Lacivita et al. [31] and Mahmoud et al. [32] also discovered that X-irradiation extends the shelf life of various fruits and other foods they investigated using different doses. The Effect of X-irradiation on the Proximate Composition of Tomatoes Tables 1, 2, 3, 4 and 5 reveal that percentage ash content of the control and the X-irradiated samples of Plum (Lycopersicon esculentum L. -Oval-shaped tomato of Italian origin), Juliet (Lycopersicon esculentum L. -1999 All American Selection Winner), Better Boy (Lycopersicon esculentum L. of USA origin), Giulietta F1 (Lycopersicon esculentum L. -a hybrid from France) and Cherry (Lycopersicon esculentum var.cerasiforme) tomatoes had pvalues of 0.895, 0.085, 0.616, 0.051 and 0.824 respectively.No significant difference between the control and xirradiated samples was observed at 5 % (α=0.05) level of significance.Similarly, the effect of X-irradiation on moisture content of Plum (Lycopersicon esculentum L. -Oval-shaped tomato of Italian origin), Juliet (Lycopersicon esculentum L. -1999 All American Selection Winner), Better Boy (Lycopersicon esculentum L. of USA origin), Giulietta F1 (Lycopersicon esculentum L. -a hybrid from France) and Cherry (Lycopersicon esculentum var.cerasiforme) tomatoes is presented in Tables 1, 2, 3, 4 and 5 show p-values of 0.935, 0.306, 0.551, 0.062 and 0.109 respectively.These p-values provide strong support for the null hypothesis that Xirradiation as no significant effect on the moisture content of these tomatoes mentioned above.In the same vein, the fat content of the control (non-irradiated) and X-irradiated Plum (Lycopersicon esculentum L. -Oval-shaped tomato of Italian origin), Juliet (Lycopersicon esculentum L. -1999 All American Selection Winner), Better Boy (Lycopersicon esculentum L. of USA origin), Giulietta F1 (Lycopersicon esculentum L. -a hybrid from France) and Cherry (Lycopersicon esculentum var.cerasiforme) tomatoes were not significantly affected as observed in the respective pvalues of 0.434, 0.434, 0.833, 0.704 and 0.489.Furthermore, Analysis of Variance (ANOVA) carried out to determine the effect of X-irradiation on the protein content of Plum (Lycopersicon esculentum L. -Oval-shaped tomato of Italian origin), Juliet (Lycopersicon esculentum L. -1999 All American Selection Winner), Better Boy (Lycopersicon esculentum L. of USA origin), Giulietta F1 (Lycopersicon esculentum L. -a hybrid from France) and Cherry (Lycopersicon esculentum var.cerasiforme) tomatoes presented in Tables 1, 2, 3, 4 and 5 produced p-values of 0.130, 0.019, 0.009, 0.010 and 0.128 respectively.These pvalues imply that the protein contents of Juliet, Better Boy and Giulietta F1 were significantly decreased (P<0.05) while that of Plum and Cherry tomatoes were not significantly affected (P>0.05).The variations in protein content may be associated to aggregation or cross-linking as a consequence of X-irradiation affecting nitrogen solubility.The significant changes (P˂0.05) in protein may also be due to free radicals that might be formed in association with splitting of the peptide bonds, deamination and decarboxylation reactions of amino acids followed by chains of chemical reactions forming other new radicals [33].More so, the effect of X- 1, 2, 3, 4 and 5 were 0.953, 0.400, 0.179, 0.160 and 0.037 respectively.This means that X-irradiation has no significant effect (P>0.05) on the carbohydrate contents of these tomatoes.However, a slight (insignificant) increase was observed in all irradiated samples of plum tomatoes when compared to the control.This agrees with the work of Lima et al. [34] who reported that carbohydrates are less sensitive and relatively stable when exposed to radiation doses not more than 10 kGy. In a nutshell, it was discovered that X-irradiation had no significant effect on the proximate composition of Plum, Juliet, Better Boy, Giulietta F1 and Cherry tomatoes. Conclusion The results of this research show that X-irradiation doses of 0.30 mGy and 0.61 mGy are adequate for extension of shelf life of Plum (Lycopersicon esculentum L. -Oval-shaped tomato of Italian origin) tomatoes by 7 days.0.30 mGy is effective in extending the shelf life of Juliet (Lycopersicon esculentum L. -1999 All American Selection Winner) and Better Boy (Lycopersicon esculentum L. of USA origin) tomatoes by 5 and 6 days respectively while 0.61 mGy is effective in extending the shelf life Giulietta F1 (Lycopersicon esculentum L. -a hybrid from France) and Cherry (Lycopersicon esculentum var.cerasiforme) tomatoes by 6 and 7 days respectively.X-irradiation had no significant effect on the proximate composition of tomatoes (p>0.05)commonly grown in Benue state.Therefore, X-irradiation doses in the range of 0.30 mGy -0.61 mGy are adequate for extension of shelf life of tomatoes commonly grown in Benue State without significantly affecting the proximate composition. Recommendations The present research reveals that X-irradiation doses in the range of 0.30 mGy -0.61 mGy are adequate for extension of shelf life of some varieties of tomatoes without significantly affecting the proximate composition.However, future research should be carried out to determine the effect of Xirradiation on vitamins, minerals, antioxidants, phenolics and organic acid content of tomatoes.We also recommend that further research should be carried out on the effect of Xirradiation on the shelf life and proximate composition of some varieties of tomatoes using higher doses of Xirradiation and other types of irradiation. Figure 1 . Figure 1.Effect of X-Irradiation on the Shelf Life of fully Ripe Plum (Lycopersicon esculentum L. -Oval-shaped tomato of Italian origin) Tomatoes. Figure 2 . Figure 2. Effect of X-Irradiation on the Shelf Life of fully Ripe Juliet (Lycopersicon esculentum L. -1999 All American Selection Winner) Tomatoes. Figure 3 . Figure 3.Effect of X-Irradiation on the Shelf Life of fully Ripe Better Boy (Lycopersicon esculentum L. of USA origin) Tomatoes. Figure 4 . Figure 4. Effect of X-Irradiation on the Shelf Life of fully Ripe Giulietta F1 (Lycopersicon esculentum L. -a hybrid from France) Tomatoes. Figure 5 . Figure 5.Effect of X-Irradiation on the Shelf Life of fully Ripe Cherry (Lycopersicon esculentum var.cerasiforme) Tomatoes. Figure 6 . Figure 6.Comparative Analysis of the Effect of X-irradiation on the Shelf Life Some Varieties of Tomatoes. Table 1 . Proximate Composition of the Control (Non-Irradiated) and X-Irradiated Plum (Lycopersicon esculentum L. -Oval-shaped tomato of Italian origin) Tomatoes. Values are of mean ± standard deviation of duplicate Values with same superscript are not significantly different (P > 0.05) at α = 0.05 (Duncan multiple range test) Table 2 . Proximate Composition of Non-Irradiated and X-Irradiated Juliet (Lycopersicon esculentum L. -1999 All American Selection Winner) Tomatoes. Table 3 . Proximate Composition of Non-Irradiated and X-Irradiated Better Boy (Lycopersicon esculentum L. of USA origin) Tomatoes. Table 4 . Proximate Composition of Non-Irradiated and X-Irradiated Giulietta F1 (Lycopersicon esculentum L. -a hybrid from France) Tomatoes. Tomatoes Commonly Grown in Benue State, Nigeria irradiation on the fibre content of Plum (Lycopersicon esculentum L. -Oval-shaped tomato of Italian origin), Juliet (Lycopersicon esculentum L. -1999 All American Selection Winner), Better Boy (Lycopersicon esculentum L. of USA origin), Giulietta F1 (Lycopersicon esculentum L. -a hybrid from France) and Cherry (Lycopersicon esculentum var.	2023-07-10T23:48:07.629Z	2023-05-17T00:00:00.000	{ "year": 2023, "sha1": "43a323fdbc4926ee8010502ded50b0a3d63119fd", "oa_license": "CCBY", "oa_url": "https://article.sciencepublishinggroup.com/pdf/10.11648.j.rst.20230902.11.pdf", "oa_status": "HYBRID", "pdf_src": "Anansi", "pdf_hash": "e1ad3e89fd5dc813833c2544a006ce19767938c5", "s2fieldsofstudy": [ "Agricultural And Food Sciences" ], "extfieldsofstudy": [] }
59503082	pes2o/s2orc	v3-fos-license	MCAM and its Isoforms as Novel Targets in Angiogenesis Research and Therapy Melanoma cell adhesion molecule (MCAM) (CD146) is a membrane glycoprotein of the mucin family. It is one of the numerous proteins composing the junction of the vascular endothelium, and it is expressed in other cell types such as cancer cells, smooth muscle cells, and pericytes. Some recent works were designed to highlight its structural features, its location in the endothelium, and its role in angiogenesis, vascular permeability, and monocyte transmigration, but also in the maintenance of endothelial junctions and tumor development. MCAM exists in different splice variants and is shedded from the vascular membrane by metalloproteases. Studies about MCAM spliced and cleaved variant on human angiogenic physiological and pathological models permit a better understanding on the roles initially described for this protein. Furthermore, this knowledge will help in the future to develop therapeutic and diagnostic tools targeting specifically the different MCAM variant. Recent advances in research on angiogenesis and in the implication of MCAM in this process are discussed in this chapter. Introduction Angiogenesis is the process of new blood vessel formation from preexisting vessels.It contributes to physiological processes such as development and wound healing, but also to pathological processes, such as tumor angiogenesis.The identification of new targets involved in angiogenesis remains an important challenge to fully understand the involved mechanisms and to generate new therapeutic tools.Recent studies have highlighted CD146, an endothelial junctional molecule, as a key factor in angiogenesis.This molecule that displays different isoforms and that is present on different cell types could hence constitute a novel target for therapy.Different reviews have underlined its structural features, localization, and functions in the endothelium.This chapter thus mainly addresses the differences in CD146 isoforms with a special focus on their role in angiogenesis and the therapeutic tools targeting the molecule. Historically, CD146 was discovered in 1987 by Professor J.P. Jonhson for the first time.It was identified as a marker of melanoma progression.These data were obtained by using an antibody generated by mouse immunization with a cell lysate of metastasizing melanoma.This antibody (MUC18) allowed the identification of a 113 kDa transmembrane protein.MCAM (melanoma cell adhesion molecule) described as a marker of metastasizing melanoma [1]. In 1991, the team of Professor.F. Dignat-George identified Sendo-1 antigen as a marker of circulating endothelial cells in the blood by flow cytometry.This was made possible through the generation of a mouse monoclonal antibody named Sendo -1 [2] obtained by mice immunization with a HUVEC cell lysate.Sendo-1 was able to stain the human endothelium whatever the vessel size and its anatomical location within the vascular tree [3,4].Gicerin and HEMCAM refer both to the avian homologues of the molecule [5]. Genomic description The specific location of the CD146 gene is on the arm q23.3 of the chromosome 11 in humans and on the chromosome 9 in mice (www.ensembl.org).The gene encoding the CD146 protein extends over 14 kb.It consists of five immunoglobulin-like domains, two variable domains, and three constant domains of C2 type, as well as a transmembrane domain and an intracytoplasmic portion [6].The extracellular part of the molecule, including the five immunoglobulin domains, is encoded by 13 exons; the transmembrane domain and the intracellular domain are encoded by three exons. The promoter of CD146 presents different putative binding sites and motifs including AP1, AP-2, CRE, SP1, CArG, and c-myb.Analysis of this DNA segments suggests that the four SP-1 sites, the two AP-2 domains, and one response element to AMPc-(CRE) form the minimal promotor of CD146 [7].Specific sites play a role in CD146 expression.The AP-2 sites, which are located at −131 and −302 by relative to the initial ATG, inhibit the expression of CD146 by 70 and 44%, respectively.Moreover, when mutated, the CRE site inhibits by 70% the transcription of the genes.Therefore, AP-2 [8] and CRE sites [9] have been described to modulate CD146 expression in melanoma cells, leading to an increase in tumor growth and metastatic potential in these cancers.In fact, the AP-2 binding site located in the promoter (located at −23 bp) is an inhibitor of the transcription of CD146 while the other AP-2 sites (located at −131 and −302, respectively) are transcription activators [8]. The size of CD146 mRNA is around 3.3 kb and has been first identified in human melanoma cancer cells [10].Its encoding region is about 1940 bp.A large homology in the mRNA sequences exists between human and mouse, but differences can be noted.Thus, in humans, there is a lengthening of the 3' and 5' UTR region as wells as a loss of 6 pb in exon 2. The encoding regions and 5'UTR have a homology of about 80 and 72%, respectively, between the murine and human genes and there is only 31% of homology for the 3'UTR fragment.Finally, the protein sequence shares about 76% of homology between these two species [1,10,11]. Proteic structure and isoforms The proteic structure of CD146 is composed of a signal peptide of 28 amino acids (AA), five immunoglobulin domains (including two variable domains and three constant domains), a hydrophobic transmembrane region (AA 561-585), and an intracellular region.The protein sequence derived from the coding region of CD146 has a theoretical molecular weight of about 72 kDa.However, CD146 has a molecular weight of about 113 kDa.This difference is due to the glycosylation sites present on the protein sequence.Indeed, glycosylations represent about 35% of the total molecular weight of CD146 with mainly N-glycosylations.The presence of sialylation has also been shown [12]. CD146 has many similarities with other immunoglobulin family members such as BCAM (B-cell adhesion molecule) and ALCAM (activated leukocyte cell adhesion molecule), including the same number of immunoglobulin-like domains, similarity of functions and expression on tumor and endothelial cells.Thus, the ALCAM protein plays a role in CD4+ T lymphocytes and in tumor invasion [13,14]. A short and a long isoform generated by alternative splicing have been identified as the two isoforms of membrane CD146.They have not been identified simultaneously.The long isoform was the first discovered in human melanocytes in 1987 and the short isoform was identified as a complementary DNA from chicken more recently [15].In addition, a soluble form of CD146 was also identified in endothelial cell culture supernatant (HUVEC) and in bloodstream in patient [16]. Concerning the extracellular sequence, it is common to both isoforms.The difference is located in the intracytoplasmic portion [15].The two isoforms are the result of an alternative splicing on exon 15 causing a reading frame shift.The short isoform displays a shorter intracytoplasmic domain containing a phosphorylation site for protein kinase C (PKC) and an interaction site for the protein with PDZ domain while the long isoform displays two domains for phosphorylation by PKC and an endocytosis signal sequence [15]. The intracytoplasmic domain sequence is similar to mice and human at 95 and 93% for the short isoform and long isoform, respectively.This conservation across species is in accordance with the important functions carried by the intracytoplasmic domain of CD146. Finally, a soluble CD146 isoform with a molecular mass around 100 kDa, was identified for the first time in 1998 [16].This isoform is detectable in human plasma and serum [17] and is generated by a metalloprotease-dependant shedding of the extracellular domain of CD146.The use of nonspecific inhibitors of metalloproteinase (GM6001) inhibits the formation of soluble CD146 [18]. CD146 localization All the data concerning the expression of the different isoforms of CD146 and their functions are summarized in Figures 1 and 2. Localization in cancer cells CD146 has been identified for the first time in melanoma where it plays an important role in disease progression.Thereafter, CD146 has been shown to be expressed in various cancers, such as pancreatic/breast/prostate/ovarian/lung/kidney cancers, osteosarcoma, Kaposi sarcoma, angiosarcoma, Schwann cell tumors, or leiomyosarcoma (Figure 1).The mechanism of this neo-expression is still largely unknown but, in prostate cancer, it was reported that high expression of CD146 resulted from hypermethylation at the promoter of the CD146 gene [19]. However, almost nothing is known on the differential expressions and localizations of the different isoforms of CD146 in these cells.A recent study has shown that many cancer cells expressing CD146 were able to secrete soluble CD146 through a metalloprotease-dependant shedding [20]. Vascular localization CD146 is expressed on the whole vascular tree whatever the vessel anatomical location and caliber.The localization of the long and short isoforms of CD146 is different.The induction of long CD146 expression in the CHO cell line (which does not constitutively express CD146) results in the expression of the protein at the intercellular junctions.Costaining of CD146 with VE-cadherin, focal adhesion kinase (FAK), PECAM, and the complex catenin/cadherin shows no colocalization, suggesting that CD146 is not located in the adherent junctions, tight junctions, or focal adhesions sites [21,22]. Overexpression of the long form of CD146 in the MDCK cell line (Madin-Darby canine kidney) leads to a basolateral localization of the protein.A dileucine motif on its intracytoplasmic peptide sequence is necessary for this localization [21].An immunohistochemical staining of long CD146 in endothelial colony-forming cells (ECFC) confirmed this junctional localization of the protein.In addition, the presence of a cytoplasmic pool of long CD146 that can be redistributed to the cell membrane was also described in Ref. [23]. The short isoform of CD146 does not share the same cellular localization.Transfection shows an apical localization of the protein in MDCK cells [21] that was confirmed in ECFC in a culture with a specific antibody generated against this isoform [23].The confluence state of endothelial cells appears to regulate the spatial distribution of the two isoforms.Indeed, the long CD146 isoform was not detected at the junction in nonconfluent endothelial cells.Under this condition, the long CD146 isoform was intracytoplasmic and the short CD146 isoform was essentially nuclear and at the migration front [23].In other experiments performed in chickens, it was shown a preferential localization of the long isoform of CD146 in the microvilli where the protein plays a role in their formation.Overexpression of CD146 increased the size of these microvilli [24]. Localization on immune cells On peripheral blood of healthy patients approximately 1% of blood mononuclear cells express CD146.An analysis by flow cytometry of different lymphocyte populations showed an expression of CD146 on B and T lymphocytes in humans [25]. Research has shown that about 1% of B lymphocytes cells express CD146 and its expression is upregulated by a factor 5 following stimulation with IL-4 and CD40.Moreover, CD146 can be neo-expressed on some cell populations after stimulation [25].The generation of two antibodies by rat immunization using cells from the T lymphocytic cell line HUT102 deepened these studies and shown that 2% of CD3+, CD3+/CD4+, and CD3+/CD8+ lymphocytes express CD146. Moreover, stimulations with IL-2 [25] and PHA (phytohemagglutinin) [26] increases the amount of CD146+ T lymphocytes.The cells are also found in vivo in the synovial fluid of patients with rheumatoid arthritis [26]. In mice, a leucocytes screening was carried out which demonstrated that CD146 is not detectable on T/B lymphocyte populations, monocytes, and dendritic cells while 30% of neutrophils and 60% of NK cells express CD146.CD146 expression was correlated with an increased expression of CD11b and CD27 reflecting the maturity of NK.These CD146+ NK cells have a decreased cytotoxicity and produce gamma interferon in smaller quantities [27]. Bone marrow environment In adults, hematopoiesis takes place in the bone marrow located in long bones of the human body.It is composed of a dense network of discontinuous capillaries allowing easy passage of cells produced in the bone marrow into the blood.A vascular sinus network which is mainly composed of stromal cells (reticular, endothelial, adipocyte, and osteoblast) serves to support the hematopoiesis process. In one particular study, a subpopulation of bone marrow stroma cells was shown to express CD146 and to display characteristics of mural cells.They were characterized as a subpopulation of advential reticular cells which are abundant in the bone marrow and are able to generate bone tissue and a hematopoietic environment after isolation and implantation into an immunodeficient mouse [28]. Furthermore, angiopoietin -1 is regulated by CD146+ stromal cells.A decrease in the expression of CD146 by siRNA or FGF-2 (CD146 and Ang-1 regulator) reduces the capacity of these cells to participate in the remodeling and the assembly of pseudovascular structures in vitro and to form hematopoietic microenvironment in vivo.From data on the spatial location of adventitial reticular cells and the expression of Tie-2 (the angiopoietin-1 receptor), it was suggested that CD146 and angiopoietin-1 are involved in the interaction between endothelial and stromal cells [29]. Localization in the central nervous system (CNS) CD146 is found in the central nervous system (CNS).It is expressed during fetal development of the embryo but decreases after birth.Studies performed in chickens and rats have shown an expression of CD146 in the cerebellum, hippocampus, Purkinje cells, and sensorimotor cells of the spinal cord [30].In chickens, CD146 binds NOF (neurite outgrowth factor), causing neurite extension [31], and increases the extension of the optic tectum process [32]. The different functions of CD146 CD146 was reported to be involved in many physiological processes.It has been described to play a role during the vascular development but also during the angiogenic process.As others junction molecules, it was described to be an actor during inflammation by modulating the migration of leucocytes through vascular endothelium. CD146 during the vascular development The role of CD146 was studied during vascular development.To this end, a model of CD146 inactivation by antisense morpholino-oligonucleotides was developed in zebrafish.Authors observed a decrease in intersomitic vessels followed by a decrease in blood flow and a reduction in vessel lumen observed by microangiography after CD146 inactivation [33].It was also shown an inhibition of the VEGF-dependent angiogenesis [34]. Permeability and leucocytes migration CD146 has been shown to be involved in endothelial permeability [33].Using both a monocyte cell line, THP-1, and freshly isolated monocytes, it was also showed that it modulates monocytes transmigration.Junctional CD146 was shown to bind monocytes through a heterophilic interaction to increase their transmigration.In addition, an increased transmigration was observed following the binding of soluble CD146 on monocytes [33].Another study showed that neo-expression of CD146 on lymphocytes induced new cellular properties.Indeed, an increase in the adhesion of CD146+ T lymphocytes effectors was observed after stimulation with IL-1 beta.This effect was blocked by the addition of anti-CD146 blocking antibodies [34].An increase in the adhesion of CD4+/CD146+ T lymphocytes on endothelium was also observed after an inflammatory stimulus.In this study, in vitro transfection of the long isoform of CD146 in NKL.1 cell line induced a reduction of rolling cells and an increased adhesion to the endothelial monolayer.Moreover, these phenomena were accompanied by an increase in microvilli in T lymphocyte cell membrane.Another study showed an increased permeability of HMVEC (human microvascular endothelial cells) following incubation with an anti-CD146 antibody (P1H12) [35].Finally, CD146 is coexpressed with CCR6 on a population of TH17 lymphocytic cells [36]. Angiogenesis Angiogenesis is an important mechanism, both in fetal life and at adulthood.Endothelial cells with angiogenic capacities are able to proliferate, migrate, adhere, and generate new capillaries from a preexisting one. The injection of an anti-CD146 antibody (AA98) led to a decrease of 70% in the number of vessels in a membrane model, chorioallantoic membrane model, in chicken.Furthermore, in mice, this antibody reduced the number of vessels in different models of xenografted tumors (hepatocellular carcinoma, pancreatic, and leiomyosarcoma) [37], demonstrating a role of CD146 in tumor angiogenesis. The recent discovery of the existence of two isoforms of CD146 and the description of a soluble form of CD146 led to study their implications in the angiogenic process.Specific siRNA directed against these two isoforms has shown that the absence of the short CD146 decreased the proliferation, migration, and adhesion of endothelial cells, whereas its overexpression led to the reverse phenomena.These experiments showed that the long CD146 was also necessary to generate pseudocapillaries in Matrigel in vitro by stabilizing the junctions of neovessels.It thus appears that both the short and long isoforms of CD146 display complementary effects to generate neovessels.The effects of the short CD146 were confirmed in vivo by the transplantation of endothelial colony-forming cells (ECFC) overexpressing this isoform in a mouse model of hind limb ischemia.Indeed, it increased the incorporation of ECFC in ischemic muscle and favored the generation of neovessels [23].A study of the mechanism showed that the short CD146 is associated with VEGF-R2 [38], but also angiomotin and VEGF-R1 at the endothelial cell surface [39,40].This association is essential for these different pathways.Indeed, the absence of the short CD146 isoform decreases the phosphorylation of VEGF-R2 in endothelial cells and prevents the proangiogenic effect of vascular endothelial growth factor (VEGF). Soluble CD146 is also able to increase the formation of pseudocapillaries in vitro and to induce neovascularization in a rat model of hindlimb ischemia.In addition, subcutaneous injection of Matrigel containing soluble CD146 in mice increased the recruitment of both mature and immature endothelial cells, as well as smooth muscle cells, resulting in the formation of capillary-like structures [41].Of interest, it was reported that soluble CD146 stimulates the short CD146 isoform through its binding on angiomotin [39] and that the angiogenic properties of soluble CD146 are additive to those of VEGF [41].The roles of the different forms of CD146 are summarized in Figure 2. Cancer cell growth and dissemination CD146, which is neo-expressed on cancer cells, modulates their growth and dissemination.In prostate cancer, CD146 expression was observed in different cell lines.CD146 overexpression increased their invasiveness and metastatic potential [42].CD146 overexpression was also observed in biopsies of patients.Its expression was correlated with a poor prognosis.In ovarian carcinomas, CD146 expression was also correlated with the increase of the metastatic potential.In addition, inhibition of CD146 protein expression in ovarian cancer cell lines led to inhibition of invasiveness, tumor spread and induced cancer cell apoptosis.This was explained by the fact that a lack of CD146 induced a decreased activity of Rho GTPase [43] involved in the invasion, proliferation, and metastatic spread of cancer cells. It was also demonstrated that CD146 expression is increased in osteosarcomas as compared to nonpathological osteoblasts [44].Injection of antibodies against CD146 decreased the amount of lung metastases in an immunodeficient mouse model injected with cells derived from human osteosarcoma [45]. In breast cancer, it was reported that CD146 would act as a tumor suppressor [46] while other studies have described CD146 as a poor prognosis marker [47].Indeed, CD146 overexpression in a breast cancer cell line induced an increased motility and tumorigenicity [48].Recent studies have also shown that CD146 induces the epithelial-mesenchymal transition (EMT) in so far as its expression is correlated with markers of EMT in gastric cancer [49].Moreover, in triple negative breast cancers, an increase of CD146 expression in epithelial cells correlates with a loss of epithelial markers in favor of mesenchymal markers, increasing their invasiveness, migration, and the number of mammospheres.In addition CD44 expression increases and CD24 expression decreases on the cell surface suggesting that cells acquire phenotypic characteristics of cancer stem cells [50]. At present, there is no data on the differential expression and roles of the two membrane isoforms of CD146 on cancer cells.However, recent studies have shown an important role of soluble CD146 in tumor development.First, an increase of soluble CD146 concentration was described in blood of cancer patients with nonsmall cell lung cancer as compared to patients with respiratory inflammatory disease and healthy subjects [51].In this chapter, we showed that association between an increased soluble CD146 concentration and an increased number of circulating endothelial cells (CEC) constitute a poor prognostic factor [51]. Recently, a study showed that human cancer cells that express membrane CD146 on their surface have also the ability to secrete the soluble form of CD146 [20].This was described in melanoma, colorectal and pancreatic cancer cell lines.The authors demonstrated that soluble CD146 secreted by cancer cells could display autocrine effects on cancer cells and paracrine effects on vascular endothelial cells.Indeed, in vitro stimulation of cancer cells with recombinant soluble CD146 increased their proliferation and the production of protumorigenic factor such as VEGF.They also demonstrated that this stimulation protected cancer cells from apoptosis induced by H 2 O 2 and decreased cancer cell senescence.In particular, the c-myc signaling pathway appeared to be upregulated by soluble CD146.Soluble CD146 secreted by cancer cells also increased the proliferation of surrounding endothelial cells, stimulating tumor angiogenesis.These effects were confirmed in vivo in different models of xenografted mice and an antisoluble CD146 antibody was able to block these effects.Thus, soluble CD146 was described as a proangiogenic factor and seems to have a major role in tumor development. Ligand and cell signalization Historically, the first molecule interacting with the extracellular portion of CD146 is NOF (neurite outgrowth factor).A stable transfection of complementary DNA encoding for CD146 induces an adhesion of neuronal cells on a NOF matrix [32].More recently, laminin-8 has been identified as a new vascular ligand of CD146 expressed by TH17 lymphocytes.In this study, it has been demonstrated that the laminin-411/CD146 interaction favor adhesion and tissue transmigration of these lymphocytes, leading to an increased inflammation [52].Furthermore, one study showed that CD146 DNA transfection in the CD146deficient melanoma cell line Mel-888 induced an increased aggregation between these cells and cells which do not express CD146 suggesting that there are other still unidentified partners [53]. The existence of a homophilic interaction for CD146 is controversial.One study showed that CD146 transfection in neuronal cells induced their aggregation, suggesting that CD146 could create homophilic bonds [32].Another in vitro study demonstrated that the neurite growth of PC12 cells is increased when cells are in a chimeric CD146 protein substrate.In addition, under these conditions, the use of an anti-CD146 antibody blocks neurite growth.This inhibition would be associated with an inhibition of CD146-CD146 homophilic interaction [54].CD146 dimerization at the cell membrane following stimulation with an activating CD146 antibody (clone AA98) was also demonstrated using fluorescence resonance energy transfer (FRET) and pull-down.The use of an NFkB signaling pathway inhibitor reduced this dimerization [55].Finally, a recent study highlighted dimerization after stimulation with VEGF [56]. Conversely, other studies could not replicate the homophilic interaction, in particular, between soluble CD146 and CD146-Fc [33]. Recently, new ligands for CD146 were identified.A direct and strong interaction between CD146 and VEGFR-2 was demonstrated in endothelial cells and this association was important for VEGFR-2 phosphorylation by VEGF.These results were confirmed in a CD146 KO mouse model where the absence of CD146 inhibited vessel formation induced by VEGF.Experiments in mouse models of pancreas and melanoma cancer cell xenograft have shown that the combined use of anti-VEGF antibody (bevacizumab) and anti-CD146 antibody (AA98) displayed a synergistic effect on tumor development [57]. Another work identified galectin 1 as a new CD146 ligand on the endothelium [58].This protein induced apoptosis of endothelial cells and specifically bound to CD146 via extracellular glycosylations.This interaction is specific for galectin 1 since it is not found with galectin 2. Using siRNA or antibodies able to block CD146 resulted in an increased cell apoptosis, suggesting a protective role of CD146 against apoptosis. Different factors have been shown to modulate CD146 expression: -A stimulation with TNF-alpha increases the amount of CD146 present at the endothelial cell surface [33]. -TGF-beta administered to hepatocyte cells pretreated with an inducer of acute hepatitis (tetrachloride carbonate) increases the amount of CD146 mRNA and the regenerative capacity of these cells [59]. -HSP27 (heat shock protein 27), a chaperone molecule involved mainly in tumor differentiation and tumorigenesis, inhibits the migration and invasion of melanoma cells and thus acts on the tumor phenotype [60].Overexpression of HSP27 has been shown to decrease the expression of CD146 and increase the expression of E-cadherin in melanoma cell lines.These variations in protein expression determine, among other, malignant phenotype of melanoma cells [61]. -AKT activation by PD98059 and Wortmannin increases CD146 expression at the cell membrane in melanoma cell lines.Conversely in these cell lines, overexpression of CD146 increases AKT which inhibits BAD (Bcl-2-associated death promoter), increasing cell survival [62]. Membrane CD146 activates multiple signaling pathways, leading to the activation of the NFkB pathway.CD146 dimerization has been described in the membrane of endothelial cells following the addition of culture medium of tumor cells (A375 cell line).Inhibition of the NFkB pathway (by BAY11-7082 compound) causes a reduction of the nuclear translocation of NFKB but also inhibits the dimerization of CD146 [63,64].The junctional molecules involved in adhesion such as VE-cadherin or claudins are also involved in a phenomenon of actin cytoskeleton reorganization.CD146 is also connected to the actin cytoskeleton. Indeed, CD146 targeting with the S-ENDO1 antibody led to FAK phosphorylation and an increase in the release of intracellular calcium and extracellular calcium entry.This mechanism of action of calcium flux was mediated by the recruitment and activation of Fyn leading to the phosphorylation of PLC gamma.Calcium entry also caused the recruitment of PYK2 and p130.On the other hand, FAK activation led to signaling pathways involved in the reorganization of the actin cytoskeleton and also modulated transcription factors involved in cells survival and migration.In these studies, there was no evidence of direct interaction between CD146, paxillin, and FAK.It seems therefore important to identify the intermediate partners [65,66]. Recently, a study confirmed the role of CD146 in the migration and induction of signals related to the actin cytoskeleton.Indeed, CD146 displays direct physical interaction with the ezrin-radixin-moesin (ERM) proteins, allowing the recruitment of ERM at the protrusions of melanoma cells.This phenomenon induces the elongation and expansion of microvilli at these protrusions [67]. Recruitment by CD146 allows the sequestration of a RhoA inhibitor (Rho guanine nucleotide dissociation inhibitory factor 1) leading to RhoA activation and an increased cell motility. Another study showed that CD146 is redistributed in a polarized structure named W-RAMP (Wnt5a-mediated actin-myosin receptor-polarity) in subconfluent melanoma cells stimulated with Wnt5a.W-RAMP is involved in membrane retraction and the direction of cell migration with an intervention of Rho-A [68]. In another study that focused on the priming of ECFC with soluble CD146 in order to improve the therapeutic potential of these cells in vivo, the authors showed that a priming of these cells with soluble CD146 did not modify the number of engrafted ECFC in the ischemic muscle but improved their survival capacity leading to an enhanced revascularization [39].They showed that in ECFC, it exists a signalosome that is located in a particular region of cell membrane called lipid rafts.This signalosome contains soluble CD146, the short isoform of CD146 (shCD146), presenilin-1 but also the two VEGF receptor called flt1 and flk1.The mechanism of action is characterized by a sequential proteolytic cleavage, induced by soluble CD146, with an extracellular shedding of the short CD146 followed by an intramembrane cleavage which is mediated by both the ADAM/matrix metalloproteases (MMP) and the gamma-secretase protein.The consequences of this shedding involved a nuclear translocation of the new intracellular peptide of shCD146 which binds to the transcription factor CSL and is associated with a modulation of gene transcription leading to angiogenesis (eNOS) and cell survival (FADD, Bcl-xl).The association between CD146 and VEGFR2 was described in a previous paper and based on these results the authors showed that the effect of soluble CD146 on EFCF is dependent on VEGFR2 but also VEGFR1 which are phosphorylated by soluble CD146.All these findings show that the stimulation of this cell by soluble CD146 and the proteolytic cleavage of shCD146 is a promising pathway to increase the regenerative properties of endothelial progenitor cells for the treatment of cardiovascular diseases (Figure 3). CD146 in pathology 6.1. Obstetrical pathologies About 2% of fertile women are affected by spontaneous fetal loss.The mechanism of this fetal loss is not yet understood. One study showed that CD146 is highly expressed during the implantation window. During the following steps, the level of CD146 decreased rapidly and CD146 blocking with an antibody caused abortion [69].CD146 is expressed by the intermediate trophoblasts (or extravillous) in humans but is not detected on the syncytiotrophoblasts and cytotrophoblasts [70]. After this work, soluble CD146 was described as a novel physiological factor with angiogenic properties involved in the regulation of placenta vascular development by acting on extravillous trophoblast (EVT).Using placenta explants, soluble CD146 was demonstrated to inhibit the growth of extravillous trophoblasts and the ability of EVT to migrate and form pseudocapillary tubes on Matrigel.A clinical study on the role of soluble CD146 in 50 pregnant women was also conducted.A physiological decrease of plasmatic soluble CD146 was observed in pregnant women as compared to nongestational women.These results inspired the authors to study the effects of prolonged administration of soluble CD146 in a pregnant rat model.Repeated systemic injection of soluble CD146 after mating caused a significant decrease in the pregnancy rate and the number of embryos.Histological studies of placenta showed a decreased migration of glycogen cells (cells that are similar to the EVT in rat) in female rat treated with soluble CD146. In mice the use of a specific antibody blocking CD146 (AA98) caused a decrease in the blastocysts adhesion on a uterine epithelium cell monolayer and a decrease in the growth of trophoblastic cells.In addition, injection of this antibody in the uterine horn of the mouse at 3.5 dpc (days post coitum) resulted in a decrease of embryo implantation at 7.5 dpc.Histological analysis showed that the embryos were present but smaller and in poor condition [69].Two clinical studies were in line with these observations.A first clinical study showed that the rate of membrane CD146 expression was lower on intermediate trophoblasts in the placenta of preeclamptic patients when compared to patients with nonpathological pregnancies [70]. In a second study, two populations of women have been used to compare the blood level of soluble CD146.In this study, the authors used 100 blood samples which were taken 2 months after the last obstetrical events between women with no pregnancy lost which have at least one living child and 100 blood samples from women with at least two consecutive losses at/ or before 21 weeks of gestation.They found an increase in the level of soluble CD146 in the second population compared to the first control population [71].In this study, the two populations used are age matched. Thus, in view of these results soluble CD146 may represent an attractive biomarker of vascular placental development as well as a therapeutic target in obstetric complications. Inflammatory diseases Endothelial functions are altered in inflammatory diseases. In inflammatory kidney disease, biopsies of patients with nephropathy show an increased expression of membrane CD146 on endothelial cells, but also on the mesangial cells and a neo-expression of CD146 on tubular cells [72].In addition, there is a correlation between CD146 expression and proteinuria, endocapillary proliferation and inflammatory syndrome. The serum level of soluble CD146 is also modulated.Thus, an increase in CD146 secretion was observed in chronic renal failure which was correlated with the severity of this disease in type 2 diabetic nephropathy patients [73]. In a second type of inflammatory disease, CD146 has also been identified in primary cultures of keratinocytes while its expression was not observed on healthy skin.An increase in the expression of CD146 has been observed in various skin diseases.For example, CD146 is expressed in suprabasals keratinocytes of psoriasis patients [74].CD146 is also detected in Kaposi's sarcoma, lichen planus, on the skin overlying skin neoplasms or in chronic and acute chronic dermatitis [74].On the other hand, the expression of CD146 is not increased in other skin diseases such as lupus erythematosus. Tumor pathologies CD146 is expressed in many cancers, such as melanoma, prostate cancer, breast cancer, pancreatic cancer, lung cancer, Kaposi sarcoma, angiosarcoma, Schwann cells tumors, or leiomyosarcoma. The role of tumor CD146 was first studied in melanoma.A direct correlation has been demonstrated between the increase in metastasizing capacities and the increased expression of CD146 [75].The level of expression of CD146 by human melanoma cell lines has been shown to correlate with their ability to form tumors and metastasis in a mouse xenograft model in immunodeficient nude or SCID mice [76].In addition, CD146 increases the number of lung metastases following intravenous injection of melanoma cells in nude mice in vivo [77].These observations were confirmed through the use of interfering RNA directed against CD146 leading to a decrease in migration, proliferation, and invasion in vitro [78]. CD146 immunohistochemistry staining was performed on human primary melanoma tissue, showing a CD146 expression on tumor-associated endothelium and on smooth muscle cells [79].The role of CD146 in tumor angiogenesis has been described in particular thanks to the use of the AA98 antibody [37,64] that is able to block tumor angiogenesis and decrease tumor growth of human melanoma xenograft model in immunodeficient mice. Currently, mechanisms involved in melanoma progression are unclear.A study showed that a particular population of B lymphocyte cells, the B1 lymphocytes, has a prometastatic potential.Indeed, depletion of this population caused a decrease of tumor growth and metastasis dissemination in mice in an experimental metastasis model, following a B16 cell line injection.The decrease in metastases dissemination involved homophilic interactions between B1 and B16 cells thanks to CD146 [80].In addition, coculture of B1 cells with melanoma cells increased the expression of CD146 at the cell membrane of cancer cells, increasing the number of metastases in vivo. A clinical study was conducted on a cohort of patients with skin cancer.Patients were divided into two groups: early and late stage melanoma, in order to analyze the presence of different commonly used cancer markers including CD146.Analysis in the blood of patients showed that CD146 was the only protein correlated with the advanced stages of the disease [81].Another study confirmed this finding by demonstrating that CD146 is a marker of poor prognosis and survival in melanoma patients.CD146 constitutes a better marker than biopsies analysis of sentinel lymph node [82]. Angiogenesis-related diseases and therapeutic approaches Recent studies revealed that both isoforms of CD146 are involved in angiogenesis with a promigratory and a proproliferative role of the short CD146 and a vessel stabilization role of long CD146, which is also described in this chapter.Soluble CD146 secreted by both endothelial and cancer cells is also able to stimulate angiogenesis.These different forms are involved in physiological angiogenesis but also in pathological angiogenesis, in particular in tumor angiogenesis. Therefore, different antibodies have been generated to block its functions.The first one was ABX-MA1, an antibody recognizing the human form of this molecule.This antibody was able to inhibit the formation of spheroids containing melanoma cells, reducing metastasis, tumorigenicity, and vascularization of the tumor in vivo.This reduction was related to the inhibition of MMP-2 expression which is heavily involved in metastasis formation [83]. Another team-generated monoclonal antibody specifically directed against the vascular endothelium of tumors.During the screening of these antibodies, the authors focused on the AA98 antibody.This antibody recognizes CD146 localized in the intratumoral vasculature but not recognizes CD146 expressed on blood vessels in healthy tissues [37].This antibody inhibits both in vitro and in vivo angiogenic properties of CD146 in human tumors xenografted in immunodeficient mice.In addition, it was demonstrated that the AA98 antibody is a potential diagnostic and therapeutic agent in vascular and cancer diseases.Following this work, it was shown that AA98 antibody inhibits phosphorylation of p38/MAPK, suppresses NFkB activation, and inhibits MMP-9 and ICAM-1 expression.This suggests that deleting NFkB is a pivotal point of the inhibitory effects of the antibody on endothelial cell migration, angiogenesis, and development of tumor metastases [64]. Of interest, this antibody displays additive inhibitory effects when used in combination with the anti-VEGF antibody bevacizumab in xenografted models of human pancreatic tumors and melanoma [57].In addition, it reduces significantly the chronic inflammation in the colon in a mouse model and prevents the development of cancer associated with this chronic inflammation [38]. Recently, a novel antibody was generated against the soluble form of CD146 [20].The authors demonstrated that this antibody was able to decrease tumor angiogenesis and growth but to also induce apoptosis of human melanoma and pancreatic tumors xenografted in immunodeficient mice.Of interest, this antibody cannot bind membrane CD146, a property that should limit the side effects that could be observed with antibodies targeting the membrane form. Figure 1 . Figure 1.Summary table for the different isoforms of CD146 expressed in several organs and cells related to their functions, pathologies, and references associated. Figure 2 . Figure 2. Expression, cell localization, and functions of the different isoforms of CD146 by endothelial cells (EC) and blood circulating cells. Figure 3 . Figure 3. Mechanism of actions of endothelial progenitor cells (EPC) activation leads to their recruitment after inflammatory or angiogenic stimulation. Functions of the different CD146 isoforms and the inhibitory antibodies associated are summarized in Figure 4. inhibits spontaneous pulmonary metastasis of osteosarcoma cells in vivo.Clin Cancer Res.2003 Dec 15;9(17):6560-6566.	2018-12-30T04:27:33.542Z	2017-04-05T00:00:00.000	{ "year": 2017, "sha1": "1b2fc6af79b57a60fa1e49067a138411c385dc9f", "oa_license": "CCBY", "oa_url": "https://www.intechopen.com/citation-pdf-url/53335", "oa_status": "HYBRID", "pdf_src": "ScienceParsePlus", "pdf_hash": "4db6e6ef5bd95578e93ac3161fd2690e51f7577a", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Biology" ] }
270794543	pes2o/s2orc	v3-fos-license	Research trends and hotspot analysis of age-related hearing loss: A bibliometric analysis from 2019 to 2023 Background: Age-related hearing loss (ARHL) — also termed presbycusis — is prevalent among older adults, leading to a range of issues. Although considerable progress in the understanding of ARHL over the decades, available reports lack data from recent years and do not comprehensively reflect the latest advancements and trends. Therefore, our study sought to assess research hotspots and trends in ARHL over the past 5 years to provide the basis for future research. Materials and methods: The Web of Science Core Collection database was searched and screened from January 1, 2019 to October 21, 2023, according to the inclusion criteria. CiteSpace (5.8.R3), VOSviewer (1.6.19), and Microsoft Excel 2019 were employed for bibliometric analysis and visualization. Results: 3084 articles from 92 countries led by the United States and China were included. There has been a steady upward trend in the number of publications from 2019 to 2023. The most productive institutions, authors, and journals are Johns Hopkins University ( n = 113), Lin FR ( n = 66), and Ear and Hearing ( n = 135), respectively. Trend topic analyses revealed that “ cochlear synaptopathy ” and “ dementia ” were the predominant foci. Keywords, including “ individuals ” and “ national health ” , began to appear. Conclusion: Over the past 5 years, the annual number of publications has increased significantly and will continue to do so. Research on the mechanism of ARHL, represented by “ oxidative stress ” , is a continuing focus. Emerging topics such as “ individual differences ” and “ national health ” may be potential future hotspots in this field. Introduction Age-related hearing loss (ARHL) -also known as presbycusisis a multifactorial illness characterized by aging-related bilateral, symmetrical, and progressive hearing loss (Gates and Mills, 2005).ARHL is closely associated with age-related degeneration of inner ear structures (Bowl and Dawson, 2019).According to the World Health Organization, the number of people with ARHL is soaring annually, almost one-third of individuals aged over 65 years are experiencing hearing loss worldwide (Löhler et al., 2019).By 2050, it is estimated that 900 million older adults will experience severe hearing impairment due to ARHL (Patel and McKinnon, 2018).Additionally, ARHL ranks as the third most common cause of years lived with disability diseases globally (GBD 2017 Disease andInjury Incidence andPrevalence Collaborators, 2018).Accumulating evidence has shown that ARHL is closely linked to depression (Rutherford et al., 2018), social isolation (Mick et al., 2014), and a decline in cognition (Slade et al., 2020a).In the context of older people's health, the absence of psychological problems, along with maintaining a good level of functional abilities and functional autonomy, are key determinants that influence the degree of satisfaction with their health status (Agustí et al., 2023), as opposed to economic issues such as financial situation or housing (Parra-Rizo, 2017).Moreover, ARHL poses a significant financial burden to society.For instance, the USA healthcare spending on ARHL is projected to reach $30 billion by 2030 (Stucky et al., 2010).ARHL is increasingly becoming a major public health concern worldwide (Lv et al., 2023).Thus, a deeper understanding of the current state of ARHL research is imperative, which is critical for developing new prevention and treatment strategies for the disease, coordinating research around the world, and planning health systems. Bibliometrics is a subfield of informatics that focuses on quantitative and qualitative literary analyses, using the literature system and bibliometric properties as the research object.It can quantitatively estimate the outline, association, and clustering of the study field (Ma et al., 2021a).This approach is now widely used to evaluate the impact, credibility, and quality of academic work (Ellegaard and Wallin, 2015;Rondanelli et al., 2016).In particular, bibliometrics can evaluate the contributions and impacts of various authors, nations/regions, organizations, fields, and journals; it can also appraise the state, directions, and frontiers of research endeavors (Smith, 2008;Chandra et al., 2013;Pu et al., 2016).CiteSpace and VOSviewer software are the most frequently used bibliometric tools for data processing and visualization (Chen, 2004;van Eck and Waltman, 2010).They can help readers understand structured content more intuitively and are useful tools for assessing the thematic evolution of structured content (Cobo et al., 2014). In recent years, bibliometric analysis has been extensively employed in various fields, including ARHL (Lv et al., 2023;Cui et al., 2022).We highly approve of their work, however, limited by earlier publication dates, available ARHL bibliometric studies did not include the latest ARHL studies that had mushroomed globally in recent years, and therefore did not reflect the latest field directions.Therefore, the purpose of this study is to combine the latest research results in the field of ARHL in the past 5 years to reflect the current research status in the field more systematically and to provide references and directions for researchers.In this study, we illustrated new hotspots in the ARHL field that have emerged in recent years such as national-health and individual differences, and updated the latest progress of previous hot research directions such as the use of hearing aids and the relationship between noise and ARHL.In addition, we updated and extended the ideas of previous studies based on the latest literatures, such as past study (Lv et al., 2023) has mentioned cochlear synaptopathy as a pathogenesis of ARHL, but has not mentioned the treatment options for it while our study presented this aspect based on the most recent studies.The Web of Science Core Collection (WoSCC) database was utilized for in-depth analysis and update of bibliometrics-based ARHL research.Descriptive statistics were carried out following the retrieval of pertinent bibliometric data (annual articles, countries/regions, authors, institutions, disciplines, journals, references, and keywords) from each ARHL study.The present study applied CiteSpace and VOSviewer to create knowledge maps that illustrate research hotspots and growing trends in ARHL research, to provide a reference for future studies. Data sources and search strategy Relevant studies on ARHL published between January 1, 2019 and October 21, 2023 were retrieved from the WoSCC database.The WoSCC database encompasses about 12,000 prominent, top-notch journals and a broad range of bibliometric indicators, and thus it is ideal for bibliometric analysis.Besides the general literature search, it has a crucial feature called citation index searching, which is useful for evaluating the academic value of literature on a certain topic (Wu et al., 2021a).The following search terms were used: TS = ("age-related deafness" OR "old* adults hearing loss" OR "old* adults hearing impairment" OR "agerelated hearing disorder" OR "age-related hearing loss" OR "old* adults deafness" OR "presbycusis" OR "presbyacusis" OR "presbyacousis"). Screening criteria The literature search and screening process is displayed in Fig. 1.The screening process was done independently by 2 coauthors (QW and TM) using the following criteria: (1) published in "English language"; (2) the publication type is "article" and "review"; (3) the timespan is between 2019 and 2023.Early access, proceeding paper, editorial material, letters, retraction, book chapters, and data paper were eliminated.Controversial articles were discussed in team meetings before a decision was made.Finally, a total of 3084 publications were included in the final analysis. Statistical analysis The full record and all cited references of the included studies were downloaded and exported to ; Chaomei Chen).CiteSpace analysis included the following items: overlay of journals and cited journals, the cluster network graph and the timeline view of co-cited references, the annual publications, the centrality value of authors, countries, and institutions, and the keywords with the strongest citation bursts.Scientifically-based knowledge networks, including publication nations, institutions, journal clusters, and keywords, were analyzed using VOSviewer. Publication activity and citation analysis Overall, 3084 ARHL-related articles were published between January 1, 2019 and October 21, 2023.The volume of ARHL-related papers has been rising (Fig. 2).Generally, the number of citations in this field has increased annually during the past 5 years (Fig. 2).Specifically, the annual number of publications and cumulative citation frequency per year peaked at 725 publications and 8076 cumulative citations in 2022, respectively. Contributions by authors A total of 12,329 authors contributed to the identified studies.The 10 most prolific authors are listed in Table 1.Lin FR from the Johns Hopkins Bloomberg School of Public Health was the most productive author, with 66 publications, followed by Reed NS (n = 59) and Deal JA (n = 53) Eight different clusters of authors were identified (Fig. 3).The active cooperation occurred within the same clusters, such as that between Lin FR and Deal JA, Dubno JR and Anderson S, and Dawes P and Lozupone M. Additionally, active cooperation was observed among clusters, such as between Dubno JR and Humes LE, between Reed NS and Golub JS, and between Jayakody DMP and Thomas JP. Contributions by institutions Overall, 3337 institutions published ARHL-related articles.The top 10 most productive institutions between 2019 and 2023 are depicted in Table 2. Johns Hopkins University in the USA was the most productive institution, with 113 publications, followed by Johns Hopkins Bloomberg School of Public Health (n = 88), University of Toronto (n = 73), The University of Manchester (n = 73), and University College London (n = 62).University System of Ohio had the highest centrality (0.16), followed by the State University System of Florida (0.16) and the University of Toronto (0.15).The centrality of these institutions was over 0.1, highlighting their impact on ARHL research.A network of cooperation among institutions is shown in Fig. 4 University of Toronto (192), indicating a history of continuous close collaboration. Contributions by nations/regions A total of 92 countries/regions released publications on ARHL between 2019 and 2023.As shown in Table 3, the USA published the most articles (n = 1133), followed by China (n = 418), England (n = 296), and Australia (n = 240).The centrality score is a key indicator to quantitatively evaluate the significance of nodes in a network (Lei et al., 2022).England (0.23) and USA (0.11) had the highest centralities, underscoring their leading role in ARHL research.Other nations/regions had a relatively lower centrality, suggesting that most of them are not highly influential and have not collaborated closely with other nations. Furthermore, VOSviewer was utilized to create a country/region coauthorship overlay visualization map (Fig. 5).Every node signifies a unique country/region, and the size of each node corresponds to the quantity of publications generated by that country/region.The higher the number of publications produced in a country/region, the larger the node corresponding to that country/region.A smaller distance and a thicker connecting line between two nodes indicate a stronger relevance and link strength, respectively.The color of each node corresponds to the average appearing year (AAY) of the country/region (Fan et al., 2023).Early entrants into the ARHL field (e.g., the USA and Canada) are denoted by a blue or purple color, and new entrants into the field (e.g., Greece and Turkey) are denoted by a yellow or green color. Contribution by journals A total of 787 journals published 3084 ARHL-related papers.The top 10 most productive journals in this field in the past five years are listed in Table 4. Ear and Hearing published the most articles (n = 135), followed by the International Journal of Audiology (n = 95) and Frontiers in Aging Neuroscience (n = 84) (Table 4).Co-citation analysis of journals was carried out using VOSviewer (Fig. 6A).Hearing Research had the highest cooperation with other journals, with the highest total link strength of 481,023, followed by Ear and Hearing and Journal of the Acoustical Society of America.Each article was labeled with one or more subject categories to facilitate rapid search in the WoSCC database.All citations of journals were clustered into four categories: Otorhinolaryngology, Audiology Speech Language Pathology, Neuroscience, and Geriatrics Gerontology. Fig. 6B shows the overlay of the journals and cited journals.The cited journals are displayed on the right side of the dual-map, with the citing journals on the left.There were four primary citation paths.The orange paths indicate that molecular/biology/genetics journals were generally cited by molecular/biology/immunology journals.The grey paths imply that molecular/biology/genetics journals were generally cited by dentistry/dermatology/surgery journals.The cyan paths imply that psychology/education/social journals were generally cited by psychology/education/health journals.The green paths demonstrate that molecular/biology/genetics journals were generally cited by medicine/ medical/clinical journals. Analysis of co-cited references Co-citation is a research method that measures how closely related publications are to one another.Co-citation occurs when two or more publications are cited simultaneously in one or more papers (Ma et al., 2021b).The top 10 most cited references in the field of ARHL are listed in Table 5.The most frequently cited articles were Haile LM et al. (2021), Wu PZ et al. (2019), andKotwal AA et al. (2021).The paper published by Haile LM et al., titled "Hearing loss prevalence and years lived with disability, 1990-2019: findings from the Global Burden of Disease Study 2019", was published in the Lancet and had the highest impact factor (168.9). Co-occurrence analysis of keywords is another popular method for identifying popular hot research topics in bibliometrics.The number of publications that simultaneously include two keywords determines how connected they are in a co-occurrence analysis (Wu et al., 2021b).VOSviewer was used to analyze author keywords extracted from 3084 articles.After some similar-meaning keywords were combined (such as "older-adults", "older-people" and "older people"), VOSviewer parameters were as follows: the minimum number of occurrences of a keyword: 30.There were 9457 keywords, with 186 keywords meeting the thresholds.Then, the co-occurrence network graph for the keywords was dawn (Fig. 7B).Keywords were divided into four clusters, representing four primary research directions for ARHL.The relative distance between two nodes indicates the intensity of their association, and the size of the nodes is proportionate to the frequency of keywords.The frequency of two nodes occurring together increases with the thickness of the lines connecting them (Zhou et al., 2022). The red cluster was primarily associated with mechanisms of ARHL and contained keywords such as "mechanisms", "individual-differences", "noise", "auditory cortex", "inner ear", and "mutation".The green cluster was related to mental health and prevalence and included keywords such as "anxiety", "depression", "mental health", "stress", "prevalence", "risk factors", and "balance".The blue cluster concerning older people and their quality of life mainly consisted of keywords such as "older adults", "aids", "health-care", "cochlear implantation", "quality of life", and "outcomes".The yellow cluster mainly involved cognitive issues in ARHL patients and the keywords included "dementia", "cognitive impairment", "Alzheimer's disease", and "decline". The overlay visualization map of the most popular terms in ARHLrelated studies is shown in Fig. 7C.In addition, each of these keywords was color-labeled using VOSviewer based on the average year of publication.More recent keywords are highlighted in red and those that emerged earlier are denoted by a blue color.Keywords such as "auditory cortex", "mouse model", "inner ear", and "noise" characterized the main theme in the previous phase, whereas keywords such as "health-care", "prevalence", "epidemiology", "prevention", and "intervention" showed a relatively latest average publication year, indicating that this topic may have been more eye-catching recently and may soon become a research hotspot. Next, emergent word detection for keywords was performed using the co-citation network of keywords.The strongest citation bursts for the top 30 keywords in ARHL are shown in Fig. 7D.The time axis is represented by the blue line in Fig. 7D, and burst detection is denoted by the red segment on the blue time axis, which shows the start year, end year, and burst duration.It was found that "national health" (6.54) had the strongest citation bursts, followed by "national-health" (6.48), "selective attention" (5.9), "reliability" (5.88), and "symptoms" (5.75).The terms "gene", "stria vascularis", "spiral ganglion neurons", "cognition", and "Alzheimer's-disease" emerged early and were early-stage subjects, according to the initial time of appearance."Individual-differences", "social isolation", and "national health" are the hotspots in ARHL and entering an explosive period. General information According to the WoSCC database, 3084 publications on ARHL were published between 2019 and 2023 in 376 academic journals by 12,329 authors from 3337 institutions across 92 countries/regions.For the past five years, there has been a steady growth in the number of publications on ARHL, suggesting increasing interest in this field. According to visualization analysis of contributions by countries/ regions, the USA predominates in this field, producing 1133 papers, which accounts for 36.7 % of all the publications.Notably, a majority of the top 10 most productive countries were industrialized nations.This might be explained by the degree of economic growth and monetary commitment to science made by these nations.Among the top 10 nations, England (0.23) had the highest centrality score (0.36), followed by the USA (0.11) and Australia (0.10), suggesting that they played an essential role in promoting global collaboration in ARHL research. Three of the top 10 universities were based in the USA, two were in Canada, three were in Australia, and two were in England.The most productive university was Johns Hopkins University.A collaboration network map showed that some universities collaborated in ARHL research.However, more collaboration among countries and organizations is warranted to foster ARHL research. Identifying leading researchers and their contributions in a certain field may provide a path and guidance for other scholars interested in the field (Zhou et al., 2022).Lin FR, Reed NS, and Dubno JR have substantially contributed to the ARHL field, as evidenced by their numerous high-quality papers.Distinguished researchers alike are more inclined to produce influential papers continually and carry out collaboration/viewpoints exchange.However, network density suggested that there is still room for collaboration among top authors. Journal analyses showed that Ear and Hearing, with the highest number of articles and citations, was the most significant journal in this discipline.Overall, the top 10 journals on ARHL research were mainly in the fields of audiology, otorhinolaryngology, and neurosciences domains.This implies that ARHL is a significant global problem for otolaryngologists, neurologists, and audiologists.Furthermore, the top 10 co-cited studies were published in journals with a higher impact factor, such as the Lancet and JAMA Network Open. Emerging hotspots Burst keywords are important indicators of new research directions and trends (Wu et al., 2021a).Research hotspots and frontiers in ARHL were identified after the examination of the top 30 keywords with the strongest citation bursts.Its primary contents are as follows: Individual differences in ARHL patients A deeper understanding of person-level determinants of changes is crucial to explain heterogeneity (Acuff et al., 2022).Ramlackhan et al. (Crawford et al., 2023) found that most people with hearing loss ignore this health issue, despite the ample data supporting the advantages of hearing therapies.Therefore, they investigated individual factors impacting judgments concerning the management of hearing loss and found that patients with a greater trait susceptibility to experiencing boredom were inclined to have a more aversive subjective experience of hearing loss and lower uptake of treatments, yielding a notable disparity between subjective measures of hearing loss (e.g., self-report hearinghandicap scales) and objective audiometric assessments (e.g., audiograms).Ultimately, these people were more inclined to believe that their hearing problems had not been resolved after treatment.Souza et al. (Souza et al., 2019) proved that differences in individual characteristics, including age, working memory capacity, and level of hearing loss, can cause different listeners to respond differently to changes in the same signal from hearing aids.For example, individuals with a lower working memory display low speech intelligibility in noise with a mild signal processing fitting, and individuals with a higher working memory present better speech intelligibility under the same conditions. The effect of individual differences on hearing is also evident compared with the hearing status of older people of different ethnicities, such as the Mabaan tribe living in the Sudanese desert who retained their hearing in old age (Rosen et al., 1962). National-health concerns for ARHL patients ARHL is a major and rapidly growing public health concern.In the past few years, several studies have analyzed the risk factors of ARHL from a national health perspective and proposed action initiatives based on a national perspective.Kim et al. (Kim and Chung, 2019) obtained data from the 5th Korean National Health and Nutrition Examination Survey (KNHANES) to investigate the possible effects of dietary nutrients on ARHL and found that the recommended intakes of riboflavin, niacin, and retinol, respectively, may contribute to the reduction in ARHL prevalence in older adults.Similarly, using the same database, Choi et al. (Choi et al., 2021) found that dietary antioxidants or anti-inflammatory foods may help reduce ARHL.Man et al. (Man et al., 2021) examined the magnitudes and temporal trends of ARHL burden and its influencing factors at the national, regional, and global levels based on data from the Global Burden of Disease Study 2019, which is important in establishing more effective preventive measures and lessening the strain of ARHL.The National Health Service (NHS) is a vital component of a country that works to enhance the health and lives of its people (Kadel et al., 2022;Sinclair, 2022).It increases life expectancy, enhances the quality of life of the population, and better manages mortality caused by various diseases.Roberts et al. (Roberts et al., 2021) suggested that to curb the epidemic of unhealthy aging, a national nutrition roadmap should include essential elements such as improved public health messaging about nutrition and aging, routine screening, and medical referrals for age-related illnesses that can be treated with nutrition prescriptions. Social isolation issues in ARHL patients Social isolation is an objective and quantifiable measure characterized by a limited network of relationships and social connections (Holt-Lunstad et al., 2015).It is associated with falls, re-hospitalization, heart disease, cancer, and nutritional risk (Nicholson, 2012) and increases mortality in older people (Holt-Lunstad et al., 2015).Communication barriers due to hearing loss limit active participation in social and cultural activities, leading to loneliness (Strawbridge et al., 2000) and social isolation (Pronk et al., 2011) Q. Wu et al. supported that using hearing aids/hearing implants could help lessen loneliness, social isolation, and depression. It is well established that exposure to noise results in CS and eventually neuronal degeneration.Studies on humans and animals have demonstrated that CS is the earliest contributor to presbycusis (Lv et al., 2023). It is challenging to assess CS or hidden hearing loss since standard audiological testing is not sensitive enough.It has been suggested that high-frequency audiometry might be utilized for early detection of CS, although more research is still needed to determine the relevant technical details (Lokwani and Prabhu, 2022).Additionally, previous studies attempted to use the auditory brainstem response (ABR) to identify cochlear synaptic illness in people; however, the results were inconclusive, perhaps because of the large variability in the ABR wave-1 amplitude.As a result, a method that uses ABR wave curvature was proposed and examined while ignoring the amplitude of ABR wave 1 to identify cochlear synaptic loss (Bao et al., 2022).This method may help overcome the existing problem of ABR variability and can be used in the clinical diagnosis of hearing disorders. With the ongoing quest for CS treatment, various results have been reported.Tziridis et al. (Tziridis and Schulze, 2022) found that the Ginkgo biloba extract EGb 761 prevented noise damage-induced cochlear ribbon synapse loss as opposed to restoring it.Cassinotti et al. demonstrated that ARHL is caused by an age-related decline in cochlear neurotrophin-3 (Ntf3) expression and Ntf3 supplementation may be a treatment for this common condition (Cassinotti et al., 2022b).Manickam et al. found that macrophages play a role in the healing of IHC synapses after CS caused by noise (Manickam et al., 2023). Oxidative stress. Numerous studies link oxidative stress to presbycusis etiology (Prasad and Bondy, 2020;Alvarado et al., 2021;Fujimoto and Yamasoba, 2019).Early ARHL is specifically induced by chronic inflammation, programmed cell death, and changed antioxidant enzyme levels in the cochlea, as well as decreased activity of Complexes I, II, and IV (Someya et al., 2010;Someya and Prolla, 2010;Menardo et al., 2012;Rousset et al., 2020).Nuclear factor erythroid 2-related factor 2 (Nrf2) -a key regulator of antioxidant capacitytriggers the development of multiple oxidative stress-associated acute and chronic diseases.Thus, it is currently a new strategic goal for the creation and application of medications for hearing protection.Although research on Nrf2-targeted hearing protection is ongoing, it has been mostly conducted in vitro on animals and cells (Li et al., 2021).Interestingly, Choo et al. (Choo et al., 2022) found that atorvastatin administration significantly prevented mitochondrial damage and dysfunction and overproduction of reactive oxygen species and their consequent oxidative stress and intrinsic apoptosis in ARHL.This suggests that atorvastatin is a promising marketed drug candidate for slowing the progression of ARHL.The study explored the effects of repurposing commercially available drug candidates on hearing in aging mice. Pathophysiological alterations in the auditory cortex. Studies have reported that the pathophysiological changes in the auditory cortex are a major factor in the pathogenesis of presbycusis (Jayakody et al., 2018a).Moreover, ARHL modifies the auditory cortex.Guerreiro et al. (Guerreiro et al., 2023) found that compared with younger persons, only older adults with hearing loss showed a reduction in neuronal distinctiveness in the auditory cortex; however, older adults without hearing loss did not show this reduction. However, research into the molecular mechanisms involved in the auditory cortex is in its infant stage.Previously, Binghan Xue et al. (Xue et al., 2023) found that excitatory and inhibitory intralaminar cortical circuits were diminished in aged C57BL/6 J mice compared to young adult mice.Elsewhere, Yue Liu et al. (Liu et al., 2023) showed that Bcl2 deficiency in auditory cortex affected lipid metabolism, thereby disrupting synaptic function and neurodegeneration through in vitro and in vivo tests as well as metabolomics studies.They also found that targeting Bcl2 may be an effective approach to treat ARHL. Recently, extensive research has been directed as discovering the significance of hearing aids on the auditory brain.Stephanie Rosemann et al. (Rosemann et al., 2021) found that wearing hearing aids, even for a brief period of time, could alter the audiovisual integration and functional brain connectivity between auditory and visual cortices (Rosemann et al., 2021).Similarly, Minqian Gao et al. (Gao et al., 2023) discovered that hearing aids promote the reconstruction of the impaired functional connection between the auditory and cognitive cortexes. ARHL and cognitive decline Accumulating evidence has implicated depression and dementia in the development of ARHL (Davis and Smith, 2013), and the three are closely related (Slade et al., 2020b;Chern and Golub, 2019;Jayakody et al., 2018b).Moreover, age-related hearing loss age differences in cognitive function should account for age-related hearing loss to avoid overestimation (Guerreiro et al., 2023).Among cognitive conditions linked to ARHL, Alzheimer's disease (AD) is the foremost common cause of dementia in old individuals (Förster et al., 2022), and dementia is a prevalent problem in this population (Lv et al., 2023). Assessing the cognitive functioning of individuals with ARHL poses challenges.Recent research suggests that multidimensional EEG features could serve as valuable biomarkers for this purpose.In a study by Zhao R et al. (Zhao et al., 2023), the ARHL group displayed a prolonged delay, a reduced alpha-beta rhythm energy ratio, a smaller wavelet packet entropy value, and a significantly lower P300 peak amplitude.The researchers established correlations between these specific indicators and subjective scale findings in the ARHL group, indicating their potential utility in evaluating cognitive capacities associated with ARHL. In older adults, hearing aids may slow cognitive decline.A metaanalysis by Brian Sheng Yep Ye et al. (Yeo et al., 2023a) found that the rate of long-term cognitive decline was reduced by 19 % in individuals with hearing loss using hearing restoration aids.Furthermore, a strong correlation was found between the use of these devices and a 3 % rise in short-term general cognitive test scores.Marcia Regina Cominetti (Stucky et al., 2010) et al. investigated the impact of ARHL on cognitive decline in older persons enrolled in the Irish Longitudinal Study on Aging (TILDA).They also reported that the use of hearing aids lowered the lower risk of cognitive decline. Exploring the link between ARHL and cognitive decline has led to the emergence of new therapeutic concepts.Carola Y. Förster et al. (Cominetti et al., 2023) proposed a hypothesis based on their research, suggesting that amyloid-beta might negatively impact the blood-labyrinth barrier similar to its effects on the blood-brain barrier, contributing to ARHL pathology.To potentially improve cognitive outcomes and potentially prevent the development of AD and related comorbidities, individuals with ARHL might consider treating the condition by replenishing the cochlea with specific neurotrophic factors. ARHL and noise Earlier research discovered that noise produces a stressful atmosphere that intensifies the body's pro-oxidative and pro-inflammatory Q. Wu et al. processes (Tang et al., 2023;Fuentes-Santamaría et al., 2022), which leads to oxidative stress and inflammation, eventually ARHL.Given that acoustic trauma and aging have been shown to cause hair cell loss at the base end of the cochlea, a study showed that human temporal bone specimens had consistent, significant hair cell loss throughout the course of their lifetimes (Kujawa and Liberman, 2019).Similarly, elderly animals raised in calm settings did not exhibit hair cell loss until much later in life, and even then, the loss was minimal.This suggests that aging and noise exposure are important regulators of presbycusis. Bamini Gopinath et al. (Gopinath et al., 2021) analyzed the data on hearing loss and occupational noise exposure from 1923 participants aged 50 years and above.They found that hearing loss had a greater 10year incidence in patients who had previously reported exposure to workplace noise (35.5 %) compared with those who had not (29.1 %).This highlights the need for preventive steps to reduce noise exposure at work are therefore minimize the impact of ARHL. Furthermore, researchers should explore the molecular pathways driving ARHL development in the context of noise to reveal new approaches for patient treatment.Adelaida M. Celaya et al. (Celaya et al., 2021) found that insulin-like growth factor 1 (IGF-1) haploinsufficiency lead to an age-related chronic subclinical proinflammatory state, and increased susceptibility to stressors.This was indicated a more severe degree of noise-induced damage, characterized by increased oxidative stress, apoptosis, and inflammation.Another investigation (Zhang et al., 2021b) showed that the therapeutic benefits of SOD2 overexpression in the treatment of ARHL were mediated by the PI3K and MAPK signaling pathways following noise/kanamycin exposure. ARHL and hearing aids Air conduction hearing aids have been proposed for patients with mild to severe hearing impairment owing to their ability to improve speech comprehension, hearing-specific quality of life, and the general standard of living (Thomas and Völter, 2023).The human brain can be directly affected by hearing aids.Margreet Vogelzang et al. reported that both hearing impairment and the application of hearing aids affect brain activation regions involved in language processing (Vogelzang et al., 2021).These effects can be detected by examining complicated sentences, instead of basic ones.Furthermore, as was previously mentioned (Yeo et al., 2023b), hearing aids can improve short-term and long-term cognition. Several factors have been reported to influence the outcomes of using hearing aids in community-dwelling individuals with dementia and hearing loss (Hooper et al., 2022).These include: degree of hearing aid handling proficiency, experiential consequences of patients, degree of hearing aid comfort or fit, interactions between patients and environment and social reinforcement for patients. Hearing aids are beneficial for individuals with hearing impairment.However, a study conducted in 2023 (Chibisova et al., 2023) indicates that 85 % of the global population may still lack access to necessary hearing aids.In mainland China (Zheng et al., 2023), this disparity is attributed to factors such as the reluctance of older individuals with hearing loss to seek treatment, the perception that hearing aids are unnecessary, and prevalent negative attitudes and misconceptions about their efficacy.This situation highlights China's challenges in terms of health literacy and insufficient infrastructure for providing hearing healthcare.Conversely, in the United States, affordability stands out as a significant barrier to the accessibility of hearing healthcare, as emphasized by Anna Marie Jilla et al. (Jilla et al., 2023) Although the mechanisms by which hearing aids affect the human brain have been investigated, and many hearing aids have been updated to meet the needs of patients, some patients do not have access to such aids, which deserves further attention from the government and society. Practical implication The study highlights two main aspects of practical significance: (1) Researchers can efficiently acquire essential knowledge about agerelated hearing loss (ARHL) by reading this paper.They will gain insights into the research hotspots and trends in this field, significantly enhancing their work.This focus on emerging and valuable scientific questions will help reduce the waste of effort and resources caused by the repetitive exploration of well-studied topics.(2) Through keyword burst analysis and keyword co-occurrence analysis, policymakers can intuitively grasp current research trends and unresolved issues related to ARHL.This enables them to develop targeted policies and allocate financial resources effectively to address the challenges in this field, which is crucial for improving the standard of living for older adults.For example, laws can be amended to increase penalties for noise-producing businesses and individuals to reduce noise damage at the source.Additionally, more funds can be allocated to provide hearing aids to older adults in need, enhancing the availability of these devices.Furthermore, investment can be increased in promising scientific studies to accelerate research in this area. Limitations This study has some limitations specific to the nature of bibliometrics that need to be mentioned.Firstly, we only included studies from the WoSCC database which may have caused bias and inaccurate results.Furthermore, only articles published in English were included, which may mean non-English writing papers were underestimated, thus our conclusions might not be all-inclusive.Nevertheless, considering the large amount of data generated by numerous publications included in this study, this bias should be minimal.Finally, because WoSCC data is constantly updated, our search results may not match the current body of literature.In future, modifications should be implemented to overcome these constraints. Conclusion The surge in publications within the last five years underscores the increasing global interest in ARHL.Notably, research on ARHL is predominantly led by the United States.Various prominent themes have emerged as this research area has evolved, with "cochlear synapses," "oxidative stress," and "dementia" currently standing out as significant focal points.Moreover, new avenues of study are emerging in the realms of "public health" and "individual differences."In summary, this pioneering bibliometric analysis provides unbiased and systematic insights into recent ARHL research over the past five years, serving as a valuable reference for future investigations in this field. Research projects of provincial universities and colleges in Heilongjiang Province (2023-KYYWF-0157) and the Doctoral Fund of the First Affiliated Hospital of Harbin Medical University (2024B08).Tianyu Ma receives funding from Heilongjiang Provincial Nature Foundation (YQ2022H013).We would like to thank all the reviewers who participated in the review, especially MJEditor (www.mjeditor.com),for its linguistic assistance during the preparation of this manuscript. Fig. 7 . Fig. 7. A. Top 10 high-frequency keywords on ARHL from 2019 to 2023.B. Clustering co-occurrence map of keywords.C. Keyword distribution based on the average appearance time.D. Top 30 keywords with the strongest citation bursts. Johns Hopkins University.Meanwhile, Lin FR ranked first in total citations, with 11,788 citations, followed by Dubno JR (n = 6618) and Humes LE (n = 5116).The influence of authors based on the number of publications, citations, and H-index was also assessed, these authors were the most influential scholars in ARHL research.The annual output of the top 10 authors between 2019 and 2023 is displayed in Table1. Fig. 2. Annual distribution of ARHL-related articles and cumulative citations from 2019 to 2023.Q.Wu et al.of Table 1 Top 10 productive authors from 2019 to 2023. Table 2 Ranking of top-10 institutions from 2019 to 2023. Table 3 Top 10 most productive countries/regions from 2019 to 2023. Q.Wu et al. Table 4 Ranking of top-10 journals from 2019 to 2023. Q.Wu et al.	2024-06-29T06:17:18.967Z	2024-06-25T00:00:00.000	{ "year": 2024, "sha1": "f76a29f9f8743159705ecd66bcb491e28eeec526", "oa_license": "CCBYNC", "oa_url": "https://doi.org/10.1016/j.exger.2024.112489", "oa_status": "HYBRID", "pdf_src": "ScienceParsePlus", "pdf_hash": "988f17210e743ab969d773dfb4a4c38ea4e0408e", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
42438773	pes2o/s2orc	v3-fos-license	Wild-type p53-mediated induction of rat mdr1b expression by the anticancer drug daunorubicin. The expression of P-glycoproteins encoded by the mdr gene family is associated with the emergence of the multidrug resistance phenotype in animal cells. mdr expression can be induced by many extracellular stimulants including cytotoxic drugs and chemical carcinogens. However, little is known about the mechanisms involved. Here, we report that the expression of the rat mdr1b can be induced by anticancer drug daunorubicin. Further analysis identified a bona fide p53-binding site spanning from base pairs -199 to -180 (5'-GAACATGTAGAGACATGTCT-3') in the rat mdr1b promoter that is essential for basal and daunorubicin-inducible promoter activities. In addition, our results show that wild-type p53 can up-regulate not only the promoter function but also endogenous expression of the rat mdr1b. To the best of our knowledge, this is the first report showing that a specific p53-binding site is involved in the transcriptional regulation of mdr gene by wild-type p53. Since p53 is a sensor for a wide variety of genotoxic stresses, our finding has broad implications for understanding the mechanisms involved in the inducible expression of mdr gene by anticancer drugs, chemical carcinogens, UV light, and other DNA-damaging agents. Multidrug resistance (MDR), 1 a major obstacle to the effective chemotherapy of many human malignancies, is characterized by the increased survival of cells in the presence of cytotoxic drugs with unrelated structures. A major mechanism for the development of MDR phenotype is overexpression of Pglycoproteins which are encoded by the MDR gene family (for reviews, see Refs. 1 and 2). The MDR gene family contains two members in humans and three in rodents. However, only one human (MDR1) and two rodent (mdr1a and mdr1b) mdr genes are functionally related to the MDR phenotype. High mdr mRNA levels are seen in certain tumor types before chemotherapy and, in some cases, are associated with relapse following chemotherapy (for reviews, see Refs. 1 and 3). Increased mdr gene expression occurs in cultured cells selected by continuous exposure to both anticancer drugs and other cytotoxic agents, in which gene amplification is believed to be often associated with the overexpression of mdr genes (4,5). However, increased mdr gene expression preceding gene amplification has been observed in early passages of drugselected cells (6). Transient exposure of cells to different cytotoxic agents such as antitumor drugs (7)(8)(9)(10), chemical carcinogens (11)(12)(13)(14)(15)(16)(17)(18)(19), and UV irradiation (20), etc. is also able to activate mdr expression, indicating that increased mdr expression is mediated by complex mechanisms. The precise mechanisms of the induction of mdr gene expression by anticancer drugs, chemical carcinogens, UV, and other DNA-damaging agents remain unknown. It has been suggested that both post-transcriptional and transcriptional mechanisms are involved (7). A possible role for the cytoskeleton in posttranscriptional stabilization of mdr1 mRNA in rat hepatocytes treated with certain agents was suggested (21). On the other hand, in rat liver cells, it was found that doxorubicin-mediated mdr1 mRNA induction was fully inhibited by actinomycin D, suggesting that transcriptional regulation is involved (10). Nuclear run-off and transfection analyses showed that AAF-, methylcholanthrene-, aflatoxin B1-, methyl methanesulfonate-, or mitoxantrone-induced mdr1 expression is also associated with increased rates of transcription (9,11,15). Here, we show that the expression of the rat mdr1b can be induced by anticancer drug daunorubicin. Further analysis demonstrates that a bona fide p53-binding site (5Ј-GAACATG-TAGAGACATGTCT-3Ј) located within bp Ϫ199 to Ϫ180 of rat mdr1b promoter is essential for not only basal but also daunorubicin-inducible promoter functions. We also provide evidence indicating that both the promoter activity and endogenous expression of the rat mdr1b could be modulated by wild-type p53. Although the modulation of mdr expression by either mutant or wild-type p53 has been noted, no p53-binding sites have been identified previously (22)(23)(24)(25)(26)(27). The present report represents the first evidence that a specific p53-binding site is involved in the transcriptional regulation of the mdr gene. Since p53 is responsive to a variety of genotoxic stresses (for reviews, see Refs. 28 and 29), which also induce mdr gene expression, our finding has important implications for understanding mechanisms involved in the inducible expression of drug-resistant genes by DNA-damaging agents. Cell Culture, DNA Transfection, and Chloramphenicol Acetyltransferase (CAT) Assay-The rat hepatoma H-4-II-E cells were purchased from the American Type Culture Collection (ATCC 1548). Human osteosarcoma SAOS-2 cells, low-passage rat embryonic fibroblasts (REFs), A1-5 cells, and T101-4 cells were generously provided by Dr. G. Lozano. All the cell lines were maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal calf serum (Life Technologies, Inc.), 1 mM glutamine, and 50 g of neomycin/ml in a humidified incubator containing 5% CO 2 . Prior to treatment, cells were grown in the medium to 70 -80% confluence. Then cells were treated with daunorubicin (7 g/ml) for various periods of time and harvested for the preparation of nuclear extracts and RNA. The calcium phosphate precipitation method (33) was used to transfect cells with DNA. In brief, 2 h before transfection, cells in the exponential growth phase (approximately 70 -80% confluence) were plated in Corning six-well plates. DNA-CaPO 4 precipitate was added to the medium and incubated for 5-6 h. After cells were shocked with 15% glycerol for 30 s, washed with phosphate-buffered saline, cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Stable transfectant A1-5 and H-4-II-E cells were established by co-transfecting the cells with the reporter constructs and pcDNA3 (Introgen, Carlsbad, CA) at 5:1 ratio followed by selection with G418 (400 g/ml, Life Technologies, Inc.). Pools of G418-resistant cells were collected and used for further analysis. In transient transfection assays, cells were directly treated with daunorubicin (7 g/ml) 24 h after transfection. After 20 -24 h of drug exposure, cells were harvested. CAT activities in the cell extracts were measured by a previously described method (34) using total protein extract (measured by the Bio-Rad protein assay kit) as a reference. Relative CAT activity levels were calculated by a PhosphorImager (model 400S, Molecular Dynamics) in terms of the conversion of [ 14 C]chloramphenicol into acetylated chloramphenicol. Preparation of Nuclear Extracts and GMSA-Nuclear extracts were prepared from H-4-II-E cells by the method of Digman et al. (35) with modifications as described previously (31). GMSAs were performed with approximately 5 g of nuclear proteins in a total volume of 20 l of binding mixture containing 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.5 mM dithiothreitol, 10% glycerol, 0.2% Nonidet P-40, 3 g of poly(dI-dC)⅐poly(dI-dC), and radiolabeled DNA probe at room temperature for 20 min. RNase Protection Assay-A 162-nucleotide antisense RNA probe (Ϫ37 to ϩ125) was synthesized using T7 RNA polymerase as described previously (30). Of total RNA from cells, either 20 g (for the mdr1b probe) or 1 g (for the 18 S rRNA probe) was hybridized with 32 Plabeled antisense RNA probes (2 ϫ 10 5 cpm) and subjected to RNase protection assays as described previously (17,30). The protected RNA products were analyzed on a 7% denaturing polyacrylamide gel and quantified using a Personal Densitometer SI (Molecular Dynamics). Reverse Transcriptase-PCR Amplification and DNA Sequencing-Two micrograms of total RNA isolated from H-4-II-E cells was used for reverse transcriptase reaction. On completion of the reverse transcriptase reaction, the enzyme was inactivated by heating to 94°C for 56 min. Ten picomoles of each 5Ј primer (5Ј-CCTGAAGACTGGATA-ACTGTCATGGAGGAT) and 3Ј primer (5Ј-AGAGGGGGCCGAGTAC-TATCTACAAGGTAA) were used in a PCR to amplify rat p53 cDNA (30 cycles of 1 min at 94°C, 2 min at 45°C, and 3 min at 72°C). PCR products were electrophoresed on an agarose gel, purified, and subjected to automated sequencing (ABI PRISM). Daunorubicin Induces mdr1b Expression in Rat Hepatoma Cells-To investigate whether the expression of the rat mdr1b gene expression is regulated by the anticancer drug daunorubicin, we treated rat hepatoma H-4-II-E cells with daunorubicin (7 g/ml). At various time intervals, cells were harvested and mdr1b mRNA levels were measured by the RNase protection assay. As shown in Fig. 1, the steady-state mdr1b mRNA levels in these cells were elevated after exposure to daunorubicin for 12 and 24 h. Increases of about 3-5-fold were seen in three independent experiments. Similar results were obtained in cells treated with adriamycin and chemical carcinogen 2-AAAF (data not shown). These results demonstrated that rat mdr1b expression can be induced by these cytotoxic agents in rodent cells. Rat mdr1b Promoter Responds to Daunorubicin Treatment-To investigate the possible involvement of transcriptional regulation in the induction of the rat mdr1b gene expression by daunorubicin and, if so, to identify DNA sequences responsible for the daunorubicin induction of mdr1b expression, we generated a set of 5Ј deletion mutant CAT constructs and transfected them into H-4-II-E cells following treatment with or without daunorubicin. When Ϫ1288 RMICAT, Ϫ243 RMICAT, and Ϫ214 RMICAT reporter constructs containing 1288, 243, and 214 bp of the rat mdr1b upstream sequences, respectively, plus 125 bp downstream from the transcription start site, were transiently transfected into H-4-II-E cells, CAT activities increased an approximately 1.6 -1.9-fold in daunorubicin treated versus untreated cells (p Ͻ 0.05) (Fig. 2). However, when Ϫ163 RMICAT, which contains additional deletion to Ϫ163 bp was transfected, basal transcriptional activities were reduced more than 80%. More importantly, the deletion also abolished daunorubicin inducibility (Fig. 2). Together, RNase protection analyses of rat mdr1b transcripts in H-4-II-E cells. Cells were treated with daunorubicin (7 g/ml) for different times as indicated. RNA was extracted and then subjected to RNase protection assays as described under "Materials and Methods." An 18 S rRNA probe was used as a reference. The autoradiograph is representative of results from three independent experiments. p53-mediated Regulation of Rat mdr1b Expression these results indicated that the rat mdr1b promoter can respond to daunorubicin treatment and that the sequence from bp Ϫ214 to Ϫ163 is essential for the promoter's daunorubicin responsiveness. We previously identified a NF-B-binding site (bp Ϫ167 to Ϫ158) involved in basal and insulin-induced promoter function (31). Since it was reported that daunorubicin can induce the NF-B activity in human fibrosarcoma HT1080 cells and HL-60 promyelocytes (36,37), it is possible that NF-B was also responsible for the inducible promoter activity of the rat mdr1b by daunorubicin in the H-4-II-E cells. To test this possibility, we transfected Ϫ243 RMICAT-m, in which the NF-B-binding site was mutated (31), into H-4-II-E cells following daunorubicin treatment. As shown in Fig. 2, although basal activity was reduced when compared with the wild-type Ϫ243 RMICAT, Ϫ243 RMICAT-m still retained daunorubicin responsiveness. Besides, we did not observe an obvious increase of NF-B binding activity after daunorubicin treatment using GMSAs (data not shown). These results suggested that NF-B may not be directly involved in the daunorubicin-inducible promoter function of the rat mdr1b in H-4-II-E cells. Ϫ214 to Ϫ177 bp Is Sufficient to Confer mdr1b Promoter Inducibility by Daunorubicin-To further substantiate the above observations, we generated two additional constructs by inserting sequences from bp Ϫ214 to Ϫ127 (containing NF-B site) or Ϫ214 to Ϫ177 (containing no NF-B site), respectively, into a pBLCAT 2 vector containing the tk basal promoter and a CAT reporter gene. These constructs were then transiently transfected into H-4-II-E cells, and CAT expression was measured. As shown in Fig. 3, both constructs are capable of responding to daunorubicin treatment, giving rise to comparable levels of induction, whereas daunorubicin did not have effects on the tk promoter. These results suggested that NF-B site is dispensable for the inducibility of mdr1b promoter, and that the sequence from bp Ϫ214 to Ϫ177 may contain important cis-acting elements responsible for the induction of mdr1b promoter activity by daunorubicin. Daunorubicin Induces Formation of a Specific Protein-DNA Complex Within bp Ϫ201 to Ϫ177-To test whether daunorubicin treatment could induce protein DNA binding at sequences within bp Ϫ214 to Ϫ177, we prepared nuclear extracts from H-4-II-E cells treated with or without daunorubicin and performed GMSAs. As shown in Fig. 4A, a major DNA-protein complex was formed in the daunorubicin-untreated nuclear extracts when a double-stranded oligonucleotide spanning bp Ϫ214 to Ϫ177 was used as the probe (lane 1, C1). The binding activity of this complex remained largely unchanged after daunorubicin treatment (lanes 2-5 versus lane 1). However, a slow migrating protein-DNA complex was induced 1 h after treatment (C2, lane 2). The binding activity of this induced complex remained elevated but gradually reduced throughout the 12-h induction period (lanes 2-5). To further characterize the sequence specificity of this daunorubicin-induced DNA binding activity, double-stranded oligonucleotides covering the left and right regions of bp Ϫ214 to Ϫ177 (fragments A and B, Fig. 4C) were used in competition GMSA. Fig. 4B shows that both the unlabeled probe (lane 2) and fragment B (bp Ϫ201 to Ϫ177) (lane 4) could compete for daunorubicin-induced DNA-protein complex, indicating that the induced protein binding required the sequence residing within fragment B. To further define the binding sequence of the induced protein complex, two site-directed mutant oligonucleotides (M1 and M2) containing mutations on 5Ј-or 3Ј-end of fragment B, respectively ( Fig. 4C), were used as competitors. However, neither mutated oligonucleotides could compete for daunorubicininduced DNA-protein complex (Fig. 4B, lanes 5-6), suggesting that the daunorubicin-induced protein binding required both the 5Ј-and 3Ј-sequences of fragment B (bp Ϫ201 to Ϫ177). Daunorubicin-induced DNA-binding Protein Is p53-In examining the DNA sequence of bp Ϫ201 to Ϫ177 (fragment B), we found within it a sequence (bp Ϫ199 to Ϫ180) strikingly similar to the p53-binding consensus sequence 5Ј-PuPuPuC(A/ T)(A/T)GPyPyPy-3Ј (38), with only 2 base pair mismatches. A comparison of the putative mdr1b p53-binding site with the p53 consensus sequence and the p53-binding site from the gadd45 third intron (39) is shown in Fig. 5B. To determine whether the sequence located between bp Ϫ199 and Ϫ180 was indeed a p53-binding site, we carried out GMSAs using a double-stranded oligonucleotide spanning bp Ϫ214 to Ϫ177 as the probe and nuclear extracts prepared from daunorubicintreated H-4-II-E cells in the presence of various unlabeled oligonucleotides as competitors. As shown in Fig. 5A To further strengthen this observation, antibodies were used in GMSAs. As shown in Fig. 5A, the daunorubicin-induced protein-DNA complex was supershifed by p53 antibody PAb421 (lane 6), whereas c-Jun and NF-B p65 antibodies did not affect the formation of induced DNA-protein complexes (lanes 7-8). Taken together, these results strongly suggested that the rat mdr1b promoter sequence located between bp Ϫ199 and Ϫ180 (5Ј-GAACATGTAGAGACATGTCT-3Ј) is a p53-binding site. To determine whether the rat p53 in H-4-II-E cells is a wild-type or mutant form, we amplified cDNA copies of the rat p53 by reverse transcriptase-PCR and sequenced it directly (see "Materials and Methods"). The result showed that H-4-II-E cells has a wild-type rat p53 mRNA (data not shown) with a sequence consistent with that published previously (42). p53-binding Site Is Required for the Daunorubicin-inducible promoter Activities-To characterize the functional role of the FIG. 3. Sequences from bp ؊214 to ؊177 confer the inducibility by daunorubicin. H-4-II-E cells were transfected with Ϫ214/Ϫ127 tk-CAT and Ϫ214/Ϫ177 tk-CAT constructs, and CAT activity was assayed as described under "Materials and Methods." Values shown are the averages for three representative experiments in which each transfection was performed in duplicate. S.D. values are represented by the bars. 2 g of DNA was used in each transfection in the absence or presence of daunorubicin (7 g/ ml), and the extracts were normalized to the same protein concentration. In the schematic diagram, the position of NF-B-binding site is indicated. FIG. 4. Induction of a protein-DNA binding complex by daunorubicin. A, GMSA using 5 g of nuclear extracts from H-4-II-E cells treated with or without daunorubicin (7 g/ml) for the indicated times. Extracts were assayed for binding to the labeled double-strand oligonucleotide (bp Ϫ214 to Ϫ177) shown in panel C. Note that a new DNA-protein complex (C2) was induced in daunorubicin-induced nuclear extracts (lanes 2-5). B, GMSA using nuclear extracts prepared from H-4-II-E cells treated with daunorubicin (7 g/ml) for 6 h. Extracts were assayed for binding to the labeled double-stranded Ϫ214 to Ϫ177 oligonucleotide in the presence or absence of an 100-fold molar excess of the indicated competitors shown in panel C. The autoradiographs are representative of the results from three independent experiments. C, the mdr1b promoter sequence from bp Ϫ214 to Ϫ177 and oligonucleotides used for competition in panel B. In oligonucleotides, mutated bases are indicated in boldface. FIG. 5. Identification of a p53-binding site in the rat mdr1b promoter. A, GMSA using nuclear extracts prepared from H-4-II-E cells treated with daunorubicin (7 g/ml) for 6 h. Extracts were assayed for binding to the labeled double-strand oligonucleotide (bp Ϫ214 to Ϫ177) in the presence or absence of an 100-fold molar excess of the indicated competitors (lanes 2-5), p53 (PAb421), anti-c-Jun, and anti-p65 antibodies (lanes 6 -8), respectively. Note that the slower migrating complex was completed by gadd45 p53-binding sequence (lane 2) and supershifted by PAb421 (lane 6). The autoradiograph is representative of the results from three independent experiments. B, comparison of the sequence from bp Ϫ199 to Ϫ180 with the p53 consensus sequence and the p53-binding site from the gadd45 third intron. Two mismatch base pairs are indicated in lowercase. In the schematic diagram, P represents G or A; W represents A or T; Y represents T or C. p53-mediated Regulation of Rat mdr1b Expression identified p53-binding site, the same mutations in Fig. 4C were introduced within the context of the wild-type Ϫ214 RMICAT construct, and resultant recombinants were designated Ϫ214 RMICAT-m1 and Ϫ214 RMICAT-m2. These mutant constructs were then transfected into H-II-4-E cells following treatment with or without daunorubicin. As shown in Fig. 6A, both mutations abolished the daunorubicin responsiveness. Similar results were obtained when the same mutations were introduced into heterologous (tk) promoter constructs (Fig. 6B). These results suggested that the integrity of p53 binding is essential for the daunorubicin inducible-promoter function of the rat mdr1b. It has been reported that promoters containing p53-binding sites, in some cases, essentially showed no obvious DNA damage responsiveness in transient transfection assays after the treatment of UV or other DNA-damaging agents, whereas higher levels of the induction of the same reporters were seen in stable transfectants (43). Consistent with these observed only low levels of inductions of mdr1b CAT activities by daunorubicin were observed in our transient transfection assays (Figs. 2, 3, 6, A and B). To test whether the rat mdr1b promoter can respond to daunorubicin more dramatically in stable transfectants than in transient transfected cells, we stably transfected both Ϫ214 RMICAT and Ϫ214 RMICAT-m1 into H-4-II-E cells. As expected, wild-type CAT reporter (Ϫ214 RMICAT) exhibited more significant responsiveness to daunorubicin (4-fold, Fig. 6C), which is comparable with the induction levels of mdr1b mRNA by daunorubicin (Fig. 1). As a control, Ϫ214 RMICAT-m1 in stably transfected H-4-II-E cells failed to respond to daunorubicin (Fig. 6C). These results further strengthened the notion that the p53-binding site is required for the promoter's daunorubicin responsiveness. Why the fold induction is different between transient and stable transfectants is unclear but could be due to the participation of chromatin proteins or structure in p53-mediated gene expression, since studies have indicated that transiently transfected DNA, unlike stably transfected templates, are not efficiently packed into chromatin (44). Consistent with this finding, a recent report showed that high mobility group protein-1, an important component of chromatin, is a coactivator of p53 (45). In another set of experiments, wild-type p53 (pCMVp53) or mutant p53 (pCMVp53 248 ) expression vectors were co-transfected with reporter constructs into SAOS-2 cells, which contain a homozygous deletion at the p53 gene locus and do not produce a p53 protein (47). As shown in Fig. 7B, co-transfection FIG. 6. Requirement of p53-binding site for both basal and daunorubicininducible promoter activities. A, CAT assays of wild-type Ϫ214 RMICAT, mutants Ϫ214 RMICAT-m1, and Ϫ214 RMI-CAT-m2 which contain mutant p53-binding sites (see schematic at left) were transiently transfected into H-4-II-E cells following treatments with or without daunorubicin (7 g/ml). In the schematic diagram, mutated bases are indicated in boldface. B, CAT assays of Ϫ214/Ϫ177 tk-CAT wild-type and mutant constructs after transient transfection into H-4-II-E cells following treatment with or without daunorubicin (7 g/ml). Mutated bases (indicated by X) are the same as shown in panel A. 2 g of DNA was used in each transfection. Results shown are the averages for three representative experiments after normalization to the protein concentration of the cellular extracts. S.D. values are represented by the bars. C, CAT assay of Ϫ214 RMICAT and Ϫ214 RMI-CAT-m1 after stable transfection into H-4-II-E cells in the presence (ϩ) or absence (Ϫ) of daunorubicin (Dau) (7 g/ml). The autoradiograph shown is a representative of the results from one of three independent pools for each stable Ϫ214 RMICAT and Ϫ214 RMICAT-m1 cell line. Fold induction refers to that in transfectants not treated with daunorubicin. p53-mediated Regulation of Rat mdr1b Expression of pCMVp53 trans-activated CAT activity in cells transfected with the wild-type reporter (Ϫ214 RMICAT) but not in cells co-transfected with reporters containing mutated p53 site (Ϫ214 RMICAT-m1 or Ϫ214 RMICAT-m2). Moreover, co-transfection of mutant p53 expression vector also failed to activate wild-type as well as mutant reporters (Fig. 7B). Similar results were obtained when heterologous reporter constructs (Ϫ214/ Ϫ177 tk-CAT and mutant Ϫ214/Ϫ177 tk-CAT-m2) were used in co-transfection assays (Fig. 7C). These results, collectively, demonstrated that wild-type p53 can trans-activate rat mdr1b promoter activity specifically via the identified p53-binding site. Endogenous mdr1b Expression Is Modulated by Wild-type p53-To assess the regulation of endogenous mdr1b expression by p53, we examined mdr1b mRNA levels following temperature shift in A1-5 cells. We reasoned that if the mdr1b is a true p53 target gene, its expression should increase following wildtype p53 induction after temperature shift. As a control, we also measured mRNA levels in REFs and T101-4 cells following temperature shift. REFs has an endogenous wild-type p53, whereas T101-4 cells, like A1-5 cells, are derived from REFs but carry a non-temperature-sensitive p53 mutant (46). RNase protection assays revealed mdr1b mRNA levels increased a 3-6-fold in A1-5 cells after temperature shift from 37°C (mutant p53) to 32.5°C (wild-type p53) (Fig. 8, compare lanes 3 and 4 to 1 and 2). This induction was unlikely due to a nonspecific phenomenon by the temperature change, because no induction of mdr1b expression was observed in control REFs or T101-4 cells (compare lanes 7 and 8 to 5 and 6, and 11 and 12 to 9 and 10). These results indicated that the up-regulation of the mdr1b in A1-5 cells after temperature shift was induced by wild-type p53, and that p53 is indeed capable of modulating endogenous mdr1b gene expression. DISCUSSION In this study, we identified an authentic p53-binding site located from bp Ϫ199 to Ϫ180 of the rat mdr1b that is important for both basal and daunorubicin-inducible promoter activities. We also provided evidence showing that both the promoter function and endogenous expression of the rat mdr1b can be modulated by wild-type p53. A bona fide p53 response gene should fit the following criteria (28): (i) the existence of p53-binding sites that can be specifically recognized by p53; (ii) the ability of these sites to act as a p53 response element, activating basal transcription in a wild-type p53-dependent manner; (iii) the response of the element to p53 in the endogenous genomic promoter context; and (iv) the induction of the target genes after cellular stress, such as DNA damage, in cells containing wild-type but not mutant forms of p53. The results presented in this study suggested that the rat mdr1b meet all these criteria. Therefore, like p21/WAF1, mdm-2, GADD45, cyclin G, bax, and IGF-BP3, etc., the rat mdr1b can be considered as a genuine p53 response gene. Studies on the role of p53 in the regulation of the mdr gene family have been quite controversial. Previous studies have demonstrated that co-transfection with several mutant p53 expression vectors activated the human MDR1 and hamster pgp-1 promoters, whereas co-transfection of a wild-type p53 expression vector had no effect or repressed the promoter activity (22)(23)(24)(25)(26). Yet, no bona fide p53-binding sites were elucidated in these studies. It has been shown that p53 can indeed repress activities of many promoters without specific p53-binding sites (for review, see Ref. 28). The repression usually involves promoters containing the TATA box, presumably through sequestering TATA-binding protein, transcription activating factors, or interacting with other transcriptional activators by p53. Paradoxically, the human MDR1 promoter is a TATA-less promoter, therefore mechanisms involved in the repression of the MDR1 promoter by wild-type p53 are un- FIG. 7. Activation of mdr1b promoter by wild-type p53 but not mutant p53. A, CAT assay of Ϫ214 RMICAT and Ϫ214 RMICAT-m1 after stable transfection into A1-5 cells. Cells cultured at 37°C were either shifted to 32.5°C or continuously cultured at 37°C for 24 h and then harvested for CAT assays. The autoradiograph shown is representative of the results from one of three independent pools for each cell line. Fold induction refers to that in cells cultured at 37°C. B and C, p53-null SAOS-2 cells were transfected with 2 g of wild-type (Ϫ214 RMICAT or Ϫ214/Ϫ177 tk-CAT), p53-binding site-mutated (Ϫ214 RMI-CAT-m1, -m2, or Ϫ214/Ϫ177 tk-CAT-m2) mdr1b promoter reporter alone or in combination with 1 g of wild-type p53 (pCMVp53) or mutant p53 expression vector (pCMVp53 248 ) as indicated. Empty control vector (pCMV) was used to normalize the amounts of the transfected DNA to a total 3 g of DNA in each transfection reaction. Each column represents the mean of relative CAT activities from three independent experiments after normalization to the protein concentration of the cellular extracts. S.D. values are represented by the bars. p53-mediated Regulation of Rat mdr1b Expression known (26). Similarly, it is also unclear how the p53 mutants gain the functions to activate the human MDR1 promoter (24). In addition to repressing it, wild-type p53 was also shown to stimulate the MDR1 promoter in p53-null cell line in a transfection assay (27). The reasons for the discrepancies among these studies are still unknown but there are many plausible explanations: (i) p53 is a multiple functional protein whose functions are regulated by a complex network (48), its regulation of gene expression may differ not only among cell types but also among physiological conditions under which assays are performed; (ii) p53 can also bind transcriptional coactivators such as CBP/p300 (49 -51), which interacts with a battery of other transcriptional regulators such as NF-B, Jun/Fos, nuclear receptors, and their coactivators (for review, see Ref. 52). The abundance of these transcriptional regulators may differ among different cell settings and thereby influence the overall expression of transfected genes; (iii) different p53 expression vectors, mdr reporter constructs, and time of analysis, may affect the overall results. It should also be noted that even the transfection procedures themselves may perturb endogenous p53 levels (53), affecting results of transient transfection assays. These considerations, taken together, may explain the discrepancy of the transfection results described above. In this regard, the identification of an authentic p53-binding site in the mdr1b promoter region as described herein is of particular importance, since it is the first time a specific p53-binding site was elucidated to be implicated in the transcriptional regulation of mdr gene expression. Our observation of the involvement of wild-type p53 but not mutant p53 in the regulation of the rat mdr1b expression may be relevant to the increased expression of the mdr1b during hepatocarcinogenesis. Although the expression of mdr1 is highly activated, mutation of p53 does not always occur during hepatocarcinogenesis, at least in its early stage of liver tumor development (54). In addition, it has been known that the mdr1b expression in rat liver can be rapidly activated by chemical carcinogens such as 2-AAF and aflatoxin B1 (12,13), however, in rat hepatocellular carcinomas induced by these carcinogens, p53 mutations do not always occur (55,56). More importantly, van Gijssel et al. (57) recently reported that p53 activity can be also induced by 2-AAF and aflatoxin B1 in rat liver. When rat hepatoma H-4-II-E cells (contain wild-type p53) were treated with 2-AAAF, p53 activity was also been induced. 2 These results, taken together, suggested that the activation of the rat mdr1b during chemical hepatocarcinogenesis may be due to the elevated wild-type p53 activities. In broader prospects, it has been known that p53 is a universal sensor of genotoxic stress (58), and can be induced by a wide variety of DNA-damaging agents such as UV, ␥-irradiation, carcinogens, and cytotoxic drugs (for reviews, see Refs. 28 and 29). Strikingly, many of these agents are also known inducers of mdr gene expression, suggesting that p53 may contribute to the induction. The p53-binding site identified in this study lies in the previously identified murine mdr1b enhancer region (59). It overlaps a palindromic sequence recognized by two peptides (41 and 49 kDa) (30), and adjoins a downstream NF-B-binding site which is also important for the promoter function (31). It is believed that most inducible cis-acting elements contain multiple, distinct transcription factor-binding sites that are part of a combinatorial mechanism that relies on cooperative binding, interaction of transcriptional activator proteins, and transcriptional synergy (60). Our study of site-directed mutations demonstrated that the full promoter activity of the rat mdr1b requires the integrity of both the p53-binding site (bp Ϫ199 to Ϫ180) and NF-B-binding site (bp Ϫ167 to Ϫ158) (Fig. 2) (31), suggesting that cooperative mechanisms between these two cis-acting elements are implicated in the regulation of the rat mdr1b expression. More recently, coactivator CBP/p300 was shown to interact with both p53 (49 -51) and NF-B (61,62), and enhance p53-and NF-B-dependent transactivation, respectively. The activity of the rat mdr1b promoter was also found to be enhanced by CBP/p300. 2 Taken together, these may suggest that the binding of p53 and NF-B to the mdr1b promoter may recruit CBP/p300 and basal transcriptional machinery to form a higher order transcription enhancer complex, similar to that proposed in interferon-␤ and E-selectin promoters (61,63), which modulates inducible expression of the rat mdr1b. However, since our knowledge is rather limited at this moment, the validity of this model still needs to be further tested. Finally, we would like to stress that, although our present results clearly demonstrated the direct involvement of p53 in the rat mdr1b gene regulation, the roles of p53 in the evolution of drug resistance in cancers remain to be critically evaluated. In clinical setting, the loss of functional p53 has been reported to be well correlated with de novo resistance to radiation and anticancer drugs, and some tumors with wild-type p53 respond well to chemotherapeutic drugs (Refs. 64 -66, for review, see Ref. 29). However, it is unknown whether the correlation of drug resistance and p53 mutations is directly due to the activation of mdr by mutant p53, or other mechanisms such as alterations in drug targets, transporters, metabolisms, or the expression of genes regulating cell death and/or survival. Further studies are required to elucidate the molecular insights into how p53 regulates clinical drug sensitivity in cancer chemotherapy.	2018-04-03T05:21:20.927Z	1998-06-19T00:00:00.000	{ "year": 1998, "sha1": "f3fa4e40acc96bc05901531deee514dae5a98332", "oa_license": "CCBY", "oa_url": "http://www.jbc.org/content/273/25/15387.full.pdf", "oa_status": "HYBRID", "pdf_src": "Adhoc", "pdf_hash": "5a15261b878336beb0efa398598484e76d5fe2ea", "s2fieldsofstudy": [ "Biology", "Medicine" ], "extfieldsofstudy": [ "Medicine", "Biology" ] }
244837935	pes2o/s2orc	v3-fos-license	Numerical and Physical Modelling of the Performance of the Pro-vortex Vanes in Shaft Spillways The paper focuses on the analysis of hydraulic conditions in the proximity of the intake part of high shaft spillways equipped with pro-vortex vanes and discusses recent enhancements in modelling of the shaft spillways and compares the acquired results of the performance of the spillway after complete removal or rehabilitation of the vanes in context of capacity and overall hydraulic conditions. Increasing requirements on safety of embankment dams during floods with respect to anticipated effect of the climate change scenarios on parameters of design floods demand further assessment of capabilities of outlet structures to meet the updated needs. Such dam safety assessments often conclude in the need of designing additional measures to improve existing structures. Despite different approach to the evaluation of the uncertainties and subsequent risk assessment the goal of improving safety of large dams remains consistent in the effort of all developed countries. Adjustments of the intake part of shaft spillways can present a valid design option for increasing capacity of the complex spillway/tunnel structure if supported by solid analysis of hydraulic conditions inside these structures. As the governing idea of the pro-vortex vanes is to ensure spiral flow inside the shaft and to minimize the pressure fluctuations the paper presents results from physical model of several designs of the pro-vortex vanes which approximated possible adjustment of tower like shaft spillway of existing large dam in Czech Republic and also results from CFD modelling illustrating the importance of combination of both modelling approaches. For the CFD part, different turbulence models are discussed. Introduction The shaft spillways often present a technically and economically viable option when considering a design of an outlet structure for large earthfill or rockfill dams. Therefore the hydraulic performance and conditions for different flow regimes near the inlet part, which influence significantly the capacity, has been studied in the past [1], [2] especially with respect to pressure fluctuations of aerated spiral flow in vertical shafts and transition elements. In contrast to the straight spillways for shaft spillways with circular (or another closed shape) the coefficient of discharge depends not only on the actual thickness of the overfalling nappe but also to a ratio of the thickness of the overfalling nappe to the radius of the circular plan [3], [4]. When the cross section of the spillway, i.e. the intake part of the shat spillway, vary from the original shape presented in the literature the effect on discharge coefficient should be evaluated using physical or numerical models especially in case where local morphological conditions lead to shapes allowing negative pressures [5]. The transition between free flow and submerged region at the inlet part is accompanied with the creation of a vortex which can be stable or unstable based on the local conditions and influence of additional elements near the intake part such as pro-vortex vanes. The transition process is also accompanied with significant pressure fluctuations which might induce unwanted loading on the shaft body and adjacent structures. Especially for dams with clay core, the potential of increased seepage along the shaft or tunnel and subsequent internal erosion evolution is considered unacceptable. When assuming symmetrical inflow conditions, i.e. without influence of the reservoir topology or position of the intake part with respect to dam or banks, and no streamlining elements, the flow in the shaft can be considered axial in free-flow and transition regions, while unstable vortex will arise under submerged conditions. Ensuring stable spiral flow decreases the pressure fluctuations [6] at the cost of decreasing capacity which on the other hand is unacceptable and prevented by anti-vortex elements for spillways passing through solid rock. The applicability of physical modelling using Froude's model law and the limits was tested and successfully verified on scaled models by Sikora [7] who also described the coefficient of aeration of the pressure flow in outlet tunnels from shaft spillways. Decreasing calculating costs, led to significant progress in numerical simulations in the capacity curve estimation [8]. The Two-phase CFD simulations using RANS method with k-e SST turbulence model and non-homogeneous velocity field can provide sufficiently accurate results [9], also presented in this paper. The remaining issue is selection of correct boundary conditions with respect to reservoir topology, complete geometry of the spillway combined with still demanding calculation costs under unsteady state condition. Assuming steady state, the of pressures fluctuations and possible periodic loading cannot be identified nor quantified as shown in [10] but as the periodic loading with potentially dangerous frequencies can occur during certain discharge range, the inherently unsteady physical modelling cannot be omitted. Physical model A physical model of a real shaft spillway structure was constructed in the laboratory in order to study the performance of different design and setup of streamlining elements. In this paper the spillway of Jirkov dam in Czech Republic was used as prototype for laboratory model scaled 1:20. The outlet structure of this 55.6 m high rockfill dam with a clay core consists of tower like shaft spillway and two bottom outlets 800 mm in diameter each. The spillway consists of circular (9.4 m in diameter) nappe shaped overflow structure -the inlet part, with four very unusual pro-vortex vanes, followed by vertical shaft (3 m in diameter), a sharp bend, short lowered section, aerated sudden raise of the ceiling profile and an outlet tunnel with 2% bed slope ensuring free-surface supercritical flow conditions. The bottom outlets discharge behind plunger valves into the lowered section of the outlet tunnel from shaft spillway just before the sudden raise of the flow profile. The cross section of the inlet part is non-vacuum free shaped overflow section for nappe thickness 0.5 -1.1 m. The current pro-vortex vanes are only 0.4 high but 9.0 m long, see following figures. In order to study the effect of the complex structure the laboratory model was constructed including the outlet tunnel and several meters of the channel downstream of the dam toe. Furthermore new potential designs of pro-vortex vanes with more common shape were tested for pressure fluctuations and hydraulic performance. Tested alternatives Apart from the current setup with four specific vanes following alternatives were tested. Table 1. Setup alternatives tested at the laboratory model Alternative description Alternatives Var 4a, 4b, 5a and 5b were tested also with the hydraulically smooth transition added at the bottom of the shaft and are denoted " + smooth bend" further in the text. Measurement The experiments were conducted under the steady inflow into the large upper reservoir in the laboratory for 10 different discharges corresponding with floods with different reoccurrence intervals from Q1 to Q10000 or full capacity, i.e. submerged regime at the intake part. The model was equipped with 16 pressure probes with measuring frequency 1 Hz to 1 kHz. Four probes were positioned along the bottom part of the vertical shaft and 6 probes on the bottom just downstream of the bend. The discharge was measured by induction flowmeter at the inlet pipe to the upper reservoir. Also the water level was measured in the reservoir and along the outlet tunnel and in the channel downstream. The pressure fluctuations were assessed from two points of view. Firstly, the fluctuations were assesed with respect to the probability of exceedance for particular discharge. It is important to stress out, that the results vary significantly along the shaft and the width of the outlet tunnel bottom. Therefore, the comparison must take into account position of the probe, overall magnitude of the pressure fluctuation and the probability of occurrence of the particular discharge conditions. Secondly, the possible oscillations caused by fluctuations with dominant frequencies were analyzed as such loading can potentially damage the structure and increase the risk of internal erosion. Numerical modelling The aim of the numerical model was to analyse the discharge -water level relationship under different mesh density and turbulence model. The results were compared with the physical model and results commented for intake part without streamlining elements. ANSYS CDF with RANS method and k-e and Shear Stress Transport (SST) turbulence models were used for numerical modelling. The non-structured mesh generated by the Delaunay algorithm with significant refinement around the intake part, boundary of the domain and water surface was applied for discretization of the model. Axisymmetric geometry of the spillway was taken into account and the 3D model was created by rotating cross section, see figures 4 and 5 . Boundary conditions (BC) and turbulence models parameters To represent the inflow into the solved domain the Inlet-Total Pressure BC with defined hydrostatic (depth governed) distribution was used. The free water level surface was defined via Opening pressure BC with average relative pressure 0 kPa, which allows for water level variations. Results and discussions Following figures present selected results from both numerical and physical model. At first the problem of flow regimes and their boundaries was analysed from the physical model. It is clear from the following rating curve of the outlet structure, that while the relatively higher hydraulic smoothness of the improved bend can be seen to have rather positive impact in terms of capacity and submergence occurrence, the streamlining elements reduce the capacity in the free-flow region by approximately 4 -5%. Also the current pro-vortex vanes cause earlier transition to the submerged regime. The newly designed pro-vortex vanes in Var 5b setup, however, allow for higher discharges before transition into submerged regime occurs. The figure 8 presented above clearly demonstrates the effect of streamline elements, even the atypical current ones, on the pressure fluctuation. Also the effect of the hydraulically smooth transition element at the bottom of the shaft is well demonstrated. The removal of the current pro-vortex vanes would lead to increased capacity, se figure 7, but without implementing hydraulically smooth bend, the pressure fluctuations will rise. Implementation of more typical design of pro-vortex vanes will ensure spiral flow mode along the shaft and significantly reduce the pressure fluctuations even further. There is, however, a risk of oscillations. As presented in figure 9, some of the probes at certain discharges shown dominant frequencies and therefor a significant dynamic loading on the structure. It should be stress out that the FFT analysis only discovered dominant frequencies at certain probes and only for very high discharges which are rather unlikely to occur during the remaining service life of the dam. The benefit of the lowered fluctuations is evident event for discharges with higher probabilities of occurrence, e.g. Q20. The results from the numerical models as presented in figure 10 demonstrate good agreement between both physical and numerical model. The geometry scaling effect was insignificant and the difference between turbulence models is approximately 10% with physical model results directly between different turbulence models. The numerical calculations were still rather demanding and it is quite clear that gaining similar pressure fluctuation results for all tested alternative setups and discharges would present an enormous task if compared with relatively less time consuming experiments in the laboratory due to inherently unsteady flow conditions. Conclusions Specific local conditions and design decisions lead to construction of complex outlet structures for which the general recommended values of discharge coefficients are not applicable. The hydraulic interaction between the inlet part, shaft and outlet tunnels also presents a challenge when assessing the capacity curves without help of modelling and can end up with significant under or overestimation of the spillway capacity thus adding undesirable uncertainties to the safety assessment. Physical modelling of the shaft spillways remains a valid option for description of hydraulic conditions in the entire outlet structure system from both time and economy point of views despite recent computing capabilities. The physical model allows analysis of the results of any topological adjustments in a real time, which is especially useful for optimization of shape of designed elements, while every change in topology of the numerical model requires new calculation process. The limits of the physical models are well defined and the numerical models can provide answers to those questions beyond such limits.	2021-12-03T20:09:35.817Z	2021-11-01T00:00:00.000	{ "year": 2021, "sha1": "038b1ea95c18beaacc2eda9f41ed58a4144d9c1b", "oa_license": null, "oa_url": "https://doi.org/10.1088/1757-899x/1203/3/032082", "oa_status": "GOLD", "pdf_src": "IOP", "pdf_hash": "038b1ea95c18beaacc2eda9f41ed58a4144d9c1b", "s2fieldsofstudy": [ "Engineering", "Environmental Science" ], "extfieldsofstudy": [ "Physics" ] }
198901506	pes2o/s2orc	v3-fos-license	Relativistic redshift of the star S0-2 orbiting the Galactic center supermassive black hole General Relativity predicts that a star passing close to a supermassive black hole should exhibit a relativistic redshift. We test this using observations of the Galactic center star S0-2. We combine existing spectroscopic and astrometric measurements from 1995-2017, which cover S0-2's 16-year orbit, with measurements in 2018 March to September which cover three events during its closest approach to the black hole. We detect the combination of special relativistic- and gravitational-redshift, quantified using a redshift parameter, $\Upsilon$. Our result, $\Upsilon=0.88 \pm 0.17$, is consistent with General Relativity ($\Upsilon=1$) and excludes a Newtonian model ($\Upsilon=0$ ) with a statistical significance of 5 $\sigma$. General Relativity predicts that a star passing close to a supermassive black hole should exhibit a relativistic redshift. We test this using observations of the Galactic center star S0-2. We combine existing spectroscopic and astrometric measurements from 1995-2017, which cover S0-2's 16-year orbit, with measurements in 2018 March to September which cover three events during its closest approach to the black hole. We detect the combination of special relativistic-and gravitational-redshift, quantified using a redshift parameter, Υ. Our result, Υ = 0.88 ± 0.17, is consistent with General Relativity (Υ = 1) and excludes a Newtonian model (Υ = 0 ) with a statistical significance of 5 σ. General Relativity (GR) has been thoroughly tested in weak gravitational fields in the Solar System (1), with binary pulsars (2) and with measurements of gravitational waves from stellarmass black-hole binaries (3,4). Observations of short-period stars in our Galactic center (GC) (5)(6)(7)(8)) allow GR to be tested in a different regime (9): the strong field near a supermassive black hole (SMBH) (10,11). The star S0-2 (also known as S2) has a 16 year orbit around Sagittarius A* (Sgr A), the SMBH at the center of the Milky Way. In 2018 May, it reached its point of closest approach, at a distance of 120 astronomical units (au) with a velocity reaching 2.7% of the speed of light. Within a 6 months interval of that date, the star also passed through its maximum (March) and minimum velocity (September) along the line-of-sight, spanning a range of 6000 km s −1 in radial velocity (RV - Fig. 1). We present observations of all three events and combine them with data from 1995-2017 (Fig. 2). During 2018, the close proximity of S0-2 to the SMBH causes the relativistic redshift, which is the combination of the transverse Doppler shift from special relativity and the gravitational redshift from GR. This deviation from a Keplerian orbit was predicted to reach 200 km s −1 (Fig. 3) and is detectable with current telescopes. The GRAVITY collaboration (9) previously reported a similar measurement. Our measurements are complementary: i) we present a complete set of independent measurements with 3 additional months of data, doubling the time baseline for the year of closest approach, and including the third turning point (RV minimum) in September 2018, ii) we use three different spectroscopic instruments in 2018, which allows us to probe the presence of instrumental biases, iii) we perform an analysis of the systematic errors that may arise from an experiment spanning over 20 years to test for bias in the result, and iv) we publicly release the stellar measurements and the posterior probability distributions. We use a total of 45 astrometric positional measurements (spanning 24 years) and 115 RVs RV measurements were obtained from the W. M. Keck Observatory, Gemini North Telescope, and Subaru Telescope. All our RV observations were taken using AO. We supplement our observations with previously reported RVs from Keck from 2000 (7) and the Very Large Telescope (VLT) from 2003-2016 (8). This work includes data from a total of 2 imaging instruments and 6 spectroscopic instruments (13). We scheduled our 2018 observations using a tool designed to maximize the sensitivity of the experiment to the redshift signal (13). Using this tool, we predicted that, given the existing data , spectroscopic measurements at the RV maximum and minimum in 2018 would provide the most sensitivity to detect the relativistic redshift (see Fig. 3). While they are less sensitive to the effect, imaging observations of the sky position of S0-2 in 2018 also slightly improve the measurement of the relativistic redshift. The RVs of S0-2 are measured by fitting a physical model (which includes properties of the star such as its effective temperature, surface gravity, and rotational velocity in addition to RV), to its observed spectrum (13). The same procedure is applied to the new and archival observations; in the latter case this spectroscopic method improves the precision by a factor of 1.7 compared to previous analyses (14,15). We also characterized additional sources of uncertainties beyond the uncertainties in the fit- ii) Re-examination of the spectroscopic data showed that one spectroscopic instrument had additional systematic bias from its optical system, which resulted in a systematic offset in RV compared to other instruments. We include an RV offset parameter in the orbit fit to account for this systematic offset. iii) We assessed systematic uncertainties by observations of bright RV standards stars of the same spectral-type as S0-2 (Table S3). This systematic error is 1.3±1.2 km s −1 , smaller than the statistical uncertainties and about 6 times smaller than previous RV observations of S0-2 (15). When these sources of systematic error are included in the analysis, the average RV uncertainty of S0-2 is found to be 20 km s −1 for the Keck and Gemini observations. The astrometric positions of S0-2 with respect to Sgr A are placed into a common absolute astrometric reference frame using a multi-step cross-matching and transformation process. We adopted an improved methodology for obtaining precise astrometry and a more accurate absolute reference frame compared to previous work (7). This resulted in an average astrometric uncertainty for S0-2 of 1.1 milliarcsecond (mas) for speckle imaging, and 0.26 mas for AO imaging. The astrometric and RV measurements are combined in a global orbital model fitting using a standard post-Newtonian approximation which includes the first-order GR corrections on the Newtonian equations of motion, the Römer time delay due to variations in the light propagation time between S0-2 and the observer, and the relativistic redshift. For the astrometric observables, we ignore the negligible effect of light deflection by the SMBH but include a 2D linear drift of the gravitational center of mass. This drift accounts for systematic uncertainties in the construction of the astrometric reference frame. The RV observable is fully derived in (13). To our level of accuracy, it is: where c is the speed of light in a vacuum, v z 0 is a constant offset introduced to account for systematic uncertainties within our RV reduction, V Z,S0−2 is the Newtonian line-of-sight velocity of S0-2, V 2 S0−2 /2c is the transverse Doppler shift predicted by special relativity depending on S0-2's velocity V S0−2 and GM/cR S0−2 is the gravitational redshift predicted by GR incoporating the SMBH gravitational parameter GM (the graviational constant G, and SMBH mass M) and on the distance, R S0−2 , between S0-2 and the SMBH. Υ is a scale parameter introduced to characterize deviations from GR: its value is 0 in a purely Newtonian model and 1 in GR (13) (for more details see S1.3.3 in supplementary material). The model has 14 parameters: 6 orbital parameters for S0-2, the gravitational parameter of the SMBH (GM ), the distance to the Galactic center R 0 , a 2-D linear drift of the SMBH parametrized by the 2-D position (x 0 ,y 0 ) and velocity (v x 0 , v y 0 ) of the black hole from the center of the reference frame, an offset for the RV v z 0 , and the redshift parameter Υ. Several statistical tests are performed to assess systematic effects, using two different information criteria estimators to compare models: the Bayesian evidence and the expected logarithm predicted density (13). We examine several sources of systematic uncertainties in the orbital fit: (i) potential offsets in RVs and astrometric positions from different instruments and (ii) potentially correlated uncertainties in astrometric measurements. Based on Bayesian model selection, we find that NIRC2 spectroscopy requires a RV offset with respect to other instruments (likely due to optical fringing). No other instruments require an RV or astrometric positional offset. We include a parameter for the NIRC2 RV offset in the model so it is fitted simultaneously. Based on the model selection criteria, we also find spatial correlation in the as- We developed an orbit modeling software package to model the orbits. The software uses Bayesian inference for model fitting, using nested sampling to estimate the posterior probability distribution via the multinest package (16,17). We also perform Monte Carlo simulations to evaluate our fitting methodology and to show that the statistical uncertainties are robust (see supplementary materials). We initially compare a purely Newtonian model with a purely relativistic (Υ fixed to 1) model. We use the Bayes factor model selection criterion to show that the relativistic model is preferred by the data with high confidence. The difference of the logarithm of the Bayesian evidence between these two models is 10.68. Expressed as an odds ratio, the GR model is 43,000 times more likely than the Newtonian model in explaining the observations. We then fitted the more general model that includes the Υ redshift parameter as a free parameter. The estimated values for the 17 fitted parameters are in Table 1 (the posterior dis-tributions are shown in Figs. S10-S13). The estimation Υ = 0.88 ± 0.16 and its marginal posterior is shown in Fig. 3c. We estimate the systematic uncertainties due to the astrometric reference frame construction by performing a jackknife analysis on stars used to construct the reference frame. This adds a systematic uncertainty on the redshift parameter of ∼ 0.047, which when added in quadrature with the statistical uncertainties, results in a total uncertainty σ Υ = 0.17. The measured redshift parameter is therefore 0.88 ± 0.17, consistent with GR at the 1σ level while the Newtonian value Υ = 0 is excluded by > 5σ. Our estimation also agrees at the 1σ level with the measurement by the GRAVITY collaboration (9). Our experiment is independent from theirs, using a different set of measurements that includes the third turning point. We examined additional sources of systematic error that were previously not considered. The best-fitting model to the RV and the fit residuals is presented in Fig. 2. A fit using a parameter encoding deviations from GR only at the level of the gravitational redshift gives α = −0.24 ± 0.32, where α = 2 (Υ − 1) is the standard gravitational redshift parameter (13) (see Supplements and Section 2.1.3. from (1)). Our observations also constrain two other parameters: the mass of the black hole (M BH ) and the distance to the Galactic center (R 0 ). From our model with Υ as free parameter, the 68% marginalized confidence interval for M BH = (3.984 ± 0.058 ± 0.026) × 10 6 M and R 0 = 7, 971 ± 59 ± 32 pc, where the first uncertainty is the statistical uncertainty and the second uncertainty is the systematic error σ from the jackknife analysis (see Table 1). If we assume GR is true, then M BH = (3.964 ± 0.047 ± 0.026) × 10 6 M and R 0 = 7, 946 ± 50 ± 32 pc (see Supplemental texts for discussion). The nested sampling chains are provided in the Data Supplements. The gravitational redshift is a direct consequence of the universality of free fall and of special relativity (18), hence of the Einstein equivalence principle, a fundamental principle of GR, which provides a geometric interpretation for gravitational interactions. Violations of the equivalence principle are predicted by some theories of modified gravity motivated by the development of a quantum theory of gravitation, unification theories, and some models of dark energy (19). While the gravitational redshift has been measured with higher precision within in the Solar System (20,21), our results and those of the GRAVITY collaboration (9) extend the measurements to higher gravitational redshift and around a massive compact object, a SMBH. Sgr A* has a mass ∼ 4 × 10 6 times larger than that of the Sun. This constrains modified theories of gravitation that exhibit large non-perturbative effects around black holes, but not around non-compact objects like those in the Solar System (see (22)(23)(24) Competing Interests The authors declare no competing interests. Data and material availability: All (other) data needed to evaluate the conclusions in the paper are present in the paper or Figure 2: GR orbit modeling of S0-2. A: Astrometric measurements of the short-period star S0-2 in orbit around the SMBH (Sgr A) overlain with our best-fitting projected orbit in the plane of the sky. The origin of the coordinate system coincides with the fitted SMBH center of mass (13). The x and y axes corresponds to offsets in right ascension and declination from the SMBH. 45 astrometric measurements from 1995-2018 of which 11 are new observations (black) and 34 rederived measurements (grey). The best-fitting SMBH linear drift has been removed from the measurements. The line of nodes (dashed line) shows the intersection of the orbital plane with the plane of the sky (this line also passes through the position of the black hole). S0-2 moves clockwise in this projection; the star is behind the black hole below the line of nodes and in front of the black hole above the line of nodes. The color and intensity used in the best-fitting orbital plot represent the direction and magnitude of the line-of-sight velocity with colors corresponding to panel B. B: RV measurements and the best-fitting RV model (colored line) using 115 RV measurements from 2000-2018. 42 measurements were previously reported (empty circles), 45 were rederived for this work with improved methodology (grey dots), and 28 are new observations (black dots). The color of the best-fitting orbit represents the value and sign of the line-of-sight velocity. C: residuals from the best-fitting RV model. D & E: Observations around the three turning points, 1 at the closest approach to Sgr A in the plane of the sky (D) and 2 RV turning points (maximum and minimum RV, E) provide the greatest sensitivity to the relativistic redshift. Figure 3: Measured deviation from Newtonian predictions. The fitted deviation from Newtonian prediction, overlaid with the best-fitting orbit model (red line) corresponding to Υ = 0.88. The inset shows the posterior probability distribution for Υ; 0.88 is the median value. The red shaded areas show the model 68% and 95% confidence intervals. The observed RVs are shown as black points after removing the Newtonian part of the model. For comparison, we show the RV deviation expected for a purely relativistic signal (Υ = 1, dotted blue line) and for a purely Newtonian model (Υ = 0, dashed blue line) for an orbit with the same orbital parameters. Our measurement is consistent with the GR model at the 1σ confidence level while the Newtonian model is excluded at > 5σ confidence. Supplementary Text Figs. S1 to S18 Tables S1 to S13 Other Supplementary Materials for this manuscript include the following: Data S1 to S4 (Astrometry Measurements, RV Measurements, Nested Sampling Chains, S0-2 points in jackknife analysis) (Table S1). Table S2 presents all the S0-2 RV measurements used in this work. (27). We use the same spectra as in (14), except for 2003 Jun 08 and 2003 Jun 09. In previous publications, the spectra for those two nights were combined. In re-examining the data, we found that the wavelength solution varied between the two nights. By not combining the nights, we reduce interpolation errors from shifting the spectra and thereby better capture intrinsic orbital variations in the RV. Keck OSIRIS Spectroscopy The Keck OSIRIS instrument is an integral-field spectrograph that can sample two spatial dimensions and one spectral dimension simultaneously. It has a resolving power of R = λ/δλ = Table S1) with the Keck I Laser Guide Star AO system (12,29). The spectra of S0-2 and other stars in the data cubes are extracted using a circular aperture centered on the star on each spectral channel, with an annulus around the star to estimate sky background. Blank sky and standard stars are observed during the night to correct for sky emission and atmospheric absorption lines. Further details are available in (30). We produce a combined spectrum from all the data cubes taken each night. Gemini NIFS Spectroscopy The Gemini NIFS instrument (R = 5000) is also an integral-field spectrograph, which produces data similar to OSIRIS. All the NIFS data were obtained in 2018 using the natural guide star adaptive optics system Altair (31) and with the K grating and the HK filter. The NIFS data were reduced using the data reduction package Nifty4Gemini (32), following standard methods (e.g. (33)). Similar to OSIRIS, blank sky and standard stars are observed during the night to correct for sky emission and atmospheric absorption lines. We use the software package molecfit (34) to fit and remove the atmospheric features. We use the same method for spectral extraction as with OSIRIS. Subaru IRCS Spectroscopy We carried out Echelle spectroscopic observations of S0-2 using the IRCS (35) New and Re-derived RV Measurements We present new radial velocity measurements from spectra with NIRC2, OSIRIS, NIFS, and IRCS instruments. While the RV measurements from NIRC2, OSIRIS, and IRCS taken before 2017 were presented previously (7, 14, 38), here we re-derive all NIRC2, OSIRIS, and IRCS RVs using an improved method. We use a synthetic spectral grid and Bayesian inference to model the spectra using a physical model that includes the physical properties of the star (e.g. effective temperature) along with its rotational velocity and radial velocity. We use the BOSZ spectral grid (39) which has synthetic spectra calculated over the range of wavelength of the observations as well as the reported physical properties of S0-2. This grid reaches high effective temperatures (up to 35,000 K), which covers previously reported temperatures for S0-2 and other stars within 0.04 pc of the SMBH (40). We use the StarKit spectral fitting software package to perform the parameter estimation (41). StarKit simultaneously models the physical properties of the star (effective temperature, surface gravity, metallicity, alpha-elemental abundance), the continuum (modeled as a second-order polynomial), rotational velocity, radial velocity, instrumental broadening, and wavelength sampling in order to compare the model with the data directly. We use a Gaussian likelihood to compare the model to the observed spectrum and the uncertainty on the flux. The flux uncertainty was estimated using the standard deviation of the flux in the continuum of the star. To account for potential mis-estimation of the flux uncertainties, we also included an additive flux uncertainties term in the spectroscopic fit. We find that this additive term is smaller than the flux uncertainties. Parameter estimation is done via Bayesian inference with sampling of the posterior using the nested-sampling algorithm MultiNest (17). More details on StarKit and its application to Galactic center data are given in (42-44). We use the median and 1 sigma central credible interval of the marginalized posterior as the radial velocity and its uncertainty. The spectroscopic observable V is defined as: where λ obs is the observed wavelength, λ em is the emitted wavelength of the spectral fea- We find that when the SNR in the continuum of spectrum falls below 20, the Bracket gamma feature becomes too noisy to be robustly measured by this technique; we therefore only include measurements with SNR > 20. This criteria excludes the RV measurement from 2007 July 21 (SNR = 16) that was previously included in (7). Using observations of stars that are RV standards on the same night as observations of S0-2, we can also evaluate the accuracy of the radial velocity measurements. We selected radial velocity standards to be stars that have spectral type similar to that of S0-2 and which have been previously observed as radial velocity standards. We extract these stars using the Set of Identifications, Measurements and Bibliography for Astronomical Data (SIMBAD) database, selecting ones that do not have significant variations in radial velocities between multiple previous measurements. Measurements of these stars are included in Table S3. Figure S2: Comparisons of the RV uncertainty for S0-2 RV measured using a Gaussian fit to the Bracket Gamma line (grey triangles) and using the full-spectrum fitter StarKit (black circles). RV uncertainties are shown as a function of SNR. On average, StarKit uncertainty estimates are about 1.7 times more precise than using a Gaussian. These measurements are also on average about 2.2 times more precise than average RV values reported by (8) using SINFONI on VLT. When the Gaussian fitting method was used, the weighted average difference from the reported SIMBAD values was 8.3 ± 1.2 km s −1 , but when using StarKit, radial velocity measurements of the standard stars had a weighted average difference from the reported SIMBAD velocities of only 1.3 ± 1.2 km s −1 (Fig. S3). We attribute this improvement to StarKit's ability to fit the non-Gaussian absorption lines. This result shows the robustness of the StarKit method and a reduction in systematic uncertainty. Star Figure S3: A: Histogram of the differences between radial velocity measurements and reference values of standard stars given in Table S3. B: Differences between radial velocity measurements and reference values compared to their reference velocity for each individual measurement. There is a velocity bias from the presence of these lines, but the bias is smaller than the uncertainty. The simulations of the effect of sky and gas subtraction suggest there should be an additive error to the RVs. To better assess this, we fit the RVs of 3 stars near S0-2, which have similar brightness and are of similar spectral-type so that their spectral features are comparable to S0-2. These three stars, S0-9, S0-14, and S0-15, only show a linear trend in RV so we include three parameters in the fit: a baseline RV value, an acceleration in RV, and an additive error to be added in quadrature that is simultaneously fitted with the two model parameters. The posterior probability distribution function (PDF) of the systematic uncertainty resulting from independent fits of these three stars are presented in Fig. S5 and are consistent with each other. A combined analysis in which we fit the three stars simultaneously is shown in Fig. S5. The 68% confidence interval for this systematic uncertainty is 11 +4.8 −4.1 km s −1 . We also check for an additive error for S0-2 RVs by including an additive error parameter in the S0-2 orbit fit. Figure S4: Histogram of the velocity offsets from fits to simulated data compared to their reference values. The goal of these simulations are to evaluate the effect of residuals from background subtraction on the spectral features of S0-2 and how it affects the fitted velocities. These simulations suggest that the RV uncertainties of S0-2 should be larger than the fitted uncertainties. In this simulation, the uncertainty should be larger by about 14 km s −1 added in quadrature to the fitted uncertainties. This fit is similar to that from the three other stars and leads to an estimate of the systematic uncertainty of 11.9 +4.1 −3.7 km s −1 . These values are also consistent with the simulations from the gas and sky emission line subtraction residuals (see above). Based on these results, we include an additive error of 11 km s −1 for S0-2 RV measurements with OSIRIS and NIFS. This error is smaller than the RV uncertainties for NIRSPEC, NIRC2, and SINFONI, so we do not include it for those instruments. We also examine two additional sources of systematic uncertainties in the RV measurements: uncertainty in the wavelength solution and optical fringing in the NIRC2 spectra. The wavelength solutions for OSIRIS and NIFS are derived from Ar, Ne, Xe arclamp lines, while the wavelength solution for NIRC2 is derived using the OH skylines. We measure the accuracy of the wavelength solution by comparing the observed centroid of the OH lines from observations of the sky to their vacuum wavelength values. We estimate the systematic error in the radial velocity using the standard deviation of these differences. Using this measure, the wave- Based on the Bayesian information criteria, such an offset very significantly improves the fit compared to offsets applied to the other spectroscopic instruments (See Section 1.6). New spectroscopic observations are reported in Table S1. Table S2 presents the RV measurements both before (v z ) and after correction for the local standard of rest velocity (v lsr ). The RV uncertainty (σ vz ) includes the additive error for each epoch of observation. We also include literature measurements from (8) used in the orbit fitting. Imaging and astrometric measurements The sky positional measurements of S0-2 are made using 2 instruments: speckle imaging with NIRC on Keck I (1995)(1996)(1997)(1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005) and AO imaging with NIRC2 on Keck II (2005-2018). The data reduction and point source detection methods are described in detail in (14,53). Here, we summarize the data and methods used to place the measurements of stellar positions in a common reference frame. Table S4 present new astrometric observations and Table S5 presents the astrometric measurements used in the S0-2 orbit fit. We also transform the positions into separation and position angle (defined to increase East from North) from the origin of the reference frame in each epoch. Using the separation and angle is not straightforward (due to the fit for drift in the reference frame), so we use the coordinates in all our fitting procedures. Speckle imaging Speckle imaging consists of very short exposure images (t exp = 0.1s) designed to be shorter than the atmospheric turbulence time scale. The individual images are combined and postprocessed to produce a deep image for each epoch of observation using a speckle holography technique; this process can reconstruct images that are at the diffraction-limit of the telescope and is described in (7,54), with additional improvements described in (55). The speckle images have a field of view of ∼ 5 × 5 and have a magnitude limit for detection of stars of K = 16.6 mag as defined by the brightness above which 90% of the stars are detected. In total, 27 epochs of speckle data were re-derived and used in this analysis. Adaptive optics imaging Two types of adaptive optics imaging data were obtained with NIRC2 on Keck II for this work: We switched to a shorter wavelength filter in 2018 to avoid astrometric biases due to the infrared source associated with Sgr A* itself, which is very red (57). The central AO data is 2.4 magnitudes deeper than with speckle imaging, with a limiting magnitude of Kp = 19.0 mag averaged across the 10"×10" field. In total we use 18 AO measurements for S0-2, with 9 new measurements from 2017-2018. Reference frame construction All the stars in the NIRC2 field of view are moving; thus establishing a common astrometric reference frame for observations over 20 years is difficult. We have developed a Sgr A-rest frame in which the SMBH is assumed to be at the origin and at rest. This reference frame is Alignment of 24 years of data We place the speckle and AO astrometric observations of the central region into to a common reference frame using a subset of the sample of secondary astrometric standards described above (see 1.2.3). This process, which places stars such as S0-2 in the Sgr A-radio rest reference frame, is described detail in (53), with improvements in the methodology described in (55). if the nearby source is not detected (14). Orbital fit The astrometric and radial velocity measurements presented in the previous sections are simultaneously combined to fit an orbit. This section discusses our orbital fit: the orbital modeling, the likelihood, a brief description of the sampler, and the statistical tools for model comparison. Modeling of the observations The modeling of the astrometric and radial velocity measurements is described in two parts: (i) the orbital dynamics, and (ii) the modeling of the observables. Orbital dynamics The orbital dynamics of the star S0-2 is determined by integrating the post-Newtonian equations of motion derived from a Schwarzschild metric in harmonic coordinates (59) where R is the position of S0-2 with respect to the BH, V its velocity, R = \|R\| and V = \|V \|. The black hole spin and the quadrupole moment that arise respectively at the 1.5 and 2 post-Newtonian orders in the equations of motion (59) can be neglected. The initial conditions used to integrate these equations of motion are computed from a set of orbital parameters: the period P , the eccentricity e, the time of closest approach T 0 , the inclination i, the argument of periastron ω and the longitude of ascending node Ω. A detailed description of these orbital elements can be found in (60). Any orbital modeling beyond a pure Newtonian 2-body interaction will lead to a time dependence of these orbital parameters, requiring these parameters to be considered as oscillating elements. In particular, the post-Newtonian perturbation from Eq. (S2) induces a time variation of the orbital elements P , e, ω and T 0 (61). We use the J2000 epoch (t J2000 ) as a convention for reporting the initial conditions. The set of 6 orbital parameters are therefore transformed into a cartesian initial position and velocity for the epoch J2000 and used to integrate (forward and backward) the equations of motion. The coordinate system used in this integration is centered on the BH. The Z-axis of the coordinate system is defined by the vector pointing from the Solar System to the Galactic Center, and the X and Y axes are defined such that the X-Y plane is parallel to the plane of the sky, with the X-axis pointing East and the Y-axis pointing North. The units used in our calculations are astronomical units (au) and Julian years, defined as 365.25 Julian days. The transformation between the oscillating elements and the initial conditions is a classic Newtonian transformation: the eccentric anomaly E is computed by solving the Kepler equation where n = 2π/P . This leads to the position and velocity of the star in its orbital plane where a = (GM/n 2 ) 1/3 is the semi-major axis, ξ is the projection along the major axis, and η is the projection along the minor axis. The initial conditions in our coordinate system are obtained after applying the three Euler rotations: where A, B, C, F , G, H are the classic Thiele-Innes constants: A = cos Ω cos ω − sin Ω sin ω cos i , (S11) B = sin Ω cos ω + cos Ω sin ω cos i , (S12) C = sin ω sin i , (S13) F = − cos Ω sin ω − sin Ω cos ω cos i , (S14) G = − sin Ω sin ω + cos Ω cos ω cos i , (S15) These initial conditions are then used to integrate the equations of motion to provide R(t) = iteratively (e.g. (63)) using the following iteration scheme: For our purposes, only one iteration is required: This leads to a modulation of the light propagation time between -0.5 days at closest approach and 7.5 days at apoastron. A second iteration would lead to a correction of < 20 minutes, which is negligible at our level of accuracy. We also neglect the Shapiro time delay, which yields a maximum correction of ∼5 minutes. Astrometric observable: The astrometric observations are given in terms of angular positions x and y, which are modeled as where R 0 is the line-of-sight distance to the Galactic center and x 0 , y 0 , v x 0 and v y 0 model a 2D offset and linear drift of the gravitational center of mass with respect to the center of our reference frame. These four parameters are included to model systematics that appear at the level of the construction of the reference frame (56). We neglect the gravitational light deflection of the SMBH, which produces an effect on the order of 20 µas on the astrometry at closest approach (64), smaller than our observational uncertainty. Spectroscopic observable: The spectroscopic observable V is given by Eq. (S1), Using a regular post-Newtonian expression for the frequency shift (e.g. (63,65)), the spectroscopic observable becomes where U em/obs is the gravitational potential at the emission/observation of the light signal, V em/obs = V em/obs is the norm of the velocity of the emitter/observer at the emission/reception of the signal, and N = Xem−X obs \|Xem−X obs \| is a unit vector pointing in the direction of the line-of-sight. In Eq. (S22), the derivative of the Shapiro time delay is neglected (terms of the order of O(1/c 3 )). The maximal contribution from this term arises at closest approach and remains below 5 km/s (64), below the current measurement uncertainty. Eq. (S22) can also be written as where RV is the observed radial velocity corrected for the Velocity of the Local Standard of Rest (V LSR = N · V obs ). All V em are evaluated at t = t em , such that the Römer time delay is taken into account. The first term on the right hand side of this equation N · V em is the standard Newtonian line-of-sight velocity of the star S0-2, which is V Z (t) from Eq. (S10). The second term (N · V obs ) 2 /c is a second order term proportional to the square of the VLSR. The contribution from that term to the radial velocity remains below ∼ 30 m/s so is neglected. The (N · V obs )(N · V em )/c term is a cross term that remains below 0.1 km/s and can be neglected as well. The last terms comprise the relativistic contributions to the redshift. The U obs /c term is the gravitational redshift contribution related to the observer and V 2 obs /2c is the transverse Doppler shift predicted by special relativity. In our case, these two terms comprise several contributions, the main ones being from the Galactic potential, from the potential of the Sun, of the Earth and of the Moon. The order of magnitude of all these terms is below ∼ 0.1 km/s (66) and can safely be neglected as well. The last terms are the second-order transverse Doppler effect from special relativity and the gravitational redshift from the star S0-2. The combination of these two terms is the signal we are seeking to measure. Dropping all negligible terms, we model the RV as: where v z 0 is a constant velocity offset that accounts for possible systematic effects in the radial velocity measurement or in the VLSR correction. The second term of this equation is the relativistic transverse Doppler shift predicted by special relativity, while the third term is the gravitational redshift predicted by GR. The signatures produced by the second and third contributions are highly correlated. Indeed, using a Newtonian orbital model, one gets where a is S0-2's semi-major axis, [RV ] spec is the contribution from special relativity to the RV and [RV ] grav is the contribution from general relativity, i.e. the gravitational redshift. Since an offset v z 0 is fitted to our data, the constant term GM 2ac is unobservable and both signals from special relativity and from the gravitational redshift are completely degenerate and the data is only sensitive to the sum of the two. To quantify possible deviations from the predicted relativistic signal, we introduce a dimensionless parameter Υ whose value is 0 for a purely Newtonian model and 1 in GR (see (67)). The expression for the radial velocity used in our orbital fit is given by Alternatively, we can assume that special relativity is correct and only search for a deviation from the gravitational redshift prediction. Such a deviation is usually parametrized by a parameter α whose value is 0 in GR (see Eq. (6) from (1)) and the radial velocity are then modeled as All our estimations of the Υ parameter can be translated into an estimation of the α parameter through: and the uncertainty on α is given by σ α = 2σ Υ . The observable depends on the SMBH gravitational parameter GM , which is the parameter that is fitted (and not the mass directly). We express the SMBH GM in units of the Sun's gravitational parameter GM where M is the mass of the Sun and whose value is precisely measured from planetary ephemerides (see table 8 from (68).) Bayesian samplers and software Parameter exploration was done using the Nested Sampling package MultiNest (16,17). The analysis was also done independently using the STAN modeling language and NUTS sampler (69). The MultiNest sampler used in this analysis was preferred because it allowed for efficient calculation of the Bayesian evidence and has been successfully used in previous GC orbit analyses (7,70,71). The STAN implementation of our analysis was primarily used to confirm our results. Model selection and information criteria We base model selection on two criteria. The first is the Bayesian evidence that is a direct output of the nested sampling algorithm. Here, the subscript k denotes that the probability is conditioned on Model k being true (P k (. . . \| . . .) ≡ P(. . . \| . . . , Model k )), {d j } represents the dataset, θ the estimated parameters, P k ({d j } \| θ) is the likelihood and P k (θ) the prior. The evidence is used to infer the probability of the different models via Bayes theorem (P(Model k \| {d j }) ∝ E k P(Model k )) assuming that the a priori probability, P(Model k ), is even over all the models under consideration. Sometimes, this assumption can artificially bias probabilities toward fine-tuned models (72,73) and may give inconsistent results when the true model is not included in the comparison (74). The ratio of evidences under the assumption of uniform priors is known as the "Bayes factor" or odd ratio: The logarithm of the ratio of evidences is often compared to roughly judge the strength of one model over another with a log ratio (log(E 0 /E 1 )) value under 1 considered "barely mentioning", 1 to 2.5 being "positive", 2.5 to 5 having "strong evidence", and greater than 5 having "very strong evidence" of one model over another (75)(76)(77). We also use the expected log probability density (elpd) as an additional model selection criteria (78): which has been shown to be, asymptotically, a good approximation to the elpd (82). In principle, the probability of observations d i given the dataset that excludes ) needs to be reevaluated for each data point since the posterior probability, P k (θ \| {d j } j =i ), is unique for each data set, {d j } j =i . We avoid this by reweighting the posterior probability given the full data set (P k (θ \| {d j })) to match the distribution P k (d i \| {d j } j =i ). Using this approximation, Equation S31 becomes (83): where {θ k } and {w k } are deviates, and corresponding weights, of the posterior P k (θ \| {d j }). The inverse sum in Equation S32 is usually numerically unstable because infrequent deviates will correspond to low P k (d i \| {d j } j =i , θ k ) values and thus be weighted higher (83). This is avoided if a nested sampling algorithm, such as MultiNest, is used to sample from the posterior. In this case, the inverse divergence behavior is avoided because nested sampling weights are proportional to the likelihood, P k ({d j } \| θ) (16,17). As a summary, in this analysis a selection for a more complex model is decided when both the difference of the evidence E and the elpd are larger than 2.5 in favor of the more complex model. Likelihoods We consider several likelihoods in our analysis to capture different sources of errors. For the first one, we assume the astrometric positions ({x i }, {y i }) and radial velocity ({RV i }) to be normally distributed about the astrometric ({x(t astro,i )}, {y(t astro,i )}) and radial velocity (RV(t RV,i )) predicted values and with their dispersions equal to their uncertainties: Here we define x ∼ N (µ, σ) to mean that variable x is normally distributed about µ with a dispersion σ. To determine whether we have under-estimated the uncertainties, we also explore likelihoods that include an additional additive error for the astrometry (σ astro ) and the radial velocities (σ inst ): To account for potential correlations in the uncertainty of astrometric measurements, we also consider a likelihood with separate covariance matrices for the astrometric positions (x ≡ {x i } and y ≡ {y i }) corresponding to times t astro ≡ {t astro,i }: where x ∼ N (µ, Σ) denotes that the vector x is normally distributed around the vector µ with a covariance matrix of Σ. We model the covariance matrices by a single correlation matrix, ρ, where the covariance matrix is given by where d ij is the 2D projected distance between point i and point j ( This matrix introduces a correlation length scale Λ characteristic of the correlation and a mixing parameter p, both of which will be fitted simultaneously with the model parameters. Priors In the orbit fitting, we used uniform priors on all fitted parameters. While this choice is common, it has been shown that uniform priors can potentially bias estimated parameters (84,85). With this in mind, we used simulated data to understand the impact of our fitting procedure by identifying possible biases in the estimated parameters and assessing the accuracy of the confidence intervals obtained in our analysis. Here, we summarize the methodology to test for fitting biases (for a full description, see (85)). We generated 1000 mock datasets by simulating measurements at epochs corresponding to our observations. Each mock dataset was generated by drawing a random measurement from a normal distribution distributed about an assumed true value, with a dispersion taken to be the actual measurement error at that epoch. We then fit these 1000 mock datasets using the same procedure that is used to fit the real data. For these 1000 fits, we computed the redshift bias, or the difference between the estimated redshift parameter and the input redshift parameter. The distribution of the 1000 bias values is shown in Fig. S6. This distribution is centered around zero and indicates the bias on the redshift parameter is negligible. We next consider the accuracy of our confidence intervals. In the classical definition of a confidence interval, for a sufficiently large number of experiments, the confidence interval inferred from each experiment will contain, or cover, the universally "true" value a prescribed fraction of the time (confidence level × 100%) (86). For example, by this definition, given 100 possible observed (or randomly drawn) datasets, a 68% confidence interval requires that about 68 out of 100 fits produce a confidence interval that covers the true value. Thus, the statistical efficiency, defined as the ratio of effective coverage (the experimentally determined percentage of datasets in which the inferred confidence or credible interval covers the true value) to stated coverage (68% for a 1-σ confidence interval), is a powerful performance diagnostic that can be used to investigate the accuracy of calculated confidence or credible intervals (85). By Figure S6: Distribution of the bias values on the redshift parameter. This distribution is obtained by considering fits of 1000 mock datasets generated using the same epochs of measurements as our real dataset. The specific bias factor is defined as the difference between the estimated redshift parameter and the input redshift parameter. This distribution shows that the procedure used in our fit does not induce any substantial bias on the estimated redshift parameter. definition, a statistical efficiency of one would indicate exact coverage. The statistical efficiency for the redshift parameter determined from the 1000 mock datasets is 1.002 ± 0.02 (or 0.682 ± 0.015 coverage for a 68 % confidence interval). This shows that the confidence interval on the redshift parameter we derive represents a robust estimate of the statistical uncertainty. The bias analysis shows that using uniform priors in this analysis does not lead to any substantial bias in the estimation of the redshift parameter. In addition, the statistical efficiency demonstrates that the confidence intervals used in this analysis are well defined and have close to exact coverage. Accounting for instrumental systematic effects on the RV measurements in the orbital fit We assessed two potential sources of systematics in the RV measurements: (i) systematic RV offsets between the different instruments and (ii) possible additive uncertainties for the different instruments. The RV measurements used in our analysis have been performed by seven different instruments: NIRSPEC, NIRC2, OSIRIS Kbb, OSIRIS Kn3 at the Keck Observatory, NIFS with Gemini, SINFONI at the VLT and with IRCS at SUBARU. Since the different instruments work differently and are sensitive to different systematics, it is important to cross-validate the different datasets. In order to do so, we added 7 parameters to our orbital fit: one offset per instrument. We performed 8 different fits: one reference fit with all the offsets forced to 0 and 7 where we fitted for one offset at a time simultaneously with the model parameters. We then used the model selection criteria described in section 1.4 to assess which offsets, if any, are significant. The results from these fits are presented in Table S6. Using the threshold on the information criteria presented in section 1.4 (∆E > 5 and ∆ elpd >5), we conclude that the NIRC2 dataset presents a significant offset. This conclusion, which is obtained based purely on statistical arguments, is reinforced by the fact that NIRC2 measurements are affected by fringing, as explained with more details in Section 1.1.1. For these two reasons, in all our orbital fits, we include an offset for the NIRC2 dataset that is fitted simultaneously with the other model parameters. Table S6: Results from orbital fits that include an offset per spectrograph instrument. The third and fourth columns are the differences between the log-evidence and the elpd of the fit with the offset and the reference fit where no offset is considered. A more complex model is adopted when both differences are larger than 5. In addition to considering instrumental offsets in the RV dataset, we have also considered additional possible instrumental systematic uncertainties. We use the likelihood defined by Eqs. (S36-S38) and to introduce 7 additional parameters: one systematic uncertainty for each instrument. The methodology is the same as the one followed for the instrumental offsets: we performed different fits by considering one additional systematic uncertainty at a time and assessed the significance of each instrument systematic uncertainty by using our model selection criteria described in Section 1.4. The results of these fits are presented in Table S7. This analysis does not show any evidence for any additional systematic uncertainty. This means that our RV uncertainties are not underestimated and in the following, no additional systematic uncertainty in the RV is included. Accounting for systematic effects on the astrometric measurements and analysis of correlation within the astrometric dataset at the level of the orbital fit At the level of the orbital fitting, we have assessed our astrometric dataset by considering three effects: (i) an additional possible systematic uncertainty on the astrometric measurements (ii) a possible offset between the different instruments and (iii) the correlation within the astrometric measurements. Possible additional systematic uncertainties for the astrometric measurements For the study of the systematic uncertainty, we followed the same methodology as the one presented in the previous section for the RV measurements. We used the likelihood defined by Eqs. (S36-S38) and considered two additional systematic uncertainties in the orbital fit: one for the Speckle measurements and one for the AO measurements. We compared three different fits: a reference fit where no additional systematic uncertainty is considered, one fit where the Speckle systematic uncertainty is fitted and one fit where the AO systematic uncertainty is fitted. We used our model selection criteria described in section 1.4 to identify if these additional systematic uncertainties are relevant. The results of these fits are presented in Table S8. The difference in our information criteria are below the threshold presented in section 1.4 which leads to the conclusion that no additional systematic uncertainty needs to be added to our astrometric dataset. Possible additional offset between the different astrometric instruments We also assessed the possibility of an offset between the Speckle and AO measurements. We added a 2D offset to the fit and compared the log-evidence and elpd of a fit that includes these additional 2 parameters to a fit where no offset is considered. Tab. S9 shows the result of these fits and shows that no additional systematic offset between the two instruments need to be considered Correlation within the astrometric measurements The second effect assessed regarding our astrometric dataset is the presence of correlation in the measurements. Such correlations are expected because of detected and undetected source confusions and because of the presence of correlated systematic effects that arise during the construction of the reference frame. Assuming all the measurements to be statistically independent will therefore lead to uncertainties that are overoptimistic for the estimated parameters. In order to study correlations within our dataset, we have used two different methods: (i) we modeled the correlation and we fitted for the related parameters simultaneously with the model parameters and (ii) we used a Monte Carlo approach where we kept only one measurement per correlation length scale. For the first method, we used the likelihood defined by Eqs. (S39-S41). This likelihood introduces a correlation matrix between the astrometric measurements. We considered the form for the correlation matrix given by Eq. (S42), which assumes that the correlation within the astrometric dataset is related to the 2-D projected distance between measurements. This likelihood introduces two additional parameters that are fitted simultaneously with the model parameters: a correlation length scale Λ and a mixing parameter p. We used our model selection criteria described in Section 1.4 to compare orbital fits that consider this correlation with a fit that uses the regular uncorrelated likelihood and the difference in the log-evidence is of 7.3 while the difference of the elpd is of 13.5, showing a strong evidence in favor of the model that includes correlations. In this analysis, we therefore use this correlation matrix and fit for the two Figure S7: Posterior probability distribution function for the two parameters that parametrized the correlation matrix: Λ a correlation length scale and p a mixing parameter. The marginalized 68% confidence interval for the correlation length scale is Λ = 28 +24.6 −13.6 mas, which corresponds roughly to half the Keck diffraction limit. parameters Λ and p simultaneously with the model parameters. Fig. S7 shows the posterior distribution for these two parameters. The fitted correlation length scale is around 30 mas, which corresponds to half the Keck diffraction limit. In order to validate the analysis presented in the previous section, we used a second method to assess the correlations in our dataset. In this second method, we used a Monte Carlo approach and generated 500 mock astrometric datasets by choosing randomly one observation per 2D projected length scale of 30 mas. We then performed 500 fits by considering that the observations of these datasets are independent. As a result, we obtained a distribution of estimated parameters. Fig. S8 shows the distribution of the redshift parameter Υ for these 500 fits. The mean of these estimated redshift parameters is 0.88 and their dispersion, which is an estimation of the uncertainty due to correlations is 0.13. The total uncertainty, which is the quadratic sum of the statistical uncertainty obtained when considering all the astrometric mea-A B Figure S8: Estimation of the redshift parameter for 500 datasets generated by choosing randomly one astrometric measurement per correlation length scale of 30 mas. The top panel shows the 1σ estimated values for the redshift parameter for these 500 fits (and the red errobar indicates the estimation obtained using all the astrometric measurements and assuming their error to be statistically independent). The bottom panel shows the distribution of the 500 redshift parameter best-fitting values. surements as independent (which is given by 0.89 ± 0.13) and the correlation uncertainty is This result gives confidence in both methods. In our analysis, we include the correlation in our model and fit for the two parameters Λ and p simultaneously with all other model parameters. Systematic effects arising from the construction of the astrometric absolute reference frame The Sgr A-radio rest astrometric reference frame is defined by the positions of seven masers ( (56)). The small number of reference stars may lead to systematic uncertainties in the construction of the reference frame. In order to estimate these systematics, we have undertaken a jackknife resampling method. The details of the jackknife method are described in (7) and (56). We construct seven reference frames by dropping one different maser for each of them. Each is applied to the cross-epoch alignment following the same methodology as described in Section 1.2. As a result, 7 sets of S0-2's positions are derived (see Table S11). We used these 7 sets of S0-2's astrometric measurements combined with the RV measurements to fit for S0-2's orbit following the same methodology as above. The resulting estimations of the redshift parameter are presented in Fig. S9 and in Table S10. These estimations can then be used to infer the systematic uncertainty due to the construction of our reference frame by evaluating where n is the sample size (in this case 7), x n−1,i is the redshift estimator derived by excluding the ith maser andx n−1 is the average of these subsets. The estimations of the redshift parameter from Table S10 give an estimation of the systematic uncertainty of 0.0466. This systematic uncertainty is independent of the statistical uncertainty determined in the orbital fit and needs to be added in quadrature. The systematic uncertainties for the other parameters are obtained in the same way and are presented in Table 1. The systematic uncertainty is larger than the statistical one only for the Black Hole position and velocity parameters. Results and discussion Here we summarize the orbital fitting methodology described in detail above. We use a Gaussian likelihood for the RV observations and include an offset for the NIRC2 measurements in the fit. The likelihood for the astrometric part is given by a multivariate distribution characterized by a correlation matrix that is parametrized by a length scale Λ and a mixing parameter p, see Eq. (S42). The fit includes 6 orbital parameters for S0-2 and 7 parameters related to the SMBH: the GM , the distance to the GC R 0 , the linear drift of the SMBH x 0 , y 0 , v x 0 , v y 0 , a RV offset v z 0 . This amounts for 16 fitted parameters. Different fits using different models were done in this analysis: 1. a fit using a purely Newtonian modeling. 2. a fit using a purely GR modeling. 3. a fit using a modeling that include the correction from special relativity but no effects from GR. 4. a fit using a pure GR modeling and including the presence of an extended mass modeling a possible population of compact objects near the SMBH, such as neutron stars and stellar mass black holes. 5. a fit introducing an additional parameter Υ to encode deviation from special and general relativity at the level of the redshift as described in Eq. (S26). 6. a fit similar to the previous one but including the extended mass. 7. a fit introducing an additional parameter α to encode a deviation from General Relativity at the level of the gravitational redshift as descirbed in Eq. (S27). Table S12 lists the log evidence and the elpd for these 7 fits. These quantities are useful to assess the models favored by the measurements (see section 1.4). The chains sampling the posterior probability distribution from the fits 1, 2 and 5 are available as Data S3. Similarly, the difference in maximum of the log-likelihood between the two fits is 10.4. To assess the significance of the corrections from General Relativity, we compared fit 2 (GR model) to fit 3, which includes only corrections from special relativity but not General Relativity. The difference of log-evidence and elpd between these two fits is 4.23 and of 4.21, respectively, indicating a clear detection of the general relativistic model over a model that includes special relativity only. In terms of Bayes factor, this means that the pure GR modeling is 70 times more likely than the modeling using special relativity given the measurements. The results from fit 2 include the estimation of the SMBH mass and R 0 . The 1D marginalized 68 % confidence interval on these two parameters are: M BH = (3.964 ± 0.047 ± 0.026) × 10 6 M and R 0 = 7, 946 ± 50 ± 32 pc, where the first uncertainty is the statistical uncertainty and the second uncertainty is the systematic σ from the jackknife analysis. Figure S10 presents the posterior distribution for these two parameters. The estimated BH mass and R 0 differs at the level of 2σ with those measured by the GRAVITY collaboration (9). Their systematic uncertainty was given only for the redshift parameters and not for the other parameters (9). Fit 4 includes the extended mass in the context of General Relativity. The extended mass distribution is modeled by a power-law as in (7): where γ is a powerlaw parameter, M 0 is the extended mass enclosed inside r 0 and where r 0 has been taken as 0.011 pc such that it encloses S0-2 apoapse. Figure S10: Posterior pdf for the global parameters GM and R 0 for a fit that assumes General Relativity to be correct (Fit 2 in Section 2.1). stars and stellar mass black holes. The posterior distribution for the extended mass enclosed within S0-2's orbit is presented in figure S11 and the 68% (respectively 95%) upper confidence limit is M 0 < 5.5 × 10 3 M (< 12.7 × 10 3 M ). Fit 5 is our canonical solution presented in the main text. Here we discuss it in further detail. The estimated parameters for this fit are presented in Table 1. The posterior pdf for the global parameters is presented in Fig. S12 while the posterior pdf for the coefficients of the astrometric correlation matrix is presented in Fig. S7. The redshift parameter is correlated with several other fitted parameters. Fig. S13 presents some joint-posterior distributions of the parameters that show the greatest correlation with the redshift parameter. The full RV residuals are presented in the main text. The 2018 RV residuals that are presented in Fig. S14. The astrometric measurements, best fit model and residuals are presented in Fig. S15. The separation between Sgr A and S0-2 is presented in Fig. S16 and allows us to compare our astrometric measurements with the one from the GRAVITY collaboration (9). . This fit assumes a power law slope for the density profile of any extended mass, with index γ = 1.5 (M 0 is not significantly sensitive to this value, see (7)). No significant extended mass has been detected, but this analysis has resulted provides a improved limit compared to previous works (7). Our model predicts an angular separation of 22.6 mas at the end of March, which seems in agreement with their figure 1 (although no uncertainty is given in (9)). The results presented in Table 1 have been obtained assuming that the extended mass resulting from massive non-luminous object such as stellar remnants is negligible. We tested the impact of adding an additional parameter to the fit to include an the hypothetical presence of such an extended mass (see fit 6). We use a power law density-profile modeling for the extended To aid in understanding the geometry of the orbit of S0-2, we show the orbit using different projections in Figure S17. The last fit (fit 7) is similar to fit 5 (parameterized redshift model), but here we only allow the gravitational redshift to vary. This is parametrized by the conventional parameter α (see Eq. (S27), or the section 2.3.1. from (1)). The resulting estimation of the α parameter is given by α = −0.24±0.32±0.047 in agreement with Eq. (S28) above. This parametrization provides constraints on theories of modified gravity. Several modified theories of gravitation violate the equivalence principle so can be characterized with this parametrization (see e.g. (88)). One example of a theory that violates the equivalence principle around a black hole is the quadratic Einstein-Gauss-Bonnet theory where the scalar field is non-minimally coupled to the standard matter fields (22)(23)(24). The quadratic Einstein-Gauss-Bonnet theory is a tensor-scalar theory with a specific coupling between the scalar field and the Gauss-Bonnet invariant. These Figure S17: The orbit of S0-2 in different projections, centered on the supermassive black hole (black circle). The solid line indicates the orbital motion, with the darker portions more recent in time. The X-Y projection is on the plane of the sky. Z is along the line of sight with positive Z directed away from Earth and negative Z toward Earth. The orbit of S0-2 is tilted toward the Earth, with its closest approach to Sgr A* lying behind the black hole in this projection. Also labeled are the location along the orbit of RV maximum (red cross in April) and RV minimum (blue cross in Sept). These two points, along with the 2D closest approach (white circle in May) provide the most sensitive observations for the redshift parameter estimation. The 3D closest approach position (grey dotted line) is very close the projected 2D closest approach. Also marked are the location of S0-2 in Jan, 1 2018 and Jan 1, 2019. This figure was produced with the aid of the Rebound code (87). recently studied theories are (amongst others) motivated by string theory and by unification scenarios (22)(23)(24). These theories can present large deviations from General Relativity around black holes while they will behave exactly as GR around non compact objects, such as those in our solar system (22)(23)(24). Black holes would acquire a large scalar charge that characterizes the 1/r behavior of the scalar field at large distances (22)(23)(24). If this hypothetical scalar field is non-miniminally coupled to the standard matter fields, it will produce a violation of the Einstein equivalence principle. A well-studied prototype for the Lagrangian modeling the interaction between the scalar field and the standard matter is given by the Lagrangian from Damour and Donoghue (89,90). In this modeling, the scalar field presents a dilatonic coupling to the standard matter fields that leads to a dependency of the constants of nature (the fine structure constant, the mass of the fermions and the quantum chromodynamic energy scale) to the scalar field. Such a coupling is known to break the equivalence principle and leads to violations of the universality of free fall (see e.g. (89)) and of the gravitational redshift (91). It can be shown (see e.g. (88, 91)) that the deviation from the gravitational redshift in such a theory will be parametrized by the α parameter introduced in Eq. (S27). This parameter is directly related to the hypothetical scalar charge of the SMBH and to the dilatonic coefficients that characterize the coupling between the scalar field and the standard matter fields (88). This example illustrates how the current constraint on the gravitational redshift of S0-2 around Sgr A* can probe some effects beyond Solar System tests (because the scalar field will vanish around non compact objects) or with gravitational waves (because violation of the gravitational redshift is currently not testable with gravitational wave measurements). Adaptive scheduling tool In order to maximize the use of telescope time in 2018 and to enhance our chance of a successful detection of the relativistic redshift, we developed an adaptive scheduling tool to help us to plan our observations. This tool is fully described in (92) and here we briefly outline its principles and results regarding the redshift measurement. This tool determines what type of measurements and which nights of observation are optimal to measure a given parameter. This scheduling tool (or cadence tool) is based on the computation of the Fisher matrix using the assumption that the posterior probability density functions are Gaussian. This method is computationally fast to consider a large number of possible measurements to find the optimal ones to measure a signal. The Fisher matrix is given by where P is the matrix of the partial derivatives of the model with respect to the parameters: where M (t i , p) is the expression of the model of our observables (in practice, we have two types of observables: the astrometry and the RV), p is the set of fitted parameters and σ i corresponds to the uncertainty of the ith measurement. In practice, we used the modeling presented in The cadence tool iteratively determines the future optimal observations (the optimal scheduling and the optimal type of measurement) in a given observational window assuming that we already have past measurements available. More precisely, we start the procedure with existing data and search within a given window for the measurement that most increases the information entropy related to a given parameter. Once this optimal measurement is found, it is added to the set of "existing" data and we iteratively search for the next most important measurement. This procedure furnished a sequence of optimal measurements that maximizes the signal to noise ratio of a given parameter. This sequence of measurements depends on the parameter that we are trying to optimize, on the set of fitted parameters (because of correlation), on the past measurements, on the expected uncertainty for the future observations, and on the future observational window that is considered. In practice, the optimization procedure relies on a brute force method in the sense that we compute the covariance matrix for every additional measurement included in our future observational window. However, it remains sufficiently fast for two reasons: (i) it requires only one evaluation of the model and of its partial derivatives for the past measurements and over the full future observational window and (ii) instead of inverting the full Fisher matrix after each additional measurement, we update the covariance matrix by adding one observation. In other words, if Σ (n) is the covariance matrix related to a set of n measurements, the covariance matrix for a set which includes an additional measurement at time t k will be given by where U (k) is the update matrix which depends on the measurement at time t k and is given by whereP (k) is a column vector containing the partial derivatives of the measurement at time t k with respect to the fitted parameters p: P (k) i = 1 σ k ∂M (t k ,p) ∂p i . In practice, since we are optimizing on the uncertainty of one single parameter, only one term of the update matrix needs to be computed and this avoids the need to invert the full Fisher matrix a large number of times. This adaptive scheduling tool has been used in the planning of our measurements in 2018. We ran this tool by considering the measurements before 2018 as "past observations" and searched for the optimal sequence of observations in 2018 in order to measure the redshift parameter Υ. The result was the sequence presented in Table S13, under the assumption that new RV uncertainties would be 20 km/s and new astrometric uncertainties 0.9 mas. For this simulation, we assumed that all the model parameters are fitted. Correlations between the parameters are substantial. 9 out of the 12 optimal measurements are RV observations. This makes sense considering that the redshift impacts directly the RV but not the astrometry. In addition, the adaptive scheduling does not favor nights at the maximum of the redshift signal. Rather, it favors measurements that are at the RV turning points (i.e. the maximum and minimum of the RV curves, see Fig. S18). This is due to correlations between the redshift parameter and the other model parameters, in particular with T 0 and with the SMBH GM . These correlations are maximal exactly when the redshift signal is maximal, therefore that epoch is not optimal in order to measure Υ. In other words, the redshift signal can easily be absorbed by a small change in T 0 or in the SMBH GM . To demonstrate the effect of correlations, we ran a test case and searched for the optimal measurements if we fit for all the model parameters except for T 0 and GM . Fig. S18 shows the 12 optimal observations that are all located close to the maximal of the redshift signal. In conclusion, our analysis has shown that, due to correlations, the optimal measurements to detect the relativistic redshift are RV measurements taken at the epochs corresponding to the maximum and minimum of the RV and not at the maximum of the relativistic signal. This conclusion is highly sensitive to the assumed uncertainties for future measurements (in this analysis 20 km s −1 for RV and 0.9 mas for the astrometry). The optimal measurements sequence is also highly dependent on the parameter we are optimizing for (here the redshift parameters). Another optimal sequence of measurements would be obtained for optimal measurement of A B Figure S18: Representation of the RV measurements sequence in 2018 optimized to detect the relativistic redshift (see Table S13). The top panel represents the optimal RV measurement to detect the relativistic redshift. The black curve is the RV model. The bottom panel shows the relativistic contribution from the same measurements with the redshift signal. In these simulations, all measurements prior to 2018 are used (black points). The red points show the optimal measurements sequence under the assumption that all the model parameters are fitted simultaneously. The optimal RV measurements are located at the RV turning points and not at the maximum of the redshift because of correlations with T 0 and GM . The observations in orange are the optimal measurements under the assumption that T 0 and GM are not fitted. These optimal measurements are located at the maximum of the redshift signal.	2019-07-24T21:26:20.000Z	2019-07-24T00:00:00.000	{ "year": 2019, "sha1": "6fc1b55901447d833a3d9fbc53503c1ce7a478cb", "oa_license": null, "oa_url": "https://www.science.org/cms/asset/9b318de9-5652-4b20-9eab-f6eadab49e35/pap.pdf", "oa_status": "BRONZE", "pdf_src": "Arxiv", "pdf_hash": "fc8ecd65bdea8988366e8f51d8a60933e955c2d7", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics", "Medicine" ] }
250692739	pes2o/s2orc	v3-fos-license	On the convergence speed of iterative methods for linear inverse problems with sparsity constraints In this article we report on different iterative algorithms for the minimization of Tikhonov type functionals which involve sparsity constraints in form of lp-penalties. We summarize results on the well known iterated soft thresholding, the iterated hard thresholding and a semismooth Newton approach. While the first two converge globally but very slow, the last one converges only locally but superlinearly. Furthermore we propose a combination of soft and hard thresholding. While this method has theoretically the same convergence rate than the soft thresholding (namely it converges linearly), our numerical experiments show that the combined approach is almost always better than both methods alone. Introduction Recently sparsity constraints have gained a lot of attention in different fields of mathematics. Due to the influential paper [4] sparsity constraints are by now a very popular tool for the regularization of inverse problems, see e.g. [14,18,6,1]. The most popular algorithm for the minimization of regularizing functionals with sparsity constraints is the iterated soft thresholding algorithm which was introduced in [7,17]. It is now a folk wisdom that this algorithm converges very slowly. The aim of this article is twofold. First we outline different approaches to the minimization of regularizing functionals with sparsity contraints by means of generalized gradient methods and a semismooth Newton approach and analyze the convergence behavior of the obtained algorithms. Second we present different numerical experiments that illustrate the convergence speed of the different methods. To fix notation for this article let K : 2 → H be a linear and bounded operator mapping the sequence space 2 to some Hilbert space H. The regularizing functional we consider is where f ∈ H, w k ≥ w 0 > 0 and 1 ≤ p ≤ 2. The sequence w = (w k ) plays the role of the regularization parameter and regularizes each component of u independently. The problem above may be motivated as follows. We are given a linear operator equation Au = f with a linear and bounded operator A : H → H and possibly noisy data f . Often the inversion of A is ill-posed and practically impossible. Hence regularization is required. A special 4th class of regularization methods is, as motivated in [4], the regularization by sparsity constraints. Let (ψ k ) k be an orthonormal basis of H and assume that the unknown solution u * is composed of a few components in this basis. A way to use this knowledge as a regularization is to minimize the functional With the help of the synthesis operator B : 2 → H given by Bc = k c k ψ k and the operator K = AB we can rewrite this precisely as (1). The outline of the paper is as follows. The Section 2 introduces the generalized gradient methods and show how they can be applied to the minimization of the functional (1). Section 3 shows briefly how Newton methods may be used and Section 4 treats the problem how to check if an algorithm for the minimization of (1) has converged without knowing the true minimizer. Section 5 presents numerical experiments on the different methods and the last section gives a short conclusion. Generalized gradient methods This section introduces generalized gradient methods for the minimization of functionals of type (1). These methods can be motivated as follows. In the theory of optimization an important class of problems are constrained minimization problems where Ω is a convex and closed subset of a Hilbert space H and F : H → R. Two well known algorithms for the solution of these problems are the gradient projection method and the conditional gradient method. Both methods will be described in the following subsections. Since the idea for their generalization is the same for both of them, we give it now: The constrained minimization problem (2) can be rephrased as where I Ω is the indicator function of the set Ω, i.e. it is zero for u ∈ Ω and infinity otherwise. Note that I Ω is a convex and lower semicontinuous functional (the latter since Ω is closed) and hence we can consider a generalization of constrained minimization problems by for a general convex and lower semicontinuous functional Φ. The gradient projection method and its generalization Assume that the functional F is differentiable and that F is Lipschitz continuous with constant L. The gradient projection method for the minimization of (3) reads as follows: (i) Choose an initial value u 0 ∈ Ω and a stepsize 0 < s < 2/L. (ii) Update by the iteration where P Ω denotes the orthogonal projection onto the set Ω. Hence, the gradient projection method does a gradient descent step and projects the result back onto Ω. To motivate the generalization we make the observation that the projection onto Ω is characterized by If we now deal with the problem (4) we introduce the mapping as a generalized projection (where we omitted the dependence on Φ). Note that the mapping P s is also called proximal mapping in the context of variational analysis [15]. We state the generalized gradient projection method as: (i) Choose an initial value u 0 such that Φ(u 0 ) < ∞ and a stepsize 0 < s < 2/L. (ii) Update by the iteration u n+1 = P s (u n − sF (u n )). Gradient projection methods have been used for minimization problems with sparsity constraints also in [5,8]. We now turn to the concrete problem of minimizing (1), i.e. in this case we have F (u) = Ku − f 2 /2 and hence F (u) = K * (Ku − f ) and L ≤ K 2 . The functional Φ(u) = k w k \|u k \| p is known to be convex and lower semicontinuous. The corresponding generalized projection is and is known to be a shrinkage operator. It is given by where the component functions S w,p are where G w,p (y) = y + wp sgn(y)\|y\| p−1 , see [4,12] for detailed explanations. Hence, the generalized projected gradient method applied to the minimization of (1) now resembles the iterated soft thresholding: (i) Choose an initial value u 0 such that Φ(u 0 ) < ∞ and a stepsize 0 < s < 2/ K 2 . (ii) Update by the iteration Note that the stepsize s = 1 as in [4] is ok as soon as K < √ 2 which is a little weaker than K < 1 as needed in [4]. The paper [2] shows that the iterated soft-thresholding with p = 1 converges with linear speed towards the minimizer as soon as the operator K is injective. This means that there is a constant C > 0 and a λ ∈]0, 1[ such that for the iterates u n of the iterated soft-thresholding and the minimizer u * it holds This is a remarkable result since in the case p = 1 the iteration is only non-expansive and not contractive (i.e. the Lipschitz constant is exactly one) and linear convergence can not be obtained by Banach-like arguments. As argued in [2] the constant λ is typically very close to one and hence the convergence is in practice very slow. For non-injective K similar results are expected. The paper [5] deals with the possibility to speed up the iteration be choosing larger stepsizes while [8] uses Barzilai-Borwein stepsizes and a backtracking line search. The conditional gradient method and its generalization The conditional gradient method for problem (3) reads as follows: Here the generalization to problem (4) is straightforward: We calculate the descent direction as Note that v n can be written as v n = (∂Φ) −1 (−F (u n )) and hence we need that the subgradient ∂Φ is invertible. To apply this algorithm to the problem of minimizing Ψ from (1) we have to do a modification because the subgradient of Φ(u) = k w k \|u k \| p is not onto for p = 1. There are at least two different possibilities. The first one is described in [3] and uses the splitting As shown in [3] this again leads to the iterated soft thresholding. Note that is this case one has to deal with a non-convex F . The second possibility is described in [1] and uses the following observation. Let u * be a minimizer of (1). Then one can estimate and hence, one has to a-priori bound for the coefficients of the minimizer. Consequently the minimizer of the functional does not change if we change the penalty term for values which are larger than S 0 . We do this by introducing the function The function ϕ p is a C 1 quadratic extension of the p-th power. We now consider the functional which has the same minimizers as Ψ from (1). It can be shown that the splitting has all the properties that ensure convergence of the generalized conditional gradient method, again, see [1]. It is an easy calculation to show that the descent direction v n has the form where we formally set Note that for p = 1 the function H 1 is the hard-shrinkage function. In [1] it is shown that the stepsize guarantees convergence of the algorithm. Moreover it is shown in [1] that the iterated hardthresholding converges linear for 1 < p ≤ 2 and like u n − u * ≤ Cn −1/2 for p = 1. This is theoretically slower than the iterated soft-thresholding but in practice the hard-thresholding is often faster especially when the sparsity is high. Higher order methods The previous section showed that iterated soft as well as hard thresholding are gradient type algorithms and hence a slow convergence can be expected. In this section the applicability of higher order methods for the minimization of (1) is discussed. We restrict ourself to the case p = 1. The minimizer u * of Ψ = F + Φ is characterized by Since the subgradient ∂Φ is a maximal monotone operator we can reformulate this characterization for every γ > 0 as The advantage of (10) over (9) is that it is an equation and not an inclusion and furthermore it may be "smoother" as will be seen below. In the special case (10) is The function S γw,1 defined by (5) and (6) is the well known soft thresholding operator. The paper [9] analyzes a semismooth Newton method for the solution of the equation (10) and hence, minimizing (1). It turns out that the semismooth Newton algorithm is an active set strategy (which is not surprising, see [11]) and has the following form: (iii) Calculate the active and the inactive set: (iv) Update according to Here we denoted with K * K\| A→A and K * K\| I→A the operator K * K restricted to the indicated sets. Note that step (iv) involves the inversion of the operator K * K\| A→A which is in general finite dimensional and hence, for injective K, only an ill-conditioned problem and not ill-posed. In [9] it is shown that the semismooth Newton method converges locally superlinear, i.e. if u 0 − u * is small enough, it holds How to check for convergence? Many algorithms for the minimization of (1) come without a justified stopping rule. Two possibilities are described below: (i) Motivated by (11) we can define the residual r n = u n − S w,1 (u n − K * (Ku n − f )) and terminate the iteration while the residual is smaller that some tolerance tol, i.e. r n < tol. with a proper, convex and lower semi-continuous Φ which also fulfills that Φ(u)/ u → ∞ for u → ∞. It is shown in [1] that the value is an upper bound for the distance to the minimal value, i.e. Here we denoted with Φ * the Fenchel dual of Φ. Furthermore it is shown that D(u n ) → 0 if u n is a sequence converging to a minimizer of Ψ. Hence, the value D(u n ) can serve as a stopping criterion for any iterative procedure minimizing functionals of the form (12). Note that the condition on Φ is fulfilled for the functionals (8) for 1 ≤ p ≤ 2. Numerical experiments In this section we illustrate the performance of the iterated soft thresholding from [4,7,17] and the iterated hard thresholding from [1]. Moreover we used a combined approach where iterated soft and hard thresholding are applied alternatingly. We also show an experiment on the semismooth Newton method. Performance for different regularization parameters In this experiment we analyzed the performance of the different algorithms for a fixed problem but for different regularization parameters. The problem under consideration is inverse integration (or differentiation [10, 16, 1]), i.e. the operator K : L 2 ([0, 1]) → L 2 ([0, 1]) given by The data f is given as (f (t k )) k=1,...,N with t k = 1 N k. We discretized the operator K by the matrix The minimization problem reads The true solution u 0 is given by small plateaus and hence the data f δ = Ku 0 + δ is a noisy function with steep linear ramps. Figure 1 shows our sample data. The results of the 1 minimization after 1000 iterations of the soft, hard and combined thresholding is shown in Figure 2 while Figure 3 and Table 1 report the obtained functional value and the residual norm. Table 2. Comparison of the CPU time in seconds and the number of iterations to reach a given residual norm. The problem under consideration is inverse integration with 5% noise and a regularization parameter w k = 5 · 10 −3 . Table 2. Left: CPU times in second, right: number of iterations. Note that for a larger regularization parameter the iterated soft thresholding performs well concerning the residual norm, while for smaller values it gets worse. The combined approach is always good concerning the residual norm while for the smallest regularization parameter the iterated hard shrinkage gives the smallest residual norm. Another observation is that the functional value gives different results. Here the iterated hard thresholding is always good and the iterated soft thresholding performs comparably bad. Scaling of the algorithms In this experiment we assess how the computational cost and the number of iterations grow with the size of the problem. Again we considered the inverse integration problem from Subsection 5.1 with problem sizes from 32 to 512. We stopped the different algorithms when the residual norm r n falls below the value 5 · 10 −3 . Table 2 and Figure 4 report the results. The combined approach is always better than the soft and the hard thresholding, both for CPU time and the number of iterations. We emphasize that the stopping criterion was the residual norm. It turned out in our examples that the iterated hard thresholding reduces the functional value much faster than the iterated soft thresholding, see Figure 5 and Table 3 for an illustration of this effect. Figure 5 also reports the evolution of the estimator D from (13). Note that this value decays much faster for the soft thresholding and the combined approach than for the the hard thresholding. We can conclude that neither the residual norm nor the estimator D estimates the decay of the functional value properly: a small functional value may lead to a large residual norm and vice versa. Table 3. Number of iterations to reach a given functional value Ψ for the different methods. The problem is the same than in the last line of Table 2. The minimal value of Ψ in this problem is 3.94. Deblurring in a Haar basis As an example for the performance of the semismooth Newton method from Section 3 we consider a blurring operator A : L 2 ([0, 1]) → L 2 ([0, 1]) given by Au = k * u with the kernel k(x) = c (1 + x 2 /λ 2 ) −1 with λ = 0.01. We choose c such that k dx = 1 and consider u to be extended periodically to R in order to evaluate the convolution integral. In this example we work with a synthesis operator B : 2 → L 2 ([0, 1]) mapping coefficients (c k ) to a function u = k c k ψ k where the (ψ k ) form the orthonormal Haar wavelet basis [13]. Hence, the operator under consideration K = AB is a blurring after a Haar wavelet synthesis. We start with a given function u which is piecewise constant. The data f is computed as f = Au + noise such that we have 25% relative error, i.e. f − Au / f = 0.25. The Haar coefficients of u have been reconstructed by minimizing (1). As a comparison we also computed the classical Tikhonov regularization, which amounts to the minimization of w k \|c k \| 2 . Figure 6 and Table 4 show the results of both 1 and the above 2 regularization where we discretized the problem to 1024 Haar wavelets. The parameters w k have been tuned by hand to produce optimal results. Since the original data is quite sparse in the Haar wavelet basis, the 1 -reconstruction leads to much better results. It also turned out that the algorithm is robust with respect to different initial values u 0 . We tested several initial values (starting at zero, at K * f or at a random position) and the observed convergence behavior was very similar in all cases. The SSN method converged in six iterations and in 0.3 seconds (for comparison: soft thresholding converged in 329 iterations in 2.8 seconds, hard thresholding did not converge within a reasonable time and the combined approach needed 412 iterations and 3.3 seconds). n Ψ(u n ) r n 1 3.3920e+01 2.9676e+06 2 1.3905e+02 3.2499e+04 3 1.3326e+01 6.2647e+05 4 7.9347e+00 1.7517e+04 5 6.0006e+00 8.0510e-02 6 5.9823e+00 5.7542e-09 Table 4. Illustration of the performance of the SSN method for deblurring in a Haar basis. The second column shows the decay of the function value Ψ while the third column shows the norm of the residual. The data is the same than in Figure 6. Conclusion We have seen that the well known iterated soft thresholding for minimization problems with sparsity constraint can be seen as either a generalized projection gradient method or a generalized conditional gradient method. Furthermore we summarized results on iterated hard thresholding as a generalized conditional gradient method and a semismooth Newton method for the same problem. While for both soft and hard thresholding one only needs to apply the operator and its adjoint once for each iteration in the semismooth Newton method a finite (probably small) linear system of equations has to be solved. In our numerical experiments we also investigated an approach which combines the soft and the hard thresholding by simply using them alternatingly. It turned out that this combination almost always improves the convergence when measured in different terms like the residual norm, the functional value on the estimator D from (13). The semismooth Newton method converges fast but no global convergence result is available at the moment.	2022-06-28T01:43:30.810Z	2008-01-01T00:00:00.000	{ "year": 2008, "sha1": "d3e5245a9be7bd734d6e0775006b195f28a80ecd", "oa_license": null, "oa_url": "https://doi.org/10.1088/1742-6596/124/1/012031", "oa_status": "GOLD", "pdf_src": "IOP", "pdf_hash": "d3e5245a9be7bd734d6e0775006b195f28a80ecd", "s2fieldsofstudy": [ "Mathematics", "Computer Science" ], "extfieldsofstudy": [ "Physics" ] }
267162958	pes2o/s2orc	v3-fos-license	Heterogeneous integration of high-k complex-oxide gate dielectrics on wide band-gap high-electron-mobility transistors Heterogeneous integration of dissimilar crystalline materials has recently attracted considerable attention due to its potential for high-performance multifunctional electronic and photonic devices. The conventional method for fabricating heterostructures is by heteroepitaxy, in which epitaxy is performed on crystallographically different materials. However, epitaxial limitations in monolithic growth of dissimilar materials prevent implementation of high quality heterostructures, such as complex-oxides on conventional semiconductor platforms (Si, III-V and III-N). In this work, we demonstrate gallium nitride (GaN) high-electron-mobility transistors with crystalline complex-oxide material enabled by heterogeneous integration through epitaxial lift-off and direct stacking. We successfully integrate high-κ complex-oxide SrTiO3 in freestanding membrane form with GaN heterostructure via a simple transfer process as the gate oxide. The fabricated device shows steep subthreshold swing close to the Boltzmann limit, along with negligible hysteresis and low dynamic on-resistance, indicating very low defect density between the SrTiO3 gate oxide and GaN heterostructure. Our results show that heterogeneous integration through direct material stacking is a promising route towards fabricating functional heterostructures not possible by conventional epitaxy. R ecent advances in producing ultrathin freestanding singlecrystalline membranes have enabled heterogenous integration of dissimilar crystalline materials in a single electrical or photonic device, opening a path towards creation of devices with enhanced performance and functionalities 1,2 .The focus of recent studies was mainly on the membrane generation method and a rough demonstration of a prototype device fabricated via heterogeneous integration [3][4][5] .However, to advance this field to the next stage, it is pivotal to demonstrate the possibility of creating a device with state-of-the-art performance.Many aspects of heterogeneous integration of dissimilar materials are unknown, but perhaps the most important is the interface quality between the transferred single-crystalline membrane and the host heterostructure.To verify this, we have fabricated gallium nitride (GaN)-based high-electron-mobility transistors (HEMT) with a heterogeneously integrated SrTiO 3 gate oxide layer and characterized its performance.We find that with careful fabrication and transfer of the gate oxide membrane, the device performance in regard to the oxide/HEMT interface show excellent quality, matching or exceeding that of conventionally deposited amorphous gate oxides as well as in-situ grown SiN oxides [6][7][8] . GaN-based HEMT is one of the most promising structure for high-power and RF applications owing to its superior properties, such as high electron mobility and high breakdown field [9][10][11] .To further improve the performance and reliability beyond conventional GaN HEMTs with Schottky metal gate, metal-oxidesemiconductor (MOS)-HEMTs structures have been proposed to suppress gate leakage and passivated the GaN surface from ambient.In this regard, MOS-GaN HEMTs with various dielectric materials such as Al 2 O 3 and HfO 2 have been demonstrated [12][13][14] .In MOS-HEMT, the crystallization and interfacial quality of the gate dielectric material play a crucial role in determining the performance 15 . Among various dielectric materials, complex-oxide materials have attracted considerable interest due to their diverse functional properties such as high dielectric constant (high-κ), ferroelectricity, magnetism, and superconducting properties making complex-oxides an attractive material system for developing nextgeneration devices 16,17 .However, epitaxial limitations in monolithic growth of dissimilar materials make it difficult to integrate single-crystalline complex-oxide materials with other material platforms such as GaN.The epitaxy of complex-oxides on substrates with different lattice constants and thermal expansion coefficients results in growth of polycrystalline films with inferior material properties.In other words, epitaxial limitations make it difficult to integrate complex-oxide materials onto conventional semiconductors while maintaining its excellent functional properties 18 . To address this challenge, recent advances in fabricating singlecrystalline freestanding complex-oxide membrane techniques have paved the way to seamlessly integrate complex-oxides with any arbitrary semiconductor platforms [19][20][21] .In this work, we demonstrate state-of-the-art GaN HEMTs utilizing heterogeneously integrated single-crystalline strontium titanium oxide (SrTiO 3 , abbreviated as STO) gate dielectric films.Epitaxial liftoff and direct stacking of freestanding membrane enables integration of crystalline complex-oxide materials on GaN HEMT platforms.STO, a representative perovskite material, exhibits ultrahigh dielectric constant (κ ~300 at room temperature) and comparable breakdown field with conventional dielectric materials 22,23 .This material was recently utilized to demonstrate 2D-FETs successfully 24 .The fabricated devices show excellent electrical characteristics such as negligible hysteresis (ΔV), low subthreshold swing (SS) close to the Boltzmann limit, and low dynamic on-resistance.We conclude that the pristine interface between the transferred complex-oxide membrane and GaN is attributed to the superior performance of the devices. Results Structure of the STO/GaN HEMTs. Figure 1a-c shows a 3D schematic illustration, photograph, and optical microscope image of the fabricated STO/GaN HEMT device.Centimeter-scale freestanding STO membrane with a thickness of ~25 nm was transferred onto the AlGaN/GaN HEMT heterostructure as the gate insulator.The channel width, channel length, and gate length are 60 µm, 30 µm, and 4 µm, respectively.Figure 1d shows the energy band diagram of the fabricated device (the characterization and fabrication procedure are shown in Supplementary Figs. 1, 2, respectively).With an estimated valence band offset of ~0.5 eV and conduction band offset of ~0.1 eV, the STO gate insulator forms a type-I straddling band alignment with AlGaN [25][26][27][28][29][30][31] .Typically, the type-I negative band alignment of gate dielectric and AlGaN can potentially lead to injection of electrons from the gate toward the interface of the gate insulator and AlGaN due to the low energy barrier to electrons of negative band offset 32 .Furthermore, the injected electrons may be readily captured by trap states near the interface (interface and border traps) that originate from the poor interface quality and defects in the gate insulator.These electron trapping processes may negatively impact the performance of the HEMT device by causing reliability issues, such as decrease in current level and an increase in dynamic on-resistance as a result of decreasing electron density in the 2DEG at the subsequent on-state after the off-state bias stress 33 .However, a clean interface quality and high crystallinity of the gate insulator results in low trap states near the interface, which can compensate for the adverse effects of the type-I negative band alignment and enable highly reliable operation of the devices even with type-I negative band alignment.Electrical characteristics.Figure 2 shows the electrical characterizations of the fabricated device.Figure 2a shows the bidirectional transfer characteristics (I DS -V GS ) normalized with channel width for a drain voltage (V DS ) of 0.1, 1 and 5 V.The inset shows a zoomed in subthreshold region of the transfer curve, showing negligible drain induced barrier lowering (DIBL) for increasing V DS .Figure 2b shows an identical in linear scale, confirming stable device operation with negligible hysteresis, which imply very low defects at the oxide/HEMT interface.To confirm the output characteristics, we measured the output curve (I DS -V DS ) normalized to the channel width for a V GS range from −4 to 1 V, as shown in Fig. 2c.Regarding the pulse operation of digital circuit systems, a large hysteresis during the on-and off switching of the transistor critically degrades the stability and power efficiency of the circuit.Therefore, demonstrating a device with negligible hysteresis is crucial for ensuring stability and increasing power efficiency in digital circuits. As shown in the Fig. 2d, the device exhibits the ON/OFF ratio ( ~2.6 × 10 7 ) at V DS = 5 V and the hysteresis (ΔV) of the device is nearly zero ( ~0.04 mA) at 1 µA mm −1 .Since the hysteresis behavior in MOS-HEMT structures is typically caused by the trapping and detrapping mechanism due to border traps in the gate dielectric 34 , negligible hysteresis of the STO/GaN MOS-HEMT implies that the STO/GaN HEMT is free from border traps owing to the single-crystalline nature of the STO membrane and a clean STO/GaN interface.Furthermore, Fig. 2e shows the subthreshold swing (SS) extracted from the bidirectional transfer characteristics, showing an ideally low minimum SS (SS min ) value of approximately 62 mV dec −1 in both forward and reverse direction, which is close to the Boltzmann limit at room temperature (60 mV dec −1 ).By utilizing the following equation, the interface trap density (D it ) can be estimated from the SS value: where k is Boltzmann constant, T is temperature and C STO is the capacitance of STO 23,24 .The estimated D it at room temperature is approximately 6.33 × 10 11 eV −1 cm −2 , which is even lower than MOS-HEMTs with conventional amorphous-based gate dielectric materials 13,35 .Thus, the steep SS of our device, which originates from the clean interface of the STO/GaN HEMT, indicates excellent gate controllability, and shows that it's an advantageous structure for low power consumption applications.Consequently, owing to the pristine interface of the STO/GaN and the high crystallinity of STO, the STO/GaN HEMT exhibits outstanding characteristics in terms of hysteresis and SS min compared to the other works, as shown in Fig. 2f [36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51] . The STO/GaN HEMT shows the high off-state breakdown voltage (V BR ) of 414 V as shown in Supplementary Fig. 3.The breakdown field (E BR ) of device is calculated to be ~2 MV cm −1 .Supplementary Fig. 4 shows the gate leakage characteristics of the STO/GaN HEMT.It should be noted that the gate leakage in forward bias is considerably affected by the band alignment and barrier height.As shown in Fig. 1d, STO gate insulator forms a type-I straddling band alignment with AlGaN, resulting in higher gate leakage in forward bias region due to the low energy barrier to electrons by negative band offset. Dynamic on-resistance degradation.To further investigate the interface quality of our STO/GaN HEMT device, we measured the dynamic on-resistance (R on,D ) by varying the quasi-drain voltage (V DSQ, ), which indicate the reliability of HEMTs under high quasi-bias stress.Figure 3a, b illustrates the trap states near the interface (interface and border traps) and the mechanism of the dynamic on-resistance effect, respectively.The R on,D indicates the ratio of R on before and after applying a stress voltage, V DSQ , at the off state.Due to the trapping of electrons at the interface between the GaN and gate dielectric layer (interface and border traps) during the bias stress, the current degrades after the bias stress, thereby increasing the R on 52 .While applying a high DSQ at the off state (V GS < V TH ), the potential differences between the gate and drain electrode generate the electric field in the vertical direction, inducing electrons injection from the gate toward the interface of the gate insulator and AlGaN as shown in Fig. 3b (left).The injected electrons from the gate may be readily captured by trap states near the interface of the GaN and gate dielectric layer that originate from the poor interface quality and defects in the gate insulator, collapsing the current level due to the decreasing electron density in the 2DEG during the on state (V GS > V TH ) as shown in Fig. 3b (right).Accordingly, this allows us to investigate the interface quality of the STO/GaN by analyzing the degradation of on-state current level and R on,D 36,53 .The periodic drain voltage for high quiescent (V DSQ , V GSQ ) and ON state was applied using a pulsed I-V system.The pulse width of V DSQ , V GSQ , and ON state are 3 ms and 2 ms, respectively.The V GSQ was kept at −6 V, while V DSQ was varied from 0 to 40 V, and the ON state drain voltage swept from 0 to 10 V as shown in Fig. 3c. Figure 3d exhibits the output characteristics for various V DSQ and V GSQ combinations, showing only 5 % degradation of the on-state current level compared to the initial state (V GSQ = V DSQ = 0 V) during the bias stress.As the amplitude of V DSQ increases, the electric field generated by the potential drops between the gate and drain electrodes is enhanced, leading to the increases of the electron trapping into the interface trap at the GaN/dielectric layer. To evaluate the influence of R on,D as a function of the quiescent bias (V DSQ ), we calculated the R on,D by increasing the V DSQ from 0 V to 40 V with 5 V steps as shown in Fig. 3e.Owing to the high crystalline quality of STO and the clean interface of the STO/ GaN, the maximum R on,D only increases up to 7 %, which is much lower compared to conventional GaN MOS-HEMTs utilizing deposited amorphous oxides 54 . TEM characterization of STO/GaN interface.Finally, to directly investigate the interface of the STO/GaN HEMT, a cross-sectional TEM measurement was conducted.Figure 4a shows the top view SEM image of the fabricated STO/GaN HEMT device and the investigated area of the TEM sample.The TEM was performed at the AlGaN/STO interface, (including a GaN capping layer) as illustrated in the left image of Fig. 4b. Figure 4b (right image) shows the TEM image of at the STO/GaN/AlGaN interface, which showed no defects, airgaps, unexpected interfacial layers or residues at the STO/GaN interface.We suspect that the dangling bonds of the transferred STO membrane may form atomic bonding with the underlying substrate (in our case, GaN HEMT heterostructure) by the thermal annealing process, which consistent with the previous works [55][56][57][58][59] (the mechanism of the interface bonds formation is schematically illustrated in Supplementary Fig. 5).Moreover, the selected area electron diffraction (SAED) pattern (inset) of the STO region, verified the crystalline nature of the transferred STO membrane.We believe these results, along with the electrical analysis of the gate oxide interface, strongly support that the reliable operation and excellent performance of the fabricated STO gate oxide HEMT is a result of a pristine interface between highly crystalline STO gate oxide and GaN. Conclusions In summary, we heterogeneously integrated crystalline complexoxide material on AlGaN/GaN HEMT as a gate dielectric by epitaxial layer transfer approach.Using STO with ultrahigh-κ properties as the gate oxide, we fabricated a MOS-HEMT device.The fabricated devices exhibited a negligible hysteresis (ΔV) at a drain current of 1 µA/mm, and a minimum SS value of 62 mV dec −1 .We attribute these results to an extremely clean interface between the STO and GaN, free from interface and border traps.The dynamic on resistance measurements were performed for further interface quality analysis, in which our device only showed a maximum resistance increase of 7%. Finally, we confirmed through TEM that no unwanted interfacial layer, residues, or airgaps between STO/GaN exists, and that the transferred STO maintains its crystalline nature.Our results demonstrate the potential of heterogeneous integration of complex-oxide materials with mature semiconductor technologies, substantially expanding the possibility of creating high performance electrical and photonic devices with novel functionalities. The integration of STO with AlGaN/GaN HEMT structure begins with the successive epitaxial growth of a water-soluble strontium aluminum oxide (Sr 3 Al 2 O 6 , abbreviated as SAO) sacrificial layer, followed by the growth of STO gate oxide on a single-crystalline STO (001) substrate by pulsed-laser deposition (PLD).It has been reported that the epitaxial growth of oxide film on the SAO template allows the transfer of single-crystalline STO membranes without fundamental thickness limitation 60 .The single-crystallinity of the epitaxially grown STO film on SAO/ STO substrate is confirmed using X-ray diffraction (XRD) and electron backscatter diffraction (EBSD) map, as shown in Supplementary Fig. 1a-c.After deposition of a poly(methyl methacrylate) (PMMA) supporting layer on the as-grown STO/ SAO/STO substrate via spin-coating, the stack was immersed in deionized (DI) water for ~24 hours to completely dissolve the SAO sacrificial layer 61 .The freestanding STO membrane with a thickness of ~25 nm was then transferred onto the AlGaN/GaN HEMT structure followed by removal of the supporting layer by acetone and rinsed by isopropyl alcohol (the complex-oxide membrane transfer procedure schematically illustrated in Supplementary Fig. 2). The transferred membrane was patterned through standard photolithography and etched using a combination of ion milling and etching in diluted hydrofluoric acid (HF) solution.We believe that the HF etch removes most of the defects caused by the ion milling in the gate oxide membrane, leading to excellent characteristics.Mesa isolation was conducted by inductively coupled plasma reactive ion etching (ICP-RIE) using a mixture of BCl 3 /Cl 2 gas.A Ti/Al/Ti/W (20/120/20/30 nm) stack was deposited for the source/drain electrode via e-beam evaporation, followed by rapid thermal annealing at 500 °C for 2 min in N 2 environment.Finally, a Ni film ( ~500 nm) was deposited as the gate electrode via plasma sputtering (the low magnification crosssectional image of the fabricated device is shown in Supplementary Fig. 6). Electrical and material characterizations.The electrical properties of the fabricated devices were measured using a semiconductor parameter analyzer (Keithley−4200A-SCS) with a 4255-RPM module to apply pulsed signals.The structural and interfacial analysis was performed using a scanning electron microscope (SEM), focused ion beam (FIB), and transmission electron microscopy (TEM).SEM measurements were performed using JEOL high-resolution SEM (IT-500HR), ZEISS SEM with an EBSD detector.The cross-sectional TEM specimens of the fabricated devices were prepared using a Ga-focused ion beam milling (ZEISS crossbeam 540) technique.TEM measurements were performed using a JEOL ARM 200 F (NEOARM). Fig. 1 Fig. 1 Structure of the STO/GaN HEMTs.a Schematic illustration, b photograph, c optical microscopy image and d energy band diagram of fabricated AlGaN/GaN high-electron-mobility transistors (HEMT) with SrTiO 3 (STO) gate dielectric. Fig. 2 Fig. 2 Electrical characteristics.a Normalized bidirectional transfer curves of the device at drain-source voltage (V DS ) = 0.1, 1 V, 5 V, respectively in log scale and b in linear scale.c Normalized output curves in gate-source voltage (V GS ) range of -4 to 1 V. d Normalized bidirectional transfer curves at V DS = 5 V.The inset shows hysteresis (ΔV) at I DS = 1 µA mm −1 .e The subthreshold swing (SS) versus I DS curve for forward and reverse direction sweep.f Benchmark of minimum SS (SS min ) and ΔV of the SrTiO 3 (STO)/GaN high electron mobility transistors (HEMT) compared to previously reported works of metal-oxide-semiconductor (MOS)-HEMTs. Fig. 3 Fig. 3 Dynamic on-resistance degradation.a Schematic illustration of trap states near the interface.b Schematic illustration of physical mechanism of the dynamic on-resistance (R on,D ), as a result of decreased electron density in the two-dimensional electron gas (2DEG) at the subsequent on-state after the off-state bias stress.c Synchronous pulse signal of gate-source voltage (V GS ) and drain-source voltage (V DS ) scheme for R on,D measurement.d Normalized output curve measured with pulsed I-V system for the quiescent drain-source voltage (V DSQ ) range of 0 to 40 V, under the quiescent gate-source voltage (V GSQ ) of -6 V.The inset shows only 5% degradation of the on-state current level compared to the initial state during the bias stress.e R on, D /R on ratio as a function of V DSQ . Fig. 4 Fig. 4 TEM characterization of STO/GaN interface.a Plan-view scanning electron microscope (SEM) image of HEMT device, black box represents region of focused ion beam (FIB) milling for transmission electron microscope (TEM) analysis.b Cross-sectional TEM image of SrTiO 3 (STO)/GaN interface.The inset shows selected area electron diffraction (SAED) pattern of the transferred STO.	2024-01-24T16:56:38.547Z	2024-01-19T00:00:00.000	{ "year": 2024, "sha1": "f245c296b930b72b621bb2ca1bfe6bcd13f4f374", "oa_license": "CCBY", "oa_url": "https://www.nature.com/articles/s44172-024-00161-z.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "bca76d5f1b839d9c12d033a10348781a2a39df67", "s2fieldsofstudy": [ "Engineering", "Materials Science", "Physics" ], "extfieldsofstudy": [] }
248038260	pes2o/s2orc	v3-fos-license	Reactive oxygen species (ROS)-responsive size-reducible nanoassemblies for deeper atherosclerotic plaque penetration and enhanced macrophage-targeted drug delivery Nanoparticle-based therapeutics represent potential strategies for treating atherosclerosis; however, the complex plaque microenvironment poses a barrier for nanoparticles to target the dysfunctional cells. Here, we report reactive oxygen species (ROS)-responsive and size-reducible nanoassemblies, formed by multivalent host-guest interactions between β-cyclodextrins (β-CD)-anchored discoidal recombinant high-density lipoprotein (NP3ST) and hyaluronic acid-ferrocene (HA-Fc) conjugates. The HA-Fc/NP3ST nanoassemblies have extended blood circulation time, specifically accumulate in atherosclerotic plaque mediated by the HA receptors CD44 highly expressed in injured endothelium, rapidly disassemble in response to excess ROS in the intimal and release smaller NP3ST, allowing for further plaque penetration, macrophage-targeted cholesterol efflux and drug delivery. In vivo pharmacodynamicses in atherosclerotic mice shows that HA-Fc/NP3ST reduces plaque size by 53%, plaque lipid deposition by 63%, plaque macrophage content by 62% and local inflammatory factor level by 64% compared to the saline group. Meanwhile, HA-Fc/NP3ST alleviates systemic inflammation characterized by reduced serum inflammatory factor levels. Collectively, HA-Fc/NP3ST nanoassemblies with ROS-responsive and size-reducible properties exhibit a deeper penetration in atherosclerotic plaque and enhanced macrophage targeting ability, thus exerting effective cholesterol efflux and drug delivery for atherosclerosis therapy. Introduction Atherosclerosis is a chronic inflammatory disease, the underlying cause of many life-threatening cardiovascular events [1][2][3]. Intimal macrophages play a critical role in atherosclerotic progression [4][5][6]. At early stages, monocytes are recruited to the intima and differentiate into macrophages. Within the intima, those macrophages engulf excessive amounts of oxidized lipoprotein particles and turn into pathological macrophage/foam cells. Foam cells secrete inflammatory factors to induce a maladaptive inflammatory response, eventually leading to the destabilization of atherosclerotic plaques. Delivering drugs to intimal macrophage/foam cells to suppress plaque inflammation is a potential intervention strategy for atherosclerosis therapy [7][8][9]. Nanoparticles have been widely utilized as drug and gene carriers for atherosclerosis therapy [10][11][12]. Nanoparticles must penetrate a thick fibrous cap-mainly composed of vascular smooth muscle cells and collagenous fibers-through leaky vasculature and subsequent inflammatory cell-mediated sequestration (ELVIS) effect [13,14] before they reach pathological macrophage/foam cells [15,16]. Although particles in the range of 100-200 nm typically have prolonged circulation in the blood, Peer review under responsibility of KeAi Communications Co., Ltd. their penetration in atherosclerotic plaque is limited [17,18]. Instead, nanoparticles of smaller size (sub 40 nm) can effectively penetrate plaque tissue and reach intimal macrophage/foam cells [19], but their plaque accumulation is insufficient due to short circulation time [18,20]. To effectively end up in atherosclerotic lesions, nanoparticles are desired to have extended blood circulation and deep atherosclerotic plaque penetration. In atherosclerotic plaque, excess reactive oxygen species (ROS) are produced by pathologic resident cells [21][22][23]. Although ROS-responsive materials have been developed to utilize oxidative microenvironment for biomedical applications, including atherosclerosis therapy [24][25][26][27][28][29][30], using ROS stimulus to design a smart delivery system for more efficient delivery of drugs to atherosclerotic plaques has not been attempted before. In this study, we developed a ROS-responsive size-reducible nanocarrier on the basis of multivalent host-guest interactions of β-cyclodextrins (β-CD)/ferrocene (Fc) for targeted treatment of atherosclerosis (Scheme 1). In this delivery system, simvastatin (ST) was selected as a model statin drug to suppress plaque inflammation and promote cholesterol efflux [31][32][33]. β-CD-anchored discoidal recombinant high-density lipoprotein (rHDL), termed as NP 3 ST , was utilized as a core as it was shown to possess not only deep plaque penetration property by virtue of its small size but also plaque targeting and cholesterol removal ability [34,35]. Fc was grafted onto hyaluronic acid (HA), and the resulting HA-Fc conjugates were utilized to crosslink NP 3 ST through multivalent host-guest interactions between β-CD/Fc to form ROS-responsive nanoassemblies (HA-Fc/NP 3 ST ) [36,37]. We engineered the particles of nanoassemblies to have the size in the range of 100-200 nm in order to have extended circulation time in the blood. HA was expected to guide the nanoparticles to accumulate in the plaque lesions area by targeting the CD44 receptors that are highly expressed in endothelial cells of plaque lesions [38][39][40]. Upon entering the plaque intima, the hydrophobic Fc was expected to be oxidized into hydrophilic ferrocenium ion (Fc+) under the action of excess ROS [41,42], leading to disintegration between β-CD/Fc and the disassembly of HA-Fc/NP 3 ST . The released NP 3 ST was expected to play an essential role in atherosclerosis by penetrating through plaque, targeting the macrophage/foam cells, and mediating cholesterol efflux. We characterized the structure, properties and functions of HA-Fc/NP 3 ST both in vitro and in vivo. We tested and confirmed the delivery efficiency and efficacy of HA-Fc/NP 3 ST in an animal model of atherosclerosis. Materials Sodium hyaluronic acid (HA, molecular weight 150 kDa) was purchased from Freda Biochem Co., Ltd. ( α-tocopheryl polyethylene glycol 1000 succinate (TPGS)-block-β-CD conjugates (TPGS-βCD) was synthesized by our lab [34]. The apolipoprotein A-I (apoA-I; 97% purity) was isolated from the industrial waste during the production of albumin in our lab [43]. Lecithin cholesterol acyl transferase (LCAT) was isolated from the human plasma by using agarose gel chromatography from human plasma [44]. Methanol and acetonitrile of chromatographic grade were used for HPLC analysis. All other reagents were of analytical grade. Synthesis and characterization of HA-Fc conjugates HA-Fc conjugates were synthesized by grafting Fc-NH 2 onto the HA backbone through amidation reaction [45]. The synthesized HA-Fc conjugates were characterized by using 1 H-NMR analysis in D 2 O at 500 MHz (Bruker AVANCE AV-500, Germany). The grafting degree (GD) of Fc in HA-Fc, defined as the number of Fc per 100 sugar residues of HA-Fc, was calculated by iron content (P, %) of the product shown in equation (1). The P was measured by Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES, PerkinElmer Optima8000, USA) under the conditions shown in Table S1. Besides, amantadine (Am) was utilized as a surrogate of Fc-NH 2 to synthesize HA-Am. The GD of Am in HA-Am conjugates was obtained from the 1 H-NMR spectrums by comparing the integrals of signals at δ 1.65-1.85 (peak III) and δ 2.10 (peak 3) in Fig. S1 [46]. Preparation of nanoparticles To prepare NP 3 ST , ST-loaded liposome (NP 1 ST ) was prepared by using ethanol injection method [35]. Then TPGS-block-β-CD (TPGS-βCD) conjugates were inserted into NP 1 ST via post-insertion method in water bath under a rotation rate of 100 rpm at 37 • C for 5 h to anchor β-CD on the surface of NP 1 ST (NP 2 ST ). Finally, NP 3 ST was obtained by incubating NP 2 ST with apoA-I (6 mg/mL) in the presence of sodium cholate under stirring at room temperature for 8 h [47]. HA-Fc/NP 3 ST was prepared by incubating HA-Fc with NP 3 ST . Briefly, HA-Fc was dissolved in Tris-HCl buffer (0.01 M, pH 8.0) and then dropwise added into NP 3 ST solution under stirring at room temperature for 8 h (molar ratio of Fc/β-CD was 1:1). After that, the mixture was incubated in an ultrasonic bath at room temperature for 3 h. All the operation was processed in dark. Finally, the mixture was sterile-filtered through 0.22 μm millipore filter, and HA-Fc/NP 3 ST was obtained. Besides, HA-Am/NP 3 ST , served as ROS-unresponsive control nanoassemblies, was prepared by incubating HA-Am with NP 3 ST under the same operations. Particle size, zeta potential, entrapment efficiency (EE), and drug loading (DL) measurements The nanoparticles' particle size and zeta potential were measured by dynamic light scattering (DLS) (Brookhaven Instruments, U.S.A) at 25 • C. The EE and DL of drug-loaded nanoparticles were quantified by using the mini-column centrifugation method [35,48]. Stability assessments The storage stability, dilution stability and LCAT resistance capacity of the nanoparticles were assessed by measuring the change of particle size and EE. Briefly, the nanoparticles were incubated in Tris-HCl buffer at 4 • C for 14 days, diluted by 50 times with Tris-HCl buffer and incubated with 4.67 μg/mL LCAT (mimicking plasma LCAT level in the examined subjects [49]) for 2 h and 24 h, respectively. The changes of particle size and EE were measured. The drug leakage ratio was expressed as the percentage of EE reduction after storage relative to the EE before storage [50]. Fluorescence resonance energy transfer (FRET) assay Nanoassemblies between HA-Fc and NP 3 ST was verified by FRET analysis. Rho 123 and RITC, used as FRET pairs, were conjugated onto HA-Fc and apoA-I, respectively [51]. Then, Rho 123-labeled HA-Fc and/or RITC-labeled apoA-I were utilized to prepare the fluorescence-labeled HA-Fc/NP 3 ST . The excitation and emission wavelengths of these channels were listed in Table S2, and the fluorescence intensities were measured by fluorescence spectrophotometer (RF-5301PC, Shimadzu, Japan). FRET index was calculated by using equation (2) [52]. Where F b and D b mean the fluorescence intensity of dual-labeled nanoassemblies in the channel F and D, F d and D d mean the fluorescence intensity of Rho 123-labeled nanoassemblies in the channel F and D. ROS-responsiveness assessment The H 2 O 2 -triggered oxidation of Fc was first assessed as described in Supporting Information. The ROS-responsiveness of the HA-Fc/NP 3 ST was then evaluated by using the FRET method. Briely, the dual fluorescence-labeled Rho123-HA-Fc/NP 3 ST -RITC was incubated in Tris-HCl buffer supplemented with 0.02 mM of H 2 O 2 (mimicking the ROS concentration in normal tissue [53]) or 1 mM of H 2 O 2 (mimicking the ROS concentration in atherosclerotic plaque [54]). The FRET index was determined at 0, 1, 2, 4 and 8 h post-incubation. Besides, ROS-responsiveness of HA-Am/NP 3 ST was evaluated. Furthermore, the morphologies of HA-Fc/NP 3 ST before and after treatment by 1 mM of H 2 O 2 for 2 h were imaged by transmission electron microscopy (TEM) (H-7650, Hitachi High-Technologies Corporation, Japan) [35]. Finally, nanoparticles were mixed in Tris-HCl buffer supplemented with 1 mM H 2 O 2 . After incubation for 0, 1, 2, 4 and 8 h, the mixture were diluted by 20 times, and particle size and zeta potential were determined. Verification of multivalent host-guest interaction Multivalent host-guest interaction property of the HA-Fc/NP 3 ST was evaluated by FRET method. The dual fluorescence-labeled Rho123-HA-Fc/NP 3 ST -RITC was incubated in Tris-HCl buffer supplemented with 0, 1, 5 or 50 equivs of free β-CD. The FRET index was determined at 0, 1, 3, and 12 h post-incubation. In vitro drug release studies Dialysis method was utilized to study in vitro release kinetics of ST. Briefly, the nanoparticle was mixed with 1 mM H 2 O 2 , and 3 mL of the mixture was loaded into dialysis tube (molecular weight cutoff at 3500 Da). The dialysis tube was immersed in the medium consisting of 100 mL PBS (0.01 M, pH 7.4) supplemented with 1 mM H 2 O 2 as well as 0.5% sodium dodecyl sulfonate (SDS). The dialysis tube was incubated at 37 • C water bath under a rotation rate of 100 rpm for 120 h. The released ST was sampled from the release medium at pre-determined time points and measured by using HPLC [35]. Drug release profiles in the absence of H 2 O 2 were determined for comparison. Cell viability assay Murine aortic endothelial cells (MAECs) and RAW 264.7 cells were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin sulfate at 37 • C and 5% CO 2 . The cytotoxicities of the formulated nanoparticles on MAECs and RAW 264.7 cells were assessed by using MTT method [35]. Construction of cell models MAECs (5.0 × 10 4 ) were seeded on a Transwell polyester membrane cell culture insert in 24-well plate (pore size 0.4 μm, diameter 6.5 mm, Corning Incorporated, Corning, NY, USA). The transendothelial electrical resistance (TEER) values of the endothelium monolayer were measured by using an electrical resistance meter (Millicell®-Electrical Resistance System, Millipore, USA), and a continuous endothelium monolayer formed until the cell monolayer exhibited a steady TEER value [55,56]. Macrophage spheroids were prepared with RAW 264.7 cells using the hanging-drop/agarose method [57,58]. In addition, macrophage monolayer was obtained by seeding RAW 264.7 cells in the lower chamber of Transwell. To construct injured endothelium-macrophage spheroid model, the obtained macrophage spheroids were handpicked and translocated to the lower chamber of Transwell. Then tumor necrosis factor-α (TNF-α) (10 ng/mL) was added to the upper chamber to damage the intact cell monolayer. In order to evaluate the change of the endothelium cell monolayer, TEER values were monitored and the expression of CD44 on MAECs was quantified using a flow cytometer (MACSQuant, Miltenyi Biotec, Gladbach, Germany). In addition, an injured endotheliummacrophage monolayer model was constructed via replacing the macrophage spheroids with macrophage monolayer [51]. Cellular drug uptake study In the injured endothelium-macrophage monolayer model and injured endothelium-macrophage spheroid model, the culture medium in the upper chamber was replaced with fresh medium containing nanoparticles and the culture medium in the lower chamber was replaced with fresh medium supplemented with 1 mM of H 2 O 2 . After incubation at 37 • C for different lengths of time, the culture medium and cells in the lower chamber were collected for quantification of the drug content by using HPLC and protein content via BCA assay (Beyotime Institute of Biotechnology, China), respectively. The amount of drug uptake was indirectly determined by subtracting the amount of drug in the culture medium from the total amount of the added drugs. The amount of cellular drug uptake was normalized based on the cell protein content of each group and expressed as μg mg − 1 protein [35,47]. Transendothelial investigation Transport of nanoparticles across the endothelium monolayer was evaluated by measuring the apparent permeability coefficient (Papp, cm/s) of ST [51,59]. The effect of free HA or sodium azide on ST permeation was investigated via pretreatment of the endothelial cells with free HA (10 mg/mL) for 2 h or sodium azide (1 mg/mL) for 1 h. In addition, permeation of nanoparticles across the continuous endothelial monolayer was determined. Penetration study Penetration of nanoparticles in the macrophage spheroids was evaluated by using confocal laser scanning microscope (CLSM). Briefly, the obtained macrophage spheroids were handpicked and translocated to 15 mm glass-bottom dishes. Next, hydrophobic fluorescent dye C6 was loaded into nanoparticles as a surrogate of ST, and the macrophage spheroids were treated with 1 mL of C6-loaded nanoparticles supplemented with 1 mM of H 2 O 2 for 8 h. After that, the macrophage spheroids were rinsed with PBS thrice and imaged by CLSM (LSM700, Carl Zeiss, Germany) using Z-stack scanning from the top of the macrophage spheroid to the equatorial plane [60]. The fluorescence intensity in the center of macrophage spheroids at equatorial plane was analyzed by Image-Pro Plus 6 to represent the penetration ability of nanoparticles [61]. Besides, penetration of nanoparticles in macrophage spheroids was evaluated in the absence of H 2 O 2 . Macrophage targeting studies Macrophage targeting behavior of nanoparticles was assessed using the injured endothelium-macrophage monolayer model. The C6-loaded nanoparticles were added to the upper chamber and the medium in the lower chamber was changed to fresh medium supplemented with 1 mM of H 2 O 2 . After incubation for 8 h, the fluorescence intensities of C6 in macrophages were determined by flow cytometry. To investigate the influence factors in macrophage targeting behavior of nanoparticles, the overexpressed CD44 on MAECs were blocked by pretreatment with free HA (10 mg/mL) for 2 h [51], the medium in the lower chamber was changed to the fresh medium without H 2 O 2 or the overexpressed SR-BI on macrophages were blocked by pretreatment with free apoA-I (50 mg/mL) for 2 h [62]. For fluorescent microscopic analyses, macrophages were cultured on the glass coverslips in the lower chamber of transwell. After incubation, the macrophages were rinsed with PBS thrice, counterstained with Hoechst 33342 (10 mg/mL) for 15 min and finally imaged under CLSM. Cholesterol efflux assay Cholesterol efflux assay was investigated in the injured endotheliummacrophage monolayer model. The RAW 264.7 cells in the lower chamber of Transwell were incubated with oxLDL (60 μg/mL) and NBDcholesterol (5 μM) for 24 h. Then, the cells were washed and treated with fresh medium supplemented with 1 mM of H 2 O 2 . In the meantime, nanoparticles were added to the upper chamber and incubated for 24 h. Finally, the amount of NBD-cholesterol in the media and macrophages lysates were quantified by a microplate reader at an excitation of 469 nm and an emission wavelength of 537 nm. Cholesterol efflux (%) was expressed as the percentage of fluorescence intensity in the media relative to the total fluorescence intensity in the media and cell lysates [63]. The effects of nanoparticles on the cholesterol content, permeability and fluidity of cell membrane of macrophages in the injured endothelium-macrophage monolayer model were evaluated [35]. RAW 264.7 cells in the lower chamber of Transwell treated with DMEM alone, or oxLDL alone were tested as control groups. Intracellular lipid deposition Intracellular lipid deposition was assessed in the injured endothelium-macrophage monolayer model. The RAW 264.7 cells in the lower chamber of Transwell were incubated with oxLDL (60 μg/mL) for 24 h. Then, the cells were washed and treated with fresh medium supplemented with 1 mM of H 2 O 2 . In the meantime, nanoparticles were added to the upper chamber and incubation for 24 h. Finally, the intracellular lipid deposition in the macrophages was imaged under inverted fluorescence microscope (IX53, Olympus, Japan) and the positive staining area (%) was quantified by Image-Pro Plus 6 [47]. RAW 264.7 cells in the lower chamber of Transwell treated with DMEM alone or oxLDL alone were tested as control groups. Macrophage phenotypic analysis RAW 264.7 cells seeded in the lower chamber of Transwell were incubated with oxLDL (60 μg/mL) for 24 h. Then, the cells were treated with fresh DMEM medium supplemented with 1 mM of H 2 O 2 . In the meantime, nanoparticles were added to the upper chamber. The cells were incubated for 24 h and then collected for macrophage phenotypic analysis [34]. RAW 264.7 cells in the lower chamber of Transwell treated with DMEM alone or oxLDL alone were tested as control groups. Inflammatory cytokine inhibition assays RAW 264.7 cells in the lower chamber of Transwell were incubated with oxLDL (60 μg/mL) for 24 h. Then, the cells were treated with fresh medium supplemented with 1 mM of H 2 O 2 . In the meantime, nanoparticles were added to the upper chamber and incubation for 24 h. After that, the medium in the lower chamber was centrifuged at 12000 rpm for 10 min at 4 • C. The supernatants were collected to measure the concentrations of interleukin-6 (IL-6), TNF-α and chemokine ligand 2 (CCL2) using ELISA kit (Dakewe Biotech Co., Ltd, China), respectively. RAW 264.7 cells in the lower chamber of Transwell treated with oxLDL alone were tested as a control group. Atherosclerosis model The animal experiments in this study were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. The protocol was approved by Institutional Animal Care and Use Committee of China Pharmaceutical University. Male wildtype C57BL/6 mice (6 weeks) were purchased from Qinglong Mountain Animal center (Nanjing, China). Male apoE-deficient mice (5 weeks) were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China) and utilized to develop an atherosclerosis model via feeding a high-cholesterol diet (21% fat and 0.15% cholesterol) for 16 weeks [51,64]. Pharmacokinetic study in wild-type C57BL/6 mice Hydrophobic fluorescent dye DiR was loaded into nanoparticles as a surrogate of ST. The wild-type C57BL/6 mice were randomly divided into 4 groups of three (n = 3) and injected with DiR-labeled Nanoparticles via tail vein. At fixed time points, an equal volume of blood samples was collected into 96-well black plates. An IVIS system (FX PRO, Kodak, USA) was utilized to measure the fluorescence intensity of blood samples at 0, 1, 2, 4, 8, 12 and 24 h post-injection. Tissue distribution in atherosclerotic mice The atherosclerotic mice were randomly divided into 4 groups of three (n = 3) and injected with DiR-labeled nanoparticles via tail vein. After injection for 4 h or 24 h, the mice were sacrificed. Major organs (heart, liver, spleen, lung, kidney) and aortic trees were collected and then imaged under an IVIS system using 720 nm excitation and 790 nm emission filters [64]. Plaque penetration and macrophage colocalization in atherosclerotic mice The atherosclerotic mice were randomly divided into 2 groups of three (n = 3) and injected with DiR-labeled nanoparticles via tail vein. At 4 h after injection, the mice were sacrificed. The aortic roots were collected and 8 μm thick paraffin sections were prepared, which were further processed with CD31 [65] or CD68 [66] immunostaining. Finally, the slides were observed by CLSM. Pharmacodynamics study in atherosclerotic mice The atherosclerotic mice were randomly divided into 5 groups of six (n = 6). All groups were treated with two injections per week (3-4 days apart) via tail vein for 12 weeks: (ⅰ) saline, (ⅱ) saline, (ⅲ) NP 3 ST , (ⅳ) HA-Am/NP 3 ST , (ⅴ) HA-Fc/NP 3 ST . Besides, the second group received ST orally per day during the treatment. The ST dose in each group was 15 mg/kg. Notably, the mice were fed with high-cholesterol diet during the therapeutic period. Finally, the mice were euthanized, the serum levels of inflammatory factors (IL-6, TNF-α and CCL2) were determined by using ELISA kit, and the aortic roots were collected for hematoxylin and eosin (H&E) staining, oil red O staining, CD68 immunostaining and CCL2 immunostaining [51]. Microscopic images of the aortic root sections were digitized and quantified with Image Pro Plus 6.0 software. Safety study in atherosclerotic mice The atherosclerotic mice were randomly divided into 2 groups of six (n = 6), which received saline or HA-Fc/NP 3 ST (15 mg/kg ST) two injections per week (3-4 days apart) via tail vein for 12 weeks. After that, blood samples were gathered for biochemical analysis of alanine aminotransferase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) via ELISA kit (Dakewe, China). Besides, main tissues (heart, liver, spleen, lung, kidney) were collected for H&E staining and imaged under an inverted fluorescence microscope. Statistical analysis All values were expressed as mean ± standard deviation (SD). Statistical analysis was performed with mann whitney u test or One-way ANOVA by SPSS 19.0 (IBM Corporation, USA). Significance was reported as p < 0.05, p < 0.01 or *p < 0.001. Characterization of HA-Fc conjugates and nanoassemblies HA-Fc and HA-Am conjugates were synthesized via amidation reaction. The 1 H-NMR spectra confirmed the presence of characteristic proton peaks of HA (glucoside H) and Fc-NH 2 (c) in HA-Fc, and the characteristic proton peaks of HA (glucoside H) and Am (III) in HA-Am, verified the successful synthesis of HA-Fc and HA-Am (Fig. S1). The GD of Fc and Am in the synthesized HA-Fc and HA-Am conjugates was 14.3 ± 1.0% and 15.3 ± 1.0%, respectively. Particle size, zeta potential, EE and DL of the prepared nanoparticles were measured (Fig. 1A-D). The particle size of NP 3 ST significantly increased after being crosslinked by HA-Fc and HA-Am. The zeta potentials of nanoassemblies became more negative because of the presence of negatively charged of HA. DL of the HA-Fc/NP 3 ST utilized in this work was 1.80 ± 0.01%. Nonetheless, HA-Fc/NP 3 ST with a higher DL could be prepared by increasing the mass ratio of ST/SPC during the preparation process (Table S3). Besides, the nanoparticles showed high stability after storage for 14 days or being diluted by 50 times because negligible changes of particle size and drug leakage were observed (Fig. S2). Compared with NP 3 ST , both nanoassemblies showed higher stability in the presence of LCAT for even 24 h, attributing to the shield effect of HA polymers [48]. The oxidation properties of Fc were investigated. Given •OH production in the Fenton reaction triggered by the oxidation of Fc, degradation of MB after capture of •OH was utilized to characterize the H 2 O 2triggered oxidation of Fc [67]. The results showed that degradation of MB was H 2 O 2 concentration-dependent and suggested that Fc was promptly oxidized by 1 mM of H 2 O 2 (Fig. S3). Consequently, 1 mM of H 2 O 2 , a typical ROS concentration close to the oxidative stress in atherosclerotic plaque [23,54,[68][69][70][71], was chosen as ROS-responsiveness condition in the following experiments in this work. FRET phenomenon was observed in Rho123-HA-Fc/NP 3 ST -RITC (Fig. 1E). The emission intensity of Rho123 at 524 nm decreased and that of RITC at 575 nm increased in Rho123-HA-Fc/NP 3 ST -RITC, indicating the energy transfer from fluorescence donor of Rho123 to acceptor of RITC and the coexistence of both components on the nanoassemblies. The FRET index was calculated as 1.768 ± 0.085, which was utilized to reflect FRET efficiency. Therefore, FRET method was utilized to confirm ROS-responsiveness of HA-Fc/NP 3 ST . The FRET indices of Rho123-HA-Fc/NP 3 ST -RITC in the presence of 1 mM of H 2 O 2 significantly decreased over a course of 8 h-incubation while Rho123--HA-Am/NP 3 ST -RITC was unresponsive (Figs. S4A and 1F). The ROS-triggered disassembly of HA-Fc/NP 3 ST was visually illustrated by TEM (Fig. 1G). NP 3 ST exhibited as small-sized discoidal particles and aligned as rouleaux [35], which changed into large-sized spheres with multi-seed structure surrounded by a white rim after being crosslinked by HA-Fc. However, HA-Fc/NP 3 ST was disassembled after incubation with 1 mM of H 2 O 2 for 2 h characterized by the loose and irregular shape as well as release of the small-sized particle. To directly verify the ROS-triggered size-reducible property of HA-Fc/NP 3 ST , the changes of particle size and zeta potential after mixing the nanoassemblies with 1 mM of H 2 O 2 were measured at predetermined time points. The particle size and zeta potential of HA-Fc/NP 3 ST after incubation with 1 mM H 2 O 2 for 8 h were close to that of NP 3 ST (Fig. 1H and I), which demonstrated the H 2 O 2 -triggered disassembly of HA-Fc/NP 3 ST and release of small-sized NP 3 ST . Furthermore, the multivalent host-guest interactions in HA-Fc/NP 3 ST were also verified by using FRET method (Figs. S4B and S5). FRET indices of Rho123-HA-Fc/NP 3 ST -RITC in the presence of 0, 1 and 5 equivs of free β-CD were almost unchanged over a course of 12 h-incubation while that promptly decreased against 50 equivs of free β-CD [72]. Subsequently, drug release in response to ROS was studied. Only 38.2% of the drug was released in 72 h in the absence of ROS (Fig. 1J), while almost 36% of drug was released within 4 h in the presence of 1 mM of H 2 O 2 and 55% of drug was released after 72 h-incubation (Fig. 1K). In contrast, drug release profiles of HA-Am/NP 3 ST in the presence or abscence of H 2 O 2 were almost no different. Taken together, we demonstrated that small-sized NP 3 ST were assembled into large-sized HA-Fc/NP 3 ST by HA-Fc conjugates based on multivalent host-guest interactions between β-CD/Fc. Upon encountering oxidative stress microenvironment mimicking the plaque intima, the hydrophobic Fc was oxidized into hydrophilic ferrocenium ion (Fc+) under the action of excess ROS, hence leading to disintegration between HA-Fc/NP 3 ST shows efficient transendothelial behavior, deeper penetration in macrophage spheroids and specific macrophage targeting ability HA-Fc/NP 3 ST at the ST concentration of 5-40 μg/mL exhibited no cytotoxicity on MAECs and RAW 264.7 cells (Fig. S6). The TEER change and CD44 expression of the endothelial monolayer after TNF-α stimulation were observed (Fig. S7), suggesting the successful construction of an injured endothelium model. Drug leakage ratio of nanoparticles during the process of transendothelial transport was negligible (Fig. S8). Therefore, the measured Papp values of the drug could represent the transport behavior of nanoparticles. Besides, the TEER of the endothelial monolayer after incubation with nanoparticles for 8 h stayed almost unchanged (Table S4), indicating that endothelial monolayer was not damaged by the nanoparticles. In the transendothelial transport assessment, NP 3 ST exhibited the highest transendothelial capability studied in the model of continuous endothelial monolayer [73], but HA-Fc/NP 3 ST and HA-Am/NP 3 ST showed higher Papp values in the model of damaged endothelial monolayer ( Fig. 2A). The pretreatment of the damaged endothelial monolayer with 10 mg/mL of free HA for 2 h [51] or 1 mg/mL of sodium azide for 1 h [74] significantly reduced transendothelial transport of HA-Fc/NP 3 ST and HA-Am/NP 3 ST . This observation suggests that the nanoassemblies crosslinked by HA polymer specifically accumulate to the damaged intercellular space and leak into the lower chamber with the help of HA-CD44 receptors affinity and that they cross the endothelial monolayer through energy-dependent transcellular transport pathway [51]. In fluorescent C6-loaded nanoparticles penetration assessment, macrophage spheroids were utilized to mimick three-dimensional structure of atherosclerotic plaque tissue and the penetration behaviors were evaluated by using CLSM (Fig. 2B and C). NP 3 C6 showed the highest penetration ability among all the tested groups probably ascribed to its small size. ROS-responsive HA-Fc/NP 3 C6 nanoassemblies penetrated deeper in macrophage spheroids than HA-Am/NP 3 C6 . Nonetheless, such behavior difference in penetration was not seen in the absence of H 2 O 2 . The result ascertains that HA-Fc/NP 3 C6 rapidly disassembled in response to the presence of H 2 O 2 and released smaller NP 3 C6 that penetrated deeper in the macrophage spheroids. Macrophage targeting behavior of nanoparticles was studied in the injured endothelium-macrophage monolayer model by using CLSM and flow cytometry. The C6 signal intensity increased in the following order: free C6, NP 3 C6 , HA-Am/NP 3 C6 and HA-Fc/NP 3 C6 (Fig. S9, 2D and 2E). Uptake of HA-Fc/NP 3 C6 by macrophages was significantly inhibited by the pretreatment of the endothelial cells with free HA, removal of H 2 O 2 in the lower chamber or pretreatment of the macrophages with free apoA-I, suggesting that HA-Fc/NP 3 C6 competes with free HA or apoA-I for targeting to macrophages and shows ROS-responsiveness. Cellular drug uptake profiles of nanoparticles in the macrophages of injured endothelium-macrophage monolayer model and injured endothelium-macrophage spheroid model were determined (Fig. S10). HA-Fc/NP 3 ST showed higher cellular drug uptake amount than either NP 3 ST or HA-Am/NP 3 ST attributing to its efficient transendothelial and macrophage spheroid penetration capacity as verified above. Taken together, HA-Fc/NP 3 ST shows efficient transendothelial behavior, a deeper penetration ability in macrophage spheroids and macrophage targeting in vitro. HA-Fc/NP 3 ST exhibits effective anti-atherosclerotic effects in vitro Cholesterol efflux from macrophages in the lower chamber of Transwell was assessed by measuring the reduction of NBD-cholesterol fluorescence intensity. Cholesterol efflux capacity increased gradually in the order: DMEM, free ST, HA-Am/NP 3 ST , NP 3 ST , HA-Fc/NP 3 ST . Free ST is reported to upregulate the expression of cholesterol efflux receptors on macrophage/foam cells [75], and NP 3 ST , the core particle, possesses favorable cholesterol removal ability [35]. HA-Fc/NP 3 ST showed higher cholesterol efflux capacity than free ST by 5.37-fold, NP 3 ST by 1.85-fold and HA-Am/NP 3 ST by 2.25-fold (Fig. 3A). Considering the majority of intracellular cholesterol accumulating in the cell membrane, the effect of nanoparticles on cell membrane property was evaluated. As observed, HA-Fc/NP 3 ST reduced cholesterol content (Fig. 3B) as well as improved permeability (Fig. 3C) and fluidity (Fig. 3D) of macrophage membrane. Oil red O staining was carried out for intracellular lipid deposition assessment. The capacity of nanoparticles to reduce intracellular lipid deposition was consistent with their cholesterol efflux capacity (Figs. 3E and S11). The changes of macrophage phenotypes were determined by flow cytometry. [76][77][78][79], but HA-Fc/NP 3 ST significantly polarized the macrophages to M2 phenotype ( Fig. 3F and G), which could be explained by cellular cholesterol efflux capacity of rHDL particles [34,35] and ST intervention [80,81]. We went on to study the inhibitory effect of nanoparticles on inflammatory cytokine IL-6 ( Fig. 3H), TNF-α (Fig. 3I) and CCL2 (Fig. 3J) secretion. We found that despite the stronger cholesterol efflux capacity of NP 3 ST than HA-Am/NP 3 ST , the latter was more potent in inhibiting the inflammatory cytokine secretion, presuming its higher efficiency bringing drugs to the cell. Compared with the untreated group, HA-Fc/NP 3 ST reduced the secretion of IL-6 by 56%, TNF-α by 57% and CCL2 by 54%. HA-Fc/NP 3 ST shows improved accumulation in plaque and stronger anti-atherosclerotic effects in vivo To verify the long circulation time of HA-based nanoassemblies, pharmacokinetics of the nanoparticles was evaluated in male wild-type C57BL/6 mice. After i.v. injection of DiR-labeled nanoparticles, DiR fluorescence intensity of blood samples colleted at a certain time interval was measured to represent the residual content of nanoparticles. Compared with free DiR or NP 3 DiR , fluorescence imaging indicated that HA-based nanoassemblies gained extended blood circulation (Fig. 4A and B). Nevertheless, the majority of nanoparticles were cleared from the blood in 24 h following injection. Tissue distribution of DiR-labeled nanoparticles was also assessed in the atherosclerotic mice after i.v. injection for 4 h and 24 h (Fig. 4C-E and S12). Compared with NP 3 DiR , HA-based nanoassemblies showed much higher accumulation in lesionprone aortic arch but reduced accumulations in the major organs (heart, liver, spleen, lung, kidney). The percentage of nanoparticle in plaques was calculated as the DiR fluorescence intensity in aorta/(total DiR fluorescence intensity of the main organs + DiR fluorescence intensity in aorta) × 100% [64]. HA-Fc/NP 3 DiR was found to accumulate in atherosclerotic lesions more efficiently than HA-Am/NP 3 DiR . We attributed it to the ROS-triggered disassembly and release of small-sized NP 3 DiR enabling deeper plaque penetration. Subsequently, distribution of the two nanoassemblies in atherosclerotic plaque was assessed. HA-Fc/NP 3 DiR (Red) penetrated deeply in the atherosclerotic plaque evidenced by the high fluorescence intensity in the deep of plaque tissue (CD31 labeled with green) (Fig. 4F). Meanwhile, HA-Fc/NP 3 DiR (Red) colocalized with the macrophages or de-differentiated vascular smooth muscle cells (VSMCs) (CD68 labeled with green) more prominently (Fig. 4G). Quantification analysis demonstrated that HA-Fc/NP 3 DiR penetrated deeper in atherosclerotic plaque by 1.48-fold and more effectively target to macrophages by 1.83-fold than HA-Am/NP 3 DiR (Fig. 4H). After that, in vivo anti-atherosclerotic efficacy of HA-Fc/NP 3 ST was confirmed more effective than either NP 3 ST or HA-Am/NP 3 ST ( Fig. 5A and B). Compared with the saline group, HA-Fc/NP 3 ST reduced plaque size by 53%, decreased plaque lipid deposition by 63%, lowered CD68-positive cells content (macrophages or de-differentiated VSMCs) by 62% and resulted in reduction of inflammatory factor CCL2 by 64%. Meanwhile, HA-Fc/NP 3 ST reduced serum inflammatory factor levels of IL-6 by 52% (Fig. 5C), TNF-α by 59% (Fig. 5D) and CCL2 by 48% (Fig. 5E) than the saline group, which suggested its potent inhibitory effects on systemic inflammation. Finally, safety study was performed in atherosclerotic mice. HA-Fc/NP 3 ST did not cause toxicity to the liver and kidney as similar levels of ALT, AST and ALP were observed in the saline and HA-Fc/NP 3 ST groups (Fig. 5F). Besides, HA-Fc/NP 3 ST did not cause pathological damages to the major organs (Fig. 5G). Collectively, HA-Fc/NP 3 ST exert extended blood circulation, accumulate in atherosclerotic lesions, penetrate deeply in plaque and target to the macrophages. Moreover, HA-Fc/NP 3 ST exhibit effective antiatherosclerotic activity but show almost no safety problems to the atherosclerotic mice. That is to say, HA-Fc/NP 3 ST has potential clinical use for the treatment of atherosclerosis via alleviating inflammation and lowering lipid deposition in plaque, while its complicated preparation process can be a challenge for large scale production. Furthermore, the effectiveness and in plasma from the atherosclerotic mice (n = 6), and (G) H&E histopathological staining of major organs (heart, kidney, liver, spleen, lung). Scale bar: 1000 μm for H&E, oil red O and CD68 as well as 200 μm for CCL2 in A, 200 μm for G; p < 0.05, p < 0.01, *p < 0.001 biosafety have been demonstrated in atherosclerotic mice only. Given that there exist obvious pathological differences between humans and mice, HA-Fc/NP 3 ST should be thoroughly evaluated in large animal models of atherosclerosis to better predict its efficacy and safety and then tested in clinical trials. Conclusions In this work, ROS-responsive size-reducible HA-Fc/NP 3 ST nanoassemblies were developed successfully for targeted atherosclerosis therapy. HA-Fc/NP 3 ST were assembled via multivalent host-guest interactions between β-CD/Fc with ROS-responsiveness in the atherosclerotic lesions. HA-Fc/NP 3 ST effectively crossed the injured endothelium via the recognition of HA-CD44 receptors, disassembled rapidly caused by ROS, penetrated deeper in macrophage spheroids and promoted macrophage-targeted cholesterol efflux. HA-Fc/NP 3 ST specifically accumulated in an atherosclerosis-prone region, effectively penetrated through the plaque and highly colocalized with the atherosclerotic plaque-associated macrophages. HA-Fc/NP 3 ST exhibited much improved antiatherogenic effects. In summary, rHDL-based ROSresponsive HA-Fc/NP 3 ST nanoassemblies represent a new drug delivery system for deeper penetration into atherosclerotic plaque and enhanced macrophage targeting, effectively enhancing cholesterol efflux and exerting anti-inflammatory therapy for atherosclerotic diseases. Declaration of competing interest There are no conflicts of interest to declare.	2022-04-09T15:08:34.672Z	2022-04-07T00:00:00.000	{ "year": 2022, "sha1": "c2d7a0a3196fbd67b03111954106211caf54d187", "oa_license": "CCBYNCND", "oa_url": "https://doi.org/10.1016/j.bioactmat.2022.03.041", "oa_status": "HYBRID", "pdf_src": "PubMedCentral", "pdf_hash": "d46f68d63bbde9723333c524aa7de0acdd48b010", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [] }
233394506	pes2o/s2orc	v3-fos-license	Consistency issues in Gaussian Mixture Models reduction algorithms In many contexts Gaussian Mixtures (GM) are used to approximate probability distributions, possibly time-varying. In some applications the number of GM components exponentially increases over time, and reduction procedures are required to keep them reasonably limited. The GM reduction (GMR) problem can be formulated by choosing different measures of the dissimilarity of GMs before and after reduction, like the Kullback-Leibler Divergence (KLD) and the Integral Squared Error (ISE). Since in no case the solution is obtained in closed form, many approximate GMR algorithms have been proposed in the past three decades, although none of them provides optimality guarantees. In this work we discuss the importance of the choice of the dissimilarity measure and the issue of consistency of all steps of a reduction algorithm with the chosen measure. Indeed, most of the existing GMR algorithms are composed by several steps which are not consistent with a unique measure, and for this reason may produce reduced GMs far from optimality. In particular, the use of the KLD, of the ISE and normalized ISE is discussed and compared in this perspective. I. INTRODUCTION Gaussian Mixtures (GMs) are a powerful tool often used to approximate probability distributions in very different application areas. In some contexts, such as in filtering and estimation, GMs approximate time-varying distributions that must be updated in real-time, and very often the number of components of the GM increase with time. For instance, in the field of target tracking in clutter [6], [15], [17], the Bayesian recursion produces a number of components that exponentially grows with time. In cases like this, if no action is taken, the problem becomes computationally intractable after few steps. To address such a drawback, many algorithms have been proposed in the literature in the last three decades that aimed at reducing the components of a given GM, while maintaining a substantial similarity with the original one. Most algorithms, such as those in [1], [4], [10], [11], [12], [13], [18], [19], [20], [21], [22], combine greedy coarse reduction steps with refinement steps, all with the common underlying principle of minimizing a given dissimilarity measure (D-measure, for short) between the original mixture and its reduced version. In general, the problem of Gaussian Mixture Reduction (GMR) can be cast into a nonlinear constrained optimization problem, where the optimization variables are the parameters of the reduced GM (weights, means and covariances). The underlying idea is that most of the information contained in the original GM should A. D'Ortenzio and C. Manes are with Department of Information Engineering, Computer Science and Mathematics, University of L'Aquila, 67100 L'Aquila, Italy, alessandro.dortenzio@graduate.univaq.it, costanzo.manes@univaq.it. This work has been supported by the Italian Government under CIPE resolution n. 70/2017, Centre of excellence EX-EMERGE be conveyed in the reduced GM (minimal loss). However, a lot of issues arise when tackling the GMR problem. First of all, although there are many D-measures that can be used, none of them has theoretical or practical features that make it preferable to others in all applications. A common problem shared by all D-measures is the presence of a large number of local minima in the GMR optimization problem. Some of the most used D-measures are the Kullback-Leibler Divergence (KLD) [9], the Integral Squared Error (ISE) [14] and its normalized version (NISE), and the Squared 2-Wasserstein distance [1], [5]. Among these, only the ISE and NISE have closed forms when applied to GMs, while only the KLD allows a closed form computation of the barycenter of a mixture, which is an important quantity in most reduction procedures. Considering that each D-measure has its own pros and cons, most existing reduction algorithms mix up heterogeneous actions inspired by different D-measures, driven mainly by the ease of computation and weakly supported by theoretical considerations. In this work the use of KLD, ISE and NISE in GMR algorithms is analyzed and discussed. The features of these D-measures are discussed and tested on simple but informative examples, and some light is shed on the issue of consistency of all steps of a GM reduction algorithm with a single D-measure. Indeed, inconsistent choices, often made in practice, may lead to solutions quite far from optimality. The notion of Best Single Gaussian Approximation of a submixture is also introduced and compared with the concept of Barycenter of a mixture. The paper is organized as follows: in Sec. II the GMR problem is formulated; Sec. III introduces some D-measures; Sec. IV presents the basic steps of a GMR algorithm; Sec. V introduces the concepts of BSGA and of Barycenter of a GM; Sec. VI summarizes the features of KLD, ISE and NISE; Sec. VII reports numerical tests. Conclusions follow. Notations. In this paper R N + denotes the set of nonnegative vectors in R N , the symbol 1 N denotes a vector of ones in R N , and will be used to represent summations in compact form: denotes open cone of symmetric positive definite matrices. The symbol N (·\|µ, Σ) denotes a multivariate d-dimensional Gaussian density with mean µ ∈ R d and covariance Σ ∈ S d : II. THE GAUSSIAN MIXTURE REDUCTION PROBLEM A Gaussian Mixture (GM) is a convex combination of N d-dimensional Gaussian densities of the form: all parameters of the mixture. The number N of Gaussian components is the size of the mixture. Note that a given value of Θ ∈ H N uniquely identifies a GM, but different values of Θ can identify the same GM. Indeed, considering that any permutation of the parameters in Θ yields the same mixture, then there are at least N ! equivalent parametrizations of a given GM. 1 Roughly speaking, the reduction of a given GM f (x\|Θ h ) of size N h (the superscript h stands for hypothesis mixture) is the process of constructing a GM g(x\|Θ r ) with lower size N r (reduced mixture) that is similar, in some sense, to the original one. The symbol D(· ·) will denote a generic dissimilarity measure (D-measure, for short, or deviation) between two d-dimensional distributions, so that D(f h g r ), where f h and g r are shorthands for f (x\|Θ h ) and g(x\|Θ r ), respectively, denotes the dissimilarity between the original and reduced GMs. In general, D-measures must satisfy the two following requirements, for all pairs of distributions p and q: (3) D(p q) = 0 ⇐⇒ p = q, (identity of indiscernibles). (4) If, for any three distributions, p, q, h, also the following are satisfied D(p q) = D(q p), (symmetry); (5) D(p q) ≤ D(p h) + D(h q), (triangle inequality), (6) then the dissimilarity D(· ·) is a distance and defines a metric in the space of distributions. The Gaussian Mixture Reduction (GMR) problem with respect to a given D-measure is the problem to find the GM g(x\|Θ r ), of a given size N r , that minimizes the dissimilarity from the hypothesis GM f (x\|Θ h ), with N r < N h . The solution is formally given by This is a complex, non-convex nonlinear constrained optimization problem, which in general does not admit a closed form solution. In addition to the presence of (a lot of) local minima, there are at least N r ! global minima. This happens because, as discussed below equation (2), any permutation of the parameters of a solution Θ r * is an equivalent solution. Note also that any parameter permutation of a local minimum is still a local minimum. Another issue with the optimization problem (7) is that few D-measures can be computed in closed form for Gaussian Mixtures. Indeed, the evaluation of most D-measures requires a volume integration over the whole domain R d . III. DISSIMILARITY MEASURES In this section some D-measures that are used in most existing GMR algorithms are introduced. A. Kullback-Leibler Divergence Given two continuous distributions, p and q on R d , the Kullback-Leibler Divergence (KLD) of q from p (also known as differential relative entropy) is defined as [9] where the integral is over supp(p) (the support of p), so that the KLD is well defined when supp(p) ⊆ supp(q). When considering GMs, the support is R d and the KL-divergence is always well-defined. The KLD measures the information loss when p is approximated by q and is a D-measure, because it satisfies (3) and (4), but is not a distance, since (5) and (6) are not satisfied. The KLD of two Gaussians N (·\|µ i , Σ i ) and N (·\|µ j , Σ j ) admits the following closed form When applied to GMs, the first argument of the KLD is the original GM while the second argument is the reduced one: The main issue with the KLD (10) is that it does not have a closed form, neither its gradient w.r.t. the parameter set Θ r , useful in numerical minimization, has a closed form. One way to compute the KLD between two GMs is through numerical integration of (10) over the whole domain R d , a computationally demanding procedure, especially when d is large. Also MonteCarlo integration methods turn out to be computationally intensive [7]. A positive feature of the KLD, that will be discussed further on in this paper, is the simplicity of the computation of the barycenter of a GM (see Sec. V). B. The ISE dissimilarity Another widely used D-measure between two distributions p and q is the Integral Square Error (ISE), called also Integral Square Difference [14], defined as the square of the L 2 -error (The name ISE is used when q is aimed at approximating p, so that the difference p − q is in fact an error.) In addition to satisfying the properties (3)-(4), like the KLD, the ISE satisfies also the symmetry (5), but fails to satisfy the triangular inequality (6), and therefore is not a true distance between p and q (note that the square root of D ISE (p q), which is the L 2 -error norm p − q 2 , satisfies all properties (3)-(6), and therefore is a true distance and defines a metric in the space of distributions). When applied to GMs f h and g r the ISE has a closed form: J hh and J rr are called self-likenesses of f h and g r , respectively. J hr is called cross-likeness between f h and g r . From Similar expressions hold for J hh and J rr where Thus, the ISE (12) can be written as Exploiting the following identity [19], [20]: the components of H hh , H hr , H rr can be easily computed Thus, the computation of the ISE between two GMs is an easy and straightforward task. In the [19], [20] it is shown that also the computation of the gradient of the ISE with respect to the parameters in Θ r , useful when pursuing the numerical solution of (7), is straightforward. The availability of closed forms for the computation of the ISE and of its gradient is one of the main advantage over the KLD, for which analogous closed forms do not exist. C. The NISE dissimilarity A dissimilarity measure closely related with the ISE is its normalized version, denoted NISE, defined as When applied to the GMs f h and g r , considering the expression (12) for the ISE, we have Like the ISE, the NISE satisfies the properties (3)-(5), and not (6). The property of the NISE of being confined in the interval [0, 1] makes it attractive, in many contexts, like in target tracking, as a measure to evaluate the accuracy of an approximation [4], [13]. Like the ISE, also the gradient of the NISE can be computed in closed form, because the expressions (11) and (20) are made of the same building blocks J hh , J hr and J rr . D. Other dissimilarity measures Many other D-measures have been considered in the literature for addressing the GMR problem, but are not considered here due to space reasons. Among these, we mention the Bhattacharyya and the Kolmogorov variational distances [19], [20], the Cauchy-Schwarz distance [8], the Wasserstein distances [1], [3], [5] and the class of Composite Transportation Distances (CTD) [3], [22]. Among these, only the Cauchy-Schwarz admits a closed form the evaluation of the cost and of its gradient, while the CTDs can be easily computed solving a Linear Programming problem. IV. GENERAL STRUCTURE OF GMR ALGORITHMS Many algorithms have been proposed in the literature with the aim of providing approximate solutions of the GM reduction problem with low computational complexity, so that they could be used in real-time applications. Most of them make use of two basic steps: greedy reduction and refinement [4]. A. Greedy Reduction This step consists in performing an iterative reduction of the original GM, until a GM with the prescribed number of components is achieved with some degree of similarity with the original one, and, hopefully, not far from the optimal solution of (7). Thus, the reduced GM can serve as a good starting point for subsequent refinements. The basic operations involved at each iteration are pruning and merging of GM components. 1) Pruning: is the process of removing one or more components from a given Gaussian Mixture according to some criterion. Common approaches consist in eliminating components according to criteria based on: • magnitudes of the weights w (thresholding, k smallest, out of prescribed percentile,...), • cost in terms of increment of the chosen D-measure. Pruning according to weights-based criteria is typical in target-tracking contexts [2]. The GM components to be removed can be those whose weights are below a fixed or adaptive threshold, or the components associated to the k smallest weights, with k fixed or varying at each step, or those associated to the smallest weights whose sum is below a prescribed value (typically 0.05). This approach is faster than using cost-based criteria, so it can be used to speed up the reduction phase. On the other hand, it can lead to a significant worsening of the final approximation, especially in some critical situations 2 . Cost-based pruning criteria aim at removing from a GM those components whose elimination would give the least increment of the chosen D-measure. Thus, for a GM of size N the dissimilarity measure must be computed N times (one computation for each elimination hypothesis), and these computations must be repeated at each pruning step, until the desired number of components N r is reached. It is clear that pruning according to cost-based criteria is usually slower than pruning according to weights-based criteria, but for some dissimilarity measures it can consistently improve the result. However, it is important to note that applicability of cost-based criteria is limited, because many dissimilarity measures, like KLD, cannot be computed in closed form for Gaussian mixtures. In such cases, approximations [7] or upper bounds [11] of the dissimilarity measures can be used, at the price of worsening the final result. Whatever the criterion used, after each pruning step the weights must be normalized, so that they sum up to one. 2) Merging: is the process of replacing a specific subset of Gaussian components (a sub-mixture) of a given GM with only one Gaussian component, according to some criterion. For a given GM p(x\|Θ) of order N , we use p(x\|Θ) to denote a sub-mixture of orderN < N , where Θ denotes a subset of the parameter set Θ with normalized weightsw ∈ ∆N −1 , while p(x\|Θ u ) denotes its unnormalized version, i.e. the submixture whose weightsw are a subset of the weights w of the original mixture, so thatw T 1N < 1. Of course, w =w/(w T 1N ). Using this notation, the problem of merging theN components of the sub-mixture p(x\|Θ) can be formulated similarly to (7), as the problem of finding the Gaussian N (·\|µ, Σ) with minimal dissimilarity from p(x\|Θ): N (·\|µ * , Σ * ) is the Best Single Gaussian Approximation (BSGA) of the sub-mixture p(·\|Θ) for the given D(· ·). The BSGA can be used to replace in p(·\|Θ) the components of the sub-mixture p(·\|Θ u ), yielding a reduced GM of size N −N + 1. The weight assigned to the new component N (·\|µ * , Σ * ) is the sum of the weights of the merged components,w T 1N , so that the sum of the weights of the resulting mixture remains one. As a matter of fact, in the literature the merging action has not the interpretation the we have given here. In most papers, merging consists in replacing a sub-mixture with its barycenter, defined as the Gaussian density that minimizes a weighted sum of pairwise dissimilarities [16]. Stated in formulas, the parameters of the barycenter of a sub-mixture 2 A critical situation for pruning is the case of a mixture where a group of very similar Gaussian components is associated to very small weights, below the pruning threshold. In this case, the merging of the group into a single Gaussian would be better than pruning the components of the group. (22) (the barycenter can equivalently be defined considering in (22) the unnormalized sub-mixture p(·\|Θ u ).) In general, for a given D-measure, the barycenter and the BSGA of a submixture do not coincide. Nonetheless, the KLD-barycenter and KLD-BSGA coincide [11], and it is not difficult to prove that also the ISE-barycenter and ISE-BSGA coincide, while this is not true for the NISE. However, only the KLD barycenter/BSGA admits a closed form solution (see Sect. V). The choice of which components to merge in a mixture can be made using local or global cost criteria. A local criterion consists in checking, at each step, the dissimilarities between all pairs of components of the given GM, and then merging the pairs with the least dissimilarity (a variant of this approach is to create clusters of more than two similar components, and perform the merging of the least dissimilar cluster). A global criterion consists in checking the effects of all possible merging of pairs of components on the dissimilarity with the original GM, and then choosing to merge the components which yield the least dissimilarity. 3) Combining pruning and merging: at each step of an iterative greedy reduction algorithm either pruning or merging is applied, the choice depending on criteria involving, in general, both the weights and the evaluation of the dissimilarity between the GMs before and after the reduction, as in [19]. B. Refinement The greedy reduction step can provide a suitable starting point for a subsequent tuning of the parameters of the reduced mixture. Most refinements in the literature can be roughly divided in the following three classes: • optimization (e.g. [19], [20]); • clustering (e.g. [1], [13]); • optimization-clustering (e.g. [21], [22]). Refinement procedures in the first class consist in using standard numerical methods for solving the optimization problem (7). The ISE and NISE are suitable for this approach, because closed forms exist for their computation, together with their gradients, while the KLD is not. Of course, if a descent method is applied, in general the refinement procedure will provide a local minimum close to the starting point. For this reason, it is extremely important that the greedy reduction provides a good starting point for the refinement. When the adopted D-measure admits a closed form only between pairs of Gaussians, like the KLD and the 2-Wasserstein, then clustering can be used to decrease the dissimilarity between the original and reduced GMs. In some cases [21], [22], clustering and optimization are combined together. C. Consistency issues in the pipeline of reduction operations It is important to stress that, due to the abundance of local minima for the GMR problem (7), the outcome of all the mentioned refinement approaches strongly depends on the starting point of the algorithm, which is the reduced GM provided by the greedy reduction step. For this reason, we believe that it is important that in the pipeline of reduction steps (greedy pruning/merging operations + refinement by clustering/optimization) the same D-measure should be used (consistency issue). If the initial point of an algorithm aimed at optimizing a given D-measure is provided by a greedy reduction performed on the basis of a different D-measure, the result of the refining phase can be far from optimality. Indeed, most GMR algorithms proposed in the literature are made of a pipeline of steps in which different D-measures are considered (inconsistency), and a further D-measure is used for evaluating their performances (typically, the NISE is used for final assessment). The reason is that, depending on the chosen D-measure, some reduction steps may have or not a closed form or can be completed with simple or cumbersome computations. To the best of our knowledge, the issue of consistency among the steps of a reduction procedure has never been raised before. We refer to Sections VI and VII for a further discussion on this issue. V. KLD AND ISE BSGA AND BARYCENTERS One attractive point of the KLD as a dissimilarity measure is that the BSGA of a sub-mixture and its barycenter coincide [11], and can computed in closed form. Given an unnormalized submixture p(·\|Θ u ) of sizeN of a given GM p(·\|Θ), with Θ u = {w,μ, Σ} ⊂ Θ, the mean and covariance of the barycenter are computed as follows: In the merging step of a GMR algorithm, the Gaussian component N (·\|µ * , Σ * ) replaces the unnormalized sub-mixture p(·\|Θ u ) in the GM p(·\|Θ), with assigned weight w * = w T 1N . This replacement is also called moment-preserving merging, because it preserves the mean and the covariance of the original GM [11]. The ISE barycenter and the ISE BSGA coincide as well, although they can not be computed in a closed form, due to trascendental equations. Therefore, most GMR algorithms compute the merging actions using KLD-barycenters, through equations (23) (the KLD barycenter is also frequently used in clustering steps, e.g. when updating the centroids in a k-means scheme). However, it is important to stress that the numerical computation of the ISE barycenter is eased by the availability of a closed form of the ISE gradient. VI. SUMMARY ON KLD, ISE AND NISE FEATURES This section lists some features of the KLD, ISE and NISE that explain the reason why most GMR algorithms jump from one D-measure to another in the various steps [4]. Features of the KLD: a1) KLD BSGA and KLD barycenter coincide (moment preserving merging of sub-mixtures); a2) closed form (23) available for the KLD barycenter; a3) closed form for the KLD and KLD gradient not available: -numerical computations are needed; -closed-form only for KLD of two Gaussians (9); a4) pruning operations may dramatically increase the KLD (KLD is prone to merge components rather than cut them); Although properties (a1),(a2) make attractive the KLD for merging, (a3) makes it unsuitable in GMR optimization problems (7). Property (a4) is obvious when looking at (10): if a rather isolated component of f h is pruned then there are points where the ratio f h (x)/g r (x) in the logarithm of (10) can take on high values (indeed, the KLD between two Gaussians (9) grows quadratically with the distance between the means). Features of the ISE dissimilarity: b1) ISE BSGA and ISE barycenter coincide; b2) a closed form is not available for the ISE barycenter (numerical computation is needed); b3) closed forms for ISE and ISE gradient are available; b4) in some situations, optimizing the ISE resembles pruning. Property (b2) makes the ISE less appealing than KLD for merging, although thanks to (b3), it can be used as a criterion for merging components of a GM. Indeed, in [19], [20], during the greedy reduction phase, at each step, the pair of Gaussians with the lowest impact on the ISE between the original and the reduced GMs are merged. However, the KLD barycenter is used for merging (inconsistency). In general, property (b3) makes the ISE attractive as a cost function to be numerically optimized, as in [19], [20] during the refinement phase. The property (b4) will be illustrated in the example section VII. Features of the NISE dissimilarity: c1) NISE BSGA and NISE barycenter do not coincide (they are close when the ISE is close to 0); c2) closed forms for BGSA and barycenter not available (numerical computations are needed); c3) closed forms for NISE and NISE gradient are available; c4) in some situations, optimizing the NISE resembles pruning. c5) the NISE takes values in [0, 1] ([0, 1) for GMs); c6) in most cases the NISE tends to yield reduced GMs with lower covariance, w.r.t. the ISE. The ISE and NISE share the properties (c2),(c3),(c4), and thus the same comments are appropriate. Like the ISE, merging with NISE can be done by numerically solving the optimization problem (21) or (22), although obtaining different solutions in the two cases. Due to property (c5) the NISE is often used to compare results of different reducing algorithms (e.g. [4], [13]), although it is never used as the only cost function inside the reduction pipeline. Property (c6) directly comes from the NISE expression (20), by considering that the value of J rr in the denominator tends to be large when the covariances of the reduced mixture are small. VII. NUMERICAL TESTS ON CONSISTENCY The features and issues listed in the previous section are discussed here on a couple of case studies. To make things clear, the very simple reduction problem of a GM of size two (N h = 2) to a GM of size one (N r = 1) is considered. This is equivalent to the problem of finding the BSGA of a submixture made of two Gaussian components. denote the parameters of the components of the original GM f h . In conditions of perfect symmetry of the components in , the mean of the KLD-barycenter (23) is the arithmetic mean µ * KLD = (µ h 1 + µ h 2 )/2. A rather challenging and enlightening situation is when the original GM is not perfectly symmetric, i.e. when the covariances are the same, but the weights are slightly different. Below, we compare the KLD, ISE and NISE BSGAs for a set of four GMs of size N h = 2 with parameters It is interesting to analyze the behaviors of the three BSGAs when µ h 2 moves away from µ h 1 , so that the overlapping of the two components of the original GM progressively decreases and the covariance of the GM increases (quadratically with µ h 2 − µ h 1 , as in (23)). First thing to note is that the mean µ * KLD is always roughly in the middle between µ h 1 and µ h 2 , since it is the weighted mean of µ h 1 and µ h 2 (23), while the means µ * ISE and µ * NISE vary considerably when µ h 2 moves away from µ h 1 . Only when µ h 2 = 1, close to µ h 1 , the shapes and locations of the three BSGAs are all similar (Fig. 1). For µ h 2 ∈ {2, 4, 10} the NISE BSGA is very similar to the Gaussian component of the original GM with the highest weight. Indeed, the mean value is almost the same, µ * NISE ≈ µ h 2 , while the covariance Σ * NISE is a bit larger than Σ h 2 . The ISE BSGA has an intermediate behavior: when µ h 2 ∈ {1, 2}, rather close to µ h 1 , the mean µ * ISE is close to µ * KLD , while the covariance Σ * ISE is larger than Σ * KLD (Fig.s 1-2). When µ h 2 ∈ {4, 10}, rather far from µ h 1 , the ISE BSGA is more similar to the NISE BSGA, and hence to the Gaussian with the highest weight in the original mixture (Fig.s 3-4). Indeed, µ * ISE ≈ µ * NISE ≈ µ h 2 , while the covariance is larger, Σ * ISE > Σ * NISE , so that the peak values of the original GM and that of the ISE BSGA are very close. On the contrary, due to the smallest covariance, the peak of the ISE BSGA is considerably higher than the peak of the original GM. Note that the effect of ISE and NISE optimization, when µ h 2 ∈ {4, 10}, is the same as pruning, while the effect of KLD optimization is the same as merging (see the properties (a4),(b4),(c4) in the previous Section). It is important to stress that the ISE and NISE BSGAs have been computed by numerically solving (21), due to properties (b2),(c2), and we have found that in general local minima are present. For instance, the surface plot in Fig. 5 shows that the NISE has a local minimum with the mean coinciding with µ h 1 = −1, and this is true for all the four cases considered. Moreover, we have found that when µ h 2 ∈ {4, 10} the ISE has a local minimum close to the KLD BSGA (μ ISE = 2.0419, Σ ISE = 11.1479). Indeed, a descent algorithm that is initialized with the KLD BSGA, which is easy to compute using (23), will converge toward this local minimum, failing to reach the global one. These considerations should warn from choosing as initial point for the ISE optimization a mixture obtained by a greedy reduction carried out according to KLD-based criteria or KLD merging, as done in [19], [20]. (7) strongly depends on the choice of the Dmeasure. In particular, looking at Fig.s 3-4 it is apparent that the GMR carried out minimizing the KLD, yields a reduced GM whose shape can be very different from the original one, although preserving the mean and the covariance. In contrast, the GMR carried out minimizing the ISE yields a reduced GM that rather faithfully overlaps with the most significant portion of the original GM, while pruning the least significant components. The larger covariance, with respect to that of the corresponding portion of the original GM, is due to the uncertainty added by the reduction process. In this respect, it is not advisable the use of the NISE in the GMR problem (7) since it yields reduced GMs with artificially small covariances (see property (c6)). To further analyze the issue of consistency, a GMR problem taken from [19] is considered below, where a onedimensional GM of size N h = 5 has to be reduced to size N r = 2. The parameters of the original GM are: Using the ISE-consistent merging a different sequence of reduction steps comes up: the components 3 and 4 (out of 5) are merged first, followed by components 2 and 3 (out of 4), and finally 1 and 2 (out of 3), without any case of pruning. Thus, merging components into the ISE-barycenters has led the algorithm to prefer merging over pruning in the second iteration, unlocking a different reduction solution in the iterative process. It can be noticed that the final score is almost halved in magnitude w.r.t. to the KLD-barycenter case. We got similar results applying the modified ISEconsistent William's algorithm to much more complicated situations, not reported here, so that we ascertain that the choice of carrying out a pipeline of actions consistent with a unique D-measure, has a tendency to provide more accurate reduced GMs, according to the chosen D-measure. To further support our arguments about consistency, the performances of fully consistent algorithms are compared on the same test GM (25). As KLD-consistent algorithm we consider the one proposed by Runnalls in [11], where an upper bound on the KLD of the GMs before and after the moment-preserving merge has been proposed and used as a cost criterion for merging. Runnalls' algorithm does not consider pruning because only a bound on the KLD-cost of merging actions has been developed in [11]. This is not a real drawback because the KLD naturally does not foster pruning (property (a4)). By comparing the Runnalls' algorithm with the modified ISE-consistent Williams' algorithm, we get the result of Fig. 8. The KLD values have been computed through numerical integration here, due to (a3). As expected, by solving the GMR problem in a consistent way, fairly different solutions are found. The KLD-consistent reduced GM is such to preserve the mean and the covariance of the original GM, but the shape is rather different from the original one. On the contrary, the ISE-consistent reduced GM has a shape that quite accurately traces a good portion of the original GM (the components with larger weights), and prunes the remaining (the thin peak on the left). By looking at the examples of Fig.s 1-4 and of Fig. 8, we ascertain that in applications aimed at computing maximum a posteriori (MAP) estimates, GM reductions according to ISE measure should be preferred (ISE-consistency), while in applications where minimum mean square error (MMSE) estimates are needed, the KLD measure is the right choice. Nevertheless, the computational burden introduced by using a specific reduction pipeline can be the main hindrance. In general, algorithms based on global dissimilarity measures require more computational power and, if working in realtime scenarios, one might prefer a trade-off between accuracy and efficiency by recurring to lighter solutions; on the other hand, if time is not a constraint, or the available computational power is significant, one should consider GMR pipelines fully consistent with a unique D-measure of interest. VIII. CONCLUSIONS In this work, after the presentation of a general view of the GMR problem and algorithms, the features of the most used D-measures (KLD, ISE, NISE), have been reviewed in some detail and illustrated on simple but informative case studies. In particular the issue of consistency in the pipeline of actions taken in a GMR algorithm has been discussed. The analysis and the tests performed reveal that all actions should be consistent with a single D-measure in order to obtain a reduced GM hopefully close to the optimal one. Unfortunately, the considered D-measures have complementary characteristics, in terms of existence of closed forms and ease of computation, and for this reason most GMR algorithms inconsistently combine their use, thus yielding results that in some situation can be far from optimality. In a future work we intend to include other D-measures in the analysis.	2021-04-27T01:16:20.398Z	2021-04-26T00:00:00.000	{ "year": 2021, "sha1": "bb647e267e571709f19c1c21b25d405e3d56212a", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "bb647e267e571709f19c1c21b25d405e3d56212a", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [ "Computer Science", "Mathematics" ] }
139468893	pes2o/s2orc	v3-fos-license	Visualization of High-Speed Impact of Projectile in Granular Sheet with Destructive Collision of Particles The impact and penetration of a projectile in a particle-laden space, which are expected to have frequently occurred during the formation of the solar system and will occur in the case of an impact probe for future planetary exploration, were experimentally simulated by using the ballistic range. A two-dimensional sheet made from small glass beads or emery powder was formed by the free-falling device through a long slit in the test chamber evacuated down to about 35 Pa. A polycarbonate projectile of a hemisphere-cylinder or sphere shape with the mass and diameter about 4 g and 25 mm, respectively, was launched at the velocity up to 430 m/s, and the phenomena were observed by the high-speed camera at 20,000 fps. From a series of images, the bow-shock-wave-like laterally facing U-shaped pattern over the projectile and the absence of particles in the trail behind it were clearly seen. At the impact of the particles on the projectile surface, fine grains were formed due to the destructive collision and injected outward from the projectile. The images obtained by different lighting methods including the laser light sheet were compared. The effects of the particle diameter, its material and the impact velocity were also investigated. Introduction Impact in a particle-laden space in a vacuum is not an unusual event.For example, it is well known that the destruction and aggregation of objects at the impact played an important role during the formation of the solar system [1].An artifi-cial impact is expected to reveal the interior structure of a celestial object at the lunar and planetary exploration.In HAYABUSA 2 mission conducted by the Japan Aerospace Exploration Agency (JAXA), the impactor is planned to hit the surface of the asteroid Ryugu [2] for the in-situ observation of its interior structure.To reveal the mechanism of the phenomena in such cases, the fundamental understanding about the impact in a particle-laden space is essential. When the particles are packed in the space, the dynamics of their motions has been intensively and extensively investigated in the field of the terra-dynamics. The numerical method using virtual particles called DEM (Discrete Element Method) [1] [3] is known to appropriately simulate the behavior of the granular material both microscopically and meso-scopically.From a macroscopic viewpoint, the fluid-dynamics-like model is expected to work well.For example, the compressible and non-expanding fluid model successfully describes the nature of irreversible compression of granular materials [4]. When the particles at the undisturbed state are separately located at some distance, the situation seems similar to the rarefied gas dynamics with relatively large mean free path.In the presence of the atmosphere, various studies have been numerically and experimentally conducted in the framework of the two-phase flow.For example, the combination of the Eulerian description of the dynamics of the fluid and the Lagrangian description of the motion of the floating solid particles is known to reasonably simulate the dusty flow around a body at a supersonic speed [5].The phenomena around a circular cylinder in the stream of small spheres rolling down the slope of an inclined flat plate were experimentally observed, assuming that the aerodynamic force acting on the particles is negligible in comparison with the force due to collisions [6].In most of the studies made so far, the destructive collision, which may frequently occur at the high-speed impact, was not taken into account.Considering the application to the planetary science or planetary exploration engineering mentioned above, the impact velocity is expected to be high, and the fundamental understanding about the phenomena involving the destructive collision into finer grains is necessary based on the experimental study in the absence of the atmosphere. In the present experimental study, a body was shot into a particle-laden space as seen in [3].For convenience of observation and simplicity of analysis, we experimentally simulated the two-dimensional on-plane impact and penetration of a projectile in a sheet of particles.A projectile with the diameter much larger than that of the particle was launched by the ballistic range in the direction on the plane of the granular sheet, which was formed by the free-falling device through a narrow slit [7] [8] in the low-pressure test chamber.The phenomena were observed by the high-speed camera in the direction normal to the particle sheet, as illustrated in Figure 1.It should be noted that the visualized image strongly depends on the lighting method.The images were obtained by various lighting methods including the laser light sheet.The effect of the experimental condition was investigated with respect to the impact velocity, the diameter of particles and the material of the particles.The major objectives in the present study are 1) to reveal the characteristic features of the granular flow around a projectile penetrating on the sheet of particles, 2) to clarify the presence of the destructive collision of particles at the projectile surface and its role in the granular flow field, and 3) to investigate the effect of the impact velocity, the diameter and the material of particles. Ballistic Range and Particle Sheet Generator The ballistic range in the authors' laboratory [9] shown in Figure 2 projectile was launched and penetrating on the particle sheet in this period.The projectile was finally caught in a semi-hard-landing manner by the projectile catcher made from the sponge layer and the oil clay. As discussed later, the obtained image strongly depends on the lighting method and the viewing area of the camera.We used three types of arrangement of the lighting and viewing area of the camera as shown in Figure 4.The arrangement (a) was selected as the nominal setting and was used to observe the phenomena both just after the impact of the projectile and during the penetration on the particle sheet.The back-lighting was selected for clearness of the obtained Journal of Flow Control, Measurement & Visualization image.After the penetration proceeded for some distance, the granular flow field around a projectile is expected to be in a steady state.The arrangement (b) was used for the observation after long penetration.We have to be careful to judge the presence of matter from a visualized image, because the dark image can be produced by not only the absence of matter but also the absence of light.To reduce the risk of misunderstanding, the arrangement (c) with the front-lighting was tested and compared with the arrangement (a).For the lighting, the high-intensity metal halide lamp MID-25 FC (Lighterrace Inc.) with the maximum power 250 W was used.To capture the images of the impact phenomena, the high-speed monochrome camera Phantom Miro 310 (Nobby Tech.Ltd.) was used.The sensitivity is 12 bit.The frame rate, exposure time and the spatial resolution were set to be 20,000 fps, 1 μs and 512 by 256 pixels, respectively, in the present experiment. Particles and Projectiles Three types of the glass beads (Types #40, 60 and 80) with different size were used for the particle sheet.They are originally supplied as the grinding powder with the particle size controlled under the industrial standard, that is, 355 -500 μm for Type #40, 250 -355 μm for #60 and 180 -250 μm for #80.The magnified image of the particles by the microscope showed that the shape of a glass bead was not a smooth sphere but a rugged irregular ball [7] [8].In addition to the glass beads, the emery powder with the particle size about 400 μm was used for the particle sheet.The emery powder is mainly made from the aluminum oxide Journal of Flow Control, Measurement & Visualization and iron oxide and is harder than the glass beads.The difference in the hardness of the particle is expected to change the nature of the destructive collision at the projectile surface and to affect the behavior of the granular flow around a body. In the case of type #40 glass beads, the number density of the particles was estimated as 5 × 10 8 1/m 3 from the mass flow rate through the slit [7].The falling speed of the particles was estimated as about 3 m/s from two continuous snapshots and the frame rate [7].It was negligible in comparison with the flight velocity of the projectile. Two types of the projectile shapes were tested, that is, the hemisphere cylinder and the sphere, as shown in Figure 5.Both types of the projectiles were made from the polycarbonate for its strength and low mass.The diameter of the body was 25.75 mm, which is the same as the inner diameter of the acceleration tube. Consequently, the projectile was launched without the sabot by the ballistic range.Such launch method without the sabot enables us to avoid significant disturbance added to the trajectory and attitude of the projectile at the sabot separation [9].To obtain a high velocity from the ballistic range, the projectile mass was reduced by the hollow structure with the thickness 2 mm.The average mass of the hemisphere cylinder model and the spherical model is about 3.9 g and 4.3 g, respectively. Uncertainty and Repeatability of Experimental Results The projectile velocity was estimated from the frame rate and the difference in the projectile position between two continuous snapshots.The uncertainty in the estimated velocity was mainly caused by the blur of the projectile image in the exposure time [9], and it was less than 5%. Thanks to the launching of the projectile without the sabot, the uncertainty in the flight condition was relatively small.The free flight of the projectile before reaching the edge of the particle sheet was stable.The fluctuation in the velocity and the path angle during the free flight was smaller than ±10 m/s and ±1.5 degrees, respectively [7].The repeatability of the particle sheet generator was good Two-Dimensionality of Phenomena The present experimental setup was designed under the assumption that the motion of the particles mainly occurs on the plane of the particle sheet, because the plane of symmetry of a projectile coincides with that of the sheet.To confirm that, the setup of the lighting and the camera shown in Figure 6(a) was additionally tested.Thanks to the mirror put in front of the projectile catcher, the oblique frontal view of the projectile was obtained.The cloud seems to expand more in the vertical direction than in the horizontal direction.These pictures imply that the granular motion on the particles sheet was more significant than the out-of-plane motion.The fine grains were produced by the destructive collision of the particle sheet at the projectile surface. Though the flight velocity cannot be estimated from the oblique view, it was expected to be about 370 -380 m/s from the experimental data of the spherical projectile at the same pressure of 0.6 MPa charged at the breech of the ballistic range. To check the two-dimensionality of the phenomena, the visualization using the laser light sheet was also conducted.Two types of the laser light sheets, that is, the horizontal sheet and the vertical sheet, were tested as shown in Figure 7(a) and contrast.In both pictures, the projectile moved from left to right, and the cross section curve of the projectile surface was clearly seen.Though the projectile velocity could not be estimated because of unclear images, it was expected to be about 400 m/s from the experiments in the similar condition.This fact indicated that the out-of-plane granular flow was not so significant to fully cover the projectile surface. Consequently, the two-dimensional behavior of the particles and fine grains around a projectile was expected to be visualized in the present experimental setup as illustrated in Figure 1. Formation of Granular Flow Field around a Projectile The typical pattern of a snapshot taken by the back-lighting arrangement (a) in The above features were commonly observed in both cases of the sphere model and the hemisphere cylinder model.However, the diffusion of the fine grains into the trail was much weaker in the case of the hemisphere cylinder model as shown in Figure 9.This was expected to be caused by the presence of the sharp corner at the rear end of the body as pointed out in [8].This fact indicates that the properties of the fine grains produced from the glass beads at the collision with the projectile surface depend on the initial particle size. Effect of Particle Size, Material and Impact Velocity The effect of the material of the particle sheet on the granular flow field around a hemisphere cylinder model is shown in Figure 12, where the images taken at 0.15 ms after the impact with the sheet of the glass beads #40 and that of the emery powder are compared.The images were intensified by 20% in the brightness and contrast.The impact velocity is almost the same for both cases at about 440 m/s.The formation of the laterally facing U-shaped fine grain zone and the zone of absence of the particles or fine grains behind projectile was clearly seen in both cases.The dark zone in the laterally facing U-shaped structure becomes narrower for the emery powder than for the glass beads.This fact indicates that the properties of the fine grains formed at the particle collision at the projectile surface also depend on the particle material. Finally, the effect of the impact velocity was shown in Figure 13 ing U-shaped layer of the fine grains was seen in front of the projectile than at the velocity 330 m/s.In addition, the luminous zone of the fine grain layer in front of the projectile becomes wider in the case of higher impact velocity.The mass flux of the colliding glass beads increases with the impact velocity and the destruction of the colliding glass beads becomes more significant at higher impact velocity.The velocity of the fine grains injected in the forward direction at the projectile surface is expected to increase with the impact velocity.As a result, higher production rate and higher injection velocity of the fine grains are obtained at higher impact velocity.Consequently, the zone of the fine grains spreads more quickly in front of the projectile at higher velocity. Conclusions The impact and penetration of a projectile in a particle-laden space were experimentally investigated by using the ballistic range.A thin sheet made from small glass particles or emery powder was formed by the free-falling device through a long slit over the trajectory of a projectile in the test chamber.A polycarbonate projectile of a hemisphere cylinder or sphere shape with the mass and diameter about 4 g and 25 mm, respectively, was launched at the velocity from 310 m/s to 430 m/s.The phenomena were observed by the high-speed camera at 20,000 fps.To reduce the effect of the flow of the residual air in the test chamber, it was evacuated beforehand.From the obtained images using various lighting and camera arrangement including the laser light sheet, the two-dimensionality of the phenomena was discussed.The bow-shock-wave-like laterally facing U-shaped pattern in front of the projectile and the zone of absence of the particles or fine grains in the trail behind it were clearly observed in the pictures.The process of the formation of the granular flow field around a projectile was characterized by the destructive collision of the glass beads into fine grains at the projectile surface, the removal of the particles or fine grains behind the projectile by the sweeping effect, the shock-wave-like propagation of the laterally facing U-shaped zone of the fine grains in front of the projectile, and the diffusion of the fine grains into the trail of the projectile.The similar pattern was observed irrespectively to the projectile velocity, the size and material of the particles.However, the production rate and the spread speed of the fine grains depend on these conditions.The above results suggest that the present phenomena can be numerically simulated by the model including the appropriate description for the motion of the original particles, the formation of the fine grains at the projectile surface and the flow of the fine grains.The numerical analysis using such model is expected to be quite useful for understanding of the formation of the celestial objects in the solar system, designing the impact probe for planetary exploration in the future and so on. Figure 1 . Figure 1.Schematic view of experimental setup. was used for the present experiments.It can launch a projectile with the mass about 5 g at a speed up to about 500 m/s, depending on the charged pressure in the high-pressure chamber (breech) at 0.4 MPa to 0.8 MPa[7].Before the shot, the projectile was inserted at the rear end of the acceleration tube with the length 6 m.The muzzle of the acceleration tube was open to the test chamber without the diaphragm.The test chamber and the acceleration tube were evacuated down to about 35 Pa to reduce the effect of the residual air as much as possible.The photo of the test chamber interior is shown in the inset of Figure2.The particle sheet generator was set above the trajectory of the projectile launched from the muzzle.The particle sheet generator was composed of the particle reservoir having the triangular cross section and the narrow slit with the width and length 2 mm and 600 mm, respectively, at the bottom of the reservoir.The long lid was set at the exit of the slit.It opened by receiving the electric signal from the outside of the test chamber.As shown in Figure3, almost the uniform particle sheet was generated before the impact of the projectile by the present device except the local clustering of the particles, which became more evident for the case of smaller particles.The sheet of free falling particles continued for about 10 s.The Figure 2 . Figure 2. Photos of ballistic range facility and test chamber interior. Figure 3 . Figure 3.Snapshot of particle sheet before impact of a sphere model with exposure time 1 μs. Figure 4 . Figure 4. Arrangement of lighting and viewing area of camera (top view of test chamber). Figure 5 . Figure 5. Hemisphere cylinder model and sphere model used for a projectile. Figure 6 ( Figure 6(c) were intensified by enhancing the brightness and contrast by 20% using the image processing function of Microsoft  Power Point  2008 for Mac. Figure 7 ( b), respectively.For the light source, Kentech model LDB2W blue laser was used.The maximum power, the wave length and the width of the laser sheet were 2 W, 450 nm and 2 -3 mm, respectively.If the granular flow around the projectile had a significant three-dimensional out-of-plane structure, the laser light sheet would be scattered by the presence of the thick cloud of the particles and fine grains, and the cross section curve of the projectile surface could not be visualized by the laser light sheet.Figure8(a) and Figure 8(b) show the snapshots of the sphere model in the horizontal and vertical laser light sheets, respectively.These images were intensified by 20% in the brightness and Journal of Flow Control, Measurement & Visualization Figure 6 . Figure 6.Oblique frontal view of motion of a sphere model and granular flow around it.(a) Setup of lighting and camera view; (b) Snapshot just after impact of projectile at edge of particle sheet; (c) Snapshot at 0.1 ms after impact. Figure 4 Figure 9 . Figure 4 is shown in Figure 9(a).The particle sheet was made from the grass beads #40.The hemisphere cylinder model was used for the projectile, moving from left to right in the picture.The projectile velocity was estimated as 436 m/s.The snapshot was taken at 0.2 ms after the impact at the edge of the particle sheet.The bow-shock-wave-like laterally facing U-shaped structure over the projectile and the dark zone in its trail are clearly seen.The motion of the particles in the other area seems undisturbed in the picture.The dark zone behind the body indicates the absence of the particles due to the sweeping effect of the Figure 11 Figure11shows the effect of the particle size on the granular flow field around a sphere model.The images were intensified by 20% in the brightness and contrast.The images obtained in the particle sheet of the grass beads #40 (coarse), #60 (medium) and #80 (fine) are compared.The picture of the glass beads #80 is the same as the snapshot 4 in Figure10.Qualitatively, these patterns seem almost the same.However, the region behind the projectile was more significantly . The hemisphere cylinder model and the glass beads #40 were used for the projectile and the Journal of Flow Control, Measurement & Visualization Figure 12 .Figure 13 . Figure 12.Effect of particle material on formation of granular flow field around a hemisphere cylinder model.(a) Glass beads #40; (b) Emery powder.	2019-04-30T13:07:48.925Z	2018-06-29T00:00:00.000	{ "year": 2018, "sha1": "d310ee0313938aa6470d22b5f9830ba2d6e3f1f5", "oa_license": "CCBY", "oa_url": "http://www.scirp.org/journal/PaperDownload.aspx?paperID=85748", "oa_status": "GOLD", "pdf_src": "ScienceParseMerged", "pdf_hash": "d310ee0313938aa6470d22b5f9830ba2d6e3f1f5", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Materials Science" ] }
241852165	pes2o/s2orc	v3-fos-license	Parametric Modeling of Solid Mechanics in Engineering Research Based on Cloud Computing Model Traditional parametric models of solid mechanics have disadvantages such as poor lightweight level of cloud data and low integrity of operations of solid mechanics. To solve the above problems, a parametric model for solid mechanics in engineering research based on cloud computing models is designed. By calculating the number of cloud data copy optimizations, the upper limit of resources required for engineering research is estimated, and the specific values is assessed by continuously cycling the demand, so as to complete the basic conflict analysis, and set up a cloud computing engineering research environment. On this basis, the modeling index is selected. By analyzing the sensitivity of the index, the purpose of optimizing the modeling parameters is achieved, and a new solid mechanics parametric model is constructed. Analyzing and contrasting the experimental data shows that after applying the parametric model of solid mechanics in the engineering research based on the cloud computing model, the lightweight level of cloud data can be increased by up to 24 levels, and the operational integrity of solid mechanics can be increased by about 30%. materials and the original material reaches the limit of their application, the application of nonlinear models becomes more and more widely [3,4]. Solid mechanics is a branch of early formation, strong theoretical and extensive application in mechanics. It mainly studies the displacements, motion, stress, strain, and destruction of deformable solids produced by various internal points under the action of external factors (such as load, temperature, humidity, etc.). The study of solid mechanics has both elasticity and plasticity branches; it has both linear and nonlinear branches. In the early studies of solid mechanics, most of the hypothetical objects were uniform continuous media, but the composite mechanics and fracture mechanics developed in recent years expanded the scope of research. The non-uniform continuities and non-continuous bodies containing cracks are studied. The traditional solid mechanics parametric model obtains the state of engineering data in the cloud environment by NSGA-II method, then uses the elliptic basis function neural network method to obtain the standard approximation model, and substitutes all the data that conforms to the operation rules into the model, obtaining a series of value. Then, the data is sorted by the corresponding binary tree based on size to achieve the application value of the model. However, with the advancement of scientific and technological means, this traditional parametric model has gradually produced cloud data with a lower lightweight level and lower computing integrity. In order to solve the above problems, a new parametric model of solid mechanics in engineering research based on cloud computing model was designed. The establishment of the basic operating environment was accomplished through the optimization of cloud data copy quantity and demand estimation of engineering research resource, and the practical value of the model was proved by comparing experimental data. Establishment of cloud computing engineering research environment The research environment of cloud computing engineering is the basis for the operation of the new solid mechanics parametric model. The specific operation flow can be performed as follows. Optimization of cloud computing data copy quantity With the operation of the cloud computing engineering research environment, the amount of application data is increasing, and different cloud data copy groups have also become different. With continuous customization and data accumulation between copies, the number of users accessing data online at the same time may become more and more, resulting in higher visits of certain rows in the node's basic table. In order to avoid problems caused by increased access to certain cloud data, unbalanced model loads, and increased average request waiting time, the number of copy must be properly adjusted to disperse the heat to improve the availability of cloud computing data to ensure the stability of the operating environment of the model [5][6]. In the sharing architecture of cloud computing shared database, all copy data are stored in the shared basic table and extended table mode. Each group of engineering computing units uses metadata mapping to achieve data isolation in the replica view, that is, the users of each copy see as if the cluster is only servicing them. Therefore, user requests of each copy are independent of each other, and the requests of the entire cluster are also independent of each other. Assuming that represents the number of request ( ) of cloud data copies that arrive within time interval . Let denote the probability of arrival of ( ) , that is: Among them, represents the upper limit of cloud computing data copy, represents the existence of data request, and represents the total amount of data that can reach stably. In a sufficiently small unit time, the frequency of the data request is independent of time, and is approximately proportional to the length of the unit's interval, ie, there is no sudden increase in access in each sufficiently small unit time. In high-order infinitesimal of unit time, two requests won't arrive at the same time, that is, one copy only responds to one request at the same time, and other requests enter the waiting queue. According to the above description, the probability formula is drawn: is the high-order infinitesimal of , is the probability that the data request of cloud computing copy enters into the waiting queue, and when , there is: According to formula (3), it can be derived as follows: When , that is: Where represents the natural variable, represents the optimization constant of cloud computing data copy, represents the random natural number, and represents the optimization order of magnitude. Demand estimation of engineering research resource Demand estimation of cloud computing environment engineering research resource refers to the resource demand for a single copy in the shared table mode of shared database, which is related to the number of user requests. However, in the multi-engineering application program, redundant resource demand is required to handle the isolation and security processing of multiple resources. The storage usage of a resource is independent, proportional to the frequency of data access requests for each user [7][8]. The storage usage of all resources is proportional to the total number of users of data nodes. Let be the number of all users on the engineering research Among them, represents the resource demand setting, represents the request quantity relationship between users, represents the number of connection requests, represents the original demand estimation, and represents the number of users to be connected. Taking into account the SLA agreement between the project demand resource and the user, the SLA estimation model is shown in Figure 1, this model reflects the estimation form of satisfaction rate of the cloud computing engineering research. When the actual response time is higher than that specified in the SLA agreement, the request provided by the application service meets the engineering research requirements, and the request response time does not meet the agreed upper limit. On the contrary, engineering research needs cannot be met. Figure 1 SLA estimation model for cloud computing engineering research The demand estimation of cloud computing environment engineering research resource is a multi-objective optimization problem. For ease of calculation, some reasonable simplifications are made based on a greedy method. For example, R = {0.2s, 0.3s, 0.4s, 0.5s} corresponds to the required values of the response time of the estimation result A, the estimation result B, the estimation result C, and the estimation result D in their respective SLAs, and they are all at data node x. When an insufficient problem occurs on data node x, first, all data nodes are divided into two sets A and B according to resource usage. At this time, x belongs to set A, and the shortage of x is the most serious. Thus, x can be the data node that migrated first. Set B has data nodes a, b, , and a has the most abundant remaining resources. According to the idea of greedy algorithm, a data node is selected as the destination data node, and then the tenant with smaller value is migrated to this node as mentioned in the previous example. According to this idea, the data of the estimation results are successively transferred until the data node a is no longer insufficient. Conflict analysis of cloud data engineering research The conflict analysis of cloud data engineering research is related to the storage characteristics. This study is based on the storage method of the basic table and the extended table in the multi-copy shared data mode of shared database. The basic business data of the application is stored in the basic table, and the copy customized or extended data is stored in the extended table [9][10]. From the characteristics of the transaction request, the request number and frequency of transactions for the basic table far exceeds the request for the extended table. In addition, when a data record in the extended table is converted into data in the basic table, it can only be an attribute in one of the records. Thus, if the same amount of logical data is requested, the physical data amount of the extended table far exceeds the amount of physical data in the basic table. In a single transaction access, the amount of data access D in the extended table is greater than the basic table. Finally, the data in the extended table depends on the metadata table for metadata query to obtain conversion information of the data in the extended table, and the query efficiency is much lower than that of the basic table. Let represent the results of the conflict analysis of cloud data engineering research. Formula (6) can be used to express this result: Among them, represents the basic conflicting request efficiency, represents the research parameter of the cloud computing engineering, represents the stability factor of the cloud environment operation, is the stable operation cycle, and represents the conflict request frequency. The calculation result of formula (7) is utilized to use conflict analysis results of the cloud data engineering research, and the specific cycle operation method is shown in Figure 2. Figure 2 Operation method of conflict analysis for cloud data engineering research 3 Methods Based on the research environment of cloud computing engineering, in order to ensure the smooth operation of the new solid mechanics parametric model, it is necessary to complete the construction of the remaining applications of the model through steps such as selection of modeling index. Selection of modeling index of solid mechanics Selection of modeling index of solid mechanics is a key step in establishing a new parametric model. When using the center-difference equation to solve the solid-mechanical equations, the nonlinear convergence of the model itself can be used to improve the high degree of vectorization and parallelization of modeling index [11][12]. In general, the common solid mechanics modeling index includes four types, and the mutual constraints between them can be expressed as Figure 3. , and represent the four types of solid mechanics modeling index, respectively, and the specific determination method is shown in formula (8). Among them, , , and represent the solid mechanics parameters affecting the four modeling index, respectively, represents the impacting coefficient among indexes, represents the original quantity of stable modeling, and represents the minimal value of the product of influence coefficient and original quantity of stable modeling. During the collision of modeling indexes, the relevant influencing parameters of each index change significantly. At this time, to ensure that the index itself is not affected by the modeling event, fixed number processing can be performed for each index [13]. The specific processing results are shown in Table 1. Index sensitivity analysis Relying on the relative sensitivity to filter the modeling index may cause the overall performance of the model to drop too much. Therefore, a compromise is adopted in the selection of design variables, that is, the method considering the direct sensitivity and the relative sensitivity at the same time. First of all, the index with large relative sensitivity is excluded from relative sensitivity of each performance [14][15]. From the rest of the indexes, the indexes for weight loss are selected with certain rules. In the selection, the indexes with the quality parameters of 50%-70%, and the impact of indexes with quality parameters lower than 50% on the model is almost negligible. Finally, a new round of screening is performed on the excluded indexes. First, some indexes with quality parameters of high sensitivity are excluded, such as the first type of indicators, and then a small number of indexes with high direct sensitivity is selected. If selecting indexes with higher relative sensitivity and lower direct sensitivity, the quality parameter of this index may be small. Although its relative sensitivity is high, its direct sensitivity is small, and it is difficult to effectively improve stability even with a large performance enhancement process. The advantages of selecting relative sensitivity and direct sensitivity are that the lightweight selection of indexes can effectively stabilize the performance of the model's running structure [16][17]. Let indexes, , and represent the sensitivities of the modeling indexes respectively, and their specific expressions are shown in formula (9). (9) Among them, represents the relative sensitivity of the index, represents the direct sensitivity of the index, and represents the quality parameter of the index. On the basis of the above calculation results, the specific sensitivity analysis results of each index can be expressed as Table 2 through the direct analysis method. Parametric optimization algorithm Based on the above calculation results, the model indexes are edited by a parametric optimization algorithm. The specific editing results are shown in Figure 4. Figure 4 Editing effect of parametric optimization algorithm on model indexes Under the constraint of the figure, using the formula (9), the parametric optimization results of the four model indexes can be expressed as: Among them, represents the primary level of parametric optimization, represents the optimization authority, and represents the fixed result of the permission setting. The solid mechanics genetic algorithm is a highly parallel, random and adaptive global optimization probability search algorithm that is formed by simulating the biological evolution process in nature. Multi-objective genetic algorithm is based on this. The basic principle is: first, a group of randomly generated populations is searched for as the initial value. Each individual in the population is called a chromosome, and the smallest element of the chromosome is a gene, which corresponds to a certain characteristic of the solution, that is, a design variable. After successive iterations, the offspring chromosomes are obtained from previous generations through crossover or mutation operations. The chromosomes of each generation are measured by the degree of fitness, and in the process of the formation of a new generation of chromosomes, some of the offspring chromosomes are selected and eliminated according to their fitness to keep the population size constant [18][19]. The probability that the chromosome with high fitness is chosen is high. After many iterations, the algorithm converges to a better chromosome, which may be the optimal solution to the multi-objective problem. Under the influence of the algorithm, the parametric model of solid mechanics in the engineering research based on cloud computing model achieves the purpose of improving the computational integrity of the model by continuously narrowing the level of its own optimization parameters, and makes use of stable engineering research properties to make lightweight level of cloud data reach the rated standard [20]. To organize the parametric optimization results of the four model indexes, the parametric model of the solid mechanics in the engineering research based on the cloud computing model can be expressed as: Among them, represents the parametric model constant for solid mechanics in the engineering research based on the cloud computing model, represents the operational integrality factor of the model, is normalized parameter, and represents the upper limit of the lightweight level of the cloud data. Experiment To verify the practical value of the parametric model for solid mechanics in engineering studies based on cloud computing model, the following comparative experiments were designed. Two computers equipped with Windows 10 operating system and running memory of 256G were used as experimental objects. One of the computers was equipped with a traditional parametric model as an experimental group; the other computer was equipped with a new parametric model as an experimental group. Other variables were kept the same, at the same time, the changes of the relevant operating data of the two sets of parameterized models were recorded. Experimental parameter setting Before starting the experiment, the relevant experimental parameter settings was completed according to the following table. The experimental parameters are set as shown in Table 3. In the above table, CES parameter represents the stability of the cloud environment, ORG parameter represents the level of engineering research objects, MPS parameter represents solid mechanical parameter, MSC parameter represents model stability coefficient, TDL parameter represents the lightweight level of target cloud data, and OTM parameter represents the integrity of solid mechanics operations of the target. In order to ensure the absolute fairness of the experiment, the data of the experimental group and the control group were always consistent. Comparison of lightweight level of cloud data Under the premise of ensuring other experimental conditions remain unchanged, using 16 minutes as the experimental time, the changes in the lightweight level of cloud data after applying the experimental group and the control group in this period of time were recorded, in order to avoid the impact of sudden events. The experiment was divided into three parts: the operation state of the low mechanical parameters, the operation state of the middle mechanical parameters, and the operation state of the high mechanical parameters. The specific experimental results were shown in Figure 5, Figure 6, and Figure 7. Figure 5 shows that when the model was in operation status of low-level mechanical parameter, with the increase of running time, the minimum value of the cloud data lightweight level is 40 files after the application of the experimental group model, and the cloud data lightweight level 84 files was reached when the running time was 10 minutes, and the difference between them was 44 files. After applying the control model, the minimum value of the cloud data lightweight level was 8 files, and when the running time was 8 minutes, the cloud data lightweight level reached a maximum of 36 files. The difference between the two was 28 files, far lower than the experimental group. Figure 6 shows that when the model was at the operation status of middle mechanical parameter, with the increase of the running time, after applying the model of experimental group, the minimum value of the cloud data lightweight level was 52 files, and when the running time was 6, 7, and 8 minutes, the lightweight level reached the maximum of 80 files, and the difference between them was 28 files. After applying the control group model, the minimum value of the cloud data lightweight level was 12 files, and when the running time was 7, 13 minutes, the cloud data lightweight level reached the maximum value of 28 files, the difference between the two as 16 files, much lower than the experimental group. Figure 7 shows that when the model is at the operation status of high mechanical parameter, with the increase of the operation time, the minimum value of the cloud data lightweight level was 48 files after the application of the experimental group model, and the cloud data lightweight level reached the maximum value of 88 files, when the running time was 8 minutes, and the difference between them was 40 files. After applying the control group model, the minimum value of the cloud data lightweight level was 4 files, and when the operation time was 7, 14, and 16 minutes, the cloud data lightweight level reached the maximum value of 20 files, the difference between the two was 16 files, much lower than the experimental group. Comparison of integrity of solid mechanics computing Under the premise of ensuring that other experimental conditions were not changed, 16 minutes was taken as the experimental time, and the changes of integrity of the solid mechanics operation were recorded after applying the experimental group and the control group model. In Experiment Control order to avoid sudden events, the experiment was divided into three parts: operation status of low mechanical parameter, operation status of middle mechanical parameter, and operation status of high mechanical parameter. The specific experimental results were shown in Table 4, Table 5, and Table 6. Analyzing Table 4 shows that when the model was at operation status of low mechanical parameter, with the increase of the operation time, the computing integrity showed a stepwise increasing trend after the application of the experimental group model. When the operation time was 15 and 16 minutes, the computing integrity of the mechanics reached a maximum of 78.91% after the control group model was applied, and the computing integrity presented a rising trend. At an operation time of 16 minutes, the computing integrity of reached a maximum of 43.05%, which was much lower than the experimental group. Analyzing Table 5 shows that when the model was at operation status of middle mechanical parameter, with the increase of the operation time, the computing integrity showed an increasing trend and then stabilizing and then rising after the application of the experimental group model. When the operation time was 16 minutes, the computing integrity of the mechanics reached a maximum of 82.64%; after the control group model was applied, and the computing integrity presented a stepwise decreasing trend. At an operation time of 16 minutes, the computing integrity of reached a maximum of 50.04%, which was much lower than the experimental group. Analyzing Table 6 shows that when the model was at operation status of high mechanical parameter, with the increase of the operation time, the computing integrity showed a trend of floating back and forth after the application of the experimental group model. When the operation time was 8, 10, 12, 14, 16 minutes, the computing integrity of the mechanics reached a maximum of 84.09%. After the control group model was applied, and the computing integrity presented the trend of decreasing first, then stabilizing, and then rising. At an operation time of 1 minutes, the computing integrity of reached a maximum of 51.33%, which was much lower than the experimental group. Results and Discussion The parametric model of solid mechanics in engineering research based on cloud computing model is based on retaining the advantages of the traditional model application, effectively improving the design for the inadequacies, and improving the application stability of the new model by optimizing the solid mechanics operation parameters and other links. In the future, all major academic institutions in China can use this model as a starting point to gradually improve research in solid mechanics related fields. (NSGA) Non-dominated Sorting Genetic Algorithm Availability of data and material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.	2020-08-20T10:12:41.839Z	2020-08-19T00:00:00.000	{ "year": 2020, "sha1": "fc246ecaa3472c8dc6cf05383e5ce60c4086b4c3", "oa_license": "CCBY", "oa_url": "https://jwcn-eurasipjournals.springeropen.com/track/pdf/10.1186/s13638-020-01849-4", "oa_status": "GREEN", "pdf_src": "Adhoc", "pdf_hash": "8cf5dfa5a33af66fe7e9a92357ec1f1848e70a53", "s2fieldsofstudy": [ "Engineering", "Computer Science" ], "extfieldsofstudy": [] }
19598774	pes2o/s2orc	v3-fos-license	EFFECTIVENESS OF INFLUENZA VACCINATION IN ELDERLY OUTPATIENTS IN SÃO PAULO CITY , BRAZIL To investigate the effectiveness of the influenza vaccine in a population of elderly outpatients, we compared the occurrence and frequency of influenza like illness (ILI), respiratory illness and hospitalization in vaccinated and non-vaccinated subjects. All the outcomes were clinically defined. The two groups were similar with respect to demographics, health situation and habits. The influenza vaccine contributed to the protection of the elderly investigated, since the vaccinated subjects referred a significantly lower number of ILI than the non-vaccinated. There is a need for more studies about the effectiveness of the influenza vaccine in our country in elderly and other groups of our population. INTRODUCTION Influenza is an acute respiratory illness characterized by fever, myalgia, cough and headache.The aetiologic agent is an Orthomyxovirus, and three serotypes, A, B and C, are known to cause human illness.The disease lasts, in median, for three days, but cough and malaise can persist for weeks.Complications of influenza include bronchitis and pneumonia, besides otitis media and exacerbation of chronic respiratory disease 15 . The virus consists in an RNA core and a protein envelope.It has a marked capacity to mutate its external antigenic composition, thus escaping the host immune defenses.Minor changes in virus antigenic composition cause local epidemics.Pandemics arise when major changes in antigenic composition occur.Pandemics are thought to originate at the Far East, where humans, pigs and avian live in close proximity 6,9 . Climate, humidity and life-style are other factors associated to the occurrence of the influenza. The excessive morbidity and mortality, expressed as pneumonia, excessive hospitalization and death among elderly and patients with chronic medical conditions are described as the most serious result of influenza epidemics. The World Health Organization leads a worldwide Program of Prevention of Influenza, through a network of laboratories that provides surveillance and identification of influenza virus isolates from different continents. The Regional Group of Observation of Influenza (GROG) started its activities in 1995, in Brazil.Besides WHO, the GROG, now designated as "Grupo de Vigilância Epidemiológica da Gripe (VIGIGRIPE)" is responsible for the surveillance of the Influenza in Brazil.The number of specimens isolated and the viral identification is improving every year.In 1999 the VIGIGRIPE collected 1599 specimens and identificated 239 type A and 21 type B influenza virus 16 . In order to formulate the vaccine, viruses from all over the world are evaluated and, in February for the northern hemisphere, and in September for the southern hemisphere, the WHO publishes which strains of Influenza virus will likely predominate in the next season. The influenza vaccine is effective in elderly and patients with chronic medical conditions, as it has been demonstrated by different studies, many of them from the northern hemisphere 2,11 . The aim of this study was to investigate the level of protection conferred by the influenza vaccine in elderly outpatients from the city of São Paulo, Brazil. SUBJECTS AND METHODS This retrospective study was held during the 2000 annual campaign of vaccination against influenza.Individuals 60 years or older who sought the Immunization Center, located at the "Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo", in São Paulo city, for influenza vaccine, were enrolled.A questionnaire containing information on demographics, health conditions such as diabetes mellitus, systemic arterial hypertension (sah), chronic obstructive pulmonary disease (copd), asthma, bronchitis, pulmonary emphysema, previous respiratory disease, hematological disease, neoplasm, therapeutic with corticosteroids, tobacco and alcohol use and influenza vaccination in 1999 was applied.The same two investigators applied all the questionnaires.All the answers were registered. The primary outcomes measured refer to the comparative occurrence, in the previous 12 months, of influenza like illness (ILI), characterized as fever, cough, myalgia and headache, frequency of ILI, respiratory illness defined as any other disease than ILI compromising the respiratory system, hospitalization and hospitalization for respiratory diseases.All outcomes were clinically defined.No laboratory tests were applied to confirm reported episodes of ILI. Statistical analysis: Subjects vaccinated against influenza in 1999 were compared with those non-vaccinated with respect to all variables described above.The Pearson c 2 and the t-test were used to assess differences in proportions and medians of discrete and continuous data. A confidence level of 95%, with p < 0.05 was considered statistically significant. The vaccinated and non-vaccinated subjects did not differ with respect to sex, age, presence of underlying diseases, tobacco and alcohol use (Table 1).The only significant difference was in the number of episodes of influenza like illness (ILI).The vaccinated subjects referred less episodes of ILI than the non-vaccinated ones (Table 2). DISCUSSION Our study focused on the association of vaccination against influenza and the occurrence and frequency of clinically defined influenza likeillnesses, respiratory diseases, use of antibiotics for any disease and for The accepted method to assess the severity of the influenza season is estimating the excess of pneumonia and influenza (P&I) mortality.There is a linear correlation between excess of P&I hospitalizations and mortality rates, in USA 14 .The effect of influenza in mortality varies in different countries.REICHERT et al. states the role of influenza in mortality in Japan is much greater than in United States 12 .This difference is probably associated with the environment and life style.It has been hypothesized that the efficacy of the vaccination is related to the match between the vaccine and the epidemic strains 8 .A study held in Argentina showed a partial match between the circulating influenza virus and the vaccinal strains, from 1994 to 1998 13 . We found an association of previous vaccination against influenza with a reduction in the number of episodes of ILI, but not in other measured outcomes.The use of clinically defined ILI can account, at least partially, for the results. The efficacy of the influenza vaccine is well established in elderly 1 .In a population of working healthy adults, the efficacy and costeffectiveness of the vaccination against influenza are still not established 3 . The vaccines are more effective in reducing the laboratory confirmed cases of influenza than in the clinically defined cases 5 .Many cases of ILI, clinically defined, as we use as an outcome, may represent, in fact, infections caused by other agents.Laboratory-confirmed influenza represents just part of the clinically defined ILI. Our study can be compared to that by NICHOL et al. 10 , which targeted elderly people living in the community.But, differently from that study, we did not find an association of influenza vaccination and reduction of hospitalizations for respiratory diseases.This difference may be attributed to the fact that in the study by NICHOL et al. the vaccine recipients reported a higher proportion of coexisting diseases than the non-vaccinated. We did not find an association between the vaccination against influenza and the use of antibiotics for respiratory diseases, what makes our results different from that of CONNOLLY et al. 4 , who found, in a study from 1989, bronchitis and pneumonia the most frequent complications of influenza. In Brazil the annual campaign of influenza vaccination, held since 1998, is sponsored by the government.It provides free influenza vaccine for all persons 60 years or older, health care professionals and patients with chronic medical conditions, HIV infection and Aids.Since the influenza vaccination in Brazil has been available for large scale use for a few years, we can not compare our results with those found of HOSKINS et al. 7 , who suggested a decreased protection after a number of annual vaccinations.Besides, we do not have data on excess of mortality or hospitalization associated with influenza epidemics, before 1998. In Brazil the annual coverage of influenza vaccination in elderly is reducing, probably because large numbers of vaccinated did not perceive a reduction in the occurrence of influenza.Moreover misinformation attributes to the vaccine improbable side effects. There is a need to raise the coverage of influenza immunization in elderly, since the vaccination is associated with a protective effect in this population.More studies are necessary to define the role of the vaccine in the protection of other groups in our country.This is a retrospective study, based on information referent to the previous year, given by elderly subjects who may not have accurately remembered all possible influenza like illness episodes, what may have biased the results.Despite this facts, this study suggests that inactivated vaccine against influenza had a role in protection against influenza in elderly Brazilians. Table 1 Demographic characteristics, chronic diseases and habits of the patients Table 2 Measured outcomes according with vaccination against influenza or not in 1999 VAI: vaccinated against influenza in 1999; ILI*: influenza like illness; m = median; ¶ :p significant treatment of respiratory disease, hospitalization in general and for respiratory causes.	2017-06-24T12:23:14.742Z	2001-12-01T00:00:00.000	{ "year": 2001, "sha1": "5b78215b2baa7309074d1fe5a063a6b9389f2592", "oa_license": "CCBYNC", "oa_url": "https://www.scielo.br/j/rimtsp/a/CTmGkj9CW9wZNLqR7g3Wk5f/?format=pdf&lang=en", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "5b78215b2baa7309074d1fe5a063a6b9389f2592", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
233353653	pes2o/s2orc	v3-fos-license	Mediating Effect of Work Self-Efficacy on the Relationship Between Psychosocial Safety Climate and Workplace Safety Behaviors Among Bank Employees After Covid-19 Lockdown Purpose: Studies find that psychosocial safety climate is positively associated with workplace safety behaviors. However, the mechanism through which psychosocial safety climate exerts its effect needs further investigation. Therefore, this study investigated the indirect effect of work self-efficacy in the relationship between psychosocial safety climate and workplace safety behaviors. Methodology: Participants, who were 155 bank workers (F=66.5%; mean age= 33.9, SD=6.4), responded to an online survey of workplace safety behavior scale (WSBS), psychosocial safety climate scale (PSC-12), and work self-efficacy scale (WSES). Results: Results from correlational analyses revealed that psychosocial safety climate positively related to workplace safety behaviors. Moreover, work self-efficacy positively correlated to workplace safety behaviors. The mediation analysis using Hayes Process Macros indicated an indirect effect of work self-efficacy in the relationship between psychosocial safety climate and workplace safety behaviors. Creating a psychosocially safe climate may enhance bank workers’ safety behaviors in the period after the Covid-19 pandemic. Introduction Since the outbreak of the global Covid-19 pandemic, relevant agencies and governments have continued to make strategic efforts at containing the virus's spread. One of such efforts is the closure of banks to avoid physical contact. However, since banks cannot be closed forever due to ripple effects on the economy, there is a need to partially re-open them for business with stringent measures to ensure that further virus' spread is curtailed (see Nigeria Centre for Disease Control, NCDC, 2020). Thus, safety behaviors of bank workers may be relevant in order to protect them and their customers from contracting the virus. Safety behaviors were originally defined by Salkovskis (1991) as behaviors implemented -either overtly or covertly -in specific situations to prevent feared outcomes; these are preservative behaviors implemented to prevent anticipated negative consequences (Salkovskis, 1991). In the context of work, Griffin and Neal (2000) highlight that safety at work consists of safety compliance, which revolves around the use of appropriate personal protective equipment and following standard procedures that guide daily operations, along with safety participation (citizenship) that includes activities extending beyond the employees' formal work role (Chughtai, 2015) to include participation in safety-related campaigns and the promotion of safe work ethics among co-workers (Toppazzini et al., 2017). These elements can be strengthened by a safety initiative (Curcuruto et al., 2019) that agrees with the proactive responsibility of fostering changes that can lead to better safety practices. This is similar to the safety participation construct of Griffin and Neal (2000). Both situational and personal factors may affect safety behavior at work (Christian et al., 2009;qtd. in Toppazzini and Wiener, 2017). Situational factors such as motivation to practice safety behaviors (Curcuruto et al., 2018), transformational leadership (Conchie, 2013;Chughtai, 2015;Muchiri et al., 2019), and ethical leadership (Chughtai, 2015) can provide the inspiration needed for subordinates to observe safety regulations and make extra efforts toward a safe workplace. Person-related factors refer to specific individual characteristics that affect workplace safety behaviors. For instance, Toppazzini et al. (2017) report that conscientious personality types are more likely to observe safety rules and practices. Similarly, organizational commitment was also found to be associated with workplace safety behavior (Curcuruto et al., 2018). The present study examined the indirect effect of work self-efficacy in association between PSC and workplace safety behaviors among bank workers after a Covid-19 lockdown. Bond et al. (2010) define PSC as a "shared belief held by workers that their psychological safety and wellbeing is protected and supported by senior management," which is considered to minimize the risk of psychological and social harm that workers may experience 2012) in the course of discharging their duties. Empirical studies on the link between PSC and workplace safety behaviors abound. For instance, PSC is reported to predict employee's health and safety status (Ansah et al., 2018), but also correlate with well-being (Ishola, 2017), improved productivity (Becher et al., 2016), conducive work environment, better work engagement, and the reduction of psychological distress and emotional exhaustion (Dollard et al., 2012). Psychosocial Safety Climate (PSC) and Workplace Safety Behavior Related studies report a link between safety culture and safety performance (Otitolaiye, 2016;Nadhim et al., 2018). In the Covid-19 lockdown, PSC is expected to facilitate safety behaviors as Lyu et al. (2018) establish among construction workers. The same is reported among the staff of analytical laboratories in Nigeria (Agaja, 2012). Previous studies on PSC's links were conducted during normal operational activities in selected organizations. Employees with a high perception of safety climate tend to display a high level of safety behaviors while both safety climate and safety behaviors may result in reduced workplace accidents (Beus et al., 2010;qtd. in Toppazzini and Wiener, 2017). Hence the need to examine the role of PSC in workplace safety behaviors among bank employees after Covid-19 lockdowns. Therefore, we propose that: Hypothesis 1: Psychosocial safety climate will be positively associated with workplace safety behavior. Workplace Self-Efficacy and Workplace Safety Behavior Workplace self-efficacy is a personal factor that may impact bank employees' workplace safety behaviors in the lockdown following the Covid-19 pandemic spread. Bandura's (1997) views on the application of self-efficacy to the workplace captured "an individual's conviction (or confidence) about their abilities to mobilize the motivation, cognitive resources, and courses of action needed to successfully execute a specific task within a given context" (Stajkovic and Luthans, 1998, p. 66). Self-efficacy reflects the ability to increase effort and persist in a challenging task, which eventually increases the chance of success (Chughtai, 2015). Past studies establish an association between self-efficacy and success at work (Loeb, 2016), workplace well-being (Singh et al., 2019), improved job performance (Towler,CEMJ 5 Mediating Effect of Work Self-Efficacy on the Relationship Between Psychosocial Safety Climate 2019), and organizational citizenship behavior (Alessandri et al., 2021). Individuals who have high self-efficacy can assume more challenging goals in a persistent manner to achieve success (Towler, 2019), especially during the pandemic and public health challenges caused by Covid-19. These studies hint at a strong relationship between workplace self-efficacy and workplace safety behaviors (Chen and Chen, 2014). The higher the perceived self-efficacy of employees, the more likely they maintain workplace safety behaviors (Chen and Chen, 2014). Similarly, Nykanen, Salmela-Aro, Tolvanen, and Vuori (2019) confirm that safety intervention can lead to an increase in both safety-related self-efficacy and internal locus of control, while Curcuruto, Parker, and Griffin (2018) report that safety initiative can enhance self-efficacy beliefs and a future-focused approach to safety. Therefore, we propose that workplace self-efficacy will play a role in employees' readiness to follow safety rules and guidelines prescribed by the NCDC (NCDC, 2020) to combat Covid-19. Thus, we hypothesize that: Hypothesis 2: Work self-efficacy will be positively associated with workplace safety behavior. Mediating Role of Work Self-Efficacy We proposed that PSC positively influences bank employees' workplace safety behaviors through workplace self-efficacy. A work environment with a psychosocially safe climate enhances employees' health, wellbeing, and productivity (Becher et al., 2016;Ishola, 2017;Ansah et al., 2018), which is a result of employees' perception of management commitment to and prioritization of their safety concerns. Such a work environment may motivate employees to participate and show commitment to issues that border on their own and others' safety. Bandura (1997) opines that self-efficacy is a positive personal characteristic that may positively influence behavioral outcomes. The Covid-19 pandemic has stretched demands for workplace safety beyond specific occupations to all work settings. Employees in all occupational settings are now required to adhere to specific safety standards in order to keep themselves and others safe at work. Bank workers are also expected to actively participate in safety concerns because their inability to do so may have implications for their own and customers' health. While management is expected to commit to a psychologically safe workplace (Dollard et al., 2012;Yulita et al., 2016), employees' characteristic -such as work self-efficacy -may enhance their compliance and participation. Previous studies show that self-efficacy mediates the relationship between transformational leadership and work engagement (Prochazka et al., 2017). Hence, we hypothesize that: Material and Method Participants and Procedure Due to the restrictions by the current global Covid-19 pandemic, this survey was conducted online via Google forms. Participants were employees of selected banks in Nigeria. Their consent to participate in the study was sought before they proceeded to complete the questionnaire. Out of the 157 responses from bank workers who responded to the online survey, 155 results (F=66.5%; mean age= 33.9, SD=6.4) were found useable. The response rate was 98.7% and the results were gathered from May 30 to July 8, 2020. The distribution of participants by status revealed that there were 35 (22.6%) junior staff, 72 (46.5%) middle level staff, and 48 (31%) of the senior cadre. Measures Workplace safety behaviors were assessed using the safety behavior questionnaire by Vinodkumar and Bhasi (2010). This study made use of the safety compliance and safety participation components of the scale, with eight items. Participants responded to a five-point Likert format ranging from "strongly disagree" (1) to "strongly agree" (5). The responses were summed to arrive at a global score of safety behavior at the workplace. The scale demonstrated strong internal reliability of the study (α = .91). Psychosocial safety climate was measured using the 12-item psychosocial climate scale (PSC-12) developed by Hall, Dollard, and Coward (2010). The scale has four subscales Work Self-efficacy WSB PSC 0.21 0.31 0.12** that can either be summed or treated separately: management priority, management commitment, organizational communication, and organizational participation. This study employed a sum total to examine direct and indirect effects while the subscales were also utilized in a correlational analysis. PSC-12 had a five-point Likert response from 1 = "strongly disagree" to 5 = "strongly agree." The sum total showed a Cronbach alpha coefficient internal consistency of α = .70 Work self-efficacy was measured with a six-item work self-efficacy subscale from the original psychological capital questionnaire developed by Luthans et al. (2007). The subscale has a five-point Likert format response format, viz. 1 = "strongly disagree" to 5 = "strongly agree." The scale showed a strong internal consistency for this study (α = .91). Results The results gathered in Table 1 revealed a significantly positive association among the dimensions of psychosocial safety climate, viz. management commitment (r = .29, p < .01), management priority commitment (r = .29, p < .01), management communication commitment (r = .35, p < .01), management participation commitment (r = .36, p < .01), and work self-efficacy. Moreover, the results revealed a positive significant relationship between management commitment and workplace safety behaviors (r = .37, p < .01), management priority and workplace safety behaviors (r = .37, p < .01), management communication and workplace safety behaviors (r = .42, p < .01), along with management participation and workplace safety behaviors (r = .48, p < .01). Overall, the results showed a positive significant association between psychosocial safety climate and workplace safety behaviors (r = .48, p < .01). Furthermore, PSC is significantly and positively associated with work self-efficacy (r = .36, p < .01). Finally, work self-efficacy is significantly and positively associated with workplace safety behaviors (r = .52, p < .01). Mediation Effect of Work Self-Efficacy The IBM SPSS process macro by Hayes (2018;v.3.4) was used to conduct the mediation analysis; the tool was recently used by researchers to estimate the mediation model (Cero and Sifers, 2013). Moreover, the capacity for the process macro to estimate model coefficient with confidence interval through bootstrapping approach was found to be beneficial (Hayes, 2013). Table 2 showed a statistically significant total effect of psychosocial safety climate on workplace safety behaviors (β = 0.31, SE = .05, p < .001), but also a significantly direct effect of psychosocial safety climate (β = 0.21, SE = .05, p < .001) and work self-efficacy (β = 0.68, SE = .12, p < .001) on workplace safety behaviors. These results indicated that psychosocial safety climate and work self-efficacy independently predict workplace safety behaviors. Furthermore, the standardized indirect effect of psychosocial safety climate and workplace safety behaviors via work self-efficacy is significant (β = 0.15, SE = .08, 95% CI = [0 .004, .306]). The overall proportion of the total effect was 60%. Discussion The study examined the association between PSC and workplace safety behavior, along with the indirect effect of work self-efficacy in the link between PSC and workplace safety behaviors among bank workers after the Covid-19 lockdown. As hypothesized, our findings revealed that PSC has a positive association with workplace safety behavior. Thus, management priority, commitment, communication, and participation in advancing a safe environment for daily operational activities assist employees to display safety behaviors so as to curtail the spread of the virus. Previous findings establish similar positions that PSC facilitates safety behavior (Otitolaiye, 2016;Nadhim et al., 2018;Lyu et al., 2018). This is due to the capacity of PSC to create a conducive work environment (Ishola, 2017) that enables employees to ensure adherence to safety guidelines as proposed by the Nigeria Centre for Disease Control (NCDC, 2020). Moreover, such an environment facilitates initiatives among workers (Curcuruto et al., 2018), hence when employees know that they can use their initiatives, they are likely to reciprocate by performing safety behaviors at work. Furthermore, the study established a positive association between work self-efficacy and employee safety behaviors, which agrees with Bandura's (1986) position that a reasonable belief in one's ability to perform a task will enhance ultimate success in the pursuit of such tasks. This study suggested that bank workers after the Covid-19 lockdown show significant capacities to ensure that the spread is curtailed, thus preventing the further spread of the virus. This finding agrees with previous studies that find self-efficacy to be associated with success in tasks (Loeb, 2016;Towler, 2019) and that safety initiative is to be driven by self-efficacy (Nykanen et al., 2019). Plausibly, bank workers' beliefs in their capacities to actively follow safety measures can lead to better workplace safety behaviors and, consequently, the combating of the spread Finally, in confirmation of the study prediction, we found that work self-efficacy mediates the relationship between PSC and workplace safety behaviors. Bandura's (1997) proposition that self-efficacy can improve chances of success was established through its role in the interplay between PSC and workplace safety behavior. Prochazka et al. (2017) establish that employees' engagement can improve with good management through self-efficacy. Thus, the possible safety behaviors that bank workers now display in the partial reopening of the economy during the Covid-19 pandemic in a psychosocially safe climate are enhanced by work self-efficacy. The implications of the findings from this study are that practitioners and researchers may need to investigate workplace safety beyond the supposed high-risk occupations, especially after the experience of the Covid-19 lockdown. At the managerial level, this study confirmed the importance and necessity for positive PSC because when employees feel that their employers show commitment and give priority to safety concerns at the workplace, the employees reciprocate by complying with safety rules and participating in the safety behaviors, thus reducing the spread of the pandemic. Similarly, management may need to take certain practical measures to ensure that safety equipment -both for personal and customer use -is available in sufficient quantity and quality. Moreover, an appropriate monitoring procedure is essential to guarantee enforcement so employee initiatives should also be encouraged so as to enhance their self-efficacy because when employees believe that their efforts are recognized and permitted, it boosts their self-efficacy. One of the few limitations of this study is the online nature of the survey, meaning that we were unable to precisely establish the number of people who received the link we shared against those who eventually responded to the survey. Hence, we encountered difficulties in ascertaining the response rate. We had to employ this method due to the necessity of a social distancing protocol, which forbids close contact with people during the Covid-19 pandemic. Another limitation was the cross-sectional nature of the data, which requires others to treat the results with caution. A longitudinal design would have been more appropriate but was difficult for implementation during the pandemic. Furthermore, data for this study came from employees in the banking sector in Nigeria. Thus, we call for caution when transposing our findings onto other sectors that may not meet the conditions under which this study was conducted. Therefore, we suggest that similar studies should be conducted in other sectors such as educational institutions, the health sector, and retail. We conclude that PSC plays a significant role in employees' workplace safety behaviors. This is because the study confirmed our hypothesis that PSC is positively associated with workplace safety behaviors. The current study was able to contribute to the expansive body of knowledge not only by establishing the positive relationship between PSC and work self-efficacy but also by enabling -through work self-efficacy -the association between PSC and workplace safety behavior. Hence, this initial study establishes an indirect effect of work self-efficacy in the association between PSC and workplace safety behavior, particularly after the Covid-19 lockdown in Nigeria.	2021-04-23T13:13:49.290Z	2021-03-01T00:00:00.000	{ "year": 2021, "sha1": "7de4e82dc4323c369560a41557ca94044ce53b07", "oa_license": "CCBY", "oa_url": "https://journals.kozminski.edu.pl/system/files/Akanni.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "43cb5056bc09540a547019130728b21b13571fb0", "s2fieldsofstudy": [ "Psychology", "Business" ], "extfieldsofstudy": [ "Medicine" ] }
56613611	pes2o/s2orc	v3-fos-license	Laboratory Diagnosis of Antiphospholipid Syndrome Antiphospholipid syndrome (APS) is a heterogeneous autoimmune disorder characterized by arterial or venous thromboembolic events and obstetric complications in association with the persistent laboratory evidence of antiphospholipid antibodies (aPL). As the clinical manifestations of APS lack specificity, the diagnosis of APS is essentially dependent on the detection of circulating aPL. These aPL are autoantibodies directed against a complex of phospholipids and phospholipid-binding proteins, not directly binding to phospholipids. aPL associated with APS include lupus anticoagulant (LA), anticardiolipin (aCL), and anti-β2 glycoprotein-I (aβ2GPI) IgG or IgM antibodies. LA is a subset of aPL that binds to phospholipid-associated proteins in coagulation complexes and disrupts phospholipiddependent coagulation tests. aPL such as aCL and aβ2GPI antibodies are traditionally detected by ELISA, however newer automated platforms with various solid analytic and detection systems are now available in the diagnostic market. The international classification criteria of APS were developed and revised by international experts and societies, publishing international consensus guidelines on the recommended best practices for technical and performance requirements. To make a definitive diagnosis of APS, the presence of at least one clinical feature (vascular thrombosis or pregnancy morbidity) and one laboratory abnormality must be observed (Table 1). Persistent positivity of laboratory tests is important and the laboratory abnormality must be present on two or more occasions at least 12 weeks apart. A remote test could avoid false positive results from the interfering events, but positive tests separated more than five years from a clinical manifestation also risk misclassification. Despite these attempts to produce consensus guidelines, some issues related to laboratory testing still remain unresolved. Poor assay reproducibility, variable sensitivity and specificity, and a lack of standardization and harmonization comprise methodological problems. But progress has been made and reference calibrators for aβ2GPI immunoassays was newly developed. In this review, we want to give an overview of laboratory testing for APS with summary of recent international consensus guidelines for the detection of aPL. Also a brief review of prognostic significance of aPL profile and potential future diagnostic assay is presented. Introduction Antiphospholipid syndrome (APS) is a heterogeneous autoimmune disorder characterized by arterial or venous thromboembolic events and obstetric complications in association with the persistent laboratory evidence of antiphospholipid antibodies (aPL). [1][2][3] As the clinical manifestations of APS lack specificity, the diagnosis of APS is essentially dependent on the detection of circulating aPL. These aPL are autoantibodies directed against a complex of phospholipids and phospholipid-binding proteins, not directly binding to phospholipids. aPL associated with APS include lupus anticoagulant (LA), anticardiolipin (aCL), and anti-β2 glycoprotein-I (aβ2GPI) IgG or IgM antibodies. LA is a subset of aPL that binds to phospholipid-associated proteins in coagulation complexes and disrupts phospholipiddependent coagulation tests. [1][2][3][4][5][6][7] aPL such as aCL and aβ2GPI antibodies are traditionally detected by ELISA, however newer automated platforms with various solid analytic and detection systems are now available in the diagnostic market. The international classification criteria of APS were developed and revised by international experts and societies, publishing international consensus guidelines on the recommended best practices for technical and performance requirements. [8][9][10][11][12][13][14] To make a definitive diagnosis of APS, the presence of at least one clinical feature (vascular thrombosis or pregnancy morbidity) and one laboratory abnormality must be observed (Table 1). Persistent positivity of laboratory tests is important and the laboratory abnormality must be present on two or more occasions at least 12 weeks apart. 9 A remote test could avoid false positive results from the interfering events, but positive tests separated more than five years from a clinical manifestation also risk misclassification. Despite these attempts to produce consensus guidelines, some issues related to laboratory testing still remain unresolved. Poor assay reproducibility, variable sensitivity and specificity, and a lack of standardization and harmonization comprise methodological problems. 6,7,[15][16][17] But progress has been made and reference calibrators for aβ2GPI immunoassays was newly developed. 17 In this review, we want to give an overview of laboratory testing for APS with summary of recent international consensus guidelines for the detection of aPL. Also a brief review of prognostic significance of aPL profile and potential future diagnostic assay is presented. Lupus anticoagulant LA is quite heterogeneous group of antibody and does not show uniform aPL activities, therefore laboratory detection of LA is limited to phospholipid-dependent clotting assays. 1-3,6-8 Consensus guidelines from different societies have been introduced and updated to guide the testing and interpretation process. In 2009, an update of the International Society on Thrombosis and Haemostasis (ISTH) guideline was published, 10 and followed by a updated guideline from the British Committee for Standardization in Haematology (BCSH) in 2012. 11 Recently, the Clinical Laboratory Standards Institute (CLSI) also published LA detection guideline in 2014. 14 General agreements exist on sample preparation, use of dilute Russell's viper venom time (dRVVT) in diagnostic repertoires, use of normalized ratios, phospholipid-dependent calculations, mixing test interpretation, and interpretative reporting. 2,7,15,18,19 Recommendations differ on reference interval cut-offs and all three cover testing of anticoagulated patients ( Table 2). The current ISTH guideline stresses that LA testing should be limited to patients who have a significant probability of having APS, or who have unexplained prolonged activated partial thromboplastin time (aPTT) in the course of routine laboratory testing. 10 Double centrifugation to obtain platelet poor plasma is advocated by each guideline. There is evidence that no single assay system is sensitive enough to detect all LA and multiple assays are required to ensure weak LA detectable and to improve specificity, although one positive test is regarded as having a LA. [9][10][11]14 All guidelines agree on the use of dRV-VT as the primary test for LA. The ISTH guideline recommends dR VVT for its specificity and aPTT with low phospholipid concentration because of its sensivity. 10 BCSH states that the second test can be an aPTT with proven LA sensitivity, a modified aPTT, or a dilute prothrombin time (PT). 11 The CLSI guideline 14 recommends aPTT as the second test, but suggesting to employ additional tests. Recent studies using two different LA methods provided more objective LA reporting with additional diagnostic information. 20,21 Sensitivity and specificity of assays also depend on the cut-off values and can be improved by establishing local reference ranges. ISTH recommends the cut-off values above the 99th percentile of the distribution, whereas both BCSH and CLSI recommend 97.5th percentile of normally distributed data. [9][10][11]14 Mixing test is the second step in LA three-step procedure (screening, mixing, and confirmation) and improves the specificity. CLSI considers the mixing step as the last one and unnecessary in specific circumstances. 14 All guidelines agree that mixing tests should be performed with a 1:1 proportion of patient and pooled normal plasma (PNP). Test results are interpreted based on the cut-off value of clotting time or the calculated index of circulating anticoagulant (ICA). Confirmatory test must be performed by increasing the phospholipid concentration and in addition, BCSH suggest platelet neutralization procedure or LA insensitive reagent. 10,11 Phospholipid dependence is calculated as the percentage correction or LA ratio (screen/confirm). 15,18,19 To improve the performance of LA assays, the conversion of clotting times for screen, mixing, and confirm tests to normalized ratio using the PNP value is advocated by ISTH and BCSH. 1,2,10,11,15 Integrated tests include screening and confirmation (low and high phospholipid concentration) in a single procedure on every patient irrespective of the abnormal screen test results. 10,14,18,19 This approach is advanta- 1. Vascular thrombosis One or more episodes of arterial, venous, or small vessel thrombosis, in any tissue or organ 2. Pregnancy morbidity (a) One or more unexplained deaths of a morphologically normal fetus at or beyond the 10th week of gestation, or (b) One or more premature births of a morphologically normal neonate before the 34th week of gestation because of: (i) eclampsia or severe preeclampsia defined according to standard definitions, or (ii) recognized features of placental insufficiency, or (c) Three or more unexplained consecutive spontaneous abortions before the 10th week of gestation, with maternal anatomic or hormonal abnormalities and paternal and maternal chromosomal causes excluded Laboratory criteria 1. Lupus anticoagulant present in plasma, on two or more occasions at least 12 weeks apart 2. Anticardiolipin antibody of IgG and/or IgM isotype in serum or plasma, present in medium or high titer (i.e. > 40 GPL or MPL, or > the 99th percentile), on two or more occasions, at least 12 weeks apart 3. Anti-β2 glycoprotein-I antibody of IgG and/or IgM isotype in serum or plasma (in titer > the 99th percentile), present on two or more occasions, at least 12 weeks apart Modified from Miyakis et al. 9 Antiphospholipid syndrome is present if at least one of the clinical criteria and one of the laboratory criteria are met. GPL, IgG phospholipid units; MPL, IgM phospholipid units. geous on the detection of weaker LA, but still raising concern on the performance of mixing tests. 22,23 All three guidelines concur that it is not ideal to test for LA while the patients are being treated with anticoagulant therapy. 2,7,10,11,14,18,19 ISTH recommends that LA test can be performed on undiluted plasma if the international normalized ratio (INR) is less than 1.5. Alternatively, if the INR is between 1.5 and < 3.0, a 1:1 dilution of patient plasma and PNP can be considered. 10 BCSH and CLSI suggest LA test is possible on 1:1 mixtures with PNP for higher INR values. 11,14 ISTH and CLSI urge caution when interpreting LA results on patients receiving unfractionated heparin (UFH), whereas BCSH specifies that LA tests should not be performed, if the patient is receiving therapeutic doses of UFH, because it may cause erroneous results. 11 Only CLSI covers potential interferences by new anticoagulants, information on which is still awating. 14,18,19 LA results should always be considered in the context of a full laboratory aPL profile. 10 Three guidelines all state that quantitative results should be accompanied by an interpretative comment that indicating whether the findings are consistent with the presence or absence of LA. 10,11,14,18,19 Each supports the recommendation to retest at least 12 weeks apart for the evidence of antibody resistance. Anticardiolipin and anti-β2 glycoprotein-I antibodies In 2006, ISTH updated the clinical and laboratory criteria for APS. 9 According to the revised criteria, APS requires the presence of IgG and/or IgM aCL antibodies exceeding 40 IgG (GPL) or IgM (MPL) phospholipid units or 99th percentile, or IgG and/or IgM aβ2GPI antibodies at titers exceeding 99th percentile, on two or more occasions, at least 12 weeks apart. IgA isotype of aPL was not included in the current laboratory criteria. Both aCL and aβ2GPI ELISA should be performed following standardized procedures (Table 1). A new guidance from ISTH for the testing aPL with solid phase assays was recently introduced in 2014. 13 To be useful in the standardization of the assays, it published ten recommendations covering from patient selection to results interpretation and reporting (Table 3). Various international workshops and the emergence of new platforms and technologies also have contributed to reduce laboratory variabilities. 8,12,[24][25][26] Testing for aPL by solid phase assays should also focus on patients who are likely to have APS. [1][2][3][4][5]8,9,[11][12][13]15 Collection, storage and handling of samples are less critical compared to LA test. Serum or plasma can be used. The manufacturer should state the sample type recommended and plasma should be platelet poor by double centrifugation. 12,13 aCL antibody assays use the complex of cardiolipin plus β2GPI (human or bovine) as antigen. Because not all human aβ2GPI antibodies bind to β2GPI from other sources, whole molecule of β2GPI of human origin should be used. 12,13 As no gold standard exists, new assays should be validated before implementation. Linearity, precision, limit of detection, internal and external quality control, and clinical performance should be checked. The possible interfering factors such as cryoglobulins, rheumatoid factors, hemoglobin, bilirubin and triglycerides may cause bias on results. 9,11-13 To avoid errors, duplicate testing is recommended especially for manual ELISA tests, but technical progress with automated platforms may allow single testing for patient samples and controls. In contrast to ELISA, newer automated platforms does not require calibration curves for every run either, unless the reagent lot is changed. 12,13 The test signals are converted to antibody units derived from the calibration curve. Development of an international standard will facilitate the uniformity in reporting results of aCL and aβ2GPI antibodies. 13,27 Normal cut-off values should be determined in healthy subjects using the 99th percentile. [9][10][11][12][13] If this is not feasible, manufacturer's cut-offs may be acceptable after adequate verification process. 12,13 aβ2GPI assay shows higher specificity than aCL for APS diagnosis and the use of 99th percentile cut-offs seem to be more sensitive than the > 40GPL units. 2,9,13,15 Results of aCL and aβ2GPI antibody tests should be interpreted with LA results in view of clinical context. Each test result above cut-off should be regarded as positive and positive results need to be confirmed after 12 weeks. Inclusion of interpretative comment is strongly recommended. 12,13 Mostly aCL and aβ2GPI antibodies are measured by ELISA, although chemiluminescence (CLIA) and fluorescence enzyme immunoassays (FEIA) have recently been introduced to the market. 16,17,[27][28][29][30][31][32][33] In most of the cases, fair to moderate agreement was found among different assays. 16,17,29,[31][32][33] de Moerloose et al. 28 observed far higher agreement between three well-established commercial assays. However, on the contrary Gutensohn et al. 30 found moderate to poor accordance between five different assays in women with a history of miscarriage. The low agreement may be due to the selection of study population, low number of positive results, and different cut-off values. Different detection methods, antigen epitope variations, different assay designs, test interferences, and pre-or post-analytical issues all can be further reasons for the variability. Most samples showing imperfect agreement had values around the borderline or low-positive ranges. Utilizing an automated system can improve reproducibility and reduce interlaboratory variation. [15][16][17][27][28][29][30][31][32][33] Willis et al. 17 also analyzed that when the assays were calibrated using the recently developed reference IgG materials, the correlations for aβ2GPI antibodies were more improved. Anti-β2 glycoprotein-I antibody against domain I β2GPI is a 326 amino acid polypeptide synthesized by hepatocytes, endothelial cells, and trophoblasts. It contains five homologous domains of approximately 60 amino acids each, domain I at the N-terminus though to domain V at the C-terminus. 1,2,24 The β2GPI molecule circulates in plasma as a closed globular conformation and hides a cryptic epitope on domain I, thus preventing antibody binding. When β2GPI is immobilized on a negatively charged phospholipid surface via domain V, the configuration changes to an open form. This interaction induces exposure of the cryptic domain I to which aβ2GPI antibodies can then bind. 1,2,8,24,34,35 Recent studies demonstrated that aβ2GPI antibodies against domain I correlate well with thrombosis and obstetric complications compared to other domain-binding antibodies. [1][2][3]5,8,13,15,24,26,[34][35][36][37] Because not all of the patients with aPL in their plasma suffer from thrombosis and/or pregnancy morbidity, it is suggested that there exist different populations of aPL. de Laat and de Groot 34 found that about half of patients with aβ2GPI antibodies showed reactivity toward domain I. Besides, the presence of anti-domain I antibodies showed better association with thrombosis at odds ratio (OR) of 18.9 and 95% confidence interval (6.8-53.2), compared with aβ2GPI antibodies toward other domains (OR 1.1, range 0.4-2.8). Through an international multicenter evaluation, they could confirm better correlation of aβ2GPI domain I antibodies with thrombosis and also found a better association with pregnancy morbidity (OR 2.4, range 1.4-4.3). Mondejar et al. 36 and Pengo et al. 37 show that CLIA technology Patient selection Generalized search for aPL is discouraged to prevent incidental findings Testing should focus on younger patients ( < 50 years) with unprovoked venous/arterial thromboembolism, thrombosis at unusual sites or thrombotic/pregnancy complications associated with autoimmune disease 2. Blood collection Serum or citrated plasma (0.109 M sodium-citrate) platelet-poor ( < 10,000 platelets per uL) plasma Assay specifications should be validated if a different sample type to the one indicated by the manufacturer is used Samples stored at 2-8˚C and tested within 2-3 days or at -20˚C or below for longer storage. Avoid freeze-thawing cycles 3. Choice of assays Perform a β2 glycoprotein-I (β2GPI)-dependent anticardiolipin (aCL) and an anti-(aβ2GPI) assay Human β2GPI should be used as β2GPI source Performance characteristics Between-run imprecision should be < 20% (ELISA) and < 10% for automated systems Internal quality control material (included in the kit or non-kit material) that is negative and another with potency around cut-off should be included in every run At least one non-kit negative and positive control (commercial or patient material) material should be included in every run A run should be rejected if one control sample is out of the allowed rage Participation in an external quality control scheme is strongly recommended Detection limits should be determined in a negative sample with the same matrix as the patient samples Samples with values above the analytical measuring range of the test should be diluted and re-tested or reported as 'higher than the upper measurement range value' Values below detection limits should be considered as 'lower than the lower limit of detection' The imprecision of the assay should be considered for interpretation of results around cut-off Whenever feasible assays should be evaluated for clinical performance in detecting thrombotic/pregnancy complications Interferences Rheumatoid factor can produce falsely elevated IgM aCL and aβ2GPI Avoid icteric, hemolytic and lipemic samples Heterophile antibodies, human anti-animal antibodies and high levels of (monoclonal) immunoglobulins may produce false-positive results Triple anti-phospholipid antibody positivity Current guideline advises investigators to classify APS patients into four different risk categories according to one or more positive aPL tests. 9 It is because certain issues of specificity and sensitivity of laboratory assays remain unresolved, while there are evidences that multiple aPL positivity is associated with increasing rate of thrombosis. Several clinical studies indicate that triple positive APS patients are at high risk to develop recurrent thrombotic events. 24,26 Pengo et al. 38 also showed that 98% of initial triple positivity was confirmed after 12 weeks, while double or single positive cases were confirmed in 84% and 40%, respectively. Similarly during a two to four years of follow up for 136 initial positive samples, we could find that 100% of initial triple positivity confirmed aPL presence, whereas double or single positive cases confirmed positivity in 75.0% and 38.3%, respectively (unpublished data). Further study is required to determine whether aPL profile will affect APS classification and then influence clinical decision making. Conclusion APS requires the combination of at least one clinical and one laboratory criterion. Testing for aPL should be limited to patients who have a significant probability of having APS. Persistent positivity of laboratory tests is important. Solid phase aCL and aβ2GPI antibody tests should interpreted together with coagulation LA tests to assess the clinical significance. ELISA detection of aPL has been widely used domestically as well, although CLIA and FEIA methods have recently been introduced. 31, 33 We also follow the revised international guidelines to monitor repeat positivities in Korea. 40,41 Several studies have shown that triple positivity correlates more strongly with both thrombosis and pregnancy morbidity than the presence of double or single positivity. IgG aβ2GPI domain I antibody was mainly present in triple positive patients and associated with thromboembolic events. However, we should be aware of interassay and interlaboratory variability and the performance characteristics of the assays.	2018-12-22T14:03:46.140Z	2017-05-02T00:00:00.000	{ "year": 2017, "sha1": "34e90fb1170a49bd84985557f79cf3736709050a", "oa_license": null, "oa_url": "https://doi.org/10.14345/ceth.17002", "oa_status": "GOLD", "pdf_src": "MergedPDFExtraction", "pdf_hash": "62b7951f36c5f4a7e649354be22e08b665fc8d96", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
6637654	pes2o/s2orc	v3-fos-license	Atoll-scale patterns in coral reef community structure: Human signatures on Ulithi Atoll, Micronesia The dynamic relationship between reefs and the people who utilize them at a subsistence level is poorly understood. This paper characterizes atoll-scale patterns in shallow coral reef habitat and fish community structure, and correlates these with environmental characteristics and anthropogenic factors, critical to conservation efforts for the reefs and the people who depend on them. Hierarchical clustering analyses by site for benthic composition and fish community resulted in the same 3 major clusters: cluster 1–oceanic (close proximity to deep water) and uninhabited (low human impact); cluster 2–oceanic and inhabited (high human impact); and cluster 3–lagoonal (facing the inside of the lagoon) and inhabited (highest human impact). Distance from village, reef exposure to deep water and human population size had the greatest effect in predicting the fish and benthic community structure. Our study demonstrates a strong association between benthic and fish community structure and human use across the Ulithi Atoll (Yap State, Federated States of Micronesia) and confirms a pattern observed by local people that an ‘opportunistic’ scleractinian coral (Montipora sp.) is associated with more highly impacted reefs. Our findings suggest that small human populations (subsistence fishing) can nevertheless have considerable ecological impacts on reefs due, in part, to changes in fishing practices rather than overfishing per se, as well as larger global trends. Findings from this work can assist in building local capacity to manage reef resources across an atoll-wide scale, and illustrates the importance of anthropogenic impact even in small communities. The dynamic relationship between reefs and the people who utilize them at a subsistence level is poorly understood. This paper characterizes atoll-scale patterns in shallow coral reef habitat and fish community structure, and correlates these with environmental characteristics and anthropogenic factors, critical to conservation efforts for the reefs and the people who depend on them. Hierarchical clustering analyses by site for benthic composition and fish community resulted in the same 3 major clusters: cluster 1-oceanic (close proximity to deep water) and uninhabited (low human impact); cluster 2-oceanic and inhabited (high human impact); and cluster 3-lagoonal (facing the inside of the lagoon) and inhabited (highest human impact). Distance from village, reef exposure to deep water and human population size had the greatest effect in predicting the fish and benthic community structure. Our study demonstrates a strong association between benthic and fish community structure and human use across the Ulithi Atoll (Yap State, Federated States of Micronesia) and confirms a pattern observed by local people that an 'opportunistic' scleractinian coral (Montipora sp.) is associated with more highly impacted reefs. Our findings suggest that small human populations (subsistence fishing) can nevertheless have considerable ecological impacts on reefs due, in part, to changes in fishing practices rather than overfishing per se, as well as larger global trends. Findings from this work can assist in building local capacity to manage reef resources across an atoll-wide scale, and illustrates the importance of anthropogenic impact even in small communities. PLOS Introduction the advent of small motorboats, and the loss of traditional management frameworks, have likely all contributed to changes in reef health and fish abundance on Ulithi. These patterns need to be better understood in order to document climate change impacts and the effects of endemic human drivers (including resource extraction and management), as well as to facilitate mitigation planning, adaptation, and local capacity building. Ulithi Atoll therefore presents a unique opportunity to study an isolated social-ecological system whose ecosystem services have been compromised, at least in part due to subsistence level extraction activities in the relatively recent past despite continuous use for over 2000 years [25]. This study characterizes atoll-scale patterns in shallow coral reef habitat and fish community structure by identifying patterns and correlations between environmental characteristics, human communities, and biological systems on Ulithi Atoll. Specifically, we addressed: 1) reef fish and benthic community structure variability across the Atoll, 2) characteristics that define reef types, and 3) factors influencing this variation. Ethics statement We received specific permission to sample reefs from Philip Paiy, Chief of Falalop, Lipipi Clan, representing Council of Ten (CO-X), Ulithi Atoll. We also received a formal invitation to conduct the research from the Council of Tamol (Branch of Yap legislature representing the outer islands), and received stated support from Yap Marine Resources. We had formal meetings with the Governor's office (Yap) and the Legislature to brief them on the work. Finally, we received on-site permission from village Chiefs (or designee) prior to each sampling visit before any inwater work was conducted. All data and findings were shared with each village on every island. No institutional Animal Care and Use committee approval was needed; no animals were disturbed, collected or sacrificed. However, all sampling procedures were reviewed and approved by the local communities as part of obtaining permission to complete this research. Study location The Ulithi lagoon encompasses approximately 500 km 2 making it one of the largest atolls on Earth (Fig 1). The Atoll consists of ca. 44 low-lying (max. elevation ca. 10 m) islets and islands (total land area ca. 4.3 km 2 ). The diversity of reef types (and associated biodiversity) is high due to both physical factors and human settlements [27]. Permanent residents of Ulithi number about 900, with approximately another 350 people from other neighboring islands living on the Atoll during the school year (August-May). The 4 inhabited islands are Falalop (which lies just outside the main Atoll) with a population of between 500-700 people, MogMog (Northern part of the Atoll), with a population of approximately 150, Asor (North-East part of the Atoll), with a population of approximately 70, and Federai to the southeast, with a population of approximately 150 (Fig 1). The populations of these islands have remained relatively stable, with the exception of Falalop, which has increased by about 450 people since 1949 (this is largely due to the building of an airstrip during World War II, and the construction of the Outer Islands High School in 1961) [24]. Site selection During three consecutive years (2012-2014), we sampled reef fishes, benthic composition, coral morphology, and coral distribution at 32 sites throughout the Atoll and associated nearby islands (Fig 1, Table 1). We selected sites across a broad spectrum of biotic and abiotic factors. A major goal of our larger project is to address concerns of the local communities regarding reef health, so we considered input from local people about the degree of fishing pressure, perceived reef degradation, and historical and current use in order to aid in site selection. Surveys were conducted during June-July of each year. Sites covered a range of anthropogenic disturbances, including fishing pressure, boat use (launching, anchoring), nutrient input (human and livestock waste), and physical disturbance from World War II (dredging, physical barrier construction, use of amphibious landing craft, coral mining for construction). Site selection was based on proximity to human settlements, management (restrictions on human access to reef resources), and jurisdiction (number of human dependents in a management area). We also considered environmental factors unaffected by human influence such as exposure of reefs to deep water (e.g. oceanic or lagoonal site). Lastly, we sought broad coverage of the Atoll and nearby islands. Due to logistical constraints and diving conditions, 'oceanic' sites were primarily on leeward sides of islands, but exposed to very deep water, and outside the lagoon. Because local inhabitants have preferentially occupied the more sheltered locations inside the lagoon, there were few 'inhabited oceanic' sites, and they were mostly limited to the Leeward side of one island (Falalop; Fig 1). This was unfortunate from an experimental design perspective, but unavoidable. The remote location and logistical considerations necessitated the use of snorkel gear rather than scuba gear for all surveys. Over the course of the study, the same team of researchers surveyed the reef crest and the reef table in shallow sites at depths between 1.5 and 5 meters. In general, the reefs dropped off quickly below this depth, as the Atoll is surrounded by very deep water. Even the lagoonal sites dropped off rapidly to sand or deeper reefs. Benthic characterization Benthic community structure was evaluated using 0.25m 2 quadrats placed randomly on the reef crest area at each site between 1.5-5 m depth. Twenty quadrats per site each year was the Fig 2, whether the site was located within the Ulithi lagoon or exposed directly to the ocean, the island with jurisdiction for reef and fishery management, sampling (fish transects f, benthic quadrats b), straight line distance (km) between the site and the boat launch site for the island with jurisdiction, the human population of that island, and the GPS coordinates. *UAR4 and Yea3 were ca. 15 modal level of effort (sample sizes for all years and all sites are provided in S1 Table). Quadrat locations were selected by using a random number generator to set the distance between quadrats and direction of swim within the reef crest corridor. Percent cover of key organisms was determined within each quadrat (counts were used for larger mobile invertebrates and giant clams). Each quadrat was documented photographically. A total of 10 functional group categories were used to assess benthic cover: stony coral, octocorals, hydrocorals, macroalgae, algal turfs, encrusting algae, cyanobacteria, bare substrate and non-coral sessile and mobile invertebrates. Stony corals and hydrocorals were categorized into one of 12 morphological groups and identified to genus when possible (S2 Table). Instances of disease, paling, and bleaching within each quadrat were noted. Stony coral colony sizes were measured by recording maximum length, width, height, nearest live coral neighbor and coral functional group for each coral that intercepted a 50 m transect line (the same transect as used for the fish counts). Fish community characterization All fish were counted along 50 m transects, in the same habitat and area (1.5-5 m depth) as the quadrats, and were identified to the species level. The transect count area was along a 50 meter long transect, and extended from the sea floor to the surface of the water column. We conducted two to three transects each year at each site. For all transects, the same diver first counted mobile fish on a 5 m wide swath, before returning along the same transect and counting cryptic benthic fishes on a 1 m wide swath. The total lengths of mobile fishes were estimated to the nearest decimeter, and nearest centimeter for cryptic benthic fishes [31]. For analysis of fish community structure, fish species were classified into one of five trophic guilds: 1. herbivores, 2. planktivores, 3. corallivores, 4. carnivores, and 5. piscivores (S3 Table lists all of the species observed on the transects and the trophic category in which they were placed). Species that have a wider trophic range (omnivores) were categorized by their main food preference according to the 5 categories mentioned above. Biomass was estimated using the published length/weight relationships most appropriate for the region [31][32][33]. Sharks and large rays were occasionally seen on transects, but their overall low abundance makes band transects a poor approach to estimate their actual numbers and bias their contribution to biomass [34,35]. Therefore, elasmobranchs were recorded, but not included in our biomass calculations. Statistical analyses We used agglomerative hierarchical clustering (Ward's minimum variance method; hclust in the R Statistical Package, [36]) based on the Bray-Curtis dissimilarity index (for benthic data) and Cao dissimilarity Index (for fish data), following published recommendations [37] to look for patterns in benthic and fish community data. We used the site groups identified in this manner as the basis for our subsequent statistical analyses. To compare biomass, abundance, and the total length of fishes between site groups,we used analysis of variance (ANOVA) to compare fish biomass, abundance and total length after checking that our data met standard assumptions of normality and homoscedasticity. We examined the effects of anthropogenic and physical environmental factors on fish community structure using permutational multivariate analysis of variance (PERMANOVA) based on distance matrices of the fish diversity at each site. To do so, we used adonis, in the package vegan [38], which partitions distance matrices among potential sources of variation. We fit linear models to these distance matrices, and evaluated the pseudo-F ratios with a permutation test. The number of permutations for all of these tests was set at 999. The model for the fish assemblages (fish~exposure + distance + population), was selected by comparing models with all possible combinations of the following factors and variables related to site characteristics, each stratified by year to control for potential inter-annual differences: exposure (lagoonal or oceanic), distance (distance in kilometers from the site to the village with jurisdiction), population (number of human inhabitants of the village with jurisdiction) and index (a continuous measure of the site's orientation with respect to the prevailing northeast trade winds). Similarly, we used PERMANOVA to examine the relationship between benthic cover characteristics and the same anthropogenic and physical environmental factors used to explore fish community structure. The model selected, stratified by year to control for potential inter-annual differences, was: benthic~exposure + distance Ã population. Also using PERMANOVA, the relationship between fish community composition and benthic cover was examined with the model: fish~Acro_thicket + Acro_table + Mound1 + Mound2 + Branch1 + Branch1 + Encrust + Foliose + Solitary + Columnar + Montipora + Coral − Monti + Soft + Fleshy + Macroalg + Turf + CCA. Results We discovered a high prevalence of the coral identified as Montipora sp. [28] and reference it frequently hereafter as 'opportunistic' to distinguish it from species of Montipora that appear more slow growing (evidenced by the spatial advancement of this Montipora even over the course of this study). Sites with the opportunistic Montipora sp. were generally lagoonal sites, sites where water movement appeared low, and all sites where small boats landed frequently and/or human influence was high (near villages) (Fig 1, Table 1), identified in this paper as cluster 3 sites (Figs 2 and 3). Two other sites sampled on Falalop, farther from villages and boat landings, and exposed to deep water (RunS and FPS; Fig 1, Table 1, identified in this paper as Cluster 2 sites), had smaller amounts of this Montipora. Very small amounts of the same species of Montipora were found on the western, oceanic facing sites of Sohng and Piglelei (identified as cluster 1 sites), but none of these colonies appeared to be expanding rapidly or overgrowing neighboring coral colonies as was the case at other sites. We conducted two separate cluster analyses: one for the benthic data, and one for the fish data. Comparing the two clusters visually (Fig 2), both dendrograms show three distinct clusters. Due to the high degree of similarity between the benthic and fish clusters, results were comparable when analyzed based on either cluster analysis. For the remainder of this paper, data are presented and discussed following the site clusters characterized by the benthic data (Fig 2 -benthos, left panel). Site clusters and characterization-Benthos Most sites clustered according to their proximity to villages and high fishing activity areas, as well as relative exposure to deep water (Table 1, Fig 2); cluster 1 sites 'uninhabited and oceanic' were exposed to deep water, and either located outside the Atoll on neighboring islands, (e.g., Geilob Wall, 'Giil', Table 1), or on the Atoll (e.g. Yealil Outside, 'Yeal', Table 1), and were farthest from villages, with the least human impact; cluster 2 sites 'inhabited and oceanic' were generally near villages but were exposed to deep water (mostly the island of Falalop); and cluster 3 sites (with the exception of a few sites-see Fig 1) 'inhabited and lagoonal' were generally lagoonal with lower water movement, and were close to villages (e.g., Mog Mog Landing, 'Mogc', Table 1). Benthic cover (% cover) compared across sites from cluster 1 (uninhabited, oceanic), cluster 2 (inhabited, oceanic), and cluster 3 (inhabited & uninhabited, lagoonal) (a-f). The relative abundance of hard coral morphotypes is shown for the 3 site clusters in e1-e3. Note that the opportunistic Montipora sp. is considered separately from all other stony corals for both cover and morphotype. The box-and-whisker plots show the median value (dark horizontal bar); the box length is the interquartile range, the upper whisker marks the smaller of the maximum value and quartile 3 +1.5 interquartile range (IQR), and the lower whisker marks the larger of the smallest value and quartile 1-1.5 IQR. Outliers are not shown. Plots produced using the R package graphics, version 3.3.1. Site clusters and characterization-Fishes A total of 102 (50m x 5m) fish transects were performed during our study period (2012-2014), which corresponded to a total of 22,153 counted and sized individuals, representing 276 species. Values were remarkably consistent over the years, with the mean number of fish and mean number of species per transect ranging from 208.6 to 226.9 and 34.9 to 42.6, respectively. Cluster analysis of fish community structure revealed three major groups, with the sites clustering into groups comparable to the benthic community clusters (Fig 2). The following analyses were based on the clustering of sites by benthos (Fig 2, left panel). Sites in cluster 1 (least human influence) (Fig 1, Table 1) had the highest average fish biomass at 79.0 g/m 2 (+/-77.1 SD, all trophic groups combined), more than double the fish biomass of cluster 3 sites (mean: 28.5 g/m 2 +/-24.9 SD) which were generally close to villages and within the Atoll lagoon ( Fig 4) (pairwise t-test, p = 0.003 with a Bonferroni adjustment; other pairwise biomass comparisons were not significant). Cluster 2 sites were generally inhabited with greater opportunity for direct human impact, but with similar oceanic exposures as cluster 1 sites. Here, fish biomass (mean: 63.0 g/m 2 +/-73.2 SD) was approximately 20% lower than cluster 1 sites, but more than twice that of cluster 3 sites (Fig 4). Biomass at cluster 3 sites was lowest for all groups of fishes (28.5 g/m 2 +/-24.9 SD) (Fig 4). The biomass of specific fish trophic groups varied significantly between the three clusters of sites (Fig 4, ANOVA: F (2,99) = 5.805, p = 0.004). Fish biomass in cluster 1 sites was dominated by piscivores, macro-carnivores and large herbivorous species (parrotfish and surgeonfishes), while biomass in cluster 2 and cluster 3 sites was dominated by herbivorous species (Fig 4). Numerically, the clusters did not differ significantly in fish abundance (ANOVA: F (2,99) = 0.5453, p = 0.581), although herbivores were more scarce in cluster 1 sites, compared to cluster 2 and cluster 3 sites (Fig 4). Overall, the mean fish length differed significantly between the three clusters (ANOVA: F (2,99) = 15.446, p<0.0001), and a pairwise t-test showed that cluster 1 (highest mean length of fishes) and cluster 2 means were significantly different from cluster 3 means (p<0.0001 and p = 0.004, respectively; cluster 1 vs. cluster 2, p = 0.194, Fig 4). With the exception of the corallivores, individual fish in the different trophic groups tended to decrease in mean length across clusters (i.e., cluster 1 vs. 2 vs. 3, Fig 4). For some groups, such as the herbivores, a greater abundance of smaller fishes appeared to maintain comparable biomass levels between cluster 1 and cluster 2 sites, and a reduction in the number of the highest trophic level piscivores was offset by an increase in the number of smaller carnivores and herbivores, especially damselfishes (Fig 4). Size structure did, however, differ among clusters, with larger piscivores and herbivores found in clusters 1 and 2, and smaller individuals in cluster 3 (Fig 4). Body size (TL, mm) differences for piscivores differed significantly among site clusters (ANOVA: F ( Fig 4). Both human and environmental factors were correlated with differing fish community structure. PERMANOVA results support the hypothesis that fish assemblages surveyed at sites within the lagoon differed significantly from oceanic sites, and that these assemblages also differed as a function of the size of the human population with jurisdiction over that site and their distance from the inhabited island (Table 2). PERMANOVA results show that the same human and environmental factors appeared to affect benthic composition, perhaps even more directly ( Table 3). The reef benthic composition differed as a function of site exposure (lagoonal vs. oceanic), distance from the nearest human community (greatest effects) and the human population size of the community with management jurisdiction. There was a significant interaction between human distance and population size ( Table 3). PERMANOVA results relating aspects of the benthic cover to the composition of the associated fish communities suggest that those features that contributed positively to habitat complexity (e.g., Acropora sp. and the abundance of branching and mounding corals) were also related to differences in fish community structure ( Table 4). The abundance of macroalgae, turf and the opportunistic species of Montipora were also among the benthic cover categories with a (marginally) significant effect on fish community composition (Table 4). Linked social-ecological systems This study has demonstrated that coral reef community structure shows distinct patterns across the scale of Ulithi Atoll, with benthic and fish communities clustering into comparable groups. The drivers of this clustering include a combination of anthropogenic factors, and reef exposure and location. Although a strong human signature may appear unusual given the low human population density, subsistence fishing, and large size of Ulithi Atoll, this study adds support to other studies demonstrating that even small populations can have substantial impacts on their ecosystems [17,[39][40][41], resulting in a linked social-ecological system that probably goes back many centuries on Ulithi Atoll, as on other Pacific atolls [30]. An understanding of the ecological patterns and their drivers can support management planning for the communities of Ulithi and would likely be applicable to other remote, tropical islands, especially in the face of climate change and associated stressors [7,20,42]. Environmental factors that contributed to the site clustering (exposure, weather, and prevailing wind and waves) also may have contributed to historical choices for ideal village sites, linking the effects of reef location and anthropogenic impact [43][44][45]. Cluster 3 sites, all of which are near villages, are also located in the least exposed areas (primarily lagoonal). Inhabited lagoonal sites appear to be particularly vulnerable to reef degradation, evidenced by decreased coral cover, lower fish biomass, smaller fish sizes, and high levels of the opportunistic Montipora sp. (Figs 3 and 4). This could be due in part to reduced water movement and higher residence times for runoff from the villages, as well as elevated temperatures on these shallow reefs [17,46,47]. Therefore, reefs in these lagoonal habitats may exhibit reduced resiliency [7,11]. Increased fishing pressure adds to this reduced resiliency, and likely exacerbates rates of reef degradation [42,44,45]. On Ulithi Atoll, high fuel costs (keeping fishers close to shore), the loss of sailing canoes (which has resulted in greater reliance on motor boats and fishing from shore), and 'new' fishing methods such as night spearfishing for herbivorous fishes, have led to increased fishing pressure on reefs close to villages, likely contributing to the patterns we observed. The opportunistic Montipora coral is largely associated with cluster 3 and lagoonal sites near villages and boat landings (Fig 2). While dominance by a single coral species has not been documented in other studies to the degree found here, decreased coral diversity with high coral cover has been observed in sheltered sites (notably patch reefs) on other Pacific islands [48]. Residents of Ulithi Atoll have reported decreased yield of food fish and invertebrates on Montipora-dominated reefs (John Rulmal Jr, personal observation). Given the importance of structural complexity for fishes and mobile invertebrates, and in particular the importance of branching corals [49], observed reductions in fishes and invertebrates at these Montiporadominated sites (also reported by local fishermen) may be due to a synergistic effect of benthic structure and fishing pressure [50]. Abiotic factors-Exposure and resiliency While villages of the Atoll islands are positioned facing the lagoon (Asor, Mogmog, Federai), the villages of Falalop island, outside the Atoll, are an exception. Falalop is comparatively exposed and surrounded by deep water (although village sites are on the lee side of the island). Despite exposure to deep water, the sites adjacent to the villages (including Falalop Men's House) group with cluster 3, suggesting the influence of anthropogenic factors [17]. We note however that with regard to fish community structure, Falalop Men's House is in cluster 2, and had higher biomass than cluster 3 sites (Figs 2 and 4). This suggests an effect of abiotic factors [17], and potentially more resiliency in these sites exposed to higher water movement and deeper water [7,8,31,42]. Cluster 1 sites, westerly or southerly facing reefs and reefs outside of Ulithi Atoll (coded in blue in Fig 1), lack permanent human settlements, but are occasionally fished, especially for celebrations and community events. These sites were dominated by stony corals with high morphological complexity, extremely low or no incidence of the opportunistic Montipora sp., and relatively high cover of macroalgae and crustose coralline algae, consistent with data published previously [27,51]. These least impacted reefs should be carefully considered in management planning as they could serve as important recruitment pools for adjacent, more highly impacted reefs in cluster 2 and especially cluster 3 [52,53]. Fishing pressure and site characteristics While differences in benthic community structure were correlated with a combination of exposure to deep water and proximity to villages, fish community structure was strongly driven by distance to villages and the population size of the community with jurisdiction ( Table 3). Sites near human settlements, where fishing pressure is high due to easy access, and exposure to waves and wind are reduced, clustered together based on fish diversity and biomass (lower biomass) (Fishes, cluster 3, Fig 2). These sites also had 'lower quality' benthic communities, perhaps exacerbated by fishing methods and depressed fish populations [4,17,40,43]. The most remote sites (Fishes, cluster 1, Fig 2, Turtle Islands, Fig 1), although included in the fishing jurisdictions of two of the four villages, had high fish biomass, lacking a strong signal of fishing pressure. In addition to lower fishing levels at these sites, fishing methods were often different, with less spearfishing and net casting (targeting herbivorous fish), and more hook and line fishing (targeting a larger diversity of fish at different trophic levels) [6,20,23,43]. Although outside the scope of this study, a historical understanding of fishing methods, landings, and traditional management is key to understanding the current patterns of coral reef community structure, and will be examined in future work [39,40,54,55]. Herbivorous fish are often the preferred catch at cluster 3 sites near villages since they can be caught from shore by spear and cast nets. Pressure on herbivorous fish was indicated by smaller sizes at these sites compared to cluster 1 sites (Fig 4). Herbivores as a functional group have been shown to have a significant role in maintaining reef habitat, as well as in facilitating reef recovery [31,[56][57][58]. The observed reduction in herbivorous fishes is likely compromising their role in maintaining settlement and growth substrate for corals [59][60][61] and contributing to the decreased coral cover (not including the opportunistic Montipora) and morphological diversity observed at these sites. Although we found the biomass of herbivorous fish to be low at sites near villages, they were numerically abundant, with assemblages dominated by small individuals (Fig 4), consistent with trends of fishing pressure impacts on fish communities [62,63]. However, the high numbers of juveniles also indicate that these sites continue to have robust recruitment, suggesting that these sites could respond quickly to reductions in fishing pressure [64]. The correlation between benthic habitat composition and fish community structure supports previous studies suggesting that changes in one can alter the structure of the other [49,59,[65][66][67][68]. Changes in community structure can be the result of destructive extraction activities, nutrient loading and physical perturbations, as well as the positive effects of habitat rehabilitation resulting from protection [62,69,70]. The opportunistic Montipora is a potentially important driver of community structure on these reefs into the future-possibly reducing the diversity and abundance of both corals and fishes, and affecting the productivity (from a human extraction perspective) of this system. The interplay between fish and benthic structure has important implications for management, as fishing pressure is likely to have differential impacts in these linked ecological systems. Conclusion Atoll-scale patterns documented here can facilitate management planning by determining potentially resilient sites, sites under more anthropogenic pressure, and sites that are situated in locations that may predispose them to greater future degradation under multiple stressors. Finding compromised sites as well as 'healthier' sites can facilitate a deeper understanding of these systems and how to manage them [9]. Uninhabited, oceanic sites tended towards greater diversity and complexity of fish and benthic communities, and may be important sources of food for people while the more impacted areas are managed for recovery, so rotation strategies may be appropriate. Establishment of no-fishing zones and gear restrictions to limit pressure on specific trophic guilds, especially closer to villages, could also provide spill-over to fished areas [71,72]. Management strategies need to be carefully planned and assessed to ensure effectiveness [73,74]. Ultimately, the people of Ulithi, with enhanced knowledge about the system from studies such as this, should be supported in their management efforts within their own cultural and historical context. The patterns in reef community structure presented here are currently being used by the people of Ulithi Atoll to develop more effective management strategies. For example, given our finding that sites that cluster as uninhabited and oceanic also have the highest densities and biomass of targeted fish, managers are utilizing those sites more during good weather and times of ample fuel in order to reduce pressure on sites near villages. Sites closer to villagesimportant sources of food on a regular basis-are being managed as rotating closures to enhance spillover. If communities on Ulithi and other outer islands act now by implementing traditional methods informed by scientific data, management may prove effective in a relatively short time period. Supporting information S1 Acknowledgments This work is dedicated to John Rulmal Sr. who catalyzed this effort with his vision for a healthy community and a healthy ecosystem: One People One Reef. We would like to thank the people of Ulithi for welcoming us for many years, sharing their knowledge of the reefs, and for being dedicated citizen scientists. Mario Dohmai provided invaluable help with fish naming and translation. Sara Cannon provided help in the field and preparing outreach materials, she has been an inspiration to many. We thank Kelsey Doyle for documenting this work in her films, Kate Crosman, Sam Nelson, Amalia Bernardi, and Alessio Bernardi for their help in the field, and the wonderful undergraduate and high school students who joined us on expeditions and community events. Thanks to PMA (Pacific Missionary Airways) for keeping us sober, and to the Mnuw for not.	2018-02-27T08:57:21.961Z	2017-05-10T00:00:00.000	{ "year": 2017, "sha1": "fbe944f6506d5c0d462ce6655ecd9a42f568f852", "oa_license": "CCBY", "oa_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0177083&type=printable", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "fbe944f6506d5c0d462ce6655ecd9a42f568f852", "s2fieldsofstudy": [ "Environmental Science" ], "extfieldsofstudy": [ "Geography", "Medicine" ] }
16295286	pes2o/s2orc	v3-fos-license	The Regulation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-Induced Lung Tumor Promotion by Estradiol in Female A/J Mice Epidemiological studies indicate that women are at a higher risk developing lung cancer than men are. It is suggested that estrogen is one of the most important factors in lung cancer development in females. Additionally, cigarette smoke, and environmental pollutants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), may play salient roles in female lung carcinogenesis. However, the mechanisms responsible for the interaction of these factors in the promotion of lung cancer are still poorly understood. The present study was designed to explore two ideas: first, the synergistic lung tumorigenic effects of 4-(methylnitrosamino)-1-(3-pyridyl)-butanol (NNK) combined with TCDD, 17β-estradiol (E2) or both through a long-term treatment experiment, and second, to identify early changes in the inflammatory and signaling pathways through short-term treatment experiments. The results indicate that A/J mice given E2 had strong effects in potentiating NNK-induced activation of MAPK signaling, NFκB, and COX-2 expression. In the long-term exposure model, E2 had a strong tumor promoting effect, whereas TCDD antagonized this effect in A/J mice. We conclude that treatment with NNK combined with either E2 or TCDD induces lung carcinogenesis and the promotion effects could be correlated with lung inflammation. E2 was shown to potentiate NNK-induced inflammation, cell proliferation, thereby leading to lung tumorigenesis. Introduction The tobacco-specific compound nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-butanol (NNK) is the most powerful tobacco carcinogen [1]. NNK and its metabolites induce lung cancer and cancer cell growth through genotoxic and epigenetic mechanisms. Among these mechanisms, NFkB is reported to be the key mediator of NNK exposure in the development of lung cancer [2]. Through activation of NFkB-related pathways, NNK increases the release of inflammatory cytokines including TGF-b, IL-1, IL-8, and G-CSF [3]. These inflammatory mediators may further promote neoplasia by inducing preneoplastic mutation, proliferation, resistance to apoptosis, invasiveness, angiogenesis, and secretion of immune suppressive factors [3]. Aside from the tumorigenic effects of NNK alone, emerging data suggested that other environmental toxicants are also considered to play significant roles in the development of lung cancer [4]. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a member of the polyhalogenated aromatic hydrocarbon family and is one of the most toxic environmental compounds produced by industrial combustion and chemical manufacturing processes [5]. We have recently reported that the relationship between TCDD exposure and cigarette smoking could be synergistic in the development of lung cancer in female A/J mice [6]. Lung cancer is the leading cause of cancer deaths worldwide in men and women. Exposure to tobacco smoke remains the most prominent risk factor for lung cancer development, with 90% of female lung cancer deaths being attributed to smoking [7]. However, independent of tobacco exposure, women are at greater risk of lung cancer than men [5]. Epidemiological and clinical data also suggest a gender difference in the etiology of lung cancer [8]. Therefore, additional lung cancer risk factors in females should be taken into consideration [9]. Estrogen is a recognized factor in the pathogenesis of breast, endometrial, and ovarian cancers [10]. Fu et al. reported that significantly more women under 50 years old were diagnosed with lung cancer when they are largely premenopausal, than men who were younger than 50 years old [11]. These observations suggest that estrogen could be an important factor in lung carcinogenesis [10]. Strong correlation between estrogen and lung cancer development have been reported before. For example, estrogen governs many physiological functions including cell growth, differentiation, and development through estrogen receptor (ER)-mediated signaling pathways in cancer cells [12,13]. Upon activation by estrogen, ER causes the proliferation of lung cancer cells through the activation of several growth factor genes, such as tumor necrosis factor-a (TNF-a), epidermal growth factor (EGF), and insulin-like growth factor-1 (IGF-1) that are known to mediate cell division in lung neoplasia [14]. 17b-estradiol (E2) and aromatase, the enzyme that converts testosterones to E2 are elevated in lung cancer tissues [15]. In addition to the ER-dependent activity, the estrogen metabolites such as OHE2 increased the NFkB and COX-2 expression in lung cells [16]. Therefore, female mice exposed to cigarette smoke showed more vigorous inflammatory reactions and generated more toxic oxidative stress in their airways than did male [17]. Accordingly, these findings suggest that cigarette smoke, estrogen, and other environmental pollutants play salient roles in female lung carcinogenesis that is associated with chronic inflammation. Hence, we intend to investigate the possible influence of TCDD, 17b-estradiol (E2), or both on the regulation of NNK-induced lung carcinogenesis in A/J mice that has been shown to be the strain most sensitive to cigarette smoke exposure [18]. The present study was designed to explore the lung tumorigenic potential of NNK combined with TCDD, E2 or both through a long-term treatment experiment and the possible contribution of inflammatory signaling pathways in lung tumorigenesis through a short-term treatment experiment. Surgery, chemical treatments, and tissue preparations Six-week old female A/J mice, acquired from the Laboratory Animal Center of the National Cheng Kung University Medical College, were housed (five mice per cage) in a pathogen-free environment, maintained on Lab Diet 5010 (PMI Feed, Inc, USA) at 2462uC and, 50%610% relative humidity, and were subjected to a 12-h light/12-h dark cycle. Animal studies were approved by the Laboratory Animal Center of the National Cheng Kung University Medical College and performed according to the local guidelines for animal care and protection (Permit number: 98112). The experimental design for the treatment groups and the duration of exposure is shown in Figure 1. At 7 weeks of age, mice were sham operated (Sham) or ovariectomized (OVX) by bilateral dorsal incision under anesthesia (0.15 ml/0.02 kg b.w. of a 1:1 solution of hypnorm and dormicum diluted a further 1:1 in sterile water s.c.). Analgesia (0.1 ml 10% buprenorphine/0.02 kg b.w.) was given during the recovery from surgery. The surgically treated animals were housed singly and given 2 weeks to recover. The exposure experiments were performed out at 9 weeks of age for all animals. The mice were randomly divided into 7 groups, in which groups 1-3 were the sham-operated mice, and groups 4-7 were OVX mice. Treatment of the 7 groups was as follows: (1) Sham Control group, i.p. corn oil 0.1 ml every two days until the end of the experiment; (2) Sham NNK group, a single i.p. injection of NNK followed by i.p. corn oil 0.1 ml every two days until the end of the experiment; (3) Sham NNK+TCDD group, a single injection of NNK followed by given 0.1 ml of corn oil every two days for 1 week. At the 3 rd week, mice were given a loading dose of TCDD followed by the weekly maintenance doses of TCDD for 3 weeks; (4) OVX, NNK group, a single i.p. injection of NNK followed by i.p. corn oil 0.1 ml every two days until the end of the experiment; (5) OVX, NNK+TCDD group, mice were given a single dose of NNK at the first week, followed by giving corn oil for 1 week. Mice were then given a loading dose of TCDD followed by the weekly maintenance doses of TCDD for 3 weeks; (6) OVX, NNK+E2 group, mice were given a single dose of NNK then given E2 subcutaneously from the 2 nd week until the end of the experiments; and (7) OVX, NNK+E2+TCDD group, mice were given a single dose of NNK followed by E2 at the 2 nd week until the end of the experiments. At the 3 rd week, mice were given a loading dose of TCDD followed by the weekly maintenance doses of TCDD for 3 continuous weeks. NNK (1 mg/0.1 ml/mouse) was given intraperitoneally at the 1 st week of the experiment (Group 2-7). At the 2 nd week, E2 (2 mg/kg/b.w.) was given subcutaneously every 2 days until the end of the experiment (Group 6, 7). The loading dose of TCDD (5 mg kg/b.w./i.p.) was given at the 3 rd week of the experiment, then the maintenance dose of TCDD (1.42 mg/kg/b.w./i.p.) was given 3 times a week for an additional 3 weeks (Group 3, 5, 7). The doses of TCDD were modified from those described in published studies, which showed successful enhancement of lung tumorigenesis in mice [6]. To delineate the effects of NNK combined with E2, and/or TCDD in the inflammatory response, we designed a short-term exposure experiment (terminated at the 7 th week after NNK treatment) and a long-term experiment (terminated at the 24 th week after NNK treatment) to evaluate lung tumor induction. At the end of the experiments, all mice were euthanized under ether anesthesia, and serum was obtained from cardiac blood. Lung tissues were excised, weighed, and examined for the presence of gross tumors at necropsy and were then sectioned and fixed in 10% paraformaldehyde or frozen in liquid nitrogen. Histology and immunohistochemistry (IHC) Lung tissue sections (4 mm) were sliced from paraffin-embedded, formalin-fixed lungs and stained with hematoxylin and eosin (H&E). For immunohistochemistry, tissue sections were deparaffinized and hydrated. Antigen retrieval was performed in a microwave for 20 min with citrate buffer (pH 6.0), and then the slides were immersed in 3% H 2 O 2 for 20 min to block the activity of endogenous peroxidase. After blocking, the slides were incubated overnight at 4uC using primary antibodies at a 1:100 dilution and then incubated with the appropriate secondary antibody for 1 hr at room temperature. The slides were developed with the STARRTREK Universal HRP detection Kit (Biocare medical, Concord, CA, USA) according to the manufacturer's protocol, and the slides were counterstained with hematoxylin. Determination of the TGF-b and TNF-a Levels Mouse serum was used to determine the levels of TGF-b and TNF-a using an ELISA kit according to the manufacturer's instructions (R&D systems, Inc, Minneapolis, MN, USA). Western blot analyses Randomized frozen lung samples from 3 individual mice from different groups were homogenized, and the lysates were subjected to gel electrophoresis and immunoblotting. Cytoplasmic extracts and nuclear extracts were performed by using Buffer A (10 mM HEPES, pH 7.8, 10 mM KCl, 2 mM MgCl 2 , 1 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF), Buffer B (500 ml buffer A containing 0.5% NP-40), and Buffer C (50 mM HEPES, PH7.8, 50 mM KCl, 300 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF, 20 ml glycerol). Briefly, lung tissues were homogenized with Buffer A. Following centrifugation, the supernatant were collected as cytoplasmic extracts, and the pellet were washed with Buffer B then re-suspended with Buffer C. Following extraction, the nuclei were removed by centrifugation, and the supernatants were collected as the nuclear extracts. Immunoreactive proteins were visualized with a chemiluminescent detection system (PerkinElmer Life Science, Inc. MA, USA) and BioMax LightFilm (Eastman Kodak Co., New Haven, CT, USA) according to the manufacturer's instructions. Statistical analyses The results are expressed as the mean 6 standard error of the mean (SEM). The experimental data were analyzed using Student's t-test. Data of tumor multiplicity were analyzed by Fisher exact test. Differences were considered to be statistically significant when the p value was less than 0.05. General health status of the short-term and long-term treatment groups To test the role of inflammation associated with lung tumorigenesis, sham-operated or ovariectomized A/J mice were exposed to NNK alone, NNK combined with E2, TCDD or both, Figure 1. Experimental design. Female A/J mice were divided into 7 groups in which groups 1-3 were sham-operated (Sham) and groups 4-7 were ovariectomized (OVX). Mice were OVX 2 weeks before given NNK. Group 1 is the sham-operated control group in which mice were given corn oil i.p. 3 times per week until the end of the experiment. Groups 2-7 were pretreated with NNK (1 mg/0.1 ml/mouse, i.p.) at the 1 st week of the experiment. Groups 3, 5, and 7 received a loading a dose of TCDD (5 mg kg/b.w./i.p.) from week 3 and the maintaining dose of TCDD (1.42 mg kg/ b.w./i.p) 3 times weekly from week 4 for 3 weeks. In week 2, groups 6 and 7 were exposed to 17b-estradiol (E2, 2 mg/kg/b.w.) subcutaneously every 2 days continuously until the end of the experiments. The experiments were terminated after the 7 th week as a short-term exposure model to detect the inflammatory response, and terminated at the 24 th week as a long term exposure model to evaluate lung tumor formation. doi:10.1371/journal.pone.0093152.g001 and the experiment was terminated at the 7 th week after NNK injection (short-term experiment, Figure 1). The effects of NNK combined with TCDD, E2, or both on lung tumor promotion were examined in the long-term experiment, in which A/J mice were sacrificed at the 24 th week after NNK injection ( Figure 1). The changes in body weight and lung weight are summarized in Table 1. The mean body weights (BW) and lung weights (LW) increased with time among all groups, but the LW/BW ratio did not display significant changes at any point through the experimental period (Table 1). Tumor-Promoting effects of E2, TCDD and both on NNK-induced lung adenoma formation The lung adenoma formation observed in the long-term experiments showed that lung tumors with white coloration were located in the subpleural area (Figure 2a, sub panel). The lung tumors were composed of cuboid cells with hyperchromatic nuclei. This histological feature was consistent with adenoma ( Figure 2b, right panel). The tumor incidence and multiplicity of control of each treatment group are listed in Table 2. The sham-operated control mice showed a 5.6% occurrence of spontaneous lung adenomas (group 1), whereas treatment with NNK (group 2) significantly increased tumor incidence (19.4%) and multiplicity (0.1760.04), suggesting that NNK significantly induced lung tumor formation in female mice. Combination treatment with NNK and TCDD in the sham-operated A/J mice significantly increased tumor incidence (34.6%) and multiplicity (0.2560.03) compared to the control group (group 3 vs. group 1). Similar results were also observed in the OVX mice received NNK combined with TCDD (group 5) that tumor incidence (33.3%) and multiplicity (0.3860.17) were higher than single NNK injected in OVX mice (group 4). The results confirmed that TCDD enhances lung tumorigenesis induced by NNK; however, the role of E2 in the lung tumorigenesis did not clarify among these groups. To explore the role of E2 in potentiating lung tumorigenesis, the E2-attributable effects on NNK-induced lung tumorigenesis were analyzed. OVX mice treated with NNK (group 4) showed lower tumor incidence compared to sham NNK group (group 2), suggested that female hormone may regulated lung tumorigenesis. Addition of E2 combined with NNK in the OVX mice (group 6) showed the highest tumor incidence (55.6%) and multiplicity (2.0660.24) among all groups following 24 weeks of exposure ( Table 2). The results suggested that E2 has a strong enhancing effect on NNK-induced lung tumors. Compared with the effect of TCDD and E2 in NNK-induced lung tumor, the OVX mice supplemented with E2 (group 6) showed significantly higher tumor multiplicity (2.0660.24) than OVX mice treated with NNK+TCDD (group 5, 0.3860.17) indicating that tumor promoting effect was significant in the E2+NNK group (group 6) than TCDD+NNK group (group 5). However, simultaneous administration of E2 and TCDD to NNKtreated mice (group 7) resulted in significantly decreased tumor incidence (50%) and multiplicity (0.5560.04) compared to the NNK+E2 group (group 6) ( Table 2). The results indicated that E2 possesses greater tumor-promoting effects than TCDD; TCDD did not potentiate but somehow antagonized the E2 tumorpromoting effects in A/J mice ( Table 2). The effect of interactions between NNK and E2, NNK and TCDD on the inflammatory response in a short-term experiment Based on the previous reports, which showed a link between lung inflammation and carcinogenesis [19], we evaluated the expression of inflammatory cytokines using mouse serum. Our results indicated that tumor multiplicity of OVX NNK group (group 4) was similar to sham NNK group (group 2), therefore, mouse serum and lung tissues selected from group 1-3 (shamoperated groups), and group 5-7 (OVX groups) were used for further analysis. OVX NNK group (group 4) was excluded in the following experiments. As shown in Figure 3, compared with the sham control group, TGF-b levels increased in the sham-operated mice receiving NNK, NNK+TCDD, or OVX mice receiving NNK+TCDD, NNK+E2, and NNK+E2+TCDD in the shortterm experimental study (Figure 3a, left panel). OVX mice exposed to E2 did not show differences in TGF-b levels compared to the other OVX groups (Figure 3a, left panel). The results indicate that NNK combined with TCDD, E2, or both had similar effects on the induction of TGF-b levels. Interestingly, in the longterm treatment groups, the levels of TGF-b were not significantly changed, except for the NNK+E2 groups, suggesting that E2 continuously stimulates the production of TGF-b. In contrast, TNF-a levels were significantly elevated only in the sham and OVX mice receiving NNK, NNK combined with TCDD, E2 or both in long-term treatment groups (Figure 3a, right panel). The results implied a different expression pattern of TGF-b and TNFa, showing that TGF-b was affected by the treatment in the shortterm experiment whereas the expression of TNF-a is increased after long term treatment. We then determined whether administration of E2, TCDD or both affected NNK's ability to induce an inflammatory response in lung tissues using COX-2 expression and the NFkB pathways as inflammatory markers. The results showed that sham and OVX mice exposed to NNK+TCDD, the expression of phospho-IkB, nuclear p65, nuclear p50, and IkB degradation were all significantly increased compared to sham NNK group (Figure 3b). In OVX mice treated with NNK+E2 or NNK+E2+TCDD, the expression of phospho-IkB, nuclear p65, nuclear p50, COX-2, and IkB degradation were also significantly increased while the expression of cytoplasmic p65 and p50 were decreased. The most significant increases in expression of the nuclear NFkB subunits p65 and p50, as well as COX-2 were found in the NNK+E2 groups (Figure 3b). The immunohistochemistry results of the short-term experimental groups for COX-2 expression further demonstrated that the distribution of COX-2 positive stained bronchial epithelial cells in OVX mice treated with NNK+E2 was more widespread than in other treatment groups (Figure 3c). These results suggested that the pro-tumorigenic effects of NNK and the potentiated tumorigenic effects when combined with TCDD or E2 are convergent on a persistent/ dysregulated inflammatory response. Nevertheless, co-administration of NNK+E2+TCDD induced less COX-2 expression than the NNK+E2 group, implying an antagonistic response could have occurred between TCDD and E2. Alteration of MAPK signaling pathways It has been reported that NNK promotes the activation of MAPK pathways in cancer cells [1]. E2 can also activate MAPK pathways, leading to the proliferative and anti-apoptotic effects observed in a variety of cell types [20]. We hypothesized that the interaction between NNK and E2 in promoting inflammation may be through the co-activation of MAPK signaling pathways. Three major MAPK signaling pathways were detected by western blot analyses, and the results revealed that ERK1/2, p38, and JNK were significantly phosphorylated and activated upon challenged with NNK combined E2 (Figure 4a). Moreover, we also revealed that the combined treatment induced overexpression of cyclin D1, which is a key regulator of cell cycle progression (Figure 4a). The immunohistochemistry results further confirmed that NNK combined with E2 induced potent overexpression of the cellular proliferation as determined by the proliferative marker PCNA in the bronchial epithelium ( Figure 4b). Nevertheless, NNK combined with TCDD also induced activation of MAPK pathways, cyclin D1 and PCNA expression but to a lesser extent (Figure 4a and 4b). The results indicated that NNK combined with TCDD or E2 induces lung tissue inflammation and cellular proliferation and may require the activation of MAPK signaling pathways. NNK combined with E2 was more prone to activate MAPK pathways, cyclin D1 and PCNA. Alteration of cancer cell proliferation, inflammation, and progression We next examined whether lung inflammation and proliferation could also be observed in lung tissues after a long-term experiment. We found that the expression of COX-2, iNOS, MMP-9, and PCNA were increased in groups treated with NNK combined with TCDD, E2, or both, in which E2 had strong effects on promoting NNK-induced inflammation and proliferation in the long-term exposure experiment (Figure 5a). Similar to the above results, co-administration of TCDD in the NNK+E2 group rendered the inflammation and proliferation effects of NNK combined with E2. The results suggested that A/J mice given TCDD or E2 over a long-term period can potentiate the NNKinduced activation of MAPK signaling that mediates an increase in inflammation and cell proliferation, leading to lung tumorigenesis. E2 combined with NNK showed the most potent lung tumorigenic effects among all treatment groups (Figure 5b). Discussion NNK and TCDD are both well-known human carcinogens that have been proved to induce lung cancer formation in animal studies. Our previous study indicated that TCDD promoted NNK-induced lung tumor in female A/J mice, implying that estrogen and environmental toxicants such as TCDD, NNK could cooperate the mechanisms of lung tumorigenesis [6]. It has been reported that chronic inflammation induced by environmental toxicants is an important mechanism in lung tumorigenesis [16]. Therefore, the short-term exposure model in the present study was performed to identify whether NNK combined with TCDD or E2 induced inflammation. To delineate the relationship between inflammation and lung tumorigenesis, we performed the long term exposure model. Tumor promoting effects of E2 and TCDD when The results are expressed the means 6 SEM (n = 3 in each group). *p,0.05 compared to sham control groups. # p,0.05 compared to short-term sham NNK groups. { p,0.05 compared to long-term OVX, N+T groups.`p,0.05 compared to long-term OVX, N+E2+T groups. Short term: experiment terminated at the 7 th week. Long term: experiment terminated at the 24 th week. (b) Lung homogenates were used to detect the expression and activation of pIkB, IkB, cytoplasmic p65, nuclear p65, cytoplasmic p50, nuclear p50, COX-2 and GAPDH by using specific primary antibodies. (c) Immunohistochemistry for COX-2 protein in A/J mice lung tissues. Arrows indicated COX-2 positive stained bronchial epithelium (magnification, 6100). Sham: sham-operated, OVX: ovariectomized, C: control, N+T: NNK+TCDD, N+E2: NNK+E2 (17b-estradiol), N+E2+T: NNK+E2+TCDD. doi:10.1371/journal.pone.0093152.g003 combined with NNK was also investigated. Moreover, the possible interaction in mice exposed to NNK in combination with TCDD, E2, or both, in inducing lung tumorigenesis was investigated. Figure 5b proposes the mechanistic model, showing that E2 possesses a strong enhancing effect on NNK-induced lung inflammation through activation of the MAPK and NFkB pathways, leading to COX-2 overexpression. This inflammatory response could be correlated with the potentiated effects of E2 on lung tumor promotion. On the contrary, co-administration of TCDD with NNK and E2 decreased inflammation, tumor incidence and multiplicity when compared with NNK+E2. Our current findings suggest that NNK and E2 together significantly induce lung tumor formation in OVX mice, implying that the induction of lung tumor formation in intact NNK-treated mice is hormone-related. Besides, TCDD may antagonize lung tumorigenic effect induced by E2. In this report, we used a murine model of lung adenocarcinoma induced by NNK, and have demonstrated the lung tumor promoting effects of E2 was more potent than TCDD. We have shown that the tumor multiplicity and the inflammatory response were more obviously by E2 treatment, indicating that E2 can strongly potentiate inflammation and tumorigenesis induced by NNK (Table 2). These results are consistent with previous studies showed that estrogen not only promotes the malignant development of breast cancer, but also acts on non-target organs, including lung cancer through various molecular mechanisms [21,22]. In addition to the solitary effect of estrogen, females exposed to other environmental chemicals such as benzo[a]pyrene that may interfere with the physiological functions of estrogen leading to increasing susceptibility of cancers [23][24][25]. However, our present study indicated that NNK combined with E2 and TCDD did not show an addictive effect in lung tumor formation, instead, the tumor multiplicity of NNK+TCDD+E2 groups was lower than NNK+E2 groups ( Table 2). Those results are consistent with others, suggesting that TCDD exposure antagonize a wide spectrum of estrogen induced responses, including cell proliferation [26]. A number of mechanisms have been suggested, including that TCDD down regulation of the estrogen receptor (ER) in the uterus and other organs by activation of aryl hydrocarbon receptor (AhR) [27]. Mechanistic study indicated that TCDD up-regulates estrogen metabolizing enzymes, such as the CYP1 family, to enhance the metabolism of estradiol to inactive metabolites, thereby decreasing the half-life of active estrogen [28]. TCDD induced inhibition of estrogen-stimulated growth in MCF-7 cells could be due to the block entry into S phase by inhibiting the activation of cdk2, cdk4, and Rb [29]. Those above findings can provide evidence, at least in part, to support our finding that TCDD antagonized the lung tumor promoting effects of estrogen. The precise mechanisms by which estrogens or TCDD influence the lung tumorigenesis is unknown. One recent study indicated that the induction of an NFkB-dependent inflammatory response in lower airways after prolonged exposure to cigarette smoke could be involved in lung tumorigenesis in animals [30]. Our previous study and others showed that cigarette smoke modulate the expression of genes involved in estrogen metabolism that increases accumulation of 2OHE2 and 4OHE2 in lung cells, and lead to NFkB activation, COX-2 expression, and lung carcinogenesis [16,31]. Thus, we examined inflammatory response in the lung tissue and revealed that NNK combined with E2, TCDD or both significantly increased translocation of NFkB subunits (p65 and p50) from cytoplasm to nucleus, evidenced by the findings that the expression of cytoplasmic p65 and p50 were decreased, while nuclear p65 and p50 were increased (Figure 3b). Meanwhile, the expression of COX-2, which is an NFkB downstream inflammatory mediator, was increased. Interestingly, NNK+E2 groups had strengthened the expression of these inflammatory proteins (Figure 3). The results were consistent with tumor incidence and multiplicity observed in the long-term study that E2 had potent lung tumorigenic effect when combined with NNK. Although the mechanisms of inflammatory induced by E2 or TCDD are not well understood, continuous to inflammation is reported to increase lung cancer development. Thus, a continuous inflammatory response driven by E2 or TCDD could potentiate lung tumorigenesis by NNK ( Figure 5). However, TCDD coadministered with NNK+E2 induces less lung tumor formation as well as inflammation responses compared to NNK+E2 group ( Figure 3 and Table 2). As reported earlier, TCDD may interact with NFkB pathway through AhR-mediated signaling that AhR and NFkB physically interact, thereby causing mutual repression [32][33][34]. Nevertheless, our results showed that NNK combined with TCDD did not inhibit the expression of inflammatory-related proteins ( Figure 3); thus, it is possible that TCDD co-administrated to NNK+E2 induced less inflammation compared to NNK+E2 could be due to the antagonistic effect of TCDD and E2. MAPK pathway (includes ERK1/2, JNK, and p38 kinases) is the most extensively investigated intracellular signaling cascade involved in NFkB activation and the pro-inflammatory [35]. Our results further implied that NNK combined with E2 appeared prone to activate MAPK pathways (Figure 4). In agreement with our findings, Majidi et al. reported that E2 significantly enhances cell proliferation in response to NNK through NFkB and MAPK signaling pathways. The report suggested cooperation between NNK-induced b-AR and b-ER mitogenic signaling, leading to the up-regulation of the Ras/Raf/ERK1/2Elk1 signaling pathway in lung epithelial cells [36]. Upon activation of MAPK signaling pathways, various pro-inflammatory cytokines including TGF-b, TNF-a, IL-6, and IL-8 were released. Our present results indicate that TGF-b production increases in the short-term experiment then declined, whereas, in the long-term experiment, TNF-a significantly increases after treatment with NNK and in the combined treatment groups (Figure 3a). Consistently, Biseli et al. indicated that TGF-b levels were increased in the lung airway and lung parenchyma after a 2-month exposure to cigarette smoke condensates(CSC) and may be the stimulus responsible for collagen fiber deposition in lung parenchyma [37,38]. Another study indicated that TGF-b increased after a single CSC exposure for 2 h then declined over time after 6 months of CSC exposure. The apparent down-regulation of TGF-b expression with longterm exposure may be more complex, TGF-b may switched on and off in different cell populations at different times [39]. Loss of the TGF-b response has been shown to be associated with tumor development and/or tumor progression in some cancer cell lines [40,41]. Therefore, these reports support our finding that TGF-b increases in response to the short-term exposure model that may be involved in regulating the inflammatory response; however, the decline in TGF-b in the long-term exposure model may be correlated with tumor progression or metastasis. An unexpecting finding of our experiments was that the expression of TNF-a increased in the long-term exposure model but not in the short-term model. One recent study also found that TNF-a release was increased after a 3 month exposure to cigarette smoke in lung tissues [42]. The molecular mechanisms of TNF-a in tumor progression are due to its ability to activate multiple signaling pathways, including NFkB, that play critical roles in mediating cell proliferation and survival [43]. Therefore, Karabela et al. reported that TNF-a neutralization is effective against urethane-induced lung oncogenesis in mice [44]. These reports indicated that increased TNF-a may contribute to inflammationassociated tumorigenesis. Current knowledge gives new insight into the etiology of lung cancer development is a complex pathway mediated not by a single carcinogen, but rather by the synergistic interaction between environmental chemicals, hormones and cigarette smoke carcinogens [4]. This interaction raises public health concerns and challenging questions [1]. Until now, to delineate the complex relationship between exposure to multiple hazardous chemicals and lung carcinogenesis is still difficult. The present study is the first to explore the differential mechanisms of combined exposure by both short-term and long-term exposure animal models. This finding is of particular important to women as they are at risk for lung cancer development due to cigarette smoking exposure. It is appears that E2 stimulates NNK-induced activation of MAPK signaling, which correlates with increase in inflammation, cell proliferation, and tumorigenesis. Furthermore, NNK may increase the accumulation of carcinogenic E2 metabolite 4-OHEs and decreases the protective 2-OMeE species in lung tissues [45]. However, TCDD co-administered with NNK+E2 may antagonize tumor promoting effect by E2. Further studies are needed to determine the exact mechanisms of how E2 promote NNKinduced lung cancer and the contradictory mechanisms of TCDD and E2.	2017-04-13T15:28:47.066Z	2013-08-28T00:00:00.000	{ "year": 2014, "sha1": "7118e6ec80d29251bae6b338b0be9625e7fe1e7b", "oa_license": "CCBY", "oa_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0093152&type=printable", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "7118e6ec80d29251bae6b338b0be9625e7fe1e7b", "s2fieldsofstudy": [ "Biology", "Chemistry" ], "extfieldsofstudy": [ "Medicine" ] }
210934987	pes2o/s2orc	v3-fos-license	Glucocorticoids mobilize macrophages by transcriptionally up-regulating the exopeptidase DPP4 Glucocorticoids are potent endogenous anti-inflammatory molecules, and their cognate receptor, glucocorticoid receptor (GR), is expressed in nearly all immune cells. Macrophages are heterogeneous immune cells having a central role in both tissue homeostasis and inflammation and also play a role in the pathogenesis of some inflammatory diseases. Paradoxically, glucocorticoids have only a limited efficacy in controlling the resolution of these macrophage-related diseases. Here, we report that the transcriptomes of monocyte-like THP-1 cells and macrophage-like THP-1 cells (THP1-MΦ) have largely conserved gene expression patterns. In contrast, the differentiation to THP1-MΦ significantly altered the sensitivity of gene transcription to glucocorticoids. Among glucocorticoid-regulated genes, we identified the exopeptidase dipeptidyl peptidase-4 (DPP4) as a critical glucocorticoid-responsive gene in THP1-MΦ. We found that GR directly induces DPP4 gene expression by binding to two glucocorticoid-responsive elements (GREs) within the DPP4 promoter. Additionally, we show that glucocorticoid-induced DPP4 expression is blocked by the GR antagonist RU-486 and by GR siRNA transfection and that DPP4 enzyme activity is reduced by DPP4 inhibitors. Of note, glucocorticoids highly stimulated macrophage mobility; unexpectedly, DPP4 mediated the glucocorticoid-induced macrophage migration, and siRNA-mediated knockdowns of GR and DPP4 blocked dexamethasone-induced THP1-MΦ migration. Moreover, glucocorticoid-induced DPP4 activation was also observed in proinflammatory M1-polarized murine macrophages, as well as peritoneal macrophages, and was associated with increased macrophage migration. Our results indicate that glucocorticoids directly up-regulate DPP4 expression and thereby induce migration in macrophages, potentially explaining why glucocorticoid therapy is less effective in controlling macrophage-dominated inflammatory disorders. Glucocorticoids exert a wide array of systemic and tissuespecific effects, by signaling through the cognate glucocorticoid receptor (GR; 2 NR3C1) in numerous tissues and cell types to systematically influence development, homeostasis, metabolism, and inflammation (1). One of the most important effects of both endogenous and exogenous glucocorticoids is immunomodulation, exerted mainly by suppressing transcription of pro-inflammatory genes and/or induction of anti-inflammatory genes (2). Synthetic glucocorticoids are commonly prescribed anti-inflammatory and immunomodulatory agents. Their therapeutic activity is substantial in a wide spectrum of diseases, including acute and chronic inflammation, autoimmune disorders (3), organ transplantation (4), and hematological cancers (5). Contrary to well-known anti-inflammatory effects of glucocorticoids, there is emerging evidence of pro-inflammatory effects during inflammation (6 -8). For example, glucocorticoid signaling in macrophages has been reported to up-regulate the expression of NLRP3 inflammasome component and to enhance the ATP-dependent secretion of cytokines such as TNF␣ and interleukin-6 (9). These findings suggest that glucocorticoids likely play a dual role regulating the innate and adaptive immune response differentially. These effects may depend on the type of inflammatory stimulus (10) and/or the timing of treatment (11), thus modulating the balance of the cellular state toward a net pro-inflammatory or anti-inflammatory state (7). These macrophage-intrinsic properties may explain why glucocorticoids are less effective in macrophagemediated diseases (12), such as chronic obstructive pulmonary disease (11), ulcerative colitis (13), systemic lupus erythematosus (14), and rheumatoid arthritis (15). cro ARTICLE Macrophages are involved in all phases of the inflammatory response, including alarm, mobilization, and resolution phases, and are able to drive either the propagation or resolution of inflammation (12,16). The ontogeny of macrophages is still not fully understood; however, it is accepted that they can be grouped as tissue-resident macrophages (established independently of hematopoiesis) or infiltrating macrophages derived from circulating monocytes that are established following an inflammatory response (17)(18)(19). Thus, macrophages may contribute to the pathophysiology of several diseases, including inflammatory disorders (20,21), cancer (22), and also states of low-grade inflammation such as obesity (23,24). The migratory capacity of macrophages has been studied in tumor-associated macrophages, because they have a critical role in different stages of tumor progression (25). In other inflammatory pathologies, such as multiple sclerosis, differences in activation and polarization of macrophages promote their migratory properties toward chemoattractants (26). This migration is associated with cytoskeleton rearrangements and also has been proposed to depend on the levels and type of integrin expression (26). For example, the expression of the chemokine receptor CXCR4 on mouse and human mature macrophages has been associated with migration toward lymph nodes during murine peritonitis resolution (27). Finally, in human macrophages (differentiated from CD14 ϩ monocytes), CXCR4 expression and cell motility (showing a longer distance traveled) was induced upon dexamethasone stimulation. However, little attention has been directed to the identification of genes essential for macrophage movement (28). Dipeptidyl peptidase-4 (DPP4), encoding for a membrane glycoprotein with exopeptidase activity, was recently described as being involved in the inflammatory macrophage profile associated with type 2 diabetes, obesity, and atherosclerosis (29,30). This protein is multifunctional, with both enzymatic and nonenzymatic activities. The extracellular domain of DPP4 presents the catalytic site, primarily associated with inactivation of incretin hormones, such as glucagon-like peptides 1 and 2, and the gastric inhibitory polypeptide, as well as catalysis of chemokines (CCL2/MIP-1a, CXCL12/SDF-1, and CCL5/Rantes, among others) (31). DPP4 has also binding sites for adenosine deaminase and fibronectin, and their binding is associated with pro-inflammatory responses (32). In this study, using THP-1 cells, we show that the monocyteto-macrophage differentiation was associated with both higher GR levels and greater sensitivity to glucocorticoids on macrophage-like THP-1 cells (THP1-M⌽) compared with monocyte-like THP-1 cells. These changes resulted in modifications of the glucocorticoid-dependent macrophage transcriptome. During the monocyte-to-THP1-M⌽ differentiation, cells undergo chromatin remodeling, thus enhancing GR accessibility to glucocorticoid-response elements (GREs) within the DPP4 promoter. Furthermore, we show that DPP4 is a novel glucocorticoid-responsive gene specifically in human and mouse macrophages, but not regulated in monocytes. An in vitro migration assay using THP1-M⌽ and M1 polarized bone marrow-derived macrophages (BMDMs) reveals that glucocorticoids regulate the macrophage movement via a DPP4-dependent process. Transcriptome analysis of monocyte-like THP-1 cells and macrophage-like THP-1 cells reveals a high conservation of gene expression between both cell types We investigated whether the state of cell differentiation among monocytes and macrophages modifies the gene expression, with profiles of monocyte-like THP-1 cells (THP-1) and macrophage-like THP-1 cells (THP1-M⌽) analyzed by genome-wide microarray (Fig. S1). Regulated genes were evaluated through a Venn diagram (Fig. 1A), which showed that a large number of genes are commonly expressed in THP-1 and THP1-M⌽ (6,637 genes, corresponding to 82.4% of the total expressed genes). In contrast, 608 genes were only found in THP-1, whereas 803 were restricted to THP1-M⌽ (Fig. 1A). Provocatively, a volcano plot comparing genes expressed in THP1-M⌽ with monocyte-like THP-1 cells revealed that the gene NR3C1/GR was highly expressed in THP1-M⌽ (Fig. 1B). These data were validated by qRT-PCR and Western blotting showing a 6-fold increase in the GR mRNA and a 3-fold increase in GR protein in THP1-M⌽ compared with undifferentiated monocyte-like THP-1 cells ( Fig. 1 (C and D), respectively). Interestingly, this phenomenon also was observed in primary murine BMDMs using M-CSF, where we found an increase in GR mRNA in macrophages post-differentiation from bone marrow monocytes (BMM) (Fig. S2). Monocyte-to-macrophage differentiation enhances their responsiveness to glucocorticoids in macrophages Due to the different expression levels of GR in monocyte-like THP-1 cells and THP1-M⌽, we investigated whether the state of cellular differentiation alters the sensitivity to glucocorticoids. For these experiments, monocyte-like THP-1 cells and THP1-M⌽ were treated with dexamethasone (Dex) for 6 h, and total isolated mRNA was subsequently analyzed by a genomewide microarray. Principal component analysis demonstrated considerable separation between treatment (control versus Dex) in both cell types. However, Dex-treated monocyte-like THP-1 cells and THP1-M⌽ were dramatically separated, indicating differential glucocorticoid-regulated transcriptomes in these cells (Fig. S3A). To identify unique and common genes regulated by Dex among the cell types, the gene list was sorted by Venn diagram, revealing only 741 genes commonly regulated by Dex in monocyte-like THP-1 cells and THP1-M⌽ ( Fig. 2A). Moreover, the number of genes uniquely regulated by Dex in THP1-M⌽ (4,222 genes) was 7-fold higher with respect to monocyte-like THP-1 cells (529 genes) ( Fig. 2A). The most significant genes commonly and uniquely regulated by Dex in monocyte-like THP-1 cells and THP1-M⌽ were further plotted on a volcano plot and validated by qRT-PCR (Fig. S3, B-G). To elucidate the significance of these findings in both cell types, gene sets were analyzed by Ingenuity Pathway Analysis (IPA) software. Provocatively, cellular movement was overrepre- Glucocorticoids mobilize macrophages via DPP4 sented in THP1-M⌽ compared with monocyte-like THP-1 cells (Fig. 2B). Moreover, cell-mediated immune response, immune cell trafficking, and inflammatory response pathways were enhanced in macrophages compared with monocytes ( Fig. 2B and Fig. S4), suggesting that THP1-M⌽ are more responsive than monocyte-like THP-1 cells to signaling by glucocorticoids. Glucocorticoids enhance macrophage-like THP-1 cell migration Pathway analysis of the microarray data revealed that cell movement was the top biological function following Dex treatment in both monocyte-like THP-1 cells and THP1-M⌽, although cell movement annotation was more dramatically regulated in Dex-treated THP1-M⌽. The number of genes involved in cell movement and differentially regulated according to IPA was 1,102, 44.3% of which were induced, whereas the remaining 63.7% were inhibited in THP1-M⌽. Heat map analysis of the top 25 genes revealed that the magnitude of Dexinduced gene expression was greater in THP1-M⌽ (Fig. 2C). Based on these findings, we evaluated how GR could impact THP1-M⌽ migratory properties in a cell migration assay based on the Boyden Chamber principle. Spontaneous cell migration assay with cells seeded on the insert and serum as the chemoattractant at the bottom revealed that Dex treatment did not impact the monocyte-like THP-1 cell migratory properties at 6 and 24 h (Fig. 2D). However, Dex treatment of THP1-M⌽ increased their migratory properties after 24 h (Fig. 2D). This effect was blocked by 1-h pretreatment with the GR antagonist, RU486 (Fig. 2D). This finding was corroborated by knocking down GR expression via siRNA. THP1-M⌽ transfected with siRNA GR had ϳ75% of GR silenced (Fig. 2E) and a significant reduction in Dex-induced cell migration compared with cells transfected with nontargeting control (NTC) siRNA (Fig. 2F). These results suggest that glucocorticoids regulate the migration of macrophage-like THP-1 cells. The DPP4 is a new glucocorticoid-responsive gene regulated in macrophage-like THP-1 cells Data from our transcriptome analysis suggested that the pathways of cell migration and inflammation were highly regulated by glucocorticoids; thus, to substantiate this finding, we turned to the use of nanostring analysis. Among the 594 genes present in the code set Human Immunology, 194 genes were regulated by Dex, specifically 134 in monocyte-like THP-1 cells and 157 in THP1-M⌽ (Fig. 3A). The expression pattern of genes related to the immune response sorted through a Venn diagram showed that 97 genes were commonly regulated by Dex in THP-1 and THP1-M⌽ (corresponding to 50% of the total regulated genes) (Fig. 3A). Additionally, the number of genes regulated exclusively by Dex in THP-1 and THP1-M⌽ was 37 and 60, respectively, indicating that almost twice as many genes were differentially regulated by Dex in THP1-M⌽ (Fig. 3A). Among these genes, DPP4 was the most up-regulated by glucocorticoids in THP1-M⌽ and also was part of the 100 genes mostly induced by microarray ( Figs. 2A and 3B). Interestingly, using a small cohort of RNA samples (n ϭ 9) from human monocyte-derived macrophages (38) that were treated with 100 nM dexamethasone for 6 h, we found that glucocorticoids significantly induced the mRNA expression of DPP4 in 6 of 9 samples analyzed (Fig. S5). Furthermore, DPP4 was not regulated by glucocorticoids in monocyte-like THP-1 cells (Fig. 3B). Glucocorticoid-dependent regulation of DPP4 was confirmed by qRT-PCR, through a dose-response and time-course analy- Glucocorticoids mobilize macrophages via DPP4 sis indicating that high levels of DPP4 mRNA were exclusively up-regulated in THP1-M⌽ by 10, 100, and 1000 nM Dex (Fig. 3C) and maximally induced 12 h after Dex treatment (Fig. 3D). Additionally, Dex-induced DPP4 up-regulation was observed at the protein level by Western blotting and flow cytometry, observing a double immunoreactive band of ϳ100 and 114 kDa (also induced by 10, 100, and 1,000 nM Dex) (Fig. 3E) and higher fluorescent intensity induced by Dex exclusively in THP1-M⌽ (Fig. 3F). Finally, the functionality of GR in DPP4 induction was evaluated through pharmacological and genetic inhibition, The graph shows that Dex treatment induces migration only in M⌽-THP-1, and this phenomenon is reversed by using the GR antagonist RU486. E, THP1-M⌽ were transfected with NTC or GR siRNAs. 24 h after transfection, GR protein knockdown was evaluated by Western blotting (75% reduction). On the left, a representative immunoblot of GR and ␤-tubulin expression is shown. Right, densitometry analysis of GR normalized to ␤-tubulin. F, in vitro migration assay of THP1-M⌽ transfected with NTC or GR siRNAs that have been treated for 24 h with or without 100 nM Dex. The histograms show that GR knockdown abolishes Dex-induced macrophage migration. Cell migration was calculated as percentage relative to vehicle-treated groups. Data are mean Ϯ S.D. (error bars) and are representative of three independent experiments. *, p Ͻ 0.001; , p Ͻ 0.0001; two-tailed unpaired Student's t test (E) and one-way ANOVA statistical test with Tukey's multiplecomparison test (D and F). Glucocorticoids mobilize macrophages via DPP4 using a pretreatment with GR antagonist RU-486 and transfection with GR siRNA, respectively. DPP4 induction by Dex in THP1-M⌽ was blocked in the presence of RU486, both at the transcript level (6 h) (Fig. 3G) and protein level (24 h) (Fig. 3H), and inhibited by GR silencing, at the protein level at 24 h (50% down-regulation with respect to NTC (Fig. 3I). Glucocorticoids regulate DPP4 expression by binding to GREs in regulatory regions of the DPP4 gene Through the determination of nascent RNA levels, we observed that glucocorticoids directly regulated the induction of DPP4, with an increase within just 30 min of treatment ( Fig. 4A), thus indicating that DPP4 is a direct transcriptional target of the GR. In silico analysis of the human DPP4 gene revealed the presence of numerous putative GREs located in the regulatory region between 1.5 and 6.5 kb upstream of the transcrip-tion start site, each one with a score higher than 80% in relation to the consensus GRE sequence (Fig. 4B). Therefore, monocyte-like THP-1 cells and THP1-M⌽ treated with 100 nM Dex or vehicle for 2 h were evaluated by ChIP coupled to real-time PCR (ChIP-qPCR), using anti-GR antibody and IgG isotype as negative control. In THP1-M⌽ treated with Dex, an enrichment of GR was observed in GRE Ϫ4,200/Ϫ4,185 (3.6-fold) and GRE Ϫ1,782/Ϫ1,767 (3.2-fold) of the regulatory region of DPP4, suggesting a direct transcriptional control of the DPP4 gene by GR (Fig. 4D). In Dex-induced monocyte-like THP-1 cells, this effect was not observed (Fig. 4C). As a control to evaluate whether there is a differential specificity of GR in the binding to the DPP4 promoter, we analyzed the recruitment of liganded GR to the GRE located in the promoter region of the glucocorticoid target gene GILZ. We observed an enrichment of GR to the GRE of GILZ in both monocyte- and are representative of n ϭ 3 to 6 independent experiments. , p Ͻ 0.01; *, p Ͻ 0.001; **, p Ͻ 0.0001; two-way ANOVA test with Tukey's multiplecomparison test (C and D) and one-way ANOVA test with Tukey's multiple-comparison test (G and I). Glucocorticoids mobilize macrophages via DPP4 like THP-1 cells and THP1-M⌽ following Dex treatment (6and 13-fold, respectively, Fig. 4 (C and D)). These data suggest that chromatin structure reorganization occurs during the monocyte-to-macrophage transition, thus allowing GR binding and serving as the mechanism through which DPP4 is differentially regulated in monocyte-like THP-1 cells and THP1-M⌽. To address this question, we evaluated chromatin accessibility using a formaldehyde-assisted isolation of regulatory elements (FAIRE) assay in the same regions of the DPP4 gene where GR occupied sites according to ChiP-qPCR in macrophages. Upon differentiation and Dex treatment, we observed an increase in DNA accessibility in both regions of the DPP4 gene flanking GRE Ϫ4,200/Ϫ4,185 and GRE Ϫ1,782/Ϫ1,767, respectively, in THP1-M⌽, whereas there was limited accessibility of them in monocyte-like THP-1 cells (Fig. 4E). As control, DNA accessibility was also observed in the site flanking GRE of the GILZ promoter, without differences between THP1-M⌽ and monocyte-like THP-1 cells. The increase in FAIRE enrichment seen at the two GREs of DPP4 gene suggests that the chromatin remodeling during the monocyte-to-macrophage transition is required for GR to initiate Dex-induced DPP4 gene transcription. Glucocorticoids enhance DPP4 enzymatic activity in THP1-M⌽, and this effect is blocked by DPP4 inhibitors sitagliptin and linagliptin The effects mediated by DPP4 in different cell types have been associated with both enzymatic and nonenzymatic functions. Based on this classification (and having shown that liganded GR up-regulates mRNA and protein DPP4 levels), we evaluated whether glucocorticoids could also modulate the DPP4 enzymatic activity. For these studies, monocyte-like THP-1 cells and THP1-M⌽ were stimulated with Dex for 24 h or were pretreated with RU-486 or co-stimulated after 3 h with two concentrations of specific DPP4 inhibitors, sitagliptin and linagliptin. Cell lysates of each experimental condition were evaluated by fluorometric assay, showing no DPP4 activity from monocyte-like THP-1 cells under evaluated conditions (Fig. 5A). Conversely, in THP1-M⌽, we observed that Dex increased DPP4 enzymatic activity, and this effect is mediated by GR because it was blocked by RU486 pretreatment (Fig. 5A). In addition, using sitagliptin (25 and 50 nM) and linagliptin (1 and 10 nM), we found that both inhibitors completely block DPP4 enzymatic activity induced by Dex (Fig. 5A). Notably, treatment with both DPP4 inhibitors directly blocked DPP4 enzymatic DPP4 promotes macrophage-like THP-1 cell migration The multifunctionality of DPP4, enzymatic activity to regulate chemokine actions or nonenzymatic via interaction with other proteins, could also regulate, directly or indirectly, macrophage mobility. To investigate this idea, we silenced DPP4 in THP1-M⌽ using siRNA or DPP4 inhibitors, evaluating cell migration behind glucocorticoid effects. Expression of DPP4 induced by Dex was decreased by 80% at the protein level in THP1-M⌽ transfected with siRNA DPP4 with respect to NTC (Fig. 5C). Using this strategy to evaluate GR participation in cell migration, we compared the migratory potential of THP1-M⌽ primed with Dex in the presence of DPP4 inhibitors. Cells were seeded in the insert, and medium with serum was added in the lower chamber as chemoattractant. Interestingly, the Dex-mediated increase in the migratory potential was inhibited in the presence of either 10 nM linagliptin or 50 nM sitagliptin, suggesting that this effect depended on DPP4 catalytic function (Fig. 5D). Finally, NTC and DPP4 siRNA macrophages, primed or unprimed with Dex for 24 h, were used to evaluate DPP4 participation in cell migration. An increase in the migratory potential induced by Dex in the NTC was suppressed by DPP4 knockdown (Fig. 5D), suggesting that DPP4 activity is necessary for enhancing macrophage migration. Interestingly, CXCL12 was able to promote the migration of cells treated with Dex for 24 h, and this effect was inhibited by both GR and DPP4 knockdown (Fig. 5E). The induction of the Dpp4 gene by glucocorticoids is an exclusive mechanism in pro-inflammatory M1 macrophages Our findings in THP1-M⌽ indicate that glucocorticoids promote cell migration in part by inducing DPP4 expression. To examine whether this is a conserved mechanism, we assessed the effects of Dex treatment on mouse BMM and BMDMs. After inducing macrophage differentiation with M-CSF (M0, unstimulated macrophages), cell cultures were activated by treatment with lipopolysaccharide/interferon-␥ (M1) or with interleukin-4 (M2). The phenotype of unpolarized (M0) macrophages as well as macrophages polarized to M1 and M2 was evaluated by confocal microscopy, showing the polarizationdependent morphological differences and the "classical" marker CD68 (Fig. 6A). Moreover, qRT-PCR analysis revealed that M1 macrophages exhibited the characteristic up-regulation of Nos2/iNOS, Ccl5/Rantes, and Tnf mRNA, whereas M2 macrophages exhibited increased expression of Arg1, Retnla, and Chil3 (Fig. 6B). To assess the impact of macrophage activation on glucocorticoid responsiveness, BMM, M0, M1, and M2 macrophage cultures were treated with 100 nM Dex for 6 h. Quantitative RT-PCR revealed that expression of M1-associated genes Nos2 and Tnf were suppressed 6 h after Dex treatment, whereas expression of M2-associated genes was not affected (Fig. 6B). Interestingly, whereas Dex treatment induced equivalent expression of the classic glucocorticoid target genes Gilz in each group and the induction of the chemokine receptor Cxcr4 in all groups of the macrophage populations, Dpp4 was exclusively induced only in M1 macrophages ( Fig. 6C and Figs. S6 and S7). Finally, we examined the effects of glucocorticoid treatment on BMDM macrophage migration. For this purpose, M0, M1, and M2 macrophages were treated for 24 h with Dex, and their spontaneous migration was assessed. Consistent with our finding in THP1-M⌽, we found that Dex treatment enhanced spontaneous migration of M1 macrophages but did not affect the migration of M0 or M2 macrophages (Fig. 6D). Interestingly, M1 macrophages treated with Dex for 24 h and then pretreated or not for 1 h with 50 nM sitagliptin were used to evaluate the CXCL12-induced migration. We observe that the migration of M1 macrophages could also be induced by CXCL12, and this effect was blocked by pretreatment with sitagliptin (Fig. 6E). Finally, we evaluated glucocorticoid-induced effects in peritoneal macrophages that naturally show an ambulatory or motile phenotype. Together with Gilz induction, we observed Dpp4 induction upon Dex treatment (Fig. 6F) and higher cell migration induced by glucocorticoids (Fig. 6G), suggesting that glucocorticoid-induced migration in macrophages is mediated by DPP4. Discussion The glucocorticoid receptor is expressed in almost all immune cells and mediates the actions of both endogenous or exogenous glucocorticoids, acting as potent regulators of inflammation (39). Interestingly, glucocorticoids have complex and different pleiotropic effects on monocytes and macrophages, but their contribution toward systemic anti-inflammatory effects is not yet fully understood. Here, we evaluated whether the process of monocyte-to macrophage differentiation modified glucocorticoid responsiveness. Transcriptome analysis of monocyte-like THP-1 and macrophage-like THP-1 cells revealed a higher GR expression in macrophage-like THP-1 compared with monocyte-like THP-1 cells. In addition, we report the identification of the pro-diabetic and pro-inflammatory exopeptidase DPP4 as a new glucocorticoid-responsive gene exclusively regulated in macrophages. Provocatively, DPP4 promotes the migration of macrophages that is induced by glucocorticoids. Glucocorticoids suppress inflammation through the induction of potent anti-inflammatory effects and are frequently used to treat chronic inflammatory diseases involving lymphocytes, although they are less effective in suppressing macrophage-mediated diseases (11, 13,14). Within macrophages, glucocorticoid action depends on the context and the timing, and they also have the capacity to mediate pro-inflammatory activities, such as enhancing leukocyte trafficking and pro-inflammatory cytokine production mainly during the first steps of the immune response (9). One theory explaining this dual effect on immune cells gene regulation is that glucocorticoids initiate opposing forces simultaneously inducing pro-and anti-inflammatory pathways as a pro-resolutive strategy to quickly recover the cellular and tissue homeostasis (7,40). The effect of GR activation is highly gene-, cell-, and stimulus-specific, as is evident from our transcriptome data. For example, the inhibition of CSF1 (colonystimulating factor 1) and its receptor CSF1R could be one possible reason why glucocorticoids alone would not induce the differentiation of monocytes into macrophages. Glucocorticoids mobilize macrophages via DPP4 The process of differentiation of monocytes to macrophages involves major structural and biochemical changes in the cell. However, transcriptome analysis between monocyte-like THP-1 cells and THP1-M⌽ demonstrated a high number of genes commonly regulated between both cell types. These associations observed could be due to different gene expression levels, rather than de novo transcription of uniquely expressed genes, as is the case for GR. Moreover, higher GR levels Glucocorticoids mobilize macrophages via DPP4 observed in macrophages could be related to greater sensitivity toward glucocorticoids after differentiation of monocytes. Genes involved in cell movement, trafficking, and chemotaxis were overrepresented among up-and down-glucocorticoid-regulated genes in THP1-M⌽, with DPP4 induced and differentially regulated by Dex exclusively in THP1-M⌽ and primary murine polarized M1 macrophages. DPP4, also known as CD26, was originally described as a marker of T cell differentiation and activation (41). This study provides the first evidence that DPP4 expression is directly regulated by glucocorticoids, making it a promising candidate for glucocorticoid effects in human pro-inflammatory macrophages. Additionally, the presence of a highly conserved GRE motif in the position Ϫ4,200/Ϫ4,185 in humans and mice indicates possible shared mechanisms between species. The importance of DPP4 for the medical community lies in the approval of the use of its inhibitors for treatment of type 2 diabetes, as monotherapy, or in combination with other oral anti-diabetes drugs (42) and the benefits in decreased risk of major cardiovascular events (43,44). DPP4 inhibitors have antiinflammatory effects, playing a critical role in obesity-induced inflammation and insulin resistance limiting macrophage infiltration in chronic inflammatory mouse models and regulating M1/M2 balance by mediating the reversion of one to the other (24). Previously, Zhong et al. showed that DPP4 expression was increased during monocyte differentiation into dendritic cells/ macrophages and that nonenzymatic DPP4 function was associated with inflammation during obesity (32). The exopeptidase DPP4 involved in the regulation of the immune system cleaves dipeptides from the N-terminal region of peptides and proteins (with a residue of Ala or Pro in the penultimate position) as well as various chemokines (45). The loss of two amino acids resulting from DPP4 enzymatic action can cause 1) increased or reduced biological peptide/protein activity, 2) increased specificity toward the receptor, 3) ligand inactivation, or 4) generation of receptor antagonists (46). Therefore, as chemokines direct leukocyte migration under homeostasis and inflammation, DPP4 proteolytic processing could have relevant consequences for correct functioning of the immune response. Our findings in macrophage-like THP-1 cells and murine polarized M1 macrophages indicate that glucocorticoids enhance spontaneous and CXCL12-induced migration in part by inducing DPP4 expression. Noncatalytic DPP4 functions have also been related to adhesion and migration processes and interaction with extracellular matrix proteins (fibronectin and collagen). Moreover, DPP4 inhibitors ameliorate atherosclerosis by preventing monocyte recruitment and chemotaxis via modulation of RAC-1 (21). Additionally, DPP4 in T cells interacts with the chemokine receptor CXCR4 (41), selectively binding the chemokine CXCL12 (45). This binding could promote internalization of the complex DPP4/CXCR4 in the membrane, regulating local and temporal CXCL12 activity (41,47). Regarding the nonenzymatic activity of DPP4 regulating the macrophage mobility, Hiromura et al. (48) have shown that DPP4 inhibitors affect DPP4 and caveolin-1 (CAV-1) interaction, resulting in the suppression of inflammation in mouse and human macrophages. In addition to this, it is well-known that CAV-1 activation of the GTP-binding protein RAC-1 plays a role in cell migration (49,50). Thus, DPP4 inhibitors could block the interaction of the DPP4/CXCR4 axis, the activation of CXCR4 by CXCL12, and finally the consequential activation of CAV-1/RAC-1 in the promotion of macrophage mobility. Interestingly, we observed that CXCR4 and CXCL12 genes were enriched in the cell movement and migration pathways by IPA. Our data show that glucocorticoids also increased the expression of these genes in macrophage-like THP-1 cells and primary mouse macrophages and that they could be another downstream regulator of migratory capacity mediated by GR activation. Finally, enzymatic DPP4 activity induced by Dex was completely blocked by specific DPP4 inhibitors (sitagliptin and linagliptin), suggesting the possibility that synthetic glucocorticoids would present a low efficacy in the resolution of macrophage-induced inflammation. Alternatively, these data also may indicate that glucocorticoids, through DPP4 induction, potentiate the retention and egress of macrophages from inflamed tissues, perhaps contributing to their anti-inflammatory properties of glucocorticoids. Mouse colony maintenance All studies were performed with approval by the NIEHS, National Institutes of Health, animal care and use committee. The mice used for these studies were C57BL/6J purchased from the Jackson Laboratories (Bar Harbor, ME). Mice were maintained in a pathogen-free facility with 12-h day/night cycles. Standard mouse chow and water were provided ad libitum. Cell culture The human monocytic cell line THP-1 (ATCC-TIB-202, American Type Culture Collection, Manassas, VA) together with their derived macrophages were maintained in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units of penicillin/streptomycin, 25 M HEPES (pH 7.0), and 50 M ␤-mercaptoethanol (complete medium) at a ratio of 2.5 ϫ 10 5 cells/ml under conditions of humidity at 5% Glucocorticoids mobilize macrophages via DPP4 CO 2 and 37°C. Monocyte-like THP-1 cells were differentiated into THP1-M⌽. Briefly, monocyte-like THP-1 cells (2 ϫ 10 6 cells/well) were activated with 0.5 M PMA in serum-free medium supplemented with 25 M HEPES for 3 h. Subsequently, adherent cells were washed with PBS and cultured first for 24 h with recovery medium without PMA (complete RPMI) and then for another 24 h in RPMI supplemented with 10% charcoal-stripped serum before being treated with dexamethasone. The success of the differentiation protocol was evaluated using phase-contrast microscopy and flow cytometry using a fluorophore-conjugated panel of antibodies against markers of monocyte and macrophage lineages (CD15s-BV510, CD11b-PE-Cy7, and CD11c-BV421) and Cell Tracker and 7-aminoactinomycin D for viability (BD Biosciences) (Fig. S1). Where indicated, cells were pretreated with 1 or 10 M RU-486 for 1 h prior to the addition of Dex or pretreated with Dex for 3 h prior the addition of DPP4 inhibitor (linagliptin (1 and 10 nM) or sitagliptin (25 and 50 nM)). Generation of knockout macrophage-like THP-1 cells by siRNA For siRNA experiments, 3-day macrophage-like THP-1 cells completely differentiated were seeded in 6-well plates at a density of 1 ϫ 10 6 cells/ml and then transfected with 25 nM NTC or with a 25 nM concentration of a mixture of four siRNAs provided as a single reagent of siRNA against GR or against DPP4 (ON-TARGETplus siRNA, Dharmacon) and DharmaFECT-1 in a mixture of Opti-MEM and medium without antibiotic. The transfection reaction was maintained at 37°C in 5% CO 2 for 24 h. The transfected cells were recovered in complete medium for another 24 h and maintained in charcoal-stripped medium for an additional 24 h prior to adding 100 nM Dex for 6 h for RNA or 24 h for protein analysis. The efficiency of the transfection was evaluated by Western blotting, and cells with GR or DPP4 silencing were used for functional analysis. Microarray analysis Following Dex stimulation, cells were collected and lysed for total RNA extraction using a Qiagen RNeasy minikit (Qiagen, Hilden, Germany). Gene expression analysis by microarray was carried out using Agilent whole human genome 4 ϫ 44 multiplex format oligonucleotide arrays (014850) (Agilent Technologies, Santa Clara, CA) following the Agilent one-color microarray-based gene expression analysis protocol. Starting with 500 ng of total RNA, complementary RNA labeled with the Cy3 probe was synthesized according to the manufacturer's protocol. For each sample, 1.65 g of Cy3-labeled complementary RNA was fragmented and hybridized for 17 h in a rotating hybridization oven. The oligonucleotide arrays were washed and then scanned with an Agilent scanner. Data were obtained using the Agilent Feature Extraction software (version 12), performing the error modeled, adjusting for additive and multiplicative noise. The resulting data were processed using the Omic-Soft Array Studio software (version 7.0) and visualized by principal component analysis. To identify the differentially expressed probes and to determine statistical differences between the means of the groups, an analysis of variance (ANOVA) was used. In addition, we used a multiple-test correction ANOVA and Benjamini-Hochberg with a value of p Ͻ 0.05 to reduce the number of false positives. NanoString analysis The analysis of gene expression using the NanoString platform (NanoString, Seattle, WA) was carried out using the Human Immunology code set (NS_Immunology_C2328), which measures 547 endogenous RNAs and 14 housekeeping genes. 50 ng of each total RNA sample was used according to the manufacturer's instructions. RNA expression was quantified in an nCounter Digital Analyzer, and raw counts were generated and normalized with nSolver software (version 3.0). The data were normalized using the manufacturer's positive and negative control probes as well as two housekeeping genes (HPRT1 and PPIA). All samples passed the initial quality assurance/quality control of nSolver, and the replicates were wellcorrelated (R Ͼ 0.98). The raw and normalized compiled data (log 2 of counts) were reanalyzed in Partek for statistical analysis (finding 159 probes with an average expression of less than 4 counts that were excluded), with 388 probes finally subjected to ANOVA in the treatment groups with p value corrected by false discovery rate post-hoc Benjamini-Hochberg for each comparison group. qRT-PCR analysis The analysis of the gene expression in dose response and time course using monocyte-like THP-1 cells and THP1-M⌽, human monocyte-derived macrophages (38), murine bone marrow monocytes, bone marrow-derived macrophages, and peritoneal macrophages by qRT-PCR was carried out using 50 ng of total RNA and the One-Step RT-PCR kit (Bio-Rad) together with sets of predesigned and validated TaqMan primer/probes for each analyzed transcript Glucocorticoids mobilize macrophages via DPP4 Chil3/Ym1 (Mm00657889_mH), and Ppib (Mm00478295_m1) for mouse genes. The samples were run in duplicate in the real-time thermocycler model CFX96 from Bio-Rad. The Ct values from each transcript were normalized to the housekeeping gene PPIB and expressed relative to the level of the transcript in the unstimulated condition. As a positive control of the effect of Dex, the glucocorticoid-responsive genes FKBP5 and GILZ were used. Additionally, the activity of GR and levels of each transcript regulated by Dex were evaluated in the presence or absence of RU-486. Analysis of canonical pathways using IPA The lists of significantly regulated genes were annotated using IPA. Enrichment or overlap of canonical pathways and the top biological functions were determined by IPA, using Fisher's test (p Ͻ 0.05). Gene networks involved in the inflammatory response, cell movement, and chemotaxis were constructed using the Pathdesigner tool of IPA. Protein analysis by Western blotting and flow cytometry Total proteins were extracted in radioimmune precipitation buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% SDS) supplemented with an inhibitor mixture of proteases (Roche, Rotkreuz, Switzerland). Equal amounts of protein were loaded and separated in precast Novex 10% Tris-glycine minigels (Thermo Fisher Scientific) and transferred to nitrocellulose membranes under a semidry rapid transfer system (Bio-Rad) and blocked with blocking buffer (LI-COR, Lincoln, NE) for 60 min at room temperature. Subsequently, the membranes were incubated overnight at 4°C with primary antibodies anti-GR (1:1,000 dilution) and anti-DPP4 (1:1,000 dilution) in 5% skimmed powdered milk in TBS-T and 5% BSA in TBS-T, respectively. Blots were washed and incubated with goat anti-rabbit IRDye680-conjugated secondary antibody (LI-COR) for 1 h at room temperature and visualized with a LI-COR Odyssey Imaging scanner system. The obtained immunoreactivity was normalized to ␤-actin and/or ␤-tubulin proteins as a loading control and was expressed relative to the protein level of the unstimulated condition. To determine the expression at the protein level of activation markers in monocyte-like THP-1 cells and THP1-M⌽ by flow cytometry, the cells were stimulated for 24 h with 100 nM Dex, fixed in paraformaldehyde for 10 min at room temperature, and permeabilized according to the surface or intracellular staining evaluated. The immunostaining process was performed using a panel of antibodies (BD Biosciences) conjugated against CD15s-BV510, CD11b-PE-Cy7, and CD26/DPP4-PE and their respective isotypes according to the manufacturer's specifications, prior to blocking Fc using a commercial blocker. The samples were evaluated in triplicate in the LSR II cytometer (BD Biosciences) and analyzed through the software FACSDiva version 6.1.3. Determination of the enzymatic activity of DPP4 by fluorometric assay Monocyte-like THP-1 cells and THP1-M⌽ were treated with the GR antagonist RU486 before the stimulation of Dex for 24 h or with Dex during the first 3 h before adding two concentrations of the specific inhibitors of DPP4, sitagliptin and linagliptin. The cells were collected and lysed with lysis solution according to the manufacturer's instructions using the commercial kit DPP4 Activity Assay (Sigma-Aldrich). The results were plotted as pmol/ml/min (microunits/ml), where 1 unit of DPP4 is the amount of enzyme that hydrolyzes the DPP4 substrate to produce 1.0 mol of AMC/min at 37°C. In silico analysis of GREs in the human DPP4 gene Analysis in silico using the JASPAR software database revealed the presence of putative GREs in the promoter region. These GREs were mapped and analyzed by multiple alignments against the consensus sequence using the STAMP software, demonstrating a likelihood of GR binding in those regions of the DNA. According to this, primer probes flanking each of the GREs found in the promoter region of DPP4 were used for ChIP-qPCR and FAIRE analysis. ChIP-qPCR and FAIRE analysis Monocyte-like THP-1 cells and THP1-M⌽ seeded at a density of 1 ϫ 10 6 cells/ml stimulated with or without 100 nM Dex for 2 h were collected and evaluated by ChIP using the EZ-Magna ChIP TM A/G chromatin immunoprecipitation kit with immunomagnetic beads (EMD Millipore). For this, centrifuged and pelleted cells were cross-linked using 1% formaldehyde for 10 min at room temperature followed by quenching of the reaction with 1ϫ glycine for 5 min and then lysed and homogenized with a Dounce homogenizer for isolation of the nuclear fraction in a solution containing cOmplete TM protease inhibitor mixture (Sigma-Aldrich). The nuclear fraction isolated was sonicated in a Bioructor with a controlled-temperature high-pressure cooling system (Diagenode, Sparta, NJ). A fraction of the fragmented chromatin was used to evaluate the quality of chromatin through agarose gel electrophoresis. DNA fragments sized between 0.2 and 0.5 kb were immunoprecipitated using 3 g of anti-GR mAb (Cell Signaling) or the same concentration of anti-IgG as isotype control (EMD-Millipore). Subsequently, the immunocomplexes were isolated using magnetic beads of protein A/G-agarose; washed with solutions of low and high concentration of salts, LiCl solution, and TE buffer; and treated with RNase, proteinase K, and temperature to dissociate them for recovery and elution of the DNA. Aliquots of each DNA sample recovered were purified using columns, analyzed by quantitative PCR, using primers-probes flanking the two GREs: GRE Ϫ4,200/Ϫ4,185 (forward, 5Ј-CCTAGTGGAGCTGTGA-GAAGA-3Ј; probe, 5Ј-TCCAGTTACACGGAACAAGCTG-TCC-3Ј; reverse, 5Ј-CAGGCTGGCGTTGAGTATATG-3Ј) and GRE Ϫ1,782/Ϫ1,767 (forward, 5Ј-GCACAGGGTGTGA-AGATATTTG-3Ј; probe, 5Ј-TGCCCTCCAGAGAACAAAT-TGACCT-3Ј; reverse, 5Ј-GAGGCTGGCTGACATCTAC-3Ј). The Ct values of each of the samples analyzed in triplicate were compared with respect to the initial input and normalized to the IgG isotype values and expressed as the -fold enrichment of Glucocorticoids mobilize macrophages via DPP4 the stimulated condition compared with the control. Additionally, GRE located in the promoter of the GILZ gene was used as a positive control. FAIRE analysis was performed according to Simon et al. (51) using the same set of primers-probes previously analyzed for ChIP-qPCR. Experimental setup for mouse peritoneal macrophages and bone marrow-derived macrophages Peritoneal macrophages (PM) and BMDMs were isolated from 8 -12-week-old C57BL/6 mice by flushing the peritoneal cavity with 5 ml of ice-cold complete medium and flushing the femur and tibia with complete medium, respectively. The BMM were purified by negative selection using the EasySep TM mouse monocyte isolation kit (Stemcell Tech, Vancouver, Canada) and resuspended in complete medium supplemented with 100 ng/ml M-CSF (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells at a density of 5.0 ϫ 10 5 cells/well were incubated for 6 days at 37°C and 5% CO 2 with medium change every 3 days. The BMDM phenotype was analyzed by phase-contrast microscopy and confocal immunofluorescence using anti-CD68 antibody (Biolegend, San Diego, CA); by flow cytometry of the surface markers Ly6C-PerCP/Cy5.5, CD11b-FITC, F4/80-APC and DPP4-PE, as well as the M1 and M2 markers CD80-BV421 and CD206-PE/Cy7, respectively (Biolegend); and by the gene expression profile using qRT-PCR. For polarization to M1 and M2, BMDMs unpolarized (M0) were stimulated for 24 h with 10 ng/ml murine recombinant interferon-␥ (Miltenyi Biotec) and 50 ng/ml lipopolysaccharide (Sigma) and with 10 ng/ml murine recombinant interleukin-4 (Miltenyi Biotec) to the M1 and M2, respectively, in 10% charcoalstripped serum. For the experimental setup, M0, M1, and M2 macrophages were stimulated with 100 nM Dex for 6 h for the gene expression profile and until 24 h for protein analysis. Analysis of migratory capacity of monocyte-like THP-1 cells, THP-1-derived macrophages, peritoneal macrophages, and BMDMs unpolarized and polarized to M1 and M2 Monocyte-like THP-1 cells, 6-day THP1-MO untransfected (mock) and transfected (NTC, GR siRNA and DPP4 siRNA), were treated with or without 100 nM Dex for 24 hours and then evaluated for their migratory properties. For some experiments, cells were preincubated with 10 M RU-486 for 1 hour prior to the addition of Dex. PM and BMDM (unpolarized and polarized to M1 and M2) were treated in the same manner as described above Immediately after the stimulation, the supernatant was collected, and the cells were washed and detached with fresh and warmed 10 mM PBS-EDTA, collected in serumfree medium (without chemoattractant molecules) or in the presence of DPP4 inhibitors, counted, and reseeded at a density of 4 ϫ 10 5 cells/ml in the insert of a QCM TM Chemotaxis 5-m 24-well migration assay (with a 5-m pore size for monocyte/ macrophage movement) (EMDMillipore, Burlington, MA), and medium with 10% FBS or 100 ng/ml CXCL12/SDF-1a (human or mouse) as a chemoattractant in the lower chamber After 6 and 24 h, the migratory cells that adhered to the lower surface of the insert in the chamber were detached, lysed, and quantitated by the incorporation of a fluorescent probe CyQUANT GR dye in a plate reader, according to the man-ufacturer's instructions. Each migration assay was repeated three times. The percentage of cells migrated was calculated in relation to unstimulated or untransfected conditions. Immunofluorescence staining M0, M1, and M2 macrophages were grown in glass-bottom culture dishes (MatTek Corp.). Then cells were washed with warm PBS, fixed with warm 4% paraformaldehyde for 20 min at room temperature, and permeabilized in PBS containing 2% BSA and 0.1% Triton X-100 for 30 min at room temperature. Cells were then blocked for 1 h with PBS containing 5% goat serum and 0.1% Triton X-100 at room temperature prior to incubation of the specimens at 4°C overnight with anti-CD68 (Biolegend, San Diego, CA) antibody. The following morning, samples were washed with 1ϫ PBS containing 0.1% Tween and incubated with the secondary antibody goat anti-rat AF594 for 1 h at room temperature. Samples were then washed, air-dried, and mounted with ProLong gold antifade mountant with 4Ј,6diamidino-2-phenylindole (Thermo Scientific). A Zeiss laserscanning confocal microscope (LSM 880; Carl Zeiss) was used to analyze CD68 expression. Statistical analysis GraphPad Prism version 7.0 was used to analyze the data. To determine the statistical significance of the results, the twotailed unpaired Student's t test and one-or two-way ANOVA statistical test were performed with the ad hoc post-test according to the distribution of the data. Those comparisons whose value was p Ͻ 0.05 were considered statistically significant. In all of the experiments, the samples were analyzed in duplicate, and each experiment was performed at least three times independently.	2020-01-29T14:04:56.358Z	2020-01-27T00:00:00.000	{ "year": 2020, "sha1": "f9ec2582db7e0db733f2560be1bfa76be9252dfd", "oa_license": "CCBY", "oa_url": "http://www.jbc.org/article/S0021925817495214/pdf", "oa_status": "HYBRID", "pdf_src": "Highwire", "pdf_hash": "33b9550963b75cd7957cf25ce1ceaff8aa331e9b", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Medicine", "Chemistry" ] }
250116467	pes2o/s2orc	v3-fos-license	Caspase-Dependent Apoptosis Induced by Simarouba Glauca on Human Non-Small-Cell Lung Cancer, A549 Cells Background: The leaves of Simarouba glauca (S. glauca) have been used as a potential source of anticancer agents in traditional medicine. Attempts have been made to isolate anticancer agents from the leaves of S. glauca. The objective of the present study was to demonstrate the anticancer and apoptotic effect of the leaf extract of petroleum ether (LPE) on human non-small-cell lung cancer A549 cells. Methods: MTT assay was used to investigate the effect of LPE on the viability of A-549 cells. The apoptotic effect of human lung cancer cells was evaluated using fluorescence staining, acridine orange/ ethidium bromide staining, Hoechst staining, flow cytometry analysis, annexin V staining, and caspase assay. Results: The results showed a direct correlation between the dose and the rate of cytotoxicity. Fluorescence staining revealed apoptotic features, such as blebbing and chromatin condensation. Flow cytometry analysis and annexin V staining revealed phosphatidyl serine externalization. Caspase assay confirmed that the extract inhibited cell death. Caspase 3 expressions indicated that the cell death occurred either through the mitochondrial pathway or the death receptor. Conclusions: The study revealed that the LPE induced the apoptosis of human non-small-cell lung cancer, A549 cells, either through mitochondrial or death receptor pathway. Introduction Medicinal plants are studied for pharmacological and biological properties in recent years. To understand their mechanism of action, the researchers have done molecular and phytochemical analysis. Medicinal plants have been found to be very effective against experimental as well as clinical cases of tumors. Undesired side effects sometimes occur during chemotherapy. Plant-derived products may reduce adverse side effects associated with cancer treatments (Jose et al., 2018;Jose et al., 2020). This type of experimental research has led to the commercialization of a number of important plant-based drugs used in chemotherapy. Currently, a lot of plant products are being used to treat cancer. Scientists all over the world are concentrating on medicinal plants to boost the immune cells of the body against cancers. They are also a significant source of synthetic and herbal drugs. So far, pharmaceutical companies have screened more than 25,000 plants to discover anticancer drugs. The herbal system of medicine has been practiced for thousands of years . Drug discovery and development from plant-based compounds are more RESEARCH ARTICLE Caspase-Dependent Apoptosis Induced by Simarouba Glauca on Human Non-Small-Cell Lung Cancer, A549 Cells cost-effective than conventional synthetic compounds (Rayan et al., 2017). Medicinal plant extracts have a large diversity of chemical structures, which might prove to be suitable for specific medicinal applications (Vikas et al., 2019). There are only limited suggestions about how plants tightly regulate their cell cycle machineries endogenously even after enormous exposure to hazardous components. Plant-derived compounds, such as vinblastine, topotecan, and other compounds have been used as anticancer drugs (Brahmer and Ettinger, 1998;Qu et al., 2015;Carrato et al., 2020). Simarouba glauca (S. glauca) has been used for medicinal purposes in several counties. S. glauca is an evergreen edible oil tree, which is commonly known as 'Lakshmi taru' or 'paradise tree' belonging to the family Simaroubaceae. S. glauca is one of the important herbal drugs used against various diseases (Jibi et al., 2016;Yeo et al., 2016;Osagie-Eweka et al., 2021). The leaf extract of Simarouba is known for its pharmacological properties, such as haemostatic, antihelmenthic, antiparasitic, antidysentric, antipyretic, and anticancerous (Rivero-Cruz et al., 2005;Mathew et al., 2016;Balu et al., 2020). Several quassinoids from S. glauca have exhibited cytotoxic effects against human oral epidermoid carcinoma (KB) cells, including glaucarubin, glaucarubol, glaucarubinone, and glaucarubolone (Kupchan et al., 1976;Polonsky et al., 1978). In Brazil, Simaroubaceae is represented by the genera Picrolemma and Quassia within the Amazon, Picrasma, and Castela to the South, and Simaba, Simarouba, and Picrolema, which can be seen throughout the country (Almeida et al., 2007;Alves et al., 2014).T wo hundred quassinoids have been isolated and identified (Vieira and Braz-Filho, 2006). The chloroform extract of S. glauca exhibited significant cytotoxicity against several human cancer cell lines (Likhitwitayawuid et al., 1993;Seo et al., 2001;Jose et al., 2018;Prajapati et al., 2018). The objective of this study was to demonstrate the anticancer and apoptotic effect of the leaf extract of petroleum ether (LPE) on human non-small-cell lung cancer A549 cells. Plant The leaves of S. glauca were collected from the Trivandrum district and authenticated with voucher specimen 95212 and deposited at JNTBGRI herbarium. The plant leaves were then shade dried and powdered. This powder was used for sequential solvent extraction using petroleum ether (LPE). Cell lines and culture conditions A-549 cells of human non-small-cell lung cancer cells were obtained from National Centre for Cell Sciences (NCCS) Pune, grown in DMEM media and supplemented with 10 % FBS, HEPES buffer, penicillin (100 units/ml), and streptomycin (100 μg/ ml). Cells were maintained at 370 C containing 5% CO2. Cytotoxicity assay using MTT assay MTT (3-(4,5 Dimethylthiazol-2yl)-2,5-Diphenyl Tetrazolium Bromide) assay relies on the ability of a mitochondrial dehydrogenase enzyme from healthy cells to cleave the tetrazolium rings of the pale yellow MTT and form impermeable, dark blue formazan crystals, which accumulate within healthy cells (Mossman et al.,1983). A detergent was added causing solubilization of the cells, and consequently the liberation of the crystals, which were solubilized. The color was quantified using a multi-well plate reader. The in vitro response was studied using an MTT assay. The lung cancer cell line was maintained in a DMEM medium with 10% FCS. Briefly, cells were harvested, counted, and seeded (5×10 3 cells/well in 100 μl) in 96 well titre plates (Axygen), and PBS was added to the outer wells (200μl/well). After 24 hours of incubation at 37°c and 5% CO2 to achieve cell attachment, media were removed and cultures were treated with varying concentrations of drugs (12.5-800 μg/ ml) diluted with the medium. The negative controls were also kept (i.e. cells and media). The plates were further incubated for 24 and 48 hours. Following the completion of the incubation, media were removed without disturbing the cells. Then, 100μl of 5mg/ml stock solution of MTT was added to each well, and plates were further incubated for 2 hours in dark at 37ºC and in a CO 2 incubator. Next, 100 μl of lysis buffer was added to each well. The plates were then incubated for 4 hours in dark and in a CO 2 incubator. The absorbance was recorded using multiplate reader. Three replicates were set up for each concentration. The concentration required to reduce absorbance by 50% lC 50 in comparison to control cells was determined. The IC 50 values were derived by substituting the percentage inhibition values of drugs for their respective curves. Morphological determination of the apoptotic cells Analysis of cell death (apoptosis) using dual acridine orange/ethidium bromide fluorescent staining, visualized under a fluorescent microscope: For the assessment of apoptosis after treatment of A-549 cells for 48 hours, dual acridine orange/ethidium bromide fluorescent staining of unfixed A-549 cells was used. The mixture of fluorescent dyes consisted of acridine orange at 5μg/ml in PBS. This stain makes visible the DNA-condensed chromatin of apoptotic cells. Slides were observed under a fluorescent microscope. Acridine orange was observed using standard narrow band FITC excitation (excitation wavelength 450-490nm and barrier filter 520-560nm). Ethidium bromide only stains cells in the late stages of apoptosis and secondary necrosis when membrane integrity has been lost. Early apoptotic cells are impermeable to the dye. The early stages of apoptosis are readily detectable using acridine orange (Haque et al., 1997). Cells were cultured in test tubes 1x 10 6 . A-549 cells were incubated in DMEM medium with 10% FCS, and incubated with LPE in a CO2 incubator at 37°C for 48 hours (Kirsch-Volders et al., 1997). For the assessment of apoptosis 48 hours after exposure to different concentrations of leaf extracts, dual acridine orange/ethidium bromide staining of unfixed A-549 was used. These dyes stained the DNA, allowing the chromatin of apoptotic cells to be observed. The medium was removed, and the gel was gently pelleted. Then, 1μl of acridine orange and 100g of ethidium bromide in 1ml waswere added to cells and immediately washed once with buffer saline (PBS) and re-suspended in 10ml of 10% glycerol in PBS. The slides were analyzed by fluorescent microscopy. The number of cells manifesting morphologic features of apoptosis, such as chromatin condensation, and the loss of nuclear envelope (Kerr et al., 1994) were counted as the function of total number of cells presenting in the field. Nuclear counter stain for fluorescence microscopy Hoechst 33342 nucleic acid stain is cell-permeant nuclear counterstain that emits blue fluorescence. One advantage of Hoechst 33342 is that it is membrane-permeant and stains live cells. Hoechst 33342 binds to adenine-thymine-rich regions of DNA in the minor groove. On binding to DNA, the fluorescence greatly increases. Cells were cultured in test tubes. 1x106 cells were incubated in DMEM medium with 10% FCS containing LPE in a CO 2 incubator at 37ºC for 48 hours. For the assessment of apoptosis, 48 hours % of Growth Inhibition = 100 -absorbance of the drug-treated cells X 100 The absorbance of untreated control cells line at lower concentration of 12.5μg/ml, the percentage of cytotoxicity at 24 and 48 hours were 19% and 26%. At the concentration of 800μg/ml, the extracts showed maximum activity of 65% and 81%, respectively (Fig.1). The IC50 value of LPE at 24 hours on A-549 cells was 159μg/ml. LPE-induced maximum cytotoxicity in A-549 was 81% at the concentration of 800μg/ml and following 48 hours of incubation Acridine orange/ ethidium bromide dual staining Acridine orange/Ethidium bromide dual staining was used for detecting apoptosis of the 10µg/ml and 15 µg/ ml LPE treated A549 cells for 48hours. While examining under inverted fluorescent microscope, viable cells were seen as fluorescent green; whereas, the apoptotic cells were seen as fluorescent orange. The findings showed nuclear fragmentation, membrane blebbing, and membrane invagination (Figure 3). Hoechst staining In Hoechst staining the control cells, A-549 cells, appeared light blue in color and the apoptotic cells appeared fluorescent blue in color, showing chromatin condensation and DNA fragmentation after 48 hours of incubation of LPE at the concentrations of 10 µg/ml and 15 µg/ml (Figure 4). Annexin V status A number of annexin-positive cells were observed after 48 hours of treatment with LPE at concentrations of 10 µg/ml, indicating an apoptosis induction of 67% in A-549 cells ( Figure 5). Caspase 3 expression After treating with LPE at the concentration of 10 µg/ml, the A-549 cells showed 63% caspase expression at 48 hours, compared to untreated control cells ( Figure 6). Statistical analysis after exposure to different concentrations of LPE, cells were stained with 5 µM Hoechst 33342. These dyes stain the DNA and allowed the visualization of condensed chromatin of apoptotic cells. Detection of apoptotic cells by annexin V-FITC and PI staining The cells were centrifuged with cold PBS at 1500-2000 rpm for 10 minutes. Then, 100µl binding buffer (1X) was added to the pellet. Next, 5µl of annexin V FITC and 5µL of propidium iodide was added. The cells were gently vortexed and kept for incubation at room temperature in the dark for 15 minutes. About 400µl of 1X binding buffer was added to each tube. Then, the tubes were analyzed by flow cytometry. Caspase 3 expression Caspase-3 is a key protease that is activated during apoptosis. The cells were treated with 10µg/ml LPE. After 48 hours of incubation, the cells were washed twice with cold PBS and prepared for acquisition using FITC conjugated monoclonal active Caspase-3 antibody apoptosis detection kit. The cells were fixed in cytofix solution at a concentration of 1x10 6 cells/0.5 ml. The cells were then fixed in ice for 30 minutes, resuspended in perm wash buffer containing the antibody, and incubated for 30 minutes at room temperature. Cells were then analyzed by flow cytometry. Finally, 10,000 cells were acquired, and the results were interpreted using Diva software analysis. MTT assay on A-549 cells Different concentrations of LPE (12.5μg/ml, 25μg/ml, 50μg/ml, 100μg/ml, 200μg/ml, 400μg/ml), 800μg/ml)) were treated on A-549 cell lines and were assayed to check the cytotoxicity at 24 and 48 hours. It was seen that when treated with LPE, the cytotoxicity varied depending on the concentration and time. In A-549 cell The results are represented as the mean + SD. The data were analyzed by using excel and Easy plot. Discussion From ancient times, medicinal plants posse pain-relieving and healing capabilities and today we still rely on the curative properties of plants. Plant-based-medicines have a vital role in the prevention and treatment of cancer. In addition, medicinal plants are economical and readily available. A great deal of pharmaceutical research done in technologically advanced countries has considerably improved the quality of the herbal medicines used in the treatment of cancer. All over the world, scientists are concentrating on medicinal plants to boost the immune cells of the body against cancer. They are also an important source of synthetic and herbal drugs. So far, the pharmaceutical companies have screened more than 25,000 plants for their therapeutic properties . In this study, the anticancer and apoptotic properties of the leaf extract of S. glauca was assessed against human non-small-cell lung cancer, A549 cells. Many compounds isolated from different members of this family have been previously reported to have anti-cancer properties. For example, early cancer screening in S. glauca indicated that an alcohol extract had cytotoxic activity against cancer cells at a low dosage. Following that, scientists discovered that several of the quassinoids in S.glauca had anti-leukemic action against lymphocytic leukemia in vitro Jose et al., 2018). Researchers found that yet another quassinoid present in S. glauca, holocanthone, also possesses antileukemic and anti-tumorous properties (Narayanan, 2011). In the present study, petroleum ether extract (LPE) obtained from S. glauca was screened for cytotoxicity using an MTT assay and the findings indicated that the viability of the MTT assay conveniently provided drug sensitivity information. Appropriate cytotoxicity was proved for cancer cell lines treated with S. glauca petroleum ether leaf extract. We evaluated the apoptotic and anti-proliferative properties of S. glauca as a member of the Simaroubaceae family. Various compounds isolated from different members of this family have been previously reported to have anti-cancer properties. For example, the most potent quassinoids from S. glauca with such antitumor properties, such as bruceantin, bruceantinol, glacarubinone, and simalikalctone D, deserve special attention (Guo et al., 2005). Using human non-small-cell lung cancer, A549 cells, the extract of the plant demonstrated remarkable cytotoxicity in the present study. Different fluorescence staining techniques were applied to A-549 cells treated 10 µg/ml and 15 µg/ml LPE in order to determine apoptotic cell death and apoptotic characteristics. In the acridine/ ethidium bromide staining, acridine orange enters the living cell and intercalates the G-C base pairs of the DNA. At the A-T base pairs, ethidium bromide enters the dead cell and intercalates the DNA. The viable cells fluoresce green, while the dead cells fluoresce orange. The apoptotic feature like membrane blebbing, chromatin condensation and DNA fragmentation were observed in this study. Analyzing the data of Annexin using flow cytometry revealed early and end stages of apoptosis. Caspase 3 using flow cytometry determined that cell death occurred through either mitochondrial or death receptor pathway. In a nutshell, it was discovered that S. glauca possessed the potential to induce apoptosis in cancer cells. In conclusion, this study yielded that LPE had remarkable anticancer activity via apoptotic induction through mitochondrial and death receptor pathways. This substantiated that S. glauca posed potential anticancer properties. Further research is recommended to develop a drug for treating malignancies.	2022-06-30T06:16:48.050Z	2022-06-01T00:00:00.000	{ "year": 2022, "sha1": "7de9b426bf21d2877a5e6dde8d84c3ee1230a445", "oa_license": "CCBY", "oa_url": "http://journal.waocp.org/article_90139_2375a6a29364cf2d75eaec93250a05fb.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "65d0fcc467360f71d25b6bd68c2f0ab14e326f23", "s2fieldsofstudy": [ "Biology", "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
237971140	pes2o/s2orc	v3-fos-license	Genetic diversity and population structure analysis – a prerequisite for constructing a mini core collection of Balkan Capsicum annuum germplasm Abstract Pepper (Capsicum annuum L.) is one of the most important vegetable crops worldwide. The Balkans, including Bulgaria, are considered as the secondary centre of pepper diversity especially for C. annuum, where local forms with diverse phenotypes and qualities have formed due to the specific agro-climatic conditions and breeding traditions. Evaluation of the genetic diversity and structure of a pepper collection is an important tool for further development of new varieties and the maintenance of sustainable agriculture. In this study, a set of 179 C. annuum accessions collected from different locations in the Balkan Peninsula was genotyped with 21 simple sequence repeat (SSR; microsatellite) markers. In total, 146 alleles were amplified among which the majority were with low frequencies (<5%). The mean He, Ho and PIC for the 21 SSR loci in the whole set were 0.531, 0.249 and 0.483, respectively. Model-based structure analysis divided the collection into 3 main clusters (K = 3) that grouped accessions with distinct fruit traits like shape, size, pungency. Further genetic structure analysis at increasing Ks suggested the presence of sub-clustering within the three main clusters. A Balkan C. annuum mini core collection was constructed based on the allelic diversity and the inferred genetic structure. As far as the mini core collection captured substantial part of the allele richness, genetic and phenotypic diversity of the analysed 176 non-redundant accessions, while maintaining good representativeness, we believe it will be of high interest to pepper breeders and germplasm conservation specialists. Supplemental data for this article is available online at https://doi.org/10.1080/13102818.2021.1946428 . Introduction Pepper (Capsicum spp.) is one of the most broadly cultivated and consumed vegetables worldwide with harvested area and production in the last five years reaching 3.7 million ha and over 40 million tonnes, respectively (www.fao.org). Its production continues to progressively grow because of the high nutritional value of pepper fruits. Pepper fruits have various applications in the human diet like fresh and processed vegetables, flavourings in food products, spice, but also in pharmaceutics, cosmetics and even as an ornamental plant [1,2]. The high content of bioactive compounds, such as capsaicinoids, carotenoids (capsanthin, capsorubin, β-carotene, β-cryptoxanthin, lutein, zeaxanthin, etc.), vitamins (C, E and provitamin A), dietary fibres and some essential mineral oils have made this vegetable crop an excellent source for protection against various chronic degenerative diseases and human health protection [3,4]. Capsicum genus is comprised of as many as 36 species [5] of which five (C. annuum L., C. frutescens L., C. chinense Jacq., C. baccatum L. and C. pubescens Ruiz et Pav) are cultivated ones [6,7]. Among the domesticated Capsicum species, pungent (chilli or hot pepper) and non-pungent (sweet pepper) forms of Capsicum annuum L. are most popular and have a worldwide commercial distribution [8]. Because Capsicum is of economic and nutritional importance, breeders have improved some agronomic traits, such as pungency, fruit shape, abiotic and biotic stress resistance. However, this leads to reduced genetic diversity of breeding lines, so some useful genes in the landraces (local forms) are lost due to the breeding activities [3]. Conservation and sustainable utilization of genetic resources are keys to the continuous improvement of peppers in order to respond to climate change and increasing global food demand in the successive decades. Conservation of genetic diversity is an essential prerequisite to enhance plant breeding programmes and develop new varieties with desirable agronomic traits and to broaden the genetic basis of this economically important crop. There are a number of pepper germplasm collections around the world (the uSA, South America, Asia and Europe). However, many of them are difficult to maintain due to their large size and a lack of adequate information about population structure and genetic diversity at the interspecific and intraspecific level. Selecting a representative core collection is a proven and effective tool for overcoming the expenses and difficulties of managing the huge genetic resources in the gene banks [9]. The core collection is a subset of the germplasm collection that represents the genetic diversity of the entire collection, has no redundant accessions and is small enough to be easily managed [10,11]. Different types of descriptors like passport data, geographic origin, morphological, agronomical, biochemical and DNA markers can be used for phenotypic and genetic diversity evaluation and construction of core collections [12][13][14]. There are already some core collections of Capsicum using phenotypic data [15], genotypic data [16,17] as well as combined phenotypic and genotypic data [18][19][20]. Core collections for disease resistance against northern root-knot nematode and Potato virus y (PVy) have been constructed [21,22]. Hanson et al. [23] developed Capsicum core collection to analyse the antioxidant (Ao) content and antioxidant activity (AoA) in accessions from AVRDC-the World Vegetable Center. The cultivated Capsicum species capture a broad diversity generated by evolution and natural selection, as well as domestication in different primary and secondary centres of diversity, and artificial selection in distinct agricultural environments [24,25]. The Balkans, including Bulgaria, are considered as the secondary centre of pepper diversity especially for C. annuum, where local forms (landraces) with diverse phenotypes and qualities have formed due to the specific agro-climatic conditions and breeding traditions [26,27]. Locally maintained and well adapted pepper landraces can still be found in small farms and villagers' yards of Bulgaria and other Balkan countries. They have been maintained for centuries by passing down from generation to generation and preserving most important features including orientation, shape, size, colour and taste of the fruit, productivity and content of valuable bioactive components. Some of them in addition show good tolerance to biotic and abiotic factors and have been used in various breeding programmes to develop nutritionally improved and high yielding cultivars [28,29]. The high biodiversity of pepper represents a unique resource that could be used in future breeding programmes to increase the diversity and identify forms with increased resistance to a number of abiotic and biotic stress factors. Given the new priorities, it is relevant to study the biodiversity of Balkan pepper in the above-described aspects. Its in-depth characterization is of fundamental importance in order to avoid genetic erosion and to ensure maximum use of genetic variability in breeding programmes. The Capsicum collection was created, developed and maintained as an initial step in the pepper breeding work at the Maritsa Vegetable Crops Research Institute (MVCRI), Plovdiv, Bulgaria many decades ago [28]. It includes over 1500 pepper accessions which are collected in various ways: personal contacts, different expeditions, national and international exchange, etc. In recent years, the participation in different national and international projects enabled its increase with additional Balkan varieties, local forms (landraces) and breeding lines most of which have been collected from a large number of locations in six countries on the Balkan Peninsula (Bulgaria, Serbia, North Macedonia, Albania, Romania and Greece) under the SEE-ERA.NET PLuS project ERA 226. An important task of the research is the study and conservation of Balkan Capsicum resources. The large number of accessions of this collection greatly impedes the research process and the choice of genetic material to be included in the breeding programmes. This requires the creation of the core collection that includes a limited number of genetically diverse accessions to represent genetic diversity within the entire collection. This will ensure a more effective study of available pepper germplasm and the assessment of other important traits like productivity, the content of biologically active substances, minerals and trace elements, resistance to diseases and pests and a number of other biological characteristics that are relevant to the breeding process. up to now, part of the Capsicum annuum accessions with diverse phenotype have been characterized by conventional phenotyping, according to various Capsicum descriptors [30,31]; high-throughput fruit phenotyping using tomato analyser in combination with conventional analysis [32], and by compilation of conventional and high-throughput phenotypic, biochemical and virus resistance analyses [33]; testing for fungal [34] and some important pest infestation [35,36]. However, a large part of this subset of accessions has never been analysed at the DNA level. To date only a small number of Bulgarian pungent small fruited red pepper landraces have been analysed by simple sequence repeat (SSR; microsatellite) markers [37] and some commercial pepper varieties were recently analysed with inter simple sequence repeat (ISSR) markers [29]. Therefore, more precise analysis using codominant microsatellite (SSR) markers is necessary to determine the genetic diversity and infer the population structure of Balkan peppers. This is a very important prerequisite for the next step aimed at development of a core collection with application in future breeding programmes and association mapping of important nutritional and environmental adaptation traits. owing to the above considerations and the importance of more detailed pepper germplasm characterization, the aim of the present study was to evaluate the genetic diversity and to determine the genetic structure of 179 Balkan C. annuum accessions using 21 SSR markers and construct a mini core collection on the basis of these data. Plant material The plant material consisted of 179 diverse Balkan pepper accessions, including local forms/landraces, varieties and 4 breeding lines (Capsicum annuum L.). The passport data for all 179 Balkan pepper accessions were described in previous studies [32,33]. They were maintained and phenotypically evaluated in Maritsa Vegetable Crops Research Institute (MVCRI), Plovdiv located in South-Central Bulgaria (42°10′35.3″N 24°45′50.5″E). Accessions were collected from Bulgaria, Serbia, North Macedonia, Albania, Romania and Greece. However, over 63% of them originated from Bulgaria. Some accessions collected from different regions of Bulgaria, Serbia and North Macedonia are known locally by the same name but appear to differ in their morphological characteristics and have been labelled differently (Supplemental Table S1). In addition, accessions showing segregation for some of the analysed phenotype traits were separated as distinct biotypes that were labelled with capital letters (A and B) in order to be genetically analysed and distinguished. Each accession was represented by 30 plants in three replications (10 plants/replication) in field trials at MVCRI in Plovdiv. The tested pepper accessions were divided into seven varietal groups according to their fruit shape, with conical, elongate, pumpkin shape, bell or blocky, conical to blocky, elongate to bell or blocky and round fruit types [33]. DNA extraction All DNA samples were extracted from freeze-dried leaf tissue (10-15 mg) of field-grown plants (10 individual plants/genotype) using a DNeasy PowerPlant ProHTP 96 Kit (qiagen). The concentration and the purity of DNA samples were determined with a NanoDrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, uSA). Primer synthesis Published primers for pepper SSRs [38][39][40], http://solgenomics.net (Supplemental Table S2) referred to as locus-specific primers (LSPs) were extended with generic non-complementary nucleotide sequences tagF 5 ' -A C G A C G T T G T A A A A -3 ′ a n d t a g R 5'-CATTAAGTTCCCATTA-3′, respectively, at their 5′ ends as described in Hayden et al. [41]. Primer aliquots (50 pmol/ µL) were prepared by mixing equimolar amounts of forward and reverse primers in miliq H 2 o and were referred to as stock primer sets for each locus. In addition, two generic primers complementary to the LSP extension sequences, tagF' 5′-ACGACGTTGTAAAA-3′ and tagR' 5′-CATTAAGTTCCCATTA-3′, were also synthesized. The tagF' primer was labelled at its 5′ end with one of the following fluorescent dyes: FAM, ATTo565 (PET) or ATTo550 (NED) to allow direct detection of alleles on the automated capillary sequencer (ABI3130, Thermo Fisher Scientific, Wilmington, DE, uSA). All primers were synthesized by Microsynth (Microsynth AG, Balgach, Switzerland). PCR was performed on a Veriti 96 Thermal Cycler (Thermo Fisher Scientific, Wilmington, DE, uSA) using the following PCR conditions, depending on the melting temperature of the locus-specific primers. These were performed according to [41][42][43] with some minor modifications: 1. The PCR programme 50°С included a denaturing step at 95°С for 3min, followed by 7 cycles of amplification, each including a denaturing step at 92°С for 30 s, an annealing step at 50°С for 1.30 min, and a synthesis step at 72°С for 1 min; SSR analysis Electrophoresis and visualization of SSR alleles was performed on an ABI3130 DNA analyser. A standardized multi-pooling procedure was used to prepare SSR products for electrophoresis. After PCR, a 3-fold initial dilution of the PCR products and subsequent mixing (1:1:0.5), respectively, FAM: PET: NED, was performed up to a final dilution of 1/25× to 1/75×. Seven mixed pools were developed according to the length of the PCR products (CAMS142_FAM, CAMS398_VIC and EPMS331_NED; HPMS2-24_FAM, GPMS29_PET and HPMS1-5_NED; HPMS1-6_FAM, CAMS405_PET and CAMS811_NED; CAMS153_FAM, EPMS376_PET and EPMS397_NED; HPMS2-13_FAM, EPMS335_PET and CAMS606_NED; HPMS1-143_FAM, CAMS234_PET and CAMS199_NED; CAMS864_FAM, EPMS418_PET and HPMS1-1_NED). In cases where the intensity of the amplified products was strong, a change in the mixing ratio of the labelled products was performed. Subsequently, the diluted products were mixed with labelled internal standards GeneScan™ 500 LIZ™ dye Size Standard (Thermo Fisher Scientific, Wilmington, DE, uSA) and formamide, denatured and electrophoresed on an ABI3130 DNA analyser. SSR allele sizing was performed with the Gene Mapper v4 software (Thermo Fisher Scientific, Wilmington, DE, uSA). Data analysis Allele number, frequency of alleles, observed heterozygosity (Ho), expected heterozygosity (He) and Polymorphic Information Content (PIC) were calculated with Power Marker v3.25 software [44]. The chord distance matrix [45] was used to construct a phylogenetic tree with the unweighted pair group method with arithmetic mean (uPGMA) module of Power Marker v3.25. The resulting tree was visualized and annotated by using the Evolview v3 webserver [46]. The clades in the phylogenetic tree were visually identified as monophyletic groups that could be separated from the main tree with a single cut according to the clade definition provided in [47]. Genetic structure analysis A model-based population structure analysis was performed in STRuCTuRE 2.3 [48] using an admixture model with correlated allele frequencies. The tested number of possible K was set from 1 to 15 with 15 runs for each K. Each run had a burn-in period of 100,000 and 1,000,000 MCMC iterations. The most probable value of K was determined using the ΔK [49] implemented in Structure Harvester v. 6.93 [50]. The genotypes were assigned to a respective group if showing membership coefficient ≥70%. The graphical representation of the cluster analysis was done using the pophelper package in R version 4.0.4 [51]. The degree of differentiation between the resulting genetic clusters was evaluated using pairwise Jost's D [52] and analysis of molecular variance (AMoVA) in R version 4.0.4 [51]. In order to analyse the presence of patterns manifested by quantitative phenotypic traits in the groups established by the clustering, we performed a principal component analysis (PCA). The results from the PCA were visualized using colour codes for the structure clusters and geometric form codes for the fruit shape. For the analysis we used data for plant height, stem height, fruit length, fruit weight, fruit width and fruit wall thickness. The phenotype data used for the PCA are published in [33]. The analysis was done and visualised in R version 4.0.4 [51]. The SSR marker data were used for construction of a mini core collection with the Core Hunter 3 package in R [53]. For this purpose, we tested two optimization strategies. The first one aimed to minimize the distance between each accession and the nearest entry from the core collection (A-NE). The second was a simultaneous optimisation of A-NE and maximization of allele richness, expressed as the percentage retained in the core collection alleles from the whole collection. Both approaches were done with the Cavali-Sforza distance matrix [45]. The proportion of retained alleles was used as a direct measure for the preserved genetic diversity by the core collections. We also compared this metric of the collections to the mean of 1000 randomly selected samples of respective sizes. Genetic diversity of Balkan Capsicum annuum accessions using SSR markers To explore the genetic diversity and population structure, we investigated the patterns of molecular diversity with 21 SSR markers in the Balkan C. annuum germplasm collection consisting of 179 accessions, phenotypically evaluated using various Capsicum descriptors. The markers were selected to cover 8 of the 12 chromosomes of pepper with a minimum of 1 marker per chromosome. In this study, standardized PCR conditions at 4 different temperatures of annealing (50, 61, 62 and 63 °C) were optimized (Supplemental Table S2), which enabled amplification of markers in multiplexed PCR reactions. Standardized PCR conditions were achieved by performing an initial optimization step with five different LSP primer concentrations (30,50,60,80 and 100 nmol/L). The adjusted LSP primer concentration enabled the PCR specificity and yield to be controlled and helped to prevent non-specific annealing of LSP during the first few PCR cycles [41]. As previously observed [42,43], this optimization step was a prerequisite for correct and efficient amplification of microsatellite alleles in all tested genotypes and eliminated the need to use complex touchdown PCR or to adjust the annealing temperatures for each locus. The standardized procedure is cost-effective by enabling multiplexing of several loci in one PCR reaction. Just a few accessions displayed undistinguishable multilocus genotypes at all analysed 21 SSR loci. These genetically redundant accessions (CAPS-021, CAPS-133 and CAPS-143) were removed from the subsequent analyses, leading to a final panel of 176 genotypes. The diversity pattern of the 21 SSR loci across the whole set of accessions revealed a total of 146 distinct alleles ranging from 2 in locus HPMS1-6 to 18 in locus CAMS864, with a mean of 6.95 alleles per locus (Table 1). Several genotypes showed null alleles in 7 out of the 21 loci tested. out of the 146 detected SSR alleles, 63 were 'common alleles' (frequency of >5% in the analysed set), 43 were 'less common alleles' (frequencies between 1% and 5%), 40 were very rare alleles (frequency of <1% in the analysed set). The fact that about a half of the alleles were with frequencies of less than 5% is evidence for the presence of a broad level of genetic diversity in the studied set of 176 C. annuum genotypes. The mean PIC for all 21 SSR markers was 0.483, with values ranging from 0.022 for marker HPMS2-13 on chromosome P1 to 0.873 for marker CAMS864 on chromosome 7. The genetic diversity index (He or GD) ranged from 0.022 (HPMS2-13) to 0.879 (CAMS864) with a mean of 0.531. The average observed heterozygosity (Ho) across all 21 loci was 0.249, with the highest values of 0.994 in loci HPMS1-1 and CAMS234, and 0.972 in locus CAMS153. Absence of heterozygous accessions was observed only in the locus HPMS2-13, which showed the lowest level of He. Genetic structure of the Balkan Capsicum annuum collection The SSR genotyping results were used to perform population structure analysis for 176 accessions under an admixture model using the STRuCTuRE software [48]. The optimal number of clusters by Evano method [49] was defined as K = 3 (Figure 1(A)). Two additional peaks were detected in the Evano graph, one at K = 4 and a lower one at K = 11. A genotype was considered a member of a particular cluster if the probability for the membership was at least 70%. For K = 3 ( Figure 1(B) Figure 1(C)). This cluster was very diverse in terms of fruit tastes, as it included accessions with non-pungent fruits and ones showing different levels of pungency. The majority of genotypes (almost 63%), however, had pungent fruit taste. The accessions that were not assigned to any of the three clusters formed a group of 24 Bulgarian, 3 Macedonian, 2 Romanian, 6 Serbian accessions and two accessions of unknown origin (CAPS-151 and CAPS-151B). The group consisted of genotypes with various fruit shapes and sizes. Fifty-nine percent of the genotypes in this admixed group were with conical fruits, 27% with elongate, about 11% with pumpkin and one genotype (CAPS-136) with bell or blocky shaped fruits. Most of them were non-pungent but 12 (34.3%) showed different levels of pungency. The presence of secondary and tertiary smaller peaks at increasing Ks pointed at underlying sub-clustering within the 3 main clusters. In order to further explore this hypothesis, we also examined the population structure at K = 4 and K = 11. Clear separation of Cluster 3 into two subclusters, Cluster 3.1 and Cluster 3.2, was evident at K = 4 (Figures 1(B) and 2). Cluster 3.1 consisted of 11 accessions having relatively smaller fruits in comparison to Cluster 3.2, which comprised 41 genotypes with predominantly elongate and conical fruits. Additional sub-clustering in the other main clusters was also observed at K = 11 ( Figure 2). The two main clusters, Cluster 1 and Cluster 2 that remained intact at K = 4, were also each split into three distinct subclusters at K = 11. The two sub-clusters . phylogenetic tree of the Balkan C. annuum germplasm collection. the tree was produced using the upgma method based on chord distance [45] computed from the allele frequencies at 21 SSR loci. the tree is drawn and annotated using the evolview v3 [46] with the following additional data starting from right to left: barplots of the group membership from the model based genetic structure analysis at K = 3, K = 4 and K = 11; fruit shapes of the accessions annotated with text and different colours; cl-1 to cl-17B represent the main clades in the tree. Different fruit shapes are additionally indicated by different shapes and colours of the tree leaves. at K = 4 derived from the most diverse main Cluster 3, were further subdivided at K = 11. Cluster 3.1 was separated into distinct subclusters, Cluster 3.1.1 and a group of admixed accessions with more than 50% membership to Cluster 3.1.2. The larger sub-cluster (3.2) was also split into two distinct sub-clusters (3.2.1 and 3.2.2) and a group of admixed genotypes sharing between 20 and 30% membership with both Cluster 3.2.2 and Cluster 3.2.3 ( Figure 2). Notably, Cluster 3.2.3 did not form distinct separation at K = 11 but only participated in admixed accessions. Genetic structure and quantitative phenotypes To analyse the distribution of the genotypes in the clusters inferred by the model-based approach, a biplot analysis of several quantitative traits was performed. The greatest contribution to the first composite axis had the fruit width and fruit wall thickness, while stem height, plant height and fruit length contribute to the second axis. The biplot graph (Supplemental Figure S1) clearly illustrated the characteristics of the genotypes grouped together, representing three consecutive zones each dominated by members of one of the inferred genetic clusters. The majority of the accessions from Cluster 1 were situated to the right end mainly in quadrant 4, but some of them fell into quadrant 1. They had wide fruits with average or below the average length, thick fruit wall and a low fruit length-to-width ratio, whose values in most of the genotypes were close to 1 or lower. The accessions from Cluster 2 are situated to the left of the genotypes from Cluster 1, in both quadrants 1 and 4. The group has a greater length to width ratio and a lower average fruit wall thickness compared to the genotypes from Cluster 1, but still higher than the whole collection mean. The genotypes from Cluster 3, a highly diverse group with small round or elongate fruits, had three features in common: the small fruit width and weight and thin fruit wall. The genotypes with elongate fruits also had low fruit length-to-width ratio. The members of the two subclusters at K = 4 (Cluster 3.1 and Cluster 3.2, Figure 2) were interspersed in quadrants 2 and 3 on the biplot figure, and there is no obvious difference between them. However, the accessions from Cluster 3.1 had more than twice as small fruit length-to-width ratio, and much lower weight. The admixed accessions were scattered in all of the four quadrants. Genetic structure and genetic diversity The analysis of genetic diversity in the clusters derived using the model-based approach at K = 3 revealed that Cluster 3 was the most diverse group ( Table 2). It had the highest number of alleles, allele richness and gene diversity followed by the group of admixed accessions, Cluster 1 and Cluster 2. All clusters had numerous private alleles: 21 in Cluster 3, 11 in both, Cluster 1 and the admixed group, and 6 in Cluster 2. Most of these private alleles were with very low frequencies and just eight of them exceeded 5% frequency in the respective cluster: four in Cluster 2 and four in Cluster 3 (Supplemental Table S3). To analyse the level of genetic differentiation between the clusters, we evaluated Jost's D index and AMoVA. These analyses were done using only the accessions with more than 70% membership to one of the three main clusters, excluding the admixed genotypes. The observed values of D index ranged between 0.15, for Clusters 1 and 2, and 0.186, between Clusters 1 and 3 (Supplemental Table S4). The results from the AMoVA showed that 15.4% of the genetic variance was partitioned among the populations, 41.1% among the samples within populations and 43.5% within the samples (Supplemental Table S5). When considering K = 4, the differentiation between the clusters was more pronounced. We estimated higher values for D, ranging between 0.15 for the cluster consisting predominantly of bell or blocky fruits and the one with conical ones (Clusters 1 and 2), and 0.29 between the new subcluster 3.1 and the one with predominantly bell and blocky fruits. According to the AMoVA results, the differentiation between the clusters also increased at K = 4, as the percentage of the between-cluster variation was estimated to be 17.6% (Supplemental Table S5). It is worth noting that the smallest group, Cluster 3.1 had 72 alleles. Phylogenetic analysis A phylogenetic tree based on chord genetic pairwise distances [45] was constructed using the uPGMA procedure. The tree grouped the accessions into 17 Clades including two subclades, Clade 17a and Clade 17b. It shows generally good congruence with the three main clusters inferred by the model-based approach at K = 3 and the substructures at K = 4 and K = 11 (Figure 2), providing finer resolution of the population structure. This is best visible in Clade 17, where most of the conical fruit accessions belonging to the main Cluster 2 of the model based clustering were grouped into two large subclades. These were further separated into smaller subclades, grouping together accessions belonging to substructure clusters at K = 11. Some of the admixed accessions that cannot be associated with a single substructure cluster were also grouped in distinct subclades in the phylogenetic tree. It is worth noting that four accessions (CAPS-009, CAPS-049, CAPS-114 and CAPS-018) were separated into single accession clades (cl.-3, cl.-7, cl.-8 and cl.-15, Figure 2) Construction of a mini core collection In order to construct a 'multi-purpose' or CC-I type of core collection, according to the classification described by odong et al. (2013) [11], we constructed two series of core collections. Each of the series consisted of six core collections with an increasing number of individuals from 5% to 30% of the whole collection with increments of five percent. The series differed by the optimization method used for the selection of the entries in the core collections. one of them was constructed using minimization of accession-tonearest-entry distance (A-NE), and the other was optimized by combining the A-NE with the maximization of allele coverage (CV). The metrics from both approaches were compared to the means of 1000 randomly selected groups of the same sizes. The selection resulted in six mini core collections consisting of 9, 18, 26, 35, 44 and 53 individuals. The genetic diversity was preserved at significantly higher levels in the collections constructed combining A-NE and allele richness as construction criteria. The approach using just A-NE minimisation as an optimization method resulted in selection of collections which retained much lower proportions from the alleles discovered in the whole collection; their numbers were commensurable with the means of the randomly selected collections of the same sizes (Figure 3(A)). on the other hand, A-NE as a sole optimization criterion provided a better representativeness of the accessions from the whole panel, as manifested by the lower average A-NE distances (Figure 3(B)). The analysis showed that the method based on the combination of the two criteria was superior for preserving allele richness while keeping the representativeness at an acceptable level. Therefore, we choose the A-NE/CV method for final sampling of the core collections. By using the combined method, samples of 9, 18, 26, 35, 44 and 53 individuals captured 62, 75, 86, 91, 95 and 99% of the alleles from the whole collection (Table 3). The final mini core collection of 44 accessions captured nearly 95% of the SSR alleles, including all common alleles, all less common alleles and 32 out of 40 very rare alleles (Supplemental Figure S2(A)). out of the 44 entries, 9 were from Cluster 1, 8 from Cluster 2, 18 from Cluster 3 and 9 from the admixed accessions (Table 3, Supplemental Table S6) and they were relatively evenly distributed among different clades of the phylogenetic tree (Supplemental Figure S2(B)). For each core collection, sample size, mean number of alleles per SSR locus (allele richness), gene diversity (he), observed heterozygosity (ho), polymorphic information content (pic), the % of retained SSR alleles (alleles %) and the distribution of selected entries among the clusters inferred from the model-based analysis are indicated. Discussion Genetic resources are an important tool for overcoming the current challenges posed by climate change and the need to provide food security for the growing human population. However, the large size of these collections is a serious drawback for their proper use in conservation and management practices. Establishment of a core collection is a primary objective of many gene banks around the world because of the reduced cost and the efforts for its conservation. one of the most important issues is to develop a subset of accessions in which the gene diversity of the whole collection is preserved. The Balkan Capsicum collection analysed in this study is a part of the large collection developed through many years of exchange of genetic material among the gene banks, breeding efforts and expeditions in Bulgaria and neighbouring countries on the Balkan Peninsula. In this study, 179 accessions of C. annuum L. most of which representing local forms adapted to the specific agro-climatic conditions and selected through many years' traditions of cultivation in small private farms on the Balkan Peninsula, cultivars and breeding lines were subjected to molecular and phenotypic evaluation as a step towards the development of a mini core collection. The number of accessions in our study is much smaller than those reported in other studies. However, it includes only C. annuum genotypes. For example, Gu et al. [17], Nicolaï et al. [18], Lee et al. [19] and Carvalho et al. [20] have used a higher number of accessions collected from wider geographic areas and included different Capsicum species like C. annuum L., C. frutescens L., C. chinense Jacq., C. eximium, C. praetermissum, C. baccatum L., C. pubescens, C. cardenasii, C. galapagoense and C. tovarii. Although the genus Capsicum has a broad genetic base among species [18], this of C. annuum is much narrower. Searching for C. annuum accessions with high levels of genetic and phenotypic diversity is a prerequisite for development of core collections with broad applicability. The studied collection consists of a large number of C. annuum landraces which constitute about 2/3 of the accessions and represent a valuable reservoir for improvement of the present pepper germplasm. Genetic diversity in the studied C. annuum collection To assess the genetic diversity and structure of the selected number of accessions, we used 21 SSR markers distributed on 8 pepper chromosomes. Most markers showed high PIC values with the highest one for CAMS864 and CAMS606 on chromosome 7 and CAMS234 on chromosome 6 which warrants their further use in pepper diversity studies in Bulgaria. The overall genetic diversity (He) of the studied C. annuum accessions is 0.531 with a mean number of alleles (MNA) of 6.952. These values are higher than the ones reported for 3 338 C. annuum accessions by Lee et al. [19] using SNP markers (He = 0.44), 222 accessions of C. annuum genotyped at 32950 SNP loci by Taranto et al. [54] (He = 0.048) and for 1 904 Capsicum spp. accessions by Gu et al. [17] using 29 SSR markers (He = 0.486), but lower than those identified by Nicolaï et al. [18] for 908 C. annuum accessions with 28 SSR markers (He = 0.59, MNA = 12.57) and by Zhang et al. [55] for 372 accessions of which 369 of C. annuum using 28 SSR markers (He = 0.63, MNA = 13.79). The mean Ho value (0.249) in our study is higher than the one observed by Nicolaï et al. [18], Lee et al. [19], Taranto et al. [54] and Gu et al. [17] (<0.085, 0.12, 0.023 and 0.119, respectively). The higher level of Ho observed in Bulgarian C. annuum collection is due to the higher natural outcrossing rate of pepper, especially in landraces which constitute about 2/3 of the studied C. annuum collection. The lower Ho values observed by other authors could be explained by maintaining and multiplication of accessions through selfing [18] or by long time inbreeding process including artificial selection, non-random mating between individuals, population structure and size as well as Wahlund effect (mixing of individuals from different genetic sources) [56,57]. Genetic structure of the studied C. annuum collection The model-based analysis of the genetic structure of the collection established that the most probable number of clusters given the marker data is three. The genotypes were combined in the resulting groups irrespective of the country of origin. Although the clusters were dominated by accessions of one or two fruit shape types, there were no clusters consisting entirely of a particular fruit type. Further analysis of the phenotypic diversity within and across clusters, using several quantitative traits, revealed that there are phenotypic features common for the genotypes that grouped together. The quantitative traits of the fruits were the most pronounced, as the patterns of the diversity were mainly imposed by the thickness of the pericarp and the width and weight of the fruits, which was very well demonstrated by the biplot chart (Supplemental Figure S1), where the genotypes from the different clusters prevailed in certain areas depending on the values of these traits. The number of detected clusters in our study is in good agreement with those reported by oh et al. [37] in a collection of 61 Bulgarian mostly pungent small fruited red pepper landraces and other authors studying the genetic diversity in collections of Capsicum annuum of larger sizes [55] and larger places of origin [18]. It should be noted that unlike us, oh et al. [37] found no relationship between the STRuCTuRE derived clusters and the morphological traits of the fruits. The lack of such a relationship is probably due to similarity in the fruit shape and size of most studied accessions. In regard to the relationship between the distribution of genotypes by clusters and the fruit morphology Zhang et al. [55] and Nicolaï et al. [18] observed very similar patterns of clustering. Several authors reported genetic structures in C. annuum collections, consisting of different numbers of underlying clusters ranging from 2 [25,58] to 6 or 8 [59]. However, González-Pérez et al. [25] found that the division into two clusters was mainly geographical and when the analysis was confined to the local Spanish accessions they also established the presence of three clusters in which the genotypes were distributed according to the fruit and plant traits. We also observed additional peaks of ΔK at K = 4 and 11. In our model-based analysis, when the K was set to K = 4, Cluster 3 was divided into two new sub-clusters of accessions with distinct fruit sizes. The division of these sub-clusters led to an increase in the values of pairwise metric (Jost' D) for population differentiation, defining sub-structure Cluster 3.1 as the most divergent group. The AMoVA also showed increased values of the between-cluster variation from 15.36% at K = 3 to 17.64% at K = 4. Although much lower than the between-cluster variation reported by Rivera et al. [59], it is close to the value between wild and domesticated populations in northwestern Mexico reported by oyama et al. [60]. These results show that the clusters could potentially be subdivided into more genetic sub-clusters as suggested by the presence of the additional peak of ΔK at K = 11. Construction of a Balkan mini core collection The Balkan Peninsula is an early centre of adaptation of the species from the so-called Mesoamerican food complex in Europe [61]. According to Andrews [61], these species found their way from the markets of the ottoman Turkish Empire through the Balkans to Central Europe. His hypothesis was supported by the results of Nicolaï et al. [18], defining the accessions from these parts of Europe as a 'distinct genetic pool' . To facilitate the conservation of the C. annuum genetic diversity represented in the current collection from the Balkans, we aimed at constructing a multipurpose mini core collection. of the two tested approaches, the one combining minimization of accession-to-nearest-entry distance and maximization of allele richness performed significantly better in encompassing the genetic diversity present in the whole collection in terms of percentage of retained alleles and the number of rare alleles captured in the mini core collection. Several C. annuum core collections have been constructed for different purposes. Nicolaï et al. [18] established a core collection of 332 entries that captured 97% of the genetic and phenotypic diversity for further association studies. Recently, Gu et al. [17] constructed a core collection based on SSR data consisting of 248 entries which covered 75.6% of the SSR alleles for further genotyping and gene-mining research. The final mini core collection of 44 entries proposed in the present study was sampled solely on the basis of the genetic data including allele diversity of 176 C. annuum accessions at 21 SSR loci and the genetic structure inferred from these data. Still, it captured nearly 95% of all alleles and 80% of the very rare alleles while maintaining good representativeness of the accessions, indicating that it could be very useful both in C. annuum improvement by breeding and in germplasm conservation. However, to construct and optimize most suitable mini core collections for specific purposes, the important phenotypic traits must also be considered. Therefore, further research, focused on optimization and testing of most suitable core collections for different purposes, based on different combinations of both genotypic and phenotypic data and comparing various sampling criteria is required to complete this task. Conclusions In the present study, a Balkan mini core collection of Capsicum annuum was constructed based on the allele information at 21 SSR loci and the genetic structure inferred from these data. The mini core collection is composed of 44 C. annuum accessions from several geographic locations on the Balkan Peninsula that retained a large proportion of the gene diversity present in the germplasm collection of 176 non-redundant accessions analysed in this study. The proposed mini core collection was also found to have good representativeness of the studied germplasm and therefore could be very useful in both C. annuum improvement by breeding and germplasm conservation. Further studies, including a larger sample of the more than 1500 Capsicum germplasm accessions currently maintained at the Maritsa Vegetable Crops Research Institute could allow selection of larger diverse core collections that will be suitable for gene mining and genome-wide association studies. Author contributions Elena G. Todorovska (EGT) conceptualized and designed the experiment, analysed data, prepared tables, prepared original draft and reviewed drafts, approved the final draft. Nikolai K. Christov (NKC) performed experiments, analysed data, prepared figures and tables, prepared and reviewed drafts of the paper, and approved the final draft. Stefan Tsonev (ST) performed experiments, analysed data, prepared figures and tables, prepared and reviewed drafts, and approved the final draft. Velichka Todorova (VT) maintained, evaluated phenotypically, described and contributed the material, reviewed drafts of the paper, and approved the final draft. Disclosure statement No potential conflict of interest was reported by the authors.	2021-08-27T17:07:28.844Z	2021-01-01T00:00:00.000	{ "year": 2021, "sha1": "5d455d1dbf83054dc11c2306bfd0cc5c2dbcfa18", "oa_license": "CCBY", "oa_url": "https://www.tandfonline.com/doi/pdf/10.1080/13102818.2021.1946428?needAccess=true", "oa_status": "GOLD", "pdf_src": "TaylorAndFrancis", "pdf_hash": "11269fa2377ddfa620457b3636f368508fad6306", "s2fieldsofstudy": [], "extfieldsofstudy": [ "Biology" ] }
8345810	pes2o/s2orc	v3-fos-license	DoGSD: the dog and wolf genome SNP database The rapid advancement of next-generation sequencing technology has generated a deluge of genomic data from domesticated dogs and their wild ancestor, grey wolves, which have simultaneously broadened our understanding of domestication and diseases that are shared by humans and dogs. To address the scarcity of single nucleotide polymorphism (SNP) data provided by authorized databases and to make SNP data more easily/friendly usable and available, we propose DoGSD (http://dogsd.big.ac.cn), the first canidae-specific database which focuses on whole genome SNP data from domesticated dogs and grey wolves. The DoGSD is a web-based, open-access resource comprising ∼19 million high-quality whole-genome SNPs. In addition to the dbSNP data set (build 139), DoGSD incorporates a comprehensive collection of SNPs from two newly sequenced samples (1 wolf and 1 dog) and collected SNPs from three latest dog/wolf genetic studies (7 wolves and 68 dogs), which were taken together for analysis with the population genetic statistics, Fst. In addition, DoGSD integrates some closely related information including SNP annotation, summary lists of SNPs located in genes, synonymous and non-synonymous SNPs, sampling location and breed information. All these features make DoGSD a useful resource for in-depth analysis in dog-/wolf-related studies. INTRODUCTION Dogs have been the dearest friends of humans as guardians, companions and working partners for thousands of years (1,2). Our natural curiosity, together with practical and aesthetical needs propelled the exploration of the genetic basis of the remarkable diversity of dog phenotypes (3)(4)(5). Furthermore, being one of the most thoroughly domesticated animals has made them a long-term focus and perfect model for domestication genetics studies (6)(7)(8)(9)(10). Researches into their parallel evolution with humans has facilitated our understanding of human evolution itself and, intriguingly, genes that cause diseases that are mutually shared, especially those related to cancer and neurological disorders (11,12). As a powerful genetic marker, single nucleotide polymorphism (SNP) plays a pivotal role in dog genetic researches referring to human selection (3,4,10), demographic studies (6), linkage and segregation analysis (5) and genome-wide association studies (13). To date, the latest and most widely used dog SNP data set is from dbSNP (build139:ftp://ftp.ncbi.nlm.nih.gov/snp/), where ∼80% of the ∼2.7M SNPs were called from two dogs, one Boxer (14) and one Standard Poodle (15). It provides the SNP list from all samples without individual lists, which limits its usability in population genetic analysis. The rapid development of next-generation sequencing (NGS) technology has facilitated the generation of massive dog/wolf genome data sets (7,16). However, SNP calling from the massive data generated by NGS is very laborious and requires a large amount of computational resources. Here, we develop DoGSD, the first web-based public database for accessing highly dense, broadly representa- tive dog/wolf whole-genome SNP data. We collected SNPs from two unpublished dog/wolf genomes, three recently published works (8,9,11) and the latest dog SNP data set (dbSNP139). A total of 34 our samples are Chinese indigenous dogs which represent a key phase in dog domestication and are not included in any existing SNP databases. DoGSD provides a powerful SNP retrieving interface for each individual samples and a non-redundant data set. We made annotation to integrate information, such as SNPrelated genes, transcripts, proteins and calculate allele frequencies. DoGSD has a functionality to search gene-related synonymous and non-synonymous SNPs. In addition, we incorporate the essential genetic statistics, viz. F-statistics (Fst) into DoGSD. Figure 1 shows the database construction pipeline. Details are described in Data sources, Data processing and Database implementation. Data sources Our data set integrates SNPs called from two newly sequenced wolf/dog samples and three published dog genetic works (8,9,11) with the newly released dog SNP data set (dbSNP139) ( Data processing Except for dbSNP139, we divided our samples into two groups. Group 1 is the two unpublished dog/wolf genomes (solidGW1 and CID1) generated by the SOLiD system and group 2 is from three recently published works (8,9,11), which were sequenced with the Illumina Solexa technology. All of the individual genomes were sequenced to an average of 15× coverage. For the two samples sequenced by the SOLiD system, two DNA mate-paired libraries were prepared for solidGW1 with 5 and 1.5 kb insert size, which generated 72.93G bp of raw data. A single mate-paired library with 500 bp insert size was prepared for CID1, producing 45.96G bp raw data. Using the Bioscope pipeline, 42.01 Gb of solidGW1 was aligned to the Boxer genome assembly (canFam2) with 17.9× coverage, while 32.99 Gb of the CID1 was aligned to the canFam2 with 14× coverage. We used two sets of methods and the camFam2/canFam3 reference genomes download from UCSC (http: //hgdownload.soe.ucsc.edu/goldenPath/canFam2 or 3/ bigZips/canFam2 or 3.fa.gz) to identify SNPs from these two groups, and integrated the SNPs from all 77 samples and the dbSNP139 data set to obtain a non-redundant SNP set. For group 1, SNPs were detected with the BioScope diBayes tool where valid adjacent two-base mismatches occurred between a read and the reference (canFam2). Depending on whether there was high or low read coverage on the reference, either a Frequentist or Bayesian algorithm was applied, respectively. The Frequentist algorithm is based on the null hypothesis that a given position is homozygous and any other valid adjacent mismatches are errors subject to a Poisson distribution. The Bayesian algorithm calculates the posterior probability of each site according to the expected polymorphism rate in the genome, GC content, coverage, position in a read and the quality value of the colour call and prior errors derived from the 6mer probe annealing error. SNP calling stringency was set as medium. Finally, we converted genome positions of all resulting SNPs from Canfam2.0 to Canfam3.0. For group 2, we used Burrows-Wheeler Aligner (version: 0.6.2-r126) (17,18) to map Illumina short reads to the dog reference sequence (Canfam3) in the first step. Picard (version: 1.87) (downloaded from http://picard.sourceforge. net) was then used to eliminate duplicated reads generated in the library polymerase chain reaction construction. After that, we used the tools in GATK (version: 2.5-2-gf57256b) (19,20) to realign reads around known indels (downloaded from Ensembl ftp://ftp.ensembl.org/ pub/release-73/variation/vcf/canis familiaris/), and recalibrate base quality score to obtain more accurate quality score for each base. The refined data from all individuals were jointly used to call a raw SNPs set by GATK Uni-fiedGenotyper, and finally, we identified a high quality set of SNPs, using the variant quality score to recalibrate procedure in GATK. For both groups and the dbSNP139 data set, we filtered SNPs with two-thirds derived alleles. SNPs were then annotated to genes, transcripts and proteins downloaded from the Ensembl FTP site (ftp://ftp.ensembl.org/pub/release-75/ fasta/canis familiaris/). Allele frequency was calculated for wolf/dog populations for each SNP from 77 sequenced samples. Also the Fst was calculated using vcftools (21) for the batch two data. Database implementation Several tables containing all resulting high-quality SNPs and their annotations were processed with Perl scripts and put into MySQL database. We use JSP/JAVA to implement data visualization, searching and downloading. GBrowse was also integrated for chromosome-based data visualization. Data characteristics In total, we obtained 19,333,098 non-redundant SNPs, approximately seven times greater than in dbSNP139. The average length of SNP intervals of dbSNP139 is 862.0 bp, with a median value of 241.0 bp, while the same statistics for DoGSD are 120.4 bp and 68.0 bp, respectively, showing that the SNP distribution of our database is denser and more uniform than that of dbSNP139. Ancestral LD/crossbreed LD blocks of the dog have an average size of 10 Kb (14). Thus, we compared the amount of intervals ≥10 Kb with no SNP of both data sets. Our results show that there were 333.4 Mb (covering 13.8% of the genome) without SNPs in dbSNP139 while only 7.4 Mb (0.3%) in DoGSD, clearly demonstrating that our database has fewer blocks without SNPs than dbSNP139 data set. This pattern is still true when calculating SNP intervals ≥10 Kb with only one (191.1 Mb/7.93% versus 1 Mb/0.04%) or two (148.6 Mb/6.17% versus 1.1 Mb/0.05%) SNPs. These superior characteristics are primarily due to the wider geographical sampling locations and the unbiased resequencing strategy. Together, higher density, more uniform distribution and fewer SNP rare blocks make our database more effective and accurate in specifying genome locations closely related to certain phenotypes and sequence sites under selection (22). In the 77 samples collected in DoGSD, 34 are Chinese indigenous dogs, which have never been provided by any other databases. The Chinese indigenous dog has been demonstrated as the first stage in dog domestication (11) and its SNP data set should undoubtedly help researchers to profoundly understand the evolutionary gap between wolf and breed dog. In addition to SNPs and their annotations, we also provide Fst for almost all SNP, which is an important and widely used population genetic statistic for selection detection. Users may browse and locate SNPs with potential selection signals. The Weir and Cocker-ham method (23) was used to calculate Fst between the wolf and dog populations using the inferred genotypes. Fst values provided in DoGSD are based on all 75 samples from the group 2 data set. USAGE AND ACCESS The DoGSD database can be accessed through a simple user interface. Online documentation is provided to help users to access the database. DoGSD has been designed with two main functionalities for data retrieval, Browse and Search. Users may browse the no-redundant and individual sample SNPs either with text format in tables (Figure 2A), or with a chromosome-based graphical GBrowse (24) interface. In the text format tables, SNP ID, sample and sequencing platform information, chromosome location, reference and derived alleles, three-fifths flank sequences are given for each SNP and annotated gene, transcript and protein, derived residual and counterparts from other samples are given if available ( Figure 2B). In GBrowse interface, users can obtain Fst values, a pie chart showing the allele frequency of the dog and wolf populations, SNP density in 300 kb windows size, related gene and transcript information ( Figure 2C). Users may type in chromosome numbers, start and end positions as input to retrieve data for either a single SNP or a group of SNPs. Comparative search of SNPs between two or more individuals were also implemented ( Figure 3A). Figure 3B shows a typical comparative search result. Users can compare the SNPs of individuals interested them within a chromosome range. Considering that SNPs locate in genes and coding sequences (CDSs), and especially since non-synonymous SNPs are often of greater interests for further study, we provide several functions within the Browse pull-down menu: SNP Hit On Gene, SNP Coding, SNP Coding Synony-mous and SNP Coding NonSynonymous. Users may obtain useful information by summaries of SNPs hit on genes or CDSs and examine whether they are synonymous or nonsynonymous. All the SNP data can be downloaded freely as tabdelimited files and bam/fastq/sra format files of the 75 cited samples are provided. DISCUSSION Nowadays, researches towards dog evolution, traits and human co-evolution are still hotspots in biological field. As the first database concentrated on dog/wolf whole genome SNPs, DoGSD stores huge amount of uniformly distributed high-quality SNPs, which compensates for the scarcity of wolf/dog SNPs provided by other databases. With high re-sequencing depth, sampling coverage and calling accuracy from 77 individual samples, our nonredundant SNP data set can be used as a dog SNP reference. Also, users can download individual sample SNP list from our database for population genetic analyses. Especially, SNPs indentified in the Chinese indigenous dog are valuable to researches about early phase of dog evolution. Fst value will also shed light on the selection signals in dog/wolf genomes, guiding researchers to in-depth investigation of these signals on a larger population scale and thoroughly explain the evolutionary driving forces between dog and wolf. We will keep up with whole-genome SNP data releases and update DoGSD in a timely manner with new released data from population studies of dogs and wolves either by our own research groups or from publicly available resources. We would add uploading functionality to DoGSD by which users could submit whole-genome sequence data or SNP list directly. Once we get the whole-genome sequence, we would identify SNPs in it by group 2 pipeline described in Data processing. Interfaces for calculating Fst and θ w will also be developed. We will add additional genetic statistical parameters, such as Tajima D, EHH and recombination maps to the database. Since detection of large chromosomal events promotes our understanding of complex traits and evolution from a perspective that complements SNP, we will further integrate structural variations of dog/wolf genomes into our database in the future.	2016-05-04T20:20:58.661Z	2014-11-17T00:00:00.000	{ "year": 2014, "sha1": "459e0cb3c9e694451745ba939830bd6ff9fa5694", "oa_license": "CCBYNC", "oa_url": "https://academic.oup.com/nar/article-pdf/43/D1/D777/7327883/gku1174.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "792b75e6c27035886cd7eaba861a3b84e8394400", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Computer Science", "Biology", "Medicine" ] }
117713711	pes2o/s2orc	v3-fos-license	Asymmetry of Superconductivity in Hole- and Electron-doped Cuprates: Explanation within Two-Particle Self-Consistent Analysis for the Three-Band Model In the hole-doped cuprate superconductors, the superconducting transition temperature $T_c$ exhibits a dome-like feature against the doping rate. By contrast, recent experiments reveal that $T_c$ in the electron-doped systems monotonically increases as the doping is reduced, at least up to a very small doping rate. Here we show that this asymmetry is reproduced by performing a two-particle self-consistent analysis for the three-band model of the CuO$_2$ plane. This is explained as a combined effect of the intrinsic electron-hole asymmetry in systems comprising Cu3$d$ and O2$p$ orbitals and the band-filling-dependent vertex correction. There have been some theoretical studies of the doping dependence of T c . The fluctuation exchange (FLEX) approximation 5 for the single band Hubbard model gives a monotonic doping dependence of T c 6 , and therefore has difficulties in understanding the doping dependence of T c in the hole-doped cuprates. Some studies considered superconducting fluctuation in FLEX to circumvent this problem 7,8 . There have also been some studies that adopt methods capable of dealing with the strong correlation effects [9][10][11][12][13][14][15] . In some of those studies, T c exhibits a dome-like doping dependence, but in those cases there would be difficulties in understanding the recent experimental results for the electron-doped case. The electronhole asymmetry of T c was studied in a two band model that explicitly considers the O2p orbital, but there, the antiferromagetic phase was obtained in a wide electron doping range 16 , in contradiction to the experiments mentioned above [1][2][3][4] . It has been suggested that the difference in the charac-ter of the mother compound (Mott insulator or not) between the hole-doped and the electron-doped systems can be attributed to the difference in the electronic structure originating from the crystal structure 4,12,17,18 . Namely, while the crystal structure of the single-layer hole-doped cuprates is composed of Cu-O octahedra (T-type), that of the electron-doped cuprates is composed of Cu-O squares (T -type) and (ideally) has no apical oxygens. Due to this difference, the copper 3d -oxygen 2p level offset in the T -type structure tends to be smaller than that in the T-structure. Since the d-p level offset is small in the electron-doped system,the on-site effective U , when mapped to the single-band Hubbard model, is also small. One might expect that this difference in the crystal structure, and hence the difference in the effective on-site U , can provide an explanation for the electron-hole doping asymmetry of T c . However, the inner layers of multilayered hole-doped cuprates also do not have apical oxygens and therefore have the same lattice structure as that of the electron doped cuprates. Still, it is known that T c exhibits a dome-like doping dependence even within the inner layers 19 . Therefore, it seems difficult to attribute the electron-hole asymmetry of the doping dependence of T c to the absence/presence of the apical oxygens. The aim of the present study is to understand the origin of this electron-hole asymmetry. Here, we stress that in the present study we focus only on the (non-)monotonicity of the doping dependence of T c , and leave the issue of the metallicity or Mottness of the mother compound to future studies. We start by demonstrating that this electron-hole doping asymmetry of T c is difficult to understand within the single band Hubbard model even when realistic band structures are considered. We perform first principles band calculation of HgBa 2 CuO 4 (a hole-doped system) and Nd 2 CuO 4 (an electron-doped system), and obtain tight-binding models constructing maximally-localized Wannier basis [20][21][22][23][24] . Instead of the typical T-type hole doped system La 2 CuO 4 , we adopt HgBa 2 CuO 4 because (i) it is known that the hybridization of the d z 2 orbital cannot be neglected in La 2 CuO lar, so that we can concentrate purely on the electronhole asymmetry. To take into account the electron correlation effect beyond those taken into account in the LDA/GGA level, the on-site interaction has to be treated by some many-body technique as has been done in previous studies [25][26][27] . In the present study, we adopt the two-particle self-consistent method (TPSC) proposed by Vilk and Tremblay 28 . In this method, the interaction vertices in the charge and spin channel are approximated as different constants, and these constants are determined so that the correlation functions satisfy their sum rules that originates from the Pauli's principle. It has been shown in ref. 14 that TPSC gives a dome-like doping dependence of T c for the single band Hubbard model with nearest neighbor hopping only. The obtained hopping integrals for the single-band models are given in Table I, and the corresponding band structures are shown in Fig.1(upper panels). The eigenvalue λ of the linearized Eliashberg equation for d-wave pairing, which is a measure of T c (see below), is shown against the band filling in Fig.2. For both the models of HgBa 2 CuO 4 and Nd 2 CuO 4 , we set the on-site repulsion as U/t = 8 and the temperature T /t = 0.08, and we take 128×128 meshes and 4096 Matsubara frequencies. As shown in Fig.2, λ varies monotonically in both the hole-and the electron-doped cases, namely, the domelike T c variance against doping obtained for the nearestneighbor-hopping-only case (inset of Fig.2 shows the doping dependence of λ for the t 1 -only model) is lost when a realistic band structure is adopted. Considering the previous studies mentioned in the introduction, it may be questionable whether we can reproduce the experimentally observed electron-hole asymmetry within the single band model even if we take into account the electron correlation effects beyond TPSC. Namely, the doping dependence of T c would be either dome-like shaped or monotonic on both electron and hole-doped cases when the same values of U are taken. Hence, we now proceed to the three-band model that explicitly considers the in-plane oxygen 2p x,y orbitals in addition to the copper 3d x 2 −y 2 orbital 29 . We first constructed the five-band model composed of the copper 3d x 2 −y 2 orbital and four in-plane oxygen 2p x,y orbitals by using the maximally-localized Wannier basis. Subsequently, we obtained the three-band model by removing two p π orbitals which are oriented in the direction perpendicular to the Cu-O bond. The obtained model parameters and the band structure are shown in Table II and Fig.1, respectively. For comparison, the model parameters for La 2 CuO 4 are also shown. The parameter values of Hg and Nd systems can be considered as quite similar, and especially the similarity of ∆ dp can be noticed if we compare the values to that of the La system, which has smaller apical oxygen height compared to the Hg system. The similarity of ∆ dp between the electron-doped and the hole-doped materials is expected to become even more prominent if we consider multilayer hole-doped cuprates, where one or both of the apical oxygens are missing depending on the layer. This means similar values of the on-site U when mapped to single band Hubbard models, as mentioned in the introductory part. To analyze the three-band model, the TPSC approach should be generalized for multi-band systems. We follow the generalization of TPSC presented in Refs. 30,31 . Let us briefly review TPSC for the multi-band Hubbard model. Hereafter we make use of the matrix form in the same way as Refs. 32,33 . The Hamiltonian of the three-band model is given as where c † µσ (r) is a creation operator of an electron with spin σ and orbital µ = d, p x , p y at site r, n µσ (r) = c † µσ (r)c µσ (r) is a number operator, ∆ dp is the d-p level difference, U µ is the on-site Coulomb interaction. The band filling n is defined as the average number of electrons per unit cell, so that n = 5 corresponds to the non-doped case. In the analysis for this model, we set the on-site interaction U d = 10eV and U p = 5eV, temperature T = 0.01eV. We employ 64×64 k-point meshes and 4096 Matsubara frequencies. In this three-band model, similar to the single-orbital case, the spin and charge susceptibilities are evaluated as where χ 0 (k) is the irreducible susceptibility and U sp(ch) is the effective interaction matrix for the spin (charge) channel. The irreducible susceptibility is given by using the bare Green's function G 0 µν (k) = [(i n + µ − H(k)) −1 ] µν , where µ is the chemical potential and H(k) is the matrix elements of the hopping term of the Hamiltonian in the momentum representation. Here we abbreviate the wave numbers and the Matsubara frequencies as k (for the fermionic case) or q (bosonic). Since we consider only U µ as the interaction, introducing the ansatz, susceptibilities can be determined from the following sum rules derived from the Pauli principle: where n µ is the particle number per site of orbital µ, obtained from − T N k G 0 µµ (k) = n µ . However the ansatz introduced here violates the electron-hole symmetry. Therefore if n µ > 1, considering the electron-hole transformation, the ansatz should be modified as where n h µσ = 1 − n µσ . Since n µ > 1 is satisfied for any band filling used in this study, we make use of the transformed ansatz. Using the obtained susceptibilities as described above, the dressed Green's function G(k) is determined by Dyson equation: and the self-energy Σ(k) is given by the eigenvalue λ and the anomalous self-energy ∆(k) are obtained. Here the singlet pairing interaction Γ s (q) is given by where U is the interaction matrix for the bare vertex. The superconducting transition temperature T c is the temperature where λ reaches unity. In the present study, we calculate λ at a fixed temperature and use it as a measure for T c . Let us move on to the calculation results of the effective three-band model for HgBa 2 CuO 4 and Nd 2 CuO 4 . The spin susceptibility µ χ sp µµµµ (k, ω = 0) and the absolute value of the dressed Green's function \|G dd (k, i n=0 )\| are shown in Fig.3 and Fig.4, respectively. As the number of electrons decrease from the electron-doped region (n > 5) to the hole-doped region (n < 5), peaks of the spin susceptibility around (π, π) and the Γ point are enhanced and the absolute value of the dressed Green's function is suppressed. It can be seen in Fig.3(a)(b) that the Green's function is particularly suppressed around (π, 0) (0, π), namely the hot spots (see the figures in the supplementary material 34 , in which the hot spots are more clearly visible). These behaviors can be explained as a combined effect of the following two factors. First, since the d x 2 −y 2 orbital is not half-filled due to the d-p hybridization, the d x 2 −y 2 orbital approaches the half-filling by decreasing the number of electrons. Because of this, to satisfy the sum rule for the spin susceptibility χ sp dddd (q) within the d orbital, the vertex U sp dddd necessarily increases. Therefore the spin susceptibility increases with decreasing the number of electrons. Secondly, since the Fermi level approaches the van Hove singularity point of the band structure as the number of electrons is reduced, the spin susceptibility around the Γ point is enhanced. The enhancement of the spin fluctuation results in the increase of the self energy, which in turn suppresses the Green's function. We show the band filling dependence of the d-wave pairing eigenvalue λ of the linearized Eliashberg equation (measure of T c ) in Fig.5. This result is consistent with the doping dependence of T c in both the electronand the hole-doped region (except near the non-doped regime, which we will discuss later). As shown in the inset of Fig.5, this feature remains at the temperature where the d-wave eigenvalue λ is above unity near the optimal doping rate (T = 0.003eV, 80×80 k-point meshes, and 8192 Matsubara frequencies). This result can be interpreted as follows. Monotonic increase of T c in the electron-doped region as the number of electrons is reduced arises from the enhancement of χ sp dddd (q) around (π, π), which works in favor of the d-wave pair scattering. As the band filling enters the hole-doped region, χ sp dddd (q) is further enhanced around (π, π), so that λ also increases. However, both the suppression of the Green's function and the enhancement of χ sp dddd (q) around the Γ point work against d-wave superconductivity, and therefore λ turns to decrease with further hole doping beyond δ h = 0.15, where δ h = 5−n is the hole doping rate. Thus, the doping dependence of the superconducting transition temperature is naturally understood in both the electronand the hole-doped cases. Since the Mott transition is not described within the formalism used in the present study, in Fig.5 we show the result near n = 5 by dashed lines (the calculations have been done also at n = 5 nonetheless). The absence of the insulating state in the non-doped case is attributed to the insufficiency of the evaluation of the local electron correlation effects. The inclusion of further electron correlation effects is left for future study. Nonetheless, we can expect that the inclusion of such effects will probably make the dome-like feature in the hole-doped region more prominent, while it should somewhat reduce the enhancement of λ in the underdoped regime of the electron-doped case, which seems rather strong in the present calculation compared to experimental observations 2,3 . Hence, the inclusion of further local correction is likely to make the doping dependence of T c even closer to those observed experimentally. Another related issue is the pseudogap problem in the underdoped regime. This has been addressed by TPSC in ref. 28 for the single band model, but the situation can be different in the case of the three-band model with realistic band structure. This also serves as an interesting future problem. To summarize, we have studied the doping depen-dence of superconductivity for the three band model of Nd 2 CuO 4 and HgBa 2 CuO 4 using the TPSC method. The eigenvalue of the Eliashberg equation λ exhibits an optimal doping around n = 4.85 (hole concentration δ h = 0.15) in the hole-doped region and varies monotonically in the electron-doped region, consistent with the experiment. It is found to be understood naturally in terms of the electron-hole asymmetry due to the d-p hybridization and the band-filling-dependent vertex correction. Part of the numerical calculations were performed at the facilities of the Supercomputer Center, Institute for Solid State Physics, University of Tokyo.This study has been supported by Grants-in-Aid for Scientific Research No.26247057 from the Japan Society for the Promotion of Science.	2015-10-30T06:26:41.000Z	2015-05-15T00:00:00.000	{ "year": 2015, "sha1": "89cc99642658dc89b0dfe5c6e8111f8e836c763d", "oa_license": null, "oa_url": "http://arxiv.org/pdf/1505.04017", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "89cc99642658dc89b0dfe5c6e8111f8e836c763d", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
259244739	pes2o/s2orc	v3-fos-license	Altering the spectroscopy, electronic structure, and bonding of organometallic curium(III) upon coordination of 4,4′−bipyridine Structural and electronic characterization of (Cp′3Cm)2(μ−4,4′−bpy) (Cp′ = trimethylsilylcyclopentadienyl, 4,4′−bpy = 4,4′−bipyridine) is reported and provides a rare example of curium−carbon bonding. Cp′3Cm displays unexpectedly low energy emission that is quenched upon coordination by 4,4′−bipyridine. Electronic structure calculations on Cp′3Cm and (Cp′3Cm)2(μ−4,4′−bpy) rule out significant differences in the emissive state, rendering 4,4′−bipyridine as the primary quenching agent. Comparisons of (Cp′3Cm)2(μ−4,4′−bpy) with its samarium and gadolinium analogues reveal atypical bonding patterns and electronic features that offer insights into bonding between carbon with f-block metal ions. Here we show the structural characterization of a curium−carbon bond, in addition to the unique electronic properties never before observed in a curium compound. Discovered by Seaborg, James, and Ghiorso in 1944 via α-particle bombardment of 239 Pu, curium (Z = 96) is one of the heaviest elements available in quantities suitable for traditional synthetic chemistry 1 . It is most stable in the +3 oxidation state, and possesses a [Rn]5f 7 electron configuration 1 . Its half-filled shell creates increased stability with respect to other 5f n configurations, and is often associated with an expected decrease in f-electron contributions to bonding, as well as high resistance to changes in oxidation state [2][3][4] . Additionally, similarities in ionic radii and trivalent stability lead to challenges in separating curium from americium and the lanthanides. This separation is an essential component of recycling used nuclear fuel owing to curium's significant contribution to the radiotoxicity of nuclear waste [5][6][7][8][9][10] . Despite the distinct electronic properties of the wide variety Cm 3+ compounds that have been prepared to date, no single-crystal structural characterization of a complex containing a Cm−C bond has been reported 6,[11][12][13][14][15][16][17][18][19][20] . Examination of this interaction could provide insights into methods for engaging the frontier orbitals of curium and other actinides in forming partially covalent bonds with selected ligands. This could allows us to gain some control over the electronic structure of these complex elements [21][22][23][24][25][26][27][28][29][30] . For example, it has been shown that the engagement of 5f orbitals in Cm-ligand bonds can be increased by using soft-donor ligands that can be further enhanced by applying mechanical pressure 3 . Quantum mechanical evaluation of a recently reported Am(III) cyclopentadienyl (Cp) complex showed that a variety of metal frontier orbitals were mixing with Cp' ligand orbitals to create partially covalent bonds, but also revealed a surprising degree of ionicity in the Am−N interactions with 4,4'-bipyridine in the same complex 31,32 . However, complexes of this type remain rare largely due to low available quantities of these isotopes (reactions are completed with <5 mg of metal content), the need for specialized research faciltities, and their exceptional air and moisture sensitivity 24,[30][31][32][33] . Owing to these difficulties, syntheses allowing the structural characterization of An−C (An = Pu, Am, Cf ) have only recently become accessible and can be applied to curium 24,[30][31][32][33][34] . These synthetic challenges also necessitate the use of lanthanide analogs that possess similar ionic radii and/or electron configurations for optimizing the chemistry and for providing benchmarks for comparisons with the 5 f series. Traditionally, lanthanide and early actinide organometallic chemistry has lead to the characterization of a suite of new oxidation states, electron configurations, and splitting of f-f transitions spectroscopically, greatly contributing to our fundamental understanding of these elements [24][25][26][27]29 . Utilizing these systems for the mid-actinides has historically provided an excellent basis for comparison to lanthanides and early-actinide counterparts on a fundamental level, leading to future studies in new oxidation states, covalency, and electronic properties of curium 24,[30][31][32] . Results Addition of 4,4′−bpy to two equivalents of a putative Cp′ 3 M (M = Sm, Gd, Cm) in toluene yields (Cp′ 3 M) 2 (μ−4,4′−bpy) (Fig. 2). All three molecules crystallized in the P 1 space group and were isomorphous, demonstrating a pseudo-tetrahedral geometry from the 4,4′−bpy and centroids of three Cp′ rings coordinated to a metal center. In each case, two metal centers are bridged by 4,4′−bpy, creating a dinuclear barbell shape with an inversion center in the center of the 4,4′−bpy. Additional synthetic and crystallographic details can be found in the Supplementary Information. Cif files for 1−Sm, 1−Gd, and 1−Cm crystal structures are provided as Supplementary Data 1, Supplementary Data 2, and Supplementary Data 3, respectively. 1−Sm, 1−Gd, and 1−Cm possess M−N distances of 2.626(3) Å, 2.592(3) Å, and 2.5962(16) Å, respectively. Expectedly, 1−Gd possesses a slightly shorter distance than 1−Sm owing to the lanthanide contraction. However, the M−N distance observed in 1−Cm is within error of the 4 f congener, 1−Gd, but is substantially shorter than its ionic radii-based analog, 1−Sm 35 . The M−N distance presented in 1−Sm is slightly shorter than that of a similar molecule, Cp 3 Sm(py) (py = pyridine), 2.656(3) Å 36 . A longer distance would be anticipated in 1−Sm based on the provided steric bulk from the trimethylsilyl groups of Cp′ competing with 4,4′−bpy. However, 1−Gd exhibits a notable longer Gd−N distance in comparison to that reported in Cp 3 Gd(NH 3 ), 2.501(6) Å, due to the significantly smaller size of the coordinated NH 3 37 . As there has been no single-crystal structural characterization of organometallic curium to date, comparison to similar coordination environments proves challenging. The Cm−N distance of 1−Cm is greater than those reported in Cm(HDPA) 3 ·H 2 O (H 2 DPA = 2,6−pyridinedicarboxylic acid) but quite similar toCm(S 2 CNEt 2 ) 3 (N 2 C 12 H 8 ) 10,38 . The average Cm−N distance of Cm(HDPA) 3 is 2.541(4) Å and 2.550(4) Å for the Λ and Δ enantiomers, respectively, and the reported Cm−N distance Cm(S 2 CNEt 2 ) 3 (N 2 C 12 H 8 ) is 2.601(6) Å 10,38 . Curium sets a trend for these bridged organometallic actinide systems in the trivalent state. Previously reported values of isostructural systems containing uranium 39 experiences the same phenomenon, exhibiting M−C distances between 2.728(2) Å and 2.9029(2) Å within the same Cp′ − ring. This substantial range indicates that coordination of 4,4′−bpy not only directly impacts M−C distances, but also the angle at which Cp′ coordinates. The average M−C distance in 1−Cm is shorter than those reported in (Cp′ 3 U) 2 (μ−4,4′−bpy) 39 and (Cp′ 3 Am) 2 (μ−4,4′−bpy) 31 , resulting from a decrease in ionic radii across the actinide series and is consistent with the observed M−N bond distance trend 35 . Coordination of Cp′ to curium leads to unique emission properties not before reported in a curium complex. Photoluminescence of putative Cp′ 3 Cm microcrystals was collected at room temperature upon irradiaton with an excitation wavelength of 420 nm (Fig. 3). A bathochromic shift and considerable splitting were observed, yielding a band centered at~670 nm (ca. 14,925 cm −1 ) with a full-width halfmaximum value of about 52 nm (ca. 1225 cm −1 ), noticeably larger than most previously reported 3,11,38,41,42 . While Cm 3+ typically phosphoresces red-orange from 590-620 nm (ca. 19,949-16,129 cm −1 ) 3,11,38,41,42 , red luminescence has been reported previously in CmCp 3 , albiet not redshifted to this degree 43,44 . After 24 hours of air exposure, no photoluminescence was observed. Surprisingly, 1−Cm exhibits no photoluminescence from known excitation wavelengths of 365 nm and 420 nm used for Cm 3+ , suggesting the change in coordination environment by the coordination of 4,4′−bpy to Cp′ 3 Cm quenches emission. We propose a possible explanation that a non-radiative deactivation mechanism attributable to the resonance between C−H vibrational modes of the 4,4′-bpy with the electronic emissive state of the curium ion (vide infra) may be responsible for this quenching. Solid-state absorption spectroscopy of 1−Sm, 1−Gd, and 1−Cm were measured from 350-1700 nm (ca. 28,571-5882 cm −1 ) (Fig. 4). Spectra of each were collected at room temperature; additionally, 1−Cm was measured at −180°C. Assignment of the Laporte forbidden f-f transitions of these molecules has been completed in terms of total angular momentum, J. Since actinides exhibit a complex interplay between electron repulsion, relativistic, and ligand-field effects, the analysis was performed under the intermediate coupling scheme 45,46 . A charge transfer (CT) band in 1−Sm is observed beginning at 575 nm (ca. 17,391 cm −1 ), masking the high energy fingerprint f-f transitions indicative of Sm 3+ ; however, lower energy f-f transitions in the range of 900-1700 nm (ca. 11,111-5882 cm −1 ) are still seen 47,48 . Similar to other organometallic f-block molecules 24,30,32 , notable splitting of these transitions is detected due to the unique coordination environment resulting from the coordination of Cp′. The CT band of 1−Gd, beginning at 600 nm (ca. 16,667 cm −1 ), masks the f-f transitions indicative of Gd 3+ that are characteristically detected between 270-320 nm (ca. 37,037-31,250 cm −1 ) (Fig. 4) 49 . Following the trend of 1−Sm and 1−Gd, the absorption spectrum of 1−Cm contains a CT band beginning at 615 nm (ca. 16,260 cm −1 ) that masks high energy fingerprint transitions ( Fig. 4) 10,12,18,20,44 . Transitions at 587 nm and 597 nm (ca. 17,036 cm −1 and 16,750 cm −1 ) ( J = 7/2 and 5/2, respectively) typically detected at lower energy in Cm 3+ spectra and two transitions from 630-650 nm (ca. 15,873-15,385 cm −1 ) ( J = 7/2) are observed, further displaying the unique Spin-orbit CASSCF along with MC-pDFT methods (hereinafter referred to as SO-pDFT) were employed to calculate the electronic states of 1−Sm, 1−Gd, 1−Cm, and Cp′ 3 Cm. Since the size of the systems represents a limitation from the theoretical perspective, a model consisting of one Cp′ 3 M unit (M = Sm, Gd, Cm) coordinated to pyridine was used instead. The assignment of the different electronic states was done by indicating the total angular momentum quantum number J along with the predominant 2S+1 L term in parenthesis. The assignment of the spectroscopic transitions was performed by considering vertical excitations from the ground state (GS) to different excited states (ES). In 1−Sm, the spin-orbit ground state corresponds to a J = 5/2 ( 6 H). The region between~1200 cm −1 (~8333 nm) to~11,150 cm −1 (~897 nm) exhibit a continuum of excited states belonging to J = 7/2−15/2 ( 6 H) and J = 1/2−11/2 ( 6 F) manifolds. If the barycenter of the manifolds is considered, the energy gap between them is no greater than~1500 cm −1 . Moving to higher energies, the first excited state being predominantly quartet appears at~18,270 cm −1 (~547 nm) and corresponds to a J = 5/2 ( 4 G). The assignment of the experimental absorption features and the detail about the calculated SOstates is shown in Table S7, S8. As observed, theoretical predictions agree with the experimental data, particularly in the range of 11,111 cm −1 (900 nm) to 5882 cm −1 (1700 nm), where most of the spectroscopic features were observed. The errors associated with the calculation are in the range of 105 and 760 cm −1 . This has also been observed in other Sm(III) complexes, where the 4f−4f transitions were observed in the same energy region with identical assignments 48,[50][51][52] . In 1−Gd, the scenario is simpler as it corresponds to a half-filled fshell. Given its 4f 7 configuration, the main transitions are expected to be in the high-energy region involving the J = 7/2-3/2 ( 6 P) and J = 9/2−1/2 ( 6 D) manifolds. In this case, the SO-GS corresponds to a J = 7/2 ( 8 S) with a splitting of~15 cm −1 . The first excited state appears at~29,344 cm −1 (341 nm) and was ascribed to the J = 7/2 ( 6 D) manifold. The energetic stabilization of the first excited multiplet has been previously reported for other f 7 systems at different levels of theory 3,53,54 . A detailed assignment of each transition is shown in Table S9, S10. Since the CT band in 1−Gd masks the fingerprint f-f transitions associated with the Gd 3+ ion, no experimental counterpart exists to assess the accuracy of the predicted transitions. Reports on other Gd(III) compounds have found the position of the first excited state to be between 32,467 cm −1 (308 nm) and 31,645 cm −1 (316 nm) [55][56][57][58] . The calculated value for 1−Gd exhibits a bathochromic shift of~2300 cm −1 (25 nm) with respect to the range of energy found in previous reports. Since Cm 3+ is the isoelectronic analog of Gd 3+ , similar electronic states at different energy ranges are expected. As observed in Table S11, S12, the SO-GS of 1−Cm corresponds to a J = 7/2 ( 8 S) with a splitting of 385 cm −1 . The first two excited manifolds J = 7/2 ( 6 D) and J = 5/2 ( 6 D), usually considered the emissive states, start to appear around 15,216 cm −1 (657 nm) and 16,650 cm −1 (601 nm), respectively. Therefore, the experimental absorption peaks observed between 15,873 cm −1 (630 nm) and 15,384 cm −1 (650 nm) can be ascribed to transitions towards these couple of excited manifolds. Regarding the Cp′ 3 Cm system, the assignment and position of the electronic states is almost identical to the ones found in 1−Cm. The SO-GS corresponds to a J = 7/2 ( 8 S) exhibiting a splitting of~351 cm −1 , whereas the two first excited manifolds are located at~15,121 cm −1 (661 nm) and 16,365 cm −1 (611 nm). As shown in the experimental section (Fig. 3), the photoluminescence spectrum of this complex shows a band between~16,260 cm −1 (615 nm) and~14,285 cm −1 (700 nm) with three prominent peaks observed at 15,504 cm −1 (645 nm), 15,152 cm −1 (660 nm) and 14,925 cm −1 (670 nm). This band can be assigned to transitions from the J = 7/2 ( 6 D) manifold to the SO-GS, where the three observed peaks can be attributed to the splitting of this multiplet (Supplementary Table 12). In general, curium emits in the range of 16,949 cm −1 (590 nm) to 16,129 cm −1 (620 nm) 3,10,11,38,41,42 . In this case, a non-negligible bathochromic shift that was accurately reproduced by the calculations, is observed. This phenomenon has usually been attributed to the nephelauxetic effect 59 where the ligand-field of Cp′ causes a unique reduction in the interelectronic repulsion between the f-electrons of curium(III). According to SO-pDFT calculations, 1−Cm and Cp′ 3 Cm systems are similar in terms of assignment and positions of the SO states. Nonetheless, while 1−Cm photoluminescence is quenched, Cp′ 3 Cm display a prominent emission band in the vis-NIR region. It is known that C−H vibrations can lead to non-radiative routes when the low quanta superior harmonics and vibrational modes of a coordinated ligand strongly resonate with the emissive state of the metal center [60][61][62] . For instance, it has been reported that the fundamental vibrational modes of the C−H stretching in 4,4′−bipyridine are found at~3000 cm −163,64 . Since C−H vibrational modes have proven to have a cumulative quenching effect with respect to the number of C−H bonds 60 , we propose that a resonance between the electronic emissive state of curium(III) and the fifth vibrational harmonic of 4,4′−bipyridine to be a possible explanation for the absence of emission in 1−Cm. Given the differences observed in the spectroscopy of curium in two similar environments, it is important to analyze the subtle differences that may arise in curium-ligand bonds when dimerizing two Cp′ 3 Cm units bridged by 4,4′−bpy to form 1−Cm. To do so, the natural bond orbital (NBO) approximation and the Quantum Theory of Atoms in Molecules (QTAIM) were relied upon. The analysis of the groundstate wavefunction in a localized formalism such as natural localized molecular orbitals (NLMOs) within the NBO analysis provide a unique and simple way to rationalize the chemical bond in the context of Lewis' chemical intuition. Conversely, the topological analyses of the electron density through QTAIM metrics lay out a more holistic picture of the interactions and energies associated with the electron density in the interatomic region. Optimized molecular geometry coordinates for 1−Sm, 1−Gd, 1−Cm, and Cp′ 3 Cm are provided as Supplementary The nature of the actinide-Cp′ interaction is something that is still under debate because of both the formal charge and delocalization of the system, which provides a unique electronic environment compared to traditional chalcogenide-based ligands. Thus, there is a need to continue exploring the trends across the actinide series. Under the NBO localization formalism, these interactions are shown as one πtype bond with the metal center per ligand (Fig. 5). It is interesting to note that among the three analog complexes, 1−Sm displays the major engagement of f-orbitals to bond formation (Supplementary Table 15). This is unexpected as we generally see a greater participation of their 5f-electrons in bonding than the lanthanide 4f-orbitals. This serves as additional evidence that the cyclopentadienyl-derived ligands have unique electronic properties that result in a differential interaction between lanthanides and actinides. This scenario differs significantly when the metal−N bpy bonds are analyzed, where the classic trend is recovered with 1−Cm showing the stronger orbital mixing (almost twice as much the lanthanide mixing) and greater participation of the forbitals in bonding compared to 1−Sm and 1−Gd. Thus, for the metal-Cp′ bonds, 1−Sm shows an increased degree of orbital mixing with respect to 1−Cm and 1−Gd, whose metal-carbon bonds have similar hybrid contributions to their NLMOs. Whereas for metal-N bpy bonds 1−Sm and 1−Gd resemble each other with 1−Cm showing higher metal hybrid contributions to the bond than the lanthanide analogs (Supplementary Table 15). On the other hand, the role of the coordination of 4,4′−bpy in orbital mixing on 1−Cm is not significant as Cm−Cp′ bonds in Cp′ 3 Cm are almost identical to those in 1−Cm. As a good complement to the view of the chemical bond given from localized molecular orbitals, analyzing the topology of the electron density provides a good opportunity to evaluate the accumulation of electron density, ρ(r), and the balance of kinetic and potential energies at the point where two basins (atomic fragments) contact each other along the bond path (bond critical point, BCP). Despite 1−Sm showing more pronounced Sm−Cp′ orbital mixing, the accumulation of electron density at the BCP is very similar among the three congener systems (Table 1). Conversely, the balance of potential and kinetic energy densities, V(r) and G(r), respectively, show that 1−Sm presents a similar stability of electron density at the BCP to that of 1−Cm. However, when the total energy density, H(r), is normalized against ρ(r) the difference in energy can be interpreted as effective differences in covalency. According to this metric, frequently referred to as covalency degree, it is suggested that Sm-C bonds in 1−Sm have a greater energetic stabilization caused by covalent interactions compared to those in 1−Cm and 1−Gd by~4 kJ mol −1 and~17 kJ mol −1 , respectively (Table 1). This supports the idea that the higher degree of mixing shown in Sm−Cp′ bonds correlate with the degree of covalency from an energetic perspective. On the other hand, metal-N bpy bonds show slightly larger ρ(r) values with Cm−N being the highest. The most striking difference is that kinetic energies associated are significantly increased, significantly increasing H(r) values and even switching to positive values as in Sm−N and Gd−N bonds. This indicates that these bonds have no covalent character and only Cm−N bpy bonds have a (low) covalent character. This is also reflected in the low bond orders estimated by QTAIM and NBO analyses through the delocalization index, δ(r), and Wiberg bond index (WBI) metrics, respectively. Both, δ(r) and WBI metrics show that Ln-N bonds are significantly smaller than those of the Cm-N bond. Moreover, these bonds can be compared to the previously reported values for 1−Nd and 1−Am, where both show positive values and set an unusual precedent for differences in Am(III)−N and Cm(III)−N bonds. A final comparison can be made between 1−Cm and Cp′ 3 Cm bonds, where from the NLMO analysis no major differences were found, but from a topological perspective we The electron densities were derived from ground state-specific CASSCF calculations. *Metrics tabulated correspond to values measured at the bond critical point, and Wiberg bond indices (WBI). The electron density, ρ(r) is expressed in e Å −3 , whereas the Laplacian in e Å −5 . Potential V(r), kinetic G(r), and total H(r) energy densities are expressed in kJ mol −1 Å −3 . Metal−Cp′ bonds are shown as averages of all metal−C bonds. see significant differences. The coordination of the 4, 4′−bpy seems to reduce the accumulation of electron density at the BCP while decreasing the magnitude of the potential energy with respect to kinetic energy resulting in less than half of the total energy density in 1−Cm (−21.9 kJ mol −1 Å −3 ) compared to Cp′ 3 Cm (−51.0 kJ mol −1 Å −3 ) ( Table 1). This suggests that direct correlations between orbital mixing and the degree of covalency cannot be assumed and must be contrasted carefully with alternative methods, where energies can be derived. Characterization of (Cp′ 3 Cm) 2 (μ−4,4′−bpy) and its lanthanide analogs introduces differences between An−C and Ln−C bonding. 1−Cm shares M−N similarities with its 1−Gd analog, but similar M−C bond lengths to 1−Sm, not demonstrating favor to one analog over the other and deviating from expected trends. The putative Cp′ 3 Cm complex shows lower energy emission compared traditional curium complexes. Additionally, coordination of 4,4′−bipyridine to Cp′ 3 Cm leads to a rare exmaple of the complete quenching of curium's fingerprint emissive states. The bonding patterns calculated from these three complexes demonstrate unusual orbital mixing observed in Sm−Cp′ bonds when compared to the curium and gadolinium complexes. Overall, coordination of 4,4′−bpy does not significantly affect the orbital mixing in Cm−Cp′, but it reduces the electron density accumulated in the interatomic region that ultimately translates to lower degrees of covalency. The synthesis and characterization of (Cp′ 3 Cm) 2 (μ−4,4′−bpy) leads to future studies in Cm−C bonding and necessitates further study into the unique electronic and emissive properties of this element. Methods Caution! 248 Cm (t 1/2 = 348,000 y) presents serious health hazards due to α-emission (5.078 MeV, 75%) and spontaneous fission (~8.4%), resulting in considerable neutron emission (80 mrem/h). All reactions and handling of 248 Cm were completed in a Category II radiological facility with HEPA-equipped fume hoods and gloveboxes utilizing strict safety controls. All reactions were completed using Schlenk line and glovebox techniques in an argon atmosphere with exclusion of air and water unless noted otherwise. All handling of 248 Cm was completed in a HEPA filter-equipped fume hood and negative pressure glovebox attached to the HEPA line. (Cp′ 3 Sm) 2 (μ−4,4′−bpy), 1−Sm. Using a scintillation vial (20 mL), the addition of 4,4′−bpy (2.6 mg, 0.017 mmol) to Cp′ 3 Sm (18.7 mg, 0.033 mmol) in toluene (2 mL) resulted in the immediate formation of an orange-yellow precipitate. The slurry was stirred vigorously overnight at room temperature, dried under reduced pressure, rinsed with hexane to remove any unreacted Cp′ 3 Sm, and once again dried under reduced pressure. The powder was dissolved in toluene and heated to 120°C (with the cap removed) while gentle stirring, resulting in an orange solution. Upon reaching a gentle boil, stirring was ceased and the sample was manually cooled down 70°C in 10°C increments over a period of 20 minutes. At 70°C the cap was reapplied to the vial carefully so as not to agitate the sample. The heat was then turned off and the vial was slowly cooled to room temperature, resulting in the growth of yellow crystals suitable for single-crystal x-ray diffraction studies after 3 hours. UV-vis-NIR (toluene): λ max nm (cm −1 ) = 944 (10,593) (Cp′ 3 Gd) 2 (μ−4,4′−bpy), 1−Gd. In a slight modification to the literature procedure 28 , Cp′ 3 Gd was synthesized by the addition KCp′ (115 mg, 0.65 mmol) to anhydrous GdCl 3 (50 mg, 0.19 mmol) in toluene (2 mL) and stirred at 70°C overnight. The green slurry was centrifuged and KCl was filtered off, followed by rinsing the pellet with toluene (3 × 1 mL). Toluene was evaporated under reduced pressure and the resulting powder was taken up in hexane to extract any unreacted KCp′. The slurry was again centrifuged, rinsed with hexane (3 × 1 mL), and dried under reduced pressure, resulting in a light green powder (89.4 mg, 0.157 mmol, yield 82.7%). (Cp′ 3 Gd) 2 (μ−4,4′−bpy) was synthesized by the addition of 4,4′−bpy (2.5 mg, 0.016 mmol) to Cp′ 3 Gd (18.4 mg, 0.032 mmol) in toluene (2 mL). The bright yellow precipitate was dried under reduced pressure, rinsed with hexane, and taken up once again in toluene. Crystallization was completed analogous to 1−Sm, resulting in bright orange-yellow crystals suitable for singlecrystal X-ray diffraction studies. Following actinide drying procedures reported previously 30,32,66 , DME (1.5 mL) was added dropwise to CmBr 3 •nH 2 O. The slurry was stirred for 10 minutes, followed by the dropwise addition of bromotrimethylsilane (TMS−Br, 1.5 mL). The slurry was stirred at 50°C for 2 hours and the powder began to dissolve slowly. After cooling to room temperature, hexane (4 mL) was added, resulting in a white precipitate. The sample was allowed to settle for 15 minutes before the supernatant was pipetted away, and then was rinsed with hexane (3 × 2 mL), stirring for 5 minutes and resting for 15 minutes between each rinse, followed by drying under reduced pressure for 30 minutes. The resulting white powder was taken up in diethyl ether (1.5 mL), stirred for 20 minutes, and further precipitated out by the addition of hexane (4 mL). The slurry was stirred vigorously for 5 minutes and allowed to settle for 15 minutes before pipetting away the supernatant and drying under reduced pressure for 2 hours, resulting in a white powder of putative CmBr 3 (DME) n . KCp′ (8.2 mg, 0.046 mmol) was dissolved in toluene (1.5 mL), added dropwise to CmBr 3 (DME) n , and stirred vigorously at 70°C for 2 hours. A color change from colorless to tan was observed. The sample was centrifuged to remove the KBr byproduct and filtered, followed by rinsing the pellets with toluene (3 × 0.5 mL each pellet). Toluene was evaporated under reduced pressure and hexane (1.5 mL) was added to precipitate and extract excess KCp′. The slurry was centrifuged, supernatant filtered, and the pellets were washed with hexane (3 × 0.5 mL each pellet). About 40 µL of the resulting champagne-colored solution was isolated and diluted to 1.5 mL for solution phase absorption studies. Putative CmCp′ 3 was isolated and dried under reduced pressure, resulting in a tan oil (7.1 mg, 0.011 mmol, yield 81%). Two crystal clusters were extracted for photoluminescence studies. The crystal quality was not suitable for singlecrystal X-ray diffraction but were suitable for emission studies. An immediate color change to bright yellow was observed upon the addition of 4,4′−bpy (1.0 mg, 0.006 mmol) to CmCp′ 3 in toluene (2 mL). The solution was transferred from a 20 mL scintillation vial to a 6 mL scintillation vial and concentrated under reduced pressure until an orange-yellow powder was observed (~0.3 mL toluene). The slurry was slowly stirred and heated to 120°C resulting in the orange powder dissolving. Similar to 1−Sm and 1−Gd, the solution was slowly cooled to 70°C in 10°C increments. At 70°C the vial was capped and the hotplate was turned off, allowing the sample to slowly cool to room temperature. Around 60°C a color change from orange to an emerald green was observed, followed by a slow color change to a sapphire blue over a period of about an hour. The blue solution rested overnight at room temperature. The following morning the sample was again yellow and gold crystals suitable for single-crystal X-ray diffraction were retrieved. UV-vis-NIR (toluene): λ max nm (cm −1 ) = 587 (17,036), 597 (16,750), 631 (15,848), 641 (15,601). Data availability The structural data generated in this study have been deposited in the Cambridge Structural Database (CSD) under accession codes 2236796, 2236795, and 2236794 for 1−Sm, 1−Gd, and 1−Cm, respectively. The spectroscopic, crystallographic, and theoretical data for this study are provided in the Supplementary Information. Raw spectroscopic data generated in this study is provided in the Source Data file. Cif files for 1−Sm, 1−Gd, and 1−Cm structural data are provided as Supplementary Data 1-3, respectively. Optimized molecular geometry xyz coordinates for 1−Sm, 1−Gd, 1−Cm, and Cp′ 3 Cm are provided as Supplementary Data 4-7, respectively. All other data are available from the corresponding author upon request. Source data are provided with this paper. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/.	2023-06-26T06:16:10.006Z	2023-06-24T00:00:00.000	{ "year": 2023, "sha1": "7d1eeca3d5e4dff7e08baf92deb73670a8f38f6d", "oa_license": "CCBY", "oa_url": null, "oa_status": null, "pdf_src": "PubMedCentral", "pdf_hash": "fc0713bf6881f1b714d8c936fd85ce6abe0f8cfe", "s2fieldsofstudy": [ "Chemistry", "Materials Science" ], "extfieldsofstudy": [ "Medicine" ] }
1031767	pes2o/s2orc	v3-fos-license	Neuro-pharmacological effects of Crinum zeylanicum in mice Objectives: The aim of present study was to evaluate some effects of Crinum zeylanicum (C. zeylanicum) on central nervous system. Materials and Methods: C. zeylanicum methanolic bulb extract (250-1000 mg/kg orally), 2 mg chlorpromazine and 4 mg diazepam /kg body weight intraperitoneally respectively were tested in mice using Irwin test, pentobarbitone-induced sleep test, spontaneous motor activity, apomorphine-induced stereotype behaviour, and rota-rod performance. Results: The C. zeylanicum bulb extract significantly and dose-dependently decreased apomorphine-induced hyperactivity in mice (p<0.001). The Irwin test revealed dose-dependent central depressant effect of the extract, shortened (p<0.05-0.01) the onset of sleep and prolonged the duration of sleep. The extract produced significant (p<0.05-0.001) and dose- dependent reduction in spontaneous motor activity and apomorphine-induced stereotype behaviours in mice. The extract had no effect on performance of mice on rotarod. Conclusion: The results suggest that the extract may possess sedative principles with potential neuroleptic properties. Introduction Interest in the use of medicinal plants to treat various central nervous disorders including depression, epilepsy, and psychosis is on the increase worldwide (Kinjal Chauhan et al., 2011). Plant kingdom has remained a target of search for new drugs and lead compounds by multinational drug companies and research institutes (Mahendran et al., 2011). The World Health Organization (WHO) supported programs designed to use medicinal plants more effectively in traditional health care systems especially in developing countries (WHO, 2000) where they are readily available, easily affordable, and already integrated into the people's cultures. Crinum zeylanicum Linn. (C. zeylanicum), (Family: Amaryllidaceae) is one of such medicinal plants used in traditional treatment of ailments. It is a bulbous plant that is widely distributed in tropical Africa. In western part of Nigeria, the bulb is used externally for skin troubles, injuries, and on refractory ulcers (Adesanya, 1992). In southern part of Nigeria, the Ibinis use juice obtained from the bulb for management of general debility, childhood convulsions, epilepsy, and psychosis (Jayeoba, personal communication). Previous study in our laboratory has shown that methanolic bulb extract possesses anticonvulsant effect against leptazol-induced convulsion in mice (Tijani et al., 2010). The objective of this study was to evaluate the possible neuroleptic potential of this widely used plant in the management of neuropsychiatric disorders. Plant material The whole Extraction of plant material The bulb of C. zeylanicum was crushed and air-dried at room temperature. One hundred grams (100 g) of the dry plant material was macerated in 70% methanol for 48 hours. The resulting mixture was filtered using muslin cloth followed by Whatman filter paper (No. 1). The aliquots obtained was dried on water bath and stored at -4°C until required for use. Animals Albino mice (18-20 g) of both sexes obtained from the Animal Facility Centre of National Institute for Pharmaceutical Research and Development (NIPRD), Abuja, Nigeria were used in the study. The mice were fed standard laboratory diet, given water ad libitum and maintained under laboratory conditions of temperature (22±1°C ), relative humidity (14±1%) and 12 h light and 12 h dark cycle. All experiments were performed between 7 and 11 AM in accordance to the "Guide for the Care and Use of Laboratory Animals as adopted by the National Institutes of Health and NIPRD Standard Operating Procedures. Gross behavioural effects of C. zeylanicum methanolic bulb extract in mice This study was carried out according to the method described by Irwin (1968) and modified by Perez-Saad and Buznego (2008). Adult mice of both sexes were randomized into five groups of five mice each. Group I mice received 10 ml distilled water/kg body weight. Mice in groups II, III, IV, and V received 100, 250, 500, and 1000 mg extract/kg body weight orally, respectively. The mice were placed in separate transparent cages after one hour of extract administration and were observed behaviourally for a period of one hour; (i) Central nervous system (CNS) stimulant effects such as excessive jumping, biting, sniffing, scratching, etc., (ii) CNS depressant effects indicated by excessive reduced motor activity reduced startle response and reduced response to manual manipulation, and (iii) autonomic effect such as pupillary size, lacrymation, salivation, defecation, and urination were also observed. Pentobarbitone -induced hypnosis in mice The method of Rolland et al. (1991) was used. Mice were randomized into five groups of six mice each. Group I mice received 10 ml distilled water/kg body weight orally while those in groups II, III and IV were given 250, 500, and 1000 mg extract/kg body weight, respectively orally. Mice in group V received 2 mg diazepam/kg body weight intraperitoneally. One hour after extract and thirty minutes after diazepam administration respectively, 25 mg pentobarbitone sodium/kg body weight was administered to each mouse intraperitoneally. Each mouse was placed individually in a transparent cage and then observed for onset and duration of sleep, with the criterion for sleep being loss of right reflex on all four limbs after being gently rolled sideways. The interval between loss and recovery of righting reflex was used as the index of hypnotic effect (duration of sleep) (Ramirez et al., 1998). Studies on spontaneous motor activity The study was carried out using the method described by Amos et al., (2001). Adult mice of both sexes were randomized into five groups of six mice each. Groups I served as the control and received 10 ml distilled water/kg body weight orally. Groups II, III, and IV received 250, 500, and 1000 mg extract/kg body weight orally, respectively. The group V mice received 2 mg chlorpromazine/kg body weight intraperitoneally. One hour and thirty minutes after extract and chlorpromazine administration, the mice were transferred individually into Letical activity cages (LE 3806) consisting of four ventilated motor cages connected to a multi-counter. Activities were automatically recorded after a 1-min latency period for 6 min at 30 min intervals for a period of 120 min. Test for motor co-ordination (Rotarod Test) The study was carried out according to the method described by Perez et al., (1998). Rota rod treadmill device (Ugo Basile no. 7680, Italy) was used for this experiment. Mice trained to remain on slowly moving (16 rpm) rods of 5 cm diameter for 180 seconds or longer were selected and randomised into four groups of six mice each. Group I mice received 10 ml distilled water/kg orally. Groups II, III, and IV received 250, 500, and 1000 mg extract/kg orally, respectively. One hour after administration of extract, the mice were placed singly on the rod for 3 minutes, at 30 minutes intervals for 3 h. If an animal failed more than once to remain on the rod for 3 minutes, the test was considered positive, meaning that there is lack of motor coordination. Apomorphine -induced stereotype behavioural studies in mice The method described by Randrup and Munkvad (1967) was used for the stereotype behavioural studies. Adult mice were randomized into five groups of six mice each. Group I received 10 ml normal saline/kg body weight orally. Groups II, III, and IV received 250, 500, and 1000 mg extract/kg body weight orally, respectively. The group V mice were given 2 mg chlorpromazine/kg body weight intraperitoneally. One hour after administration of saline and extract and thirty minutes after chlorpromazine administration, all the mice were given 2 mg apomorphine/kg intraperitoneally. The signs of stereotype behaviour that included circling, jumping, sniffing, and general locomotion were recorded for a period of 2 h using a hand held tally counter (Irwin, 1968). Statistical analysis All the data were expressed as mean±SEM. Differences in means were estimated by means of ANOVA followed by Dunnet's post hoc test. Results were considered significant at p<0.05. Effect of C. zeylanicum bulb extract on Irwin test The extract produced dose-dependent decrease in motor activity. Other effects observed were paw licking, erect fur, salivation, urination, and defecation ( Table 1). Effect of C. zeylanicum bulb extract on pentobarbitone-induced sleep test in mice The extract (500 and 1000 mg/kg) significantly (p<0.05 and p<0.01) reduced the onset and prolonged duration of sleep induced by pentobarbitone. The effects are comparable to that of 2 mg diazepam/kg body weight (Table 2). Effect of C. zeylanicum bulb extract on spontaneous motor activity in mice The extract (250-1000 mg/kg) produced significant (p<0.05 and p<0.01) decrease in spontaneous motor activity in the mice at all time intervals (Table 3). These effects were dose-and time-dependent. Effect of C. zeylanicum bulb extract on motor co-ordination (Rota-rod) in mice The extract-treated mice were able to maintain their posture on the rotating rod without falling for over 180 seconds and the cut-off time on the tread mill at all doses was used. Effect of C. zeylanicum bulb extract on apomorphine-induced stereotype behaviour in mice The extract (250-1000 mg/kg, p.o.) significantly (p<0.001) attenuated apomorphine -induced stereotyped behaviour in mice dose-dependently. This effect at 1000 mg extract/kg body weight was comparable to 2 mg chlorpromazine/kg body weight (Table 4) Discussion The present study reports some neuropharmacological activities of methanolic bulb extract of C. zeylanicum in mice. The extract was found to produce alteration in general behavioural pattern, shortened onset of pentobarbitone-induced sleep, prolonged duration of pentobarbitoneinduced sleeping, significant reduction of spontaneous motor motility, and apomorphine-induced stereotype behaviour in mice. It does not have any effect on the motor coordination. The present findings suggest that C. zeylanicum possesses CNS-depressant action. The extract reduced spontaneous motor activity. The spontaneous motor activity (SMA) is a measure of the gross motor activity of the animal, and reflects the integrity of the entire neuromuscular system and its control and regulation by the central nervous system (Parshad et al., 1997). The SMA is used to evaluate gross behavioural effects of drugs in laboratory animals (Carpendo et al., 1994). It measures the level of excitability of the central nervous system (Mansur et al., 1971) which correlates well with drug effect in humans. Any agent that suppresses SMA may possess central sedative properties (Ozturk et al., 1996). Central dopaminergic mechanisms play important roles in the control of motor activity and mental functions (Costal et al., 1989) such that the reduction in SMA produced by the methanolic bulb extract of C. zeylanicum at the doses used may be due to its inhibitory actions on central dopaminergic systems. Many groups of psychotropic agents including antipsychotics (Baldessarini, 1996), anticonvulsants (McNamara, 1996), antidepressants (Lowe et al., 1978), and narcotic analgesics (Reisine & Pasternak, 1996), can diminish SMA in all species of animals including humans. The ability of the extract to suppress SMA, shorten the onset and prolonged the duration of pentobarbitone-induced sleep in mice therefore suggests that it contains active principles that are sedative in nature. The lack of inhibitory effect of the extract on motor co-ordination as observed in the treadmill suggests that the extract may not be acting via peripheral neuromuscular blockade but rather centrally thus confirming its central sedative property (Capaso et al., 1996). The extract produced significant dosedependent reduction in apomorphine-induced stereotype behaviour in mice. Apomorphine is an agonist at the dopamine receptor; it binds to D 2 receptor subtype resulting in inhibition of adenylyl cyclase which reduces potassium ion conductance, and enhances calcium ion channel activity with resulting hyperactivity of dopamine in the nigrostriatal pathway. Agents that inhibit apomorphine-induced stereotypy can antagonise dopamine receptors in the nigrostriatal system (Tarsy & Baldessarini, 1986). The reduction in apomorphine-induced stereotype behaviour suggests that the extract effect may have been mediated via inhibition of dopaminergic system which may be correlated with its neuroleptic potential. It may be concluded that C. zeylanicum methanolic bulb extract contains psychoactive principles that are sedative in nature with possible neuroleptic potentials. Further studies are planned to establish mechanism of CNS-depressant action of C. zeylanicum by using various specific agonists and antagonists.	2016-04-26T18:22:02.481Z	2012-06-01T00:00:00.000	{ "year": 2012, "sha1": "8f018e088010e75bbc62c56311ac763d651b192b", "oa_license": "CCBY", "oa_url": null, "oa_status": null, "pdf_src": "PubMedCentral", "pdf_hash": "8f018e088010e75bbc62c56311ac763d651b192b", "s2fieldsofstudy": [ "Biology", "Medicine" ], "extfieldsofstudy": [ "Medicine", "Psychology" ] }
248593106	pes2o/s2orc	v3-fos-license	What can we see from the road? Applications of a cumulative viewshed analysis on a US state highway network . In many parts of the world, motorized travel is one of the most common ways that people interact with their regional landscape. This study investigates how travelers’ understandings of place might be inﬂuenced by what landforms they can see from a vehicle. It uses a cumulative viewshed analysis on the Washington State (United States) highway network to determine which physical landscape features are most frequently visible or obscured from the road. Adapting ideas from Kevin Lynch’s The Image of the City , I propose spatial data processing methods to derive landmarks, edges, and districts that could most contribute to the mental maps of travelers and should be prioritized for labeling on print, electronic, and augmented reality maps. Other applications of the cumulative viewshed include deriving scenic byways, siting proposed construction for high or low visibility, and guiding conversations about critical toponymy and perceptions of place. Introduction: highway travel and the visible landscape In many areas of the world, motorized travel on cars, buses, motorcycles, and other vehicles is the primary way that people move between locations in their home regions. Traveling along the highway, they take in a visual panorama that includes landforms moving through the field of view in an ever-changing display (Cron, 1959, p. 88;Lowenthal, 1978). Indeed, this is one aspect of automobile travel that many motorists find enjoyable. "The view from the road can be a dramatic play of space and motion, of light and texture, all on a new scale," observed Appleyard et al. in 1964 (pp. 3-4). Even for urban commuters who get no thrill from sitting in traffic, motorized travel is still one of the most common ways to see the natural landscape. Although there are various sensory ways to learn and know a landscape, such as sounds and smells, the motorist is largely sealed off from these, primarily relying on vision. Scenes from the highway influence the ways that motorists perceive space and orient themselves. Traveling through a place can reduce ignorance of that landscape for passengers who are attentive and traveling during daylight (McKenna et al., 2008). No longer terra incognita, the roadside landscape might even take on an outsized role in motorists' understandings of place. People's mental maps tend to exaggerate the size, prominence, and frequency of features that they have seen or interacted with (Gould and White, 1974, p. 33, p. 130). That being said, mechanized travel allows only a rapid and relatively limited set of views, viewpoints, and angles compared to those that would be available if the observer could simply roam the landscape, a fact sometimes bemoaned by early rail travelers (Schivelbusch, 1986). The appearances of natural features from the roadway are likewise constrained and further affected by environmental factors such as weather and lighting (Unwin, 1975). Even with the possibility of motorized travel, our cognitive maps sometimes remain sketchy and impressionistic. As our mental maps grow through repeated exposure and experiences, so expands our set of behavioral options, sense of security, and feelings of enjoyment and meaning in the landscape (Bell et al., 1978, pp. 267-269;Chang et al., 2019). For decades, urban planners and landscape architects have sought to understand how pedestrians and motorists interpret and navigate cities from streets and sidewalks. I propose that some of these inquiries can help learn how people perceive the natural landscape of the broader region. The volume The Image of the City of Lynch (1960) posited that our environmental image is the product both of immediate sensation and of the memory of past experience, and it is used to interpret information and to guide action. The need to recognize and pattern our surroundings is so crucial, and has such long roots in the past, that this image has wide practical and emotional importance to the individual. (Lynch, 1960, p. 4) A clear mental image of a landscape is thus the starting point for further learning, exploration, and individual growth. By studying residents' mental maps and navigation habits among three US metropolises, Lynch developed a framework of five elements that contribute to people's image of the city. These are paths, edges, districts, nodes, and landmarks. Paths are channels of movement, such as roads and railways; edges are other linear elements not used as paths, such as shorelines or walls; districts are areal units with a common identifying character, such as neighborhoods; nodes are strategic foci of travel such as stations and junctions; and landmarks are point references not participating in the travel network, such as hilltops or distinctive skyscrapers (Lynch, 1960, pp. 46-48). The density at which these elements are perceived on the cityscape determines the legibility and imageability of the landscape. For example, participants in Lynch's study had a well-developed image of the city of Boston along the Charles River where edges, paths, and landmarks were abundant and markedly defined; but the details of their understanding declined with distance from the river's edge. Some neighborhoods were even difficult for their own residents to conceptualize due to irregular street matrices and scarcity of landmarks (Lynch, 1960, p. 20). At the state or provincial scale, features such as roads, ridges, basins, cities, and peaks all find corollaries in Lynch's framework. The degree to which these features actually do fill the roles of paths, edges, districts, nodes, and landmarks is partly based on how visible they are (McKenna et al., 2008). In this context, GIS-based visibility analysis becomes useful for determining which landforms and other natural features might contribute most to the legibility of the landscape. Calculating the visible landscape As computer processing power has improved over the years, some researchers have explored whether Lynch's elements can be derived from spatial databases using algorithms. Computation was viewed as an attractive circumvention of the time and cost associated with finding and interviewing residents about their mental maps. Campagna et al. (2012), for example, experimented with finding prominent paths based on size and topological connectivity of streets, but only af-ter noting that some of Lynch's elements are defined by personal, historical, or cultural meanings that might be harder to calculate. Other studies introduced visual properties into the computation of Lynchian elements. Dalton and Bafna (2003) used ideas from space syntax research, such as axial lines and isovists (eye-level cross-section polygons of the field of view). The latter were useful for detecting edges, although the authors felt that the "visual elements" of edges and landmarks played only a secondary role of fine-tuning the mental map when compared with the "spatial elements" (nodes, paths, and districts) that the subject could actually traverse. Morello and Ratti (2009) used a digital elevation model (DEM) and employed 3D isovists to calculate Lynchian elements as the subject traveled through urban space. Their work uses cumulative isovists to understand commonly visible surfaces. Filomena et al. (2019) proposed methods for computing all five of Lynch's elements, identifying landmarks by measuring building heights and the longest lines of sight. They also looked at nearby points from historic registries in an attempt to capture some of the socio-cultural meaning associated with the potential landmarks. Some have questioned how much Lynch's elements are still relevant in the digital era. Park and Evans (2018) note that digital wayfinding tools can elevate alternative routes that might have once been secondary or tertiary in nature (for example, to get around accidents or slowdowns), thereby muddying the clear spatial hierarchy of path structures advocated by Lynch. Hamilton et al. (2014) observe that as algorithms generate maps dynamically, the traveler has less need of a cognitive map or wayfinding skills. Out of Lynch's five elements, this development has the biggest effect on landmarks. Indeed, the definitive landmark in the algorithmic city may actually be the self. The present article contemplates what Lynch's elements might look like when applied to geomorphological features on a state-level scale and explores ways of identifying possible elements using visibility analysis. Since Lynch's framework was developed in cities, some aspects of it may not translate directly to rural settings; for example, in the countryside, the path network is more limited than in the city. There may only be one reasonable way to get between an origin and destination point. Similarly, nodes as critical junctions between paths may be fewer and farther between. Some elements may not be used directly for route-finding but still contribute to travelers' mental maps and understanding of relative positioning. In the natural landscape setting, the visual elements of edges and landmarks identified by Dalton and Bafna (2003) are useful toward personal orientation and confirming a sense of place. Districts are also possible to derive as polygonal areas that can be seen and comprehended. Thus, the present analysis focuses on identifying landmarks, edges, and districts. GIS offers numerous approaches for studying the areas that are visible from any particular vantage point. Gridded (raster) data are most common in these analyses, wherein each cell value represents the elevation of the terrain. The software performs geometric calculations on this "digital elevation model" (DEM) to systematically detect whether anything is blocking the view between the observer cell and all possible target cells (Travis et al., 1975;Fisher, 1991). The set of cells visible from the input observer cell is commonly referred to as the viewshed of the input cell. Viewsheds have been deployed in landscape planning (Travis et al., 1975;Fisher, 1996;Sander and Manson, 2007), tourism and recreation studies (Wilson et al., 2008;Jakab and Petluš, 2013), historical analysis (Randle, 2011), and many other fields. The most accurate method of deriving a viewshed is to calculate the line of sight between the observer cell and all other cells in the study area; however, this approach is timeconsuming. Algorithms that make relatively minor sacrifices in accuracy can shorten the processing time considerably by strategically reducing the number of sight lines calculated. Ways of doing this include calculating the lines of sight to the grid edge cells first and using the results to estimate values of inner cells, or working outward from the observer in concentric rings in a way that skips calculations on areas already estimated to be obstructed (Franklin and Ray, 1994;De Floriani and Magillo, 2003;Kaučič and Zalik, 2002;Carver and Washtell, 2012). A different method described by Wang et al. (2000) avoids sightlines and instead uses "reference planes" defined by the heights of the observer cell and two other cells just in front of the target cell. This is the algorithm employed in the free and open-source Geospatial Data Abstraction Library (GDAL) software (Warmerdam, 2008). It was chosen for the present study due to its free and widespread availability, transparent documentation, and reasonable speed. A viewshed of a single point can be recorded in a Boolean raster format using a value of 1 to denote cells within the viewshed and 0 or "no data" values for all other cells. When viewsheds are calculated from multiple points, the resulting layers can be summed to create a cumulative viewshed wherein the value of any given cell represents the number of observer points that can see that cell. For example, Wheatley (1995) used a cumulative viewshed analysis (CVA) to map how many archeological monuments were visible from any vantage point within an area of interest. The study also examined the cumulative viewshed counts at the monument points themselves to help determine if intervisibility might have been a factor in monument placement. Determining visibility from polyline features (such as a road network) is carried out in practice by placing points at a fixed interval along the lines and creating a cumulative viewshed from those points. Chamberlain and Meitner (2013) gave an example of this approach to determine scenery visible from a 6 km segment of highway, with the sample points placed about 10 m apart. They described several variations on this technique to account for the speed of the vehicle and the visual magnitude of the feature within the motorist's field of view. Lee and Stucky (1998) used cumulative viewsheds as a cost surface to determine ideal paths for a variety of scenarios such as keeping unsightly features out of view, maximizing scenic vistas, and conducting reconnaissance. Some probes of novel viewshed methods have been limited by computational capacity, especially when generating many viewsheds over a broad landscape. Most studies involve urban areas or small rural study sites. For example, when calculating visibility from a sample of 220 residential properties within a town, Sander and Manson (2007) mention limited computing time as a barrier to a more comprehensive inquiry. Today's increased computational capacity and data storage capacities can allow for broader CVA studies across states or countries, especially when the viewsheds are generated and summed through automated scripts. The present study takes advantage of automation to generate and sum many thousands of viewsheds along the highways of Washington State, a study area of approximately 184 000 km 2 in the northwestern corner of the contiguous United States. A variation on the analysis weights the viewsheds by traffic counts to get a better understanding of the features visible to the most highway travelers. The results from these cumulative viewsheds are used to construct elements from Lynch's framework, thereby getting a feel for which geographic features might contribute most heavily to motorists' readings of the landscape. I conclude the analysis by discussing different uses of the cumulative viewshed, as well as some of the limitations involved in this approach. Methods The set of 1 arcsec DEMs covering Washington State was downloaded from the United States Geological Survey (USGS) National Map downloader website (https://apps. nationalmap.gov/downloader/#/, last access: 28 April 2022). The author projected these DEMs into UTM Zone 10 N, mosaicked them, and clipped them to the official state boundary (including water). The cells' spatial resolution of approximately 30 m was fine enough to capture the visibility of major natural features, while being coarse enough to allow for the calculation of thousands of viewshed operations across long distances. For more localized analyses not involving an entire state, a higher-resolution DEM would be preferable. Highway polylines containing average annual daily traffic (AADT) counts for the year 2019 were obtained from the Washington Geospatial Open Data Portal (https://geo.wa. gov/search, last access: 28 April 2022). This dataset is maintained by the Washington State Department of Transportation and contains all numbered federal and state highways within Washington State, totaling about 11 362 km in length. The traffic counts were reported as attributes of these polyline segments. Multiple segments made up a single highway, with the counts varying up and down along each route according to how many vehicles per day were estimated to travel the road on average during 2019. More recent counts were not sought, due to the influence of the COVID-19 pandemic on typical traffic patterns. To prepare for the viewshed generation, the highways were joined by their route numbers, and a set of sample viewpoints was generated at 1 km intervals along each route. Points known to be in tunnels were discarded. The traffic counts from the original highway segments were then spatially joined onto the viewpoints. This created a dataset with 11 225 viewpoints, each containing a traffic count estimate. Procedure for generating cumulative viewsheds Using a Python script (S1) and the open-source GDAL processing library, a viewshed was generated for each viewpoint. During this computation, a 1 m vertical offset was added to each viewpoint elevation to represent the minimum reasonable height of an individual looking out of a vehicle (Smith, 2006, p. 144;Capaldo, 2012). An earth curvature coefficient of 0.85417 was applied as suggested in the GDAL documentation. Visible cells were coded as 1, and other cells were coded as 0. Additionally, a second version of the viewshed was weighted by the AADT count. In other words, if a viewpoint had an AADT count of 5000 vehicles, all visible cells were coded as 5000, and non-visible cells were coded as 0. Both the weighted and unweighted viewsheds were saved as compressed TIFs. The entire process of creating the viewshed, making a weighted copy, and compressing the results took an average of 38 s per viewpoint using an Intel i7-6700 CPU running at 3.40 GHz with 64 GB of RAM. Distribution of the task across multiple machines could reduce the processing time if needed. The unweighted Boolean viewsheds were summed one by one using the GDAL Calc operation in order to obtain the cumulative viewshed in Fig. 1. In this map layer, the value of each cell represents the number of viewpoints that can see that cell. The traffic-weighted viewsheds were also summed to create a cumulative viewshed representing how much traffic at the viewpoints could see each cell during an average day (Fig. 2). Note that these cell values do not directly translate into how many people see the cell each day, as many people's trips may span multiple points and there are often multiple people within a single vehicle; however, they do help indicate which geographic features are visible to the most travelers. Note that these methods could be used on part of the data in order to answer certain questions. For example, cumulative viewshed methods can show all the land visible from one particular highway. To demonstrate this, Fig. 3 combines all the viewsheds along Interstate 90, the main east-west thoroughfare through the state connecting its two largest cities of Seattle and Spokane. In this map, pixels are simply classified as visible or invisible to give the viewer a quick and easy feel for which features a motorist could see. Procedure for identifying landmarks and edges The resulting cumulative viewsheds are quite detailed. They can be used "as is" for a city-or county-level analysis, but visualizing patterns at the state level requires some smoothing and generalization. The following procedure was used to isolate the most visible peaks and ranges using spatial data processing algorithms, thereby serving as a guide for digitizing Lynchian landmarks and edges. 1. Each cell in the cumulative viewshed was recoded with the maximum cell value falling within a radius of 1 km. The result of this was then downsampled to a 1 km cell size for faster processing and easier visual interpretation. 2. The top 5 % of pixels with non-zero values (in this case, those visible from 153 or more points) were extracted into a Boolean raster and converted into polygons. 3. A spatial aggregation operation was performed to combine any polygons with less than a 5 km gap between them, while removing any polygons or holes of fewer than 25 km 2 . 4. (Optional) To further narrow the results, for any polygon not containing a pixel in the top 1 % of non-zero cells, the Step 1 result raster was discarded. (In this case, polygons visible from fewer than 380 viewpoints were removed.) The resulting polygons represent some of the most widely visible ridges, ranges, and peaks. They can be used as a cartographic aid for digitizing Lynchian edges and landmarks, an approach that is more objective than simply eyeballing the raw results from the cumulative viewshed. Employing the algorithmically derived patterns in tandem with the cartographer's local knowledge, experience, and supplemental map layers results in a more meaningful set of features than could be obtained by the computer or human alone. Procedure for identifying districts A similar procedure was used to derive districts as areas that the user enters "inside of" whose relatively high visibility facilitates the mental construction of a "common, identifying character" (Lynch, 1960, p. 47). Since so many of the highly visible pixels in the cumulative viewshed layer were in rugged and mountainous areas that would be difficult to traverse, the calculation of districts presented here focuses on separating out the flatter areas that are still highly visible. 1. A slope layer of the DEM was smoothed by recoding each cell with the median value within a 1 km radius. The result was resampled to a 1 km cell size for faster processing and easier visual interpretation. 2. The unweighted cumulative viewshed was also smoothed by recoding each cell with the median value within a 1 km radius and rounding it to an integer. The result was resampled to a 1 km cell size for faster processing and easier visual interpretation. 3. Using the raster layer produced in Steps 1 and 2, a new layer was made containing only cells that met both of the following criteria: number of viewpoints visible was in the top quartile of cells (in this case, over 10 viewpoints) and slope was between 0.2 % and 2 % (this captured flatter terrain while eliminating water). 4. Qualifying cells were then converted to polygons, and an aggregation operation was applied to generalize the shapes. Any polygons with less than a 5 km gap between them were combined, while polygons or holes of fewer than 25 km 2 were removed. 5. Remaining polygons with over 30 km of highway inside were then identified as district candidates, thereby ensuring that these areas were indeed locations of substantial travel. This number of kilometers could be raised or lowered as desired in order to widen or narrow results. These shapes were used as a guide for human digitizing of the final district polygons in a way that was sensitive to local topographic features. Figure 4 shows how the processing operations described in Sect. 3.2 and 3.3 guided the positioning and orientation of the nodes, landmarks, and edges digitized by the author. Results The unweighted cumulative viewshed (Fig. 1) shows that the most visible spot of ground from Washington State highways is a location approximately 4298 m high on the northwest flank of Mount Rainier, the highest mountain in Washington State. This pixel is visible from 2104 of the sample points. It might surprise some that the most visible location is not actually the summit of Rainier; however, this is consistent with the Kim et al. (2004) findings that ridges sometimes offer better visibility than peaks. All sides of Mount Rainier can be seen from state highways. Large sections of the northwest face pointing toward the Seattle area were visible from over 1000 sample points. The only other landforms reaching this threshold were several peaks in the Olympic Mountains, with the most visible being Mount Constance. The high number of viewpoints recorded for these peaks may be due to their visibility from the Seattle and Tacoma metropolitan areas, where there are the most residents and roads. These cities include gentle slopes and expanses of water that afford better views of faraway points. In the more sparsely populated central part of the state, the Wenatchee Mountains and Rattlesnake Hills are widely seen, as well as long east-west ridges from the Yakima Folds (Kelsey et al., 2017). On the far east side of the state, the Blue Mountains are the most visible. Although the major eastern city of Spokane sits adjacent to several mountain peaks, these are not prominent in the unweighted viewshed, perhaps because of the hilliness of the terrain and edge effects associated with the city being located next to the state boundary. When the viewsheds are weighted by traffic counts (Fig. 2), the general patterns are similar, although features near metro areas and busy interstate highways are emphasized. These include Mount Spokane, as well as the highlands east of Seattle sometimes locally called the "Issaquah Alps". In central Washington, the eastern slopes of the Wenatchee Mountains see high values in the weighted cumulative viewshed. These highlands are the first arm of the Cascade Range visible from Interstate 90 as westbound motorists traverse a 110 km straightaway across the Columbia Basin. In fact, these maps show just how much Washingtonians' perceptions of the Cascade Range might be influenced by travel over Snoqualmie Pass on Interstate 90. This is the main artery for personal and commercial vehicle travel between the two sides of the state. The summit sees an average of 34 000 vehicles per day. Figure 3 shows that the viewshed through the pass is generally less than 10 km wide on clear days. On winter days, one is lucky just to see the roadway. Map of landscape legibility with Lynch's elements With the aid of these cumulative viewsheds and the postprocessing procedures described above, I have demarcated some of the more "legible" features on the Washington State landscape that could fit into the Lynch (1960) framework discussed earlier. I interpreted paths as the highways and nodes as the major cities along them. Landmarks are highly visible peaks, while edges are highly visible ranges, fronts, and ridges. Districts are area features that the traveler passes through and whose visibility is relatively high from surrounding points, allowing for easy imageability. These include valleys and other bowl-like features such as basins and estuaries. The result map showing the legibility of Washington from its highways is shown in Fig. 5. Many of the geographic features discussed earlier are prominent landmarks and edges in this map. The eastern front of the Olympic Mountains and the western front of the Cascade Range clearly bound the Seattle metropolitan region. These ranges are not as easily seen from their opposite sides. That being said, central Wash-ington is generally more legible than other parts of the state due to its long folded anticlines, which are even easier to see in the open shrub-steppe environment. Travelers may find it simpler to orient themselves here than in flat regions such as the northern Columbia Basin or areas of rolling topography such as the Willapa Hills and The Palouse. What is not easily seen? The interior areas of the Olympic Mountains, including the highest point, Mount Olympus, are not highly visible from the highways. In fact, the cumulative viewsheds show just how much of Washington's mountain ranges motorists cannot see. Residents with good views of the mountains on opposing sides of the Cascade Range (such as in the cities of Seattle or Yakima) may underestimate the breadth of the mountains, as it is sometimes easy to believe that these places lie just "on the other side" of whatever is in the current view. In reality, only a small percentage of the range is visible, with much unseen land lying behind the front. Areas that are invisible from roads still exist and play important roles in human and environmental systems. Quinn (2020) described some of the activities occurring on "empty spaces" in maps of Washington State, noting that sometimes these were used for NIMBY-type activities that prefer to be kept out of sight and out of mind by urban populations. For example, a person can look at a satellite image all the way back at a 1 : 7 000 000 scale and still be able to distinguish the Roosevelt Regional Landfill, yet this burial ground for much of the region's trash is not visible from any local road. Much of the commercial forest lands in southwestern Washington that employ clear cuts are both blank on the map and invisible to motorists. Some can be glimpsed from local highways, but the topography blocks the view of much more timberland beyond. The region's economy is highly dependent on these rotational forestry approaches, yet there may be less social license for the clear-cutting practices in landscapes of high visibility. Observers may also underestimate the scope of agriculture and industry when the majority of the activity falls outside of visible areas. Travelers along eastern Washington's highways are familiar with golden oceans of wheat fields, but there is much more wheat that cannot easily be seen among the famously rolling hills of The Palouse where motorists are generally winding through gullies. Applications Beyond understanding mental maps, the highway network CVA could be applied in many fields including print and digital cartography, augmented reality development, tourism planning, and toponymic studies. Cartography As people view landforms during their travels, they may ask "What is that?", perhaps accompanied by the question "Where am I in relation to that?" A glance at popular reference maps shows that some of the most visible landmarks, edges, and districts identified for Washington in Fig. 5 are more commonly labeled than others. At the time of this writ-ing, Google Maps starts showing a few major peaks at zoom level 9. Water bodies such as the Salish Sea also begin to be labeled at this level. As the users zoom in, features and labels go in and out of the view. At zoom level 11, more peaks appear. In contrast, on OpenStreetMap.org mountains do not appear until level 11, and only then as symbols. Labels for mountains and water features in OpenStreetMap do not appear until level 13. Ranges and ridges are not labeled in OpenStreetMap or Google Maps at any level. One prominent digital map that does show these types of features is Esri's "Topographic" layer, currently the default base map in ArcGIS products. Print maps are generally better than digital maps at cramming in many labels, including for linear features. When there is only one scale to work with and the label placement is done manually rather than through automated means, cartographers can make rotations, abbreviations, and size adjustments to the text that would be more difficult to achieve through algorithmically generated cartography. This includes curving and stretching a label along the length of a range or ridge. The Rand McNally 2019 Road Atlas page for Washington labels six of the seven landmarks and four of the nine edges (excluding repeats) identified in Fig. 5 (Rand McNally, 2018). The state highway map published by the Washington State Department of Transportation labels six of the seven landmarks and eight of the nine edges (https://wsdot.wa.gov/ travel/printable-maps, last access: 28 April 2022). Both maps do not show Gold Mountain, which lies near a visually busy urban area. The state map labels more edges because it includes minor ridges in the central part of the state. Both print and digital maps fail to label many area features. The Esri Topographic base map is the only one that includes any valleys. From the districts identified in Sect. 3.3, it includes Kittitas Valley and Yakima Valley, as well as Wahluke Slope. None of the maps label estuaries. Landscape planning Highway-based CVA methods can help with decisionmaking about where to build certain features or use the land in particular ways. Some types of structures might benefit from high visibility, such as retail establishments hoping to attract passing motorists, patriotic or religious symbols (such as flags and places of worship) that wish to catch people's attention, or communication towers that require a clear line of sight for transmitting signals. When performing a CVA for the siting of these facilities, an offset representing the height of the structure could be added to each terrain cell. Other types of construction or activities might find it desirable to seek a low-visibility place. Activities that are considered unsightly or politically unpopular, such as resource extraction or the burning of fossil fuels might choose to stay out of view of the highway using methods like the ones proposed by Lee and Stucky (1998), although most motorists are burning these fuels themselves. The same goes for military activities such as certain types of combat training or weapons testing. Finally, some developers of recreational or residential facilities may want to build in places that seem distanced from the busy life of the highway, where their clients do not have to see the motorists. Tourism promotion and management The methods described in this paper can also be used to identify potentially scenic byways for further investigation. Knowing these locations could be useful for tourists, photographers, artists, and officials who want to welcome visitors while protecting the surrounding environment. Recall that each viewpoint originally resulted in a viewshed raster coded with 1 in visible areas and 0 in non-visible areas. To find scenic places, the total number of visible pixels in each raster was calculated and sorted, thereby revealing the viewpoints from which the most geographic area is visible. Figure 6 shows viewpoints whose viewsheds ranked in the top 1 % of visible land area. Several strings of adjacent points indicate good potential byways near the Salish Sea, Mount Rainier, and several basins and valleys in central Washington. A check of Google Street View for the top points confirmed that many offer scenic vistas, although some views are blocked by built structures, vegetation, and features of highway engineering as discussed in Sect. 6 of this study. Points with expansive viewsheds often occur where highways cross or overlook the edges and districts identified in Fig. 5. There are also some highland areas near coasts that offer broad water views. To identify highways that could see the most mountain peaks or other types of features, a CVA could be made based on viewsheds generated at the summits of those landmarks. The values of the cumulative viewshed raster pixels could then be evaluated along the highway sample points (using tools such as Extract Values to Points in ArcGIS or Add Raster Values to Points in SAGA/QGIS) to see which stretches of road had the highest values. A next step toward confirming the value of these potentially scenic routes would be to consider the types of land use and land cover visible from those locations and how they are perceived and preferred by observers. Strong feelings of meaning, connection, and aesthetic preference could elevate the status of a landmark or other element. Even rural landscapes are laden with symbolic meaning visible in patterns of human appropriation (Cosgrove, 1989), such as agriculture, hydropower, and commercial forestry. Geographers such as Tuan (1990) and Lowenthal (1978) have ruminated extensively on the types of landscape aesthetics preferred by humans. The latter notes that a person's reaction to a landscape may depend on mood, time of day, weather, and modality of travel. Kent (1993) studied reactions to highway scenes and found that patterns of vegetation that allowed some degree of visual penetration facilitated an appealing sense of mystery for travelers. Motorists also positively responded to features that they perceived contributed to the natural or cultural quality of the area, such as unique building architecture. More recent work has considered aesthetically valued landscapes as "cultural ecosystem services", attempting to map and understand the non-material benefits that humans derive from landscapes (Plieninger et al., 2013;Bachi et al., 2020). Analysts can use viewshed operations to maximize traveler enjoyment of these landscapes. For example, da Silva et al. (2020) suggested locations for observation towers along a nature trail with the goals of minimizing visual overlap and exposing the hiker to a diverse set of ecosystems. Highway locations with expansive viewsheds could likewise be evaluated for the types of cultural ecosystem services and the variety of landscapes visible to the traveler. Augmented reality The potential for visibility analysis to inform augmented reality applications is vast and still largely untapped. Smartphone apps such as PeakFinder and PeakVisor are popular ways for recreationists to identify what mountain they are seeing just by aiming the phone in the direction of the peak and looking at the display; still, there is an emphasis on point features, with many ridges, ranges, valleys, and basins going unlabeled. It is easy to imagine asking an onboard navigation system, "What mountain range am I seeing to the right?" and getting an educated guess based on the vehicle's current location and a database of prominent feature names derived from a highway cumulative viewshed. The use of buffers, geo-fences, and individual viewsheds might allow such notices to be actively spoken through the car's speaker system if desired ("now entering the Yakima Valley" or "look to the left and you will see Puget Sound"). One even wonders if an augmented reality windshield display could unobtrusively place labels on ridges, valleys, or water bodies as requested by the driver, thus giving the impression of traveling through the map. The circumstances under which this would be useful, and whether it could be carried out safely, might be a fruitful area for further research. Critical toponymy The landmarks identified in Fig. 5 are visible from vast and diverse areas of the state. Many of these are either stratovolcanos that rise above surrounding mountains or prominent hills seen from across the water. Although they contribute to the everyday landscapes seen by motorists, the origins of the current names of these features do not seem to be widely known by locals. In the northwestern US, it can be easy to forget the relatively recent, and sometimes contested, application of toponyms, or place names. Rose-Redwood et al. (2010) suggest that a critical analysis of the place names we encounter should go beyond individual origin stories and focus on the cultural politics of naming. The most visible features on a landscape seem a suitable place to begin that inquiry. The observation of Berg (2011) that toponyms are often involved with "settler stories" is largely true for some of the most visible landforms in Washington State, although a more precise name might be "settler government surveyor stories". The names of Mount Baker and many inland water features in the Salish Sea, such as Puget Sound, come from crew members on the ship of George Vancouver, the first known European to map out the area (Morgan, 1979). Vancouver named Mount Rainier and Mount St. Helens after powerful military and political colleagues back in Britain. Coastal surveyor George Davidson named Mount Constance and nearby peaks "The Brothers" after members of his family (Meany, 1913). In an arm of the Cascade Range highly visible from the east side of the state, George B. McClellan of the U.S. Army gave Mount Stuart the name of a deceased war buddy while making a survey of mountain passes (Meany, 1923, p. 79). The settlers followed these surveyors and inscribed their stories in more place names. They include interest in natural features (Gold Mountain, Glacier Peak), interactions with animals (Rattlesnake Hills), and homage to national heroes (Mount Adams). On maps (and later in geographic databases), these explorer and settler names replaced ones used by the region's indigenous peoples through centuries past. Although indigenous toponyms generally persist in Washington State to a more prominent degree than in many parts of the country, none of the landmark names in Fig. 5 come from an indigenous language. Failed efforts to rename Mount Rainier with some variation of the indigenous word "Tacoma" played a role in the battle for economic and cultural dominance between the cities of Seattle and Tacoma during the late 1800s and early 1900s (Morgan, 1979, pp. 293-296, 327-328). The effort has been revived by the Puyallup tribe in the wake of the successful decolonial renaming of Denali (formerly Mount McKinley) in Alaska (Sun, 2021). Regardless of the eventual outcome, this attempt will likely provoke more pub-lic thought and awareness about the history and meanings of the most visible landform in the state, as well as the ways that place names are applied and contested. Limitations and opportunities for further study Although this analysis identified some landforms that may be likely to occupy a place in the mental maps of Washington's residents, it did not verify whether the computationally identified landmarks, edges, and districts do indeed play a significant role in people's mental maps. The statewide sampling and outreach that such a verification would entail were deemed to be out of scope of this paper; however, validating computationally derived Lynchian elements with qualitative surveys or other methods such as text mining from books, articles, or conversations would be an informative exercise in future studies. In order to scale across a broad area with many thousands of viewsheds, this study built upon Boolean visible and invisible calculations carried out on a uniform DEM. The analysis reveals which features should be visible under ideal conditions, but could yield more nuanced results with additional kinds of methods and ground truthing. Features in both the built and natural environments, such as buildings and trees, can obstruct lines of sight and affect the viewshed shapes and areas. This effect can be substantial in heavily wooded areas such as those in western Washington that inspired the nickname "The Evergreen State". Models that incorporate the heights of built features and forested areas of land cover would give more accurate results, although without enormous amounts of data, they would also be subject to estimation and imprecision. A viewshed operation is only as accurate as the DEM it is conducted on. Fisher (1991) lists numerous reasons that the actual elevation of a point may differ from its DEM cell value, including problems with the original survey by field workers or photogrammetrists, mistakes by people digitizing these surveys, and poor interpolation. Limitations with the precision of the data format can also hinder accuracy. For example, elements of highway engineering such as cuts through a hillside might obstruct the motorist's view but are sometimes too small to show up in the DEMs used in this study. Higher-resolution DEMs such as those produced with lidar might yield more accurate viewsheds but could increase calculation time to impractical levels. Approaches that use high-resolution data near the highways and a coarser DEM for everything else could also improve accuracy in future experiments (De Floriani and Magillo, 2003). The downside is that these multi-resolution approaches require more data preparation on the front end. The cloudy and foggy weather associated with the temperate oceanic climate in western Washington often reduces visibility. Parts of the state on the east side of the Cascade Range are generally much sunnier, with clear visibility most of the year. Future studies could factor in weather and climate when determining the most visible features. Areas shown as invisible in the CVA should be interpreted with caution, since the analysis is based only on viewsheds taken from sample points spaced by 1 km. Stretches of roadway between the sample points might be able to see areas designated as invisible in this CVA. A denser or different set of sample points would yield a different CVA, although not likely different enough to affect the results at a statewide level. Only areas within the Washington State boundary were considered in this study. Some viewpoints in Washington can see into other states (and Canada), and some viewpoints from those locations are able to see into Washington. Consequently, the values in the cumulative viewsheds and the calculation of the top 1 % of scenic points are subject to edge effects. Finally, applying strategic weights or functions to the viewsheds might give a better picture of which landforms people see and think about the most often. For example, Mount Rainier takes up much more of the field of view in the city of Tacoma than it does in Yakima. This is because Tacoma is closer to the mountain, sits at a lower elevation, and has fewer other mountains and ridges to obstruct the vista. Approaches that calculate a "visual magnitude" for each cell based on the viewing angle and distance to the target, such as those described by Chamberlain and Meitner (2013), would help with understanding which features take up the most space in observers' fields of view. attempt to determine visual impact by using an inverse square distance function and the height of the object. Such approaches are more computationally intensive and more complex to interpret than the one described in this paper; however, they may be useful toward better understanding which visible features have the most influence on a traveler's mental map. Conclusions Using automated methods, free and open-source software, and fairly ordinary computing power, this study has demonstrated how a cumulative viewshed can be created from a regional highway network traversing well over 100 000 km 2 . The resulting layer reveals the landforms most widely visible (and invisible) to motorists along the network. Weighting the contributing viewsheds by traffic counts can give a better feel for which areas are visible to the most people. Further spatial data processing on the cumulative viewshed can assist with deriving landmark, edge, and district elements that contribute to people's mental maps as proposed by Lynch (1960). Cumulative viewsheds derived from highways can help cartographers prioritize features for labeling, especially areal features such as valleys, basins, and estuaries that are often missed. This applies to both print and electronic maps, as well as augmented reality applications that point out geographic features. Other applications of cumulative viewsheds include siting features for high or low visibility, identifying potential scenic byways, and guiding discussions about place names and perceptions. Code availability. The Supplement link associated with this paper contains the Python script used to automate the creation of the viewsheds. Data availability. The source datasets for this project were downloaded from the US Geological Survey and the state of Washington as described in the methods section (Sect. 3) of this article. The cumulative viewsheds shown in the maps were derived using the code in the Supplement. Competing interests. The author has declared that there are no competing interests. Disclaimer. Publisher's note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.	2022-05-10T16:00:18.434Z	2022-05-04T00:00:00.000	{ "year": 2022, "sha1": "a0b5e516cb2289bbf6e4e14cb19a44a1b4d22f46", "oa_license": "CCBY", "oa_url": "https://gh.copernicus.org/articles/77/165/2022/gh-77-165-2022.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "e63cf1daf8b4db4decc759ccc95e20b52210ca91", "s2fieldsofstudy": [ "Sociology" ], "extfieldsofstudy": [] }
264556922	pes2o/s2orc	v3-fos-license	Cognitive Domains Function Complementation by NTNG Gene Paralogs Gene duplication was proposed by S.Ohno (1) as a key mechanism of a novel gene function evolution. A pair of gene paralogs, NTNG1 and NTNG2, sharing identical gene and protein structures and encoding similar proteins, forms a functional complement subfunctionalising (SF) within cognitive domains and forming cognitive endophenotypes, as detected by Intellectual Quotient (IQ) tests (2). Both NTNG paralogs are associated with autism spectrum disorder (ASD), bipolar disorder (BD) and schizophrenia (SCZ), with unique nonoverlapping segregation among the other 15 cognitive disorders (CD), emphasizing an evolutionary gain-dependent link between advanced cognitive functions and concomitant neurocognitive pathologies. Complementary expression and human brain transcriptome composition of the paralogs explains the observed phenomena of their functional complementarity. The lowest identity among NTNGs is found in a middle of encoded by them proteins designated as uknown (Ukd) domain. NTNG1 contains anthropoid-specific constrained regions, and both genes contain non-coding conserved sequences underwent accelerated evolution in human. NTNG paralogs SF perturbates “structure drives function” concept at protein and gene levels. The paralogs function diversification forms a so-called “Cognitive Complement (CC)”, a product of gene duplication and subsequent cognitive subfunction bifurcation among the NTNG gene duplicates. INTRODUCTION Complex behaviors arise from a combination of simpler genetic modules that either have evolved separately or co-evolved.Many genes and proteins they encode have been found to be involved in cognitive information processing with a single variant or a single gene generally accounting for only a partial phenotypic variation of a complex trait, such as cognition.Cognition as a quintessence of brain functioning can be viewed as a product of intricately interlinked networks generated by deeply embedded into it gene-nodes with specific or partially overlapping functions.The robustness of the cognitive processing towards its single elements genetic eliminations (to study their function) and its simultaneous fragility expressed in the multiple forms of neurological disorders manifest the existence of cognitive domains interlocked but SF within a unit of cognition formed upon these domains interaction.Previously, we have described a function of a pair of gene paralogs, NTNG1 and NTNG2, involved in human IQ tests performance, and underwent hominin-specific evolutionary changes (2).Hereby, we report on these gene paralogs features focusing on underlying mechanisms of their function segregation and complementation within the cognitive domains. RESULTS The previously observed phenomena of functional complementation among the NTNG paralogs within cognitive domains (2) is also manifested in NTNG-associated human pathologies diagnosed in most cases (if only not in all) by a cognitive decline (Figure 1A-1 and A-2).Both genes are associated with BD and SCZ -devastating disorders sharing similar etiology (3) with genetic correlation by multivariate analysis of 0.590 (4), linked to human creativity (5), and characterized by impulsiveness as a common diagnostic feature (6). Recently found associations of both paralogs with ASD (7) supports the reported genetic correlation of 0.194 ASD/SCZ pair (4) and shared module eigengenes detected by PC1 among these two disorders (8).12 NTNG1-linked CDs, ranging from AD to TS, span a broad spectrum of clinical features frequently involving reduced processing speed (PS) and verbal comprehension (VC, Figure 1A-1).As for NTNG2, working memory (WM) deficit and inability "to bind" events (perceptual organization, PO) are the most prominent diagnostic traits for the SLE and TLE patients (Figure 1A-2), with PN also being characterised by indolent behavior in 90% of the cases (9).Interestingly to note that association of the synapse-expressed NTNG2 with both SCZ and autoimmune pathology (SLE) correlates with a recent finding that human complement component C4 is involved in the synapse elimination and SCZ development (10).Thus, both NTNG paralogs are associated with a variety of CDs and mostly in a non-overlapping manner, except for ASD, BD and SCZ characterized by shared and wide spectrum of cognitive abnormalities.The clinical etiology of the aforementioned diseases supports the deduced by IQ functional complementation existing among the NTNG paralogs (2) with (VC/PS) and (WM/PO) endophenotypic deficits being uniquely segregated within the associated cognitive pathologies. Since both gene paralogs are expected to have identical gene exon/intron compositions but different in their intron lengths (11) we have reconstructed both paralogs 4 transcriptomes by re-processing the publicly available RNA-seq dataset (12) from healthy and SCZ human subjects superior temporal gyrus (STG) post-mortem brain tissue (Supplementary Table 1a=ST1a).A difference is noted instantly at the total expression levels (genes, exons, individual RNA transcripts) when two gene paralogs are compared (Figure 1B-1 and B-2).NTNG2 amount (as a whole gene) is 5 times larger comparing to NTNG1; exons (2-5) are 3 times, exons (8)(9) are 18 times and exon 10 is 4 times higher expressed for NTNG2 than for NTNG1.The only two exons outlaying the prevailing amount rule for the NTNG2 mRNAs are exons 6 and 7, expressed nearly at the same absolute level as for the NTNG1 exon paralogs, making them highly underrepresented within the NTNG2 transcriptome.Next, distinct non-alternating splicing modules are formed by exons (2-5) for NTNG1 (Figure 1B-1), while exons (4-5) and exons (8)(9) for NTNG2 (Figure 1B-2).Two structurally identical RNA transcript paralogs (NTNG1a = G1a and NTNG2a = G2a) have been found to exist in both NTNG transcriptomes with G2a being expressed at 8-9 times higher level than G1a.NTNG1 is uniformly presented across the all analysed 16 human samples by 2 more protein coding RNAs (G1c and G1d, detected previously in mice brain, Nakashiba et al., 2000) and by 2 non-coding intron (9-10) derived transcripts (Figure 1B-1). At the same time, NTNG2 transcriptome is comprised of one extra potentially coding RNA (G2a-like with exon 2 spliced out but in-frame coding preserved) and 2 assumed to be noncoding RNAs with exons 6 and 7 retained along with preceding and following them introns. Quite interesting that these two latter transcripts are the only RNA species with NTNG2 exon 6 and 7 retained (Figure 1B-2).Two more coding (G1f and G1n) and 4 more non-coding for NTNG1 and 9 extra non-coding for NTNG2 RNA species have been also assembled from the available reads but due to inconsistency in their appearance across all 16 STG samples they are not presented on the figure but summarized in the table (Figure 1C, for details refer to ST1d).Summarising above, quantitative and qualitative complementary differences is a 5 prominent feature characterising the brain RNA transcriptome of human NTNG paralogs.However, no significant changes at the transcription level of neither whole genes, nor individual exons, nor reconstructed RNA transcripts have been found when SCZ and healthy subjects samples are compared. Upon calling the presence of IQ-affecting SNPs (2) across all STG samples (ST1c) it has been revealed that 15 out of 16 subjects were positive for the T-allele of rs2149171 (exon 4-nested), shown above to attenuate the WM score in SCZ patients, and making a comparison among the allele carrier vs non-carrier impossible.Four healthy and three SCZ samples carry a T-allele of rs3824574 (exon 3-nested, non-affecting IQ), and 1 healthy and 1 SCZ sample each contains a C-allele of rs4915045 (exon10, non-coding part-nested, and non-affecting IQ). Thus, among the eleven cognitive endophenotype-associated SNPs only 3 were possible to call out of the available NTNG transcriptome. Distinctly complementary nature of the NTNG paralogs segregation within neurological disorders and RNA transcriptome usage in STG (Figure 1) has prompted us to analyse both genes expression across the entire human brain.We have reconstructed both genes expression profiles in the human brain areas over the life span from conception (pcw = post-conception week) to mature age (30-40 yrs old) using the RNA-seq data from BrainSpan (www.brainspan.org).Similarities and differences are easily noted when the age-dependent phases of NTNG1 and NTNG2 expression profiles are matched (Figure 2).Based on the visual inputs three distinct classifiers have been elaborated: 1. predominantly synchronous (Figure 2A(1-4)), characteristic mostly for the cortical areas; 2. predominantly mixed and asynchronous (Figure 2B), characteristic for the cerebellar cortex and subcortical formations; and 3. anti-phasic (complementary, Figure 2C), characteristic for the MD of thalamus and hippocampus.All analysed brain areas demonstrate an elevated level of NTNG2 expression in comparison to NTNG1 except for thalamus (Figure 2C) with the largest difference observed 6 at the time of birth (35-37 pcw) or soon after (4 mo) for the synchronous classifiers (Figure 2A), oscillating increment values across the life span for the mixed (Figure 2B) and antiphasic (Figure 2C) classifiers.It is quite intriguing to note that essentially all brain areas show a trend towards the expression difference being negated between the paralogs by reaching the mature age of 30-40 yrs old (nearly or above the mean age used for the IQ testing), except MD where the expression discrepancy is increased.Thus, the observed functional complementation among the NTNG paralogs is supported by the anatomical distribution of the genes in human brain and their expression pattern modality over the human subjects lifetime. A direct comparison of the NTNG paralogs shows not only identical intron-exon gene structure (Figure 1B-1, 2B-2) but also closely matched exon sizes (Figure 3A).There are three exons of identical sizes (exons 4, 8 and 9), another three exons differed by one encoded amino acid (exons 3, 5 and 6) and there are exons of different sizes (exons 2, 7 and 10).In terms of size the largest difference among the genes is visually presented by the introns: intron (9-10) of NTNG1 is 52.7 times larger its NTNG2 paralogous intron with intron (6-7) of NTNG1 being only 1.43-times larger pointing towards non-equilibria process of non-coding elements elaborations as the process of gene paralogs SF proceeded.Nevertheless, it can be generalised that in average all NTNG1 introns are several times larger their NTNG2 analogs (Figure 3A).We have shown previously that exons 6 and 7 are differentially used within the brain NTNG transcriptome (Figure 1B-1 and B-2) and to explore their potential contribution into the paralogs SF we have built identity matrices with these exons being excluded and included (but still producing in-frame existing transcripts, Figure 3B-1 left and right panels, respectively).Exclusion of both exons from the full-lengths transcripts (thus converting NTNG1m to NTNG1a and NTNG2b to NTNG2a, respectively) increases the identity of DNA on 2% (a relatively large effect since both exons together represent only 7.22 and 9.69% of 7 the total coding part of the full-length RNA transcripts, NTNG1m and NTNG2b, respectively).This effect becomes even stronger when the encoded by these transcripts proteins are also compared (Figure 3B-2).The spliced out Ukd protein domains (encoded by the exons 6 and 7) increases the proteins identity on 3.8% thus making the middle of both genes (and encoded proteins) substantially more different among the both gene paralogs.To corroborate this observation and to explore the importance of other protein parts we have directly compared the sequences encoded by the full-length transcripts and producing Netrin-G1m and Netrin-G2b (Figure 3C).Similarly to what has been shown on Figure 3B-1 and 3B-2, the lowest identity (17.5%) is represented by the Ukd domain (encoded by the exons 6 and 7) and by the preceding it exon 5 (a 3'-part of the LE1 domain).Two other areas also show a substantially low identity, namely the N-terminus (it includes the protein secretory signal indicated by an arrow) and the outmost C-terminus responsible for the unique feature of Netrin-Gs -the GPI attachment.Thus, based on the percent identity comparisons among the Netrin-G paralogs it can be predicted that there are several potential protein parts contributing to the paralogs SF. As it has been reported by Seiradake et al. (13), identical gene and protein domain compositions result in the identical structural motif with differences only in the spatial arrangement of the loops facing the post-synaptic Netrin-G's interacting partners, NGL-1 and NGL-2, respectively (Figure 3D).Loop I binding surfaces alignment (Figure 3C Involvement of the pre-synaptically expressed axon-localised NTNGs in SCZ diagnosis supports the established view of SCZ as a product of distorted trans-synaptic signaling (14), with a recent study also proving that axonal connectivity-associated genes form a functional network visualisable by fMRI (15), and that brain connectivity predicts the level of fluid intelligence (16,17).Both NTNGs have been found to participate in the brain functional connectivity found by the parcellated connectome reconstruction (18).Most of the reported disease associations link NTNG1 to SCZ with a variety of other neurologic pathologies (15 in total, Figure 1A-1), while NTNG2 pathologic associations (6 in total, Figure 1A-2) are quite limited to those affecting WM or PO.Among them is SLE frequently characterized by WM deficit (19) and also known to represent schizoid-type abnormalities characteristic for autoimmune pathologies (20,21).Immune activation is known to lead to altered pre-pulse inhibition (a key diagnostic trait for SCZ) reversed by antipsychotics (22).The three diseases associated with both paralogs (ASD, BD and SCZ) are also a primary focus of the recently initiated PsychENCODE project (23).It is also worth to mention the resemblance of the reported disease associations with the behavioral phenotypes of Ntng1 and Ntng2 gene knockout mice (24). A gene content associated with attenuated IQ score often relates to numerous diseases, such as SCZ, ASD, depression, and others (see (25) for ref. ; 26) with several of them also undergone positive selection following the human brain evolution (27).Despite the fact that global network properties of the brain transcriptome are highly conserved, among the species there are robust human-specific disease-associated modules (28) and human accelerated regions (HARs) -highly conserved parts of genome that underwent accelerated evolution in humans (29).HARs can serve as genomic markers for human-specific traits underlying a recent acquisition of modern human cognitive abilities by brain (30) but that also "might have led to an increase in structural instability… resulted in a higher risk for neurodegeneration in the aging brain" (31), rendering our intellectual abilities genetically fragile (32) and resulting in a variety of CDs.The role genomic context, epistasis (33), plays in the evolution and pathology is manifested by frequently found disease-causing alleles present in animals without obvious pathological symptoms for the host (34).Any CD is characterized by general intellectual disability (GID) plus psychiatric symptoms.A genetic perturbation-exerted behavioral cognitive deficit (BCD) in an animal model organism is a poor match to a human CD per se due to very poor contextual resemblance between the human GID and animal BCD together with the absence of interpretable psychiatric symptoms. Usefulness of animals as psychiatric models is also compromised by the fact that transcriptome differences within species tissues is smaller than among the homologous tissues of different species (35,36).No wonder that the compounds that "cure" mice models consistently fail in human trials (discussed in (37)). NTNG paralogs brain transcriptome intrinsic complementarity and possible mechanism for the IQ-affecting mutation alleles effect.There is no global change at the mRNA level between healthy subjects and SCZ patients (Figure 1B).This conclusion is supported by previously published works stating that globally altered mRNA expression of NTNG1 or NTNG2 is unlikely to confer disease susceptibility, at least in the temporal lobe (38), and Brodmann's area (39).However, the original paper-source of the STG samples RNA-seq along with many other genes (>1,000) found that NTNG1 (but not NTNG2) falls within the group of genes with significant alternative promoter usage ((12): ST6, p<9.05E-10 at FDR <0.5) and NTNG2 (but not NTNG1) clusters with genes (>700) with significant alternative splicing change ((12): ST7, p<6.15E-12 at FDR<0.5) when SCZ and controls are compared. Such GWAS observation adds an extra layer of complementary regulation to both NTNG paralogs on a top of the described in the results section complementary usage rule for the exons, formed by unspliced splicing modules, resulting transcripts and comprising them exons (Figure 1B).Based on the available RNA-seq dataset it was almost impossible to detect RNA with the matched position of NTNG SNPs used for the IQ testing (ST2c) except for two coding exons located (rs2149171 and rs3824574) and exon 10 non-coding area located but transcribed rs4915045 (in 2 out of 16 samples).This fact points towards indirect effect of the IQ-affecting mutation alleles potentially associated with shorter (secretable) isoforms generation (Prosselkov et al., unpublished) lacking two of the most prominent NTNG features: GPI-link and the Ukd domain through an aberrant splicing factor binding. The GPI-link is a hallmark of Netrin-G family members (40, 41) and lacking it the aberrant Netrin-G isoforms are likely to mimic the action of their releasable ancestry moleculesnetrins, still being able to bind to their cognate postsynaptic ligand -NGL but without forming an axonal-postsynaptic contact and potentially dominant negative consequences.The Ukd domain of Netrin-G1, despite its so-far unknown function, is involved in lateral binding to the pre-synaptically localised LAR modulating the binding strength between NGL-1 and Netrin-G1 (42).Work is currently underway in search for a similar lateral interaction partner for the Netrin-G2 Ukd domain.Inclusion of the Ukd-encoding domain exons 6 and 7 is regulated by the Nova splicing factor (43) affecting the cortex Netrin-G1 exon 7 but not exon 6, and, simultaneously, Netrin-G2 paralog exons exhibiting an opposite pattern.In general, it is tempting to speculate that deregulation of NTNG transcripts processing may have a role in the brain-controlled cognitive abilities and associated CDs.Supporting such notion, a decreased level of Netrin-G1c mRNA (exons 6-9 excluded, Figure 1B-1) has been reported for BD and SCZ (38) with Netrin-G1d (exons 6 and 7 included but 8-9 excluded, Figure 1B-1) and Netrin-G1f (a secretable short isoform consisted of domain VI only and lacking the Ukd and GPI-link) being increased in BD, but not in SCZ, in anterior cingulated cortex (44). 12 Higher Netrin-G1d mRNA expression in fetal brain but low for the Netrin-G1c isoform in the human adult (38) indicates different functionality of these two splice variants joggling with the Ukd domain inclusion/exclusion pattern.And, according to our unpublished data, if Netrin-G1 Ukd-containing isoforms are the dominant isoforms in adult mouse brain, Netrin-G2 Ukd-containing isoforms are present only at the trace level (Prosselkov et al., forthcoming), resembling the human STG samples transcriptome pattern (Figure 1B-1 and B-2).A similar "dynamic microexon regulation" associated with the protein interactome misregulation has been reported to be linked to ASD (45). Synchronous and complementary expression of NTNG paralogs in the human brain supports the IQ-associated cognitive endophenotypes.Influential parieto-frontal integration theory (P-FIT, ( 46)) states that general intelligence ("g") is dependent on multiple brain cortical areas such as dlPFC, Broca's and Wernicke's areas, somatosensory and visual cortices (47).Despite "g" is widely accepted as the only correlate of the intelligence, its unitary nature was challenged by (48) claiming had indentified two independent brain networks (for memory and for reasoning) responsible for the task performance, the idea later criticised for the employed data processing approach (49).Higher IQ scores (a composite surrogate of "g") have been reportedly associated with the fronto-parietal network (FPN) connectivity (50,51). Higher levels of NTNG paralogs expression within the cognition intensively loaded areas of the brain and the distinct patterns of expression profiles (synchronous, asynchronous/mixed, and complementary, Figure 2A) support associations of NTNG1 and NTNG2 with the recorded cognitive endophenotypes (2).Based on the expression patterning over the human life-span, among the total 16 analysed brain areas we found two falling under the same "antiphasic (complementary)" classifier (Figure 2C): HIP and MD.Adding more to that, MD is the only brain area (out of the 16 presented) where NTNG1 expression level exceeds that of NTNG2 making it a promising candidate for the phenomena of NTNGs SF explanation.Two 13 other brain areas classified by a synchronous paralogs expression deserve a special attention, dlPFC and mPFC (Figure 2A-4).PFC circuitry has been known as a "hub of the brain's WM system" (52,53), which acts through direct HIP afferents (54) and has many connections with other cortical and subcortical areas (55).mPFC may function as an intelligence-control switchboard and lPFC, part of the FPN global connectivity, predicts the WM performance and fluid intelligence (56).Interactions of the auditory recognition information fed by the vPFC stream with the sequence processing by the dorsal stream are crucial for the human language articulation (57; 58).The fact that both NTNG paralogs are extensively expressed across PFC (Figure 2A-2 and A-4) pinpoints this area as a key for future molecular studies of NTNGs and the human-unique symbolic communications.And PFC is not only implicated in many psychiatric disorders, including SCZ ((59); see also (55) for ref. ), but is also the only brain structure unique to primates without known homologs in the animal kingdom (60). Evolution of the protein paralogs encoded by the NTNGs.Forkhead box P2 (FOXP2) -a ubiquitously expressed transcription factor that has been reportedly linked to the evolution of human language through T303N, N325S substitutions when compared to a primate ortholog (61), is 100% identical to Nea protein (62).FOXP2 regulates expression of multiple genes in human and chimpanzee (63), and among them is an M3 gene brain module representative responsible for general fluid cognitive abilities (26), LRRC4C, a gene encoding NGL-1, a post-synaptic target of Netrin-G1.Similarly to FOXP2, Netrin-G1 is a 100% conserved protein among the hominins with only 1 mutation found in chimpanzee which is absent in marmoset (and other primates) and mice proteins (2).On the other hand, extinct hominins' Netrin-G2 relative to modern human contains T346A point mutation (as per current version of hg19), also found in primates and mouse and known as rs4962173 (dbSNP missense mutation) representing an ancient substitution from Neandethal genomes found in modern humans and reflecting a recent acquisition of the novel allele around 5,300 yrs BC.Nothing 14 is known regarding the functional significance of this mutation allele but biochemically a substitution of alanine (A) on a polar threonine (T) could bring an extra point of posttranslational modification, e.g. a phosphorylation or glycosylation (NetPhos2.0(64) assigns a low probability score for the T346 to be phosphorylated but NetOGlyc4.0(65) robustly predicts it to be glycosylated, see SM).Another mutation, S371A/V, reflects a selective sweep in Netrin-G2 protein from primates to hominins within a similar to T346A functional context when a hydrophobic alanine (in chimpanzee, A)/valine (in marmoset, V) is replaced by a polar serine (S) with a strong positive predictions for glycosylation but not phosphorylation (see SM).This poses a question whether these two human-specific protein substitutions associate with advanced cognitive traits as they may represent a hidden layer of poorly studied so far protein glycosylation-associated regulatome known to affect the brain function and diseases (66,67).Adding more to this, T346 is nested on exon 5 just 20 nu away from the affecting WM score rs2274855 mutation allele (2), and, together with S371A/V, they are both located within the lowest percent identity area (exons (5-7)) of Netrin-Gs (Figure 3C) and, proposedly, contributing to the NTNG duplicates SF.There are at least three more protein parts potentially contributing to the gene paralogs specialised function subdivision (based on the low identity scores, Figure 3C): the secretory peptide, the GPI-link, and the outmost structurally elaborated unstructured loops (I-III) responsible for the reciprocal binding of Netrin-Gs to their post-synaptic cognate partners, NGL-1 or NGL-2, both containing a C-terminal PDZ-binding domain (68).An interesting finding was reported in (69) reported a presence of SH3(PSD95) domain binding site (required for the phosphatidylinositol-3-kinase recruitment) in mice Netrin-G2 (100% identical to human) but not in Netrin-G1.The detected SH3 binding site overlaps with the Netrin-G2-loop III responsible for the binding specificity to NGL-2 (12,70,71).A plausible working hypothesis would be that while internalised (and being GPI-link naïve/immature) the pre-synaptic Netrin-G2 is bound to SH3-PSD95 via loop III but as soon as being secreted extracellularly (and being attached to the membrane) it is bound to post-synaptic NGL-2.Corroborating this, in the absence of Netrin-G2 in the KO mice NGL-2 is unstable on the post-synaptic surface and gets quickly internalised (24).We can only speculate regarding the potential importance of PSD-95(SH3)-Netrin-G2-NGL-2 scaffolding loop interaction/competition but the ability for Netrin-G1 to bind to SH3 has not been reported.Following this logic, Netrin-G1 should have a similar binding partner via loop II. The overall identical structural scaffold among the Netrin-G paralogs (Figure 3D) is likely to represent an anciently preserved one of the primordial protein (encoded by a single gene in the primitive urochordate C.intestinalis) and its contribution to the process of SF among the NTNG paralogs goes against the "structure drives function" concept.It looks like that it is not the "structure" but rather the "evolution" itself that drives a selection for the best structural (or unstructural in our case) fit out of the available frameworks provided by the gene duplicates to fulfill the emerged functional demand in a new ecological niche.The intricate variability of phenotype is grounded by the conserved nature of genotype and constrained by the "structure-function" limitations of the coding DNA and is only possible due to permissive evolutionary continuing elaborations of non-coding areas able to absorb the most recently acquired elements (having a potential to become regulatory at some point, e.g.like HAR5 (30)) and carried over by the neutral drift.At the same time, the multiple protein substitutions coinciding with the SF labor segregation phenomena among the Netrin-G paralogs question their neutral nature.Both of them undergo a purifying selection from mice to human through the reduction in size of non-coding DNA (introns) and encoded proteins (the mice Netrin-G2 is 2 aa longer its human ortholog) further contributing to the hostspecific SF.Thus while the non-coding sequences are used to explore the evolutionary space in time, the restrictive boundaries of the paralogs SF are determined by the protein (unstructured) elements. Molecular evolution of the Cognitive Complement (CC). Appearance of the neural crest (72), an event that "affected the chordate evolution in the unprecedented manner" (73), multipotent progenitor cells (74), and neurogenic placodes (suggesting a chemosensory and neurosecretory activities (75)) in the first primitive urochordates/tunicates coincides with the presence of Ntng precursor gene (ENSCING00000024925) later undergoing two rounds of duplication events in fish and found to affect human cognitive abilities (2).NTNG paralogs are expressed in the human neural crest-forming cells with NTNG2 10 times stronger than NTNG1 (76), both are differentially expressed in human comparing to chimpanzee and rhesus monkey with NTNG2 expression model showing stronger probability than NTNG1 (77), and both are stronger expressed in human telencephalon comparing to chimpanzee and macaque (78).NTNG1 has been classified as a brain module hub gene "whose pattern fundamentally shifted between species" (18).Belonging to distinct modules of brain expression regulation (78,79), NTNGs are classified as "genes with human-specific expression profiles" (79).The nearby gene ~260 kbp upstream of NTNG2 is MED27 (mediator of RNA polymerase II) has been proposed to be associated with the evolution of human-specific traits (80).NTNG1 has also been reported among the "adaptive plasticity genes" (81) potentiating rapid adaptive evolution in guppies (NTNG2 was not found in the input RNA). Complementarity among the NTNG paralogs and encoded by them proteins has been reported previously: brain expression complementary pattern (in almost self-exclusive manner) defined by the 5'-UTR-localised cis-regulatory elements (82); complementary distribution within the hippocampal laminar structures (83); axon-dendrite synaptic ending resulting in differential control over the neuronal circuit plasticity (84); mutually-exclusive binding pattern to post-synaptic partners, NGL-1 and NGL-2, dictated by the protein unstructural elements (13); alternative promoter usage vs alternative mRNA splicing (12) and increased coefficient of variation (CV, ST1d) for NTNG1 expression but not NTNG2 in SCZ patients (also reported in ( 85)); KO mice behavioral phenotypes and subcellular signaling partners complementarity (24); "differential stability" brain modules expression (NTNG1 is expressed in the dorsal thalamus (M11) as a hub gene (Pearson's 0.92) while NTNG2 is in neocortex and claustrum module (M6, Pearson's 0.65)) (18); hypocretin neurons-specific expression of NTNG1 (but not NTNG2) as a sleep modulator (86); top-down vs bottom-up information flows gating in mice and differential modality (Prosselkov et al., forthcoming), and human IQ-compiling cognitive domains complementation (2).The current study reports on NTNGs complementarity association with the CDs (Figure 1A); mRNA splicing pattern complementary at the quantitative and qualitative levels via differential use of the middlelocated exons (Figure 1B); brain complementary oscillatory expression over the human life span observed in the intensive cognitively loaded brain areas (Figure 2); AE of the paralogssegregated unique non-coding elements (Figure 3A); complementary pattern of the protein orthologs (mice-to-human) protein sequence evolution.Such multi-level complementation is likely to reflect a shared evolutionary origin from a single gene in a primitive vertebrate organism 700 mln yrs ago and its subsequent functional segregation among the evolutiongenerated gene duplicates in jawless fish, such as lamprey. Occupying independent but intercalating functional niches, NTNG1 and NTNG2 do not compensate but complement each other's function forming a "functional complement" of genes.Half a billion yrs ago the doubled gene dosage led to the gradual SF and manifested in a function complementation within the cognitive domains, at least in human.We would like to coin such gene pair as a Cognitive Complement (CC). CONCLUSION The emerged functional redundancy, as an outcome of gene duplication, leads to function 18 subdivision and its bifurcation among the gene paralogs resulting in the paralogs SF.A functional compensation is known to exist among the evolutionary unrelated genes but has not been reported among the gene paralogs, more frequently characterized by the function complementation.Gene paralogs structural identity (at both, gene and protein levels) does not provide a substrate for function compensation but rather for complementation, perturbating "structure drives function" rule.A gene duplication event of a tunicate NTNG primordial gene and the subsequent process of its function specialisation (driven by the new ecological niches appearance and evolution) among the gene duplicates made them to SF into distinct cognitive domains in a complementary manner forming a CC.In our forthcoming work we are to describe how Ntng mice genes function resembles that of human orthologs (Prosselkov et al., forthcoming). MATERIALS AND METHODS Human brain NTNG transcriptome reconstruction.Relates to Figure 1B and 1-C.The original source of the dataset was produced by (( 12): E-MATB-1030) and the downloaded .bamfiles used for the re-processing are listed in ST1a.All reconstructed transcripts are presented in ST1d standalone Excel file.Two samples were excluded from the analysis due to failed "per base sequence quality" measure, and zero expression level for NTNG1a and NTNG1int (9)(10) otherwise consistently expressed throughout other samples (ST1b).SAMtools software was used for the SNPs calling from the available RNA-seq datasets (ST1c).For details refer to SM. Human brain expression profiling for NTNGs across the life span.The original source of data was www.brainspan.org.All available samples were initially included into the analysis but two of them excluded at a later stage (MD for 12-13 pcw and mPFC for 16-19 pcw) due to high deviation (6-7 times) from the mean for other replicas.The mean expression values per each brain area as RPKM were plotted against the sampling age.Profiles classification was done visually considering the trend over the all plotted points as an average.NTNG1 (NTNG1m) and NTNG2 (NTNG2b) full-length mRNA transcripts assembly.Relates to Figure 3B.Human NTNG1m brain transcript has been reported previously (125) and we have also confirmed its ortholog presence in the mice brain via full-length cloning (42).Since NCBI contains only its partial CDS (AY764265), we used the RNA-seq-generated exons (Figure 1B) to reconstruct its full-length and to generate an ORF of the encoded Netrin-G1m. Similarly, human NTNG2b was reconstructed from the RNA-seq dataset and from Ensemble as follows.Exon 5 sequence was deduced from ENST00000372179, other exons were from ENST00000467453 (no longer available on the current version of Ensemble) except for exon 6 deduced by running three independent alignments against the human genomic DNA with the mice 3'-intron (5)(6), exon 6, and 5'-intron (6)(7) 9 DISCUSSION , blue color)shows a high level of conservation (with at least 5 amino acids 100% conserved) among the Netrin-G paralogs, indicating that it is unlikely to be responsible for the cognate ligand binding specificity.Neither Loop II (Figure5C, yellow color) nor Loop III (Figure5C, orange color) display a single conserved amino acid shared among the paralogous binding interfaces (as it has originally been described in(13)).Thus the complementary pattern of the pre-postsynaptic interactions mediated via specific Netrin-G/NGL pairs is reflected in the Complementary contribution of NTNG paralogs into human cognitive pathologies. 88 Netrin-G2b 77 exon 5 +Figure 3 . Figure 3. Human NTNG paralogs DNA and protein sequence comparisons and "structure-function" rule incongruency.(A) Identical gene structures with different sizes of introns.RNA-seq data from Figure 1B were used to precisely deduce the exon/intron junction boundaries.The sizes of exons 1, 10 and introns (1-2) are not indicated due to observed among the splice transcripts lengths variability (see ST1a for details).Arrows indicate location of CNS = conserved non-coding sequences underwent accelerated evolution in human compare to mice (mCNS) and chimpanzee (chCNS), as per (124); and ASC = anthropoid-specific constrained regions in human compare to marmoset (maASC), as per (121).(B) Identical exonal composition of the longest NTNG encoded RNA paralog transcripts and corresponding proteins with relatively high percent of identity among them dependent on the included/excluded Ukd domain (B-2) encoded by the exons 6 and 7 (B-1).Notably, the protein sequence represents higher percent of the paralogs difference than encoded it DNA.The matrices were obtained by GeneJockey II (Biosoft).(C) Protein alignments for the longest human NTNG encoded proteins, Netrin-G1m and Netrin-G2b, with Loops I-III highlighting binding sites for their cognate post-synaptic binding partners NGL-1 (Lrrc4c) and NGL-2 (Lrrc4), respectively, as determined by Seiradake et al. (13).Arrow indicates a putative secretory cleavage site location, as calculated by SignalIP (122), the blue rectangle delineates the area of the lowest identity (3'-domain LE1+Ukd domain); ω -denotes a point of putative GPI-attachment, as predicted by Big-PI (123).PSD-95 interaction site via the SH3-binding domain ((69), as determined for mice Netrin-G2) overlaps with the Loop III NGL-2 binding surface.Two stars indicate a modern human (T346A) and a hominin-specific (S371A/V) amino acid substitutions (2).(D) Identical structural motif of the Netrin-G1/NGL1 and Netrin-G2/NGL2 complexes as per (13).The figure's reproduction is covered by the Creative Commons license.!	2016-11-01T19:18:48.349Z	2016-01-09T00:00:00.000	{ "year": 2016, "sha1": "25281f5aa4203d0cde9ccd15df38553b18306c8c", "oa_license": "CCBY", "oa_url": "https://www.biorxiv.org/content/biorxiv/early/2016/04/04/034645.full.pdf", "oa_status": "GREEN", "pdf_src": "Anansi", "pdf_hash": "84e56a4e1b98eceadf6a350caf0d2fdef143d428", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Biology" ] }
230576363	pes2o/s2orc	v3-fos-license	Mycotoxins Analysis in Cereals and Related Foodstuffs by Liquid Chromatography-Tandem Mass Spectrometry Techniques In the entire world, cereals and related foodstuﬀs are used as an important source of energy, minerals, and vitamins. Nevertheless, their contamination with mycotoxins kept special attention due to harmful eﬀects on human health. The present paper was conducted to evaluate published studies regarding the identiﬁcation and characterization of mycotoxins in cereals and related foodstuﬀs by liquid chromatography coupled to (tandem) mass spectrometry (LC-MS/MS) techniques. For sample preparation, published studies based on the development of extraction and clean-up strategies including solid-phase extraction, solid-liquid extraction, and immunoaﬃnity columns, as well as on methods based on minimum clean-up (quick, easy, cheap, eﬀective, rugged, and safe (QuEChERS)) technology, are examined. LC-MS/MS has become the golden method for the simultaneous multimycotoxin analysis, with diﬀerent sample preparation approaches, due to the range of diﬀerent physicochemical properties of these toxic products. Therefore, this new strategy can be an alternative for fast, simple, and accurate determination of multiclass mycotoxins in complex cereal samples. (AFG 1 ), aﬂatoxin (AFG 2 ) , diacetoxyscirpenol (DAS), fumonisin B 1 (FB 1 ), fumonisin B 2 (FB 2 ), ochratoxin A (OTA), HT-2, T-2, ZEN, and beauvericin (BEA)) by LC-MS/MS. Introduction Most people of developed and developing countries use cereals and cereal-based products as their primary source of nutrients and energy [1][2][3][4]. Nevertheless, due to rich contents of fat, protein, and minerals, they are providing a great environment for fungal growth [5,6]. Contamination of cereals in preharvest and postharvest stages with fungi can lead to the production of mycotoxins [7][8][9]. In this line, some environmental agents such as humidity, temperature, inadequate storage conditions, insect damage, and drought play important roles in the level and diversity of contamination by mycotoxins [10][11][12]. In addition, the incidence and mycotoxins concentration in cereal-based food products can be associated with some factors, such as physical and chemical food characterization (pH, composition, and water activity), production management (storage, harvesting, and conditions of processing), and weather status (humidity and temperature) [13][14][15][16]. For the determination of mycotoxins in cereals and related foodstuffs, sampling of nonhomogeneous compounds and the analytical techniques are strongly important. In this line, proper sampling techniques must be put in place to obtain representative results. erefore, sample selection, sample size, and number of incremental samples must be well recognized due to the mycotoxin heterogeneous distribution within the lots [48,49]. Since fungal growth is limited to certain locations in the lot and arbitrarily distributed, fungi contamination and mycotoxin production are considered as "spot processes" [48]. According to the Commission Regulation (EC) No. 519/2014 [50], from lots ≥50 tonnes, incremental sample number must be a minimum of 100, with a total of 10 kg of aggregate samples. For lots <50 tonnes, 3 to 100 incremental samples should be collected, with a corresponding aggregate sample weight of 1 (minimum weight) to 10 kg. In the case of lots >500 tonnes, the representative sample should be at least 10% of the lot. Analysis of mycotoxin in cereals and related foodstuffs is a decisive practice to approve food security. Several detection methods have been established, among the most common currently used are LC-MS/MS methods. When compared to other separation and detection techniques, LC-MS/MS methods present very high analytical sensitivity. Extraction procedures and suitable clean-up, providing good recoveries and reducing matrix effects, are consequently extremely important to analytical method development and optimization. In this way, aqueous solvents and/or acidic solvents are crucial for quantitative extraction of FBs or OTA, while high organic solvents are suitable for mycotoxins such as AFs, OTA, and ZEA [51][52][53]. On the other hand, clean-up procedures towards mycotoxin analyses are largely performed by solid-phase extraction (SPE) or immunoaffinity columns (IAC) [54,55]. Based on solid samples such as cereals and related foodstuff samples, SPE was used as a clean-up and/or concentration step following a prior extraction procedure [56,57]. Several SPE columns are commercially available, with different solid phases ranging from C18 materials (ion exchange) to more specific adsorbent materials [56,58,59]. IAC, a method based on the interaction between antigen and antibody, displays some advantages, including a minimal loss of mycotoxins and a maximal elimination of interfering substances [60][61][62]. Compared to SPE extraction, the utilization of IAC as a clean-up procedure could greatly improve the specificity of subsequent analysis [54]. Other comparable clean-up procedure includes the QuEChERS-like method, which offers the opportunity to extend the number of analytes to be analyzed by a less time-consuming approach [58,63]. According to Amirahmadi et al. [64], this method involves extraction with acetonitrile and partitioning clean-up after the addition of a salt mixture (MgSO 4 and NaCl). Remarkably, QuEChERS is reliable with a number of advantages, such as simplicity, minimum steps, and effectiveness in cleaning-up complex samples [65]. Consecutively, the present review presents an emphasized overview on the development, optimization, and validation of LC-MS/MS-based methodologies towards the analysis of mycotoxins in cereals and related products. In addition, clean-up and extraction procedures and chromatographic and detection parameters, as well as the analytical method performance process, were well discussed. Analytical Methods: Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) e basic principle of MS/MS is the selection and fragmentation of precursor ion and measurement of the m/z ratio of the product ions formed [73,74]. ere are two fundamentally different approaches to MS/MS: tandem mass spectrometry in space or in time [75]. e triple quadrupole (QqQ) was the frequently used space instrument tandem mass spectrometry in space. Equally, other examples of tandem mass spectrometers included quadrupole-time-of-flight (QqToF) and Orbitrap hybrid instruments [76][77][78][79]. However, tandem-in-time instruments are typically ion-trapping mass spectrometers, which comprise 3D quadrupole ion traps (QIT) [80], linear ion traps (LIT) [81,82], and Fourier transform-ion cyclotron resonance (FT-ICR) instruments [83,84]. After extraction with acetonitrile/water, QqQ LC-MS/ MS methods were examined for the quantification of TCTs and ZEA in cereals by using electrospray ionization (ESI) [85] and atmospheric pressure chemical ionization (APCI) [86,87] interfaces. Cavaliere et al. [88] presented their method for the determination of 8 TCTs, three FUM, ZEA, and alphazearalenol in corn samples and used ESI QqQ MS in both polarity modes. A positive-ion mode ESI QqQ LC-MS/MS method for the simultaneous determination of 16 mycotoxins on a cellulose filter was developed by Delmulle et al. [89]. In targeted mycotoxin determination LC-MS/MS, analytical methods using a QqQ and linear ion trap (QLIT) mass spectrometer are the most commonly used procedures [90]. e combination of QqQ MS (QqQ/QLIT) is valuable because this instrument retains the selective reaction monitoring mode (SRM) [75,91,92]. Rapid multimethods based on QqQ/QLIT approaches are able to analyze simultaneously up to 300 mycotoxins and also their metabolites or other related food contaminants depending on the length of the chromatographic run [92][93][94][95]. e sustained development of mass spectrometers, including Orbitrap-based systems as well as other instrument platforms such as the QTOF, was thus driven by aims of accelerating scan speed and increasing sensitivity [96,97]. is instrument can be defined as a triple quadrupole where the last quadrupole is substituted by an oa-TOF or as the addition of a collision cell to a TOF analyser and a quadrupole analyser [97]. To perform fragmentation with higher-energy collisional dissociation (HCD), a gas-filled quadrupole (the HCD cell) was fitted directly after the C-trap [98]. Besides, it has been stated that TOF and Orbitrap analyzers, with resolving power of 10,000-100,000 and 140,000-240,000 (full width at half maximum defined at m/z), were used respectively [75]. ese analyzers are very sensitive making easier the analytes identification giving accurate results even when we are dealing with very low levels of analytes. Some authors have exploited their potential in the quantitative analysis of mycotoxins showing higher significance for Orbitrap [99]. A new generation of hybrid techniques such as the Q-orbital ion trap (Q Exactive) instrument combines the advantages of high-performance quadrupole selection of precursor ions with those of high-resolution mass detection [100,101]. e subsequently developed Q Exactive instrument allowed precursor ion isolation on an exactive-type mass spectrometer. For isolation of precursors, a mass filtering quadrupole was utilized [101,102]. ereafter, for detection, the HCD cell voltages are ramped and ions are conveyed back into the C-trap from where they are injected into the Orbitrap. In fact, structural information can be obtained on compounds of interest and fragment ions can be used for confirmation in targeted analyses [102]. Regarding identification, metabolite ions in a full scan spectrum (MS) are subsequently isolated to generate MS/MS spectra; data-dependent acquisition (DDA) approach is the most common strategy [103,104]. ereafter, metabolite structure is elucidated through MS/MS spectral similarity corresponding to the standard metabolite spectral library. In this context, Human Metabolome Database (HMDB) [105], METLIN [106], and MassBank [107] are frequently referred to as a spectrum-centric approach. MassBank is the first public source of mass spectra of small chemical compounds for life sciences (<3000 Da) [107], while METLIN includes an annotated list of known metabolite structural information that is easily cross-correlated with its catalogue of highresolution Fourier transform mass spectrometry (FTMS) spectra, MS/MS spectra, and LC/MS data [106]. Application of DDA in analysis of mycotoxins was demonstrated in several recent studies [108,109]. Nevertheless, DDA suffers from numerous limitations. For example, in one experiment, not a limited number of ions with highest abundance detected in the full MS scan are isolated and fragmented in a product ion scan experiment [110][111][112]. Also, the selected precursor ions may be derived from many adducted ions instead of molecular ions [113,114]. If applied to the analysis of mycotoxin-contaminated foodstuff, these problems would be aggravated since these metabolites habitually occur at lower concentrations, and absolute quantification is critical for compliance with regulatory limits [109]. Technological advances have greatly increased the resolution, speed, and sensitivity of mass spectrometers. is has allowed for new types of nontargeted methods to become more practicable, precisely data-independent acquisition (DIA). It should be noted that the DIA approach depends on the width of the isolation window, and many ions can be cofragmented. Consequently, the product ion spectra are more complex compared with targeted methods and at each segment producing one multiplexed MS/MS spectrum derived from multiple precursor ions [115,116]. DIA approaches have been established on each of the Orbitrap mass spectrometer platforms to take benefit of their specific architectures. Development in the area of DIA included methods such as wide isolation window SIM scan DIA (WiSIM-DIA) on the Orbitrap fusion mass spectrometer [117]. is approach utilized an ultrahigh-resolution SIM scan for quantification, complementary with classic DIA. In proteomics, several data MS analysis methods and programs, such as DIA-Umpire [118] and Skyline [119,120], were used. In this line, DIA-Umpire, a comprehensive computational workflow and open-source software for DIA data, detects precursor and fragment chromatographic features and assembles them into pseudo-MS/MS spectra which can be identified using conventional database searching and protein inference tools without the need for a spectral library [119]. In the same way, Egertson et al. [120] described the use of DIA on a Q-Exactive mass spectrometer for the detection and quantification of peptides in complex mixtures using the Skyline Targeted Proteomics Environment. e most promising feature of DIA analysis of mycotoxins is that the data generated is ideal for retrospective analysis. Newly characterized mycotoxins can be identified in archived data by high-resolution precursor mass, retention time, and multiple product ions. High-resolution MS alone has been used to collect data that can be retrospectively analyzed for the presence of mycotoxins [109,120,121]. In this vein, Renaud et al. [109] reported the development of a powerful LC-DIA analysis method on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer for mycotoxin analysis produced by Fusarium graminearum in maize. On the contrary, Berthiller et al. [122] reported a method detection limit of 0.012 g/ml for D3G in purified sample extracts, corresponding to 0.02 g/g in contaminated cereals. ese authors also estimated their LOD from the signal intensity of their standards, based on the limited ion suppression they observed. e pigment LOQ and LOD were 4.3 and 0.0005 g/kg, respectively. Good linearity for the pigment standard curve (R 2 0.999) was also observed. In LC-MS, the majority of multimycotoxin methods used ESI interface. In fact, positive-mode ESI is exclusively applied to couple high-performance liquid chromatography (HPLC) or ultrahigh-performance liquid chromatography (UHPLC) and MS detection [73,[123][124][125][126]. is technique has been effectively used for the synchronized quantification of mycotoxins with different chemical structures [54] in one single run [89,126]. e LC/MS-MS technique has been reported by many studies in multimycotoxin determination, such as 17 different mycotoxins in barley and malt [127]. Current Methods Used for LC-MS/MS Determination of Mycotoxins in Cereals and Related Products e approaches include those used for screening and quantification in both official control and research. It should be noted that the approaches discussed mostly have been developed for the determination of EU-regulated mycotoxins in various food matrices to strictly respond to the EU legislation [128]. Despite the interesting benefits that could procure MS/MS as a very selective technique, its signal could be overestimated and lost in the case of some challenging samples leading finally to false positive results. Also, although LC-MS is considered to be a highly sensitive analytical technique, trace detection levels of some analytes seem impossible especially when compromises related to sample preparation and LC-MS/MS conditions have to be made. ese methods are developed based on the QuEChERS approach [129]. is approach was established for a very rapid extraction and purification with regard to multipesticide analysis. Its relevant principle relies on the partitioning of an acetonitrile-water mixture induced by addition of inorganic salts. In general, LC-MS/MS techniques including QuEChERS approach are ineffective for the AFs and OTA detection in baby foods at the EU limits. erefore, for these particular metabolites, specific clean-up methods with immunoaffinity columns (IACs) or combinations with another clean-up technique are used [52]. e application of immunoanalysis for a rapid screening of mycotoxins represents an attractive analytical method commonly used nowadays. e main criteria for research of such approaches include simplification and rapidity of analysis, sensitivity improvement, and matrix effect reduction. Immunoassays generally applied for rapid detection of individual mycotoxins are summarized in a review concerning immunochemical assays [52]. e common immunomethods applied for mycotoxin detection rely on binding of specific antibodies to a solid support (direct competitive ELISA format) or coated antigens (indirect competitive ELISA format). ese formats are used in all nonhomogenic methods: microtiter plate immunoassays and sensors. Homorganic methods implicate the fluorescent polarization and capillary electrophoretic immunoassays [52]. Lattanzio et al. [52] detected and quantified aflatoxins (B 1 , B 2 , G 1 , and G 2 ), ochratoxin A, fumonisins (B 1 , B 2 ), deoxynivalenol, zearalenone, T-2, and HT-2 toxins in maize. In fact, reversed-phase liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (LC/ESI-MS/MS) was used as chromatographic mobile phase, a linear gradient of methanol/water containing 0.5% acetic acid and 1 mM ammonium acetate. e method exhibited good linearity; also, matrix-coordinated calibration curves for all analytes were linear over the relevant working range with r (coefficient of correlation) values between 0.9980 and 0.9999 [52]. In addition, recoveries higher than 79% were obtained for all tested mycotoxins with relative standard deviations less than 13%. ese authors reported that method performances were quite satisfactory for all tested mycotoxins at contamination levels close to or below the relevant EU maximum permitted or recommended levels. Limits of detection (LOD) in maize ranged from 0.3 to 4.2 μg/kg [52]. ese LODs are similar with or slightly lower than those reported by other authors using MRM detection for the analyses of the same mycotoxins in maize or maize-based food extracts after SPE cleanup [86,88,89]. On the other hand, QuEChERS procedure has been used for the development of an LC-MS/MS assay for the determination of 17 mycotoxins in cereals for human consumption and infant cereals [129]. All tested matrices gave LOQs below the maximum levels except for AFLA B1 in infant cereals (maximum level � 0.1 μg/kg, LOQ � 1 μg/kg). Matrix effects were nevertheless more important in soya (LOQ for the aflatoxins B 1 , B 2 , G 1 , and G 2 � 2 μg/kg) and even more in corn gluten (pet food material). Higher LOQs were thus obtained in corn gluten (pet food ingredient) for which no regulatory limits have been established [130]. ese authors, also, have chosen the ESI + mode since the sensitivity of critical compounds with low maximum levels (i.e., aflatoxins B 1 , B 2 , G 1 , and G 2 and OTA) was visibly enhanced. In contrast, an acceptable sensitivity for ZON, as [M − H] − ion, was only obtained in the ESI-mode. At the same time, the addition of ammonium formate to the aqueous mobile phase clearly enhanced the sensitivity for both type A and B TCTs detected under their ammonium adduct [M + NH 4 ] + , whereas formic acid in both mobile phases increased the overall sensitivity, giving better peak shape for the acidic compounds, i.e., FB1, FB2, and OTA [131]. Modified QuEChERS which used acidified acetonitrile (ACN), MgSO 4 , NaCl, and citrate buffer salts, combined with dispersive solid-phase extraction (d-SPE) clean-up and followed by LC-ESI-MS/MS method, was applied for the determination of EU-regulated mycotoxins in several cereals such as wheat, maize, and rice [132]. In cereals, aflatoxins, ochratoxins, fumonisins, trichothecenes, and zearalenone were detected and quantified. e performance of the method was assessed and compared to European Commission (EC) Regulations, by studying the selectivity, specificity, LOD, LOQ, linear dynamic range (LDR), matrix effect, accuracy, precision, and uncertainty. In this context, Fernandes et al. [132] reported a good linearity (r 2 > 0.9713) for all mycotoxins investigated, and LODs (S/N � 3) and LOQs (S/N � 10) were below the tolerance levels of mycotoxins set by EC. Recoveries of the extraction process, obtained with different spiked concentrations, ranged from 72.9 to 120.6%, with relative standard deviations (RSD) lower than 23.0%. Rubert et al. [133] reported the comparison of four different extraction techniques used in the determination of 32 mycotoxins in barley. ese methods included QuEChERS modification, matrix solid-phase dispersion (MSPD: extraction MeCN/MeOH, 50/50, v/v), supported liquid extraction (SLE: extraction MeCN/water/acetic acid, 79/20/1, v/v/v), and solid-phase extraction (SPE, previous SLE extract). Accordingly, it has been shown that modified QuEChERS method was faster and easier than the other methods. Also, it enables to extract well all of the mycotoxins (from 64.1% DON-3-G to 93.4% T-2). ese authors validated the method according to the directive and guide on that subject [134]. In this regard, confirmation of identity, specificity/selectivity, linearity, lowest calibration level (LCL), ranging between 1 and 100 μg/ kg for enniatin B (ENB) and NIV, respectively was done. e precision, process efficiency, and recovery were, also, studied [135]. Remarkably, Rubert et al. [133] reported that the UHPLC-HRMS was a robust technique for validation and routine mycotoxin analysis. is latter technique showed sensitivity and selectivity to identify simultaneously 32 mycotoxins. Serrano et al. [136] studied the contents of 14 mycotoxins in samples of different cereal (rice, wheat, maize, rye, barley, oat, spelt, and sorghum) and cereal products (snacks, pasta, soup, biscuits, and flour) from four countries of the Mediterranean region (Spain, Italy, Morocco, and Tunisia). Samples were extracted with matrix solid-phase dispersion (MSPD) and determined by liquid chromatography-tandem mass spectrometry with a triple quadrupole mass analyser. e frequency of contaminated samples from Spain, Italy, Tunisia, and Morocco was 33%, 52%, 96%, and 50%, respectively. For legislated mycotoxins (AFs, FBs, DON, ZEN, and OTA), the LOQs were lower than the MLs established by the European Union (EC 401/2006) [137]. For fumonisins (FBs), the levels ranged from <LOQ-184 μg/kg for FB 1 , and from 121 to 176 μg/kg for FB 2 . e maximum FB 1 value (184 μg/kg) was found in a wheat pasta sample from Tunisia, and the maximum FB 2 value (176 μg/kg) was found in a rice grain sample from Morocco. ese results were lower than those obtained in other studies for maize, wheat, rice, and barley products [138][139][140]. Recoveries of fortified cereal samples at two spiked levels ranged between 68.7-89.6% and 72.6-87.6%; in addition, the relative standard deviations varied from 3% to 14%. ese values agree with EU criteria [141]. In addition, all mycotoxins exhibited good linearity over the working range (low concentration level at LOQ), and the regression coefficient of calibration curves was higher than 0.992 [136]. Otherwise, Lacina et al. [142] have performed different extraction methods for the simultaneous analysis of 288 pesticides and 38 mycotoxins. In fact, three different extractions were carried out for wheat and other products: aqueous acetonitrile extraction followed by a modified QuEChERS method (method A), aqueous acetonitrile extraction (method B), and pure acetonitrile extraction (method C). In these extraction procedures, different eluent modifiers were used for positive-and negative-mode ESI measurements to obtain high sensitivity and very sharp peaks. en, it has been found that pure acetonitrile extraction (method C) did not show acceptable recoveries compared to QuEChERS approach and aqueous methanol extraction that present satisfactory recoveries ranging from 70% to 120% with RSD less than 20% for most of the analyte-matrix combinations. Despite the fact that QuEChERS-like method led to lower LOQ and more coherent results, the recoveries were low especially for polar analytes (DON 3-glucoside (DON-3-Glc), NIV, T2 tetraol) due to the partitioning step. On the other hand, extraction using QuEChERS approach was selected as the most suitable procedure for the tested analytes [142]. Juan et al. [143] Equally, sensitivity was high due to the low LOD and LOQ [143]. By using gradient RP-LC with atmospheric pressure chemical ionization triple quadrupole mass spectrometry (LC-APCI-MS/MS), Berthiller et al. [86] developed a novel method for the simultaneous determination of the Fusarium mycotoxins. Nivalenol, deoxynivalenol, fusarenon-X, 3acetyl-deoxynivalenol, the sum of 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol, diacetoxyscirpenol, HT-2 toxin, T-2 toxin, and zearalenone in maize have been detected [86]. Ren et al. [146] developed an analytical method for the simultaneous quantification of 17 kinds of Aspergillus, Fusarium, and Penicillium mycotoxin contaminants in foods and feeds by ultrahigh-performance liquid chromatography combined with ESI triple quadrupole tandem mass spectrometry (UPLC-MS/MS) under the multiple reaction monitoring (MRM) mode and especially focused on the optimization of extraction, clean-up. e 10 positive ions and 7 negative ions of mycotoxins were separated by gradient elution with the retention time of 6.5 and 4 min, respectively. e LOQ of selected analytes ranged from 0.01 to 0.70 μg·kg −1 , which was lower than the criteria of EU, USA, and other countries on the determination of the minimum limiting level of various mycotoxins in foods including baby foods and feed stuffs. In this way, Amézqueta et al. [147] determined the OTA residue in cocoa beans by HPLC with the LOQ value of 0.1 μg·kg −1 . Meanwhile, Sugita-Konsihi et al. [148] quantified the DON level using HPLC method and achieved reasonable LOQ value (100 μg·kg −1 ). Papp et al. [149] validated an analytical method for the determination of AT B 1 , B 2 , G 1 , and G 2 in corns, wheat, fish, peanut products, rice, and sunflower seeds by HPLC with the LOD range of 2-10 μg·kg −1 . Ren et al. [146], also, reported high correlation coefficients (r 2 > 0.99) of 17 mycotoxins which were obtained within their respective linear ranges (0.05-20 μg·kg −1 for 10 positive ions and 0.5-50 μg·kg −1 for 7 negative ions) and reasonable recoveries (70.6-119.0%) of them were also demonstrated in different spiked levels. In 2012, Soleimany et al. [87] developed and used a LC-MS/MS method for simultaneous determination of aflatoxins (AFB 1 , AFB 2 , AFG 1 , and AFG 2 ), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisins (FB 1 and FB 2 ), T2, and HT2-toxin in cereals. One-step extraction using solvent mixtures of acetonitrile : water : acetic acid (79 : 20 : 1) without any clean-up was employed for extraction of these mycotoxins from cereals. e method exhibited good linearity over the relevant working range, and R 2 was between 0.950 for DON and 0.999 for AFB 1 . ere was significant difference among the LODs in the standard solution and in matrices. LODs of mycotoxins standard solutions were far lower than LODs in matrixes. e LODs and LOQs of standards and matrixes ranged between 0.01-20 ng/g and 0.02-40 ng/g, respectively, which are acceptable because they were far below the European Regulations for correspondent maximum levels of mycotoxins in foods. e LODs were lower than those reported by Sulyok et al. [53] and comparable to those reported by Ventura et al. [150]. Concerning recovery values, the study by Soleimany et al. [87] showed a range from 76.8% to 108.4% for all mycotoxins. e recovery results were better than those reported by Delmulle et al. [ In another study, von Bargen et al. [99] described the first application of isotopically labeled 13 C 2 -moniliformin for the analysis of moniliformin (MON) in cereals. e use of high-resolution mass spectrometry was described to be a suitable alternative technique for the detection of this compound. e developed method is based on the use of strong anion exchange columns for cleanup prior to HPLC analysis. In fact, the recovery rate was equal to 75.3%, and the LOD and LOQ were 0.7 and 2.5 μg/kg, respectively. On the other hand, Sirhan et al. [152] established a new method based on QuEChERS followed by LC-ESI-QTOF-MS/MS to determine eight type-A and type-B trichothecenes in cereal samples. e recoveries of fortified cereal samples ranged from 61.9% to 110.9%, and RSDs were lower than the acceptable 12% in all the cases. e sensitivity was determined by estimating the limit of detection (LOD) and limit of quantification (LOQ). Indeed, the LODs of type-A and type-B trichothecenes were 6.1-8.3 and 12.5-18.7 mg � kg, respectively. Habler and Rychlik [95] developed a multimycotoxin stable isotope dilution LC-MS/MS method for 14 fusarium toxins. Linearity, intraday precision, interday precision, and recoveries were ≥0.9982, 1-6%, 5-12%, and 79-117%, respectively. Method accuracy was verified by analysing certified reference materials for deoxynivalenol, HT2-toxin, and T2-toxin with deviations below 7%. e recoveries range between 86 and 109% for all analytes with RSDs below 7% and between 79 and 117% for the matrix calibration with maximal RSD of 17%. e LODs range between 0.1 and 5 μg/ kg and the LOQs range between 0.2 and 15 μg/kg, except for NIV and D3G, whose LODs and LOQs are 70 and 200 μg/kg and 10 and 30 μg/kg, respectively. e high LOD and LOQ of NIV with 70 and 200 μg/kg, respectively, are due to the low MS/MS sensitivity and are comparable with the limits reported by Ediage et al. [153]. e LODs and LOQs of the ENNs and BEA using the method presented here reveal 2-100 times higher sensitivity than those previously reported [154,155]. Journal of Food Quality 7 Journal of Food Quality 9 10 Journal of Food Quality n.d [157] Journal of Food Quality 11 12 Journal of Food Quality Journal of Food Quality 13 Some of the most common methods used for both mycotoxin identification and quantification are summarized in Table 1 in terms of chromatographic conditions (mobile phase and gradient and analytical column), detection, and quantification in each method for different cereal matrices. Hidden Mycotoxins Characterization Mycotoxin derivatives that are undetectable by conventional analytical techniques are designated masked mycotoxins [161,162]. Chemical transformations that generate masked mycotoxins are catalyzed by plant enzymes [38]. e group of masked mycotoxins comprises both extractable conjugated and bound (nonextractable) varieties. Bound mycotoxins are covalently or noncovalently attached to polymeric carbohydrate or protein matrices [38,39]. Extractable conjugated mycotoxins can be detected by appropriate analytical methods when their structure is known and analytical standards are available. Bound mycotoxins, however, are not directly accessible and have to be unconventional from the matrix by chemical or enzymatic treatment before chemical analysis. Among all modified mycotoxins, most occurrence data exist for deoxynivalenol-3-β-d-glucopyranoside (D3G), which was detected in naturally contaminated maize and wheat for the first time in 2005 [163]. Cereal contamination with D3G was reported to occur worldwide according to surveys from the UK, [164], the Czech Republic [165], China [166], and Canada [167]. Subsequent surveys showed intermittently high contaminations of D3G exceeding 1000 μg/ kg in naturally contaminated wheat [122]. D3G also has been detected in oats and barley [122,168]. D3G was noticed in wheat bread; nevertheless, the levels were below the LOQ (100 μg/kg). Using a more sensitive method, 80% of 116 flour, breakfast cereal, and snack samples from the Czech market analyzed were found to be contaminated with D3G at concentrations ranging from 5 to 72 μg/kg [164]. Interestingly, Sasanya et al. [169] reported that some wheat samples contained significantly higher values (up to 2.7 fold) of D3G compared to DON. e linearity (r 2 ) of D3G was 0.914; recovery was 70.0%, while LOQ and LOD were 1 and 0.5 1 g/kg, respectively [169]. On the other hand, Berthiller et al. [162] reported a method detection limit of 0.012 g/ml for D3G in purified sample extracts, corresponding to 0.02 g/g in contaminated cereals. Berthiller et al. [162] also estimated their LOD from the signal intensity of their standards, based on the limited ion suppression they observed. e pigment LOQ and LOD were 4.3 and 0.0005 g/kg, respectively. Good linearity for the pigment standard curve (R 2 0.999) was also observed [162]. Suman et al. [170] reported the development of a liquid chromatography/linear ion trap mass spectrometry method capable of determining D3G. Samples were extracted with a mixture of methanol/water (80 : 20; v/v) and cleaned up using immunoaffinity columns. Chromatographic separation was performed using a core-shell C 18 column with an aqueous acetic acid/methanol mixture as the mobile phase under gradient conditions. e method was in-house validated on a bread matrix as follows: matrix-matched linearity (r 2 > 0.99) was recognized in the range of 10-200 μg/kg; trueness expressed as recovery was close to 90%; good intermediate precision (overall RSD < 9%) and adequate LOD and LOQ limits (4 and 11 μg/kg, respectively) were realized. e reliability of the method was finally demonstrated in bread, cracker, biscuit, and minicake commodities, resulting in relatively low levels of DON-3G, which were not higher than 30 μg/kg [170]. Dall'Asta et al. [171] developed an LC-ESI-MS/MS method for the simultaneous detection of the main fumonisins and their hydrolyzed derivatives allowing for a simplified sample preparation without previous clean-up. e method has a very low LOQ (10 μg/kg for FB 1 , 12 μg/kg for FB 2 and FB 3 , 70 μg/kg for HFB 1 , HFB 2 , and HFB 3 in maize flour) and a very good recovery for all the analytes. e sensitivity was good for all the considered analytes being the LOD and LOQ values comparable with those from other recently published LC-MS/MS methods, although those methods required a sample purification and preconcentration step [88,89,172]. Bound fumonisins were found to be present not only in thermally treated maize-based products but also in mild processed or even raw products (pasta, bread, cakes, crisps, and flour) and they were always present in almost similar or even higher amounts than the free forms [171]. Osborne fractions of maize proteins showed that fumonisins were particularly bound to prolamins and glutelins [171]. Hu et al. [173] investigated free and hidden fumonisins in raw maize and maize-based products from China. A total of 58 samples were analyzed using LC-MS/MS. Among all the samples, 66% were contaminated with free fumonisins above limits of quantitation, and a higher percentage of 86% was found for total fumonisins (free + hidden). e response functions for FB 1 , FB 2 , HFB 1 , and HFB 2 showed that all the R 2 were greater than 0.99, suggesting good linearity. e LODs of FBs and HFBs were between 6 and 7 μg/kg, and the LOQs were between 23 and 28 μg/kg [173]; these results showed that the present method was about 4 times more sensitive than that reported by Oliveira et al. [174]. In comparison, by using isotope-labeled internal standards, Bryła et al. [175] found LOQs of 22 μg/kg for HFBs, which were similarly sensitive as the study of Hu et al. [173]. Andrade et al. [176] have validated multimycotoxin method based on extraction with acidified acetonitrile and LC-ESI + -MS/MS analysis. e LOQs ranged from 0.5 to 121 μg/kg and proved to be suitable for the multimycotoxin analysis in wheat, maize, and rice products. Bound/hidden fumonisins were determined after extraction of the free forms using the multimycotoxin method, followed by a basic hydrolysis of the unextracted bound/hidden and solid-liquid extraction with low temperature purification (SLE-LTP). Recoveries for HFB 1 , HFB 2. and HFB 3 were evaluated in six replicates fortified with the prepared standards at levels of 1.2, 1.8, and 2.5 g/kg, respectively. Recoveries were 75.6% (RSD of 6.6%) for HFB 1 , 108.0% (RSD of 10.6%) for HFB 2 , and 74.9% (RSD of 12.2%) for HFB 3 . Journal of Food Quality 15 Table 2 presents a well-detailed description of the analytical method mentioned above for masked mycotoxin from cereals and related foodstuffs. Conclusion Cereals and related foodstuffs could be contaminated by diverse toxin-producing fungal species that are linked to severe and chronic toxic effects for both humans and animals. Consequently, many successful methods, such as LC-MS/MS, have been identified in this area. LC-MS/MS continues to play a central role in the determination of mycotoxins in cereals and related foodstuffs unless a drastically different approach to distinct complex mixtures is advanced. In this context, smaller amounts of samples can be processed faster than ever. To quantify free and masked mycotoxins in cereals and related foodstuffs, separation stayed as important as ever. e great increases in sensitivity and selectivity of LC-MS instruments have made a significant contribution in qualitative and quantitative determination of mycotoxins in in cereals and related foodstuffs. In this line, the increasing use of hybrid mass spectrometers, incorporating mass analyzers that are capable of high mass resolution and accurate mass measurements, mitigates some of the problems associated with selectivity and identification, but further technological development of LC-MS interfaces is required to minimize matrix effects. However, maintaining confidence in the assignment of identity and isobaric interference are still the major limitations for LC-MS methods used for the quantification and identification of mycotoxins in cereals and related foodstuffs. Eventually, interested chemists could keep continuing research and contribute to develop and suggest new and advanced analytical techniques to ensure higher sensitivity and obtain solutions to several issues related to mycotoxins. Data Availability e data used to support the findings of this study are available from the corresponding author upon request. Conflicts of Interest e authors declare no conflicts of interest.	2020-12-17T09:13:29.672Z	2020-12-10T00:00:00.000	{ "year": 2020, "sha1": "1e641e976ba1525824d329603f0b6f0eb61e590f", "oa_license": "CCBY", "oa_url": "https://downloads.hindawi.com/journals/jfq/2020/8888117.pdf", "oa_status": "GOLD", "pdf_src": "Adhoc", "pdf_hash": "9fbceb54cb43511c4e739c41334b04a1ef22d436", "s2fieldsofstudy": [ "Agricultural And Food Sciences" ], "extfieldsofstudy": [ "Chemistry" ] }
232772464	pes2o/s2orc	v3-fos-license	The Antiviral Activities of Poly-ADP-Ribose Polymerases The poly-adenosine diphosphate (ADP)-ribose polymerases (PARPs) are responsible for ADP-ribosylation, a reversible post-translational modification involved in many cellular processes including DNA damage repair, chromatin remodeling, regulation of translation and cell death. In addition to these physiological functions, recent studies have highlighted the role of PARPs in host defenses against viruses, either by direct antiviral activity, targeting certain steps of virus replication cycle, or indirect antiviral activity, via modulation of the innate immune response. This review focuses on the antiviral activity of PARPs, as well as strategies developed by viruses to escape their action. Introduction Poly-adenosine diphosphate (ADP)-ribose polymerases (PARPs) are a family of enzymes responsible for ADP-ribosylation, a reversible and transient post-translational modification of various target proteins including histones, enzymes, transcription factors and even PARPs themselves [1]. PARPs catalyze the transfer of one (mono-ADP-ribosylation or MARylation) or more (poly-ADP-ribosylation or PARylation) ADP-ribose group(s) onto their target proteins using nicotinamide adenine dinucleotide (NAD + ) as a substrate. ADPribosylation can drastically affect the functions of target proteins by modulating their enzymatic activity and by facilitating their ubiquitination, leading to their degradation [2]. PARPs can be found in cells from humans to bacteria and possess a highly conserved C-terminal catalytic domain. Among prokaryotes, many virulence factors of pathogenic bacteria such as diphtheria, cholera and clostridial toxins possess mono-ADP-ribose polymerase activity, causing important dysregulations of host cellular processes, which can lead to cell death [3]. In eukaryotes, PARPs have been identified in, at least, 77 species across five of the six eukaryotic supergroups involved in many cellular activities, such as DNA repair or apoptosis [4]. The human genome encodes 17 PARPs, all sharing a highly conserved sequence in their catalytic domain called the "PARP signature motif", a characteristic secondary structure that binds NAD + . ADP-ribosylation of the target occurs on glutamate, aspartate, cysteine, arginine, serine and lysine residues [4][5][6]. Only five of the 17 human PARPs (PARP1, -2, -3, -5a and -5b) display PARP activity and can promote PARylation. However, most of the human PARPs (PARP6 to PARP12, PARP14, -15 and -16) lack a residue necessary to elongate the ADP-ribose chain, and therefore add a single ADP-ribose to the target, a process called MARylation [7]. Lastly, PARP13 is the only family member with an inactive PARP catalytic domain (Table 1). PARPs are divided into four subfamilies based on structural domains within the protein outside of the PARP catalytic site. PARP1, -2 and -3 belong to the DNA-dependent PARP subfamily. PARP7, -12 and -13 contain CCCH zinc-finger motifs able to bind RNA. PARP5a and -5b, also known as tankyrases, possess protein-binding ankyrin repeats. Due to their distinct functional domains, PARPs can play various roles in the cell. PARPs act as transcription regulators through ADP-ribosylation of histones. Since ADPribose is negatively charged, PARylation or MARylation of histones leads to electrostatic repulsion with DNA, which allows recruitment of chromatin remodeling factors and increases gene transcription [9,10]. DNA-dependent PARPs act as DNA damage sensors involved in DNA break repair, with ADP-ribosylation at the double-stranded breaks acting as a signal, which allows the recruitment of DNA repair enzymes to the lesion site [11]. In Viruses 2021, 13, 582 3 of 14 cases of major DNA damage, overactivation of PARP1 can induce a depletion of the NAD + pool in the cell, inhibiting ATP production and cellular metabolism, ultimately leading to cell death by necrosis. In a final example, PARP5a and -5b bind and ADP-ribosylate the telomeric repeat factor 1 (TRF1), reducing its binding ability to DNA and upregulating telomere maintenance [12]. In addition to these physiological functions, recent studies have highlighted the role of PARPs as actors in host antiviral response. In the context of viral infections, PARP expression can be induced, as reported for PARP3, -4, -5a, -5b, -7, -8, -9, -10, -11, -12, -13, and -14, in cells infected with coronaviruses [13,14]. Some PARPs are considered as interferon-stimulated genes (ISGs) and can consequently play a key role in the regulation of the innate immune response. Their antiviral activity can either be direct, by interfering with the critical steps of the viral replication cycle, or indirect, through immunomodulatory mechanisms. This review will summarize and discuss the direct and indirect antiviral properties of PARPs as well as the mechanisms brought into play by viruses to escape them. Inhibition of Viral Cycle Steps by the PARPs Every step of the viral cycle, from entry into to exit from the infected cell, represents a potential target for antiviral proteins. PARPs have been shown to target several steps of the virus replication cycle, mostly by inhibiting viral genome transcription and translation ( Figure 1). Due to their distinct functional domains, PARPs can play various roles in the cell. PARPs act as transcription regulators through ADP-ribosylation of histones. Since ADPribose is negatively charged, PARylation or MARylation of histones leads to electrostatic repulsion with DNA, which allows recruitment of chromatin remodeling factors and increases gene transcription [9,10]. DNA-dependent PARPs act as DNA damage sensors involved in DNA break repair, with ADP-ribosylation at the double-stranded breaks acting as a signal, which allows the recruitment of DNA repair enzymes to the lesion site [11]. In cases of major DNA damage, overactivation of PARP1 can induce a depletion of the NAD + pool in the cell, inhibiting ATP production and cellular metabolism, ultimately leading to cell death by necrosis. In a final example, PARP5a and -5b bind and ADP-ribosylate the telomeric repeat factor 1 (TRF1), reducing its binding ability to DNA and upregulating telomere maintenance [12]. In addition to these physiological functions, recent studies have highlighted the role of PARPs as actors in host antiviral response. In the context of viral infections, PARP expression can be induced, as reported for PARP3, -4, -5a, -5b, -7, -8, -9, -10, -11, -12, -13, and -14, in cells infected with coronaviruses [13,14]. Some PARPs are considered as interferonstimulated genes (ISGs) and can consequently play a key role in the regulation of the innate immune response. Their antiviral activity can either be direct, by interfering with the critical steps of the viral replication cycle, or indirect, through immunomodulatory mechanisms. This review will summarize and discuss the direct and indirect antiviral properties of PARPs as well as the mechanisms brought into play by viruses to escape them. Inhibition of Viral Cycle Steps by the PARPs Every step of the viral cycle, from entry into to exit from the infected cell, represents a potential target for antiviral proteins. PARPs have been shown to target several steps of the virus replication cycle, mostly by inhibiting viral genome transcription and translation ( Figure 1). (1). PARPs can also inhibit viral genome replication. PARP12 inhibits viral RNA transcription within the cell cytoplasm (2). PARP1 or PARP5 PARylate or directly interact with EBNA1, preventing EBNA1 binding to the OriP promoter and inhibiting Epstein-Barr virus (EBV) replication (3). PARPs directly interact with viral proteins. PARP9/DTX3L complex and PARP12 catalyze mono-ADP-ribosylation (MARylation) of viral proteins leading to their proteosomal degradation while PARP10, through binding to avian influenza virus NS1, prevents viral RNA replication. PARP13 binds already PARylated influenza A virus proteins leading to their proteosomal degradation (4). Finally, PARP7, -10, -12 and -13 are inhibitors of viral translation stopping the formation of the translation initiation complex on viral mRNA (5). Degradation of Viral Nucleic Acids In 2002, a screening of antiviral molecules in mammal cell showed the first evidence of the antiviral activities of PARPs. PARP13, initially called zinc-finger antiviral protein (ZAP), was identified as an inhibitor of retrovirus replication in rat cells [15]. This antiviral activity of rat ZAP was first discovered using a truncated PARP13 protein, which consists of only one of the four CCCH-type zinc-finger domains that mediate RNA binding and did not include the C-terminal PARP-like domain. Several isoforms of PARP13 have since been described in rats and humans [16,17]. Northern blots of the cytoplasmic fractions from infected cells expressing rat PARP13 showed a specific degradation pattern of the cytoplasmic viral RNA, while viral RNA within the nucleus remained intact [15]. Then, experiments of overexpression and downregulation in both rat and human cells showed that PARP13 possessed broad antiviral activity against a wide range of viral species, including alphaviruses, filoviruses (Ebola virus and Marburg virus), xenotropic murine leukemia virus-related virus (XMRV), coxsackievirus B3, Japanese encephalitis virus, human immunodeficiency virus 1 (HIV-1), Sindbis virus (SINV), hepatitis B virus (HBV) and influenza A virus (IAV) [18][19][20][21][22][23][24][25][26]. PARP13 targets cytoplasmic viral RNA, preventing transcription and translation of the viral genome. Interestingly, no impact of PARP13 overexpression on herpes simplex virus 1, vesicular stomatitis virus (VSV), yellow fever virus, poliovirus, dengue virus or Zika virus (ZIKV) replication has been found, proving that PARP13 antiviral properties are specific to certain viral species [18,24]. The mechanism of PARP13 antiviral activity was then unraveled. PARP13 forms a homodimer, which can bind viral RNA through its four zinc-finger motifs [27,28]. At the molecular level, it was recently shown that PARP13 N-terminal domain specifically binds to CpG dinucleotides in single-stranded RNAs, the interaction being further strengthened by additional guanine and cytosine [29]. Following the binding of PARP13 to viral RNA, recruitment of the exosome to the target RNA is induced leading to its degradation. Coimmunoprecipitation assays showed a direct interaction between rat PARP13 and the exosome component hRrp46p, while human PARP13 interacted with hRrp42, allowing exosome complex formation [21,30]. Recent studies have suggested that, even though some viruses have strongly CpG suppressed genomes, they can be restricted by PARP13 as it seems that, more so than their number, the localization of the CpG motifs is crucial for this restriction [31,32]. Furthermore, RNA helicases have been shown to be involved in the antiviral activity of PARP13. In 2007, Margaret and colleagues showed a synergistic inhibitory effect against SINV of PARP13 and an unknown ISG [33]. This PARP13 cofactor was then identified as p72 DEAD box RNA helicase. Since an inhibition of its helicase activity led to a diminution of RNA degradation, it was suggested that p72 facilitates PARP13-mediated exosome degradation by unwinding the viral complex RNA tertiary structure [34]. In a similar way, the DEXH box RNA helicase, DHX30, was shown to be a PARP13-interacting protein required for optimal antiviral activity [35]. During the HIV-1 replication cycle, PARP13 allows recruitment of the decapping complex to the 5 -end of viral RNAs, preventing their classical cap-dependent translation initiation and leading to their degradation [21]. PARP13 can also recognize CpG motifs in retroviral RNA and recruits host factors, including the endonuclease KHNYN, which then degrade viral RNA [36]. Finally, PARP13 has been shown to interact with poly(A)-specific ribonuclease (PARN) to remove the poly-(A) tail of HIV-1 RNA, thereby exposing the 3 -end to the exosome and leading to RNA degradation. Even if the antiviral properties of PARP13 are now well-known, there is still a discussion about which of its two major isoforms displays the most efficient antiviral activity. PARP13 exists in two major isoforms: a long, constitutive full-length isoform (named ZAP-L or PARP13L) and an interferon-inducible short isoform (ZAP-S, PARP13S) lacking the C-terminal PARP domain [18]. Evolutionary analysis showed a positive selection confined to the PARP C-terminal domain, indicating that it would be an important interface for PARP13 interaction with the genome of constantly mutating viruses [16]. In this study, it was found that ZAP-L was a more potent inhibitor than ZAP-S of murine leukemia virus (MLV) and Semliki forest virus replication by 2 to 3-fold. Another study also showed that the PARP-like domain plays a crucial role in ZAP-L's antiviral activity against IAV [37]. By comparing both PARP13 isoforms, ZAP-L was recently shown to be a primary antiviral effector against the alphavirus SINV [38]. The isoform-specific targeting of viral RNA was due to differences in the subcellular localization of the two isoforms, which were mediated by the presence or absence of a C-terminal prenylation motif and allowed the recruitment of ZAP-L to sites of SINV RNA replication at the plasma membrane and in endolysosomes. Even if the PARP-like domain of ZAP-L lacks the catalytic activity of functional PARPs, mutations in this domain leads to the loss of its antiviral activity, indicating an essential function of the PARP-like domain in restricting alphaviruses in humans [39]. PARP7 might use a similar mechanism to degrade genomic RNA of viruses belonging to Togaviridae family (SINV and rubella virus). Immunoprecipitation assays have shown that PARP7 binds SINV RNA as well as the exosome complex component 5 (EXOSC5) via its N-ter CCCH-type zinc-finger domain [40]. During SINV infection, reactive oxygen species are produced by damaged mitochondria and induce oxidation of the nucleoporin protein Nup62, leading to a cytoplasmic accumulation of PARP7 where it binds viral RNA, thereby promoting its degradation. Inhibition of Viral Replication PARP1, -2 and -9 are known to be involved in transcription regulation in the cell. They can act as scaffold proteins, modifying the chromatin structure, and then facilitating or preventing the binding of transcription factors to DNA [41]. Their ability to modulate cell mRNA transcription can also affect viral RNA transcription. Since viruses rely on cellular machinery for their replication, cells have developed many defense mechanisms to prevent this, some of them involving nuclear PARPs. In infected cells, the Epstein-Barr virus (EBV) maintains latency in the form of circular double-stranded (ds) DNA, replicating its genome once per cell cycle. This replication is dependent on the viral protein EBNA1 binding to the dsDNA at the origin of plasmid replication (OriP) site. Several studies have shown that PARP1 or PARP5 were also binding partners of OriP, competing with EBNA1, and causing a decrease in EBV DNA replication and maintenance in latently infected cells [42]. Conversely, inhibition of PARP activity increased maintenance of OriP plasmids, while inhibitors of OriP replication were also stimulators of PARP activity [42]. Both PARP1 and PARP5 were shown to interact with and modify EBNA1, remodeling protein-DNA structure and leading to negative regulation of OriP replication [43,44]. In the same way, and in a sequence-specific manner, PARP1 binds the replication origin TR of the Kaposi sarcoma-associated herpesvirus (KSHV). Moreover, PARP1 catalyzes poly-ADP-ribosylation of the latency-associated nuclear antigen (LANA), affecting maintenance of the viral genome in the latently infected cells [45]. Taken together these results showed a similar inhibition mechanism of latency maintenance of two γ-herpesvirinae viruses, KSHV and EBV, by PARP1. In addition to disrupting herpesvirus maintenance in the cell, PARP1 interferes with their reactivation. Lupey-Green and colleagues showed that PARP1 specifically bound the EBV lytic promoter BZLF1, inhibiting viral reactivation. The viral protein Zta, which also binds BZLF1, is able to antagonize the PARP1-mediated inhibition of EBV lytic reactivation [46]. Moreover, PARP1, synergistically with the Ste20-like kinase hKFC, PARylates the KSHV protein replication and transcription activator (RTA), which has a central role in the switch between latency and lytic cycle. Interaction of the PARP1/hKFC complex with RTA decreases its recruitment to promoter regions and disrupts RTA-mediated KSHV reactivation [47]. Viral transcription inhibition by PARPs also occurs through epigenetic modifications. This mechanism was reported as mediating the silencing of retrotransposable elements and inhibiting transcription of integrated retroviruses. An initial study conducted in 2005 showed that PARP1 bound HIV-1 TAR RNA. TAR is the binding site of the trans-activator of HIV-1 LTR Tat-containing complex p-TEFb, which elongates the HIV-1 RNA. PARP1 binds TAR with a higher affinity than the p-TEFb complex, leading to p-TEFb displacement from HIV-1 RNA, suppressing Tat-mediated transcriptional elongation [48]. This PARP1mediated retrovirus transcription inhibition was also shown to be efficient against MLV. It is mediated by epigenetic mechanisms that involve DNA methylation and histone deacetylation, and does not seem to require the catalytic activity of PARP1 [49]. PARP1-mediated retroviral silencing also occurs before integration in a catalytic-independent manner. It has been shown that PARP1 can repress retroviruses prior to viral DNA integration by mechanisms involving histone deacetylases but not heterochromatin formation [50]. Finally, in addition to its role in the inhibition of transcription of integrated proviral DNA, PARP1 can interfere with HIV-1 integration into cellular DNA, but this point remains controversial [51][52][53][54]. Otherwise, it has been suggested that PARP12 inhibits steps before VSV replication and secondary transcription [55]. Translation Inhibition Translation of the viral genome is a usual target of ISGs in order to block viral replication. Gao and colleagues showed that PARP13 could also inhibit HIV-1 translation. Overexpression of human PARP13 in 293TRex cells reduced production of the HIV-1 Nef protein that plays an important role in virus replication in vivo by nearly 13-fold, whereas, in the same condition, nef mRNA expression decreased only by 5-fold, suggesting an inhibition of mRNA translation. Moreover, translation inhibition by PARP13 was not related to mRNA degradation. Polysome profiling, which analyzes association of mRNA with ribosomes, has confirmed that PARP13 excludes the target mRNAs from polysome fractions without affecting global cell translation [56]. The same study found that PARP13 interacted with eukaryotic initiation factor 4-A (eIF4)-A, thereby competing with eIF4-G for binding and stopping the formation of the canonical translation initiation complex on viral mRNA. Otherwise, the E3 ligase tripartite motif-containing protein 25 (TRIM25) was recently found to be a novel PARP13 cofactor involved in viral translation inhibition through an ubiquitination-dependent mechanism targeting unidentified host proteins [57,58]. PARP13 has also been shown to inhibit the formation of the translation initiation complex on IAV mRNA in a PARP-domain independent manner [23]. Finally, although this question has not yet been elucidated, it seems logical that the decapping of HIV-1 mRNA mediated by PARP13 would impair the translation initiation process since most of HIV-1 translation occurs in a cap-dependent manner [21,59]. Though a majority of studies on the translation inhibition mediated by the PARPs focus on PARP13, recent discoveries have shown that PARP7, -10 and -12 are likewise inhibitors of viral translation [60]. PARP12 exhibits antiviral activity against many viruses including Venezuelan equine encephalitis virus (VEEV), VSV, SINV, encephalomyocarditis virus (EMCV), rift valley fever virus (RVFV) and Chikungunya virus (CHIKV) [61]. It is interesting to note that PARP10 and PARP7 overexpression also inhibit VEEV replication, which may explain why a downregulation of PARP12 does not suppress the antiviral state of the cell due to a redundancy of the roles between PARPs [60]. PARP12 shares a very similar structure with PARP13 consisting of zinc-finger motifs as well as the existence of two isoforms, with the short-one lacking the PARP domain. However, contrary to PARP13, only the long isoform displays antiviral activity, and while PARP13 is catalytically inactive, PARP12 is capable of MARylating proteins [62]. PARP12 s catalytic activity has been shown to be necessary to downregulate mRNA translation [62]. Targeting Viral Proteins Despite its PARP signature, PARP9 was initially thought to be devoid of catalytic activity, which allows ADP-ribosylation [63]. In 2003, the protein Deltex E3 ubiquitin ligase 3L (DTX3L) was identified as a PARP9 binding partner [64]. Unlike PARP9 alone, the heterodimer PARP9/DTX3L displays mono-ADP-ribosylating activity. MARylation of a protein by PARP9 leads to its ubiquitination by DTX3L causing proteosomal degradation of the target protein [65]. The PARP9-DTX3L complex is thereby responsible for the degradation by ubiquitination of many viral proteins such as the 3C proteases of EMCV and human rhinovirus, both of which are viruses belonging to the Picornaviridae family [66]. Interestingly, this does not occur with the respiratory syncytial virus NS1 protein, suggesting that ubiquitination and degradation mediated by DTX3L could be specific to Picornaviridae 3C proteases [66]. The long isoform of PARP13, ZAP-L, which does not harbor an ADPribosylation activity, has been shown to bind the PARylated IAV polymerase proteins PA and PB2, leading to their ubiquitination and proteasomal degradation [37]. An avian influenza virus (AIV) protein has been shown to be targeted by PARP10; this interaction plays a regulatory role in virus replication. Expression of AIV NS1 protein in infected cells causes relocalization of PARP10 from the cytoplasm to the nuclei and reduces endogenous PARP10 expression [67]. In addition, decrease in PARP10 expression leads to cell proliferation and promotes viral replication [67]. Finally, in addition to its ability to suppress viral translation, PARP12 restricts ZIKV infection through degradation of viral proteins. PARP12L has been shown to be able to MARylate the nonstructural ZIKV proteins NS1 and NS3, both of which are involved in viral replication and host immune response modulation. NS1 and NS3 MARylation leads to their PARylation, presumably by another member of the PARP family, which increases their Lys 48 ubiquitination and subsequent proteasomal degradation [68]. Immunomodulation The role of PARPs in immune response has been investigated in several studies, and reviewed in [69][70][71]. Given that the proinflammatory roles of PARP1, through triggering of NF-κB signaling pathway, induction of chemokine expression and activation of immune cells such as neutrophils, macrophages and dendritic cells, has been extensively described, the immunomodulation properties of PARP1 have been discussed in several other reviews and will not be discussed here [72][73][74]. However, several other members of the PARP family, including PARP7, -9, -10, -11, -12, -13 and -14, have important roles in innate immunity, and the main ones are summarized here (Figure 2). PARP11-induced MARylation of the β-transducin repeat-containing protein (β-TrCP), an E3 ubiquitin ligase, has been shown to promote ubiquitination and subsequent degradation of interferon alpha/beta receptor 1 (IFNAR1) [75]. ZAP-S, the short isoform of PARP13, also has immunomodulatory properties [38,76]. Its overexpression in human HEK293Y cells enhances IFN-β mRNA expression and oligomerization of the viral RNA sensor retinoic acid-inducible gene I (RIG-I), which leads to robust activation of downstream antiviral signaling through the interferon regulatory factor 3 (IRF3) pathway [76]. On another track, a few studies have suggested that PARP7, -10 and -11 may downregulate the inflammatory response. PARP7 has been shown to mono-ADP-ribosylate TANK-binding kinase 1 (TBK1) downstream of the RIG-I signaling pathway [77]. Consequently, the TBK1induced dimerization of IRF3 decreases, leading to impaired IFN production. PARP10 exercises negative feedback on the NF-κB signaling pathway through MARylation of NF-κB essential modulator (NEMO), thereby preventing its K63 polyubiquitination [78]. PARP12 could also enhance the signaling cascade leading to NF-κB activation [62]. Moreover, it was recently shown that ZAP-S binds to and mediates the degradation of several host IFN mRNAs, thereby acting as a negative feedback regulator of the interferon response [38]. PARP9, -12, -13 and -14 have been reported to enhance the cell innate immune response. Increased concentrations of PARP9 and DTX3L, or of the E3-ubiquitin ligase complex formed by these two proteins, have been observed in cells stimulated with IFN-γ. PARP9 and DTX3L expression depends on the same promoter, which is bidirectional and contains binding sites for STAT-1 and IRF1, both of which are transcriptional factors involved in the antiviral response [79]. IFN-induced overexpression of the IFN-signaling molecule STAT-1 leads to upregulation of the translation of PARP9 and DTX3L. In turn, the PARP9-DTX3L complex is a direct binding partner of STAT-1, enhancing STAT-1 phosphorylation and, consequently, its activation. This binding also promotes STAT-1 nuclear relocalization, increasing the transcription efficiency of ISGs, thereby leading to amplification of the innate immune response [66]. In addition, the PARP9-DTX3L complex catalyzes ubiquitination of a subset of histones from the host, notably H2BJ, increasing histone methylation, which leads to chromatin remodeling and, once again, to increased transcription of ISGs. (2), which leads to robust activation of downstream antiviral signaling through the interferon regulatory factor 3 (IRF3) pathway. On the other hand, PARPs can also downregulate the antiviral defenses. PARP7 MARylates TANK-binding kinase 1 (TBK1), decreasing IRF3 activation and leading to impaired IFN production (3). Otherwise, PARP12 enhances the signaling cascade, leading to NF-κB activation, whereas PARP10 exerts negative feedback on this pathway through MARylation of NF-κB essential modulator (NEMO) (4). PARP13 mediates the degradation of several host IFN mRNAs (5), thereby acting as a negative feedback regulator of the interferon response. The PARP9-DTX3L complex is a direct binding partner of STAT-1, promoting STAT-1 phosphorylation and nuclear relocalization, thereby increasing ISG transcription and leading to amplification of the innate immune response (6). PARP14 specifically binds to STAT-6 responsive promoters (7), preventing STAT-6-mediated transcription. The inflammatory environment influences PARP9-DTX3L and PARP14 immunomodulatory properties. In the presence of the anti-inflammatory cytokine IL-4 (8), PARP14 carries out its own MARylation by an autocatalytic process that allows binding of STAT-6 and transcription activation. PARP9-DTX3L potentiates the response to IFN-γ by enhancing phosphorylation of STAT-1, whereas PARP14, by ADP-ribosylating STAT1, decreases the response while increasing phosphorylation of STAT-6, thereby promoting the anti-inflammatory response mediated by IL-4. PARP9-DTX3L can in turn inhibit MARylation of STAT-1 by PARP14. PARP11-induced MARylation of the β-transducin repeat-containing protein (β-TrCP), an E3 ubiquitin ligase, has been shown to promote ubiquitination and subsequent degradation of interferon alpha/beta receptor 1 (IFNAR1) [75]. ZAP-S, the short isoform of PARP13, also has immunomodulatory properties [38,76]. Its overexpression in human HEK293Y cells enhances IFN-β mRNA expression and oligomerization of the viral RNA sensor retinoic acid-inducible gene I (RIG-I), which leads to robust activation of downstream antiviral signaling through the interferon regulatory factor 3 (IRF3) pathway [76]. On another track, a few studies have suggested that PARP7, -10 and -11 may downregulate the inflammatory response. PARP7 has been shown to mono-ADP-ribosylate TANKbinding kinase 1 (TBK1) downstream of the RIG-I signaling pathway [77]. Consequently, the TBK1-induced dimerization of IRF3 decreases, leading to impaired IFN production. (2), which leads to robust activation of downstream antiviral signaling through the interferon regulatory factor 3 (IRF3) pathway. On the other hand, PARPs can also downregulate the antiviral defenses. PARP7 MARylates TANK-binding kinase 1 (TBK1), decreasing IRF3 activation and leading to impaired IFN production (3). Otherwise, PARP12 enhances the signaling cascade, leading to NF-κB activation, whereas PARP10 exerts negative feedback on this pathway through MARylation of NF-κB essential modulator (NEMO) (4). PARP13 mediates the degradation of several host IFN mRNAs (5), thereby acting as a negative feedback regulator of the interferon response. The PARP9-DTX3L complex is a direct binding partner of STAT-1, promoting STAT-1 phosphorylation and nuclear relocalization, thereby increasing ISG transcription and leading to amplification of the innate immune response (6). PARP14 specifically binds to STAT-6 responsive promoters (7), preventing STAT-6-mediated transcription. The inflammatory environment influences PARP9-DTX3L and PARP14 immunomodulatory properties. In the presence of the anti-inflammatory cytokine IL-4 (8), PARP14 carries out its own MARylation by an autocatalytic process that allows binding of STAT-6 and transcription activation. PARP9-DTX3L potentiates the response to IFN-γ by enhancing phosphorylation of STAT-1, whereas PARP14, by ADP-ribosylating STAT1, decreases the response while increasing phosphorylation of STAT-6, thereby promoting the anti-inflammatory response mediated by IL-4. PARP9-DTX3L can in turn inhibit MARylation of STAT-1 by PARP14. Similarly, a study conducted in human primary macrophages showed PARP14 to be specifically bound to STAT-6 responsive promoters, preventing STAT-6-mediated transcription. However, in the presence of the anti-inflammatory cytokine IL-4, the MARylation activity of PARP14 is triggered, leading to its own MARylation by an autocatalytic process that allows binding of STAT-6 and transcription activation [80]. PARP14 thereby mediates Viruses 2021, 13, 582 9 of 14 a switch between repression and activation of ISG transcription depending on the presence or absence of IL-4. Interestingly, functional redundancy between PARP9 and PARP14 has been hypothesized due to structural similarities. Following macrophage stimulation with IFN-γ, it was shown that PARP9 and PARP14 are both upregulated but then assume opposite roles. PARP9 potentiates the response to IFN-γ by enhancing phosphorylation of STAT-1 whereas PARP14, by ADP-ribosylating STAT-1, decreases the response while increasing phosphorylation of STAT-6, thereby promoting the anti-inflammatory response mediated by IL-4 [81]. It is interesting to note that PARP9 could inhibit MARylation of STAT-1 by PARP14, restoring the proinflammatory response of the cell. In addition, PARP9 binds the STAT-6 responsive promoter in replacement of PARP14 but without triggering the same switch between activation and repression of transcription, since STAT-6-dependent transcription is still impaired in PARP14 -/-primary macrophages despite IL-4 stimulation. Finally, in addition to ISG transcription regulation, PARP14 directs T cell towards Th2 differentiation by promoting binding of STAT-6 to the Gata3 promoter [82,83]. All in all, PARPs are ISGs acting as positive or negative regulators of the inflammatory process induced during viral infection. Even without direct antiviral effects on the virus replication cycle, they can modulate host defenses, inducing either upregulation of the cell antiviral state to fight viral infection or downregulation of the immune response to prevent inflammatory damages. Strategies to Escape the Antiviral Activities of PARPs Through numerous studies, it is now obvious that PARP-induced PARylation and MARylation are important post-translational modifications leading to activation of cell innate immune factors mobilized against viral infection. Viruses have developed many ways to counteract PARP antiviral activities. The genome of several RNA viruses such as coronaviruses (severe acute respiratory syndrome-related coronavirus (SARS-CoV)-1 and -2, Middle East respiratory syndrome coronavirus (MERS-CoV)), alphaviruses (CHIKV and SINV), hepatitis E virus (HEV) and the murine hepatitis virus (MHV) encodes macrodomain-containing proteins. A macrodomain is a highly conserved sequence of 170 to 180 amino-acids, originally found in human core histones macroH2A, which is a component of chromatin [84]. Macrodomains are capable of recognizing and binding mono-and poly-ADP-ribosylated proteins, subsequently modifying the ADP-ribose residues and downstream signaling or antiviral roles [85][86][87]. Alphavirus and coronavirus macrodomains were thus shown to be essential to efficient virus replication and virulence in cells [88][89][90]. Interestingly, PARP14 exhibits macrodomains, allowing them to remove ADP-ribose from substrates and exercise regulation or feedback on their PARylation and MARylation activities [80]. Moreover, some viral proteins can directly interact with PARPs, thereby neutralizing their antiviral properties. A study conducted on IAV showed that the viral protein NS1 antagonizes PARP13-induced mRNA decay by reducing its RNA-binding capacity. In addition to this direct interaction, IAV NS1 protein can alter PARP transcription. Taken together, these results explain why only prior to viral protein expression can PARP13 alter IAV replication [23]. PARP13 is also responsible for inhibition of enterovirus (EV) A71replication [91,92]. A recent study suggests that 3C protease cleaves PARP13 at the Gln-369 residue, leading to the loss of this inhibitory activity on the viral replication [93]. Regarding DNA viruses, the RTA protein of the murine γ-herpesvirus 68, which is a reactivator of the lytic viral cycle, prevents the formation of the PARP13 homodimer required for viral RNA recognition and degradation [94]. The KSHV processivity factor (PF)-8 protein, through direct interaction, leads to PARP1 proteasomal degradation, disrupting its inhibition of lytic cycle reactivation [95]. Finally, HSV has been shown to lead to hyperphoshorylation, nuclear transportation and retention by infected cell protein 0 (ICP0), an immediate early viral protein, and to proteosomal degradation of PARP5a, thereby enhancing HSV replication [96]. Another HSV protein, UL41, which is a tegument protein involved in host mRNA degradation, has been shown to be involved in PARP13L mRNA degradation leading to increased viral replication [97]. Recent studies have reported that an excessive activation of PARP1 can induce cell death, defined as parthanatos, related to DNA damage signaling. This phenomenon is also observed during viral infections where PARP1 is cleaved and therefore activated [98]. It also occurs in ZIKV infected cells through direct interaction of the helicase NS3 with PARP1, resulting in the death of neurons from infected brain [99]. However, if PARP1-mediated cell death is involved in the pathogenicity and the clinical symptoms caused by the viral infection, it could also play an important role in eliminating intracellular pathogens. In order to preserve virus replication, the adenovirus E4orf4 protein associates with PARP1, reducing its phosphorylation on serine residues, reported to enhance its activity, and therefore preventing its excessive activation [100]. Conclusions Current knowledge about PARPs, at the interface between host and viruses, seems to point to a globally underestimated and important role of this family of enzymes in host defense. Due to their cytoplasmic and nuclear localization, PARPs are capable of inhibiting virus life cycle at several stages, from transcription to translation. In addition, PARPs can act indirectly by stimulating host innate immunity through activation of intracytoplasmic sensors of pathogen-associated molecular patterns and ISG transcription. To date, ten of the seventeen human PARPs have been shown to display antiviral properties. The other seven remain to be studied and could possibly also exhibit antiviral activities. PARPs represent potentially interesting targets for new antiviral therapeutics since PARP agonists could help to restore the antiviral state of an infected cell. Although upregulation of one PARP may not be sufficient for control of the infection, the targeting of multiple PARPs, some for their direct antiviral activity and others for their immunostimulating properties, might be an interesting strategy that remains to be explored.	2021-04-04T06:16:29.409Z	2021-03-30T00:00:00.000	{ "year": 2021, "sha1": "e3b992ab5fdc18d1d35315ad2a59a56ff8a7e9e5", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/1999-4915/13/4/582/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "8e7c15067777099b2b57d669dbef3b4a19daba4e", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
250151262	pes2o/s2orc	v3-fos-license	Numerical Solutions to the Robin Inverse Problem With Numerical Solutions to the Robin Inverse Problem With Nonnegativity Constraints Nonnegativity Constraints We present iterative numerical methods for solving the inverse problem of recovering the nonnegative Robin coefficient from partial boundary measurement of the solution to the Laplace equation. Based on the boundary integral equation formulation of the problem, nonnegativity constraints in the form of a penalty term are incorporated conveniently into least-squares iteration schemes for solving the inverse problem. Numerical implementation and examples are presented to illustrate the effectiveness of this strategy in improving recovery results Introduction Γ of the boundary in hope to extract the quantity of interest p defined on an inaccessible portion 1 Γ of the boundary.For example, in the evaluation of metal-to-silicon contact quality in semiconductor transistors (e.g.[1] [2] [3]), voltage measurements of U on an accessible 0 Γ in response to an applied current input pattern g are used to infer the contact quality modeled by p on the inaccessible contact window 1 Γ .A similar situation arises from detection of material corrosion damages (e.g.[4] [5]), where static potential measurement u on an accessible part of the material boundary 0 Γ is taken to detect possible corrosion characterized by the conductivity profile p on an inaccessible Γ . For this inverse problem, there have been many numerical studies in the literature, based either on the PDE model (e.g.[5] [6] [7] and references therein) or on an integral equation formulation (e.g.[3] [8] [9] and references therein). Most of these papers have focused on the recovery of the Robin coefficients by iterative schemes for the minimizers of some objective functional that consists of both a data fitting term and a certain form of regularization, and the nonnegativity constraint for the Robin coefficient is often ignored, except for some simple truncation strategies on the side to ensure the well-posedness of the forward solver.It is well known that the inclusion of nonnegativity constraints improves the quality of solutions to ill-posed inverse problems when the quantity of interest is known to be nonnegative (e.g.[10], Chapter 9).In this study, we propose a strategy to incorporate nonnegativity constraints in the iterative schemes, in the form of an additional term in the objective functional to penalize negativity throughout the iteration process.This approach is natural in the least-squares formulation for solving inverse problems or illposed problems in general and provides a convenient and computationally economical alternative to any constrained optimization methods.We demonstrate the simplicity of this strategy in both linear and nonlinear least-squares solution methods for the Robin inverse problem, and through numerical examples, we illustrate the effectiveness of the proposed approach in dealing with the ill-posedness, resulting in much improved recovery results. Our presentation is organized as follows.In Section Integral Equation Formulation Because the equation in ( In the operator form, (2.1) can be written as with the single and double-layer potential operators defined by d and d for . Note that the operators have the following mapping properties (e.g.[13]): : : . It is well known that the Robin boundary-value problem (2.1) admits unique solution 0 U ≥ in Ω for given nonnegative model parameters p and g on Γ , and thus the equivalent boundary integral equation (2.1) yields nonnegative solution u on Γ as well (see e.g.[11]). Alternatively, we can use single-layer potential to express the solution ( ) where the density function ϕ on Γ solves the boundary integral equation In operator form, (2.4) becomes where the dual operator ′  of  is given by In our numerical implementation, we use the direct formulation (2.1) as the model equation that relates between u and p, and only use the alternative formulation (2.3) -(2.4) for generating synthetic data 0 u which, after the addition of random noise, are used to test our numerical algorithms for recovering the Robin coefficient p. Solution Methods for the Inverse Problem A common approach to the inverse problem of finding p on 1 [9] [11] [14]).Then in the iterative scheme for solving the least-squares problem, we will introduce nonegativity constraints in the form of a penalty term in the minimizing objective functional, so to steer toward nonnegative solutions. A Linear Least-Squares Method As in [9] [11], we introduce a new variable in place of ( ) 2) becomes linear in both u and v : where , d for . That is, ( )( ) ( ) Then the given measurement 0 u of u on 0 Γ can be expressed as We recast the inverse problem of finding p from given 0 u as directly solving for ( ) ( ) ( ) 3) as a linear system: Here  denotes the zero operator from ( ) System (3.4) is a linear system for ( ) , but is ill-posed.In addition to using the classical Tikhonov regularization method to address the ill-posedness (as in [11]), we wish to incorporate an additional term to penalize negativity in w and encourage nonnegagive solutions.Thus we seek the minimizer of the following objective functional that consists of the system residual from (3.4), a re-gularization for w, and a penalty for negative part of w: ( ) with regularization parameter 0 α > and penalty parameter 0 β > .The regu- larization is defined through the operator ( ) is differentiable except at 0 s = , and when its derivative is needed, we use the derivative of its smooth approximation: for some very small 0 ε > (e.g. the machine epsilon).Hence we can arrive at the first-order optimality condition of ( ) where ′  denotes the dual of  .In (3.6), while the parts from the linear system and Tikhonov regularization are linear, the part from the penalty term is not.Hence we devise an iterative scheme to solve (3.6) using the Gauss-Newton direction as follows.At each iterate ( ) k w , the step increment ( ) k d is determined by the linear equation and the next iterate is set to be We note that, when 0 β = , i.e. when the penalty term is not present, no itera- tion is needed, and this reduces to the direct method introduced in [11].However, such a direct method may produce w that admits negative values, and thus it requires some remedy, such as simple truncation, in order to obtain the nonnegative Robin coefficient p from (3.1) (see [9] [11]).By incorporating the penalty term in the least-squares formulation, we are able to ensure the nonnegativity of the solution it produces, with only a few additional iterations, so the Robin coefficient p can be readily obtained from (3.1).Moreover, when the pe-nalty term is active, it steers the iteration to produce better solutions, especially in situations where the ill-posedness is more severe. A Nonlinear Least-Squares Method In this method, we regard both u on Γ and p on its support 1 Γ as variables to be found from given data 0 u , and cast the inverse Robin problem as finding Γ from the nonlinear system of equations consisting of both the model equation (2.2) and data fitting equation (3.3): ( ) ( ) Here p in ( ) to be solved refers to the part of the Robin coefficient on its support 1 Γ , while in the formulations p remains as defined on Γ with the understanding that its support is contained in 1 Γ .It is noted that  is linear in u and p individually, but not jointly in z. To solve the nonlinear system (3.9) for ( ) , we consider minimization of the least-squares functional of the system residual, with the addition of both a regularization term and a penalty term: with regularization  and penalty ( ) as in (3.5).Note that the case without penalty (when 0 β = ) was considered in [14].For minimization of J above, we propose the Gauss-Newton iteration scheme: at each iterate ( ) is determined by the Gauss-Newton step from J: where and ( ) is the derivative operator of ( ) given by where  denotes operator composition, and ( ) Note that the addition of the penalty term in (3.10) results in a similar iterative scheme (3.11), and it does not add much computational cost.As it steers the iterations to satisfy the nonnegativity constraints, it does improve the quality of the solutions, as numerical examples in the next section will illustrate. Numerical Examples Given a regular 1-periodic parametrization with counterclockwise orientation for the boundary Γ : so that integrals involving this singularity can be dealt with by exact integration, as we employ Nyström's method with trigonometric interpolation on regular grids (see e.g.[12], Chapter 12). In our examples, we take Ω as the elliptic region for the sake of simplicity: We should note that this choice of the aspect ratio of the ellipse gives rise to a more severely ill-posed case of the Robin inverse problem than most cases in other studies, yet as observed consistently in our numerical experiments and demonstrated in examples below, the inclusion of nonnegativity constraints in the iterative schemes does help to combat the ill-posedness effectively and produce markedly better results for p. Standard parametrization of the ellipse is used: , relative to the L 2 norm of 0 u . In both iterative methods, we stop the iteration when the L 2 norm of the Gauss-Newton step ( ) k d from (3.7) or (3.11) is less than 10 −10 .Our selection of the regularization parameter α is guided by the idea of the normalized cumulative periodogram (NCP) method (see e.g.[15]), which is based on monitoring the noise pattern in the data-fitting residual 0 0 u u −  in (3.4) or (3.9).If the residual is dominated by white noise, then α is a suitable choice; if it is dominated by high frequency noise, the current parameter α shall be increased, and if it is dominated by a low frequency signal, then α shall be reduced to a smaller value ( [16]).See also ([10], Chapter 7) for other methods of selecting a proper regularization parameter value for illposed problems. The performance of the algorithms is less sensitive to the choice of β , and they work very similarly for values of β from the range between 10 −1 to 10 2 . We set 1 β = in our experiments. We first experiment using the linear least-squares method (3.7) -(3.8), and the result of one such example is presented in Figure 1.We select We then test on the nonlinear least-squares method (3.11) -(3.12), with the result of one such example presented in Figure 2. We choose this case.Typically it takes about 10 iterations from initial ( ) 0 0 z = to satisfy the stopping criterion in our experiments.For comparison, we also carry out parallel calculations without the penalty term (by setting 0 β = ), and as expected, the resulting solution assumes some negative values due to the absence of nonnegativity constraints.As can be seen, adding the penalty term not only ensures the nonnegativity of the recovered coefficient p, but also improves its quality. Throughout our numerical experiments, we have consistently observed the effectiveness of this approach in dealing with the ill-posedness of the Robin inverse problem and in improving recovery results, which is achieved within the same iterative framework and without additional computational cost. We conclude with some final remarks: 1) It is possible to fine-tune these least-squares methods by introducing different weights between the model equation residual and the data fitting residual in (3.4) or (3.9), but at the expense of one more parameter (the weight ratio) to adjust, as studied in [14].Even in this setting, the penalty strategy proposed here remains readily applicable, and the penalty parameter β is generally less sensitive and easier to determine than the weight ratio and regularization parameter; 2) In the more common approach where the dependence of u on p is determined exactly by solving the model equation (1.1) or (2.2) and only the data fitting residual is to be minimized, it is still possible to include the penalty strategy proposed here to better facilitate the solution process for maintaining nonnegativity of p, and it is reasonable to expect the improvement of quality of the recovered p in more ill-posed situations, as we have experienced and reported here. 2 R 1 Γ Consider the Robin boundary value problem for the Laplace equation on a smooth bounded domain input function on Γ .It is assumed that the support of p (denoted by 1 Γ ) and the support of g are nonempty but non-overlapping.Then the Robin inverse problem is described as: to recover the Robin coefficient p on 1 Γ from given values of the solution 0 U u = on 0 Γ ⊂ Γ with 0 ∩ Γ = ∅ .Such problems the solution U can be measured directly on an accessible part 0 Γ from given boundary measurement 0 u of u on 0 Γ is to regard u as dependent on p through the model equation (2.2), and then solve the data-fitting nonlinear equation 0 u u = for p by least-squares methods (see e.g.[3] [7]).In this section, we consider two slightly different solution methods, each of which is based on solving ( ) , u p simultaneously from the system consisting of both the model equation (2.2) and the data fitting equation 0 u u = on 0 Γ by least-squares (see e.g. 0 L Γ .Once u on Γ and v on 1 Γ are found from (3.4), then the Robin coefficient p on 1 Γ can be found from u and v by the simple relation (3.1). D are the second-order derivative operator for periodic boundary condition and for zero boundary condition respectively, and the penalty for the negative part of w by it typically takes only a few iterations (less than 10) to sa- tisfy the stopping criterion throughout our experiments.For comparison, we also present the result without the penalty term (when 0 β = ), which is direct and does not require iterations, but followed by converting from ( ) the machine epsilon.As seen, the few iteration needed for the nonnegativity constraints indeed have helped to improve the overall quality of the recovered coefficient p. Figure 1 . Figure 1.Recovered p by the linear least-squares method with nonnegativity constraints (solid), and by the direct method with truncation (dotted), in comparison with the true profile (dashed). Figure 2 . Figure 2. Recovered p by the nonlinear least-squares method, with nonnegativity constraints (solid) and without (dotted), in comparison with the true profile (dashed).	2022-07-01T15:02:58.847Z	2022-01-01T00:00:00.000	{ "year": 2022, "sha1": "b04d06a1de92b5667ce0646339d4ea846eb95cae", "oa_license": null, "oa_url": "http://www.scirp.org/journal/PaperDownload.aspx?paperID=118240", "oa_status": "GOLD", "pdf_src": "ScienceParsePlus", "pdf_hash": "1e5234cb25c1aef002f0357782c096aaac745ef6", "s2fieldsofstudy": [ "Mathematics", "Physics" ], "extfieldsofstudy": [] }
261930379	pes2o/s2orc	v3-fos-license	Dataset of audio signals from brushless DC motors for predictive maintenance Predictive Maintenance (PdM) has a main role in the Fourth Industrial Revolution; its goal is to design models that can safely detect failure in systems before they fail, aiming to reduce financial, environmental, and operational costs. A brushless DC (BLDC) electric motors have increasingly become more popular and been gaining popularity in industrial applications, so their analysis for PdM applications is only a natural progression; audio analysis proves to be a useful method to achieve this and rises as a very pragmatic case of study of the characteristics of the motors. The main goal of this paper is to showcase sound-based behavior of BLDC motors in different failure modes as result of an experiment led by researchers at Universidad del Cauca in Colombia. This dataset may provide researchers with useful information regarding signal processing and the development of Machine Learning applications that would achieve an improvement within Predictive Maintenance and I4.0.Predictive Maintenance (PdM) has a main role in the Fourth Industrial Revolution; its goal is to design models that can safely detect failure in systems before they fail, aiming to reduce financial, environmental, and operational costs. A brushless DC (BLDC) electric motors have increasingly become more popular and been gaining popularity in industrial applications, so their analysis for PdM applications is only a natural progression; audio analysis proves to be a useful method to achieve this and rises as a very pragmatic case of study of the characteristics of the motors. The main goal of this paper is to showcase sound-based behavior of BLDC motors in different failure modes as result of an experiment led by researchers at Universidad del Cauca in Colombia. This dataset may provide researchers with useful information regarding signal processing and the development of Machine Learning applications that would achieve an improvement within Predictive Maintenance and I4.0. a b s t r a c t Predictive Maintenance ( PdM ) has a main role in the Fourth Industrial Revolution; its goal is to design models that can safely detect failure in systems before they fail, aiming to reduce financial, environmental, and operational costs.A brushless DC (BLDC) electric motors have increasingly become more popular and been gaining popularity in industrial applications, so their analysis for PdM applications is only a natural progression; audio analysis proves to be a useful method to achieve this and rises as a very pragmatic case of study of the characteristics of the motors.The main goal of this paper is to showcase sound-based behavior of BLDC motors in different failure modes as result of an experiment led by researchers at Universidad del Cauca in Colombia.This dataset may provide researchers with useful information regarding signal processing and the development of Machine Learning applications that would achieve an improvement within Predictive Maintenance and I4.0.Predictive Maintenance ( PdM ) has a main role in the Fourth Industrial Revolution; its goal is to design models that can safely detect failure in systems before they fail, aiming to reduce financial, environmental, and operational costs.A brushless DC (BLDC) electric motors have increasingly become more popular and been gaining popularity in industrial applications, so their analysis for PdM applications is only a natural progression; audio analysis proves to be a useful method to achieve this and rises as a very pragmatic case of study of the characteristics of the motors.The main goal of this paper is to showcase sound-based behavior of BLDC motors in different failure modes as result of an experiment led by researchers at Universidad del Cauca in Colombia.This dataset may provide researchers with useful information regarding signal processing and the development of Machine Learning applications that would achieve an improvement within Predictive Maintenance and I4.0. © Data Description The dataset consists of .wavfiles ordered as follows: -BLDC_sound_data • 20 0 0.wav Each .wavfile is approximately 10 s long, so during pre-processing files must be trimmed to 10 s to properly analyze them.Files were recorded at 16 kHz frequency, so once they have been loaded and trimmed each file should be stored in a 160,0 0 0 × 1 array. Experimental Design, Materials and Methods The dataset contains 43 recordings of four different A2212 Brushless DC motors under different operation modes.Motors 1 and 2 were submitted to both normal circumstances and broken propeller fault, while motor 3 and 4 provide information only for the healthy and bearing fault categories respectively.Each motor was connected to a 30 A Electronic Speed Controller, supplied by a 11.1 V-2100 mAh LiPo battery; the speed of the motor was controlled using an ESP32, and this procedure can be performed in two manners: the first, is by reading a potentiometer and writing its value to the ESC, and the second, more IoT oriented is by connecting the ESP32 to a Blynk server, using a slider to write a value to the ESC.This second method offers more stability and control over the intervals but also is very dependent on the internet latency.Ten seconds of audio at 16 kHz are recorded using Audacity as an open-source software and the speed of the motors is incremented a fixed amount according to the category.Fig. 1 provides a scheme of the experimental setup used to obtain the audio recordings. The data is then properly classified, and a Pearson correlation analysis is performed to determine which combination of statistical and spectral features could be useful to train a Machine Learning model that can predict the current operational mode of a BLDC motor.Fig. 2 shows the result of said correlation analysis.Readers of this work are expected to download the audio files ( * .wav)[ 3 ] and perform digital audio processing to extract the features presented in Fig. 2 . Limitations The data was collected in a controlled environment minimizing outside noise. Ethics Statement The production of the data collected did not involve any human subjects, animal experimentation, nor any data from social media platforms.The authors have read and follow the ethical requirements for publication in Data in Brief. 2023 The Author(s).Published by Elsevier Inc.This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ) [ 1 ]iles of approximately 10 s each, with a 16 kHz sampling frequency containing the sound of four A2212 BLDC motors submitted to different categories: healthy motors, propeller failure and bearing failure.These audio files may be useful for signal processing and Machine Learning applications in Predictive Maintenance[ 1 ].Data was obtained by connecting BLDC motors to a 11.1 LiPo battery, a 30 A ESC, and an ESP32 to control the motor's speed via Blynk to obtain the motors sound, which were recorded at 16 kHz using a MCJR-005	2023-09-16T15:07:58.619Z	2023-09-01T00:00:00.000	{ "year": 2023, "sha1": "44c326982b7b2a94980167829d713a6bc95cc3a7", "oa_license": "CCBY", "oa_url": "https://doi.org/10.1016/j.dib.2023.109569", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "4a79101f0fb78555a5f62acd0366b1fa5a348f89", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [] }
7553133	pes2o/s2orc	v3-fos-license	Non-binary Hybrid LDPC Codes: Structure, Decoding and Optimization In this paper, we propose to study and optimize a very general class of LDPC codes whose variable nodes belong to finite sets with different orders. We named this class of codes Hybrid LDPC codes. Although efficient optimization techniques exist for binary LDPC codes and more recently for non-binary LDPC codes, they both exhibit drawbacks due to different reasons. Our goal is to capitalize on the advantages of both families by building codes with binary (or small finite set order) and non-binary parts in their factor graph representation. The class of Hybrid LDPC codes is obviously larger than existing types of codes, which gives more degrees of freedom to find good codes where the existing codes show their limits. We give two examples where hybrid LDPC codes show their interest. I. INTRODUCTION Binary LDPC codes are now well recognized as capacity approaching codes for various types of channels when the size of the codeword tends to infinity, and various methods have been proposed to optimize their irregularity profile with the help of Density Evolution under Gaussian Approximation (DE-GA) [2]. Other techniques based on EXIT charts [6] are also related to DE-GA and lead to the same analysis and optimization algorithms. However, there are several issues for which the binary LDPC codes show their limits: we can cite for example coded modulations and/or coding for small or moderate block lenghts. For these contexts, it has been shown recently that non-binary LDPC codes can be a good alternative. They exhibit better performance than their binary counterparts for coded modulations [3] and for code length typically in the range N ∈ [500, 2000] information bits [7,8]. The main interest of non-binary LDPC codes actually lies in the decoder: good non-binary LDPC codes have much sparser factor graphs (or Tanner graphs) than binary LDPC codes [10], and the Belief Propagation (BP) decoder is closer to optimum decoding since the small cycles can be avoided with a proper graph construction, as proposed in [7]. In this paper, we propose to study a class of hybrid LDPC codes which aims at combining the advantages of binary and non-binary LDPC codes in the same coding scheme. The class of hybrid LDPC codes is a generalization of existing classes of LDPC codes. For hybrid LDPC codes, we allow the connectivity profile of the factor graph to be irregular, but also the order of the symbols in a codeword can be irregular, that is to say, the symbols can 1 This work was supported by the French Armament Procurement Agency (DGA). belong to finite sets with different orders. We depict in section II the structure of hybrid LDPC codes and briefly describe the decoding algorithm. In section III, we recall the existing work on optimization of non-binary LDPC codes with DE-GA, and introduce a specific modelization of the messages in the factor graph which allow an efficient optimization of nonbinary LDPC codes on the binary input Gaussian channel (BI-AWGN). In section IV, the DE-GA equations for hybrid LDPC codes are derived and the optimization procedure is presented. The analysis of hybrid LDPC codes is based on a detailed representation of the factor graph of the code [1], together with the introduction of extra parameters to describe the proportion of irregular set orders in the codeword. The parameterization of hybrid LDPC codes is therefore very rich. We have then decided to optimize sub-classes of hybrid LDPC codes, and we give two different examples that show their interest when compared to the best known existing LDPC codes. The examples and the simulation results are shown in section V. II. THE CLASS OF HYBRID LDPC CODES We define a non-binary hybrid LDPC code as an LDPC code whose variable nodes belong to finite sets of different orders. To be specific, this class of codes is not defined in a finite field, but in finite groups. We will only consider groups whose cardinality q k is a power of 2, that says groups of the type G(q k ) = Z 2Z p k with p k = log 2 (q k ). Thus each element of G(q k ) has a binary map of p k bits. Let us call the minimum order of codeword symbols q min , and the maximum order of codeword symbols q max . The class of hybrid LDPC codes is defined on the product group Z Note that this type of LDPC codes built on product groups has already been proposed in the literature [11], but no optimization of the code structure has been proposed and its application was restricted to the mapping of the codeword symbols to different modulation orders. Parity check codes defined on (G(q min ) × . . . × G(q max )) are particular since they are linear in Z 2Z , but could be non-linear in the product group. Although it is a loss of generality, we have decided to restrict ourselves to hybrid LDPC codes that are linear in their product group, in order to bypass the encoding problem. We will therefore only consider upper-triangular parity check matrices and a specific ordering of the symbol orders in the codeword, which ensures the linearity of the hybrid codes. The structure of the codeword and the associated parity check matrix is depicted in Figure 1. We hierarchically sort the different group orders in the rows of the parity-check matrix, and also in the codeword, such that q min < . . . < q k < . . . < q max . To encode a redundancy symbol, we consider each symbol that participates in the parity check as an element of the highest group, which is only possible if the groups are sorted as in Figure 1. This clearly shows that encoding is feasible in linear time by backward computation of the check symbols. In order to explain the decoding algorithm for hybrid LDPC codes, it is usefull to interpret a parity check the hybrid code as a special case of a parity check built on the highest order group of the symbols of the row, denoted G(q l ) and have a look at the binary image of the equivalent code [8]. For codes defined over Galois fields, the nonzero values of H correspond to the companion matrices of the finite field elements and are typically rotation matrices (because of the cyclic property of the Galois fields). In the case of hybrid LDPC codes, the nonzero values have no linear representation and are indeed nonlinear maps that have rectangular matrix equivalents. To be more specific, the function that connects a row in G(q l ) and a column in G(q k ) is a nonlinear function that maps the q k symbols of G(q k ) into a subset of q k symbols that belongs to G(q l ). This function has an equivalent binary representation by a matrix of dimension (p l × p k ). Note that with the above mentioned constraints, we have necessarily p k < p l . It is not very difficult to generalize the Belief propagation decoder to hybrid codes, and it has been shown that even for those very particular structures, it is possible to derive a fast version of the decoder using FFTs [9]. For lack of space reason, we do not present in this paper the BP decoder for hybrid codes, and we refer to the general algorithm described in [9] for which the decoder for hybrid LDPC codes is a special case. In the rest of the paper, we will call the message passing step through h ij extension when it is from G(q k ) to G(q l ) and truncation when it is from G(q l ) to G(q k ) since the sizes for the messages in the factor graph differ. The BP decoder steps can be followed in the factor graph representation of a single parity check as depicted in Figure 2. Let us now introduce parameters that describes the irregularity of group orders in the codeword. Letγ k be the proportion of symbol nodes in the hybrid graph which belong to G(q k ) and by definition, we take q min = 2. The code rate of an hybrid code with the specific structure presented in Figure 1 can be expressed as: x vc,k 1 x cv,k 2 x vc,l x vc,l x vc,l x cv,l Note that this expression is completely general since if we fix q r = q r+1 , then both information and redundancy can share the same group order q r . In order to optimize hybrid LDPC codes, following the strategies used to optimize binary or nonbinary LDPC codes, we need to express the density evolution of the messages under Gaussian approximation along one decoding iteration, with respect to the parameters to be optimized. In our case, the parameters are the proportions of irregular connections in the graph and the proportions of irregular group ordersγ k . In the next section, we recall some required properties of DE-GA for non-binary LDPC codes, that we will use to make the generalization to hybrid codes. III. ANALYSIS OF NON-BINARY LDPC CODES OVER GF(q) Let us first present how one analyses non-binary LDPC codes on the BI-AWGN channel using a gaussian approximation. We must quote three works from which this approach is highly inspired. In [3], Bennatan et al. have proposed a density evolution for LDPC codes on GF (q) on memoryless q-ary channels. They have derived a gaussian approximation of the densities of messages, which leads to a quite easy optimization of these codes, using EXIT charts [6]. Although very general, their approach can be improved if the channel is BI-AWGN, by choosing a more accurate initialization of the densities of the LLR messages. In our work, we took a different initialization of the decoder, which describes more precisely the BI-AWGN output messages. The model for the LLRs is the same as the one proposed in [4], where the authors analyse nonbinary LDPC codes on the BI-AWGN channel. Following well known ideas, we will track the information content of the messages (here vector messages) under Gaussian approximation, that is the mutual information of a discrete input channel with additive Gaussian noise whose output is the message in the graph. In [5], Li. et al. have also proposed a DE-GA approach for non-binary LDPC codes, but the quantity they used to follow the evolution of densities was the mean of the messages instead of the mutual information. The first necessary property that must be fulfilled is a symmetry property for the vector messages. The symmetry of q-ary log density ratio (LDR) vector W is defined in [3]. Let v be the corresponding symbol sent and W a the a-th component of W. An LDR-vector is symmetric if and only if W satisfies In [3], the symmetry was defined for codes defined on fields, but this definition clearly applies for finite Abelian groups. For the BI-AWGN channel, the bitwise log likelihood ratios (LLR) are symmetric in the sense defined in [2], which in turn, induces the symbolwise symmetry of the LLR-vector. Moreover, the symmetry property (4) is kept during the non-binary BP decoder operations [5]. In the next section, we discuss the compatibility of the symmetry property with the specific operations used in the hybrid decoder, that is the truncations and extensions. We now define some useful notations, in concordance with the previous quoted works, to express information transfer functions. LLR b denotes the bitwise LLR of a received BPSK modulated bit and m bc is the mean of LLR b . LLR s denotes a symbolwise LLR vector of a GF (q) symbol and m sc its vector mean. If B is the (q − 1) × p (with p = log 2 (q)) mapping matrix from vectors of p bits to GF (q) symbols and 1 p is the all-one column vector of size p, then we have m sc = m bc B1 p . If we call σ 2 the variance of the BI-AWGN channel, thanks to the symmetry of the channel, we know from [2] that m bc = 2 σ 2 and LLR b ∼ N (m bc , 2m bc ). As said previously, the symbolwise LLRs are then symmetric. According to [5], if the messages are symmetric and gaussian distributed as N (m, Σ), the covariance matrix Σ can be uniquely determined by the mean vector m such that Again, this property is defined in a Galois field, but remains the same in a group of order q since it only requires the use of the proper addition ⊕ in the Abelian group. The symmetry allows to make the all-zero codeword assumption. If we make the approximation that all the vector messages on the graph are gaussian, then we can see on Equation (5) [3,5] that only two scalar parameters entirely define the gaussian approximation of densities of messages on the graph: σ 2 and m cv . Since the channel is known at each step of the optimization process, only one scalar parameter remains to track: m cv . Using the one-to-one relation between the scalar mean of a vector and its mutual information given in equation 6, we can express the EXIT transfer function of one iteration of the non-binary BP decoder. Let us denote the two useful functions J v and J c (for variable node decoder and check node decoder, respectively), determined as in [4]: with v ∼ N (m, Σ) with where Σ is computed from m by the symmetry relation of Equation (5). Note that J c is a particular case of J v where all components of the vector m are equal to m. Finally, we get Equation (1) that expresses the extrinsic transfer function of the non-binary BP decoder used on a BI-AWGN channel from iteration number t to iteration number t+1. (λ, ρ) are the usual parameters that define the connectivity profile of a family of GF (q) LDPC codes, and x (t) vc is the mutual information of any check node incoming vector messages at the t-th iteration. For more details about the derivation of these equations and the associated proofs, please refer to the cited papers. IV. ANALYSIS OF HYBRID LDPC CODES In this section, we now explain how we can generalize the equations of DE-GA of non-binary LDPC codes to hybrid LDPC codes and how to introduce the extra parameters that describe the irregularity in the group orders. To properly define a family of hybrid codes, it is usefull to adopt a detailed representation of the factor graph, directly inspired from the one introduced by Kasai et al. in [1]. We define a hybrid LDPC code family by π(i, j, k, l). It is the joint probability that an edge of the hybrid Tanner graph is linked to a data node of connectivity degree i in G(q k ) and to a check node of connectivity degree j in G(q l ). We also define the following marginal and conditionnal probabilities is the proportion of edges linked to a symbol node of degree i, given that this symbol node is in G(q k ), and γ(i, k) is the proportion of edges linked to a symbol node in G(q k ), given that this symbol node is of degree i. The analysis of hybrid non-binary LDPC codes is completely based on the previous approach that assumes the densities of vector messages to be gaussian distributed, when transmitting on BI-AWGN channel. We add two steps to the non-binary analysis described in the last section, that correspond to truncation and extension of messages when passing from a data node to a check node in a higher order group, and vice versa. Thanks to Equation (6), we easily obtain the expression between the mutual information x q l , of an extended LDR message in G(q l ), built from a message in G(q k ) whose mutual information is To get the relation giving the mutual information of a message in G(q k ) built by the truncation of an LDR message in G(q l ), we need to redefine the functions J v (m) and J c (m). J v (m, q) and J c (m, q) are defined in the same way as before, with q that represents the order of the group of the vector messages whose mean is m or m1 q−1 . With these new definitions of functions J v and J c , if x q k is the mutual information of the truncated vector, we have: which corresponds to the conservation of the mean of each component after truncation. We also re-define m sc by m q sc where q is the order of the group of the symbol node whose LLR vector of size q − 1 has mean m q sc . We have also shown that the symmetry property of the messages holds for the specific transformations of truncation and extension. We do not present the proofs here and they will be reported in future publication. Following the different steps of one decoding iteration, we can derive the EXIT function of one iteration of the hybrid decoder. This EXIT function is expressed in equations (2) and (3). This function expresses x ) which is the mutual information at the t-th iteration of a vector message going out of a data (resp. check) node of degree i (resp. j) in G(q k ) (resp. G(q l )) extended (resp. truncated) to become input of a check (resp. data) node in G(q l ) (resp. G(q k )). V. OPTIMIZATION AND RESULTS As for the optimization of usual LDPC codes, we want to find the parameters of a hybrid family for a given code rate that minimize the convergence threshold. In all the simulations presented in this paper, we have considered all the check nodes of same degree and in the same group. And hence the number of parameters to be optimized is reduced from four to two (distribution π(i, k) of degrees and groups of variable nodes). The ideal optimization procedure would be to jointly optimize γ and λ, i.e., the 2-variable function π(i, k). In order to simplify the optimization, we chose to fix one of these parameters, and to optimize the other one. That says we tested two directions of optimizing hybrid LDPC codes: either we look for the optimal proportions γ k of different finite sets given a fixed connectivity of the graph λ i , or we look for the optimal proportions λ i given a fixed repartition γ k of the group orders in the codeword. For both approaches, we choose to map all the redundancy bits into symbols in the highest order group G(q max ), and to prohibit information symbol nodes that are in G(2) to be of degree 2 in order to mitigate the influence of catastrophic cycles. First, we consider the optimization of λ, when γ(i, k) is fixed. From the above remarks, it follows that we fix as a priori constraints γ(2, 2) = 0, γ(i = 2, 2) = 1. The other parameters γ(2, q) for q = 2 are determined by the proportions of information symbols in the different groups. For this simplified model, the code rate is defined by: According to this expression, the code rate maximization is equivalent to the maximization of the denominator of the second term. Moreover, since π(i, k) = λ i γ(i, k), equation (3) corresponds to the convergence criterion equivalent to a strictly increasing information content x vc,qmax . Thus the cost function and all constraints are linear with respect to λ and the optimization problem can be efficiently solved using linear programming. The hybrid code solution of the optimization problem is relatively dense since it has an average row weight of 14.3 ones, but it comes from the fact that a rate 1/2 hybrid code is obtained with a graph with higher rate. Indeed, the hybrid LDPC codes are adapted for rather low rates. In Figure 3, we give the simulation results for a code with target rate R = 1/2. The hybrid code is compared to existing good codes. The irregular binary code has been chosen from the distributions in [2] and the distribution of the irregularity for the GF (8) code has been optimized with the equations of section III. All graphs have been designed with the PEG algorithm that has been widely accepted as a good finite length code construction. First, we can see that the error floor is lowered by going from GF (2) to GF (8), and that the regular (3, 6) code in GF (8) has a worse convergence than the irregular codes, but a much lower error floor. Those results are in accordance with the usual observations on binary LDPC codes. Our hybrid LDPC code with 2 group orders G(8) − G(2) is as expected a good compromise of the joint problem convergence/error floor. The convergence region has been slightly degraded compared to irregular LDPC codes, but with the effect of no observed error floor up to a FER=5.10 −6 . We expect even better results by allowing more degrees of freedom in the optimization procedure. In the second example, we optimized γ k , with λ(i, k) fixed. In this case, we look for the best proportion of group orders for a regular hybrid graph defined by the connectivity of data nodes and check nodes (d v = 2, d c = 3). According to the code rate expression the cost function is still the denominator of the second term. We aimed with this example at designing good codes for a rather low rate of R=1/6. We obtained the optimized hybrid code with three different group orders G(256)−G(16)−G(8), and we have compared our hybrid code with various good codes presented in the literature. In Figure 4, we can see that the irregular binary LDPC code is not a good solution for such low rate and moderate block length, as it is the worst code simulated. The regular code over GF (256) designed with the methods presented in [8] is better with 0.5dB gain, but is outperformed by a very specific construction of binary quasicyclic LDPC codes especially designed for low rates found in [12]. Our hybrid code shows the best performance and is to our knwoledge the best performance observed at this rate and codelengths. This confirms the fact that hybrid LDPC codes appear to be a good solution for low rate applications. Note that the error floor of our hybrid code is likely to be lowered with similar techniques as presented in [8]. We plan to address this issue in a future work. VI. CONCLUSION This paper aims at combining advantages of having variable nodes in different order finite sets, in a bipartite graph, to build non-binary hybrid LDPC codes. First, we have presented the structure and the decoding of the class of hybrid codes. We have then explained how to optimize irregular non-binary LDPC codes over GF(q) for the BI-AWGN channel, and we have described how to generalize this technique for the optimization of hybrid codes. Finally, the most interesting results are obtained for quite low target code rates (R = 1/6): our hybrid code outperforms the best known codes for this code rate. Future work will address the problem of the finite-length optimization for this class of codes.	2007-01-09T09:50:57.000Z	2006-10-01T00:00:00.000	{ "year": 2007, "sha1": "9ad0b9dba621a39e1c555e481d24b0bd308805d3", "oa_license": null, "oa_url": "http://arxiv.org/pdf/cs/0701066", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "19a6e8a8ad7fc7e403333ded07b930a40fa54404", "s2fieldsofstudy": [ "Computer Science", "Engineering" ], "extfieldsofstudy": [ "Computer Science", "Mathematics" ] }
244415303	pes2o/s2orc	v3-fos-license	Development and validation of an expanded targeted sequencing panel for non-invasive prenatal diagnosis of sporadic skeletal dysplasia Background Skeletal dysplasia (SD) is one of the most common inherited neonatal disorders worldwide, where the recurrent pathogenic mutations in the FGFR2, FGFR3, COL1A1, COL1A2 and COL2A1 genes are frequently reported in both non-lethal and lethal SD. The traditional prenatal diagnosis of SD using ultrasonography suffers from lower accuracy and performed at latter gestational stage. Therefore, it remains in desperate need of precise and accurate prenatal diagnosis of SD in early pregnancy. With the advancements of next-generation sequencing (NGS) technology and bioinformatics analysis, it is feasible to develop a NGS-based assay to detect genetic defects in association with SD in the early pregnancy. Methods An ampliseq-based targeted sequencing panel was designed to cover 87 recurrent hotspots reported in 11 common dominant SD and run on both Ion Proton and NextSeq550 instruments. Thirty-six cell-free and 23 genomic DNAs were used for assay developed. Spike-in DNA prepared from standard sample harboring known mutation and normal sample were also employed to validate the established SD workflow. Overall performances of coverage, uniformity, and on-target rate, and the detecting limitations on percentage of fetal fraction and read depth were evaluated. Results The established targeted-seq workflow enables a single-tube multiplex PCR for library construction and shows high amplification efficiency and robust reproducibility on both Ion Proton and NextSeq550 platforms. The workflow reaches 100% coverage and both uniformity and on-target rate are > 96%, indicating a high quality assay. Using spike-in DNA with different percentage of known FGFR3 mutation (c.1138 G > A), the targeted-seq workflow demonstrated the ability to detect low-frequency variant of 2.5% accurately. Finally, we obtained 100% sensitivity and 100% specificity in detecting target mutations using established SD panel. Conclusions An expanded panel for rapid and cost-effective genetic detection of SD has been developed. The established targeted-seq workflow shows high accuracy to detect both germline and low-frequency variants. In addition, the workflow is flexible to be conducted in the majority of the NGS instruments and ready for routine clinical application. Taken together, we believe the established panel provides a promising diagnostic or therapeutic strategy for prenatal genetic testing of SD in routine clinical practice. Supplementary Information The online version contains supplementary material available at 10.1186/s12920-021-01063-1. Background Skeletal dysplasia (SD) is a heterogeneous group of genetic disorders associated with various abnormalities of bones and joints. There are 461 different conditions classified into 42 groups primarily based on the clinical, radiographic, and molecular phenotypes [1]. The clinical manifestations vary in severity from mild phenotypes to severe abnormalities with perinatal mortality due to lung hypoplasia and respiratory complications [2]. Previous studies showed that the incidences of non-lethal and lethal SD are around 1 per 5,000 births [3,4] and 0.95-1.5 per 10,000 births [5][6][7], respectively. The three most common SD types are thanatophoric dysplasia (around 29%) [8], osteogenesis imperfecta type 2 (14%), and achondrogenesis (9%) [9], which account for 40 to 60 percent of all lethal SD [6,[10][11][12]. Importantly, among the perinatal deaths of SD, 23-32% occur during the first week of life [3,6]. The unexpected loss and highly distressing event result in a hugely psychological burden on bereaved parents. Therefore, it remains in desperate need of precise and accurate prenatal diagnosis of SD in early pregnancy. Traditionally, the prenatal diagnosis of SD relies on ultrasonography, followed by confirmation using either invasive monogenic testing or post-delivery radiographs and autopsy. Although the ultrasound assessment of SD includes a number of features and criteria, the accuracy of routine ultrasound approach was reported as 40-60%, because the high heterogeneity in genetic defects and large phenotypic variability [13,14]. The diagnostic confirmation of lethality was still difficult due to the lack of systematic approach [15]. In addition, the traditional genetic testing requires invasive procedures to obtain the fetal specimen such as amniocentesis and chorionic villus sampling, and the Sanger sequencing for each individual gene is costly and time-consuming [15]. Therefore, the earlier prenatal genetic diagnosis is critical issue in maternal-fetal precision medicine. The discovery of circulating fetal cell-free DNA (cfDNA) has opened a promising opportunity for early detection of fetal genetic defects [16,17]. Given that the fetal fraction in maternal blood is estimated to be around 4%-30% depending on the gestational age and maternal weight effects [18][19][20], it requires a more sensitive platform to detect the low-frequency variants in association with the fetus using maternal plasma cfDNA. Owing to the advantages of high-throughput, parallel sequencing techniques, the non-invasive prenatal testing (NIPT) has been developed and applied to identify fetal chromosomal aneuploidies using the cfDNA extracted from a blood of pregnant woman [21,22]. Although the development is still in its infancy, the precise and flexible applications of NIPT in the detection of single-nucleotide variant (SNV) and small deletion/insertion (Indel) have been reported [23][24][25]. To detect low-frequency variants at single-base resolution, targeted sequencing (targeted-seq) that offers ultra-deep coverages on the genomic region of interest has been shown to function accurately and adequately [26,27]. Furthermore, targeted-seq is cost-effectiveness in comparison with whole genome sequencing or whole-exome sequencing [28]. Two major methods, amplicon-and capture-based that utilize multiplex polymerase-chain reaction (PCR) and hybridization by probes, respectively, have been established to enrich targeted regions of interest for downstream sequencing. Previously, an amplicon-based targeted-seq for prenatally screen of FGFR3 gene [23,24] and a capture-based targeted-seq for screening 497 genes including several SDassociated genes [29] have been developed. Due to the restrictions by fewer mutation hotspots or lower coverage depth, it limits the application of these panels in routine clinical practice. Thus it remains in need to establish an expanded targeted sequencing panel for the detection of sporadic SD in early pregnancy. The advantages of amplicon-based technique are the less requirement of input DNA and higher on-target rate when compared to the capture-based method, thus it is more attractive to be applied in NIPT where the fetal cfDNA is in limited amount and high accuracy is on-demand. Here, we report the development of an expanded amplicon-based targeted-seq panel for the detection of SD at ultra-depth level. This panel examines a total of 87 recurrent hotspots located in 5 most common genes (FGFR2, FGFR3, COL1A1, COL1A2, COL2A1) associated with 11 dominant SD, in which 5 mutations responsible for lethal SD are included. The targeted-seq workflow can be applied to genomic DNA as well as cfDNA, both achieve high sensitivity and specificity of 100%. Most importantly, the workflow is optimized to be carried out on both Illumina and Ion Torrent platforms, which account for more than 90% of instrument marketplace. Collectively, we streamline the targeted-seq workflow of two different systems and confirm its performance in clinical practice. The workflow provides timely detection of SD at early pregnancy and shows the true value of prenatal precision medicine. Human subjects A total of 59 DNA specimen, including 36 cell-free (cf ) and 23 genomic (g) DNA, were collected from 6 families and 31 pregnant women (Table 1). Among them, family 1 and 2 are with fetus that were diagnosed with suspected dwarfism and osteogenesis imperfecta by ultrasound examination, respectively, while the parents were all asymptomatic. All the others were cases collected during regular pregnancy examination to screen genetic defect at early gestational age and have been followed through the gestation period until baby was delivered. Written and signed informed consent was obtained from all subjects of this study and approved by the Institutional Review Board (IRB) of the National Cheng Kung University. Genomic DNA collection and processing Roughly 10 ml of peripheral blood was drawn from all participants and in two cases of Family l and Family 2, umbilical tissue was obtained from the deceased fetus. The buffy coat was separated from peripheral blood through centrifugation at 3000 rpm for 10 min at room temperature. DNA of umbilical cord and buffy coat were isolated by QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol. The genomic DNA was stored at -20 °C for long term storage. The standard DNA "NA11316" harboring homozygous FGFR3 mutation (c.1138 G > A) was purchased from Coriell Institute (Coriell Institute, Camden, New Jersey, USA). To generate the spike-in DNA, NA11316 DNA was mixed with normal maternal gDNA (from Family 2) in different percentage (2.5%, 5%, 10%) to mimic different fetal DNA fractions in the maternal plasma. Plasma preparation and cfDNA extraction Maternal plasma was obtained by a two-step centrifugation process. In the first step, the maternal peripheral blood was separated by centrifugation of the collection tube at 2,000 g for 15 min at room temperature and the supernatant was transferred to a 1.5 ml centrifuge tube. The sample was then centrifuged at 14,000g for 10 min at room temperature and the clear plasma was stored at − 80 °C until further processing. To isolate cfDNA, maternal plasma was centrifuged at 16,000g for 5 min at room temperature, followed by cfDNA extraction using QIAamp Circulating Nucleic Acid Kit (QIAGEN) according to the manufacturer's protocol. The quality and quantity of cfDNA were determined by LabChip GX Nucleic Acid Analyzer (Perki-nElmer, Waltham, Massachusetts, USA) and Qubit Fluorometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA), respectively. The cfDNA was stored at − 20 °C until further process. Library construction and amplicon sequencing The amplicon libraries were constructed by Ion AmpliSeq ™ Library Kit 2.0 (Thermo Fisher Scientific) according to the manufacturer's protocol, and sequenced on Ion Proton (Thermo Fisher Scientific) or NextSeq550 instruments (Illumina, San Diego, California, USA). Briefly, approximately 10 ng of gDNA, or 1 ng of cfDNA were amplified using the 51 pairs of pooled primers in a single-tube multiplex PCR setting. The amplified libraries were then subjected to partial digestion, barcode ligation, purification, and run on Ion Proton sequencer (Fig. 1A). To modify the experimental procedure for Bioinformatics pipeline After the sequencing, the low-quality bases (Q score < 20) were trimmed and the filtered reads were aligned to human reference genome (GRCh38) using the Torrent Mapping Alignment Program (TMAP, Thermo Fisher Scientific) and Burrows-Wheeler Aligner (BWA)-v0.7.17 [48] for Ion Proton and NextSeq550 platforms, respectively (Fig. 1B). The output BAM file was used to call variant by using VariantCaller (v5.0.3.5, Thermo Fisher Scientific) under default parameters. To identify lowfrequency variants in association with fetal cfDNA, a [50]. Alternative allele frequency of 2% and 20% were set as the thresholds for calling somatic and germline variants, respectively. Statistics analysis Statistical analyses, including two-way analysis of variance (ANOVA), Pearson correlation coefficient (r), Kruskal-Wallis test, and two-tailed t-test, were conducted by using GraphPad Prism version 7.05 for Windows (GraphPad Software, San Diego, California USA). Data were considered significant when the P values were < 0.05. Establishment of amplicon-based targeted-seq workflow To establish amplicon-based targeted-seq workflow (Fig. 1A, B, left), 10 ng of gDNA from 17 individuals were used for library construction and sequencing on Ion Proton platform. After sequencing, the fastq data was trimmed and then mapped to the human genome. We obtained 100% coverage of each amplicon at averaged sequencing depth of 13,160x. The uniformity and ontarget rate were 99.23 ± 0.44% and 96.39 ± 3.76%, respectively. Although the mean amplicon read ratio (define by read counts in each amplicon divided to total read counts per sample) of 1.96 ± 0.45% (ratio ranged from 0.63% to 2.96%) is closed to the expected ratio of 1.96%, the amplicon read distributions are significantly different across 51 amplicons due to the characteristic of primers and GC content of each amplicon (P < 0.0001, Fig. 1C). Nevertheless, the pattern of amplicon distribution was no difference among 17 samples (P > 0.9999, Fig. 1C) and the results demonstrated high reproducibility of established workflow. Next, we aimed to generalize the workflow to be used on NextSeq550 platform (Fig. 1A, B, right). Using slightly modified procedure and under a base coverage of 3,743x, we obtained the uniformity and on-target rate were 99.14 ± 0.92%, and 97.01 ± 0.71%, respectively. The performance is comparable to the data obtained using Ion Proton. Albeit the variation of reads distribution was slightly higher using NextSeq550 (mean ratio of 1.96 ± 1.14%, ranged from 0.49% to 5.39%) than that was using Ion Proton, common features of significant differences of read distribution across 51 amplicons (P < 0.0001, Fig. 1D) and high reproducibility among samples (P > 0.9999, Fig. 1D) were observed. Collectively, these data demonstrated the high amplification efficiency and quality in the established targeted-seq workflows. Furthermore, the flexibility of running the established targeted-seq workflow on both Ion Torrent system (i.e., Ion Proton and Ion PGM) and Illumina system (i.e., HiSeq, MiSeq, and NextSeq), the instrument implemented in the majority of diagnostic laboratories, ensure the flexibility for clinical usage. Validate the amplicon-based targeted-seq pipeline for the detection of skeletal dysplasia We then seek to validate the targeted-seq pipeline using DNAs from a family with a fetus diagnosed as dwarfism by ultrasound images at gestational age of 21 weeks (Family 1, Fig. 2A). The targeted-seq workflow was applied to the parents' and fetal genomic DNAs. Deep sequencing results clearly showed that the abortus carried a heterozygous mutation in FGFR3 (c.1949 A > T, p.Lys650Met), while no mutation was detected from both parents (Fig. 2B). The identified de novo mutation was subsequently confirmed using Sanger sequencing (Fig. 2C). The mutation in FGFR3 (c.1949 A > T) is known to cause thanatophoric dysplasia, a severe short-limb dwarfism syndrome that is usually lethal in the perinatal period (OMIM #187601), which supports the findings of targeted-seq. Taken together, the results revealed that the established targeted-seq workflow effectively identify germline mutation in association with SD. We next challenged the capability of identifying lowfrequency variant using established targeted-seq workflow. We utilized a normal maternal genomic DNA and mixed with a standard DNA sample harboring FGFR3 single base mutation (NM_000142: c.1138G > A, p.G380R) at different percentages of 2.5%, 5%, and 10%. The spike-in DNA was used to mimic the condition in which the fetal cfDNA is mixed with maternal cfDNA at various fractions. Since the read depth has impact on the sensitivity of low-frequency variant detection, amplicon libraries were prepared and sequenced at expected coverages of 10,000x, 15,000x, and 25,000x on Ion Proton platform. We obtained the mean base coverages of 12,580x, 16,059x, and 23,904x, that were not too far away from our expectations. In spite of different read depths, the uniformities of 99.32 ± 0.21%, 98.31 ± 0.32% and 99.61 ± 0.29%, and on-target rates of 98.41 ± 0.20%, 98.43 ± 0.16% and 98.43 ± 0.20% were all similar and represented good quality results. In addition, amplicon read distributions from 12,580x (ranges from 0.71% to 2.96%, mean ratio = 1.96 ± 0.55%), 16,059x (ranges from 0.66% to 2.91%, mean ratio = 1.96 ± 0.52%), and 23,904x samples (ranges from 0.72% to 2.86%, mean ratio = 1.96 ± 0.55%) showed consistency regardless the different depths among samples (P > 0.99, Fig. 2D). The total depth at the spike-in FGFR3 mutation site ranges from 5,645 to 16,835. Most importantly, we identified the spike-in FGFR3 mutation (c.1138G > A) in all samples, even in the lowest 2.5% one with the minimum depth at 12,580x (Table 2). In addition, the FGFR3 mutation (c.1138G > A) was detected in all spike-in samples at the values approximated to the modeled spike-in fractions (P = 0.11, Table 2). These data demonstrated that the workflow enables the detection of low-frequency variant accurately. We duplicated the targeted-seq procedure with various spike-in DNA samples on NextSeq550. At mean base depth of 6,821x, we achieved uniformity and FGFR3 loci (c.1949A). D The targeted-seq was performed at expected read depth of 10,000x, 15,000x, and 25,000x on Ion Proton. The ratio of read count of each amplicon to total reads count was plotted. P-value was calculated by Kruskal-Wallis test. E The targeted-seq was performed using spike-in DNAs with FGFR3 mutation (c. 1138G > A) at fraction of 2.5%, 5%, and 10% on NextSeq550. The ratio of read count of each amplicon to total reads count was calculated and compared. P-value was calculated by Kruskal-Wallis test. F The allele frequency of 5 SNPs obtained from Ion Proton and NextSeq550 at various spike-in fractions were compared and the correlation was calculated using Pearson' s correlation coefficient on-target rate of the samples were 98.67 ± 1.37%, and 98.03 ± 0.77%, respectively. Notably, similar amplicon read distributions among three different spike-in fractions were observed, suggesting cfDNA fraction (as indicated by various spike-in DNA fractions) has no effect on the dynamic of amplicon distribution (P = 0.97, Fig. 2E). As there are additional 55 single nucleotide polymorphisms (SNPs) recorded in the regions covered by the 51 amplicons (Additional file 2: Table S2) in Taiwanese population [51], we compared the results of variant calling using NextSeq550and Ion Proton systems. There were 5 SNPs identified from 3 spike-in DNA samples sequenced on two platforms. Using calculated minor-allele frequency (MAF) for these SNPs, Fig. 2F showed highly concordant results from 2 platforms (r = 0.79, P < 0.001). Taken together, these data demonstrated the accuracy and high confidence of established targeted-seq workflows for the detection of low-frequency variants, suggesting the possible application of workflow in detecting genetic defects of SD in early pregnancy. The sensitivity and specificity of established targeted-seq workflow in clinical usage Given that we have validated the targeted-seq workflow, we sought to evaluate its sensitivity and specificity using cfDNA from early pregnancy stage. Library construction and targeted-seq were performed using 36 cfDNA samples (Table 1). Among them, one sample (Family 2) was suspected to have SD at regular pregnancy examination. The fetus presented short long bones and multiple fractures using ultrasound image at 21 weeks of gestational age, which resembled a case of severe osteogenesis imperfecta (OI)-related symptoms. No mutation was identified in the cfDNA or in gDNAs from fetal umbilical cord tissue. To investigate the underlying genetic defect of the proband, we performed whole-exome sequencing and found novel compound heterozygous mutations in CRTAP gene consisting of SNV and deletion, suggesting the case was a rare autosomal recessive form of OI [52]. The findings supported the true negative result of cfDNA using the established targeted-seq assay. For the rest of 35 cfDNA samples, we found no mutation in the 87 hotspots from the tested SD panel. Concordant to the testing results, follow-up from these women throughout the gestation to the baby delivered showed no indication of fetal abnormality, demonstrating the accurate call for true negative results. Overall, there are 9 true positive detections (3 spike-in samples in 3 different read depths) and 36 true negative detections from our low-frequency variant tests. Although the sample size is still small, our SD targeted-seq assay reaches 100% sensitivity and 100% specificity thus it suggests the established workflow is ready for routine clinical application. Discussion The diagnostics laboratories constantly strive to gain a precise and extensive genetic characterization of patients at increased efficiency, robust and cost-effectiveness. Herein, we developed an amplicon-based targetedseq panel for the detection of 87 pathogenic mutations, including 82 SNVs, 2 MNVs and 3 indels distributing on 5 most common genes that lead to 11 autosomal dominant SDs. The targeted-seq pipeline can be applied to cell-free and genomic DNA, both of which achieve high sensitivity and specificity of 100%. Given that the turnaround time from blood sample collection to issue test report is ~ 4 days, it is feasible and cost-effective for routine screening for pregnant women at the early pregnancy. As such expanded panel, it can rule out the most common SDs from fetus and reduce the anxiety and stress in pregnancy. For the fetus presenting abnormal skeletal growth, this assay also can be a great help in genetic confirmation using genomic fetal DNA from amniocentesis or chorionic villus sampling. The accuracy and precision are key determinants of the detection assay used in clinical diagnosis. Several studies have provided different panels to detect monogenic disorders in NIPT recently [23,24,29,53]. For instance, two amplicon-based targeted-seq panels consisting of 18 and 22 hotspots in FGFR3 genes were proposed to screen TD and ACH [23,24], respectively. In addition, Malcher et al. also reported a capture-based targetedseq procedure for screening a large panel of 497 genes in NIPT with the mean and median coverage across all sample are 267x and 222x [29]. Since the presence of fetal fraction in the maternal cfDNA can be as small as 4-5%, it needs higher coverage to precisely detect the low-frequency mutation in association with fetal cfDNA. Under the circumstance, lower overall coverage or read depth can easily lead to false negative results. Moreover, Russo et al. developed a amplicon-based targeted-seq for 337 mutation hotspots associated with autosomal recessive and dominate disorders for NIPT, including 42 hotspots in FGFR2 and FGFR3 genes [53]. However, it was a pilot study without reporting statistics of the outcome in terms of accuracy, specificity or sensitivity [53]. In the current study, we established the targeted-seq workflow that shows great performance in deep coverage, high ontarget rate and uniformity. Most importantly, the workflow is able to detect variant with as low as 2.5% minor allele frequency. Because the panel is designed to detect autosomal dominant SD bearing a pathogenic mutation in heterozygous state, the minimal requirement of fetal cfDNA should be 5%, which has been set as minimal receiving criteria of fetal fraction in maternal plasma. In this study, all the clinical cfDNA are within the reporting range of fetal fraction (ranging from 5.3% to 30.9%, mean = 16.7 ± 6.7%), suggesting the reliable and accurate interpretation of the genetic testing. According to our assay development, we will run deep targeted-seq with 1 M total reads to reach at least 2000x base depths for all of 87 hotspots. Thus it provides most accurate and robust panel so far in identifying mutations for common dominant SD. To date, several approaches have been developed for non-invasive prenatal diagnosis of a range of inherited and/or de novo transmission disorders using non-NGS or NGS-based techniques, such as droplet-digital PCR, PCR with restriction enzyme digest (PCR-RED), real-time quantitative PCR, relative dosage haplotype dosage analysis (RHDO) as well as targeted-seq. Digital PCR is useful for precise fetal genotyping by analyzing relative mutation dosages in NIPT and shows capability to detect a monogenic disorder independently of parental origin [54]. However, it requires higher amount of input DNA to reach high sensitivity and has technical difficulty of performing high-throughput in multiplex PCR, which means only for limited number of disorder. In addition, the PCR-RED is implemented in the diagnosis of FGFR3-associated SD in NIPT and shows high accuracy in follow-up of pregnancy outcome [23]. Unfortunately, the PCR-RED relies on subjective analysis using agarose gel electrophoresis and has an inconclusive rate of around 8% [55], suggesting the impracticable for the clinical practice. The NGS-based RHDO is applied for NIPT for monogenic disorders based on analysis of SNPs in haplotype blocks, and is able to detect complex mutations in gene loci, such as CYP21A2 [56], and DMD [57]. The limitation of RHDO is the requirement of haplotype construction using parental samples, and therefore the cost is higher and prohibitive to clinical application [58]. Collectively, the above-mentioned limitations from various techniques prohibit them to be applied in NIPT for monogenic disorder detection. Intriguingly, it is reported that advanced paternal age is associated with risk of certain SDs, including TD, OI, Apert syndrome, Crouzon syndrome, and Pfeiffer syndrome [59][60][61][62][63][64], suggesting a hidden risk for transmission of deleterious variants to the offspring when aging. The paternal age-associated mutation hotspots, including FGFR2: c.755C > G, c.755C > T and c.758C > G [61], and FGFR3: c.1138G > A, c.1138G > C [64], c.1454A > G, c1948A > G and c.1949A > T [60,62], are included in the expanded gene panel. Therefore, it further puts emphasis on the routine prenatal screen even when the parents are asymptomatic and the proposed targeted-seq workflow is useful to seize the mutations. Based on our findings, the expanded gene panel for a range of 11 SDs showed true negative results for those pregnant women with normal ultrasonography, suggesting the application is ready for screening for SD in earlier pregnancy. The turnaround time of 4 days and minimum requirement of 1 ng of cfDNA make the established targeted-seq panel an excellent choice to be offered in routine clinical practice. It is of notice that the mutations result in rare autosomal recessive form of severe OI-related symptoms in family 2 [52] were not detected in the established panel. It is still challenging to detect mutations for autosomal recessive diseases in NIPT as the mutations are contributed from both parents thus it may present at various fractions in the mutation sites. Traditionally it requires invasive approach to obtain the fetal specimen to distinguish the origin of maternal mutation from the fetal cfDNA carries homozygous or compound heterozygous mutations [65]. Recently, Luo et al. reported a capture-based targeted-seq workflow that can screen for chromosomal aneuploidy of chromosomes 13, 18, and 21, microdeletions, and autosomal recessive disorders simultaneously [66]. It shows promising positive prediction rate of 100% for chromosomal aneuploidy and copy number variations, although the value for autosomal recessive disorders is still not satisfied for clinical use yet. Luo et al. adopts pseudotetraploid model instead of haplotype-based method (e.g., RHDO) to estimate the fetal genotype at specific locus, and achieved 86.4% accuracy for screening three single-gene recessive disorders [66]. Thus it is worth to develop assay and bioinformatics algorithm to enable genetic diagnosis of autosomal recessive disorders in prenatal period. Nevertheless, at current time, the combination of carrier screening for married couple and targeted-seq panel screening for pregnant women to detect mutations in association with recessive disorders in parents and dominant or do novo mutations in NIPT, respectively, shall provide indispensable value to reduce the deleterious effect of pathogenic mutations on human population. Conclusions We established an amplicon-based targeted-seq panel that covers 87 pathogenic hotspot mutations reported in autosomal dominant inherited SD. We demonstrated the workflow is not only with high sensitivity and specificity but also shows remarkable concordance between Ion Torrent and Illumina systems. In addition, the assay requires only 1 ng of cfDNA as input material and takes minimal 4 days to accomplish entire workflow. The overall performance is considered fast and costeffective for routine clinical practice. We believe the established panel provides a promising diagnostic or therapeutic strategy for prenatal genetic testing of SD in routine clinical practice.	2021-11-19T06:17:07.222Z	2021-11-01T00:00:00.000	{ "year": 2021, "sha1": "fa56cc2a246d178aa7db8d3bb4ff728cbafa7cb3", "oa_license": "CCBY", "oa_url": "https://bmcmedgenomics.biomedcentral.com/track/pdf/10.1186/s12920-021-01063-1", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "fe2788f76d655b777e7642fb4f29e13d2ee5836d", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
270404169	pes2o/s2orc	v3-fos-license	Green Adsorbents for Environmental Remediation: Synthesis Methods, Ecotoxicity, and Reusability Prospects : Adsorbent materials have long been used for remediating environmental contaminants. There is an increasing focus on developing sustainable adsorbent materials for long-term use in environmentally friendly and cost-effective remediation. “Green” or “eco-friendly” sorbent materials are generally prepared from renewable or recycled resources, have minimal toxic effects, involve synthesis processes with minor chemical or energy footprints, have high reusability, and do not contribute to additional waste or contamination. Thus, it is essential for materials to have high sorption capacity, high stability, and reusability. The literature focuses on using low-cost or waste materials to produce sorbent materials for the immobilization of contaminants from soil and water systems. The regeneration possibilities of adsorbents are used to evaluate their cost effectiveness and long-term environmental impact once they are applied at field-scale. This review evaluates sustainable sorbent materials, highlighting their green and eco-friendly qualities for a circular economy, and their contribution to the United Nations Sustainable Development Goals (UNSDG). The synthesis techniques, ecotoxicity, and prospect of reusing adsorbents are highlighted. Further, the review provides insights for researchers and practitioners interested in developing and applying green adsorbents, including bio-based carbon, char, and fibrous materials for soil and water remediation. Introduction Exposure to environmental pollutants from soil and water poses potential risks to biotic and abiotic ecosystems [1][2][3][4].These pollutants are mainly manufactured chemicals, including heavy metals, fluoroalkyl compounds, agrochemicals, pharmaceuticals, and numerous known and unknown chemicals [5][6][7].Remediating these pollutants is the priority for ensuring a sustainable ecosystem, and adsorption has become a promising technology due to its efficiency, biocompatibility, and low operating costs [5,6,[8][9][10][11].For example, adsorption has been proven to be effective for treating soil and wastewater containing toxic heavy metals, dyes, antibiotics, per-and polyfluoroalkyl substances (PFAS), agrochemicals, and other emerging pollutants [8,12,13].Through adsorption, the bioavailability of pollutants is reduced, minimizing risks to the biotic and abiotic components of the environment, which is the basis for risk-based approaches in environmental remediation.In recent decades, many synthetic and natural adsorbents have been used to remediate a wide range of organic and inorganic pollutants [14][15][16].Synthetic adsorbents are suitable for treating contaminated water and soil due to their high surface area and adsorption capacity.Activated carbon/carbonaceous materials, nanomaterials, clay composites, ion exchange resins, composite adsorbents, and polymeric sorbents are among many in that category [17][18][19][20][21]. Natural adsorbents, including agricultural and industrial waste, clays, Processes 2024, 12, 1195 2 of 24 biopolymers, and cellulose-based materials, are low-cost and environmentally friendly.These features make them attractive alternatives to synthetic materials if they have the desired adsorption capacity [22][23][24][25]. Challenges exist with the development of novel adsorbent materials, including their adsorbing performance, kinetics, stability, selectivity, regeneration, and cost effectiveness.Developing sustainable environmental adsorbents is also an important consideration, i.e., whether the sorbent meets the criteria of being "green" or "eco-friendly".A "green" or "eco-friendly" adsorbent is a material that can selectively bind or adsorb pollutants or other substances from a solution or gas phase without causing harm to the environment [26], which also aligns with the twelve principles of green chemistry [27].It is expected to be derived from renewable, biodegradable resources and has a low environmental impact during production, use, and disposal [15,28].From a material perspective, the technical challenge is to obtain all these properties in a single adsorbent or find a balanced approach that is fit for purpose.For example, if regeneration of adsorbent materials is desirable, the sorption capacity might be compromised to meet this purpose.Therefore, developing novel adsorbents with desirable properties is vital for environmental remediation. The prospective environmental applications of adsorbent materials are multifaceted and include water treatment and soil stabilization, and interest in their development has grown in recent years [15,[29][30][31].Low-cost resources, e.g., smelter slag, biochar, red mud, clays, biomass, and their derived materials, such as nanoparticles, have been increasingly used to prepare adsorbents, which is the focal point of this review considering circular economy prospects.This review also focuses on advancements in synthesizing adsorbent materials, explicitly analyzing the assessment of preparation techniques, consequences for ecotoxicity, and potential for reuse or regeneration.Various preparation techniques, including chemical synthesis, physical modification, and bio-based approaches, have been evaluated for their efficiency in improving surface characteristics and adsorption capacity.The benefits and drawbacks of every technique are discussed to offer insights into the most promising strategies for producing adsorbent materials.Additionally, this review looks at the sustainable attributes of potential sorbent materials for environmental remediation, including the toxicity, reusability, and regeneration of the specific sorbent material(s).This understanding can be extended to developing "green" adsorbents for environmental remediation. "Green" or "Eco-Friendly" Adsorbents A family of materials known as "green" or "eco-friendly" adsorbents has recently attracted much attention because of their favorable effects on the environment and their use in a range of remediation industries [32].This type of badge on adsorbents has been rewarded based on three primary considerations: (i) the source of the raw material, (ii) the fate and impact of the adsorbents on the applied medium, and (iii) the reusability of the adsorbent.The adsorbent may be derived following one or all of these considerations. Green adsorbents are frequently made from naturally occurring, easy-to-process minerals or renewable resources, such as bio-based polymers, natural fibers, agricultural waste, and industrial waste [30][31][32].Green adsorbents use these materials to lessen their dependency on non-renewable resources and help minimize their carbon footprints.Ecofriendly adsorbents are also characterized by their low toxicity and biodegradability.These adsorbents are made to have as little impact on the environment as possible while efficiently remediating pollutants or toxins from air, soil, water, or other media.Green adsorbents are made to reduce their adverse effects on ecosystems and human health, in contrast to traditional adsorbents, which may degrade and release harmful chemicals into the environment [29,30].These adsorbents are easily reusable through recyclability with some downgrading of the adsorption properties or after a low-cost regenerative process by which the original properties of the adsorbent can be restored.To improve the performance and adaptability of green adsorbents, researchers are constantly investigating the synthesis of novel materials.These adsorbents provide a promising way to address environmental issues and encourage a greener and more sustainable future. Here, we used the term "adsorbents or sorbents" to describe the materials that act in the sorption process, excluding their role as the sole catalyst.A literature search was conducted to analyze papers with relevant words.The results from searching the Scopus database for both "adsorbent" and "remediation" in the article title, abstract, or critical keyword area of the record produced 3623 records from 2017 to the present (search date 1 November 2023).Among them, 2827 original studies were identified with no further filtering or quality sorting.We selected only English-language articles (2781), further filtered "within the results", and obtained revised records.To do that, we used the following restriction: "(((TITLE-ABS-KEY ("adsorbent") AND TITLE-ABS-KEY ("remediation") AND PUBYEAR > 2017 to 1 November 2023))) AND ("eco-friendly" OR "regeneration" "ecofriendly" OR "reusability" OR "ecotoxicology" OR "green" OR "eco-toxicology") AND (LIMIT-TO (DOCTYPE, "ar")) AND (LIMIT-TO (LANGUAGE, "English"))".This specific search identified a total of 2134 articles.We exported these articles into Endnote software (v20.2.1, Clarivate™, London, UK) and carefully checked them for quality.This included removing review articles from the cohort and removing confusing terminology, such as "Malachite Green", that did not study any "green" or eco-friendly aspects.Finally, 2018 articles were clustered manually into several categories and are presented as a box chart (Figure 1).The number of publications on this topic increased during the past few years (2017-2022), i.e., it increased from 134 in 2017 to 610 in 2022. performance and adaptability of green adsorbents, researchers are constantly investigating the synthesis of novel materials.These adsorbents provide a promising way to address environmental issues and encourage a greener and more sustainable future. Here, we used the term "adsorbents or sorbents" to describe the materials that act in the sorption process, excluding their role as the sole catalyst.A literature search was conducted to analyze papers with relevant words.The results from searching the Scopus database for both "adsorbent" and "remediation" in the article title, abstract, or critical keyword area of the record produced 3623 records from 2017 to the present (search date 1 November 2023).Among them, 2827 original studies were identified with no further filtering or quality sorting.We selected only English-language articles (2781), further filtered 'within the results', and obtained revised records.To do that, we used the following restriction: "(((TITLE-ABS-KEY ('adsorbent') AND TITLE-ABS-KEY ('remediation') AND PUBYEAR > 2017 to 1 November 2023)) AND ('eco-friendly' OR 'regeneration' 'ecofriendly' OR 'reusability' OR 'ecotoxicology' OR 'green' OR 'eco-toxicology') AND (LIMIT-TO (DOCTYPE, "ar")) AND (LIMIT-TO (LANGUAGE, "English"))".This specific search identified a total of 2134 articles.We exported these articles into Endnote software (v20.2.1, Clarivate TM , London, UK) and carefully checked them for quality.This included removing review articles from the cohort and removing confusing terminology, such as 'Malachite Green', that did not study any 'green' or eco-friendly aspects.Finally, 2018 articles were clustered manually into several categories and are presented as a box chart (Figure 1).The number of publications on this topic increased during the past few years (2017-2022), i.e., it increased from 134 in 2017 to 610 in 2022.The box plot shows a significant discrepancy between the adsorbents claiming to be "sustainable" or "eco-friendly" and the standard indices, such as "reusability" (regeneration or recycling) and "ecotoxicity" assessment.Approximately 28% of the 2018 published papers reported reusability data, while only 1.5% reported ecotoxicity assessment.Both indices were left out almost entirely. It is difficult to verify the degree to which the adsorbent is "green/eco-friendly" by learning only the reported outcome.A comprehensive assessment of its chemical footprint and cost analysis are needed.For example, Feng and colleagues [33] developed a graphenebased nanocomposite that removed drug residues (diclofenac) from water.This adsorbent is deemed to be reusable and non-toxic to the water bacterium Escherichia coli.However, the formulation pathway of the adsorbent requires the following chemicals along with a series of thermodynamic energy inputs: graphene oxide, terephthalaldehyde, tetrakis (4 aminophenyl) methane, 1,4-dioxane, other salts, and solvent.A similar claim was made by Bedadeep et al. [34], indicating the chemical and energy input footprint for the synthesis of their adsorbent seems to be high.The authors emphasized the green synthesis approach, considering the growing demand for reducing toxic chemical usage where possible [35]. This argument does not suggest that one adsorbent is better than another, but the adsorbents should be designed with the least possible environmental impact while still providing the desired performance.In this review, we aim to illustrate the primary considerations when developing "green" or "eco-friendly" adsorbent materials.This includes the use of waste or low-cost materials, environmentally friendly processes for preparation, ecotoxicity considerations, and reusability and regeneration.These considerations are targeted to achieve sustainable development goals and a circular economy (Figure 2). The box plot shows a significant discrepancy between the adsorbents claiming to b 'sustainable' or 'eco-friendly' and the standard indices, such as 'reusability' (regeneration or recycling) and 'ecotoxicity' assessment.Approximately 28% of the 2018 published pa pers reported reusability data, while only 1.5% reported ecotoxicity assessment.Both in dices were left out almost entirely. It is difficult to verify the degree to which the adsorbent is 'green/eco-friendly' by learning only the reported outcome.A comprehensive assessment of its chemical footprin and cost analysis are needed.For example, Feng and colleagues [33] developed a gra phene-based nanocomposite that removed drug residues (diclofenac) from water.Thi adsorbent is deemed to be reusable and non-toxic to the water bacterium Escherichia coli However, the formulation pathway of the adsorbent requires the following chemical along with a series of thermodynamic energy inputs: graphene oxide, terephthalaldehyde tetrakis (4 aminophenyl) methane, 1,4-dioxane, other salts, and solvent.A similar claim was made by Bedadeep et al. [34], indicating the chemical and energy input footprint fo the synthesis of their adsorbent seems to be high.The authors emphasized the green syn thesis approach, considering the growing demand for reducing toxic chemical usag where possible [35]. This argument does not suggest that one adsorbent is better than another, but th adsorbents should be designed with the least possible environmental impact while stil providing the desired performance.In this review, we aim to illustrate the primary con siderations when developing "green" or "eco-friendly" adsorbent materials.This include the use of waste or low-cost materials, environmentally friendly processes for preparation ecotoxicity considerations, and reusability and regeneration.These considerations are tar geted to achieve sustainable development goals and a circular economy (Figure 2). Use of Waste Materials for the Synthesis of Low-Cost Green Adsorbents Various low-cost, renewable, and waste materials are being investigated to prepar adsorbent materials, including clays and zeolites, recycled materials, and agricultural and industrial wastes.This is an increasing trend in this research area, as evidenced by th Scopus document search (TITLE-ABS-KEY (sorbent) AND (low cost) OR (waste)).Th web search results with keywords "((waste materials) AND (green adsorbents)) AND (green synthesis)" from PubMed data show a significant interrelationship with waste-ma terials-derived green adsorbents and their utilization for organic and inorganic contami nants (Figure 3).In total, 124 articles were collected to draw the co-occurrence map con structed using the VOSviewer software (V.1.6.20).The visualization network map indi cates the degree of relatedness between keywords.It highlights the research trend of uti lizing different waste materials for environmental remediation.Research related to th Use of Waste Materials for the Synthesis of Low-Cost Green Adsorbents Various low-cost, renewable, and waste materials are being investigated to prepare adsorbent materials, including clays and zeolites, recycled materials, and agricultural and industrial wastes.This is an increasing trend in this research area, as evidenced by the Scopus document search (TITLE-ABS-KEY (sorbent) AND (low cost) OR (waste)).The web search results with keywords "((waste materials) AND (green adsorbents)) AND (green synthesis)" from PubMed data show a significant interrelationship with waste-materials-derived green adsorbents and their utilization for organic and inorganic contaminants (Figure 3).In total, 124 articles were collected to draw the co-occurrence map constructed using the VOSviewer software (V.1.6.20).The visualization network map indicates the degree of relatedness between keywords.It highlights the research trend of utilizing different waste materials for environmental remediation.Research related to the green synthesis of adsorbents from different waste materials frequently considered the sorption and degradation of different organic and inorganic contaminants from soil and water (Figure 3).Understanding remediation mechanisms by utilizing spectrometric characterization techniques remains a recent research interest, alongside bringing novel synthesis techniques from different waste materials.The rise in using lowcost waste materials for developing remediation adsorbents is also because governments, environmental regulators, and industries are overwhelmed by the disposal and recycling of these resources [36].Using waste or low-cost sorbent materials may not reduce waste as much as waste-to-energy or waste-to-building/construction materials approaches can [37,38].However, it can still provide significant benefits, including reducing waste volume, decreasing environmental pollution, and improving resource efficiency.Waste and low-cost materials can also generate value-added products to provide additional economic and social benefits. green synthesis of adsorbents from different waste materials frequently considered the sorption and degradation of different organic and inorganic contaminants from soil and water (Figure 3).Understanding remediation mechanisms by utilizing spectrometric characterization techniques remains a recent research interest, alongside bringing novel synthesis techniques from different waste materials.The rise in using low-cost waste materials for developing remediation adsorbents is also because governments, environmental regulators, and industries are overwhelmed by the disposal and recycling of these resources [36].Using waste or low-cost sorbent materials may not reduce waste as much as waste-to-energy or waste-to-building/construction materials approaches can [37,38].However, it can still provide significant benefits, including reducing waste volume, decreasing environmental pollution, and improving resource efficiency.Waste and low-cost materials can also generate value-added products to provide additional economic and social benefits.Low-cost, abundant, and degradable precursor materials can be considered for the eco-friendly production of sorbent materials.Such considerations include agricultural and industrial waste, natural and low-cost minerals, and municipal solid waste (Figure 4).The number of raw or waste materials is growing over time due to decades-long research efforts toward sustainable reuse of waste materials including agricultural waste [29][30][31], shell-based waste [39], red mud and fly ash-based materials [40][41][42][43], biosolids [43], and clay minerals [25,43].Such information is vital for continuously developing sustainable sorbent materials from renewable resources.This review highlighted some examples of potential feedstock materials with their advantages and disadvantages (Table 1 and Figure 4).Low-cost, abundant, and degradable precursor materials can be considered for the eco-friendly production of sorbent materials.Such considerations include agricultural and industrial waste, natural and low-cost minerals, and municipal solid waste (Figure 4).The number of raw or waste materials is growing over time due to decades-long research efforts toward sustainable reuse of waste materials including agricultural waste [29][30][31], shell-based waste [39], red mud and fly ash-based materials [40][41][42][43], biosolids [43], and clay minerals [25,43].Such information is vital for continuously developing sustainable sorbent materials from renewable resources.This review highlighted some examples of potential feedstock materials with their advantages and disadvantages (Table 1 and Figure 4).Table 1.Various feedstock materials with their advantages and disadvantages for the synthesis of adsorbents [44][45][46][47][48][49][50][51][52][53][54][55]. Sources of Feedstock Materials Examples Agricultural Waste/Biomass Agriculture byproducts have been considered the most significant feedstock sources [56], which provide enormous opportunities to develop various remediation agents.Various agricultural wastes/biomass have been studied to develop novel adsorbents that can remove organic and inorganic pollutants.Compared with conventional adsorbents, agricultural waste is frequently accessible at little or no cost, which decreases the overall cost of contamination cleanup [57][58][59].Due to affordable cost and ambiguous availability of agricultural waste, it is suitable for use in underdeveloped or developing nations or places with limited financial means [60][61][62][63][64]. Using agricultural waste as an adsorbent can help lessen the dependency on nonrenewable resources.The circular economy and sustainable development concepts align with this strategy, reduces resource efficiency and environmental impacts (Table 1). These materials contain various fibers, hemicellulose, cellulose, lignin, ash, biopolymer, and moisture.These functional constituents largely determine their utilization as sorbent materials.For example, natural biopolymers, such as chitosan-based adsorbents, are biocompatible and ecologically friendly, making them valuable alternatives for preparing sorbent materials.Chitosan-ethylenediaminetetraacetic acid-modified biochar [65,66] and rubber-seed-shell-based activated carbon [67] have been reported for the remediation of heavy metal(loid)s in soil and water in addition to CO 2 capture.Starch and carbohydrates are two primary components of different food waste.Starch is one of the most abundant and naturally occurring polymers that can be used to prepare sorbent materials.In addition to that, carbohydrates constitute glucose units and glycosidic linkages and possess active and replaceable hydroxyl groups that can be readily cross-linked with other functional groups.Tailoring their functionality with different physical and chemical treatment could also contribute to potential remediation agents [68].For instance, Li et al. prepared a sorbent-iron-modified bacterial biomass-to remove antimony (III), and they argued that it had a higher sorption capacity than certain previously reported sorbents [69].In addition, Zhang et al. produced biochar from wood pyrolysis under oxygen-limiting conditions (650 • C for 4 h) to immobilize cadmium (Cd) and copper (Cu) from soil [70]. The high surface area, porous structure, and functional group content of agricultural waste make it an effective adsorbent.These properties improve its capacity to adsorb impurities from soil and water matrices, such as organic pollutants, heavy metals, PFAS, agrochemicals, and dyes [57,59,60,[71][72][73][74].Adsorbents from agricultural waste often exhibit low toxicity, reducing the likelihood of secondary pollution [75,76].Furthermore, their use as adsorbents can lessen the requirement for energy-and chemical-intensive procedures, resulting in a reduced carbon footprint compared to conventional techniques [77,78].To optimize the adsorption efficiency of adsorbents made from agricultural waste, several characteristics need to be optimized, such as particle size, pretreatment techniques, pH, and contact time [72,79,80].It is imperative to standardize both synthesis processes and characterization methodologies to guarantee uniform remediation performance among diverse waste types and pollutants.Agricultural waste can have different compositions depending on several variables, including crop variety, region, and harvesting technique.This variability may impact the adsorbent capacity and effectiveness, requiring careful characterization and batch-to-batch quality control.Adsorbents made from agricultural waste can effectively remove pollutants but face challenges in their regeneration and reusability due to poor thermal and chemical stability [81].Thus, the long-term stability of agri-wastederived adsorbents should be prioritized during the development of adsorbents.More investigations and technological developments are needed to expand the use of agricultural waste as adsorbents from the laboratory to industrial or field-scale applications.Practical implementation requires large-scale manufacturing and deployment.Using agricultural waste as a green adsorbent for the remediation of pollutants has many benefits, such as its availability, cost-effectiveness, renewability, and minimal environmental impact [13,82,83].However, for this strategy to be implemented successfully, the issues of uniformity, waste variability, regeneration, and scale-up need to be addressed (Table 1).Further study and cooperation among scientists, engineers, and policymakers are required to fully realize the potential of agricultural waste as a sustainable solution for pollutant remediation. Animal Wastes Feathers, hair, manure, bones, and shells are among the most investigated animal wastes for developing sorbent materials [84][85][86][87][88][89].The keratin protein in feathers and hair, organic matter and nutrients in manures, and calcium and phosphate minerals in animal bones can be chemically or thermally modified to prepare sorbent materials [90,91].Nitrogen, phosphorus, potassium, ammonia, organic matter, carbon, sulfur, and trace minerals (e.g., copper and zinc) are the major chemical components of animal waste [16,90,92,93].However, the chemical composition varies depending on the type of manure and the food habits of particular animals [94].These components are essential for nutrient cycling and agronomical benefits in soil systems.However, the raw or aerobic degradation products of manure applied to agricultural soil might cause carbon dioxide emissions due to the low stability of carbon.Thus, understanding the chemical properties of individual animal waste is essential for sustainable waste management.In addition, studying manure waste with advanced characterization techniques provides insight into animal diets, animal health, and overall management practices [95,96].Transforming animal waste into a valuable resource lowers the need for disposal and associated environmental risks, benefiting waste management.Adsorbents from animal feces have good adsorption qualities because of their large surface area, porous structure, and organic matter content [86,97].These properties are useful to efficiently immobilize pollutants from water and soil matrices, and they can be reactive to a range of pollutants, including organic pollutants, heavy metal(loid)s, antibiotics, and other emerging pollutants [86,[97][98][99].The adsorbent and pollutants interact physically and chemically during adsorption, which results in the immobilization or degradation of the contaminants [14].Furthermore, animal dung can be an adsorbent to lessen adverse and unfavorable environmental effects [15].Adsorbents derived from animal waste are frequently biodegradable and pose negligible ecosystem hazards [14,100].More investigations and technological developments are needed to expand the use of animal waste as adsorbents from the laboratory to field-scale applications. Mineral Resources Mineral waste refers to the byproducts and residues generated during the extraction, processing, and utilization of minerals, such as tailings, slags, mine water, and rock waste.If not managed correctly, these materials may contain contaminants or impurities that could impact the environment.On the other hand, clay minerals (e.g., palygorskite, smectite, halloysite, kaolinite), other non-argillaceous minerals such as oxides (e.g., goethite), and diatomaceous earth and zeolite are among the low-cost resources that can also be used to produce sorbent materials.It is worth noting that the cost of sourcing these minerals also depends on the geological deposit and country of the facilities.These minerals' active surface and manipulable charge behavior provide sorption capacity for various pollutants.Research trends regarding these properties and applications are increasing.The surface areas, porous architectures, and functional groups of minerals contribute to adsorption [101][102][103][104]. Removing a broad spectrum of pollutants is one of the main benefits of using inexpensive mineral waste as a green adsorbent [105][106][107].Heavy metals, organic pollutants, dyes, antibiotics, and other toxins from soil, wastewater, and groundwater are among them.For example, biopolymer-clay nanocomposites are increasingly investigated for their sorption capacity for a range of contaminants, given the biodegradability and inexpensive and non-toxic properties of biopolymers [108].Polysaccharides and polypeptides are among the best candidates for the development of biopolymer-clay composites.Low-cost "eco-friendly" resources were also employed when developing clay composites, such as cellulose, starch, chitosan, and peanut hull, given their abundance, biodegradability, biocompatibility, and recyclability.However, including toxicity assay in the investigations is not always the case.Biswas and Naidu [109] utilized an Australian palygorskite clay mineral to make functionalized clay sorbent to remove phosphorus from lake water.They argued that this material could effectively bury phosphate ions from lake water.The material did not have a toxic effect on aquatic microorganisms, as demonstrated in the microbial toxicity test. Depending on the properties of the pollutant and adsorbent, the adsorption may entail physical and chemical interactions such as pore filling, van der Waals interaction, hydrophobic interaction, chemisorption, surface complexation, ion exchange, and electrostatic attraction [110,111].To improve their adsorption capabilities, they can be readily functionalized or altered using techniques such as heat treatment, chemical modification, or impregnation with organic materials [107,[110][111][112][113][114][115].Because of their adaptability, adsorbent properties can be tailored to particular pollutants or environmental systems [110,116].Moreover, recycling mineral waste into useful adsorbents advances waste-to-resource ideas and the circular economy [117][118][119].Factors such as regeneration and the possible leaching of adsorbed toxins need to be considered to ensure their long-term viability, and that they present a zero chance of creating secondary pollution. Industrial Wastes Inorganic/organic wastes generated from various industrial processes, including smelting, mining, and mineral refining, have been increasingly investigated for their application as sorbent materials.By converting industrial waste into adsorbents, we can efficiently manage environmental contamination challenges while lessening the disposal burden of tailings [12,120].Using industrial waste as a green adsorbent for contamination treatment has various benefits [121].Some examples include fly ash, red mud [84], slag, mine tailings, blast furnace sludge/slag/dust [122], carbonaceous wastes from the fertilizer industry, paper mill sludge, and biosolids from the water industries.For example, the iron and other metallic oxides in smelter slags contribute to the remediation of pollutants [122]. These waste materials contain large specific surface areas, porous structures, and an abundance of functional groups-all of which are intrinsic qualities that facilitate adsorption [121,123,124].To maximize their adsorption efficiency, the physicochemical characteristics of industrial waste can also be altered by various processes, including activation, chemical modification, and blending with other materials [12,121,125].Adsorbents obtained from industrial waste have proved to be effective in remediating various pollutants, such as pesticides, heavy metals, dyes, and other emerging pollutants [121,123,124,[126][127][128].These adsorbents employ a variety of chemical and physical adsorption mechanisms, including covalent binding, surface complexation, ion exchange, and electrostatic attraction [127][128][129]. Municipal Solid Waste (MSW) Municipal solid waste includes food and green waste, glass, metals, plastic, paper/cardboard, rubber and leather, wood, and other wastes [130][131][132].These waste materials can be used according to their composition.Current research is almost entirely confined to using thermal energy to convert these wastes into reactive materials such as char or biochar.This pyrolysis can be tailored according to the source materials and desired reactive sites of the sorbent and is an area of increasing interest, given the necessity for a circular economy.The extensive availability of MSWs makes them ideal for use as adsorbents.Since MSW is generated by communities worldwide, it is readily available and might be less expensive than other raw materials [132,133].The development of decentralized methods for contamination remediation is aided by the local availability of MSW, particularly in areas with potentially inadequate waste management infrastructure [134]. The above-listed materials and feedstocks of adsorbents are not all low-cost, sustainable materials for making sorbents.They are indeed the current lead research topics, with different materials exhibiting different active sites, functional groups, and chemical/physical properties.Continuously exploring the options and providing a case-by-case basis for implementing waste materials for the development of selective remediation agents will achieve sustainable goals and consequently achieve a long-term circular economy approach.However, using MSW-based adsorbents presents several issues that need to be resolved.Because MSW is heterogeneous, accurate characterization and processing are necessary to guarantee reliable and efficient adsorption performance [130,[134][135][136].To ensure long-term environmental advantages and safety, it is also essential to carefully analyze the stability of the materials and the leaching potential over time to understand the possibility of secondary pollution caused by the given materials [136][137][138]. Chemicals and Synthesis Processes Sustainable and green preparation processes are preferable for the preparation of novel adsorbents because they can minimize the environmental footprint, energy consumption, and waste generation.Examples of the preparation of various kinds of sorbent materials include but are not limited to hydrothermal carbonization methods, microwave-assisted pyrolysis, sol-gel methods, and electrospinning methods (Figure 4).We stress that many raw materials, such as those listed in the "Mineral Resources" section, may be sorbents without additional synthesis or use of chemicals.Here, we will look at methods that are not necessarily green.Yet, our advanced and diverse research expands our knowledge and use of these methods in a green and sustainable way when developing sorbent materials.In this section, different existing synthesis methods are highlighted, along with their advantages and disadvantages for preparing green adsorbent materials, to provide a detailed understanding of each synthesis method (Table 2). Hydrothermal Carbonization Method Hydrothermal carbonization supports biomass conversion into a carbon-rich material by applying simultaneous heat and pressure.It produces "hydrochar" and other carbon materials from a variety of feedstocks, including agricultural waste, sewage sludge, and municipal solid waste [165][166][167][168][169]. Compared to conventional pyrolysis, hydrothermal carbonization is a greener method for synthesizing bio-carbon-inspired adsorbent materials.For instance, conventional pyrolysis methods produce a large amount of toxic syngas (carbon monoxide, hydrogen, and carbon-di-oxide), hydrocarbons (methane, ethylene, propylene), tars, bio-oils, and volatile organic compounds (VOCs) [170][171][172].In addition, hydrothermal methods perform at lower temperatures (<300 • C) and pressures than traditional pyrolysis, which may have less of an effect on the environment due to limited energy usage.This phenomenon makes it more environmentally friendly than conventional pyrolysis [173].However, conventional pyrolysis methods can also be environmentally friendly because they help to convert biomass waste into valuable products (e.g., biochar or engineered biochar) while reducing greenhouse gas emissions compared to traditional waste disposal methods [174]. Hydrothermal processes can form "hydrochar" with aliphatic structures, amorphous carbon, and mineral contents depending upon the feedstock source and reaction conditions [168].Inorganic elements, e.g., sulfur (S), calcium (Ca), iron (Fe), magnesium (Mg), phosphorous (P), and potassium (K), can be released from sorbents through hydrothermal processes.This low-cost and eco-friendly hydrothermal method produces high carbon yield and can recover minerals and other inorganic elements depending on the hydrothermal conditions.However, the process may be complex, and carbonization parameters may require optimization.Additionally, potentially hazardous products can be generated, and the scalability of this process might be challenging. Since water or biocompatible solvents are frequently used in hydrothermal processes, less harsh chemicals are needed, minimizing the chemical footprint and potential risks.These techniques can be used to produce a variety of green adsorbents from a broad spectrum of natural precursors, including organic materials, biomass, and agricultural waste [139,141].The hydrothermal method is also used to prepare magnetic biochar.The morphology and structure of the resultant adsorbent materials can be altered through hydrothermal synthesis, potentially improving their selectivity and adsorption capabilities [139,143].By enabling the synthesis process to be conducted at comparatively low temperatures and pressures, this increases the energy efficiency and decreases the overall environmental impact. Implementing hydrothermal technologies in large-scale production poses some problems despite their effectiveness in the laboratory.Process optimization for adsorbent synthesis, including temperature, pressure, and reaction time, can improve the scalability and efficiency of hydrothermal techniques [141,142].The variety of green adsorbents that can be produced is increased by selecting appropriate precursors.The scalability and commercial viability of the synthesis process may be impacted by the extended reaction times required for certain hydrothermal reactions. Microwave-Assisted Pyrolysis Method The microwave-assisted pyrolysis method involves rapid heating of biomass using a microwave treatment to produce value-added products.The preparation time can be significantly reduced due to its fast heating and cooling cycles, and thus, the properties of the sorbent can be tailored [147,[175][176][177][178][179][180][181].For example, microwave-assisted magnetic biochar has been shown to have up to 10 times greater adsorption capacity, surface area, and pore volume than biochar prepared via conventional methods [63,182].The process shows high energy efficiency and yield, and reduced emissions, making it suitable for making environmentally friendly sorbents.We understand there are concerns about the equipment cost, safety, and quality assurance for the specificity of the sorbent.However, we also know that using microwaves is an emerging method for sorbent development, and there is a scope for enhancing its sustainability and scalability. Microwave irradiation of biomass produces quick and uniform heating, which reduces reaction times and increases energy efficiency.Microwave-assisted high-temperature heating can lead to increased product yields [144,178].However, microwave pyrolysis can reduce secondary reactions and undesirable byproducts, improving control over the pyrolysis process and improving the quality of the biobased products.This process enables manufacturing a wide variety of bio-based materials by providing flexibility and scalability to various biomass feedstocks [183].Variability in the characteristics and composition of biomass feedstocks can affect the uniformity and efficiency of the materials; hence, careful feedstock preparation and selection are needed.However, there may still be problems with heat transfer inside biomass particles, which could reduce the overall effectiveness of the pyrolysis process [146,184].Microwaves make it possible to heat biomass quickly and evenly.High-value adsorbents can be produced from biomass through integrated procedures that combine catalytic conversion methods and microwave-assisted pyrolysis [164,184].The application of microwave-assisted pyrolysis is a promising approach for effective and sustainable biomass conversion. Sol-Gel Method The sol-gel method has been intensively investigated over the last two decades, resulting in its frequent use for synthesizing inorganic adsorbents such as silica, alumina, or calcium oxide.The sol-gel method involves the hydrolysis and condensation of metal alkoxides in a solvent.It allows precise control of the properties of materials and the ability to incorporate different functional groups onto the synthesized adsorbents [185][186][187].However, this method is time-consuming, sensitive to impurities, and in some instances, expensive.The resultant sorbent can be brittle, which limits its durability and reusability.To synthesize adsorbents, a broad range of natural precursors and ecologically acceptable reagents can be used in the sol-gel method, allowing utilization of sustainable and renewable resources [150,152].The sol-gel process can be used with functionalization to increase the affinity of the adsorbent for target pollutants [151].By improving the uniformity of the adsorption sites, the sol-gel technique helps to generate homogenous adsorbent [149,152]. The method for synthesizing green adsorbents may require several steps and precise control over the reaction conditions [150].This can make the process complex and present difficulties for scaling up production.The cost of specific sol-gel precursors and reagents may be greater than that of other materials, and the specialized equipment needed for regulated gel formation and drying may increase production costs [153,188].The manufacturing of green adsorbents can be made more reproducible and less complex by streamlining sol-gel synthesis through ongoing research into process automation and optimization.Investigating the use of sol-gel techniques to incorporate nanoparticles and nanocomposites could produce advanced adsorbents with improved selectivity and adsorption capabilities.Sol-gel technologies are at the forefront of green adsorbent synthesis for environmental applications despite the cost and process complexity considerations [149,151,153,189]. Electrospinning Method The electrospinning method produces nanofibers from a polymer solution using an electric field widely used in science and engineering applications.This method can produce adsorbents with high specific surface area and other desirable properties [156].Both synthetic and natural polymers are used to fabricate fibrous materials, e.g., polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyacrylonitrile (PAN), chitosan, and nylon 6 [149][150][151][152][153]. The sorption capacity can be improved by functionalization through modifying or adding fillers to remove pollutants [156].Further research efforts are needed to optimize the preparation process and ensure the safe application of the adsorbents. The morphology, porosity, and structure of adsorbent materials produced by electrospinning can be precisely controlled, providing opportunities to modify their adsorption characteristics for particular pollutants [154, 155,159].The interconnected porous structure and high surface-area-to-volume ratio of electro-spun fibers can improve their adsorption abilities and efficacy in eliminating pollutants from a range of media [154,157].Electrospinning can be applied to natural polymers and biodegradable materials to create environmentally acceptable adsorbents.This approach is consistent with green chemistry and sustainability concepts.Adding particular functionalities to electrospun fibers increases their selectivity for particular pollutants and their overall adsorption performance [156,160]. The electrospinning procedures might require complex setups and precise parameter control, which present commercial application and production scaling issues.The longevity and reusability of the resultant green adsorbents may be impacted by the poor chemical and mechanical stability of specific natural polymers utilized in electrospinning [156,161].The overall economic viability of electrospinning for large-scale green synthesis can be influenced by equipment cost, energy consumption, and the selection of appropriate natural polymer precursors [161].To improve process efficiency and reproducibility, ongoing research has focused on enhancing electrospinning settings, investigating innovative collector designs, and incorporating automation.Adapting the brittle/fragile nature of electrospun adsorbents with high specific surface area offers opportunities to increase their usefulness in various environmental settings, including air purification and wastewater treatment [156,190]. Biosynthesis Method Biosynthesis processes involve using natural biochemicals or living organisms to prepare green adsorbents.Here, plant extracts, biopolymers, plant or microbial extracts, and enzymes are considered as feedstock materials for the synthesis of adsorbents.These methods are mainly used for the synthesis of nanoparticles.The green synthesis of iron (Fe)based, silver (Ag)-based, titanium (Ti), zinc (Zn), and copper (Cu) nanoparticles, as well as bimetallic nanoparticles, has been widely investigated [191][192][193][194].For example, Cynomorium coccineum extract was used for the synthesis of Cu nanoparticles for dye removal [195].The phytochemicals in plant extracts act as stabilizing and reducing agents during synthesis [196].Biosynthesis represents a cost-effective and eco-friendly approach for preparing sorbent materials, mainly nanoparticles.However, the use of "green" chemicals such as plant extracts does not readily prove that the synthesized nanoparticles are nontoxic to environmental biota [197].They need to be tested for site-specific applications [198].Some of these biosynthesis procedures could be complex, and regulatory challenges may arise when genetically modified organisms are involved in synthesis. In line with renewable and sustainable resource utilization, green biosynthesis involves the use of natural sources such as plant extracts, agricultural waste, and microbial biomass [143,164].Compared to conventional synthesis techniques, the synthesis process frequently uses benign reaction conditions, minimizing the use of harsh chemicals and lowering the environmental impact [164].A variety of biomasses can be used in green/biosynthesis processes to produce a range of green adsorbents with specific characteristics and functions.Obtaining bioactive compounds from natural sources can be difficult and requires a number of stages, which could increase the overall cost of production.Due to variations in natural sources, the properties of biosynthesized adsorbents may fluctuate, making it difficult to guarantee consistent material quality and performance. Ecotoxicity Considerations The ecotoxicity of an adsorbent depends on its chemical composition, physical properties, and potential leach of toxic substances into the soil and water environment.Although green or eco-friendly adsorbents are designed to be environmentally safe, there are still chances that they pose a risk to the environment if not properly managed or disposed.The sustainable treatment and disposal of sorbent materials after sorption requires further research [199].Potential leaching and release of sorbed pollutants or sorbent components can occur if the used materials are not adequately treated or disposed.For example, harmful chemicals (e.g., heavy metals, polycyclic aromatic hydrocarbons) might be introduced into biochar materials during synthesis [200,201].Thus, post-synthesis washing and degradation methods are important for improving the biocompatibility of these materials.Several elements must be considered when evaluating ecotoxicity.These include the physical characteristics, chemical makeup, and potential leaching of harmful substances from adsorbents.It is also necessary to consider the particular organisms being targeted, dose of the adsorbent, and duration of exposure to the environmental systems [202,203]. We understand that the "ecotoxicity assessment" of sorbents is complex, with factors including "the choice of environmental receptors", "the length of study", and "the form of sorbents likely to be available or residual" [204].Most published reports present "quick" or "fit for purpose" data on ecotoxicity by studying the "toxic" effect of the sorbent on only one or two environmental organisms.For example, Biswas and Naidu [70] argued that their synthesized clay composite was safe in water by studying only waterborne heterotrophic bacterial growth.Although such preliminary ecotoxicity considerations are helpful for developing sorbents, environmental assessments or life cycle assessments of these materials are necessary to comprehend the sorbent fully.This comprehensive assessment includes production processes, cost benefits, sensitivity analysis, C emissions, and toxic risks associated with C usage, reuse, and disposal.The primary goal of ecotoxicity studies is to ensure that the use of green adsorbents for contamination remediation does not have any negative ecological repercussions.Researchers and practitioners can come up with well-informed decisions about the environmental safety and applicability of these materials by performing thorough assessments [202,[205][206][207]. Indeed, eco-friendly modifying agents are useful for developing functional materials for the sorption of pollutants and preparing sorbent materials.Nevertheless, the use of "eco-friendly" chemicals in the synthesis process may not always be possible in obtaining the desired sorbents.Highly reactive zerovalent iron nanoparticles (nZVIs) could be a good example, where exhaustive chemical-like borohydride is a much more effective sorbent than plant-based extracts [198].Therefore, it is also prudent to understand the ecotoxic effects of any material intended to be used for water or soils and balance these effects with other considerations, such as regeneration or reusability, so that the disposal of the sorbent has less ecological footprint.To determine whether green adsorbents accumulate in organisms or endure in ecosystems, it is also essential to study the behavior and fate of these substances in the environment [205,208].Adhering to appropriate risk assessment is necessary for maintaining low environmental impact of the green adsorbents.The physical structure, chemical composition, and interaction mechanism of adsorbents with pollutants must all be fully understood to facilitate the environmentally friendly objectives [202,209]. Reusability and Regeneration Regeneration processes enable quick retrieval and reuse of the used adsorbents multiple times without a statistical difference in performance [210].Reuse and regeneration can be achieved through a technically viable process, e.g., desorption, with little input cost.The regeneration of adsorbents is only considered a vital issue once adsorbents are used as ex situ remediation agents for wastewater.For instance, when nano biochar is implemented for in situ soil management, the reusability of the adsorbents does not work due to the lack of separability of the spent adsorbents.In contrast, reusability studies could be performed on adsorbents for ex situ surface water or groundwater remediation.However, properly managing desorbed pollutants from spent adsorbents is also vital to sustainable environmental management.In this section, the regeneration processes and mechanisms will be critically evaluated based on the progress of the current research. The main benefits of feasible reusability of adsorbents are cost-effectiveness and lower material disposal requirements.However, achieving such reusability can be challenging, particularly for large-scale applications.The adsorbent may lose some of its original adsorption capacity and overall effectiveness after regeneration due to incomplete desorption, destruction of active sites, or altered properties (e.g., surface area, porosity) [210].Baskar et al. [76] summarized the main techniques used for the regeneration of adsorbents, including magnetic separation, filtration, thermal desorption and decomposition, chemical desorption, supercritical fluid desorption, advanced oxidation processes, and microbialassisted adsorbent regeneration.Pros and cons are associated with these regeneration processes.The reusability/regeneration of a sorbent is a good indicator of a sustainable approach to sorbent development.However, highlighting the sustainability of a sorbent's "reusability/regeneration" may require caution.This is mainly because of the methods used to regenerate sorbent materials, some of which will be discussed. An investigation of the reusability of green sorbents used for the removal of aniline indicated that the chemicals (e.g., hydrochloric acid and ethanol) used for regeneration are mostly toxic unless fate analysis is performed [211].Acids, chelators, alkalis, solvents, surfactants, or even water are commonly used for the regeneration of adsorbents via the sorption of heavy metals.In contrast, solvents (e.g., methanol, ethanol, and acetone) were used for the desorption of organic pollutants (e.g., dyes and pharmaceuticals).Thermal regeneration might induce the loss of adsorbents, the reaction of adsorbate molecules, and the destruction of adsorbent materials, and this process is relatively costly [212].Magnetic separation provides a simple recovery of materials, but it only works on sorbent materials with magnetic properties [213].Pollutants can be volatilized or broken down by heating the spent adsorbent to high temperatures, which frees up the adsorption sites for further use.Biological treatments help to react to the spent adsorbent in some specific cases.For example, microorganisms can break down organic pollutants from the surface of an adsorbent, thus freeing the functional sites of the adsorbent.While this could be a spontaneous process of obtaining adsorbent back into action, it only works for biodegradable adsorbates.It also involves slow regeneration processes and might cause fouling in adsorbent pores [210]. Researchers have applied various strategies to overcome these challenges, including modifying the surface properties of adsorbents, optimizing regeneration conditions, developing hybrid sorbent systems, and using renewable and sustainable precursors.The regeneration adsorption-desorption model requires "switching" the properties of the adsorbents [212].Therefore, novel regeneration processes should be considered based on different sorbent materials and their mechanisms for removing pollutants. The in situ reusability of applied adsorbents is quite complicated.These qualities, which lessen waste production and encourage effective pollutant removal, support the sustainability and economic feasibility of adsorbent materials.Green adsorbents can be efficiently recovered and reused by using suitable regeneration processes [214][215][216].This maximizes their potential for long-term pollutant remediation while minimizing the environmental impact [217,218].Moreover, post-regenerative chemicals should be managed appropriately to avoid the release of residuals and daughter compounds into the environment. Conclusions and Future Perspectives This review highlights different techniques applied for synthesizing green adsorbents from a range of feedstock materials, such as natural materials, biomass, minerals, and industrial and agricultural waste, while considering their advantages and disadvantages.The thorough investigation emphasizes the importance of sustainable synthesis methods in minimizing environmental pollution and avoiding the use of toxic or harsh substances.This critical description is an effective source for scientists and industry professionals working on environmentally friendly adsorbents for contaminated site management. It is essential to mention that some generic adsorbents, like biochar or raw earth minerals, can be commercially available and labeled as "green" or "biocompatible."However, it is possible that there are insufficient publicly available data to back up these claims; they are especially scarce when adsorbents are used to clean environmental contamination.The development of adsorbent materials that are "green" or "eco-friendly" and economically viable remains a research focus for the remediation of contaminated water and soil.These are only possible when we consider not only how to achieve high adsorption capacity but also the sustainability and circular economy of adsorbent materials.Straightforward and practical criterion guidance is needed for the preparation and application of such adsorbent materials.To achieve that, we may find positive outcomes through (1) a consensus definition of green/eco-friendly adsorbents, (2) setting criteria and thresholds for assessing and monitoring the properties, performance, biodegradation, renewability, and environmentally friendly aspects of adsorbent materials, (3) the development of alternative adsorbent materials for industrial needs that are fit for purpose and green/eco-friendly, and (4) regulatory considerations for the choice and application of sorbent materials that are safe for human health and the environment.These combined efforts would benefit the research, industry, and regulatory communities for the future development of green adsorbents and their applications. Figure 1 . Figure 1.The relative weight of the key 'eco-friendly' aspects studied in original research published between 2017 and 2023.There are a few overlapping subject matters, such as the antimicrobial properties of materials vs. environmentally bio-safe materials.These are also reported in the box plot.The values in parentheses for the reported categories are the number of original papers listed in the Scopus database.Details of the data screening and quality control procedures are provided in the main text. Figure 1 . Figure 1.The relative weight of the key "eco-friendly" aspects studied in original research published between 2017 and 2023.There are a few overlapping subject matters, such as the antimicrobial properties of materials vs. environmentally bio-safe materials.These are also reported in the box plot.The values in parentheses for the reported categories are the number of original papers listed in the Scopus database.Details of the data screening and quality control procedures are provided in the main text. Figure 2 . Figure 2. Various core aspects of the "green" or "eco-friendly" sorbent materials. Figure 2 . Figure 2. Various core aspects of the "green" or "eco-friendly" sorbent materials. Figure 3 . Figure 3. Network visualization maps show co-occurrences of the most used keywords in the 'Title/Abstract' search criteria.A total of 124 publications have been selected from 2010 to the present date (14 April 2024) with keywords searched as '((green adsorbent) and (waste materials)) and (green synthesis)'.The figure highlighted the research trend with different colors, showing keyword distribution over the highlighted timeframe. Figure 3 . Figure 3. Network visualization maps show co-occurrences of the most used keywords in the "Title/Abstract" search criteria.A total of 124 publications have been selected from 2010 to the present date (14 April 2024) with keywords searched as "((green adsorbent) and (waste materials)) and (green synthesis)".The figure highlighted the research trend with different colors, showing keyword distribution over the highlighted timeframe. Figure 4 . Figure 4. Illustration of waste-derived feedstocks for the development of green adsorbents with ecofriendly synthesis methods. Figure 4 . Figure 4. Illustration of waste-derived feedstocks for the development of green adsorbents with eco-friendly synthesis methods. Food and green waste, glass, metals, plastic, paper/cardboard, rubber and leather, wood, waste, etc. Table 2 . Summary of advantages and disadvantages of synthesis methods for the preparation of green adsorbent.	2024-06-13T15:04:05.861Z	2024-06-11T00:00:00.000	{ "year": 2024, "sha1": "8c86b3bc37bfb6792c89567dc4188e72f50f100f", "oa_license": null, "oa_url": "https://doi.org/10.3390/pr12061195", "oa_status": "GOLD", "pdf_src": "ScienceParsePlus", "pdf_hash": "6c1b7820d8f1611ba50e88b296b7b3b91a37e96e", "s2fieldsofstudy": [ "Environmental Science", "Materials Science", "Chemistry" ], "extfieldsofstudy": [] }
245714417	pes2o/s2orc	v3-fos-license	An expandable informatics framework for enhancing central cancer registries with digital pathology specimens, computational imaging tools, and advanced mining capabilities Background Population-based state cancer registries are an authoritative source for cancer statistics in the United States. They routinely collect a variety of data, including patient demographics, primary tumor site, stage at diagnosis, first course of treatment, and survival, on every cancer case that is reported across all U.S. states and territories. The goal of our project is to enrich NCI’s Surveillance, Epidemiology, and End Results (SEER) registry data with high-quality population-based biospecimen data in the form of digital pathology, machine-learning-based classifications, and quantitative histopathology imaging feature sets (referred to here as Pathomics features). Materials and methods As part of the project, the underlying informatics infrastructure was designed, tested, and implemented through close collaboration with several participating SEER registries to ensure consistency with registry processes, computational scalability, and ability to support creation of population cohorts that span multiple sites. Utilizing computational imaging algorithms and methods to both generate indices and search for matches makes it possible to reduce inter- and intra-observer inconsistencies and to improve the objectivity with which large image repositories are interrogated. Results Our team has created and continues to expand a well-curated repository of high-quality digitized pathology images corresponding to subjects whose data are routinely collected by the collaborating registries. Our team has systematically deployed and tested key, visual analytic methods to facilitate automated creation of population cohorts for epidemiological studies and tools to support visualization of feature clusters and evaluation of whole-slide images. As part of these efforts, we are developing and optimizing advanced search and matching algorithms to facilitate automated, content-based retrieval of digitized specimens based on their underlying image features and staining characteristics. Conclusion To meet the challenges of this project, we established the analytic pipelines, methods, and workflows to support the expansion and management of a growing repository of high-quality digitized pathology and information-rich, population cohorts containing objective imaging and clinical attributes to facilitate studies that seek to discriminate among different subtypes of disease, stratify patient populations, and perform comparisons of tumor characteristics within and across patient cohorts. We have also successfully developed a suite of tools based on a deep-learning method to perform quantitative characterizations of tumor regions, assess infiltrating lymphocyte distributions, and generate objective nuclear feature measurements. As part of these efforts, our team has implemented reliable methods that enable investigators to systematically search through large repositories to automatically retrieve digitized pathology specimens and correlated clinical data based on their computational signatures. Introduction The NCI's Surveillance, Epidemiology, and End Results (SEER) program is a coordinated system of 19 cancer registries that is charged with providing timely and accurate data regarding cancer incidence, mortality, treatment, and survival. Pathology datasets currently available in the SEER registries are qualitative in nature, consisting of scoring and staging data captured in normal registry abstracts and pathology reports. Such datasets are generally subject to inter-observer variability, which can result in biases in population-wide studies of cancer incidence, mortality, survival, and prevalence. The main goal of our project is to enrich SEER registry data with high-quality population-based digital biospecimen data in the form of pathology tissue images and detailed computational tissue characterizations and features (also referred to as Pathomics features) derived from the images. Examples of Pathomics data include detailed characterizations of cancer and stromal nuclei and quantification and mapping of tumorinfiltrating lymphocytes (TILs) along a supplementary histology classification generated through deep-learning algorithms. These data will augment existing registry data with quantitative features obtained directly from clinically acquired whole slide tissue images and provide detailed and nuanced information on tumor histology. The scientific premise motivating this work is that the incorporation of quantitative digital pathology into the cancer registries will result in a valuable population-wide dataset that can provide additional insight into the underlying characteristics of cancer. Next Generation Sequencing (NGS) technologies have captured much attention of the clinical community for their capacity to provide insight as to personalized choice in treatment and therapy. A major limitation of NGS technologies is that they obliterate the spatial information associated within and throughout the tumor environment. Histopathology and immunostaining localization techniques preserve this information which is invaluable in making accurate determinations. In fact, it is through the process of histopathology examination that tumor margins/volumes are determined by pathologists prior to the NGS analysis. These parameters are subsequently used to help guide decisions regarding appropriate cut-offs for allele frequencies and drive other components of the overall analysis. Pathomics features extracted from high-resolution pathology images are a quantitative surrogate of what is described in a pathology report. The important distinction is that these features are reproducible, unlike human observations, which are highly qualitative and subject to a high degree of inter-and intra-observer variability. The importance of increasing reproducibility and reducing inter-observer variability in pathology studies has been previously reported. Moreover, many studies have demonstrated that quantitative image characterizations (e.g., nuclear features, patterns of TILs) are promising biomarkers which can be used to predict outcome and treatment response, if available in a large population. [27][28][29][30][31][32][33][34][35][36][37][38][39] These biomarkers integrated with clinical and genomics data can provide new opportunities to enhance our understanding of cancer incidence, mortality, survival, along with statistical characterizations of lifetime risk, and to improve prediction and assessment of therapeutic effectiveness. Our project began as collaboration among investigators within the state cancer registries of New Jersey, Georgia, and Kentucky. The consortium of partnering sites has recently expanded to include the newly established New York Cancer Registry. In this collaborative effort, we are implementing a framework of data curation and analysis workflows, computational imaging tools, and informatics infrastructure to support the creation and management of a well-curated, integrated repository of high-quality digitized pathology images and Pathomics features, for subjects whose data are being collected by the registries. The framework is being developed in close collaboration with SEER registries to ensure that it is scalable and in-line with existing registry processes and can support queries and the creation of population cohorts that span multiple registries. In our framework, whole slide tissue images in the repository are systematically processed to compute Pathomics data and to establish linkages with registry data. The current set of Pathomics data includes (1) quantification of TILs, (2) segmentation and computational description of cancerous and stromal nuclei, (3) segmentation of tumor regions, (4) characterization of regional Gleason grade for prostate cancer, and (5) identification of non-small cell lung cancer (NSCLC) adenocarcinoma subtypes. This initial set is primarily motivated by an increasing number of scientific studies that investigate TILs and the relationships among TILs, tumors, and nuclear structure of tissue. [40][41][42][43][44][45] Such investigations can provide important information to advance our understanding of immune response in many cancer types. In the future, additional Pathomics features, such as the spectral and spatial signatures of staining characteristics exhibited by the digitized specimens, will be incorporated into our framework. The informatics infrastructure for this project is being built on open-source software and leverages modern software technologies, such as containerization and web-based applications, for a scalable, extensible implementation. 46,47 The infrastructure facilitates visualization of highresolution whole slide tissue images along with associated Pathomics datasets. User authentication and access controls are implemented to thwart unauthorized access to data. The informatics infrastructure is being expanded to include tools to support content-based image retrieval. Presently, the repository manages diagnostic whole slide tissue images and analysis results obtained from 772 prostate cases, 1410 NSCLC cases, 70 breast cancer cases, and 48 lymphoma cases from the New Jersey State Cancer Registry and from 198 breast cancer cases from the Georgia State Cancer Registry. The scientific validation of the proposed environment will be undertaken through performance studies led by investigators throughout the four collaborating sites with an overarching focus on breast cancer, colorectal cancer, lymphoma, melanoma, NSCLC, and prostate cancer. We are confident that this repository will enable effective integration of pathology imaging and feature data as an invaluable resource in SEER registries. In the rest of the paper, we describe the design and implementation of the key components of the framework: the data curation and analysis processes, the initial set of image analysis methods, and the underlying informatics infrastructure for data management and visualization. Aggregation, quality control, and linkage of image data The first component of our framework is the curation of pathology imaging data and linkage with other data from the cancer registries. Image quality control is an essential step, because specimen preparation protocols and tissue scanning procedures may result in imaging artifacts and variations in image quality. We devised and refined a workflow to facilitate the collection and quality control of digitized tissue specimens and linkage of images with correlated data extracted from the cancer registries. Here we describe the workflow deployed at Rutgers and the New Jersey SEER registry; the other sites-Georgia, Kentucky, and New York-are incrementally adopting analogous workflows as approved by their SEER registries and Institutional Review Boards (IRBs). Fig. 1 depicts an instance of the workflow. Specimen retrieval and imaging are coordinated at the Biomedical Informatics Shared Resource (BISR) of Rutgers Cancer Institute of New Jersey (RCINJ). Breast, colorectal, lung, melanoma, and prostate cancer cases suitable for the project exhibiting well-defined tumor type and diagnoses are selected by a pathologist at the RCINJ and Rutgers Robert Wood Johnson Medical School. Cases within approximately a 2-year window are retrieved from onsite storage, whereas others are requested from offsite storage with the help of BioSpecimens Repository Service of RCINJ. After a certified pathologist selects suitable slides according to requirement of each cancer type-e.g., prostate cancer specimens are selected according to the Gleason grade-the specimens are imaged with an Olympus VS120 whole slide scanner with no protected health information appearing in image filename, image metadata, or the images themselves. Team members from the BISR and NJCR perform cross-specialty review of the data for quality control. A secure, IRB-approved, Oracle-based (Redwood Shores, CA, USA) Clinical Research Data Warehouse is used at Rutgers to facilitate review of imaging and correlated clinical information on an individual patient basis or as part of large cohorts. The data warehouse has been commissioned to house multimodal data (genomics, digital pathology, radiology images). It orchestrates aggregation of information originating from multiple data sources including Electronic Medical Records, Clinical Trial Management Systems, Tumor Registries, Biospecimen Repositories, Radiology and Pathology archives, and Next Generation Sequencing services (Fig. 2). Innovative solutions were implemented in the warehouse to detect and extract unstructured clinical information that was embedded in paper/text documents, including synoptic pathology reports. The Warehouse receives objective oversight by a standing Data Governance Council. 48 An Informatica-based (Redwood City, CA, USA) extraction transformation and load interface (ETL) has been developed to automatically populate the Data Warehouse with data elements originating from the multimodal data sources. This past year our team worked closely with the Google Healthcare team to successfully create and test an instance of the Data Warehouse on the Google Cloud Platform (GCP). In May 2020, we demonstrated the scalability of the cloud-based ETL, Warehouse, and Data Mart. As part of the project, our team will expand the use of the Warehouse by configuring it to integrate digitized pathology specimens with data originating from all of the collaborating cancer registries. The images and cases are linked through deidentified ID sequences. The New Jersey State Cancer Registry receives the deidentified ID as well as case information including specific surgery number and date, so that after data retrieval and decoding encrypted fields, the deidentified ID is linked with clinical data associated with the case and, more specifically, with the diagnostic surgery. This ensures that the cancer specimen images are associated with the correct staging of the disease at the time of diagnosis so that it can be used in downstream research. The total corpus of data comprising the linked data sets encompasses more than 150 data elements, including the de-coded NAACCR data, as shown in Table 1. The de-identified images are analyzed through a set of deep-learning analysis pipelines as described in the subsequent sections. Extraction of pathomics features Development of tissue image analysis methods is a highly active area of research and implementation. A variety of analysis methods for segmentation and classification of objects, regions, and structures (such as nuclei, tumors, glands) in tissue images have been developed. Excellent overviews of existing techniques can be found in several review papers. [49][50][51][52][53][54][55] Deep-learning-based analysis approaches have become popular, because deep-learning methods have been shown to outperform traditional image analysis methods in many application domains, including digital pathology. Our current tissue image analysis library consists of deep-learning methods developed by our group to classify patterns of TILs, 56,57 segment tumor regions, classify tumor subtypes, 58,59 and segment nuclei in whole slide images (WSIs) of hematoxylin and eosin-stained tissue samples. 60, 61 We should note that the analysis functionality is not limited to methods implemented by our group only. We have started with these methods because (1) they are based on state-of-the-art convolutional neural network architectures, suchasVGG16, 62 Inception V4, 63 ResNet, 64 and U-Net, 65 (2) they have achieved high accuracy scores, and (3) they have been previously used, refined, and validated in generating large, curated Pathomics datasets. For example, the TIL models were developed in close collaboration with pathologists, who generated a large set of training data, evaluated analysis results, and helped refine the models. The final models were employed to produce and publish a TIL dataset from 5202 WSIs from 13 cancer types. 56,57 The nucleus segmentation model was developed in a similar approach with one difference. In addition to manually annotated segmentations, a synthetic data generation method, based on generative adversarial networks, 66 was used to significantly increase the diversity and size of training data. 60 The model trained with the combined manual and synthetic training data was used to generate a quality-controlled dataset of 5 billion segmented nuclei in 5060 WSIs from 10 cancer types 61 in the Cancer Genome Atlas (TCGA) repository. We plan to expand the suite of analysis methods and incorporate state-of-the-art methods developed by other groups over time. Indeed, at the time of writing this manuscript, we are in the process of integrating and validating Hover-Net 67 in the framework for segmentation and classification of nuclei. The current suite of TIL analysis models can resolve TIL distributions in a WSI at the level of 50 × 50 μm 2 patches. The characteristics of tumor regions and the relationship between tumor regions and lymphocyte cells can be used to determine cancer stage and evaluate response to treatment. Our current models can segment tumor regions in lung, prostate, pancreatic, and breast cancer types and can classify tumor and non-tumor regions at the level of 88 × 88 μm 2 patches. The model for prostate cancer can segment and label a tumor subregion with one of the three Gleason scores: Benign, Grade 3, and Grade 4+5. The lung tumor segmentation model is able to segment and label a tumor subregion with one of the six tumor subtypes: acinar, benign, lepidic, micropapillary, mucinous, and solid. Nucleus segmentation is one of the core digital pathology analysis steps. The shape and texture properties and spatial distributions of nuclei in tissue specimens are used in cancer diagnosis and staging. Our nucleus segmentation model can detect nuclei and delineate their boundaries in WSIs. After a WSI has been processed by the segmentation model, we compute a set of shape, intensity, and texture features. We use the PyRadiomics library 68 to compute the patch-level features. Management, visualization, and review of pathomics features Our data analysis workflow implements an iterative train-predict-reviewrefine process to curate robust Pathomics features. This process is based on our earlier works in curating large Pathomics datasets 57,59,61 and is carried out as part of the training and prediction phases of the deep-learning analysis pipelines. We developed a set of tools to enable the iterative process and to provide support for the management, indexing, and interactive viewing of WSIs and analysis results. The tools are implemented as a set of web-based applications and services in the PRISM and QuIP software platforms. 46,47 Using these tools, pathologists can inspect the output of a tumor or TIL analysis pipeline as full-resolution heatmap overlays on WSIs. A heatmap is a spatial representation of prediction probabilities assigned to individual image patches by the deep-learning model; the probability value indicates if a patch is class-positive (e.g., TIL-positive, tumorpositive). Fig. 3 shows example heatmaps generated from the TIL (upper figure) and tumor (lower figure) analysis pipelines. Nuclear segmentation results can be viewed as polygons, which represent the boundaries of segmented nuclei as overlays on the images in QuIP (Fig. 4). Fig. 5 shows how the iterative process is executed with QuIP. For example, after a set of WSIs are processed by the TIL and tumor segmentation models, the source WSIs and the heatmaps are loaded to QuIP for management and visualization. The heatmaps and WSIs are also transformed into feature maps. Feature maps are lower resolution representations of the heatmaps and WSIs in a four-panel image. Fig. 6 illustrates an example feature map which combines TIL results from a VGG16 model and tumor segmentation results from a ResNet model. The upper left corner of the image is the low-resolution tissue image, the upper right corner is the tumor segmentation map, the lower left corner represents the TIL map, and the lower right corner is the combined and thresholded TIL and tumor maps. Feature maps allow a pathologist to review results more efficiently than examining full-resolution images and maps. If the pathologist sees potential problems with the results during this review, they use the web applications in QuIP to visualize the WSIs and heatmaps at higher resolutions. If the review necessitates refinements to the model, additional training data are generated and added to the training dataset. They can annotate regions in an image using web-based visualization and annotation tools. Patches extracted from these annotations are reviewed and labeled to create additional training data. The model is refined by re-training the method with the updated training dataset. Results The current implementation of the framework-the curation and analysis workflows, analysis methods, and informatics infrastructure-has been successfully deployed. The workflows and analytic methods have received IRB approval at all collaborating institutions. The framework has been employed to create a repository of diagnostic images from 772 prostate cases, 1410 NSCLC cases, 70 breast cancer cases, and 48 lymphoma cases from the New Jersey State Cancer Registry and from 198 breast cancer cases from the Georgia State Cancer Registry. The repository also contains results from TIL and tumor segmentation for each image and more than 2.5 billion segmented nuclei from all of the images. For each image, there are two TIL analysis results (one generated from the VGG16 network and the other from the Inception V4 network). The images and Pathomics data are managed by an instance of QuIP running at Stony Brook for interactive visualization of images and Pathomics features. All of the results and images are also stored in Box folders to facilitate bulk data downloads. Discussion and conclusions Evaluation of cancer control interventions in prevention, screening, and treatment and their effects on population trends in incidence and mortality hinge on accurate, reproducible, and nuanced pathology characterizations. Diagnostic and treatment guidelines also specify detailed measurements of TILs, nuclear grade; i.e., evaluation of the size and shape of the nucleus in the tumor cells, mitoses, and IHC staining, which are currently not included in cancer registry data abstraction. Presently, the SEER Pathology workflow, depicted in Fig. 7, begins with normal registry abstracts and electronic pathology (e-Path) reports securely transmitted to the SEER registries. Although scoring and staging data are captured and made available through the registries, there have been numerous studies that showed a high level of inter-observer variability among the diagnostic classifications rendered by pathologists, which can potentially give rise to biases when conducting population-wide studies. As the diagnosis of cancer and its immune response to therapy is made through tissue studies, the integration of pathology imaging in SEER registries is critical to precisely classify tumors and predict tumor response to therapies. Whole slide tissue scanning technologies have advanced significantly over the past 20 years. 69 They are capable of imaging tissue specimens at high resolution in several minutes, and with advanced auto-focussing mechanisms and automated slide trays, they can process batches of tissue samples with little-to-no manual intervention. Several studies have evaluated the utility of imaged tissue data in pathology workflows. [70][71][72][73][74][75] The Food and Drug Administration has approved a number of digital pathology systems for diagnostic use. 76 We expect that digital pathology will be employed increasingly as part of routine pathology workflows at hospitals and medical research centers. As institutions adopt digital WSIs into their pathology workflows, we can envision that the images and molecular reports will also be securely transmitted to the SEER registries. Within the SEER registry, images will be automatically processed by the suite of feature extraction pipelines appropriate for the type of cancer. The SEER database will be enhanced with quantitative features and the accompanying pipeline distribution version. SEER*DMS will be used to link and integrate cancer abstracts, e-Path reports, WSIs, and Pathomics feature sets from all reporting facilities. De-identified images and annotations will then be extracted for data mining and research use. Our work on building a repository of curated WSIs and Pathomics features is an important step toward realizing this capability. Availability of tissue images and Pathomics datasets will also provide an invaluable resource for medical education and Pathology training as well as to facilitate multi-disciplinary approaches, improved quality control, and more efficient remote and collaborative access to tissue information. 77,78 The first phase of our project focussed on the collection of cases and correlated pathology specimens from the archives of New Jersey State Cancer Registries and Rutgers Cancer Institute of New Jersey and on targeted prostate and NSCLC cases. To date, we have established a repository of (1) highquality digitized pathology images for subjects whose data are already being routinely collected by the collaborating registries and (2) Pathomics features consisting of patterns of TILs, tumor region segmentations and classifications, and segmented nuclei. We have completed the initial linkages with registry data, thus enabling the creation of information rich, population cohorts containing objective imaging and clinical attributes that can be mined. As part of the second phase of the effort, we have increased the number of contributing state registries to include Georgia, Kentucky, and New York and we have simultaneously expanded the scope of cancers under study by including melanoma, breast, and colorectal cancers. We will also build upon our team's previous research efforts to design, develop, and optimize algorithms and methods that can quickly and reliably search through a growing reference library of cases to automatically identify and retrieve previously analyzed lesions which exhibit the most similar characteristics to a given query case for clinical decision support [20][21][22]25,[79][80][81][82][83][84][85][86] and to conduct more granular comparisons of tumors within and across patient populations. One of the potential advantages of this approach over purely alphanumeric search strategies is that it will enable investigators to systematically interrogate the data while visualizing the most relevant digitized pathology specimens. 32,33 As part of the next phase of our project, we plan to investigate the automated nature of the full range of algorithms and methods for their capacity to enable clinicians and investigators to quickly and reliably answer questions such as: (a) What level of morphological variations are detected among a given set of tumors or specimens? (b) What changes in computational biomarker signatures occur at onset and key stages of disease progression? (c) What is the likely prognosis for a given patient population? Software availability The QuIP software and analysis methods are available as open-source codes for use by other research groups. The QuIP software platform can be downloaded and built from https://github.com/SBU-BMI/quip_distro. The codes for the analysis methods can be accessed from links at https://github.com/SBU-BMI/histopathology_analysis. Financial support and sponsorship This work is supported, in part, by UG3CA225021, UH3CA225021, U24 CA215109, U24CA180924-01A1, and 5UL1TR003017 grants from the National Institutes of Health and generous contributions to Stony Brook from Bob Beals and Betsy Barton. Additional support was provided through funding from the U.S. Department of Veterans Affairs -Boston Healthcare System through contract, IPA-RU-092920. This work leveraged resources from XSEDE, which is supported by NSF ACI-1548562 grant, including the Bridges system (NSF ACI-1445606) at the Pittsburgh Supercomputing Center. Services, results and/or products in support of the research were generated by Rutgers Cancer Institute of New Jersey Biomedical Informatics Shared Resource NCI-CCSG P30CA072720-5917. Declaration of Competing Interest There are no conflicts of interest. Fig. 7. Pathology image workflow. WSIs are de-identified and analyzed by deep-learning analysis pipelines deployed in containers. Image data are linked to the SEER Registry database to enhance it with quantitative imaging features (such as TIL distributions and tumor segmentations) extracted by deep-learning models. De-identified images and imaging features can then be used for data mining and research purposes.	2022-01-06T16:33:53.692Z	2022-01-01T00:00:00.000	{ "year": 2022, "sha1": "48fc25383b7e1f6c6d89ded1cf662ff1a810a5d6", "oa_license": "CCBYNCND", "oa_url": "https://doi.org/10.4103/jpi.jpi_31_21", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "00a51ee443ebe664c19d7ecaa17710b3090bd1a5", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
245892433	pes2o/s2orc	v3-fos-license	A Review of the Machine Learning Algorithms for Covid-19 Case Analysis The purpose of this article is to see how machine learning (ML) algorithms and applications are used in the COVID-19 inquiry and for other purposes. The available traditional methods for COVID-19 international epidemic prediction, researchers and authorities have given more attention to simple statistical and epidemiological methodologies. The inadequacy and absence of medical testing for diagnosing and identifying a solution is one of the key challenges in preventing the spread of COVID-19. A few statistical-based improvements are being strengthened to answer this challenge, resulting in a partial resolution up to a certain level. ML have advocated a wide range of intelligence-based approaches, frameworks, and equipment to cope with the issues of the medical industry. The application of inventive structure, such as ML and other in handling COVID-19 relevant outbreak difficulties, has been investigated in this article. The major goal of this article is to 1) Examining the impact of the data type and data nature, as well as obstacles in data processing for COVID-19. 2) Better grasp the importance of intelligent approaches like ML for the COVID-19 pandemic. 3) The development of improved ML algorithms and types of ML for COVID-19 prognosis. 4) Examining the effectiveness and influence of various strategies in COVID-19 pandemic. 5) To target on certain potential issues in COVID-19 diagnosis in order to motivate academics to innovate and expand their knowledge and research into additional COVID-19-affected industries. Abstract-The purpose of this article is to see how machine learning (ML) algorithms and applications are used in the COVID-19 inquiry and for other purposes. The available traditional methods for COVID-19 international epidemic prediction, researchers and authorities have given more attention to simple statistical and epidemiological methodologies. The inadequacy and absence of medical testing for diagnosing and identifying a solution is one of the key challenges in preventing the spread of COVID-19. A few statistical-based improvements are being strengthened to answer this challenge, resulting in a partial resolution up to a certain level. ML have advocated a wide range of intelligence-based approaches, frameworks, and equipment to cope with the issues of the medical industry. The application of inventive structure, such as ML and other in handling COVID-19 relevant outbreak difficulties, has been investigated in this article. The major goal of this article is to 1) Examining the impact of the data type and data nature, as well as obstacles in data processing for COVID- 19 . 2) Better grasp the importance of intelligent approaches like ML for the COVID-19 pandemic. 3) The development of improved ML algorithms and types of ML for COVID-19 prognosis. 4) Examining the effectiveness and influence of various strategies in COVID-19 pandemic. 5) To target on certain potential issues in COVID-19 diagnosis in order to motivate academics to innovate and expand their knowledge and research into additional COVID-19-affected industries. Impact Statement-The worldwide response to the COVID-19 epidemic will rely heavily on ML, defined broadly. This article enables ML researchers to quickly connect with the range of active research effort. We identify the key difficulties, potential paths for future work, and crucial community resources in particular. Given the multidisciplinary character of the problem, this review will aid data scientists in forming cross-disciplinary collaborations. We also educate strategists and policymakers on the advantages of ML and help them understand the obstacles, possibilities, and drawbacks of utilizing data science to battle the COVID-19 epidemic. I. INTRODUCTION C ORONA virus is a genus of viruses, which infect animals, birds, and cause sickness. The term "corona" derives from the Latin word "corona," that means "crown," and alludes to the virus's outer spikes, which are club-shaped and crown-like. Corona viruses are large and live in close proximity to positivestrand ribonucleic acid (RNA) viruses. Corona virus (COV) is a member of the corona viridae family and belongs to the Ortho corona virinae subfamily. Corona viruses are classified into four generation: alpha, beta, gamma, and delta COVs, with alpha and beta COVs infecting mammals and gamma and delta COVs infecting birds, respectively [1]. COV was discovered in the late 1930s after a severe respiration virus in farm chickens was found with the infectious bronchitis virus to be infected [2]. After that in 1960s human corona viruses (HCOVs) was discovered. HCOVs can be transmitted to humans through respiratory tract infections that range from minor to fatal. Whereas a moderate illness is defined as a few cases of a common cold, a lethal infection is defined as severe acute respiratory syndrome (SARS) or middle east respiratory syndrome (MERS). Both SARS and MERS are Beta COVs, which cause a deadly and contagious respiratory disease. In December 2019, another pathogenic HCOV was discovered in Wuhan, China [3] and identified as a novel corona virus injury. With a population of almost 11 million people, Wuhan is the most important transportation hub [4]. The wild animal market in Wuhan is famous for selling bats, snakes, marmots, chickens, and other live animals, and most of the patients were discovered there before December. A total of 44 cases of pneumonia with an unknown cause were reported in Wuhan between December 31, 2019 to January 3, 2020. During the informed era, the cause of the virus was unknown. However, on January 7, 2020, WHO and the government of China detected the novel corona virus (November 2019), which is a member of the SARS virus family [5]. After January 17, 2020, Ncov-2019 was swiftly expanded in China's major cities, covering over 7000 cases. As a result, the government of China has taken severe efforts to prevent the spread of this precarious virus from 23 to 2020, including experimentally delaying publicly transit, issuing warnings to inhabitants not to leave their homes, and isolating the biggest afflicted cities. The WHO recognized the disease on January 30, 2020, as the new COVID-19 has spread to 18 nations via person-to-person transmission. As per available data from www.worldometer.com, COVID-19 has impacted 32 118 316 people worldwide as of September 24, 2020, and the WHO has labelled it a pandemic [6]- [8]. The United States has the most confirmed cases, with 7 139 553 persons worldwide affected. With 206 593 deaths, the death rate in the United States has risen. The sudden rise in active cases and death cases in the United States and India shocked the entire world. Similarly, many other nations, such as Brazil, India, Colombia, Russia, Spain, Peru, Mexico, Argentina, and Africa, are among the top ten countries that are badly affected by COVID-19, and their mortality and recovery rates are both high. Some of the statics shown in the following Figs. 1-3. Similarly, in India, active cases have reached 3 11 05 270 patients, 30 300 762 patients have been recovered, and the death cases has been reached 4 13 640 by July 19, 2021. The statistics of total cases (TC), recovery rates (RR), death rate (DR), as well as Indian positive rate (PR) is shown in Fig. 4. The lack of sufficient information on early-stage symptoms is a crucial factor in the spread of COVID-19. This has resulted in a situation where citizens are unaware that they are polluted and travel without understanding of disease transmission. As a result, the ubiquitous presence of COVID-19 necessitated the management of congested areas such as bus stops, airports, and train stations. It requires a variety of inspection tools, including thermal sensors, machine learning (ML) [9], and artificial intelligence (AI) [10]- [12]. Reverse transcription polymerase chain reaction (RT-PCR) was one of the standard procedures for diagnosing COVID-19 [13]. Short sensitivity, as well as time, cannot meet the criteria of swiftly finding and elevating positive instances, and this has become one of RT-significant PCR's drawbacks. As an alternative to this obstacle, X-ray imaging and computer tomography can give a quick screening to detect the problem. ML has lately acquired prominence and is fast developing in resolving numerous challenges such as speech and object recognition, picture classification, and so on, because of the problem's complexity in improving epidemiologic techniques. In comparison to other issue domains, the COVID-19 with ML experiments have been increased dramatically in just two months. This highlights the importance of comprehending the disease's severe character, as well as the need for high-level research employing relevant intelligent computer technologies [14]. Throughout the last decade, the rapid expansion of digital approaches has played a significant role for solving different difficulties in the health sector, as well as illness prevention. The availability of digital machinery to adequately dealing with COVID-19 is also being tracked as a part of the present global health emergency. In the field of health care, the prognosis of a patient's outcome is still a difficult topic. Predicting and diagnosing diseases has become more easier as ML approaches have grown in popularity. Self-learning ML can learn from machines and generate accurate predictions. ML has successfully predicted several diseases, including hepatitis [15], human immunodeficiency virus HIV [16], Ebola virus [17], and others. ML has also been used to combine and analyze large amounts of data from COVID-19 patients. To identify the affected individuals with personalized features, several ML algorithms are used. The success of ML is determined by the data visualization used. Although ML has demonstrated its effectiveness in identifying diseases, gathering data, organizing health records, and so on, a few systematic models based on Ml have yet to be implemented due to a number of challenges, including a lack of identifying tools, a lack of medical tools, the use of biomedical data, heterogeneity, and so on. To address these issues, feature engineering has progressed by utilizing human inventiveness. However, several health philosophies that are consumed in the generalization of information have expounded on these shortcomings. It is possible that some health sector works will need to be suggested in unique ways. This issue could be overcome by using the ad hoc technique to illustrate the health composition. Nonetheless, supervised models are unable to forecast the most recent prototypes. Nonetheless, extracting relevant data while building predictors can be simple with representation learning [18]. The pandemic's quick spread has resulted in major health-care difficulties, necessitating immediate responses to mitigate the effects. In dealing with the problem, AI has a wide range of applications [19]. COVID-19 is a worldwide pandemic disease, which has presented a threat to human existence. In systematic reviews, ML training methods and other statistical modeling are used by computers to complete many tasks without precise commands were discovered [20]. ML approach are currently employed for prognosis all around the world due to their accuracy. Ml approaches, on the other hand, face a number of obstacles, such as the new internet database's poor quality. One of the issues in training a model or selecting the optimal ML model for prediction, for example, is determining the suitable parameters. To produce predictions based on the available dataset, researchers utilized the better Ml approach, which is fit in the database [21]. Most of the ML technique could be used for data analytics and for extracting hidden patterns [22]. ML techniques are meant to find complicated patterns and interfaces in data when there are unknown and sophisticated risk factor correlation patterns [23]. The rest of this article is organized as follows. Section II describes the associated work done related to COVID-19. Section III describes the different approaches of ML. ML methodology used for COVID-19 briefly explained in Section IV. Article selection strategy and a search method for the literature describes in Section V. Different ML approach for COVID-19 explain in Section VI-I. COVID-19 data processing challenges using intelligent computing approaches describes in Section VII. Discussion and implications explained in Section VIII. Limitations and future scope are described in Section IX. Finally, Section X concludes this article. II. ASSOCIATED WORK The COVID-19 virus, which is produced by the SARS-COV-2 viruses, which is require remarkable return of unique strength and competence in above 250 nations throughout the universe. During the first four months of the pandemic, the number of people influenced fluctuated from 2 to 20 million, with 250 000 deaths. All governments around the world took severe efforts to limited the rapid spreading of the COVID-19 illness among individuals, including placing more than 100 of millions of people under quarantine [24]. All of these attempts, however, are limited due to the difficulty in distinguishing between positive and negative people available symptoms of COVID-19. As a result, SARS-CoV-2 viral detection tests are thought to be critical in identifying positive cases of infection and restricting the virus's transmission [25]. The most useful and critical modalities for diagnosing the COVID-19 stage and potential dangers to the patient's lungs are radiology and imaging, notably a chest CT scan [26]. To limit person-to-person transference and improve patient care, COVID-19 must be discovered early. Separation and quarantine of healthy persons from diseased or suspected COVID-19 carriers has recently been discovered to be the most effective technique of preventing COVID-19 transmission [27]. The role of ML techniques in COVID-19 diagnosis revealed important insights, such as whether a lung computed tomography scan is used as a first alternative or screening testing for actual reversed transcriptase polymerase chain reaction (RT-PCR), and the dissimilarity between COVID-19 pneumonitis as wee as other viral pneumonitis used a computed tomography scan of the lungs [28]. III. APPROACHES OF ML ML is the best rising technique for classification [29]. ML is a method for discovering previously unknown functions, relationships, or structures between input and output variables. Typically, explicit algorithms via automated learning processes have a hard time detecting these relationships [30]. ML algorithms are used to forecast the number of confirmed illnesses and deaths in the next months [31]. There are two parts to ML. The first section employs a genetic algorithm to determine the best weight of database fusion of different node perception outcomes and delete impractical nodes, while the second employs a fault identification neural network to find fault nodes [32]. ML is a branch of AI that includes supervised, unsupervised, and reinforcement learning [33]. Common ML models including clustering, classification, dimensionality reduction, regression, reward maximization, and anomaly detection [34]. In the supervised learning paradigm, ML algorithms are trained on labelled datasets, which means that each input has a ground-truth output (continuous or discrete). On the other hand, there is no ground-truth output in unlabeled [35], and the algorithm usually aim to discovered patterns in the dataset. Aim of Reinforcement to improve the cumulative reward, which is most suitable for successive decision-making issues [36]. Unsupervised learning, as shown in Fig. 5, involves clustering analysis and dimensional reduction, whereas supervised learning considers regression and classification. Classification and control are also a part of RL. A. ML for COVID-19 Recently, three independent perspectives on work on edge detection and computing of (COVID-19) cases have been Table I). ML algorithms for activity recognition and edge computing approaches are examples of imaging processes that can inspire ML approaches, which can assist radiologists in analyzing composite imaging and texture dataset. For the innovative COVID-19, there are different model, which are capable of analyzing medical imaging and distinguishing COVID-19 [37]. ML is one of best technologies that has been successfully considered to a variety of medical fields for the detection of the new evaluation, diagnosis, genotype phenotype associations, prognosis, sickness classification, transcriptome, and death ratio minimization. The approach of automatic categorization of COVID-19 may be employed by analyzing universal attributes extraction structure of ML to build the more accurate feature, which is a critical model of learning. Convolutional neural networks (CNN), DenseNet, MobileNet, ResNet, Xception, Inception-ResNetV2, InceptionV3, NASNet, and VGGNet were picked from a range of deep CNN. The categorization was subsequently accomplished by feeding the collected features into various Ml classifiers, which identified them as COVID-19 or other illnesses cases [35]. Progressive ML algorithms can integrate and evaluate a large amount of data from COVID-19 patients to provide the best understanding of the viral spread pattern, improve diagnostic accuracy, develop new and effective treatment options, and even identify people at chance of the disease based on physiological and genetic characteristics [38]. The prediction of future COVID-19 cases detail analysis using AI, ML, and DL summarize and the authors concluded that AI, ML as well as DL are the key technologies, which can help healthcare organizations to support decision making in real-time to control the spread of the pandemic [104]. IV. ARTICLE SELECTION AND A SEARCH METHOD FOR THE LITERATURE ML, deep learning, and other intelligent systems have been employed practically everywhere in the world. Many research articles are being conducted with the assistance of these intelligent systems on a daily basis, and various publications are being published in reputable web databases. These intelligent systems, particularly ML and DL, are used in a variety of applications including IoT, healthcare, and cyber physical systems. Following the discovery of a pandemic COVID-19 ebullition in China (Wuhan) in December 2019, the importance of these intelligent technologies in resolving COVID-19 issues has risen dramatically. Since its acknowledgment, a growing number of research projects have been launched on a daily basis. Using these criteria as inspiration, an examination of the degrees of use of ML approach for solving COVID-19 methodology is presented in this article. We employed an exclusive article searching approach for papers gathering to accomplish this. Keywords "ML technique for COVID-19" have been searched on dissimilar sources such as IEEE Xplore, Elsevier, Google Scholar, Springer, and another database. Fig. 6 depicts the technique for finding papers. Many papers connected to COVID-19 can be located using a keyword search. We only examined publications relating to ML techniques because our focus is on ML approaches. For filtering papers, we employed both inclusion and exclusion strategies. Inclusion the following are the procedures for filtering papers relevant to COVID-19. 1) Papers relating to ML methods used to solve COVID-19 challenges will be considered. 2) Papers on COVID-19 news, practice guidelines, datasets, biological processes, and other topics are being considered. Also examined are the following exclusion procedures for refine research papers relevant to COVID-19. 1) COVID-19 related papers or abstract but not accessible. 2) COVID-19 papers have been published in survey reports, reviews, and other venues. 3) Papers, which are not relevant to COVID-19. All publications that are necessary to employ in this article are considered once these inclusion and exclusion processes have been applied. For the final analysis, a total of 81 papers were considered. Out of the 81 papers, 37 are ML related, and the remaining 44 are unrelated. These 81 publications were culled from a variety of sources, such as IEEE explorer, Elsevier, science direct, Springer, MDPI, Wiley, Taylor and Francis, Hindawi, Google Scholar, Plos One, OMIC, and others. The dataset utilized, method employed, as an input type of text as well as image is used, anticipated outcomes, and utilization levels are all retrieved from each publication examined for the review. According to the exclusion and inclusion criteria, the total number of research was limited to (1132) papers from all databases. The constraint involves considering the research publication of the year (2019-2021), the article types original papers published as journal articles in english only. The publications are organized by author name, year of publication, country, dataset used, method employed, and end results in (see Table II ). V. ML APPROACH FOR COVID-19 ML is a type of ML that uses algorithms for self-learning and prediction. The affected people are identified using ML algorithms based on physiological and personalized features. Inadequate the application of ML for anticipating, screening, and identifying innovative COVID-19 associated research has been summarized in this part. Randhawa et al. [79] devised a ML technique for the identification of COVID-19 genomes. The authors developed a decision tree approach, and for performance evaluation, they used publically accessible COVID-19 data with Sarbecovirus and Betacorona virus. Classification accuracy, prediction accuracy, and other evaluation metrics are addressed, and based on experimental studies, the proposed model has a 100% accuracy rate. With the use of an advance ML technique known as CNN, Apostolopoulos, and Mpesiana created a current ML technique for the detecting of COVID-19 (CNNs) [80]. The X-ray Image dataset was used to measure the proposed ML model's accuracy, performance, specificity, and sensitivity, were regarded as performance II ML ARTICLES DESCRIBING THE DATASET, THE AUTHOR'S IDENTITY, THE NATION OF PUBLISHING, THE METHOD USED IN THE ARTICLE, AND THE FINDINGS FOR ANALYZING THE COVID-19 DISEASE TABLE II (CONTINUED.) parameters [81]- [83]. Later, experimental investigations were carried out, and the constructed CNN architecture was found to have a high detection rate. Kang et al. [86] developed a novel COVID-19 automatic analysis pipeline that may entirely change the retrieved features from CT images. The authors developed a structured latent image that may encode data from multiple feature characteristics in order to investigate them by recalling images from various investigations. To obtain matching data for the COVID-19 analysis, the authors used both K-nearest neighbor (KNN) and NB techniques. The proposed model can be used for a variety of classifiers to ensure correctness. The authors achieved a 95% accuracy rate, a 96.6% specificity rate, and a 93.2% sensitivity rate. Cheng et al. [100] developed a ML-based method for predicting COVID patients' ICU transfers. Nursing assessments, laboratory data, electrocardiograms, and time series were used as input types in the authors' random forest (RF) model. The suggested model has demonstrated the importance of shock, inflammation, respiratory failure and renal in the progression of COVID-19. It had 72.8% sensitivity, 76.3% specificity, 79.9% AUC, and 76.2% accuracy, according to the researchers. The researchers stated that the ML based forecasting methods can be used as a testing tool to recognize the scourge of COVID-19 patients and to improve hospital resources by transporting more effective care based on the results of the experiments. Khanday et al. [38] suggested a strategy for detecting pandemics based on clinical text data. Using both ensemble and standard ML algorithms, the authors were able to categorize textual clinical data into four separate classifications. The researchers used 212 clinical records that were classified into four categories: ARDS, SARS, COVID, and both COVID and ARDS. They used different feature to feed the ensemble with features such as report length, bag of words and or inverse document frequency or term frequency, as well as traditional ML classifiers. The author reported that Logistic regression and multinomial Naive Bayes delivered better results with precision, recall, f1score, and accuracy rates of 94%, 96%, 95%, and 96.2%, respectively. Assaf et al. [44] employed ML approaches to accurately estimate COVID-19 risk variables. Different medical center shows of COVID-19 patients were used in the demonstration review. The researchers have only ejected severe covid19 patients at the admission stage because to poor arterial oxygen and oxygen saturation. They compared three different ML approaches for anticipating patient descent to the APACHE-II risk computing score and currently recommended predictors. They looked at 6995 individuals and discovered 162 of them needed to be admitted to the hospital, with 25 of them suffering from severe covid19. The authors found that the predicted models outperformed 92.0% accuracy, 88.0% sensitivity, and 92.7% specificity rates, respectively, based on experimental findings. Table I also presents several other uses of ML for overcoming COVID-19 problems. VI. COVID-19 DATA PROCESSING CHALLENGES USING INTELLIGENT COMPUTING APPROACHES The breakout of the COVID-19 epidemic has had an economic and social effect on billions of people across the world. Because these methods have been widely employed in a different application ranging from bioinformatics to image processing the pandemic has encouraged scientists to employ intelligent computer methods in the detection, prevention, and evaluation of COVID-19. The successful implementation of scientific methodologies requires data, whether closed-source or open-source [77]- [78]. Open-source data deliver outstanding quality, transparency, and versatility. Open-source data are thought to be more relevant for COVID-19 detection in the current COVID-19 epidemic. As a result, diagnosing and forecasting COVID-19 from text data like COVID-19 reports and social media dataset, as well as medical imaging like chest X-rays and computer geographics scans, necessitates a mix of intelligent computation technique and open-source data. While epidemiological studies of COVID-19 case describe can be used to analyze virus transmission forecasts, intelligent computer approaches can defeat the constraints of RT-PCR examine kits in identifying COVID-19 from medical imaging. Policymakers can also benefit from social data mining while conducting socioeconomic research. Although leveraging intelligent computing methods and open source dataset to identify the COVID-19 pandemic provides effective answers, there are significant obstacles to overcome. One of the most pressing issues is improving the quality of medical images for use in clinical practice, as most studies believe that using medical images to diagnose COVID-19 is ineffective. Patients who positive tested for RT-PCR should have a normal chest scan when admitted. As a result, researchers have said that RTPCR should only be used as a primary source of verification and identification, whereas medical imaging should only be used as a secondary diagnosis approach [79]. As a result, demonstrating the association between radiography and RT-PCR testing is a challenge [80]- [81]. The progress of contact fewer work flows to safeguard medical practitioners from COVID-19 influenced individuals is another problem [80]. The majority of the datasets used in the diagnostic are small. As a result, larger datasets are necessary to provide higher observations and accuracy when employing deep learning algorithms. Textual data from online origins may be used to conduct an effective epidemiological analysis of COVID-19. Because most research only looked at a few factors, the analysis of these epidemiological findings is inaccurate. As a result, in order to produce reliable projections, the research analyst must first determine the virus's regional, national, and international spread. Furthermore, considerations such as the deployment of quarantine measures and the distinction between deaths induced by coronavirus and deaths sourced by unidentified disease must be measured when predicting COVID-19 using textual data. A. Critical Analysis ML has become a rapidly expanding mode in healthcare, as it has in other real-world applications. Sensors in wearable components display real-time patients' dataset such as health status, blood sugar level, heart rate, blood pressure, and other critical signs. Health professionals could be used this information to assess each person's health and anticipate the occurrence of disorders in the future. ML approaches are disseminating novel affectivities and chances, making it easier for researchers, physicians, and clinicians to anticipate and understand diseases and advance people's lives. B. Popular ML Methods We discovered that many ML articles were used to resolve COVID-19 disputes out of all the articles included for this study. We analyzed the utilization levels of ML approaches among them based on these ML articles. MLP, KNN, SVM, RF, ANFIS, NB, LR, LIR, and other algorithms were discovered, and their statistics are shown in Fig. 7. ML approaches, such as LR, MLP, NB, SVM, KNN, and RF, were mostly employed for COVID-19, which is the top six among other ML methods, according to the analysis done for Fig. 8. In our opinion, the main motivations for using ML approaches, as shown in Fig. 8, have been recognized and articulated. SVMs have mostly been used to classify features. SVM has also equaled employed for COVID-19 associated concerns because it has delivered an effective accuracy rate in several instances. It is also worth noting that SVM is a powerful binary classifier. ANFIS, a ML approach, was also applied. It has been determined that one of the most compelling reasons to use this strategy is that it effectively combines NN and fuzzy logic. As a result, ANFIS was used to deal with the unclear data from the COVID-19 epidemic. Furthermore, for new COVID-19 data fitting and classification, approaches, such as XGB, NB, AdaBoost, and others, are used. LASSO and RF have also been used to fit COVID-19 associated dataset because they have been shown to be helpful in addressing with fitness functions difficulties. One of the well-known approaches, decision trees, has been predominantly employed for the categorization of COVID-19 genomes because it has proven to be useful in finding modules. It is also been used to keep track of the number of clusters in COVID-19 data to minimize the number of calls. Since it has been proved to be beneficial in data-driven analysis, the K-means technique has been utilized to group heterogeneous elements into COVID-19 clusters. Other ML algorithms, including as LR and vector autoregression, have also been widely used in a variety of applications, including regression analytic of COVID-19 dataset, as well as predicting and forecasting's COVID-19 time series dataset. In a number of studies, multilayer perceptron (MLP), a type of neural network, has been found to be useful in information processing. As a result, this approach has been used to anticipate the COVID-19 outbreak. As a result, we have given essential arguments for the use of each technique revealed in this research effort, in our opinion. C. Different ML and Similar Methods Publication Analysis For searching different keyword such as ("COVID-19", "ML technique for COVID-19") were conducted in the usual online databases "IEEE Xplore, Elsevier, Google Scholar, Springer" in order to extract pertinent research articles for this article. We discovered 1132 publications connected to COVID-19 based on searched keywords. We collect 655 research papers among the 692 papers after excluding those that were unrelated to our review process. Following the exclusion, 81 articles are chosen as being related to the subject. We also acknowledged other resources, which are connected to COVID-19, for instance dataset and the kind of data utilized for COVID-19, in addition to these 81 papers. There was a total of 81 papers used in this article. Because COVID-19 emerged in such a very short duration, most researchers have focused their efforts on COVID-19 detection, identification, and prediction utilizing sophisticated ML algorithms. We discovered that important researchers publish detection-based publications first, followed by prediction based research, from all of the articles we obtained. According to our findings, the top three article categories are 31% for COVID-19 detection, 27% for COVID-19 prediction, and 17% for COVID-19 screening and assessment using intelligent methodologies. The results of this investigation are shown in Fig. 8. Various researchers have conducted and continue to perform studies in order to detect, forecast, predict, and gather data on active, recovered and death causes of COVID-19 approximately the world since the outbreak in January 2020. The majority of those research works, according to our findings in this article, are mostly focused on using ML and mathematical model. Several models have demonstrated their effectiveness in predicting and forecasting the COVID-19 utilizing existing COVID-19 related data. Even if the COVID-19 pandemic is considered as a terrifying condition in its essence, the future is uncertain. Many individuals are undecided because they are unexpected with the spreadable virus, as well as the variegated, diverse, and vivid human deeds, governments engagement, and difficult situations. The situation's final ambiguity has the potential to confound the forecast accuracy aim. VII. DISCUSSIONS AND IMPLICATIONS The COVID-19 epidemic has become a major national security concern in various countries. To get insight into the repercussions and spread of this deadly disease, accurate epidemic prediction models must be developed. This article examines a variety of clever strategies for COVID-19 prognosis. ML approaches have previously demonstrated their efficacy in better understanding viral propagation patterns. Advanced features of ML have enhanced diagnostic speed as well as accuracy, created new efficient therapy improvements, and recognized the large number of affected individuals with physiological attribute [24]. The clinical data and health conditions of COVID-19 patients were studied using ML and other approaches in order to not only recognize any consistent attribute for risk evaluation, but also to classify risk and forecast the balanced trained of present disease dealing and COVID-19 defense. To deal the constantly increasing number of COVID-19 patients around the world, a viable therapeutic technique is urgently required. Because there is no viable treatments for COVID-19, developing an efficient enhancement to create or repurpose a new clinically approved medicine against COVID-19 is becoming increasingly vital. An ML-based relocation structure was modified at the time to prioritize available drugs for COVID-19 medical trials [82]. In addition, used a drug distinguish feature based on ML to aim to bring out novel drug-like compound against COVID-19 [99]. At this time, numerous efforts have been made to improve analytical achievements using ML [83], [84]. A few examples are as follows: Using a CRISPR based virus recognition system, the ML based transmission of SARS-CoV-2 was validated with great sensitivity and speed [54]. Similarly, because COVID-19 emerged in such a short period of time, many researchers have focused their efforts on detecting and predicting COVID-19 using advanced ML techniques such as taxonomic categorization of COVID-19 genomes, among others [79]. While current research has primarily focused on detection and identification, there are many others areas to noticed and investigate encourage. Afterward the beginning of the pandemic, many doctors and researchers throughout the world have refined many strategies for predicting COVID-19 in various places. However, the bulk of strategies fail due to a lack of exact answers capable of predicting the COVID-19. Each model employs a variety of procedures, constructs a variety of claims, generates a variety of outcomes, and combats uncertainty in a variety of ways. The information on COVID-19 trends around the world includes healthcare requirements, recovered, social distancing, pressure, tainted, as well as coming cases, and much more. Furthermore, in an era when technique appropriateness is critical, there was no such recognized liability. The majority of statistic revealed that the methods for estimating the number of deaths case caused by COVID-19 were inconsistent. All predicting approaches have shown a wide range of predicted differences due to a lack of exact information. Similarly, discrepancies in prediction can be explained by the forecast of approaching results based on data from specific cases. Apart from the spread of lethal diseases, social isolation, and demographics, many additional aspects such as disability, chronic sickness, inadequate immunity, and so on must be considered. The main disadvantage for researchers is that they are unable to accurately predict pandemics. As a result, in order to generate more predictions, a new study must be conducted to improve prediction methodologies and tools for the majority of biological data. The COVID-19 epidemic is spreading day by day, despite different procedures, precautions, and research publications. As of July 19, 2021, the total number of confirmed cases was 191 231 992, with 174 185 489 recoveries and 4 105 868 death fatalities worldwide. The majority of cases, particularly in India, are expected to continue to expand as social distancing procedures are calmed before pronouncing a constant rejection in new cases. A. Various Approaches for Assisting in COVID-19 Containment Various forecast approaches, remedies, and precautions adopted by each individual can temporarily halt the pandemic's spread, assisting in COVID-19 containment. No one knows when this epidemic will come to an end. This lethal disease is unrivalled in terms of its ease of transmission, a slew of symptoms that spread from one person to the next in a poisonous manner, and the scale of the epidemic has wracked the world. The pandemic may be brought to a halt because citizens have grown tired of terror and have been taught to live with a sickness, as previous outbreaks have done in the past. Humans have been exposed to a variety of terrible diseases, including Ebola, smallpox, influenza, and many others. However, all of these diseases have been eradicated following different deaths as a result of the implementation of policy measures, quarantines, and the eradication of a few diseases as a result of the development of vaccinations. The strategies used in the early plagues have become rules for a world looking for new ways to update strength and a feeling of normalcy. COVID-19's continuations are expected to be short, but its termination will necessitate a comprehensive agreement that ensures early disease prevention, including new medicines to alleviate symptoms, social protective measures, and a vaccine. Furthermore, different researcher must work on a variety of issues in order to develop drugs. Vaccines are, thus, the long-term approach to stopping this outbreak, which could take months or even years to fully resolve. If the drug is made available all over the world, the pandemic is likely to spread to people who are not now unwell or recovering. The vaccines and natural exemptions will be grouped together to defend the bulk of the populations. So, based on our findings, we can be confident that this space may be organically fielded by detecting drugs utilizing a collection of known illness treatments, such as sars-1, Spanish flu, and others using ML approaches. The importance of ML for COVID-19 has been demonstrated in this article. We have concentrated on a thorough examination of ML approaches for COVID-19, as well as their applications. B. Limitations of ML According to our findings, various limitations of ML, such as a lack of recognizing equipment, the use of biomedical data, heterogeneity, and a lack of medical equipment, rendered ML approaches more potent and suited for predicting the COVID-19 pandemic. ML approaches are suited for COVID-19 prediction because they can aid in the improved identification of infections by health systems. Multiple researches with trustworthy data on increasing the efficaciousness of medical check-ups by anticipating adverse consequences and discovering novel ways to accomplish it have been exceeded by ML technologies. When ML was compared with COVID-19 prediction, the majority of ML attributes such as recognition, lack of human involvement, and superior performance built it a more efficient and admired approaches. This eruption has been labelled a public health emergency by the World Health Organization. Every part of life is affected by technological improvements, and the medical field is one of the most directly linked to people's daily lives [85]. AI has lately been enclosed to the medical area, and it has showed promise in health care because to the high precision of data processing that allows for precise decision-making. By incorporating AI algorithms into the disease's diagnosis, researchers from all over the world aimed to ameliorate clinical diagnosing and slow the virus's spread. This review paper evaluates the outcomes of a variety of AI algorithms used in research to determine, which method is the most accurate and enhances COVID-19 diagnosis the most. There were 37 original articles used in this article, all of which use supervised learning as the major approach, albeit the methods used dissented depending on the research goal. More than half of the study participants reported coronavirus disease-related felt stress, demonstrating a strong association between health-care workers and COVID-19-related perceived stress [50]. VIII. LIMITATIONS AND FUTURE SCOPE This article examines the use of ML methods to COVID-19. The primary goal of this survey was to examine the global impact of this epidemic, as well as the motivation for using ML approaches to COVID-19. We went through all of the ML models used in COVID-19, as well as the effective incentives. Likewise, aside from prediction and identifications, the need for more study in other areas was also highlighted previously. Based on the findings of this article, we highly suggest that many more studies be done using ML on COVID-19 data using approaches that have not previously been deployed. In order to undertake further advanced research, it is also necessary to solve the issues of COVID-19 data scarcity. COVID-19 has a significant influence on a number of industries. More than 200 nations have been recognized as having COVID-19 instances. We mainly focused on the most pressing issues present in the literatures as well as potential study paths for future research. 1) Because the evidences of COVID-19, disease and other respiratoria illnesses are so standardized, establishing an appropriate DL model that can accurately identify COVID-19 remains a problem [86]. 2) A key difficulty for COVID-19 is the lack of a highquality dataset. This can be attributed to a variety of factors, considering such as 1) different sources and unpublished data; 2) the dispersed cause of COVID-19 data; and 3) data secrecy concerns that limitation of dataset availability [87]. To extend the available different datasets and speed AI research for COVID-19, collaboration across all medical organizations around the world is critical. 3) Variability in the testing procedure between nations and hospitals is a major problem that might result in nonuniformness in the labeling operations. 4) The COVID-19 infectious is quickly mutating in dissimilar parts of the world. As a result, data obtained in one location may not be adequate for inferring interferences in another. [88]. 5) Medical personnel are the first line of defense in the fight against the epidemic. To protect children from infections, additional contact-free screening, and diagnosis methods are urgently needed. 6) The majority of current deep learning models were trained on two-dimensional (2-D) pictures. However, because the majority of CT and MRI scan pictures are 3-D, appending another attribute is necessary to maximize the effect of the image [89], [90]. 7) As a result of the similar operations of combining medical imaging dataset, the data diversity grows, necessitating the requirement to verify the resilience of ML produced models. 8) The majority of the COVID-19 datasets accessible are small. As a result, transfer discovering is a promising future research path which might aid in the detection of anomalies in tiny datasets while also producing reliable predictions and impressive outcomes [91]. 9) According to the research, there appears to be a link among COVID-19 transmission and various medicals problem. As a result, a patient's record of various diseases (kidney, diabetes, heart disease, liver, etc.) can be considered into account in both the COVID-19 forecasting and detecting processes in order to offer a precise and reliable prediction model [61], [92], [93]. 10) When equated to exploiting with IoT device, building complicated ML models, analyzing, and interpreting large data requires a lot of computing power. As a result, fog and edge computing may be useful in addressing these problems [94]. 11) To improve the interpretation of data gathered from various sensors, several preprocessing processes are necessary (i.e., quality improvement, data cleaning, outlier detection, etc.) [95], [96], [97]. 12) Recently NLP applications have restricted the value of a diagnostic scheme like this. To transfer performance to a given domain context, working with methods that assess semantic textual similarity is necessary (i.e., COVID-19) [98]. 13) Because it combines diverse data, data fusion is difficult 1 . It does, however, increase the functioning of the models that arise. In the article, there are several fusion procedures. As a result, adaptive multimodels are critical for handling data from numerous sensors [99]. 14) To improve the functioning of working on sound data and X-ray, more complex approaches are required. 15) The explain ability and interpretability of ML approaches is a major problem. Medical professionals must understand, which characteristics are used to identify COVID-19 from non-COVID-19. 2 ML should also look at methods to predict infections before symptoms develop. In COVID-19 prediction, diagnosis, screening, and other different ML methodology have showed promising results. However, the majority of these models have not been tested in a real-world setting (e.g., hospitals, emerging service) to demonstrate their efficacy in combating the COVID-19 epidemic. As a result, many challenges must be overcome in order to spread such diagnosing models, include 1) network security consistence in order to allow more authentic communications and desired data on the networks; 2) fog, edge and cloud computing adaptation; and 3) privacy and security issues relating to patient's dataset. ML is a cutting-edge method with several applications in prediction. For the COVID-19 pandemic, this method will be used to detect high-risk individuals, as well as their mortality rate and other anomalies. It can be used to better understand the virus's nature and forecast future problems. IX. CONCLUSION In this article, we mainly focused on the use of different ML applications in COVID-19 disease for different purposes using various algorithms. Based on the findings of this article, it appears that ML have been utilized mostly for COVID-19 forecasting, detection, identification, and screening from December 2019 to the present. ML methods such as LR, RF, KNN, and SVM have been employed to solve COVID-19 difficulties. However, complex ML algorithms such as encouraging, bagging, stacking, and others must be included. Other ML approaches, for example Bayesian networks such as decision tree algorithms such as C4.0, Gaussian Naive Bayes and different clustering algorithm like K-Means, Hierarchical, and K-Medians, must also be used with COVID-19 dataset for successfully classified and detecting the dissimilar symptom. It is also worth noting that ANNs like back propagation, perceptron, and radial basis function based NN must be used with COVID-19 data. CNN and its derivatives have been effectively used to resolve COVID-19 concerns in the instance of DL. This article examines the methodology of ML algorithms to COVID-19. The primary goal of this survey is to examine the global effect of this pandemic, as well as the motivation for enforcing ML techniques to COVID-19. We went over all of the ML models used in COVID-19, as well as the effective incentives. Similarly, aside from prediction and identifications, the need for more research in other areas was also emphasized above. Based on the findings of the survey, we highly suggest that many more studies be undertaken using ML on COVID-19 data using methodologies that have not yet been deployed. In order to perform further advanced research, it is also necessary to overcome the difficulties of COVID-19 data scarcity.	2022-01-13T16:28:42.495Z	2022-01-11T00:00:00.000	{ "year": 2022, "sha1": "9a787a27a9e36d91f71f35ecc65148c8efe4fad7", "oa_license": null, "oa_url": "https://ieeexplore.ieee.org/ielx7/9078688/9184921/09677942.pdf", "oa_status": "BRONZE", "pdf_src": "IEEE", "pdf_hash": "512b778809401db9e9f2fffedbe788225361224d", "s2fieldsofstudy": [ "Computer Science", "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
9549858	pes2o/s2orc	v3-fos-license	The effect of subconjunctival bevacizumab on corneal neovascularization, inflammation and re-epithelization in a rabbit model PURPOSE: To evaluate the use of subconjunctival bevacizumab on corneal neovascularization in an experimental rabbit model for its effect on vessel extension, inflammation, and corneal epithelialization. METHODS: In this prospective, randomized, blinded, experimental study, 20 rabbits were submitted to a chemical trauma with sodium hydroxide and subsequently divided into two groups. The experimental group received a subconjunctival injection of bevacizumab (0.15 m; 3.75 mg), and the control group received an injection of 0.15 ml saline solution. After 14 days, two blinded digital photograph analyses were conducted to evaluate the inflammation/diameter of the vessels according to pre-established criteria. A histopathological analysis of the cornea evaluated the state of the epithelium and the number of polymorphonuclear cells. RESULTS: A concordance analysis using Kappa's statistic showed a satisfactory level of agreement between the two blinded digital photography analyses. The neovascular vessel length was greater in the control group (p<0.01) than in the study group. However, the histopathological examination revealed no statistically significant differences between the groups in terms of the state of the epithelium and the number of polymorphonuclear cells. CONCLUSIONS: Subconjunctival bevacizumab inhibited neovascularization in the rabbit cornea. However, this drug was not effective at reducing inflammation. The drug did not induce persistent corneal epithelial defects. INTRODUCTION Ocular trauma, infection, inflammation, and degeneration result in corneal neovascularization. 1 Neovessels cause structural changes that allow the overflow of fluid to the extravasculature, blood stasis and hemorrhage, and they can reduce corneal transparency with subsequent and progressive vision impairment. 1 Corneal neovascularization is one of the greatest risk factors for corneal transplant rejection 2 because it allows leukocytes access to donor tissue antigens. Corticosteroids are the first-line treatment for corneal neovascular diseases because of their ability to reduce the inflammatory process 4 and vascular proliferation, both of which are initiated soon after the ocular trauma. 5 However, side effects related to the non-specificity of corticosteroids limits their use. Such side-effects include the increased risk of cataracts and glaucoma due to high intra-ocular pressure (IOP). 6 Vascular endothelial growth factor (VEGF) and its receptors play an important role in the neovessel formation that is observed in diabetic retinopathy, venous retinal occlusion, age-related macular degeneration, and corneal neovascularization. 7 High VEGF expression was observed in neovascularized corneas after penetrating keratoplasty in corneal inflammatory diseases 8 and in guinea pigs' corneas that were burned by alkalis during the healing process. 9 Anti-VEGF drugs have sparked a revolution in the treatment of neovascular diseases by reducing neovascularization and also by their supposed action on fibroblasts. 10 These drugs can provide beneficial effects after intra-vitreous injection in age-related macular degeneration (ARMD) neovascularization, diabetic retinopathy, and glaucoma, with minimal toxicity or side effects. 11 These effects may also include the reduced formation of new vessels in other regions of the eye. The aim of this prospective study was to investigate the effects of subconjunctival injections of bevacizumab on experimentally induced corneal neovascularization by focusing on the neovessel length, inflammation, and reepithelization. MATERIALS AND METHODS This prospective, randomized, blinded study was performed at the Instituto de Pesquisas Médicas (IPEM) of the Faculdade Evangélica do Paraná (FEPAR) -Brazil and Hospital Universitário Evangélico de Curitiba (HUEC). The Animal Experimentation Norms and Principles proposed by the Colégio Brasileiro de Experimentaçã o Animal (1994) were followed. The studied variables include the vessels' lenght, degree of inflammation/diameter, epithelium integrity, and number of polymorphonuclear cells (PMN). Intervention Twenty corneas of twenty New Zealand rabbits were studied. All rabbits were healthy male albinos, weighing between 2.300 and 2.500 kg and were three to four months old. The left corneas of the animals were exposed to 1 N sodium hydroxide (NaOH), through a 5 mm diameter filter paper that was applied to the superior cornea tangential of the limbus for 20 seconds, followed by washing with 20 ml of 0.9% saline solution. Subsequently, the control group animals received a subconjunctival injection of 0.15 ml of 0.9% saline solution, whereas the study group animals received a subconjunctival injection with 0.15 ml (3.75 mg) of bevacizumab ( Figure 1). The injections were performed superiorly and close to the burned area. One surgeon performed all of the procedures in a blinded fashion. The rabbits in Groups 1 and 2 were again anesthetized, examined and digitally photographed 14 days after the corneal burn and were euthanized by a 5-ml intramuscular sodium pentobarbital injection. Subsequently the corneas were removed, maintaining a scleral margin of 3 mm, fixed in formalin for 24 hours and submitted for histopathological study with hematoxylin-eosin. The corneal epithelium integrity, or lack thereof, and the number of PMN cells per field were observed at a magnification of 400x using an optical microscope. The most damaged field in the opacity area was utilized. Digital images were obtained to examine the vessel extent and the vessel inflammation/diameter in the frontal position using a Sony CybershotH (Sony Corporation -Tokyo/Japan) 7.2-megapixel camera, which was supported by a fixed tripod with an accessory ZeissH (Carl Zeiss AG -Oberkochen/Germany) lens with a magnification of 40x at a distance of 5 cm. Each animal was submitted to two digital photography evaluations for each variable (vessel extent and vessel inflammation/diameter). A cornea specialist performed the two evaluations of the digital pictures in a blinded and randomized fashion. The corneal neovascularization was determined using a score given by the cornea specialist according to the vessel extent and inflammation/diameter degree observed. The vessel extent was determined using a scale from 0 to 4 (0 -no vessels on the corneal limbus; 1 -vessels that advanced over the corneal limbus, covering 0-25% of the burned area; 2 -vessels that reached 25-50% of the burned area; 3 -vessels that reached 50-75% of the burned area; 4 -vessels that extended to the entire burned area). The inflammation and diameter degree on the vessels were determined using a scale from 0 to 3: (0 -no inflammation or vessels; 1 -little inflammation and vessels of small diameter; 2 -moderate inflammation and vessels of medium diameter; 3 -intense inflammation and vessels of large diameter). Statistical Analysis To validate the evaluation model, the reproducibility of these measurements was evaluated using the Kappa statistic. For each evaluation, the two groups were compared with respect to the ordinal variables by using the Mann-Whitney non-parametrical test. The group comparison for dichotomous variables was executed using the Fisher exact test. p,0.05 indicated statistical significance. RESULTS On the seventh day after the corneal burn, a rabbit from the study group presented corneal perforation and was excluded from the research. Thus, 19 animals were left in the study, including ten from the control group and nine from the study group. The digital pictures of each animal were submitted to two evaluations for each variable, as shown in Table 1 (control group) and Table 2 (study group). Animal 8 of the control group ( Figure 1) had a cornea classified as degree 3 in both evaluations (vessels reaching up to 75% of the burned area) according to the vessel extent criteria and inflammatory reaction (intense inflammation and vessels of large diameter). Animal 5 of the study group ( Figure 2) had a cornea classified as degree 1 for both vessel extent criteria (vessels advancing over the corneal limbus, reaching up to 25% of the burned area) and inflammatory reaction (little inflammation and small-diameter vessels). The concordance analysis of the vessel extent estimated by the Kappa coefficient was 0.705 (95% confidence interval: 0.514 -0.895), which indicates a high level of agreement between the two evaluations. The estimated Kappa coefficients for each of the vessel extent classifications are presented in Table 3. The concordance was better characterized at the most extreme degrees (1 and 4). Regarding the vessel inflammation/diameter, the estimated Kappa coefficient between the two blinded evaluations was 0.500 (95% confidence interval: 0.269 -0.731), which again indicates a satisfactory level of agreement between the two evaluations. The estimated Kappa coefficients for each classification of vessel diameter are presented in Table 4. Although a satisfactory level of reproducibility was found in the evaluations of large diameter vessels, the concordance seemed to be weak at Degrees 1 and 2, indicating a difficulty in distinguishing between little and moderate inflammation. The comparison among the study and control groups considered the average of the two evaluations. The null hypothesis of the same results in both groups was tested versus the alternative hypothesis of different values for the considered variable. The analysis of the vessel extent showed a significant difference between the control and the study group, with smaller vessels being found 14 days after the chemical burn in the study group (Table 5). Nevertheless, no meaningful difference was found among the same groups during the same follow-up when evaluating the vessel inflammation and diameter ( Table 6). The microscopic evaluation of the corneal epithelial integrity revealed that the epithelium of all corneas from the study group had healed at 14 days compared with 60% in the control group (Table 7). Despite the reduced number of corneas that had epithelized in the control rabbits, no statistically significant difference was found between the groups. A lower number of polymorphonuclear cells was found in the treated group compared with the control group. Figure 3 shows the polymorphonuclear infiltration in Animal 1 of the control group, whereas Figure 4 shows Animal 3 of study group, which displayed little polymorphonuclear infiltration and full corneal epithelium integrity. In spite of the discrepancies among some rabbits (Figures 3 and 4) and an overall lower number of PMNs being found in the study group, no meaningful statistical differences among the groups were encountered in terms of PMN cell number ( Figure 5). DISCUSSION Previous studies used subconjunctivally administered bevacizumab at dosages varying from 1.25 mg to 3.75 mg. 6,12,13 In this study, we chose a dose of 3.75 mg. Because we were only administering one injection during the study, we wanted a high dose that would suppress the initial angiogenic stimulus. No drug-related toxicity has been reported using this concentration. Hurmeric et al. 13 compared the effect of injected bevacizumab when it was 1 2 3 1 1 2 3 3 2 2 3 2 2 2 1 4 2 3 1 1 5 3 3 2 2 6 3 3 2 2 7 4 4 1 1 8 3 3 3 3 9 3 3 3 used immediately after the injury or at three days after the injury. Better anti-angiogenic responses were found in the group treated soon after the induced damage. Thus, we also administered the subconjunctival injection soon after the injury. Vascular growth, which is mainly analyzed using the angiogenesis parameters of vessel extent and degree of inflammation, is usually determined through photographic analysis. 12,6 However, no established model for this analysis was found. Thus, an experimental model of digital photographic analysis was established, which allowed the vessel extent and inflammation/diameter to be evaluated within the indicated time period. Other photographic evaluation methods have been applied with pixel counting or measurements. Although these methods give the impression of a mathematical certainty as they appear quantitative, the data are subjective with regard to where the vessels begin and end. In addition, the delineation of the affected area may not be accurate when it is determined mathematically. Thus, we have proposed a photographic clinical analysis, in which the evaluator, who is a cornea specialist, observes not only the vessel extent but also the vessel aspect and inflammation degree. We chose to evaluate the corneas at 14 days because angiogenesis has shown a rapid growth until the 12 th day in other models. 14 In the previous studies, the vessel extent stabilized after this initial period. Fourteen days was sufficient to produce the highest level for each variable (vessel extent and inflammation). On the seventh day, a rabbit from the study group showed corneal perforation and was excluded from the study. As only one rabbit showed perforation and the remaining rabbits in the group showed an intact epithelium by the end of the 14-day period, the perforation was unlikely to be directly caused by the drug. The rabbit may have had a bacterial infection in the wound that was acquired early in the experiment when the corneal epithelium was not fully healed. Statistical analysis confirmed that the evaluation method was excellent for identifying vessel extension at Degrees 1 and 4 and good for identifying vessel extension at Degrees 2 and 3. The method was also good for identifying vessel inflammation/diameter, as indicated by the good agreement between the two blinded evaluations. For this variable, Degree 3 (intense inflammation and vessels of large diameter) showed excellent agreement. The results obtained show that this method may be reproduced for the evaluation of other anti-angiogenic drugs intended to reduce corneal neovascularization. There was a statistically significant difference in the vessel extent between the two groups, with a lesser extent in the study group compared with the control group. These data indicate a positive effect on the chemical burn and agree with the data found in recent literature. 12,13 Neovascularization is considered to be related to a poor prognosis in corneal transplants because it interferes with corneal transparency, which can result in low visual acuity. 1 Thus, bevacizumab may improve the prognosis in cases with progressive neovessels after penetrant keratoplasty or in the case of some chemical burns, especially those that require a corneal transplant. The degree of inflammation was evaluated because although the drug does not possess direct anti-inflammatory actions, it does affect fibroblasts. 15,16 Although its action in acute inflammation is not fully understood, some authors suggest that the drug decreases neutrophil invasion within the wound. 17 The results for inflammation/diameter and the number of PMN cells showed that the drug does not have a clinically meaningful anti-inflammatory effect, which agrees with the theory that the inflammatory process has many pathways and the inhibition of VEGF alone does not significantly affect inflammation. Through these data, we suggest that this treatment may be more effective when administered along with corticosteroids, which would reduce the inflammation. Further studies are necessary to determine whether concomitant inhibition of inflammation and neovascularization leads to massive cellular death by acute hypoxia, which may occur because of the inhibition of all physiological mechanisms involved in such ischemia. The integrity of the corneal epithelium was evaluated on the 14 th day because the drug may have affected the healing process and delayed wound healing. Rapid restoration of the epithelium is fundamental to the healing process and prevents wound contamination. The comparison between the two groups did not show persistent epithelial defects in the study group at the end of the follow-up. The results showed fewer corneas with an exposed stroma in the study group compared with the control group. However, no statistical difference was observed between the groups, which suggests that the drug did not impair epithelial healing. This study suggests that the bevacizumab has potential for use during the acute healing stage, in which the epithelium is still open. Corticosteroids should be used carefully during this stage because they may impair epithelial healing. 18 Delayed epithelial healing has been described previously 19,20 and is believed to be caused by the reduced expression of surface integrins and collagens, which interferes with epithelial cell adhesion. However, these studies were performed with higher doses that were applied topically several times a day. Kim et al. showed a doserelated effect. 20 Thus, we suggest that the subconjunctival administration is safer because in our study, it did not jeopardize the re-epithelialization. Chalam et al. 21 showed that bevacizumab is not toxic to corneal cells or fibroblasts when administered in concentrations up to 25 mg/ml. This finding is supported by the current study, in which there was no inhibition of re-epithelialization. Although the neovascularization induced by the burn was suppressed, the suppression was not as complete as has been described in previous studies. 12 This is possibly because VEGF is not the only factor responsible for angiogenesis. Other pro-angiogenic factors, such as TGFbeta and fibroblast growth factor, may still be involved. 16 We suggest that bevacizumab should not to be used as a monotherapy; instead, it should serve as one aspect of a multi-factorial treatment alongside other drugs, such as corticosteroids, in acute cases. This association has not been fully studied, and some conflicting results have been reported. Using a rabbit model, Kang et al. 22 showed that a combined treatment of bevacizumab and triamcinolone acetonide was no more effective than the use of bevacizumab alone. Another study 23 examined the inhibition of neovascularization in a rat model using bevacizumab alone and with dexamethasone. However, again there was no clear advantage when dexamethasone was included. Newer anti-angiogenic drugs have also been studied over the last year with promising results. For example, suramin is a non-specific anti-angiogenic drug that has a longer half-life than bevacizumb. It has been suggested that this association could provide a more potent and longer effect. 24 Pérez-Santonja et al. 25 showed a greater inhibition of the neovessels by sunitinib (an anti-VEGF and anti-PDGF drug) compared with bevacizumab. It has been found that established neovessels, such as those in diabetic retinopathy, are re-established after the drug is discontinued, thereby requiring additional treatments. 26 The drug's effect has not been extensively studied in cases of acute neovascularization, such as that following burns or the initial neovessel growth following a corneal transplant. It is also possible that if the angiogenic stimulus is reduced by specific inhibitors such as bevacizumab in the acute stage, neovessel formation may decrease in the long-term because the angiogenic stimulus has a short duration and is not maintained by the injury's physiopathology as it is in diseases such as diabetic retinopathy. Thus, this treatment could have a positive effect in the long-term for some treatments such as subsequent corneal transplants. Anti-angiogenic drugs have a great potential for beneficial effects in severely neovascularized eyes with little or no limbal damage, which generally present with very low visual acuity. Presently, there is no available treatment for this condition. Further studies with different dosages and steroids combinations with the various VEGF inhibitors that compare not only the neovascularization itself but also the inflammatory response and side effects would contribute to the search for an ideal treatment. This association may have a synergistic effect on burns or in cases with a possibility of acute neovascularization, where the neovascular stimulus occurs as a part of the acute inflammatory response. Thus, we conclude that the subconjunctival administration of bevacizumab efficiently reduces corneal neovascularization without inducing persistent epithelial defects following a chemical burn. However, this treatment does not show meaningful anti-inflammatory effects.	2014-10-01T00:00:00.000Z	2011-08-01T00:00:00.000	{ "year": 2011, "sha1": "53f37223d74b4a6aaa7f9bd99c0e7e4fb5bc2c94", "oa_license": "CCBYNC", "oa_url": "http://www.scielo.br/pdf/clin/v66n8/23.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "53f37223d74b4a6aaa7f9bd99c0e7e4fb5bc2c94", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
260113850	pes2o/s2orc	v3-fos-license	Alterations in nasal microbiota of patients with amyotrophic lateral sclerosis Abstract Background: Links between alterations in gut microbiota composition and amyotrophic lateral sclerosis (ALS) have previously been reported. This study aimed to examine the microbiota in the nasal cavity of ALS. Methods: Sixty-six ALS patients and 40 healthy caregivers who live in close proximity with patients were enrolled. High throughput metagenomic sequencing of the 16S ribosomal deoxyribonucleic acid (rDNA) gene V3–V4 region of nasal microbiota was used to characterize the alpha and beta diversity and relative abundance of bacterial taxa, predict function, and conduct correlation analysis between specific taxa and clinical features. Results: The nasal microbiome of ALS patients showed lower alpha diversity than that of corresponding healthy family members. Genera Gaiella, Sphingomonas, Polaribacter_1, Lachnospiraceae_NK4A136_group, Klebsiella, and Alistipes were differentially enriched in ALS patients compared to controls. Nasal microbiota composition in ALS patients significantly differed from that in healthy subjects (unweighted UniFrac P = 0.001), while Linear discriminant analysis Effect Size (LEfSe) analysis indicated that Bacteroidetes and Firmicutes dominated healthy nasal communities at the phylum level, whereas Actinobacteria was the predominant phylum and Thermoleophilia was the predominant class in ALS patients. Genus Faecalibacterium and Alistipes were positively correlated with ALS functional rating scale revised (ALSFRS-R; rs = 0.349, P = 0.020 and rs = 0.393, P = 0.008), while Prevotella-9 and Bacteroides operational taxonomic units (OTUs) were positively associated with lung function (FVC) in ALS patients (rs = 0.304, P = 0.045, and rs = 0.300, P = 0.048, respectively). Prevotella-1 was positively correlated with white blood cell counts (WBC, rs = 0.347, P = 0.021), neutrophil percentage (Neu%, rs = 0.428, P = 0.004), and neutrophil-to-lymphocyte ratio (NLR, rs = 0.411, P = 0.006), but negatively correlated with lymphocyte percentage (Lym%, rs = –0.408, P = 0.006). In contrast, Streptococcus was negatively associated with Neu% (rs = –0.445, P = 0.003) and NLR (rs = –0.436, P = 0.003), while positively associated with Lym% (rs = 0.437, P = 0.003). No significant differences in nasal microbiota richness and evenness were detected among the severe and mild ALS patients. Conclusions: ALS is accompanied by altered nasal microbial community composition and diversity. The findings presented here highlight the need to understand how dysbiosis of nasal microbiota may contribute to the development of ALS. Introduction Amyotrophic lateral sclerosis (ALS), commonly called Lou Gehrig's disease or motor neuron disease, is a severe neurodegenerative illness characterized by degeneration of the motor neurons.ALS patients present with muscle weakness and progressive paralysis, with survival time averaging 3-5 years after the onset of symptoms. [1,2]Although the pathogenesis and underlying genetic or environmental factors in ALS are not well-characterized, a growing body of evidence supports a role for neuroinflammation in driving ALS development. [3,4]][8][9][10] Gut microbiota can influence distant organs by affecting host systemic immune response and inflammation, by translocation, or through metabolic products. [11,12]15] However, the vast majority of studies examining potential associations between microbiota and neurological diseases continue to focus on the gut-brain axis and largely overlook possible complex and profound relationships between nasal microbiota and the nervous system via the nose-to-brain pathway. [16,17]e nasal cavity serves as one of the major access points for bacteria entering the human body, and, as a host-microbe interface, the composition of nasal microbiota, which is shaped by host genetics and environmental exposure, can affect the mucosal immunological tone. [18][21] Other work has also shown that the onset, progression and severity of neurodegenerative diseases are affected by nasal microbiota composition and function. [16,17]Indeed, unknown infectious or otherwise transmissible agent can potentially colonize the nasal cavity and eventually enter the brain, subsequently triggering an ascending pathological process that may spread to other regions of the central nervous system (CNS). [22] particular, complex interactions between nasal microbiota and the brain can induce pathogenic effects and affect the treatment response for some neurological diseases.However, direct associations between nasal microbiota and ALS have not been investigated.We thus hypothesized that ALS patients may have alterations in nasal microbiota that might affect its progression.In this study, we use 16S ribosomal deoxyribonucleic acid (rDNA) gene sequencing to investigate the composition of nasal microbiota in the context of ALS patients. Subjects The ALS patient cohort was recruited from a registered study of ALS (www.clinicaltrials.gov, No. NCT04008329) conducted in patients admitted to the Department of Neurology, First Affiliated Hospital of Fujian Medical University.All patients met the diagnostic criteria of ALS according to the revised Escorial criteria. [23]etailed clinical profiles were recorded, including demographic characteristics, clinical manifestations, history of smoking, history of diabetes mellitus, and laboratory test results.The control group included 40 healthy caregivers (the spouses of the ALS patients) who lived in close proximity with the patients, aiming to reduce the potential impact of diet, daily schedule, pollution exposure, and other related factors.Study subjects were excluded if they had a known history of human immunodeficiency virus (HIV) infection, primary immunodeficiency, systemic inflammatory disorder, or history of intranasal drug administration, including antibiotics, immune suppressants, or probiotics within the prior 3 years, and oral administration or infusion of antibiotics in the prior 2 months.This study was approved by the Ethics Committee of the First Affiliated Hospital of Fujian Medical University and conformed to standards in the Helsinki Declaration (No. MRCTA, ECFAH of FMU [2019]191).Written informed consent was given by all study subjects before enrollment. Nasal swab Nasal mucosa was sampled by swabbing the middle meatus with a sterile flocked specimen collection swab (Copan Diagnostics, Brescia, Italy).All samples were transferred to the laboratory and stored at -80°C. DNA extraction and amplification The same procedures for DNA extraction and polymerase chain reaction (PCR) amplification were used for all samples and performed by the same laboratory staff.Microbial DNA was extracted from samples using a DNeasy PowerSoil kit (Qiagen, Hilden, Germany), according to the manufacturer's protocols.DNA concentration and integrity were measured by a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively.The DNA extracted from each sample was used as template to amplify the V3-V4 region of 16S rDNA genes.The 343F (5΄-TACGGRAGGCAGCAG-3΄) and 798R (5΄ -AGGGTATCTAATCCT-3΄) primers were used to amplify the V3-V4 region of each nasal swab. Library construction and sequencing Amplicon quality was visualized using gel electrophoresis, and PCR products were purified with Agencourt AMPure XP beads (Beckman Coulter Co., CA, USA) and quantified using a Qubit dsDNA assay kit (Invitrogen Ltd., Thermo Fisher Scientific, UK).Concentrations were then adjusted for sequencing and 250 bp paired-end reads were generated by sequencing on an Illumina NovaSeq6000 (Illumina Inc., San Diego, CA, USA; OE Biotech Company, Shanghai, China). Bioinformatics analysis Raw sequencing data were obtained in the FASTQ format.Paired-end reads were then preprocessed using Cutadapt software (http://code.google.com/p/cutadapt/) to detect and cut off adapter sequences.After trimming, paired-end reads were filtered to remove low quality sequences, denoised, merged, and removed chimeric reads by DADA2 using the default parameters of QIIME2 (2020.11). [24,25]Representative reads and amplicon sequence variant (ASV) abundance table output files were selected and analyzed using the QIIME 2 package.All representative reads were annotated and aligned against the Silva database version 138 (or Unite) (16s/ 18s/ITS rDNA) using q2-feature-classifier with the default parameters.The microbial diversity in nasal swab samples was estimated with alpha diversity indices including the Chao1, Simpson, and Shannon indexes.The UniFrac distance matrix was generated with QIIME software (http://qiime.org) and used for unweighted UniFrac principal coordinates analysis (PCoA) and phylogenetic tree construction.Linear discriminant analysis (LDA) effect size (LEfSe) was used to identify differentially abundant taxa among groups.Receiver operating characteristic (ROC) curve analysis was performed and area under the curve (AUC) values ascertained using Prism software (GraphPad Prism v6.0, GraphPad Software, Inc.San Diego, CA, USA).Functional prediction and enrichment analyses were conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.Correlations were calculated with the Spearman coefficient (r s ).P values less than 0.05 were considered statistically significant. Clinical characteristics of the study population A total of 66 ALS cases and 40 caregivers were enrolled for analysis of nasal microbiota composition and diversity.Demographic and clinical features are shown in Table 1.Three patients (3/66, 4.5%) had a positive family history of ALS, and the remaining 63 cases were sporadic.Based on the site of onset, 14 cases were bulbar-onset, and 52 cases were spinal-onset.The median ALS functional rating scale revised (ALSFRS-R) score was 35 (interquartile range [IQR, Q1-Q3]: 27-41).There were five patients who accepted noninvasive positive pressure ventilation (NIPPV) support.No significant differences in age, gender, and history of smoking and diabetes were found between ALS patients and healthy caregivers (P >0.05).Among ALS patients, 15 were classified as later stage ALS, and were defined as the severe ALS group.The remaining 51 patients were defined as the mild ALS group.The median ALSFRS-R scores for the severe and mild groups were 15 (IQR: 12-17) and 37 (IQR: 32-42), respectively (Mann-Whitney test, P <0.001). Nasal microbial diversity in ALS patients Among the 106 samples, a total of 38,625 nasal microbial operational taxonomic units (OTUs) were detected, while 15,245 OTUs were specific to the ALS group and 7541 were exclusively detected in the control group [Supplementary Figure 1,http://links.lww.com/CM9/B560]. Rarefaction curves of Chao1 index showed that the ALS cases had higher values at the same sequencing depth, indicating richer microbial diversity in ALS patients.Additionally, curves tended to plateau at higher sequence sampling depths, which indicated that the sequencing depth was sufficient to capture the diversity in these samples [Supplementary Figure 2, http://links.lww.com/CM9/B560].Calculation of alpha diversity indices showed significant differences in microbial richness and evenness between nasal microbiota of ALS patients and healthy controls (HC), as indicated by the Shannon index (P = 0.030) and Simpson index (P = 0.024), while no significant difference was found in the Chao1 index (P = 0.072) [Figure 1A-C].Unweighted UniFrac-PCoA analysis of beta diversity revealed distinct clustering of some control samples, thus confirming the differences between ALS cases and controls (Figure 1D; P = 0.001). We further explored differences in the community structure of nasal microbiota among severe and mild ALS patients.However, no significant differences in microbial richness or evenness were detected among the severe and mild groups, as analyzed by the Chao1 index (P = 0.880, Figure 2A), Shannon index (P = 0.340, Figure 2B), Simpson index (P = 0.052, Figure 2C), and unweighted UniFrac-PCoA (P = 0.559, Figure 2D). Structure of the nasal microbial community in patients with ALS To explore the composition of nasal microbiota in ALS patients, we generated a heat map of genus-level relative abundance of taxa [Figure 3A], which revealed the 10 most abundant genera [Figure 3B].Among these genera, Lactobacillus, Sphingomonas, Klebsiella, Gaiella, and Lachnoclostridium were the most abundant in ALS patients, while Lachnospiraceae_NK4A136_group, Alistipes, Polaribacter_1, Pelomonas, and Helicobacter were more abundant in HC. Subsequent LEfSe analysis indicated that, at the genus level, Polaribacter_1 was significantly more abundant in HC, while at the phylum level, Bacteroidetes and Firmicutes were over-represented, and Clostridiales was significantly enriched at the order level in the healthy control samples [Figure 3C,D].In contrast, Actinobacteria and Thermoleophilia were significantly more abundant in ALS samples than controls at the phylum and class levels, respectively [Figure 3C,D]. Function prediction and enrichment analysis To gain further insights into possible differences in functional contributions of the nasal microbiota between ALS and control subjects, we conducted functional profiling of the metagenomic data to identify enriched pathways in each group.This analysis identified 41 represented Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology (KO) modules, including 38 increasing and 3 decreasing modules.Differential genera were related to KEGG pathways involved in glycan biosynthesis and metabolism, circulatory system, and immune system in the control group, whereas enriched pathways were involved in translation, nucleotide metabolism, environmental adaptation, and replication and repair, etc. in the ALS group [Figure 5]. Discussion Our study revealed that ALS patient samples, without subdividing by severity, differed from HC, suggesting possible dysbiosis of nasal microbiota of ALS patients.The finding that specific taxa significantly differed in abundance between ALS patients and HCs further suggested that some differentially enriched bacteria could serve as signature taxa for ALS. [28][29] Numerous microorganisms surviving in the nasal cavity can perform beneficial functions in maintaining a healthy mucosal environment and preventing infection and inflammation.[30] We observed a significant increase in diversity among ALS patients, possibly due to increased exposure to hazardous airborne substances.The finding of higher species richness in ALS patient nasal microbiota aligned well with the findings in gut microbiota. [8,10]While some studies purport that higher microbial diversity is a feature associated with health, our findings of higher diversity in ALS samples suggest the need for experimental validation of the effects of diversity. The affected nasal microbes may trigger dysbiosis, and according to previous reports, several neurological diseases, such as AD and stroke, are strongly associated with alterations in the nasal microbiota. [16,17]PD patients are known to harbor dysbiosis of microbial communities in the deep nasal sinus cavity, characterized by the loss of potentially beneficial bacteria concomitant with increased abundance of potentially pro-inflammatory bacteria. [31]Seki et al [32] found that Klebsiella overgrowth in the gut is highly predictive of brain damage and is associated with a pro-inflammatory immunological response.Klebsiella is also positively correlated with clinical manifestations in PD. [33] Actinobacteria are considered important commensal bacteria that survive in the nasal cavity of healthy individuals. [34]However, recent work also implicates nasal Actinobacteria in PD, [35] and Actinobacterial resting spores can serve as infectious agents, infiltrating the olfactory epithelium through the nasal cavity, where they are then mobilized via retrograde axonal transport to associated cerebral nuclei of the nose, where they remain dormant accompanied by a-synuclein.These spores may be reactivated through age-dependent or genetically impaired macroautophagic function, ultimately leading to a neurodegenerative cascade that induces PD. [36] Collectively, the dysbiosis of the nasal microbiota creates the inflamma- tory response to a-synuclein, and the aggregation of asynuclein in the dopaminergic substantia nigra further causes neuronal loss in PD. [37,38] For most MS patients, the olfactory dysfunction occurs in the early stage of disease, which correlates with the olfactory bulb deficits and olfactory tract demyelination.[41] The chronic sinusitis and inflammation in the olfactory bulb and olfactory tract could consequently spread to the meninges and lead to cortical demyelination. [42]e clinical manifestations of ALS are reportedly affected by various undetermined environmental factors.For example, Sphingomonas/Sphingobium species ubiquitously inhabit water and soil and have been isolated from PM such as PM2.5 and PM10. [43]Environmental contaminants may also disrupt nasal microbiota, leading to dynamic shifts in microbial community structure.The human brain represents a direct potential target of contaminant PM through the nose-to-brain route, [44] and long-term exposure to traffic-related air pollution has been linked to elevated risk of ALS. [45]The Sphingomonas and Acinetobacter are both potentially trans-ported through air.Sphingomonas showed strikingly high infiltration of chronic endometritis patients, and active participation in carbohydrate metabolism-related pathways. [46]OTUs identified as Sphingomonas (in Proteobacteria) are enriched in AD patients and their abundance is correlated with cognitive measures. [47]In the present study, Prevotella-9 was positively associated with lung function in ALS patients, and some members of genus Prevotella (e.g., Otu0003 or Otu0045) are also associated with improved lung function and lower likelihood of chronic obstructive pulmonary disease. [48]The gut microbiota of ALS patients are known to be deficient in Prevotella spp.compared with those of their spouses. [49]However, the role of Prevotella spp.residing in the nasal cavity in ALS development remains unknown.Peripheral immunity and lipid metabolism have been observed to be associated with ALS clinically, [50][51][52] but it has remained unknown whether there is a causal association between special microbiota and ALS, which needs systematical exploration in further studies. Dysregulation of nasal microbes may lead to an increase in their opportunistic pathogenic bacteria, which release neurotoxins that promote inflammatory response and degenerative neuronal and axonal damage, which may explain the mechanism of the ALS affected by nasal microbes, while the correlation between nasal microbiota changes and neuroinflammation was not completely identified. [53]These degenerative effects may be partially explained by the close proximity of mucosa in the nasal middle tract to the olfactory bulb, a central location for initiating neuritis. [16,17]Moreover, some evidence suggests that toxins secreted by nasal microbes might be directly linked to pathogenic inflammatory activity and degeneration in neurological diseases. [16,17]Transported microbes and their associated neurotoxins may reach the brain through different routes, including the olfactory, the trigeminal, and paranasal submucosal pathways in the posterior sinuses. [16,17]During dysbiosis, nasal microbiota can induce inflammation, toxin accumulation, e.g., alpha-synuclein in the brain and nasal cavity, and neurodegeneration by proliferating in neural pathways. [16,17]It has also been proposed that microbial dysbiosis triggers neuroinflammation through lymphatic drainage.In this scenario, secreted bacterial toxins in the posterior sinuses enter the extracellular fluids of the CNS through lymphatic drainage. [16]e human microbiome can also strongly affect immunologic responses, for example, during development when immune cells establish their ability to discern beneficial commensals from pathogens, and in adulthood when some microbes participate in maintaining immune homeostasis. [11]Nasal microbiota composition and metabolic function may increase the risk of ALS or aggravate conditions associated with ALS by impacting inflammatory response, immune function, and production of certain metabolites.Functional analyses in our study have demonstrated ALS patients exhibit marked changes in glycan biosynthesis and metabolism and immune function, while ALS-related metabolic dysfunction in glucose and energy homeostasis have been described in both humans and ALS model mice. [54]urthermore, the upregulation of glycolysis has been mechanistically linked to the degeneration of motor neurons in several animal models of ALS. [55]Additionally, the aberrant regulation of immune response pathways is characteristic of brain and spinal tissues in ALS patients. [2,3]Neuroinflammatory glial cell activation through the canonical NF-kB pathway can promote loss of motor neurons, [4] while rates of ALS progression share an inverse relationship with the proportion of regulatory T-cells (T-regs) that can suppress inflammatory response. [56]Previous studies have revealed that the microglial component is involved in the neurotoxic and neuroprotective pathways in the pathomechansims of ALS. [57]However, microglial phenotype and function are responsive to external stimuli cues, such as those provided by commensal microbiota.Shifts in nasal microbiota composition have also been linked to host inflammatory tone, and hence dysbiosis in these communities can result in immune dysregulation which, as mentioned above, is a factor in ALS development. This study has several potential limitations.Although the sample size was sufficient to evaluate significant differences between ALS and controls, the ability to explore differences between ALS subgroups may lack adequate statistical power.Thus, the cohort size was not large enough to establish a diagnostic model and carry out validation in a separate subject group.Additionally, the sample size was also too small for risk stratification and prognosis prediction.Furthermore, the large majority of participants in our study were Chinese, potentially limiting the generalizability of our research to other ethnicities or geographically distant populations.Second, ethical guidelines preclude utilization of bronchoscopy to sample the lower airways, limiting our understanding of these microbiota and their relationship to nasal microbiota.There were only five patients receiving NIPPV therapy, and we did not determine the association between utilization of NIPPV and nasal microbiota.It would be interesting to explore the change of nasal microbiota before and after the utilization of NIPPV in future studies.Third, based on current knowledge, we cannot conclude definitively whether alterations in nasal microbiota are the cause or the consequence of ALS.Finally, we did not conduct any metagenomic analyses of fungi or viruses in the microbiome, whereas the collective genomes of all microbes, including bacteria, archaea, viruses, and fungi, contribute to the diversity and function of the human microbiome. In conclusion, this study provides the data describing dysbiosis in nasal microbiota of ALS patients.These results implied that disruption of nasal microbiota could possibly contribute to ALS pathogenesis.These findings imply a role for the nose-to-brain route in the influence of nasal microbiota in ALS.Future work will investigate the possibility of exploiting host-nasal microbiome interactions in novel therapeutic strategies for treating ALS. Figure 1 : Figure 1: Comparison of alpha and beta diversity between nasal microbiota of ALS patients and HC.(A) Boxplot of Chao1 index of OTU diversity between the ALS and HC groups (P = 0.072).(B) Boxplot of Simpson indexes comparing diversity between the ALS and HC groups (P = 0.024).(C) Boxplot of Shannon indexes comparing OTU diversity between the ALS and HC groups (P = 0.030).(D) Principal coordinate analysis based on unweighted UniFrac distances to compare microbial community structure between the ALS and HC samples ((P = 0.001).ALS: Amyotrophic lateral sclerosis; HC: Healthy controls; OTU: Operational taxonomic unit; ns: Not significant; * indicated P <0.05. Figure 2 : Figure 2: Comparison of nasal microbiota diversity among ALS patients with different disease stages (mild vs. severe groups).Boxplot of Chao1 index (A) (P = 0.880), Shannon index (B) (P = 0.340), and Simpson index (C) (P = 0.052) comparing OTU diversity between mild and severe ALS groups.(D) Principal coordinate analysis of unweighted UniFrac distances to identify differences in microbial community structure between mild and severe ALS groups (P = 0.559).ALS: Amyotrophic lateral sclerosis; OTU: Operational taxonomic unit; ns: Not significant. Figure 3 : Figure 3: Identification of ALS-related taxa in nasal microbiota.(A) Heat map of genus-level relative OTU abundance in all ALS patients and HC.(B) Boxplot of the top 10 different genera between the ALS and HC groups.(C) Bar chart of differentially abundant OTUs between ALS and HC, (LDA score >4, P <0.05).(D) Cladogram of LEfSe analysis comparing differences in nasal microbiota between the ALS and HC groups.From the inside to the exterior, circles represent phylum, class, order, family, and genus.The high abundance taxa in the ALS group are highlighted in red; high abundance taxa in HC subjects are highlighted in green.ALS: Amyotrophic lateral sclerosis; HC: Healthy controls; LDA: Linear discriminant analysis; LEfSe: Linear discriminant analysis effect size; OTU: Operational taxonomic unit. Figure 5 : Figure 5: KEGG analysis (level 2) of differentially enriched metabolic pathways between nasal microbiota of the ALS and HC groups.ALS: Amyotrophic lateral sclerosis; HC: Healthy controls; KEGG: Kyoto Encyclopedia of Genes and Genomes. Table 1 : Demographic and clinical features of the participants.	2023-07-25T06:17:26.165Z	2023-07-21T00:00:00.000	{ "year": 2023, "sha1": "6a3f0eb396d338c382ba41242ccf50770b4d9dc4", "oa_license": "CCBYNCND", "oa_url": "https://doi.org/10.1097/cm9.0000000000002701", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "ae8382e884684d5af19620c48707f2117ab76e09", "s2fieldsofstudy": [ "Biology", "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
246632851	pes2o/s2orc	v3-fos-license	Clinicopathologic features, comorbid diseases, and prevalence of pulmonary hypertension in dogs with bronchomalacia Abstract Background Reports of clinicopathologic features of bronchomalacia (BM) differ because of inconsistent definitions and frequent prevalence of comorbid cardiopulmonary disease. Pulmonary hypertension (PH) secondary to BM is poorly described. Objectives Dogs with BM will be older but of any somatotype, and increased expiratory effort, ≥1 comorbid disease, and PH will be more common than in dogs without BM. Animals Client‐owned dogs (n = 210) evaluated for respiratory signs. Methods Medical records of dogs with paired inspiratory: expiratory‐breath‐hold computed tomography, tracheobronchoscopy, or both between January 2016 and December 2019 were retrospectively reviewed. Comparisons between dogs with and without BM using Mann‐Whitney rank sum or χ 2 tests (P < .05 significant were made). Because of high numbers of variables, criteria with high prevalence (>25%) were identified (n = 10) for univariate analysis (P < .005 significant). Significant variables were submitted for multivariate analysis. Results Bronchomalacia was identified in 41% of dogs of all sizes/somatotypes; 38% were >10 kg. All dogs with BM had ≥1 comorbid cardiopulmonary disorder. Dogs with BM were significantly older (P < .001), smaller (P < .001), and were more likely diagnosed with tracheal or mainstem bronchial collapse (P < .001) or bronchiectasis (P < .001). Multivariate analysis confirmed associations with age, tracheal or mainstem bronchial collapse, and bronchiectasis. In dogs with BM, PH was more prevalent. Conclusions and Clinical Importance Although significantly more common in older, smaller dogs, BM occurs in dogs of all sizes and in all instances with comorbidities. Echocardiography should be considered in dogs with BM to identify PH. \| INTRODUCTION Static or dynamic collapse of the trachea, mainstem bronchi, lobar bronchi, or segmental and subsegmental bronchi causes a spectrum of clinical signs in dogs ranging from mild to life-threatening. In humans, tracheomalacia is defined as weakness of the tracheal cartilage leading to airway collapse whereas tracheobronchomalacia implies extension to the principal bronchi. 1,2 Definitions of bronchomalacia (BM) in dogs vary, including collapse of ≥1 of principal, lobar, segmental, or subsegmental bronchi [3][4][5] Because of these various definitions, previously published reports summarizing clinical characteristics of this condition [3][4][5] lack consistency. A recent review of BM in dogs proposed a strict definition of BM: "regional to diffuse dynamic airway collapse of segmental and/or subsegmental bronchi with associated clinical signs due to airflow limitation." 6 Using a more discrete definition aims to identify and distinguish BM from other causes of airway collapse, with relevance to clinicopathologic features, diagnostic testing, treatment, and prognosis. Bronchoscopy is the primary modality used to diagnose BM in dogs, but identification of dynamic airway narrowing can be hampered by apnea or shallow breaths. Paired inspiratory : expiratory-breath-hold computed tomography (I/E-BH CT) can capture airway caliber changes at peaks of inspiration and expiration independent of spontaneous respiration. 7 Previously, a scoring system was proposed to describe and grade the severity of BM using I/E-BH CT but no direct comparisons to bronchoscopic scoring were made. 8 Another advantage of CT is that it allows assessment of comorbid respiratory [7][8][9] and cardiac disorders, [10][11][12] something that tracheobronchoscopy cannot. Although CT has documented bronchial collapsibility in dogs, prior studies assessed lobar bronchial collapse rather than BM as defined herein. 13,14 Additionally, the technique to acquire CT images should reflect relevant physiology, which makes artificial forced expiratory maneuvers reporting a high degree of airway collapsibility in healthy dogs somewhat suspect. 14 Therefore, reliance on an expiratory series (by manual hyperventilation to induce apnea on exhalation or with ventilator-assisted expiratory breath-hold) better mimics changes occurring tidal breathing. Thus, optimal assessment of collapse of segmental and subsegmental bronchi ideally requires tracheobronchoscopy, I/E-BH CT, or both. By using these diagnostic tests as inclusion criteria, we aimed to provide a refined understanding of the clinicopathologic features and (using other diagnostic testing) to identify comorbid conditions associated with BM. In dogs, pulmonary hypertension (PH) occurs secondary to obstructive airway disease 15 and is associated with decreased survival time. 16 Treatment with sildenafil can improve clinical signs and quality of life. 3 A recent consensus statement for diagnosis, classification, and treatment of PH established a clarified definition of PH based on multiple echocardiographic and clinical criteria, allowing greater correlation between clinical signs and intermediate or high probability of PH. 15 To characterize BM in dogs using the definition of dynamic segmental or subsegmental bronchial collapse, our primary objective was to describe the signalment, clinicopathologic features, and comorbid conditions using I/E-BH CT, tracheobronchoscopy, or both. We also aimed to characterize prevalence and severity of PH using updated guidelines. 15 We hypothesized that dogs with BM would be older but \| Imaging protocols All imaging was done at the University of Missouri Veterinary Health Center according to established protocols. Thoracic radiography included both lateral views and an orthogonal projection. Respiratory fluoroscopy was performed with the unrestrained patient in 1 of 4 sizes of freestanding polycarbonate kennels 4 during quiet breathing and with induced cough. Videofluoroscopic swallow studies (VFSSs) were performed after a 12-hour fast using the same equipment with observation of ingestion of contrast-containing food of 3 consistencies (liquid, slurry, kibble) according to a previously described protocol. 13 Medical records of dogs that underwent echocardiographic evalua- Thoracic CT scans were performed during the inspiratory phase of spontaneous respiration or with I/E-BH when a mechanical ventilator was used to provide breath-holds for inspiratory and expiratory phases. If thoracic CT was performed without I/E-BH, tracheobronchoscopy had to be performed for study inclusion. The protocol for I/E-BH CT has been described elsewhere. 8 Diagnosis of BM was made using I/E-BH CT scans assessing segmental and subsegmental bronchi and the surrounding parenchyma in the expiratory compared to inspiratory series. As proposed previously, 8 CT sub-scores of 1, 2, or 3 were assigned to reflect severity of collapse of segmental and subsegmental airways: sub-score 1, subtle flattening without peribronchovascular opacification; sub-score 2, distorted circular appearance or >50% narrowing with mild to moderate peribronchovascular opacification; sub-score 3, near disappearance of airway lumens with marked peribronchovascular opacification. Dogs were included in the BM group with a CT sub-score of 1, 2, or 3. Tracheobronchoscopy was performed in a systematic fashion using a standard tracheobronchial road map. 5 Endoscopic diagnosis of BM was made if there was regional to diffuse dynamic airway collapse (decrease of ≥25% of the airway caliber) of segmental and subsegmental bronchi. 6 After tracheobronchoscopy, the endoscope was gently wedged into a distal airway at the endoscopist's discretion to collect bronchoalveolar lavage fluid. One to two 20 mL aliquots of sterile saline were instilled via a 20 mL syringe through the biopsy channel of the endoscope and gentle suction applied. Lavage samples were transported to the laboratory for cytologic evaluation (cellular differential and microscopic description) and culture (aerobic and capnophilic) with antimicrobial sensitivity. If bronchoalveolar lavage was done without tracheobronchoscopic examination (and I/E : BH-CT was used to diagnose BM), either a scope-guided sample was obtained as described above, or a sterile 8Fr red rubber catheter was sterilely inserted through the endotracheal tube and gently wedged into a distal airway, and then samples were collected as described above. \| Final clinical diagnoses The comprehensive clinical picture including signalment, history, clinical signs, and diagnostic test results was reviewed by 2 internists with special interest in respiratory disease (AVP and CR) to assign final diagnoses and assign confidence to the diagnoses (suspected or definitive) for each case (Table S1). \| Probability of pulmonary hypertension When echocardiographic data were available, probability of PH (low, intermediate, high) was classified according to the metrics established in the ACVIM consensus statement on PH in dogs. 15 \| Statistical analysis Statistical analysis was done using commercially available software (SigmaPlot, Systat Software Inc. Chicago, Illinois). Patients were divided into groups (with or without BM), and history, signalment, physical examination findings, and diagnoses were compared between these 2 groups. Data were assessed for normality using the Shapiro-Wilk test. Non-normal data were compared using Mann-Whitney rank sum tests. Categorical data were compared using χ 2 test. For univariate analysis of the categorical data, only variables (n = 10) with a prevalence >25%, but not <5% in either group, were considered ( Table 1). Because of the number of interrelated variables evaluated, a Bonferroni correction was performed and thus significance for univariate analysis was a set at P < .005. The variables that were found to be significant under these conditions (n = 4) were assessed using multiple logistical regression (P < .05). \| Animals Of 265 records screened, 210 dogs met the inclusion criteria: 86 with and 124 without BM ( Figure 2). Table 2 provides a summary of the signalment data for each group. Dogs with BM were significantly older (P < .001) and smaller (P < .001) than those without BM. However, a spectrum of weights and somatotypes was noted. \| History and physical examination findings Of the history and physical examination findings meeting the criteria for statistical analysis (chronic onset, cough, crackles, tachypnea, increased bronchovesicular sounds, and abnormal respiratory effort), none were significantly different between groups (Table 1). Other history or physical examination findings, including cyanosis, nasal discharge, respiratory sounds (stertor, stridor, pulmonary wheezes), respiratory pattern, clinical signs suggestive of PH (syncope, exercise intolerance, abdominal effusion, respiratory distress), occurred in <25% of dogs in either group and therefore were not considered for statistical analysis. Dogs with BM did not more commonly have increased expiratory effort, but only 7 (8%) dogs with BM and 9 (7%) dogs without BM had this specific respiratory pattern noted in the medical record. \| Diagnostic testing The number of dogs with and without BM having each respiratory diagnostic test is shown in Table 3. Overall, the average number of respiratory and cardiac diagnostic tests was 5 and 4 in dogs with and without BM, respectively. Fifty-one dogs with BM had thoracic radiographs performed; 38 (75%) were abnormal. Seventy dogs without BM had thoracic radiographs; 61 (87%) were abnormal. The distribution of thoracic radiographic patterns is shown in Table 4. \| Identification of co-morbid diseases Definitive and suspected final respiratory diagnoses for both groups are shown in Table 5. Overall, the mean ± SD number of definitive and suspect final diagnoses (ie, comorbid diseases) was 4 ± 1 definitive and 1 ± 1 suspect in dogs with BM and 2 ± 1 definitive and 1 ± 1 suspect in dogs without BM. All dogs with BM had at least 1 comorbid cardiovascular or respiratory disease. Eighty-two dogs without BM had only a single definitive or suspect final diagnosis. Dogs with BM were more frequently diagnosed with concurrent tracheal collapse, mainstem bronchial collapse, or both (combined because of co-linearity; P < .001) and bronchiectasis (P < .001) compared to dogs without BM. Note: Only criteria where at least 1 group had a prevalence of >25% (shaded boxes) but no less than 5% in either group were considered for univariate analysis. A Bonferroni correction was performed to account for the number of interrelated variables tested and significance (*) was a set at P < .005. Abbreviations: BOAS, brachycephalic obstructive airway syndrome; GERD, gastroesophageal reflux disease. a Due to the retrospective nature of the study, historical and physical examination findings were not uniformly reported in all dogs. If the historical or physical examination finding was present ("yes"), it was recorded in this table. Absence of the finding could have been because the dog lacked the finding, the owner did not recognize the finding, or the finding did not get recorded in the medical record. b Fever defined as temperature >103F (39.4 C). c Tracheal and mainstem bronchial collapse were combined for the purpose of statistical analysis because of concern for co-linearity. d Eosinophilic bronchitis, eosinophilic bronchopneumonopathy, and eosinophilic pneumonia (ie, eosinophilic lung disease) were also combined because of co-linearity. Multiple logistic regression analysis was performed on the statistically significant variables (age, weight, presence of bronchiectasis, and tracheal collapse, mainstem bronchial collapse, or both [ 13,18,19,24,25 The diversity in clinical signs likely supports the range in severity of BM and the high prevalence of comorbid conditions. Clinical signs strongly associated with PH specified in the ACVIM consensus statement include syncope, respiratory distress at rest, respiratory distress after exercise, and cardiogenic ascites. 15 The primary means of diagnosis of BM in dogs historically has been bronchoscopic examination, 18 13,14,26,27 Artificial forced expiration using syringes to aspirate air from intubated dogs does not mimic the physiology of tidal breathing 14,27 and differs from the paired I/E : BH CTs performed in our study. These ventilator assisted breath-holds allow comparison of inspiratory and expiratory CT scans to assess changes in airway caliber and the peribronchial CONFLICT OF INTEREST DECLARATION Authors declare no conflict of interest. OFF-LABEL ANTIMICROBIAL DECLARATION Although some dogs were treated with antimicrobials, the selection of antimicrobial was at the discretion of the attending clinician and was not a part of the study. INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION Approved by the University of Missouri College of Veterinary Medicine IACUC, number 9866. HUMAN ETHICS APPROVAL DECLARATION Authors declare human ethics approval was not needed for this study.	2022-02-08T06:22:56.585Z	2022-02-07T00:00:00.000	{ "year": 2022, "sha1": "5c8d25f36fa6aea214d72f4b7155255df213cb00", "oa_license": "CCBYNC", "oa_url": null, "oa_status": "CLOSED", "pdf_src": "PubMedCentral", "pdf_hash": "558dc3d70f1a81b72efeb7b59c06be42cb7d63e6", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
56153381	pes2o/s2orc	v3-fos-license	New radial velocity studies and curve analysis of spectroscopic binary systems CS22964-161, LV Her, RW Lac and HD 34700 Using measured radial velocity data of four double-lined spectroscopic binary systems CS22964-161, LV Her, RW Lac and HD 34700, we find corresponding orbital and spectroscopic elements via a Probabilistic Neural Network (PNN). Our numerical results are in good agreement with those obtained by others using more traditional methods. INTRODUCTION Analysis of both light and radial velocity (hereafter V R ) curves of binary systems helps us to determine the masses and radii of individual stars. One historically well-known method to analyze the V R curve is that of Lehmann-Filhés [1]. Some other methods were also introduced by Sterne [2] and Petrie [3]. The different methods of the V R curve analysis have been reviewed in ample detail by Karami & Teimoorinia [4]. Karami & Teimoorinia [4] also proposed a new non-linear least squares velocity curve analysis technique for spectroscopic binary stars. They showed the validity of their new method to a wide range of different types of binary See Karami & Mohebi [5][6][7] and Karami et al. [8]. Artificial Neural Networks have become a popular tool in almost every field of science. In recent years, ANNs have been widely used in astronomy for applications such as star/galaxy discrimination, morphological classification of galaxies, and spectral classification of stars (see Bazarghan et al. [9] and references therein). Following Bazarghan et al. [9], we employ Probabilistic Neural Networks (PNNs). This network has been investigated in ample details by Bazarghan et al. [9]. Probabilistic Neural Network (PNN) is a new tool to derive the orbital parameters of the spectroscopic binary stars. In the present paper we use a Probabilistic Neural Network (PNN) to find the optimum match to the four parameters of the V R curves of the four double-lined spectroscopic binary systems CS22964-161, LV Her, RW Lac and HD 34700. Our aim is to show the validity of our new method to a wide range of different types of binary. CS 22964-161 is a double-lined spectroscopic binary that both components are near the metal-poor main-sequence turn off and the spectral types of the stars are not too dissimilar and the orbital period is P = 252.481 days [10]. LV Her is a double-lined eclipsing binary and consists of primary and secondary components which has the highest eccentricity. The spectral type of systems is F9 V and the orbital period is P = 18.4359535 days [11]. RW Lac is a detached, eccentric, EA-type, double-lined eclipsing binary and consists of the hotter, larger, more massive, and more luminous photometric primary and the cooler, smaller, less massive, and less luminous photometric secondary components. The spectral type is G5 and G7 for the primary and the secondary stars and the orbital period is P = 10.3692046 days [12]. The young star HD 34700 is a double-lined spectroscopic binary and the components are of very nearly equal mass, temperature, luminosity and similar spectral type: G0 and the orbital period is P = 23.4877 days [13]. This paper is organized as follows. In Sect. 2, we introduce a Probabilistic Neural Network (PNN) to estimate the four parameters of the V R curve. In Sect. 3, the numerical results are reported, while the conclusions are given in Sect. 4. V R CURVE PARAMETERS ESTIMATION BY THE PROBABILISTIC NEURAL NETWORK (PNN) Following Smart [14], the V R of a star in a binary system is defined as follows where  is the V R of the center of mass of system with respect to the sun. Also K is the amplitude of the V R of the star with respect to the center of mass of the binary. Furthermore ,  and e are the angular polar coordinate (true anomaly), the longitude of periastron and the eccentricity, respectively. Here we apply the PNN method to estimate the four orbital parameters, γ, K, e and ω of the V R curve in Eq. (1). In this work, for the identification of the observational V R curves, the input vector is the fitted V R curve of a star. The PNN is first trained to classify V R curves corresponding to all the possible combinations of γ, K, e and ω. For this one can synthetically generate V R curves given by Eq. (1) for each combination of the parameters: • 100 100     in steps of 1; • 1 K 300   in steps of 1; • 0 e 1   in steps of 0.001; This gives a very big set of k pattern groups, where k denotes the number of different V R classes, one class for each combination of γ, K, e and ω. Since this very big number of different V R classes leads to some computational limitations, hence one can first start with the big step sizes. Note that from Petrie [3], one can guess γ, K, e from a V R curve. This enable one to limit the range of parameters around their initial guesses. When the preliminary orbit was derived after several stages, then one can use the above small step sizes to obtain the final orbit. The PNN has four layers including input, pattern, summation, and output layers, respectively (see Fig. 5 in Bazarghan et al. [9]). When an input vector is presented, the pattern layer computes distances from the input vector to the training input vectors and produces a vector whose elements indicate how close the input is to a training input. 10 ILCPA Volume 20 The summation layer sums these contributions for each class of inputs to produce as its net output a vector of probabilities. Finally, a competitive transfer function on the output layer picks the maximum of these probabilities, and produces a 1 for that class and a 0 for the other classes [15,16]. Thus, the PNN classifies the input vector into a specific k class labeled by the four parameters γ, K, e and ω because that class has the maximum probability of being correct. NUMERICAL RESULTS Here, we use the PNN to derive the orbital elements for the four different double-lined spectroscopic systems CS22964-161, LV Her, RW Lac and HD 34700. Using measured V R data of the two components of these systems obtained by Thompson et al. [10] for CS22964-161, Torres et al. [11] for LV Her, Sandberg Lacy et al. [12] for RW Lac, Torres [13] for HD 34700, the fitted velocity curves are plotted in terms of the phase in Figs. 1 to 4. The orbital parameters obtaining from the PNN for CS22964-161, LV Her, RW Lac and HD 34700 are tabulated in Tables 1, 3, 5 and 7, respectively. Tables show that the results are in good accordance with the those obtained by Thompson et al. [10] for CS22964-161, Torres et al. [11] for LV Her, Sandberg Lacy et al. [12] for RW Lac, Torres [13] for HD 34700. Note that the Gaussian errors of the orbital parameters in Tables 1, 3, 5 and 7 are the same selected steps for generating V R curves, i.e. [16] , the error of the decision boundaries depends on the accuracy with which the underlying Probability Density Functions (PDFs) are estimated. Parzen [17] proved that the expected error gets smaller as the estimate is based on a large data set. This definition of consistency is particularly important since it means that the true distribution will be approached in a smooth manner. Specht [16] showed that a very large value of the smoothing parameter would cause the estimated errors to be Gaussian regardless of the true underlying distribution and the misclassification rate is stable and does not change dramatically with small changes in the smoothing parameter. The combined spectroscopic elements including Tables 2, 4, 6 and 8 show that our results are in good agreement with the those obtained by Thompson et al. [10] for CS22964-161, Torres et al. [11] for LV Her, Sandberg Lacy et al. [12] for RW Lac, Torres [13] for HD 34700, respectively. Here the errors of the combined spectroscopic elements in Tables 2, 4 14 ILCPA Volume 20 Table 4. Combined spectroscopic elements of LV Her. Table 5. Orbital parameters of RW Lac. International Letters of Chemistry, Physics and Astronomy Vol. 20 Table 7. Orbital parameters of HD 34700. CONCLUSIONS A Probabilistic Neural Network to derive the orbital elements of spectroscopic binary stars was applied. PNNs are used in both regression (including parameter estimation) and classification problems. However, one can discretize a continuous regression problem to such a degree that it can be represented as a classification problem [15,16], as we did in this work. Using the measured V R data of CS22964-161, LV Her, RW Lac and HD 34700 obtained by Thompson et al. [10], Torres et al. [11], Sandberg Lacy et al. [12] and Torres [13], respectively, we find the orbital elements of these systems by the PNN. Our numerical results shows that the results obtained for the orbital and spectroscopic parameters are in good agreement with those obtained by others using more traditional methods. This method is applicable to orbits of all eccentricities and inclination angles. In this method the time consumed is considerably less than the method of Lehmann-Filhés. It is also 16 ILCPA Volume 20 more accurate as the orbital elements are deduced from all points of the velocity curve instead of four in the method of Lehmann-Filhés. The present method enables one to vary all of the unknown parameters γ, K, e and ω simultaneously instead of one or two of them at a time. It is possible to make adjustments in the elements before the final result is obtained. There are some cases, for which the geometrical methods are inapplicable, and in these cases the present one may be found useful. One such case would occur when observations are incomplete because certain phases could have not been observed. Another case in which this method is useful is that of a star attended by two dark companions with commensurable periods. In this case the resultant velocity curve may have several unequal maxima and the geometrical methods fail altogether.	2019-04-20T13:11:23.875Z	2013-10-16T00:00:00.000	{ "year": 2013, "sha1": "92eda7c129708c8221ff9593b24d3ace793f8ff8", "oa_license": "CCBY", "oa_url": "https://www.academicoa.com/ILCPA.20.9.pdf", "oa_status": "HYBRID", "pdf_src": "ScienceParsePlus", "pdf_hash": "37808e79754e5d4db2495a6e60df57e6c7ad8562", "s2fieldsofstudy": [ "Physics", "Chemistry" ], "extfieldsofstudy": [ "Physics" ] }
226262605	pes2o/s2orc	v3-fos-license	Tyrosine Side‐Chain Functionalities at Distinct Positions Determine the Chirooptical Properties and Supramolecular Structures of Pentameric Oligothiophenes Abstract Control over the photophysical properties and molecular organization of π‐conjugated oligothiophenes is essential to their use in organic electronics. Herein we synthesized and characterized a variety of anionic pentameric oligothiophenes with different substitution patterns of L‐ or D‐tyrosine at distinct positions along the thiophene backbone. Spectroscopic, microscopic, and theoretical studies of L‐ or D‐tyrosine substituted pentameric oligothiophene conjugates revealed the formation of optically active π‐stacked self‐assembled aggregates under acid conditions. The distinct photophysical characteristics, as well as the supramolecular structures of the assemblies, were highly influenced by the positioning of the L‐ or D‐tyrosine moieties along the thiophene backbone. Overall, the obtained results clearly demonstrate how fundamental changes in the position of the enantiomeric side‐chain functionalities greatly affect the optical properties as well as the architecture of the self‐assembled supramolecular structures. Introduction Self-assembled chiral materials comprised of conjugated polyand oligothiophenes (CPs and COs) have been extensively studied due to their potential for being utilized in optoelectronic devices, biosensors, and as artificial enzymes. [1][2][3][4][5][6][7][8][9][10][11] CPs with an optically active substituent in the 3-position normally display optical activity in the π-π* transition region when the polymer chains are forming supramolecular, π-stacked selfassembled aggregates in a poor solvent or at low temperature. [5,[12][13][14][15] Furthermore, when mixing optically inactive CPs and COs with small chiral molecules or synthetic peptides, an induced single-chain chirality that reflects the stereochemistry of the chiral guest can be obtained. [9][10][11][16][17][18][19][20] An analogous single-chain induced chirality have also been observed for CPs having optically active zwitter-ionic side chain functionalities, as well as in CPs functionalized with L-or D-amino acid side chains. [21,22] For the latter, the chirality of the side chain was manifested in the conformation of the polymer backbone, giving rise to right-handed or left-handed helical forms of the polythiophene chains with induced circular dichroism (ICD) patterns of the two polymers that were mirror images. [22] ICD has also been reported for chemically defined oligothiophenes having chiral substituents at distinct positions along the thiophene backbone. [23][24][25][26][27] The chiral moieties can be introduced in the β-positions of the thiophene moieties [23] or at the terminal α-positions of the oligothiophene backbone. [24][25][26][27] For α,α'-linked oligothiophenes with five, six, or seven thiophene rings, with penta(ethyleneoxide) substituents attached via ester links at the terminal α-positions, the sign of the aggregation induced Cotton effect was dependent on the position of the chiral substituent as well as the length of the oligothiophene sequence. [24] Similarly, tetrameric oligothiophenes displayed opposite Cotton effects depending on the introduction of suitable D-(+)-or L-(À )-mannosidic building blocks linked to the terminal α-positions. [25] An analogous effect was also obtained when mixing α-sexithiophene with distinct polysaccharides. [28] Lately, it was also shown that oligiothiophene-proline hybrids display specific self-assembly behaviour in an aqueous environment by forming chiral superstructures, whose helicity and Cotton effect were controlled by the configuration of the amino acid moiety. [26] Thus, a variety of studies have shown that enantiomeric substitutions can be utilized to tune the optical properties as well as the structural organization of oligothiophenes. However, the differences in optical behaviour and self-assembly between oligothiophenes having the same enantiomeric substituents in the β-positions of the thiophene moieties or at the terminal α-positions of the oligothiophene backbone have not been examined. Herein we report the synthesis of a set of novel anionic pentameric oligothiophenes having L-tyrosine or D-tyrosine introduced at distinct positions along the conjugated backbone ( Figure 1). Amino acid residues were introduced at the βposition of distinct thiophene moieties or at the terminal αpositions of the oligothiophene backbone and the effects of the structural modifications were investigated by comparing the photo-physical characteristics and self-assembling properties of the chiral oligothiophenes. The obtained results revealed how alterations in the position of the enantiomeric moiety have a large impact on the oligothiophenes optical properties as well as the molecular architecture of the self-assembled structures. Synthesis of Pentameric Oligothiophenes with Tyrosine Side Chain Functionalities To achieve a library of chiroptical pentameric oligothiophenes with tyrosine substituents in the β-positions of distinct thiophene moieties or at the terminal α-positions of the oligothiophene backbone (Figure 1), the previously reported achiral oligothiophenes, penta hydrogen thiophene acetic acid (p-HTAA) and penta formyl thiophene acetic acid methylester (p-FTAM), as well as a trimeric building block, compound 2, were selected as templates (Supporting Information, SI Scheme S1). [29,30] The two enantiomers, p-HTA-L-Tyr and p-HTA-D-Tyr, were afforded by addition of L-or D-tyrosine to the carboxylate functionalities in the β-positions of p-HTAA [29] through an amide forming reaction using hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU) followed by removal of protecting groups to have anionic side chain functionalities. A similar synthetic route was applied for p-FTAM, [29] to achieve the two enantiomers, p-FTAA-L-Tyr and p-FTAA-D-Tyr, having tyrosine functionalities at the terminal αpositions of the pentameric thiophene backbone, as well as acetate functionalities in the β-positions of thiophene number 2 and 4. The third pair of enantiomers, p-FTA-L-Tyr and p-FTA-D-Tyr, was generated by hydrolysis of the carboxylate functionalities in the β-positions of 2 [29] followed by applying the HATU mediated amide forming reaction to add L-or D-tyrosine to generate the brominated trimer 3. To yield pentameric oligothiophenes having tyrosine functionalities in the βpositions of thiophene number 2 and 4, as well as carboxylate functionalities at the terminal α-positions of the pentameric thiophene backbone, the addition of 5-carboxy-2-thienylboronic acid to 3 was afforded by palladium-mediated Suzuki-Miyaura cross coupling followed by removal of protecting groups. As a part of removing protective groups or in additional final step, all the pentameric ligands were converted to the sodium salt using sodium hydroxide (SI, Scheme S1). Absorption and Emission Characteristics Next the photo-physical characteristics of the tyrosine-functionalized oligothiophenes dissolved in 20 mM HCl or 20 mM NaOH (pH 12.3) were evaluated ( Figure 2). These acidic and basic conditions were selected, since earlier studies have shown that pH-induced conformational changes of the thiophene backbone can be afforded for both anionic oligo-and polythiophenes. [29][30][31][32][33] In addition, in 20 mM HCl (pH 1.7) all the carboxyl groups of the oligothiophenes should predominantly be protonated, whereas in 20 mM NaOH (pH 12.3), the carboxyl groups, as well as the phenolic hydroxy groups should be deprotonated. In 20 mM NaOH, p-HTA-L-Tyr and p-HTA-D-Tyr, displayed similar absorption spectra with a maximum at 400 nm, as well as comparable emission maxima around 520 nm (Figure 2A). These photophysical characteristics are similar to ones reported for other anionic pentameric oligothiophenes in alkaline conditions, suggesting that the absorptionand emission spectra from the ligands in 20 mM NaOH originates from fully dissolved single oligothiophene chains or tiny clusters of chains. [29][30][31][32][33] Both dyes also showed analogous optical characteristic in 20 mM HCl, but the absorption spectra showed a red-shift, as well as two additional peaks around 330 nm and 275 nm, compared to the spectra in alkaline conditions. A red-shift of the spectra were also observed for the emission and is most likely associated with a planarization of the backbone that are induced upon protonation of the carboxyl groups. [21,[34][35][36][37] In addition, the emission intensity decreased with almost two orders of magnitude, suggesting an aggregation of adjacent oligothiophene molecules (SI, Figure S1). [13][14][15]21] p-FTA-L-Tyr and p-FTA-D-Tyr showed an equivalent photophysical behavior as their counterparts lacking the α-terminal carboxyl groups ( Figure 2B and Figure S1). However, in 20 mM HCL, the emission spectra were strikingly more redshifted with emission maxima at 640 nm, suggesting that alternative aggregates are formed due to the α-terminal carboxyl groups. The third pair of enantiomers, p-FTAA-L-Tyr and p-FTAA-D-Tyr displayed comparable absorption maxima in acidic or basic conditions, but a small shoulder at longer wavelengths could be observed in the absorption spectra in 20 mM HCl ( Figure 2C). Furthermore, in acidic conditions the emission spectra were red-shifted, and a decrease of the emission intensity was also observed ( Figure 2B and Figure S1). Hence, similar to the other pentameric oligothiophenes, p-FTAA-L-Tyr and p-FTAA-D-Tyr seem to form π-stacked self-assembled aggregates upon protonation of the carboxyl groups, whereas fully dissolved single oligothiphene chains are obtained in alkaline conditions. However, from a photophysical perspective, these aggregates seem to be different compared to the other pairs of enantiomers, suggesting that the swapping of the tyrosine side chain functionality from the β-positions of distinct thiophene moieties to the terminal α-positions of the oligothiophene backbone is influencing the morphology of the aggregates. For instance, the addition of tyrosine to the terminal α-positions might induce some steric effects that generate an alternative aggregate formation. In order to study the emission characteristics in more detail, time-correlated single photon counting (TC-SPC) measurements were carried out to determine the decay times of the enantiomeric variants. These experiments were performed at lower concentrations (5 μM) of the oligothiophenes to ensure that inner filter effects or quenching from intermolecular energy transfer processes were minimized. As judged from the absorption spectra ( Figure 2) a laser diode operating at 403 nm was appropriate for the excitation of all oligothiophenes. Although the decays all were close to mono-exponential, the decays were analyzed allowing two decay times. By using these two decay times and the amplitude weights, an intensity averaged decay time, τ ave , was calculated. The L-and Denantiomers of p-HTA-Tyr showed averaged decay times around 110 to 140 ns in acidic condition (20 mM HCl), whereas τ ave increased to about 300 ps under basic conditions (20 mM NaOH) ( Figure 3A). In contrast, p-FTA-L-Tyr and p-FTA-D-Tyr displayed longer average decay times in 20 mM HCl (τ ave around 500 ps) compared to decay times achieved in alkaline conditions (τ ave around 270 ps) ( Figure 3B). Thus, these two pairs of enantiomers displayed opposite pH-induced effects on the average decay times. It is noted that p-FTA-L-Tyr and p-FTA-D-Tyr, for which the life-time increased, also showed a large redshift of the emission in acidic conditions ( Figure 2B). Thus, the alignment in the p-FTA-L-Tyr and p-FTA-D-Tyr aggregates seems to have more efficiently planarized molecular moieties. The third pair of enantiomers, p-FTAA-L-Tyr and p-FTAA-D-Tyr, showed longer average decay times in 20 mM NaOH (τ ave around 460 to 490 ps) compared to acidic conditions (τ ave around 230 to 245 ps) ( Figure 3C). For all pairs of enantiomers, the apparent decay time along with the analyzed decay times were essentially the same within experimental uncertainty when comparing L-and D-type enantiomers. Nevertheless, the three pairs of enantiomers displayed specific averaged decay times in 20 mM HCl or 20 mM NaOH, verifying that the emission characteristics of the oligothiophenes are highly dependent on the location of the tyrosine side chain functionalities, as well as the charge of the anionic groups. Induced Circular Dichroism To investigate the optical activity in the π-π* transition region of the pentameric oligothiophenes, we next performed circular dichroism (CD) measurements in 20 mM HCl or 20 mM NaOH. In acidic conditions, all three ligands displayed a strong split-type ICD in the π-π* transition region, whereas only minor signals lacking the bisignate Cotton effect were observed in alkaline conditions (Figure 4 and SI, Figure S2). Similar to earlier observations of chiral oligo-and polythiophenes, [5,[12][13][14][15][16][24][25][26][27] this indicates that the strong ICDs may be associated with π-stacked chiral aggregation of the oligothiophenes. This indication is also supported by the already discussed red-shifts in absorption spectra and decreased emission intensities under acidic condition ( Figure 2 and SI, Figure S1). The strongest and most pronounced ICD was obtained for the p-HTA-Tyr enantiomers ( Figure 4A), whereas the two other pairs of enantiomers displayed similar but less intense ICD patterns ( Figure 4B-C). As previously reported for optical active polythiophenes, [13,15] the ICD pattern resembled the first derivative of the absorption for the respective ligand (SI, Figure S3). All ligands functionalized with L-tyrosine showed a positive Cotton effect at lower energy and a negative Cotton effect at higher energy, whereas the ICD patterns of the ligands functionalized with D-tyrosine were mirror images compared to the corresponding enantiomers ( Figure 4). Hence, the altered chirality of the tyrosine side chain functionality is influencing the helical packing of the thiophene backbone. Similar ICD patterns have also been observed for zwitter-ionic polythiophenes having L-or D-serine in the βpositions, although the ICDs in these polythiophenes were a result of main-chain chirality instead of aggregation. [21] Interestingly, in 20 mM NaOH mirror images of the CD spectra in the UV region (210-260 nm) were also observed for all pairs of enantiomers, verifying that the chirality of tyrosine side chain is preserved (SI, Figure S2). In addition, when mixing equal amounts of the D-or L-form of the respective oligothiophenes in 20 mM NaOH, racemic mixture with no CD-signal was obtained (SI, Figure S2). To get a microscopic insight into the origin of the ICD, we performed a computational study by combining classical forcefield molecular dynamics (MD) and quantum-mechanical spectrum simulations (see the SI for further details). As target system, we adopted the protonated D-form of p-HTA-Tyr, since this ligand showed the largest ICD ( Figure 4A). We first investigated the ICD at the single-molecule level, assessing how a chiral twist-deformation of the conjugated backbone induces a CD signal in a model system. We constructed a series of models of cis-pentathiophene with varying negative dihedral angles between the thiophene groups, a conformation referred to as MMMM (M for minus). The molecular structures were either fully relaxed or the dihedral angles were locked at specific values while the rest of the molecule was relaxed. The CD spectra of these systems as obtained by time-dependent density functional theory (TD-DFT) calculations are presented in Figure 5A. The principal spectral shape is a negative/positive bisignated signal that is seen to reproduce the one found in the experiment for p-HTA-D-Tyr. Results for these simple model systems thus demonstrate that single-molecule chiral deformations of the thiophene backbone can in principle produce significant ICD signals but it does not address the effects of dynamics and conformational averaging. Next, we therefore altered the dihedral angles into the forms of trans (T), cis-minus (M), and cis-plus (P). From this conformational study presented in Figure 5B, we concluded that the two innermost dihedral angles are of dominating importance for the CD responses of the system. It is thus well motivated to adopt a more concise categorization and presentation based on these two key dihedral angles, and the MM and PP conformational classes are identified as the most important ones. However, it is clear that for these model systems, molecular dynamics would give rise to equal populations of MM and PP conformations with a vanishing CD signal as a result, so population statistics of the chiral ligands must be introduced. We performed an MD simulation for p-HTA-D-Tyr and analyzed the conformational variations of the thiophene backbone. The detailed statistics of the two dihedral angles around the central rings for the full trajectories as well as for a selection of 300 snapshots are provided in the SI. In light of the discussion above, we focus on the cis conformers and it can then be observed that the MM conformers are about twice as probable as the PP ones, showing population statistics of 9.3 % and 4.3 %, respectively. Together, the PM and MP conformers occupy about 8.6 %. This observed difference in population statistics between the MM and PP conformer classes is very interesting as it indicates that an appreciable ICD could be observed also without dye-aggregation and also be the root cause of the experimentally observed ICD signals for p-HTA-D-Tyr. From the MD simulations, we extracted 300 representative snapshots and determined the corresponding CD spectra. In Figure 5C, we show the spectra for each of the conformational classes, normalized by the number of snapshots, and X thus denotes that spectral summations are performed for all the conformations with different outermost dihedral angles. It is apparent and also expected from the model-system analysis, that the signal responses from the enantiomeric conformer classes XMMX and XPPX are much stronger than the responses from any of the others. The corresponding averaged CD spectrum for all 300 snapshots of a single molecule is presented in Figure 5D (blue solid line). A distinct negative/positive bisignated band is observed in the region between 300 to 450 nm, which stands in qualitative agreement with experiment. Finally, it remains to investigate the effects on the ICD originating from intermolecular interactions upon aggregation in solution. We performed MD simulation on systems with 4, 8,12, 16, 24, and 48 chromophores under aqueous conditions. We could not observe any ordered chromophore arrangements but rather clumps of p-HTA-D-Tyr molecules with centralized thiophene backbones in π-π and herring-bone interaction patterns and tyrosine moieties at the peripheries. An illustrative aggregate structure is provided in Figure 5D. This molecular organization is in agreement with the hydrophobicity of the thiophene backbone and hydrophilicity of the tyrosine moiety. In order to address the CD spectra of the aggregate structures, we were for reasons of computational cost forced to adopt the semi-empirical ZINDO approach. We benchmarked this more approximate method at the single molecule level by repeating the calculation of the averaged spectrum for the 300 snapshots. The resulting ZINDO spectrum is shown in Figure 5D (red solid line) and, aside from a 100 nm red-shift, the low-energy, bisignated, band well reproduces that of the TD-DFT spectrum. The spectral shift is not crucial for the sake of the argument and it is also a well-known systematic error built into this semiempirical method. More importantly, the use of the ZINDO approach allows us to determine the CD spectra of the aggregates, in which case we can also expect a favorable error cancellation as to provide a reliable trend estimate. For the system composed of four molecules, the averaged CD spectrum is presented in Figure 5D (green solid line). This spectrum is also normalized with respect to the number of molecules as to be able to compare intensities against the single-molecule spectrum. We can note a general increase of the intensity as well as a small red-shift in the spectrum of the aggregate as compared to that of the single molecule. In particular, the intensity of the positive band in the ZINDO spectral region of 400-450 nm becomes quite drastically enhanced by intermolecular interactions and the resulting band profile shows a convincing qualitative agreement with the experimental spectrum for p-HTA-D-Tyr. We are led to conclude that the microscopic origin of the observed ICD under acidic conditions are rooted in the excess of MM conformer populations but enhanced by the intermolecular chromophore interactions in the formed aggregates. Fluorescence Anisotropy Decay By controlling the polarization state of the excitation and emission in the TC-SPC measurements, one can record the time-resolved fluorescence anisotropy, a well-known technique to monitor molecular rotation diffusion. [42] This technique is widely used to study protein complexes and interactions between proteins that alter their hydrodynamic volume. A feature of the time-resolved anisotropy is that it is difficult to accurately monitor rotational correlation times longer than the fluorescence decay time. However, the start of the anisotropic decay can anyway give a clear evidence if a fluorescent labelled molecular complex is much larger than the non-complexed molecule as shown for e. g., amyloid aggregates. [43,44] Herein, anisotropy decay was employed to examine if the oligothiophene enantiomers were more prone to aggregation, at low concentrations (5 μM) in acidic or alkaline conditions. In 20 mM NaOH, all enantiomers displayed well-defined decays in the range 400 ps to 1 ns with the longest rotational decay time (θ) for p-FTAA-L-Tyr and p-FTAA-D-Tyr and the shortest time for the p-HTA-L-Tyr and p-HTA-D-Tyr ( Figure 6, right panels). Under acidic conditions all enantiomers showed a considerably longer rotational correlation time compared to the ones obtained in 20 mM NaOH, suggesting that protonation of the anionic side chain functionalities induces aggregation of the oligothiophenes ( Figure 6, left panel). The fluorescence decay time was not long enough to resolve their precise values, but anyway clearly indicated some reasonable lower limit of the size of complexes in each case. Moreover, the onset of the anisotropy at t = 0, manifested as the r 0 value, is an order parameter indicating the angle between the excitation and emission transition dipole moments; this value cannot exceed 0.4, meaning that the excitation and emission dipole moments are parallel. [42] In all of the oligothiophene variants the r 0 was approximately 0.3 when well dissolved at the higher pH, whereas at the lower pH this value was altered ( Figure 6). Thus, under acidic conditions, one might expect that the excitation and/or the emission dipoles have changed their relative directions as a result of the aggregation, comparing with the anisotropy and r 0 value of the well solvated molecule in 20 mM NaOH. This is particularly evident for p-FTA-L-Tyr and p-FTA-D-Tyr that showed increased decay-time in addition to a substantial red-shift of the emission (as discussed above). Hence, this observation further corroborates that the oligothiophene moieties are more planar in these particular aggregates. Dynamic Light Scattering To further investigate if the pentameric oligothiophenes go through a pH dependent structural organization, dynamic light scattering (DLS) measurements were performed in 20 mM HCl and 20 mM NaOH. When exposed to an acidic environment all ligands displayed longer lag times in the autocorrelation function compared to when exposed to alkaline conditions (Figure 7). This behavior can be attributed to a decrease in solution dynamics, indicating formation of larger structures in 20 mM HCl. The protonation of the carboxylic groups is hence beneficial for the self-assembly process. Both enantiomers of p-HTA-Tyr and p-FTA-Tyr formed similar sized structures in acidic conditions ( Figure 7A and B), whereas both enantiomers of p-FTAA-Tyr appears to be more solubilized in the same conditions ( Figure 7C). Thus, the position of the tyrosine functionality in the pentameric thiophene backbone greatly affects the organization within the structures, where a tyrosine substituent in the α-position of the conjugated oligothiophene appears to have a negative impact on the π-stacking, leading to smaller structures. In alkaline conditions both enantiomers of p-HTA-Tyr and p-FTA-Tyr form smaller structures compared to acidic conditions and both enantiomers of p-FTAA-Tyr appear to be completely solubilized, producing very weak intensities in the correlation functions ( Figure 7C). Overall, DLS measurements confirmed that protonation of carboxylic moieties promotes π-stacked self-assembled aggregates and that alkaline conditions produces smaller aggregates which is in line with observations from the photo-physical characterization as well as the anisotropy measurements. Structural Characterization of the Oligothiophenes To elucidate the ultrastructure of the aggregated assemblies formed in acidic conditions, samples were next studied by transmission electron microscopy ( Figure 8). Both enantiomers of p-HTA-Tyr displayed long fibers, 6-17 nm wide, with a tendency for lateral stacking and aggregation ( Figure 8A), whereas the enantiomers of p-FTA-Tyr showed aggregates of short fibers, 6-12 nm wide ( Figure 8B). Hence, oligothiophenes having tyrosine in the β-positions of distinct thiophene moieties formed fibrillar structures and longer fibrils were achieved for oligothiophenes lacking the carboxylate functionalities at the terminal α-positions of the pentameric thiophene backbone. In contrast, the enantiomers functionalized with tyrosine substituents at the terminal α-positions of the pentameric thiophene backbone, p-FTAA-L-Tyr and p-FTAA-D-Tyr showed small rather round clusters 10-30 nm in diameter ( Figure 8C). Thus, alterations in the position of the enantiomeric moiety had a large influence on the molecular architecture of acid induced selfassembled structures. Conclusions In conclusion, anionic tyrosine-functionalized pentameric oligithiophenes were shown to exhibit pH-induced optical activity in the π-π* transition region and distinct substitution patterns of L-or D-tyrosine along the thiophene backbone governed the photophysical properties, as well as the self-assembly of the oligothiophenes. Thus, subtle variations in the position of the enantiomeric side-chain functionalities had large effects on the oligothiophenes optical characteristics as well as the molecular arrangement of the self-assembled supramolecular structures. We foresee that our findings will aid in the chemical design of chiral oligothiophenes that can be employed for different optoelectronic and biosensing applications.	2020-11-05T09:05:20.683Z	2020-11-01T00:00:00.000	{ "year": 2020, "sha1": "9f5a247c2348470b8769273b013082352fa2b5cc", "oa_license": "CCBYNC", "oa_url": "https://doi.org/10.1002/open.202000144", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "4adfcd52960f2b44ab4edb1ab057b40d9ed533f8", "s2fieldsofstudy": [ "Chemistry" ], "extfieldsofstudy": [ "Chemistry", "Medicine" ] }
210126456	pes2o/s2orc	v3-fos-license	Physiological aspects of muscular adaptations to training translated to neuromuscular diseases. The high level of complexity underlying the heterogeneous pathophysiology of neuromuscular diseases is a fundamental limiting factor in understanding the role of physical activity in their onset and/or clinical evolution. To overcome this difficulty, it is essential to rely on and deep knowledge of the aetiology and on the physiological adaptations to physical exercise, in order to predict how they can impact on the clinical history of each disease. This paper illustrates the possible strategies of intervention in some neuromuscular disorders, through the analysis of their supposed pathogenic mechanisms. Nevertheless, no clear conclusions can be inferred so far. Introduction Translation of fundamental physiological aspects of skeletal muscle plasticity to neuromuscular diseases may help overcoming the high degree of complexity residing in the existence of a constellation of conditions that are very different in their pathophysiological origin. In particular, open questions regard how to link physiological aspects of muscle adaptation to physical exercise, disuse, and aging to the pathophysiology of neuromuscular diseases and their evolution. Considering the plastic nature of skeletal muscle tissue capable of a wide range of adaptations through qualitative and quantitative variations of cellular protein expression to load and nutrients, the physiological translation should pass through elements of intercorrelations between the cellular structure, from one side, and the its energy disposal, from the other, determined by its use or disuse/non-use, with the leitmotif of the aging process on the background, known to correlate with the arising of age-related diseases and consequent increased probability of death. A fundamental observation from this point of view resides in the positive correlation between activity energy expenditure (AEE), unlike resting metabolic rate (RMR), and longevity, through significant delay of age-related diseases, such as cardiovascular, metabolic diseases, and cancer. As known, the subjects' daily energy consumption includes resting metabolic rate, representing almost 50% of the total expenditure (EE), and AEE, that is the energy consumption due to exercise and daily activities, whereas the thermal effect of meals (TEM) represents a small and constant quote. Of course, the relative proportion of resting and activity expenditure is highly variable between subjects and is strictly linked to their daily habits. A really important finding that may help unraveling the role of energy consumption in longevity is the age-related decline of total energy consumption, reflected by changes in body mass (particularly from birth to 20y) and daily energy consumption (from 2550 kCal/day 18-19y to 2050 kCal/day over 60y WHO/FAO guidelines) (1). In the large part of the population, the aging process adds to the generally observed reduction in the level of physical activity to determine a progressive decline in several physiological capacities. Therefore, in order to get insight into physiology of aging, it is of major importance to try normalizing this process over the level of physical activity. In other terms, it is mandatory to exclude the impact of physical activity to understand what aging per se is. In this sense, a precious help may derive from having a deep look at the ageing processin the so called master athletes (2), who have long lasting commitment to moderate to high intensity physical activity. In these subjects, it can be reasonably considered that the ageing process is, as much as possible, unlinked from inter-subjects variations in the basal level physical activity. That said, in master athlete performance inevitably declines with age and the major determinants of this loss are the reduction in the aerobic fitness (mirrored by VO 2max decrease) (3) and the concomitant changes in the skeletal muscle functional and structural features, including variations in muscle phenotype (generally a fast-to-slow transition) (4). By comparing the aging process in sedentary subjects and master athletes, it is now clear that the maximum rate of oxygen consumption inevitably declines with age but this change is enormously conditioned by the level of physical activity maintained over time. In fact, an elder active subject may express much higher VO 2max than a sedentary young (5). Therefore, being the total energy expenditure and maximal oxygen consumption largely dependent from the basal level of physical activity, from this point of view, a young may be considered elder and vice versa. Considering the organ mass change over time and the relative contribution to total mass of each organ, the skeletal muscle appears as a fundamental contributor of this variation. This phenomenon is highly amplified with age by following an exponential, unlike linear, change due to ageing-associated muscle disuse and is predictable that an earlier exponential change may well represent the age-related variations in the skeletal muscle in presence of neuromuscular diseases, independently from their pathophysiology (6). A fundamental regulator of skeletal muscle mass and its sensing of applied load and energy status is the so called mechanistic TOR, an atypical protein kinase essential for organism survival which plays the role of a catalytic subunit of two distinct dimer multiprotein complexes termed mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (7). In these complexes mTOR acts as a multieffector protein kinase. Four major inputs regulate mTORC1 by cooperating or antagonizing each other and include nutrients (amino acids and glucose), growth factors, energy status, and mechanical stress. In particular, as chemical inhibitors of glycolysis suppress mTORC1 activity, the complex senses the cellular energy of the cell. This phenomenon suggests that changes in energy disposal may promote convergent upstream regulatory signals on mTORC1. As already known, glycolysis and mitochondrial respiration convert nutrients into energy, which is stored in the form of ATP. Glucose loss, inhibition of glycolysis, or mitochondrial respiration cause a significant reduction of the intracellular ATP levels that determines a change in the intracellular ADP/ATP and AMP/ATP ratios. This change is sensed by heterotrimeric complex AMP dependent protein kinase (AMPK) Under nutrient deprivation AMPK transmits stress signals to mTORC1 (8). This process leads mTORC1 inhibition through direct and indirect mechanisms. Importantly, mechanical load and nutrients as branched chain amino acids (BCAA) (9), although first acting at different levels, seem to have a common target on the mTOR biosynthetic pathway controlling protein synthesis. For example, eccentric type exercise is able to activate downstream mTORC1 kinase effector (S6K1) responsible for translation initiation, through changes in the membrane level of phosphatidic acid (PA) (10,11). Importantly, with age, mTORC1 possibly increases its basal level of expression and activation (12). This change appears to be paralleled by reduced responsiveness to stimuli (ie, mechanical load, BCAA), partly ascribable to increased expression of negative regulators. In a condition in which mTORC1 responsiveness is blunted, increased loss of muscle mass and concomitant increased insulin resistance appears to contribute to the arising of age-related diseases. Of importance, muscle-specific inactivation of mTOR leads to severe myopathy, resulting in premature death. In fact, mTOR-deficient muscles display metabolic changes including impaired oxidative metabolism, altered mitochondrial regulation, and glycogen accumulation associated with protein kinase B/Akt hyperactivation. Importantly, loss of mTOR exacerbates the myopathic features in both slow oxidative and fast glycolytic muscles. Moreover, mTOR loss leads to reduced muscle dystrophin content thus exerting a control of dystrophin transcription (13). Physiological muscle adaptations to exercise Muscle adaptations to exercise include morphofunctional modifications triggered by the repetition of muscular contraction bouts, leading to increased mitochondrial biosynthesis and angiogenesis, fibers hypertrophy and fundamental changes in cell metabolism, including increase lactate tolerance (Tab. 1). These adaptations follow modifications of the expression and the activation a series of subcellular signals and a series, still partially unknown, of pre-and post-transcriptional events. The primitive stimulus (exercise or training) with its quality (strength or endurance) are able to activate a large number of cellular events, in turn responsible for the activation, inhibition or modulation of cross linked signaling routes, implicated in the regulation of transcription and translation. At the center of these routes mTORC1 and peroxisome PGC1a are cross-linked master regulators of the plastic responses due to strength type and endurance type exercise respectively (Fig. 1). In particular, as regards mitochondrial biogenesis following endurance type exercise of sufficient duration (14), it is possible to identify two major governors of the process: AMPK and PGC1a AMPK regulator is the energy status of the cell defined by the AMP/ATP and Cr/ PCr intracellular levels. An increased AMP/ATP ratio due to deprivation of glucose, oxidative stress and exercised induced ATP turnover, leads to acute or chronic AMPK activation. In the first case AMKP hypercactivation determines protein synthesis inhibition and increased lipid oxidation. In the latter it stimulates mitochondrial biogenesis. Endurance exercise is also associated with NAD+ fluctuations within the skeletal muscle fibers. NAD+ is a regulator of the deacetylasisirtuine family (SIRT). An increased NAD+/ NADH ratio due to acute exercise and fasting activates SIRT1 in the cytosol and SIRT3 in the mitochondria. The subsequent deacylation of transcription factors and mitochondrial enzymes is followed by a series of adaptations, which increase mitochondrial oxidative function. This adaptation is particularly significant in the presence of low load or in the recovery time from exercise. On the contrary if the load increases, the lactate production reduces the NAD+/ NADH ratio thus blunting the upregulation of mitochondrial function through this mechanism (15). Considering the final physiological effect of endurance type exercise on mitochondrial bio-genesis and oxidative capacity, possible translation of this exercise paradigm regards spinal muscular atrophy. Spinal muscular atrophy (SMA) is caused by homozygous deletion/mutations in the survival motor neuron 1(SMN1) gene with an estimated incidence of 1 in 11,000 live births. It is characterized by motoneurons degeneration in spinal cord and brainstem causing muscle wasting and weakness. In SMA clinical severity ranges from the extremely severe type 1 to the mildest type 3. Approximately 60% of affected infants have a type 1 SMA, with onset of symptoms at 6 months of age or younger, and a median life expectancy of less than 2 years without respiratory and nutritional support. New developmental motor milestones are rarely achieved after diagnosis. The mildest phenotype begins after the acquisition of autonomous ambulation, that is usually preserved till late if the onset was later than 3 ys of age, and gets lost about 10 yrs after the diagnosis if ambulation was acquired by 3 ys of age. Even an adult very mild form has been described, usually called type 4 characterized by adult onset and quite normal motor function. Between the extreme phe- notypes there is a continuum with variable severity. The different phenotypes share the same molecular defects -a homozygous deletion or mutation in the survival motor neuron 1 which results in decreased expression of the survival motor neuron (SMN) protein and degeneration of motor neurons in the spinal cord and brain stem. A paralogous gene, survival motor neuron 2 (SMN2), also encodes the SMN protein; however, 90 to 95% of the translated protein is truncated and nonfunctional as a result of aberrant splicing. The more the SMN2 copies, the mildest the phenotype, as a general rule. Therefore, modulation of pre-messenger RNA (pre-mRNA) splicing of SMN2 to promote increased production of SMN protein has been shown to be an effective treatment strategy across the disease spectrum of spinal muscular atrophy. In addition to muscle weakness, fatigue is a common complaint in SMA. A possible explanation comes from SMA animal models and post-mortem studies, demonstrating abnormal development and maturation of the neuromuscular junction. Neuromuscular dysfunction has been found also in at least half of the patients with SMA. SMN has a role in myogenesis and normal muscle differentiation requires adequate levels of SMN, supporting the hypothesis that a delay in muscle maturation is one of the primary pathologic components of SMA. The association between normal myogenesis and increased oxidative metabolism has been demonstrated. Deregulated myogenesis and impaired mitochondrial biogenesis seem to be inversely proportional to SMN availability, being more prominent in muscle from patients with SMA-I than in muscle from patients with SMA-III. The reduced mitochondrial content makes SMA muscle unable to sustain muscle fiber maturation and contraction properly, contributing to patient weakness and hypotonia (16). Recent clinical observations reporting an incomplete response to aerobic exercise in patients with SMA also sustain this scenario (17). Considering its slow progression, SMA-III may benefit of endurance exercise training in terms of functional performance and exercise capacity through the amelioration of muscle fibers metabolic function and the interruption of further muscle functional and structural impairment induced by the vicious circle of inactivity (18). It has been shown that this exercise paradigm is able to determine positive effects on post-natal maturation of motor units and physical behavior in mouse models of SMA (19,20). Although there are several inconsistencies regarding the human studies, particularly in terms of number of subjects enrolled, optimal training frequency, and possible adverse events (21), initial evidence seems to suggest a positive effect of endurance exercise type on maximal oxygen consumption (VO2max) in SMA-III (22). Despite these premises, considering the vulnerability of patients to exercise-induced fatigue, great future attention will be required to identify the correct dose of endurance exercise to be administered. Another fundamental adaptation to exercise is related to calcium fluctuations within the sarcoplasm due to bouts repetition. As known, increased activity leads to calcium release at the sarcoplasmic reticulum through ryanodin receptors (RyR) and concomitant activation of calmodulin-dependent protein kinase (CaMK) signaling. Calpain 3 (CAPN3) is associated with the muscle triad through its interaction with RyR1. Following calcium release, phosphorylation of CaMK leads to the activation of p38 MAPK, which, in turn, stabilizes the transcriptional co-activator PGC1a, leading to increased transcription of muscle adaptation genes controlled by MEF2, PPAR and, possibly, other transcription factors. CaMK activation also phosphorylates transcriptional inhibitor HDAC, leading to its relocation from the nucleus to the cytoplasm, thus alleviating transcriptional repression. In the absence of CAPN3, as in calpain myopathy (limb girdle muscular dystrophy 2A), the levels of RyR and the amplitude of calcium release are blunted, leading to decreased activation of CaMK. As a result, both branches of downstream events are suppressed determining and the failure to up-regulate transcription of genes necessary for the adaptation to exercise arises. Mutations in calpain 3 gene leads to autosomal recessive limb girdle muscular dystrophy 2A (LGMD2A), characterized by atrophy and weakness of proximal limb and girdle muscles. LGMD2A is one of the most common subtypes of LGMD worldwide, accounting for 15-40% of LGMD cases. Recently, a CAPN3 gene heterozygous deletion(c.643_663del21) has been associated with an autosomal dominant transmission pattern in thirteen unrelated European families (23). Clinical features may include a slowly progressive, symmetrical, limb-girdle weakness and selective muscle atrophy (e.g. hip adductors and extensors, and hamstring muscles), with an onset between the ages of 12 and 20. Scapular winging, scoliosis and joint contractures may also be observed. In general, ambulation loss occurs one to three decades after diagnosis; in fact,20% of LGMD2A patients may become wheelchair dependent before their thirties. Respiratory failure in calpainopathy is known to occur in patients with an advanced stage of the disease, particularly after ambulation loss. Early respiratory insufficiency requiring nocturnal non-invasive ventilation (NIV) in a 70-year-old ambulatory man with LGMD2A has recently been described (24). Most studies, with a few exceptions, have reported the lack of cardiac dysfunction in patients with calpainopathy (25). reported that cardiac function in 33 patients was normal on electrocardiogram and echocardiography, with the exception of 2 patients who had atrial fibrillation. In mice, calpain 3 transcripts are expressed during cardiogenesis, although its expression decreases as the organ matures. The absence in mature cardiomyocytes is a possible explanation for the absence of cardiomyopathy in the majority of patients. A few case reports have suggested cardiac involvement. For example, Okere et al. reported that a 23-year-old patient with calpainopathy had cardiomyopathy (26). Considering the role of calpain and on the pathophysiology of calpainopathy, a working hypothesis in LGM-D2A includes short duration bouts of endurance exercise. Notwithstanding these premises only pilot studies are available on the effects of training in LGMD2A, only focusing on resistance type exercise (27). When the quality of training includes multi joint bouts heavy exercise, possibly with pyramiding increase of weight load and decrease in repetition number, the fundamental skeletal muscle adaptations consist in increased strength and hypertrophy appearance. It is widely known that, in this case, the hypertrophic plastic response of the muscle mostly depends on muscular damage due to the eccentric components of exercise. In fact, following eccentric contractions, a stereotyped response follows particularly in unaccustomed individuals including functional and biochemical features underpinning the structural alterations that the single muscle fibers sustain (Fig. 2). A fundamental role in muscle repair in presence of eccentric induce muscle damage is attributed to dysferlin. DYSF gene, on Chromosome 2p13.2, encodes a protein called "dysferlin," the muscle-specific member of a class of homologous proteins termed "ferlins". Diseases related to mutation in DYSF Gene -Dysferlinopathies-are characterized by a selective and progressive involvement of proximal and/or distal muscles of the limb girdles, usually transmitted in autosomal recessive mode. The age at onset of muscle weakness varies widely (from congenital 14 to 73 years), but usually occurs in the teenage years or early adulthood (on average 15-27 years) (28). Serum creatine kinase (CK) levels are usually elevated (10-100 times normal values) from the early asymptomatic stage of the disease. HyperCKemia characterizes all clinical phenotypes of dysferlinopathy and is a hallmark of the disease. Three main phenotypes are usually associated with dysferlin gene mutations: limb girdle muscle dystrophy type 2B (LGMD2B), Miyoshi distal myopathy (MM) and distal myopathy with Anterior Tibialis onset (DMAT). Links between mutation type, location and phenotype are not straight forward. Dysferlin is responsible for plasma membrane repair, vesicle fusion and membrane trafficking. Not only does dysferlin widely exist in cell membranes of skeletal and cardiac muscles, it also exists in the membranes of non-myofiber cells, such as monocytes. The ferlin protein group to which dysferlin belongs all have Ca2+sensitive C2 domains. It has been demonstrated that an influx of Ca2+through the site of membrane injury triggers dysferlin-mediated membrane repair. Patients with mutations in the dysferlin gene often have impaired membrane resealing following mechanical or chemical stress, causing an influx of Ca2+. This is particularly relevant in muscle where mechanical stress due to muscle contraction is quite common and Ca2+ regulation is highly important. Furthermore, two reports have been published documenting mitochondrial abnormalities in patients with dysferlin mutations (29,30). The first report demonstrated accumulation of subsarcolemmal mitochondria in some patients' muscle fibers including one patient with ragged red fibers and paracrystalline mitochondrial inclusions. More recently up to 10% of muscle fibers were reported to be cytochrome c oxidase (COX or complex IV) deficient, and some of these fibers have increased mtDNA copy number. In the same study, a reduced complex IV and complex III levels were observed in some patients, while others showed a significant downregulation of complex I and complex IV activities and only a mild reduction of complex III. In 2016, Vincent et al. published a work regarding mitochondrial function in dysferlinopathies (30). The authors analyzed skeletal muscle biopsies for eight patients by quadruple immunofluorescent assay to assess oxidative phosphorylation protein abundance and Long-range PCR in single muscle fibers to look for presence of clonally expanded large-scale mitochondrial DNA rearrangements in patients' skeletal muscle. They reported higher complex I and complexIV deficiency in patients than age matched controls but patients do not have rearrangements of the mtDNA. So, they hypothesized that respiratory chain deficiency could be the results of an increased cytosolic Ca2+ concentration (due to a membrane resealing defect) causing mitochondrial aberrations. Considering the crucial role of dysferlin in muscle repair to eccentric mechanical strain and possible translation regards dysferlin myopathies such as LGMD2B and Miyoshi myopathy due to dysferlin gene mutations. In fact, in dysferlinopathy it has been proposed that severity of the dystrophic process mainly depends on type of exercise (concentric/ isometric and eccentric) instead of overload. Therefore, a working hypothesis in this neuromuscular disease include bouts of concentric exercise with abolishment of the eccentric components. To date, no studies have analyzed such a training paradigm in dysferlinopathy. Another fundamental knowledge for translation regards muscle energy disposal and biochemical handling during rest and exercise. It is known that fluctuations in energy pool utilization mostly depends on duration and percentage of the maximal sustainable load that is the exercise intensity (Fig. 3). From this point of view, possible translation examples include mcArdle disease and CPT2 deficiency. The myopathic form of carnitine palmitoyltransferase 2 (CPT2) deficiency, is an inherited metabolic disorder that affects mitochondrial oxidation of long chain fatty acids (LCFA). CPT (carnitine palmitoyltransferase) II muscle deficiency is the most common form of muscle fatty acid metabolism disorders, inherited as autosomal recessive condition. It is one of the possible phenotypes related to mutation in the CPT system, consisting of two enzymes (CPT I and CPT II), involved in the transport of longchain fatty acids into the mitochondrial compartment. The enzymes are located in the outer (CPT I) and inner mitochondrial membrane (CPT II) (31). Three phenotypes of CPT II deficiency are known: a lethal neonatal form, a severe infantile hepatocardiomuscular form, and a mild myopathic form (32). It has also been reported an antenatal onset of CPT2 in a small subset of patients causing malformations including brain dysgenesis and neuronal migration defects (33). Muscle CPT II deficiency is the most frequent type of CPT II deficiency. Clinical features of the mild myopathic form are muscle weakness, myalgia, pain and rhabdomyolysis, possibly causing renal failure. Joshi et al. (2014) (34) showed that the manifestation of clinical symptoms occurred more frequently during infancy (one to 12 years old) than during adolescence (13-22 years old) and adulthood (> 22 years old). Trigger factors are prolonged exer- Figure 3. Schematic representation of the cellular energy handling versus time of exercise. From the top red: free ATP and phosphocreatine/creatine system, blue: anaerobic glycolysis (muscular glycogen), brown: aerobic oxidation (muscular glycogen, plasma glucose, liver glycogen), black: aerobic oxidation (adipose tissue triglycerides, plasma free fatty acids). In muscle CPT II deficiency, symptoms occur only intermittently. Lipid accumulation in muscle fibers can be detected (34). In approximately 90% of the patients the molecular basis is a p. S113L mutation in homozygous or heterozygous state with an allele frequency of 60-70% and more than 60 mostly private mutations (35). The normal protein content and enzyme activity allow a normal function of the CPT system in situations without stress on the fatty acid metabolism (36). The biochemical consequences of the disease-causing mutations are still discussed controversially. In former studies, CPT activities in muscles of patients with CPT II deficiency ranged from not detectable (38,39) up to normal (41,42). It is known that CPT I but not CPT II is sensitive to inhibition by malonyl-CoA. Trevisan et al. showed an almost complete inhibition of total CPT activity in patients by malonyl-CoA (43). So, it was inferred that the normal malonyl-CoA-insensitive CPT II activity is deficient. Due to the demonstration that total CPT activity is normal under optimal assay conditions but abnormal when inhibited by malonyl-CoA, palmitoylcarnitine, carnitine and Trition-X100 (non-ionic surfactant), the hypothesis raised of an abnormally regulated enzyme with a normal total CPT II concentration. CPT II with the S113L mutation, is most vulnerable to inhibition when it is most needed (44). The study of Ørngreen and colleagues (45) about fuel utilization in CPT2 patients showed that they are unable to increase long-chain FAO during long-term, low-intensity cycle exercise, and carriers of single CPT2 gene mutations also can have impaired fat oxidation during exercise, which may explain milder symptoms of CPT II deficiency in these subjects. The authors demonstrated normal FAO at rest but impaired FAO during exercise in CPT II deficiency patients. Furthermore, they showed that the CPT II patients covered their energy deficit by carbohydrate metabolism, by enhanced muscle glycogenolysis (45). McArdle disease, or glycogen storage disease type 5 (GSD5), is an autosomal recessive disease, due to myophosphorylase deficiency and represents the commonest muscle glycogenosis (46). The typical clinical picture of McArdle disease consists of acute crises of early fatigue and contractures, occasionally accompanied by rhabdomyolysis and myoglobinuria usually triggered by muscle tasks that predominantly involve (aerobic/anaerobic) glycolysis for ATP production (47,48). Disease time to onset is usually in the first two decades of life, but it can also occur in infancy with progressive weakness, hypotonia, respiratory distress, and early death (47,49), and later in adult life, with atypical symptoms, such as pain and tenderness in the masticatory muscles when eating (50) or asymmetrical, slowly progressive, limb weakness and muscle wasting which may remain focal (51). The so-called 'second wind' phenomenon, that is marked improvement in tolerance to dynamic exercise (eg, bicycling at a constant, submaximal wattage) after 6-10 min of exertion, with subsequent disappearance of previous tachycardia, is a unique characteristic of patients with McArdle disease (52). Patients lack the enzyme required to mobilize glucose-1-phosphate from skeletal muscle glycogen myophosphorilase, the only isoform of glycogen phosphorylase expressed in skeletal-muscle tissue. Myophosphorylase catalyzes the breakdown of muscle glycogen into glucose-1-phosphate in muscle fibers, preventing the patients to obtain energy from their muscle glycogen stores (53). Muscle glycolysis is not totally impaired in these patients, because the muscle fibers of McArdle disease patients can still take up glucose from the blood and convert it into glucose-6-phosphate, which then enters glycolysis (53). In McArdle disease, due to (54), causing a marked decrease in skeletal-muscle capacity for ATP synthesis through OXPHOS, and in accumulation of ADP and Pi in muscle fibers. This, in turn, can potentially inhibit the myofibrillar ATPase, the sarcoplasmic reticulum Ca2_ ATPase (SERCA1) and the Na + -K + ATPase reactions, leading to decreased contractility and premature fatigue (55)(56)(57). Deficient glycogen dependent ATP supply can result in further downregulation of Na + -K + ATPase in the skeletal muscle fibers of patients and in impaired membrane excitability and muscle cramping (54). The causes for exercise rhabdomyolysis in these patients are not completely understood: either the mechanical stress imposed by large muscle glycogen stores (48), the downregulation of muscle Na + -K + pumps (55) (which are responsible for maintaining cellular volume and integrity), or increased oxidative stress (57) have been supposed to be involved. Furthermore, elevated Ca 2+ levels in the sarcoplasm (owing to the above mentioned downregulation in SERCA1) might activate proteases, phospholipases, and other catabolic enzymes that cause structural damage (58), besides causing muscle fatigue and cramps, Haller and Vissing (59) studied the 'second wind' phenomenon. Either when occurring spontaneously, either when glucose induced, it was due to a substrate-dependent increase in muscle oxidative capacity. Also, by providing glycogen-derived pyruvate a small amount of residual myophophorylase activity normalizes the oxidative deficit of muscle phosphorylase deficiency and may eliminate the 'second wind'. The spontaneous second wind seemed to be related to the lack of fuel that is critical for normal oxidative metabolism and makes muscle oxidative capacity dependent on the changing availability of extramuscular fonts, such as free fatty acids, due to the blocked glycogenolysis. Furthermore, patients with McArdle disease have been reported to be insulin resistant in terms of glucose uptake, glycogen synthase activation, and alterations in fuel oxidation. As a result, the ability of insulin to increase fat and carbohydrate oxidation is limited, and the findings suggest that skeletal muscle glycogen levels play an important role in the regulation of insulin-stimulated glycogen synthase activity (60). The onset of McArdle disease may be at the teenage years during which decreased sensitivity to insulin has been noted, so that there may well be limited transport and oxidation of extracellular glucose and nonesterified fatty acids resulting in reduced exercise tolerance. Insulin sensitivity decreases with age which may play a part in the late onset of some cases (61). Considering the inefficient utilization of glycogen in McArdle disease and long chain fatty acids in CPT2 deficiency, a working hypothesis may be the application of repeated short duration bouts followed by appropriate rest in both cases. This approach may allow to gain the benefits of exercise through phosphocreatine consumption as the main energy substrate to fuel contraction in McArdle disease and through glycogen consumption in CPT2 deficiency in presence of a strict compliance to a dietetic regimen in which reduction of fat is adequately compensated by carbohydrates intake. Preliminary results on the feasibility of this approach in ameliorating the muscle function in McArdle disease have been recently put forward by Santalla and collaborators (54). Conclusions and perspectives Without the deep knowledge of the physiological adaptations to exercise training, declined in terms of quality, intensity, frequency and duration, it is not possible to ascertain whether it is, or not, beneficial in neuromuscular diseases. This knowledge should be translated to pathophysiology, with the aim to find out the optimal matching between potential training adaptations and clinical evolution of the diseases. Without attempting this approach, feasibility of physical interventions will be far to be established.	2020-01-10T12:40:58.507Z	2019-12-01T00:00:00.000	{ "year": 2019, "sha1": "d0a19e8db56b49c3d6a1b0c3aff7b41ae8b406cd", "oa_license": "CCBYNC", "oa_url": null, "oa_status": null, "pdf_src": "PubMedCentral", "pdf_hash": "d0a19e8db56b49c3d6a1b0c3aff7b41ae8b406cd", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
221878394	pes2o/s2orc	v3-fos-license	Analysis on the Construction of Personalized Physical Education Teaching System Based on a Cloud Computing Platform The emergence of cloud computing, the change of education methods, and the requirements of lifelong education make the traditional teaching platform face great challenges. With the rapid development of network technology and computer technology, the speed of updating knowledge is accelerating day by day, and the way of education is gradually changing. Facing the informationization of education, our physical education teaching methods and means are still stuck in the traditional words and deeds, which obviously cannot meet the needs of the development of physical education and health curriculum. In terms of the overall development of sports, school sports is the cornerstone of the country’s sports development. This research is based on cloud computing technology, breaks the framework of the traditional sports model, establishes a personalized sports teaching system according to the basic theory of physical education, and designs and discusses the future college sports model. The construction and application of digital teaching resources of physical education courses in colleges and universities can help solve the problems such as shortage of teachers and contradiction between learning and training. The construction of digital teaching resources of physical education courses in colleges and universities based on cloud computing can save costs and improve resource utilization efficiency. Introduction The education in the new century is mainly to strongly advocate and promote quality education, and the goal of education has also turned to cultivating students' innovative spirit and innovative ability [1]. With the continuous development and construction of the cloud platform and cloud hosting, the current education and teaching reform has ushered in a golden development period. In the development process of teaching in the information age, the learning methods are constantly reformed, and the learning carriers and platforms are constantly changing [2]. In developing China, sports has become a national strategy and an important strategy to promote the realization of the Chinese dream and complete the great rejuvenation of the Chinese nation [3]. Promoting the development of sports for all is the result of the progress of China's times and the inevitable choice for the development of world history. In the face of education informatization, our physical education teaching methods and means still remain in the traditional words and deeds, which obviously cannot meet the needs of the development of physical education and health curriculum [4]. Under cloud hosting, the premise of teaching makes personal space construction continuously improved and also makes the function of cloud hosting more fully explored [5]. The teaching system is the key to cultivating students' talents and the main way to promote students to become pillars. In college teaching, the traditional teaching model has been strongly impacted by informatization, and the reform of the teaching model is imperative. At the same time, the organic combination of computer networks and college teaching is gradually becoming a new development trend. The cloud space of physical education teaching in the new information age effectively makes up for the lack of teaching in our country and makes physical education keep pace with the development of the times, full of vitality and vitality [6]. The rapid development of network technology and computer technology has accelerated the update of knowledge. In order to obtain more and more timely knowledge, the way of education has gradually changed [7]. Online education must be a kind of personalized teaching that meets the needs of individualized learning. Online courses must dynamically adjust the page structure according to the learner's interests, frequency of visits, and time to better meet the needs of learners [8]. With the development of information technology and the widespread application of cloud computing technology in the field of education, the construction of cloud computing has become a hot spot in the work of education informationization [8]. In the past, under the influence of traditional concepts in China's college physical education, the emphasis was placed on the standardization of movements as well as training intensity, making physical education in colleges and universities with certain mandatory specifics [9]. In this way, students do not like and pay attention to it, and it also brings difficulties to PE teachers in teaching courses. The traditional learning platform is a website based on a single hardware facility, which provides learning content through streaming media services and other technical means. The main disadvantages of this method are large investment, long cycle, high maintenance cost, poor scalability, and so on. In order to successfully construct a teaching mode that respects the individual differences of students, combines the individual characteristics of students, and earnestly implements the principle of teaching students according to their aptitude, college physical education teachers must completely change the old traditional teaching mode [10]. With the continuous development of information technology and computer networks, information in the network era has begun to grow by leaps and bounds [11]. Haipeng and others put forward that education cloud technology is to deploy innovative educational applications on the cloud platform through the integration of relevant educational information resources, so as to achieve seamless communication, interaction, resource sharing, and processing among education authorities, universities and educational institutions, and teachers and students [12]. Chen believes that cloud computing adopts service-oriented architecture, which can better support the personalized service of basic education resources. In the actual physical education classroom teaching, many physical education teachers cannot accurately grasp the profound connotation of individualized physical education in colleges and universities [13]. As far as the overall development of sports is concerned, school sports is the cornerstone of the national sports development. Jingrui proposed that the combination of cloud computing and curriculum, and the scientific management of it, can effectively combine various informal learning means and finally promote the development of personal lifelong learning in an all-round way [14]. Haoyue and Xiuqi mentioned in their research that with the help of cloud computing technology, different regions can share high-quality teaching resources and allocate educational projects according to regional characteristics, which can not only greatly promote the reform of the teaching mode but also realize the regional equalization of educational resources as soon as possible [15]. This research is based on cloud computing technology, breaking the framework of the traditional sports model and, according to the basic theory of physical education, establishing a personalized physical education teaching system to design and discuss the future college sports model. Feasibility of Cloud Computing-Assisted Physical Education 2.1. Changes Brought about by Cloud Computing-Assisted Physical Education. With the development of information technology and the wide application of cloud computing technology in the field of education, the construction of a sports teaching service platform focusing on cloud computing is a new hot spot in the in-depth development of school sports information. Cloud computing is an Internet-based, public participation supercomputing model [16]. That is, a large amount of information and processor resources stored on personal computers, mobile phones, and other devices are centralized and work together. The existing network teaching platform has insufficient storage compatibility and cannot implement teaching well across platforms. Faced with numerous digital devices and their complicated operating systems, most physical education teachers are afraid. The cloud platform can provide users with corresponding cloud services. The platform will put the written programs into the cloud and then feed back these information and resources when customers use cloud platform services. The storage performance of cloud hosting is very high, and the personal space behind it is a massive information repository based on cloud computing technology. The new personalized comprehensive teaching platform integrates the characteristics of virtualization, cluster teaching, and experiment; provides a unified solution; and has the characteristics of integration, high density, and multiplatform. Cloud computing, with its superscale, high scalability, high reliability, virtualization, on-demand distribution, cheapness, and versatility, enables ordinary users to enjoy the storage and computing capabilities of high-performance computers on computers with simple configuration, bringing great convenience to human life and work [17]. The "school sports cloud computing" model, which emerged from the application of cloud computing technology in sports teaching, will bring many new changes and development opportunities to sports teaching. At the university stage, students' independence and autonomy are more demanding, but students at this stage are often affected by less social experience. Their independence and autonomy often manifest themselves as arbitrary, sometimes contradictory, and often lead to a loss of self. It can be seen that students at this stage need teachers' active and correct guidance. The biggest characteristic of cloud computing method should be embodied in personalization. Using the information obtained from cloud computing, materials to be learned in teaching resources should be selected and organized in a targeted way dynamically. Two hundred track and field athletes were selected as test samples for this experiment. The basic information of athletes is shown in Table 1. If physical education teachers in colleges and universities can personalize their physical education classes based on their own specialty and students' reality and rely on their own practical teaching experience, they will certainly form their own personalized classroom teaching style that students like and have their own characteristics [18]. Cloud computing-assisted physical education is to give full play to the advantages of computers and the Internet and effectively integrate excellent physical education teaching resources from various places and schools into the physical education teaching process to create an information-based teaching environment. The primary factor to promote the development of college students' sports personality lies first in the training objectives set by colleges and universities. The training objectives of personalized education can be divided into two points. First, students should meet the basic requirements of the objectives of college physical education. Second, according to the characteristics of different students' sports literacy, the designated training plan for teaching students is in accordance with their aptitude [19]. Cloud computing can realize the informatization without its infrastructure. Each unit does not need to build any facilities, as long as the network purchases the required sports teaching resources. The personalized online teaching assistant system based on cloud computing is the assistance and extension of classroom teaching, and it is a tool to help students consolidate and reduce the workload of teachers after class review. At present, the education models commonly used in higher education are unified and standardized. Students expect to meet the needs of sports skills and lifelong physical fitness in physical education. Positioning, Content, and Technical Realization of Cloud Computing-Assisted Physical Education. The goal of a personalized learning system is to recommend the knowledge and information that students are interested in and want to learn according to their personal characteristics, their learning interests, and learning needs, so as to help learners to swim out of the vast ocean of information. With the continuous development of teaching cloud hosting, teaching curriculum resources have been better decomposed. Through the construction of teaching cloud hosting, the updating and arrangement of teaching resources have made great strides towards dynamic. Especially in the update and utilization of the Internet and multimedia teaching resources, the media resource base of physical education courses has been enriched to a great extent. The construction of cloud computing-assisted physical education and the application of resources will change the mode and method of traditional physical education teaching and realize the deep integration of information and physical education [20], so as to achieve the purpose of improving teaching quality and efficiency and realize the "revolutionary influence" of informatization on school physical education. In the construction of cloud space, physical education teachers all over the world can upload some excellent multimedia resources to the education cloud, and teachers everywhere can download these multimedia resources from the education cloud at will. The communication between teachers and students is an important way to ensure that teachers master students' learning situation in time. Through the construction of cloud space, the communication of teaching becomes closer, and at the same time, the communication between teachers and students breaks the boundary, so as to realize the synchronous development of the communication between teachers and students inside and outside the school. Sports test project management refers to the management of information related to sports test projects, which includes the following functional modules: the addition of sports test projects, the modification and deletion of sports test projects, and weight setting. The database tables involved in the implementation process mainly include test project information tables. Only detailed implementation instructions will be provided here for the addition of test items. The implementation process is shown in Figure 1. The basic objectives of the course are determined according to the basic requirements of most students and refer to the objectives determined and worked hard for some students with special skills. The development goal is to continue to develop on the basis of realizing the basic goal. Based on the results of the first stage of work, the number of students who passed or failed was counted, as shown in Table 2. In different stages of the proposed different guiding ideologies of physical education, these guiding ideologies have promoted the university sports theory to play a significant change. Table 3 shows the survey and statistics of the degree of the realization of physical education objectives in physical education teaching in colleges and universities. Prompt "Data added successfully" Prompt " ere is a required field is empty" End Wireless Communications and Mobile Computing There is a multi-index evaluation system composed of n evaluated objects u 1 , u 2 , ⋯, u n and m indicators x 1 , x 2 , ⋯, x m , and x ij = x j ðx i Þ ði = 1, 2, ⋯, mÞ is the observed value of the evaluated object u i on the index x i . The evaluation data matrix can be expressed as : Among them, m, n ≥ 3, the data in A is the preprocessed standardized data, and the physical education evaluation process is described as a general transformation: The script operation module analyzes and classifies the content of the event and determines what type of event it is. Modifying the code is inevitable, because we always have to correct errors, and requirements may change. A vertex without a predecessor vertex in the correlation graph of an interface component is called a starting vertex, and a vertex without a successor vertex in the correlation graph is called a final vertex. Figure 2 is an association diagram of interface components, where "v1" is the starting vertex and "v8" is the final vertex. Automation users can create automation objects, access objects provided by the automation server, get or set object properties, or call object methods. The interaction between automation objects and automation users is shown in Figure 3. Through the use of modern cloud computing technology, teaching management can be effectively improved, and the informatization development of teaching course management, education management, student management, and teacher management is realized, thus further improving the operational difficulty and convenience of teaching management. Before learning enterprise courses, the design concept of physical education courses can be first told to the learners, so that the students can effectively participate in their own course construction and have a very strong interest in learning physical education courses [21]. For example, Table 4 is a survey and statistics on the degree to which the physical education teaching in colleges and universities has achieved the educational objectives of physical education. Table 5 is a survey of students' satisfaction with school physical education. Let a ij and β ij be the column dominance and row dominance, respectively, of the evaluated object u i ði ∈ NÞ on the index x j ðj ∈ MÞ and satisfy If λ ij = μα ij + ηβ ij ði ∈ N, j ∈ MÞ, call λ ij the autonomous superiority of the evaluated object u i ði ∈ NÞ with respect to the index x j ðj ∈ MÞ, where μ is the competitive target coefficient and η is the developmental target coefficient, μ, η ∈ ½0, 1, μ + η = 1. The column strength reflects the difference in strength between the jth index of the evaluated object and the other n − 1 evaluated objects as a whole. The row advantage quantity reflects the overall advantage difference between the jth index of the evaluated object and the other m − 1 indexes. The column advantage α ij ði ∈ N, j ∈ MÞ reflects the strength difference between the jth index of the evaluated object u i and the other n − 1 evaluated objects as a whole. The line dominance reverse β ij reflects the overall dominance difference between the jth index of the evaluated object u i and the other m − 1 indicators. Traditional online courses have the disadvantages of higher development costs and being too static, and once the content of traditional online courses is developed, it is difficult to change them. Now that the speed of people's knowledge update and the pace of life are getting faster and faster, there is an urgent need to support learners' personalized sports learning in online courses. In specific physical education teaching practice, if physical education teachers want to implement personalized teaching strategies, they must completely change the traditional teaching mode in the past [22]. The cloud computing system interface module mainly presents relevant content pages to users for different operations of different users and uses natural language processing, semantic query, cloud computing technology, etc. Figure 2: Interface component association. 4 Wireless Communications and Mobile Computing to provide an operation interface to realize human-computer interaction and realize the interaction between the user and the system. Construction of the Personalized Physical Education Teaching System Cloud computing provides a development environment as a service. Users can use middlemen's equipment to develop their own resources. Physical education teachers in colleges and universities can flexibly use the existing network platform for network teaching. Teaching students in accordance with their aptitude is an important part of modern educational philosophy. In China's traditional physical education, all students are educated by the same educational program, which will make the students' talents and potentials be ignored. University leaders and teachers must attach great importance to the current development of teaching resources and the construction of guarantee mechanisms and actively set up a special department for the construction of educational information cloud hosting to realize the good construction and development of educational resources in cloud hosting. Personalized physical education is based on the educational concept of teaching students in accordance with their aptitude. Different methods are selected for education and training of different students according to their characteristics. In doing so, it is more beneficial to the personal development of students [23]. The teacher platform mainly provides the management interface of the teaching resource database and teaching rule database, presenting the analysis information obtained by the personalized data analysis module. The student platform mainly presents teaching materials recommended by the information scheduling module for different students. The administrator platform mainly presents an interface for managing user information, user rights, and various resources. Mastering sports skills is of course a necessary condition for students majoring in physical education, but under such conditions, only students majoring in physical education can be trained but not talents. This is one of the main problems in the training of physical education professionals in colleges and universities in China. Most PE teachers and students support the introduction of outward bound training in PE teaching, as shown in Table 6. For any two evaluated objects ′Þ as the superiority of u i ′ to u i ′ ′, as follows: In the formula, the aggregate function represents the event probability: The merit matrix S of several evaluated objects can be obtained, and S ij = sðu i , u j Þ is as follows: : ð7Þ The backwardness of practical teaching content is undoubtedly the main reason for the decline of practical teaching quality. Even students majoring in physical education, as future physical education teachers, are also responsible for the healthy growth of future children. Personalized services can be divided into service organization personalization and service content personalization according to different levels. Personalization of service organization means that the platform has a set of services to realize various functions. Service composition can be realized on demand according to different roles, activities, customized contents, and permissions of users. Basic courses are mainly divided into theory and practice. The teaching of basic courses should be innovated in traditional physical education. The user data collection module is the basis for realizing personalization of the whole online teaching assistance system and is mainly responsible for collecting relevant information and data of users. The personalized resource service module of the body is based on the labeled basic education resource database, processes the service requests of users, and uses the technologies of data mining and intelligent push to deeply understand user needs and actively discover potential needs so as to better provide personalized services [24]. The personalized cloud computing analysis module uses different cloud computing algorithms to mine and analyze the information of the students' original database, and the obtained results are stored in the teaching rule base after being normalized, and the personalized information is transmitted to the information scheduling module. In the actual curriculum reform practice process, we must always take students' physical fitness and physical quality as the main line of development and actively carry out physical education courses suitable for the needs of society and students. After the intensive training period, blood ALP data before and after experimental recovery are shown in Figure 4. In the process of human body modeling and simulation, these structural characteristics of the human body, such as freedom of movement, should be fully considered. For the range of motion, the simulation is designed by imitating the structure of the human body, and the simulation can have the same degree of freedom as the human body and complete the same motion as the human body. Based on the analysis of human body structure and actual motion, it is necessary to create constraint pairs between rigid bodies in the human skeleton model to ensure that rigid bodies with relative motion in the model can perform motion simulation according to the real motion mode of the human body. The tensionvelocity relationship of the model is shown in Figure 5. The significance of developmental teaching evaluation lies in changing the old concepts, methods, and means of physical education teaching evaluation. The purpose of evaluation is to examine the students' situation comprehensively, stimulate their enthusiasm for learning, and promote their all-round development. It is also a powerful means for teachers to reflect on and improve their teaching. The students in the experimental group intervened from the beginning of training. Hemoglobin in the experimental group increased significantly. The maximum exercise capacity and anaerobic power of the experimental group increased significantly. During the intensive training period, the athletes in the experimental group are full of energy. The data index of anaerobic work before and after experimental recovery is shown in Figure 6. From the perspective of competitive sports, the latest scientific and technological achievements will soon be transformed into concrete application on the sports field, thus bringing positive factors to create new sports results. From the point of view of school physical education, the curriculum, teaching objectives, teaching content system, teaching methods, assessment methods, and standards are also constantly influenced by contemporary pedagogy theories, showing a flexible development. Under the new situation of the PE teaching reform, the reform of PE teachers' teaching evaluation is urgent. Once there is no compulsory physical education, a considerable number of students will be isolated from physical education and their physical fitness will naturally decrease rapidly. The systematicness and necessity of teachers' selection of teaching contents within the prescribed class hours are inevitably guaranteed, which increases the randomness of teachers' teaching. In addition, schools at all levels seldom consider the main needs of students in the connection of physical education teaching. The demand and problems of sports skills are the center, highlighting the cultivation of ability and quality. According to the imbalance of the overall level of students, different teaching plans of physical education courses are designed to maximize the teaching results. Physical education teachers' teaching viewpoints on students, curriculum, teaching materials, teaching methods, and other aspects will not only affect teachers' judgment and decisions in teaching activities but also affect teachers' teaching behaviors in teaching plans, teaching interactions, and teaching reflections [25]. The function of the information scheduling module is to transfer the corresponding learning contents from the knowledge base and the teaching resource base to the system interface module according to the rules obtained from the teaching rule base and the personalized data analysis module, so as to present personalized learning resources for different students and recommend corresponding exercises for students according to their weak links to strengthen and consolidate [26]. At present, there are quite a lot of learning resources in online physical education courses, including some very high-quality resources. However, these resources are effec-tively integrated in the learning process of physical education courses, thus serving the learning of this course well. In physical education classroom teaching, physical education teachers should respect and understand each student's needs and personality differences and encourage and praise students' unique opinions and different requirements. Conclusions Nowadays, with the popularization of information technology, in the daily teaching activities of colleges and universities, the traditional classroom teaching mode alone can no longer meet the requirements of both teachers' teaching and students' learning, especially there are great differences in the learning ability, learning foundation, and efforts of different students in reality. Cloud computing, as a large-scale resource integration and storage technology, provides convenience for the development and utilization of digital teaching resources. The traditional classroom teaching mode ignores the personalized features of the learning process, and the reform of the teaching mode is imperative. The development of cloud hosting has a very important impact on the development of physical education in our country and has made outstanding contributions to the construction of a benign interactive environment and the innovative development of resources. At present, the traditional class teaching system is still widely used in college physical education, but with the development of the times, this class teaching system cannot meet the internal needs of the development of education. In the process of cloud space development, teaching methods and contents are constantly changing, and the communication between teachers and students and between colleges and universities is becoming increasingly close. Physical education teachers in colleges and universities should actively explore the influence of cloud computing on network teaching methods, increase the construction of digital teaching resources of physical education courses, strive to improve the construction quality and use effect of digital teaching resources of physical education courses, and speed up the process of informatization of physical education in colleges and universities. Data Availability The data used to support the findings of this study are available from the corresponding author upon request. Exercise training Excess recovery training + + + + + + + + + + + + + + + + + + + + Figure 6: Anaerobic work data before and after experimental recovery.	2020-09-24T06:58:55.536Z	2020-09-19T00:00:00.000	{ "year": 2020, "sha1": "fd673f15d156b42df720954a990b82d1214ed135", "oa_license": "CCBY", "oa_url": "https://downloads.hindawi.com/journals/wcmc/2020/8854811.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "fd673f15d156b42df720954a990b82d1214ed135", "s2fieldsofstudy": [ "Education" ], "extfieldsofstudy": [ "Computer Science" ] }
258786196	pes2o/s2orc	v3-fos-license	Towards a Clinically Meaningful Model to Structure the Development of Interoperable Order Sets, Applicable to the Point of Care in Any EMR . Standardized order sets are a pragmatic type of clinical decision support that can improve adherence to clinical guidelines with a list of recommended orders related to a specific clinical context. We developed a structure facilitating the creation of order sets and making them interoperable, to increase their usability. Various orders contained in electronic medical records in different hospitals were identified and included in different categories of orderable items. Clear definitions were provided for each category. A mapping to FHIR resources was performed to relate these clinically meaningful categories to FHIR standards to assure interoperability. We used this structure to implement the relevant user interface in the Clinical Knowledge Platform. The use of standard medical terminologies and the integration of clinical information models like FHIR resources are key factors for creating reusable decision support systems. The content authors should be provided with a clinically meaningful system to use in a non-ambiguous context. Introduction One of the potentials of digitalizing medical records is the ability to implement clinical decision support systems (CDSS) that could help healthcare providers in a variety of decisions and patient care tasks [1].CDSS are software applications designed to assist health care professionals in decision making throughout the care process.When used at the point of ordering, CDSS can integrate evidence-based clinical guidelines with computerized physician order entry systems (CPOE) [2]. Order sets are clinical tools that deliver guidance by providing a list of recommended orderable items applicable in a specific clinical context.A clinical context is a combination of various conditions including disease, symptoms, comorbidities, stage of the problem, stage of the care, demographic patient characteristics, etc. [3] that define the applicability of an order set.Orderable items could be of various types (medication, lab test, imaging, procedure, etc.) and each type could have various attributes (timing, count, frequency, etc.).Standardized order sets can positively influence adherence to evidencebased guidelines and could be used to ensure appropriate orders are being made by health professionals and therefore reduce errors [4]. Despite ongoing development and promising potentials of CDSS in general, lack of interoperability renders many CDSS as cumbersome stand-alone systems that cannot communicate effectively with other systems [1] or may be used only in the EMRs for which they had been developed.This makes the applicability of CDSS including order sets limited to local use.Interoperability enables better workflows and reduces ambiguity among systems and could be used in various areas.For example, interoperable EMRs allow the electronic sharing of patient information between different systems and healthcare providers, improving the ease with which doctors can provide care to their patients.Interoperable CDSS could disseminate the knowledge that they represent in various systems and institutions and reduces the implementation costs through their reusability.The use of standardized formalisms for knowledge representation, like terminologies as well as the integration of semantically enriched clinical information models, contributes to the development of interoperable CDSS [5,6]. We have already presented the Clinical Knowledge Platform (CKP) as an ecosystem in which clinicians could create and share interoperable order sets [3].In this paper, we present the method that we used to define various orderable items and the relevant attributes specific to each orderable item in a clinically meaningful manner.This provides clinicians with an easily understandable platform with necessary items that enables them to create interoperable order sets. Methods We need to provide the order set authors with a tool that empowers them to find the orderable items easily and rapidly, together with the relevant attributes.For that purpose, a working group including health professionals and experts in information technology (IT) and clinical informatics was created. Defining the classification of orderable items: In order to identify the various clinically meaningful categories of orderable items, the health professionals of the working group have studied a set of 325 medical records selected randomly in different general hospitals in France and Germany.Medical records were first randomly assigned to two groups.The first group, the study group, containing two thirds of these records, was used to structure the categories of orderable items.The remaining third was used to validate the categories obtained.The orders of each medical record in the study group were listed.These orders were then analyzed to identify the categories to which they belong.Whenever an order could not be included in a category already created, a new category was added.The categories of orders found in the medical records were therefore enumerated incrementally, as they were discovered.A clear clinical definition was assigned to each category of orderable items to define the meaning and functionalities of each category. Defining the attributes of each category: We then analyzed the orders of each category, to identify the various attributes that a category could include.For example, an order for the prescription of a drug may have attributes such as pharmaceutical form, route of administration, duration of administration, frequency, etc. Mapping to FHIR: CKP uses FHIR resources and standard interrelated multiterminology servers [3] to assure interoperability.FHIR resources for orderable items were investigated by the working group to map the relevant resources to the founded categories.Then, the attributes for each category were discussed and linked to the relevant FHIR content. Developing the order set designer in the CKP: The categories of orderable items and the relevant attributes were then used to develop the user interface in the CKP environment.Various catalogs of orderable items were tagged to the relevant categories and implemented in the CKP database.The content creator could search for orderable items and assemble the desired order set. Results 13 clinically meaningful categories and 40 attributes were identified for orderable items.All the categories of orderable items found in the medical records, together with the mappings to FHIR resources and the definitions are represented in table 1. ServiceRequest Each attribute could be related to one or more categories.For example, an attribute like the day of the week which, if provided, specifies that the action happens only on the specified day, is related to all categories of orderable items.However, the attribute route of administration is related only to medication and nutrition categories.We mapped each category to the relevant HL7 FHIR resource.Various categories were mapped to service request resource which is not necessarily understood by clinicians. These categories were developed from two thirds of the studied medical records and were validated on the remaining third.The validation showed that the data model was able to represent all the orders contained in the remaining third of the medical records. Figure 1 shows some superposed screenshots of the implementation in the CKP. Figure 1.When the category is selected to medication request, the relevant attributes are automatically presented to the user.The attributes are also allocated to some groups to provide the user with a better usability. While creating an order set, the user could directly search the orderable item or find it by choosing the relevant category of orderable items.Then, according to the category to which the orderable item belongs, the relevant attributes will be displayed to the user to specify the exact application of the orderable item in question. Discussion In this study, we have proposed 13 clinically meaningful categories of orderable items together with clear definitions and relevant attributes specific to each item.The content providers and clinicians could therefore create various order sets in the CKP.Using FHIR resources and mapping clinical concepts to standard terminologies in the CKP makes these order sets interoperable and allow them to be shared with various systems. Our approach to the use of standardized formalisms for knowledge representation as terminologies as well as the integration of semantically enriched clinical information models like FHIR resources, corroborates with the results of other studies that consider these issues as key factors for reusable CDSS [5].Standardized order sets are shown to be associated with reduced hospital stays, decreased adverse patient effects, lower risk of mortality and increased cost-effectiveness [6][7][8]. Only health professionals were involved in the phase of defining the categories because the output had to be clinically meaningful.General hospitals were selected because all specialties had to be covered.However, in most hospitals, the orders are already organized by categories that may have a bias on the categories of orderable items that are used while developing an order set.The working group has merged some of the categories (like imaging and nuclear medicine).This was to improve the user experience while searching for orderable items.FHIR resources are complex, not clinically meaningful and include many attributes which are not necessarily understandable by clinicians.That's why we have mapped our categories to FHIR and gathered only functional attributes that are useful in our use case, instead of using original FHIR terms. Further optimization of this structure, by other users than the developer team, including content providers, is required to confirm its generic nature.A quantitative evaluation of the impact of the interface in terms of the ease and intuitiveness to which physicians and content providers could create order sets would be of considerable interest.If the results of the evaluations are promising, recommended order items (within the categories) would be proposed as standards to be used for recommendations.CKP including the standardized and interoperable order sets can pave the way to other possible applications for improving the quality of medical practice.These applications may be based on various clinical contexts in which a physician prescribes or makes orders that are needed to be in accordance with the recommendations.The orderable categories can be also used to standardize clinical pathways, which involve a series of orders over time.Clinical pathways play a crucial role in patient care and standardizing them, like order sets, could lead to enhanced efficiency and better quality of care. Table 1 . Categories of orderable items, definitions, and mapping to FHIR resourcesMedical imaging refers to several different technologies that are used to view the human body in order to diagnose, monitor, or treat medical conditions.It includes radiography, CT, Fluoroscopy, Mammography, MRI, Ultrasound.Nuclear medicine uses radioactive material inside the body to see how organs or tissue are functioning (for diagnosis) or to target and destroy damaged or diseased organs or tissue (for treatment). SurgeryA medical act that is performed for the purpose of structurally altering the human body by incision or destruction of tissues as a part of the practice of medicine.It includes operations, but also activities like installing an ilizarov frame, pacemaker implants, etc.	2023-05-20T06:17:10.848Z	2023-05-18T00:00:00.000	{ "year": 2023, "sha1": "cc45740c6a26db4cbbba9dbfa699e2dadd28d929", "oa_license": "CCBYNC", "oa_url": "https://ebooks.iospress.nl/pdf/doi/10.3233/SHTI230055", "oa_status": "HYBRID", "pdf_src": "Anansi", "pdf_hash": "1ee60a1266849a009ade9cae40426076da306820", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
118599200	pes2o/s2orc	v3-fos-license	On quasi-local charges and Newman--Penrose type quantities in Yang--Mills theories We generalize the notion of quasi-local charges, introduced by P. Tod for Yang--Mills fields with unitary groups, to non-Abelian gauge theories with arbitrary gauge group, and calculate its small sphere and large sphere limits both at spatial and null infinity. We show that for semisimple gauge groups no reasonable definition yield conserved total charges and Newman--Penrose (NP) type quantities at null infinity in generic, radiative configurations. The conditions of their conservation, both in terms of the field configurations and the structure of the gauge group, are clarified. We also calculate the NP quantities for stationary, asymptotic solutions of the field equations with vanishing magnetic charges, and illustrate these by explicit solutions with various gauge groups. Introduction Just a couple of years after the introduction of the concept of (non-Abelian) gauge fields, in his classical paper Utiyama [1] already intended to interpret general relativity as a gauge theory, in which the Lorentz group emerges as the gauge group of transformations taking locally defined orthonormal bases to orthonormal ones. Then Møller [2] formulated general relativity as a dynamical theory of frame fields, and in this new form Einstein's theory showed indeed remarkable resemblance to Yang-Mills theories. This similarity of the two theories made it possible to import techniques developed in one field to the other (see e.g. [3]), yielded a deeper understanding of both (see e.g. [4,5]), or motivated the search for a proper gauge theoretic foundation of general relativity (see e.g. [6,7]). In particular, the concept of charge density of non-Abelian gauge fields appeared to be similar to the (ill-defined) concept of the energy-momentum density of the gravitational field: the vanishing of the gauge-covariant divergence of the (gauge-covariant) non-Abelian charge 4-current (e.g. of the fermionic matter) in itself does not yield any gauge-covariant notion of conserved charges. To overcome this difficulty, Tod [8] introduced the notion of quasi-local charges in non-Abelian Yang-Mills theories, following the analogous twistorial definition of quasilocal energy-momentum and angular momentum of Penrose in general relativity. His construction is based on the use of the holomorphic and anti-holomorphic cross sections of a Hermitian vector bundle. Hence the original construction could give a well defined notion of charge in theories with (pseudo-)unitary gauge groups (or subgroups of them). However, the Lorentz group is not such a group, thus, to be able to see the closest analogy between general relativity and proper non-Abelian gauge theories as it could be possible, it would have to be considered the latter with the Lorentz (or its covering group, the SL(2, C)) gauge group. Therefore, we need to generalize the notion of charge of Tod to arbitrary gauge groups, or at least to the groups above. In general relativity the energy-momentum and (relativistic) angular momentum take the form of 2-surface integrals, like the charge in Maxwell theory. However, in Einstein's theory there are ten additional quantities (and in the coupled Einstein-Maxwell system 16 quantities), taking the form of 2-surface integrals on spherical cuts of future null infinity, which are absolutely conserved. These are the Newman-Penrose (or NP) quantities [9,10]. Though their meaning is still not quite well understood, in the linear approximation they appear to characterize the radiation profile of the incoming radiation, while in stationary configurations they are connected with the multipole structure of the source. Thus the analogy between Einstein's theory and the non-Abelian gauge theories raises the question if the NP quantities can be introduced in Yang-Mills theories, and whether or not they are conserved. Though there is an argumentation that the analogous NP quantities cannot be conserved [11,12], as far as we are aware no such mathematical analysis of the question has been given. Although the charges and the NP type quantities are conceptually different physical quantities, technically they are similar. Thus it seems natural to consider both. Therefore, the aim of the present paper is twofold: first, to generalize Tod's definition of quasi-local charges to arbitrary gauge group and investigate the resulting expression in the standard small sphere and large sphere limits. The second is to form the analog of the NP quantities and clarify the conditions of their conservation. Mostly to fix the notation, in section 2, we review the theoretical background and recall some basic facts that we use. Then, in section 3, we introduce the general notion of the self-dual/anti-self-dual charge integrals. We will see that the notion of electric and magnetic charges can be formulated and studied in terms of these more naturally and easier than the original charge integrals of the field strength. In subsection 3.2 we summarize the key points of Tod's construction for Hermitian vector bundles, and its generalization for arbitrary vector bundles and connections is suggested in subsection 3.3, using either anti-holomorphic or holomorphic cross sections of the vector bundle. Here it is also shown that for globally trivializable bundles the modified construction also gives vanishing magnetic charges. In subsection 3.4. the limit of the quasi-local charge integrals are calculated in three limits. First, the charges are associated with small spheres near a point. We show that in the first two non-trivial orders the electric charge integral is independent of the actual construction, i.e. has some universal nature. The solution of the field equations near a point with the necessary accuracy is given in Appendix 5.1. Then we calculate the spatial infinity limit. We show that under the standard boundary conditions for the gauge fields this limit is finite and constant in time, i.e. these define conserved total charges. In the case of the vanishing total magnetic charges the total electric charges in the holomorphic and in the anti-holomorphic constructions coincide. We also calculate the null infinity limit of the general charge integrals. We show that there is no reasonable definition for the total charges at null infinity that, for semisimple gauge groups, could be conserved in general radiative configurations. There can be charge conservation only in special (e.g. not radiative) field configurations or for gauge groups whose Lie algebra contains a non-trivial solvable ideal, provided the charges are introduced in the anti-holomorphic construction. The holomorphic construction appears naturally at past null infinity. Section 4. is devoted to the Newman-Penrose type quantities. First we show that for semisimple gauge groups no reasonable definition yields conserved NP type quantities in the generic, radiative configurations, and their conservation can be expected only in special configurations or for special gauge groups. We also calculate them in special stationary field configurations and find that they are built from the expansion coefficients of the field strength with dipole and higher moments. To be able to do these calculations we had to increase the accuracy of the asymptotic solutions of the Yang-Mills equations of Newman [3] by two orders. These solutions are given in Appendix 5.2. The signature of the spacetime metric is −2, and in connection with the spin weighted spherical harmonics we use the conventions of [13]. The theoretical background Let E(M) be a real or complex vector bundle over the spacetime manifold M with an n dimensional real or complex vector space F A as the typical fiber, let Clearly, such a frame field is not unique, and we allow the vector basis to be transformed as is a function on U taking its values in some subgroup G of GL(n, R) or GL(n, C). This yields the transformation Thus we think of the set of the frames above as a principal fibre bundle P (M, G) with structure group G, and the matrices Λ A B provide the free action of G on P . Let a connection D e be given on E(M). This can be specified by its action on the vectors E A A of the frame field, yielding the connection 1-form eB , as a locally defined matrix valued 1-form, takes its values in the Lie algebra g of the Lie group G. The curvature (or field strength), also in the local frame field on U, is the g valued 2-form These equations imply, in particular, that the spacetime and gauge-covariant divergence of j A Ba is vanishing, and hence its spacetime divergence in itself is not: . Therefore, by taking the flux integral on some spacelike hypersurface Σ, it is the ∇ e -divergence free and not gauge-covariant current J A Ba := g bc that should be used to define charge. Thus it is more convenient to use the non-gauge-covariant form of the field equations. In the rest of the paper we frequently use the matrix notation instead of writing out the boldface (matrix Lie algebra) or abstract fiber indices explicitly. For example, equation If we fix a basis {e A αB } in the Lie algebra g, α = 1, ..., dim g, then we can write We define the structure constant c µ αβ of g in this basis in the usual way by e A αB e B βC − e A βB e B αC =: c µ αβ e A µC . Then the definitions yield , and that the gauge-covariant derivative of any field e.g. with components X A B =: X α e A αB can also be written in the form The expression between the parentheses is just the gaugecovariant derivative on the vector bundle defined via the adjoint representation of G as an abstract Lie group, i.e. in which the typical fiber is g as its abstract Lie algebra. Then it is straightforward to rewrite the Yang-Mills equations in terms of these notions. Also for later use, we introduce the symmetric metric G αβ := e A αB e B βA on the Lie algebra g. It is a direct calculation to check that e A µB (which are constant functions on the domain U of the frame field {E A A }) as a field on U is constant with respect to the gauge-covariant derivative D e , too. Therefore, the connection is compatible with this metric: D e G αβ = 0. In the adjoint representation, in which the basis {e A αB } is chosen to be the structure constants c µ αβ , this metric reduces to the Killing-Cartan metric g αβ on g. g αβ is well known to be non-singular precisely for semisimple g, and this is negative definite precisely for Lie algebras of compact semisimple (and hence real) Lie groups. The concept of quasi-local charge Let S be any smooth, orientable, closed spacelike 2-surface. Let us fix a Newman-Penrose complex null tetrad {l a , n a , m a ,m a } on S (i.e. l a and n a are future pointing real null vectors orthogonal to S, m a andm a are complex null tangents of S, and they satisfy the normalization conditions l a n a = 1 and m am a = −1), and recall that the area 2-form on S can also be given as ε ab = −2im [amb] , and the area element is dS = 1 2 ε ab (see. e.g. [14]). Note that while l a and n a are globally defined on S, m a andm a are defined only on the local trivialization domains of the tangent bundle of S. Then we search for the electric and magnetic charges in the form where ε A B and µ A B are two appropriately chosen globally defined cross sections of E(S)⊗ E * (S), where E * (S) is the dual bundle, * F ab := 1 2 ε ab cd F cd is the dual of the field strength, where ε abcd is the spacetime volume 4-form. In the expressions on the right we adopted the matrix notation, in which χ 1 := 1 2 F ab (l a n b +m a m b ) is just them a m b component of the anti-self-dual part of the curvature, andχ 1 is them a m b component of the self-dual part of the curvature (see e.g. [3,14,15]). (N.B.: * F abm a m b = −iF ab l a n b . Moreover, though χ 1 is defined as them a m b component of F ab and the vectors m a andm a are only locally defined, χ 1 is globally well defined. In fact, it can be rewritten into the form where ⊥ ε ab := n a l b − l a n b is the area 2-form on the timelike 2-planes orthogonal to S. In terms of the electric and magnetic field strengths defined with respect to some timelike unit vector field t a orthogonal to S, E a := F ab t b and B a := * F ab t b , respectively, where v a is the outward pointing unit spacelike normal of S such that t a v a = 0.) For real bundle and connection χ 1 and χ 1 are complex conjugate of each other, but in the complex case these are independent. Note that for ε A B = µ A B = δ A B the first integral can be rewritten into the flux integral of the trace (in the Lie algebra indices) of the current J a on any spacelike hypersurface Σ whose boundary is S, while the second is precisely the integral of the first Chern class 1 of E(S), the pull back of E(M) to S. Thus E[ε] and M[µ] are interpreted as the electric and magnetic charges, respectively, surrounded by S and defined with respect to the fields is infinite. However, we expect that the number of the independent charges be the number of the gauge potentials, i.e. dim g. Thus ε A B and µ A B should be restricted so that the number of independent charges be the correct ones, and the resulting integrals define reasonable electric and magnetic charge multiplets. Tod's suggestion Tod's suggestion [8] for the quasi-local charges has the form (3.1)- B for appropriately chosen cross sections φ A of E(S) and ϕ † B of E * (S): suppose that E(M) is a complex vector bundle endowed with a non-degenerate Hermitian fiber metric H AB ′ , and assume that the connection D e on E(M) is compatible with this fiber metric, D e H AB ′ = 0. Thus the gauge group is assumed to be some (pseudo) unitary group (or a subgroup of such). Then by means of H AB ′ the complex conjugate bundleĒ(M) can be identified with E * (M) via the fiber mapφ A ′ → ϕ † B := H BA ′φ A ′ . Finally, the section φ A was considered to be anti-holomorphic and ϕ A to be holomorphic, i.e. on the local trivialization domains to be satisfying 1 Since we adopt the convention for the wedge product in which the wedge product of the two 1-forms α a and β a is (α ∧ β) ab = 1 2 (α a β b − β a α b ) (rather than only α a β b − β a α b ), the proper normalization factor of the kth Chern class is 1 Here ð and ð ′ are the standard edth operators on S and φ A and ϕ A are of zero-spinweight functions on the domain of the frame field (see e.g. [9,14]). If V is the space of solutions φ A of (3.4) and U is the space of solutions ϕ A of (3.5), then Tod defines the quasi-local charges to be the eigenvalues of the bilinear mappings E : In the generic case on S ≈ S 2 this construction gives n (not necessarily real) charges, independently of the dimension of the gauge group. The magnetic charges are identically vanishing for globally trivializable bundle E(S). (For the details see [8].) The modified construction Our aim is to modify Tod's construction in such a way that (1) the gauge group can be arbitrary, (2) the number of the electric and the magnetic charges (at least in the generic case) be just dim g, and (3) the charges be real for real bundles and connections; but at the same time keeping the advantageous properties of the original construction (especially the vanishing of the magnetic charges for globally trivializable bundles). In principle there are several ways to ensure the correct number of charges. The first is to consider those cross sections whose components ε A B and µ A B in a local frame field {E A A } (as real or complex n × n matrix valued functions on the domain of the frame field) take their values in the matrix Lie algebra g. In this case, we could always write them as ε α e A αB and µ α e A αB for some, still arbitrary (real or complex) valued functions ε α and µ α . Then it would be the (all together 2 dim g) functions ε α and µ α that would have to be restricted appropriately to depend only on 2 dim g parameters in the generic case. Though conceptually this appears to be the most natural approach, it turns out that this framework is less flexible as it is more difficult to prove statements (e.g. the vanishing of the magnetic charges for globally trivializable bundles) than e.g. in the third one below. Another strategy is to build the charge integrals from the curvature and the fields ε A B and µ A B in the adjoint representation, i.e. when the typical fiber F A of the bundle E(M) is g as an abstract Lie algebra, and it is the structure constants that provide the matrix representation of a basis of g. However, the kernel of the action of the Lie algebra on itself via the adjoint map is not trivial, it is the center of the Lie algebra. Thus this construction may work e.g. for semisimple Lie algebras, but it would not yield any charge e.g. for Abelian Lie algebras. The third possibility is to construct the charge integrals in a vector bundle based on a representation in which the dimension n of the fiber space is just dim g (though the representation is not a priori the adjoint one). Since typically the dimension of the classical Lie algebras grows with the square of the dimension of their defining representation, the representation space with the dimension of the Lie algebra appears to provide enough room for a faithful representation. Restricting the original construction of Tod in this way we get the correct number of charges. For the sake of simplicity we follow this strategy, but, instead of ε A B and µ A B , we formulate our conditions in terms of q A B and q A B . We search for q A B andq A B in the form φ A ψ B andφ Aψ B , respectively, where φ A and ψ B are anti-holomorphic whileφ A andψ B are holomorphic. If the bundle and the connection are real, then φ A and ψ B are anti-holomorphic cross sections of the complexified vector bundles E(S)⊗C and E * (S)⊗C, respectively, and the tilde denotes complex conjugation. In terms of their locally defined components these conditions are , by forming total derivatives and using (3.6) and where γ := m e A A eB andγ :=m e A A eB , and, for the sake of brevity, we used the matrix notation. For general anti-holomorphic q A B and holomorphicq A B we could not prove more. If, however, we use the special form of q A B andq A B given in terms of φ A , ψ B ,φ A andψ B and using (3.6) and (3.7) again, we obtain where δ a is the intrinsic Levi-Civita derivative operator on S. Thus if E(S) is globally trivializable and hence the connection 1-form A A eB can be chosen to be globally defined on S, then the right hand side of (3.8) is also globally defined, yielding identically vanishing magnetic charges. Therefore, the modified construction also satisfies one of the minimal requirement of reasonableness. Let H denote the space of the anti-holomorphic cross sections φ A of E(S), and H * the space of the anti-holomorphic cross sections ψ A of the dual bundle E * (S). Similarly, let H be the space of the holomorphic cross sectionsφ A andH * the space of the holomorphic cross sectionsψ A . Then the anti-self-dual and the self-dual charges are defined to be the eigenvalues of the C-bilinear maps respectively. If E(M) and D e are real, then the complex conjugate of an anti-holomorphic cross section is holomorphic, and henceH is the complex conjugate of H. Similarly,H * is the complex conjugate of H * . Therefore,Q is the complex conjugate of Q, yielding real electric and magnetic charges. Repeating the argumentation of Tod in [8] one can show that for globally trivializable bundles over S ≈ S 2 in the generic case the number of the electric charges is dim g. On the other hand, there are exceptional 2-surfaces for which the construction still works, but the number of charges is different. Though in general q A B = δ A B is not an element of g, it can be considered as the generator of the central extension of the matrix Lie algebra g. Since it is both holomorphic and anti-holomorphic, it is worth considering this extension. E[δ] and M[δ] can be thought of as the 'mean' electric and magnetic charge, respectively, and, as we already noted, the latter is just the first Chern class of E(S). Three limits Though the quasi-local charge integrals Q[q] are well defined even in curved spacetime, for the sake of simplicity in the rest of the paper we assume that the spacetime is the Minkowski spacetime. In particular, we calculate their small and large sphere limits in the flat spacetime. Small spheres Let p ∈ M be an arbitrary point, t a a future pointing timelike unit vector at p, and let us consider the future pointing null geodesics starting from p with affine parameter r and tangents l a satisfying l a t a = 1 at p. Then for sufficiently small r the set S r of the points on these null geodesics whose affine distance from p is r is a smooth spacelike 2-surface, which is homeomorphic to S 2 . Such a surface is called a small sphere about p. Our aim is to calculate the charge integral Q[q] for small spheres of radius r, and to determine the coefficients Q (k) in its expansion Q[q] = Q (0) + · · · + r 4 Q (4) + O(r 5 ). (Since S r is contractible to p, any bundle over S r is globally trivializable, and hence M[µ] is identically vanishing.) The strategy is to expand the curvature component χ 1 and the solutions φ and ψ of (3.6) (or of (3.7)) by powers of r. Since dS r = r 2 dS 1 , clearly Q (0) = 0 and Q (1) = 0, and to calculate Q[q] with r 4 accuracy we need to know χ 1 , φ and ψ with r 2 accuracy. Similarly, expanding the component γ 01 ′ := A a m a of the connection 1-form as a series of r, substituting this to (3.6) and taking into account that ð = 1 r 0 ð, where 0 ð is the edth operator on the unit sphere (and is given explicitly in the appendix), we see that we need to know the expansion coefficients γ (l) 01 ′ for l ≤ 2. However, as the analysis of Appendix 5.1 shows, to determine the solution of the Yang-Mills equations on the light cone (more precisely, on the null boundary of the chronological future) of p for χ 1 with r 2 accuracy, formally γ 01 ′ , γ 10 ′ and γ 11 ′ must be expanded with accuracy r 3 . The solution of the hypersurface equations (5.1)-(5.3) is given in Appendix 5.1, by means of which the equations (3.6) themselves are Since φ and ψ are multiplets of type (0, 0) scalars, To solve the third of (3.11) and (3.12) first let us recall that in terms of the spinor form of the field strength, and hence it is straightforward to integrate (3.11) and (3.12) in the coordinates (ζ,ζ): (3.14) 0 and ψ (2) 0 are arbitrary constant sections, being the general solution of the homogeneous equation. Then, using the pattern of (3.13)-(3.14), it is easy to write down the holomorphic solutions, too. Though for small spheres the solution spaces H and H * are dim g dimensional, the space of the O(r k ) accurate solutions is (k + 1) dim g dimensional. The presence of the 'extra', spurious solutions is a consequence of the lack of a natural isomorphism between the solution spaces on different two-surfaces, and hence, in particular, with different radii. These yield some ambiguity in higher accurate approximations. (For a more detailed discussion of this issue in general relativity, see e.g. [16]. Also, in the calculation of certain integrals, we will use equation (2.5) of [16].) Since φ (0) and ψ (0) are constant, by (5.11) the O(r 2 ) order term Q (2) is vanishing. For the O(r 3 ) order term we obtain where the integration is taken on the unit sphere. Thus Q (3) is the volume of the 3-ball of radius r times the timelike component of the gauge covariant current at p. This result coincides with that of Tod, too. In fact, let us observe that in Q (3) only the zeroth order terms of the cross sections φ and ψ matter (the others integrate to zero), which are constant both for the holomorphic and anti-holomorphic sections. In the fourth order we obtain Though formally Q (4) is built from the O(r 2 ) accurate cross sections, their O(r 2 ) order correction terms are integrate to zero. Hence we obtain the same result in the holomorphic and the anti-holomorphic constructions. On the other hand, in general Q (4) does depend on the O(r) order 'gauge solutions' φ (1) and ψ (1) : i.e. when j µ a t a = 0 at p. The calculation of Q (5) is much longer and technically more difficult, so we summarize only the main message of that. First, ambiguities similar to that in Q (4) , parameterized by the 'extra' solutions φ (1) , ψ (1) , ... etc, appear, unless the third and fourth order terms, Q (3) and Q (4) , are vanishing. The difference of the holomorphic and anti-holomorphic constructions can appear in this order. Thus Q (3) and Q (4) have some 'universal' nature, not being sensitive to the details of the defining equations for the cross sections φ and ψ, provided φ and ψ are constant in the zeroth and first orders. Large spheres at spatial infinity Let (t, r, ζ,ζ) be the standard spherical, complex stereographic coordinate system (at least in a neighbourhood of spatial infinity) in Minkowski spacetime, and consider the charge integral Q[q] on the metric 2-spheres S r of radius r in some t = const spacelike hyperplane. Let t a and v a be their future pointing unit timelike and outward pointing unit spacelike normals, respectively, such that t a is orthogonal to the t = const hyperplanes and t a v a = 0. Let m a andm a be the complex null tangents of the 2-spheres. Then we impose the following a priori fall-off conditions for the scalar potential and the radial and tangential parts of the spatial vector potential: , respectively, for some a, b, c > 0. In addition, we assume that the time derivative of the last three satisfy (A a v a )˙= O(r −β ),γ = O(r −γ ) andγ = O(r −γ ) also for some β, γ > 0. By technical reasons we also assume that the differentiation with respect to r reduces these orders by one, but the differentiation with respect to ζ andζ does not change these orders. Then, expressing χ 1 = 1 2 (E a + iB a )v a by the potentials, we find that χ 1 = O(r −2 ) if a = b = c = 1 and β = 2. Therefore, these boundary conditions ensure the existence of the r → ∞ limit of the charge integrals Q[q] on 2-spheres S r of radius r provided q = O(1). (If q were not involved in the 2-surface integral of the curvature, i.e. if that were only the 2-surface integral of the components of the field strength in some globally defined frame {E A A }, then one could rewrite that as the 3-volume integral of its divergence on some spacelike hypersurface Σ with boundary S. Then, using the constraint equation, one could show that even weaker boundary conditions could ensure the finiteness of the original 2-surface integral [17]. Since, however, q is defined only on S, here this strategy cannot be followed, and hence we have the above 'conservative' fall-off conditions.) Next let us determine the conditions that ensure the vanishing of the r → ∞ limit of the time derivative of Q[q], To determine the order of the second term of the integrand we use the evolution equations for the field strengths in the present geometrical situation (i.e. flat spacetime foliated by spacelike 3-planes with vanishing shift vector). These arė where D a denotes the space-and spatial gauge-covariant derivative operator (i.e. for example its action on any cross section t a is the orthogonal projection to the t = const hyperplanes). By these equationsχ 1 , then the r → ∞ limit of the time derivative of Q[q] is zero. Note that by γ = 1 + ǫ the asymptotic form of γ (and ofγ, too) is i.e. its leading order term does not depend on t. Finally, let us write q = q (0) + r −ǫ q (1) + o(r −ǫ ) and substitute into the equation of the anti-holomorphicity of the cross section q. We find Thus its solution is such that the leading term q (0) does not depend on time, and hencė q = O(r −ǫ ). Therefore, under the fall-off conditions for the vector potential above, the spatial infinity limit of Q[q] exists and is conserved in time. It might be interesting to note that here we used only the anti-holomorphicity of q A B , but did not assume that it has the dyadic product structure φ A ψ B . Note that in general γ (0) cannot be transformed by gauge transformations to zero globally unless the leading term of the v a component of the magnetic field strength is vanishing. Thus q (0) is not constant on the unit sphere, and hence the total charge depends on whether q is anti-holomorphic or holomorphic. If the connection coefficients γ (0) and γ (0) can be made vanishing globally, then the total magnetic charges are vanishing and the total electric charges introduced in the holomorphic and anti-holomorphic constructions coincide. Tr(qχ 0 1 )dS 1 , where the integration is taken on the unit sphere. Hence this integral is still a function of the retarded time coordinate. In this subsection we show that for semisimple gauge groups no reasonable definition yields conserved total charge in general, radiative configurations. In particular, neither Tod's original definition (as pointed out in [8]) nor the modified one yield conserved total charges. By integration by parts and using (5.15), for the u-derivative of Q[q] we obtaiṅ Q q = 1 2π Tr qχ 0 1 − 0 ðq + γ 0 , q χ 0 2 dS 1 . (3.19) Since in any given retarded time instant u = u 0 in a generic (radiative) configuration χ 0 1 and χ 0 2 are independent data (because χ 0 1 +χ 0 1 andγ 0 can be specified independently, see Appendix 5.2.1), the integrand can be zero precisely when the coefficient of χ 0 1 and of χ 0 2 are vanishing. In particular, q A B must be anti-holomorphic, but in addition it would have to be constant in time even though the anti-holomorphic structure might be changing from cut to cut on I + :q The condition of the integrability of the system of these equations is [q,γ 0 ] = 0. Thus the charge Q[q] with anti-holomorphic q A B is conserved in the absence of outgoing radiation, γ 0 = 0, or when q is anti-holomorphic and belongs to the center of the Lie algebra g, too. Therefore, for semisimple gauge groups in the generic radiative configurations there is no total conserved charge. Charge conservation requires the non-triviality of the radical (i.e. the maximal solvable subalgebra) of g in its Levi-Malcev decomposition. Remarkably enough, though in the presence of outgoing radiation there is no charge conservation, the structure of the Yang-Mills fields at future null infinity singles out just the anti-holomorphic cross sections q A B in a natural way. Thus our suggestion for the quasi-local charges is justified by the conservation properties of the total charges at future null infinity. At past null infinity the holomorphic cross sections would appear. Even for semisimple Lie algebras and non-stationary, but special configurations, in which χ 0 0 and χ 0 1 +χ 0 1 are not quite independent, the total charge can be conserved. Such are the Liénard-Wiechert type solutions. In [18] Tod defined them geometrically as the algebraically special solutions of the Yang-Mills equations for which the field strength components χ 0 andχ 0 are vanishing, and, in contrast to our present framework, the origin of the coordinate system (u, r, ζ,ζ) (i.e. the 'source's world line') is allowed to be an arbitrary timelike curve. He showed that there is an anti-holomorphic frame in which χ 0 1 is constant both in time and on the cut of I + , and hence that the total charge is, in fact, conserved. (For another approach to the Liénard-Wiechert type configurations, based on certain assumptions on the explicit form of the 4-potential in terms of a pointlike source, the so-called colour, see [19,20], and for a recent review of them see e.g. [21]). In the special stationary case discussed in Appendix 5.2.2 γ 0 andγ 0 are vanishing and χ 0 1 = 1 2 C µ e A µB for some real or complex constants C µ . Thus, in particular, the antiholomorphicity of q A B yields that it is constant, i.e. q A B = q µ e A µB for some constants q µ . Therefore, Q[q] = C µ e A µB q B A = C α G αβ q β , yielding the meaning of the constants C µ of the solution: they should represent the total charge of the system. In fact, if we search for q A B in the form φ A ψ B for anti-holomorphic φ A and ψ B , then their anti-holomorphicity yields that they are constant, and hence that Q[q] = C µ e A µB φ B ψ A . Thus, indeed, the charges introduced in subsection 3.3 are the eigenvalues of C µ e A µB . Newman-Penrose type quantities The Newman-Penrose conserved quantities in general relativity and in Maxwell theory (and, in fact, in any theory based on a linear zero-rest-mass field equation with any spin or on the conformally invariant wave equation) are built from the second nontrivial expansion coefficient of the tetrad component of the curvature with the highest spinweight, weighted with appropriate spin-weighted spherical harmonics [9,10]. Thus it is natural to define where ω is a cross section of (in the case of real bundles, the complexified) E(M)⊗E * (M) over the u = u 0 cut of I + with −1 spin weight so that the integrand of (4.1) is globally well defined. The (non-)conservation of the Yang-Mills NP quantities Here we show that for semisimple gauge group and any non-trivial choice for ω the quantity Ω[ω] is not conserved in generic, radiative configurations. Using (5.17) a straightforward calculation (by integration by parts) yieldṡ Since χ 0 0 = χ 0 0 (ζ,ζ) and χ 1 0 = χ 1 0 (ζ,ζ) are freely and independently specifiable functions at u = u 0 (see Appendix 5.2.1), Ω[ω] is constant in time precisely when their coefficients in (4.2) are vanishing: Using (5.14)-(5.15) of the appendix, the integrability condition of this system of equations is is conserved if ω satisfies (4.3) and there is no outgoing radiation, i.e. χ 0 2 = 0; or if ω belongs to the center of the Lie algebra g and solves 0 ð 0 ð ′ ω = 3[ω, χ 0 1 ] = 0. Therefore, the NP type quantities could be conserved in special situations (e.g. for a collection of independent Maxwell fields), but not conserved in general. However, this non-conservation of the NP quantities in non-Abelian Yang-Mills theories appears to be known for a while, as Bazanski pointed out to Newman in an unpublished private communication [11,12]. Since for the Liénard-Wiechert type solutions of the Yang-Mills equations, considered in [18], the curvature components χ 0 andχ 0 are identically vanishing, the NP quantities for these solutions are vanishing for any gauge group. Yang-Mills NP quantities in stationary configurations It is known that in stationary configurations the NP quantities in the theories based on the linear zero-rest-mass field equations (e.g. in particular in Maxwell theory or the linearized Einstein theory) are vanishing. On the other hand, in stationary spacetime the gravitational NP quantities of the full non-linear theory reduce to a nontrivial combination of the mass and the dipole and quadrupole moments of the source [9]. Thus their nonvanishing is due to the nonlinearity of the theory. This result raises the question whether the analogous quantities Ω[ω] in non-Abelian Yang-Mills theories could be non-vanishing in the stationary configurations, and if they could, then what would be their meaning? In the special stationary case discussed in subsection 5.2.2 equation (5.18) reduces to Writing ω = ω µ e A µB for some spin weight −1 functions ω µ and expanding the latter as Thus ω µ 1m can always be non-zero, and if C µ ν , the 'charge matrix' in the adjoint representation, has only one eigenvector with zero eigenvalue then ω µ 1m = C µ ω m for arbitrary constants ω m . If, in addition to C µ , there are other eigenvectors with zero eigenvalue, say Ĉ µ , ... etc, then ω µ 1m will be a linear combination of all them with arbitrary coefficients ω m ,ω m , ... etc. However, ω µ jm for j ≥ 2 can be nonzero only in the exceptional case when 1 3 (j − 1)(j + 2) is an eigenvalue of C µ ν . (For a more detailed discussion of C µ ν see subsection 5. 2.2.) Substituting these into the definition of Ω[ω], by integration by parts we obtain where the functions h µ ,h µ and k µ have been introduced via χ 0 0 =: h µ e A µB ,χ 0 0 =:h µ e A µB and χ 1 0 =: k µ e A µB , respectively. Thus, in particular, if the Lie algebra g is Abelian, then by (4.6) Ω[ω] = 0, in accordance with the known fact that the NP quantities are vanishing for (an Abelian multiplet of) stationary Maxwell fields. Thus in the rest of the paper we assume that g is not Abelian. First suppose that C µ ν is generic (see Appendix 5.2.3). Then the only non-zero expansion coefficients of ω µ in terms of spin weighted spherical harmonics is ω µ 1m , while the expansion coefficients of h µ and k µ (and in the complex case ofh µ , too) are determined in Appendix 5.2.3. Substituting those solutions into equation (4.6), using the notation of the Appendix and elementary properties of the edth operators and the spin weighted spherical harmonics (e.g. the orthogonality of these harmonics with different indices), we obtain In the special case when the only eigenvector of C µ ν with zero eigenvalue is C µ and − 4 5 is not an eigenvalue of C µ ν this reduces to zero. Thus the non-Abelian nature of the Lie algebra g in itself is not enough to have non-trivial NP quantities. If, however, − 4 5 is an eigenvalue and k µ 1m are the corresponding eigenvectors, then Ω[ω] can be non-zero. In the other extreme case when C µ ν = 0 (e.g. when the total charge is vanishing) it gives 1 2 G µν 1 m=−1 (−) m H µ 1m ω ν 1−m , which is a homogeneous quadratic expression of the coefficients h α m andh α m characterizing the dipole structure of the Yang-Mills field. We saw in Appendix 5.2.3 that for the Lie algebras so(3), sl(2, R), and sl(2, C) the charge matrix has only one eigenvector with zero eigenvalue while for so (1,3) there are two, moreover there could be exceptional configurations for the last three Lie algebras, but not for so (3). Therefore, for the Lie algebra so(3) in general and for sl(2, R), sl(2, C) and so (1,3) in the generic case the NP quantities can be non-vanishing e.g. when the total charge is zero. Finally we consider the exceptional configurations of Appendix 5.2.3. In this case the solution of (4.5) is Substituting this and the solution of (5.33) into (4.6), and using the orthonormality of the spin weighted spherical harmonics on the unit sphere, we obtain that which can also be non-zero in general, even if the charge matrix has a single eigenvector with zero eigenvalue. In this case, however, Ω[ω] is built from higher, actually the 2 J th order multipole moments h µ Jm andh µ Jm of the curvature. Appendix: The solution of the Yang-Mills equations In this appendix we summarize the structure of the Yang-Mills fields near a point (subsection 5.1) and near the future null infinity in Minkowski spacetime (subsection 5.2). For the generalization to the coupled Einstein-Yang-Mills equations in the Newman-Penrose formalism, see [15]. Following [3] we use the coordinates (u, r, ζ,ζ), where u := t − r is the standard retarded null coordinate and (ζ,ζ) are the complex stereographic coordinates on the unit sphere. The complex null Newman-Penrose tetrad {l a , n a , m a ,m a } is adapted to the u = const, r = const 2-spheres, the edth operators on the unit metric sphere will be denoted by 0 ð and 0 ð ′ and their action on a scalar η of GHP type (p, q) is 0 ðη := respectively. The components of the connection 1-form A e in the NP basis are denoted by γ 00 ′ , γ 11 ′ , γ 01 ′ and γ 10 ′ , respectively. The components of the curvature are χ 0 := F ab l a m b , 2χ 1 := F ab (l a n b + m a m b ), χ 2 := F abm a n b ,χ 0 := F ab l amb , 2χ 1 := F ab (l a n b + m amb ) andχ 2 := F ab m a n b . Note that for real connections γ 00 ′ and γ 11 ′ are real and γ 10 ′ is the complex conjugate of γ 01 ′ . Similarly, also for real connections,χ 1 ,χ 2 andχ 3 are complex conjugate of χ 0 , χ 1 and χ 2 , respectively, but for general complex connections they are independent. In the gauge γ 00 ′ = 0 of Newman [3] the expression of the components of the field strength by the components of the connection 1-forms and the field equations (2.1) together with the Bianchi identities take the form 3) The first four are only the constraint (or 'hypersurface') equations, while the remaining three are the evolution equations. In the complex case there are analogous equations for the independent curvature componentsχ 0 ,χ 1 andχ 2 , too. The solution of the hypersurface equations near a point In the small sphere limit calculations of subsection 3.4.1 we need to know only χ 1 and γ 01 ′ on the light cone of the point p ∈ M. Thus we need to solve only (5.1)-(5.3) with the accuracy required by the small sphere calculations. Hence we expand the components of the curvature and the connection 1-form as χ = χ (0) + rχ (1) + r 2 χ (2) + O(r 3 ) and γ = γ (0) + · · · + r 3 γ (3) + O(r 4 ), respectively, as well as j = j (0) + rj (1) + O(r 2 ). Then the solution of (5.1) and its tilded version is 10 ′ = 1 4χ (2) 0 , (5.9) The sum of (5.2) and its tilded version yield 1 +χ The difference of these and their tilded version reproduces the expressions for χ 2 + · · · can be derived from (5.4) in an analogous way. The asymptotics of Yang-Mills fields near null infinity In [3] Newman solved the source free Yang-Mills equations near the future null infinity, and determined the freely specifiable functions of such solutions. In subsection 5.2.1 we present the results of the analogous analysis with the higher accuracy of the asymptotic expansion required by the analysis of section 4. In subsection 5.2.2 the asymptotic field equations are solved in certain special stationary configurations. In subsection 5.2.3 we discuss some examples with specific gauge groups. Special stationary configurations Let the Yang-Mills field be stationary with respect to u, i.e. all the dot-derivatives are vanishing. Newman and Penrose showed [9] that in the analogous gravitational case the asymptotic shear can be made zero by an appropriate supertranslation of the origin cut. However, now the integrability condition of the local existence of a gauge transformation Λ(ζ,ζ) taking γ 0 andγ 0 to zero is the vanishing of the corresponding curvature, which is proportional to the right hand side of (5.19). Hence, for example, in the presence of magnetic charges (see equation (3.2)) there is no such a gauge transformation. Thus in the rest of this subsection we consider those special stationary solutions of the asymptotic field equations in which γ 0 = 0 andγ 0 = 0 can be ensured even globally, and hence χ 0 1 =χ 0 1 holds. Thus the total magnetic charges are vanishing. (If the connection is real, then χ 0 1 is also real.) Then equation (5.16) immediately gives that χ 0 1 is constant on S, and hence has the form χ 0 1 = 1 2 C µ e A µB for some constants C µ . These constants are real for real bundles and connections, while in the complex case they are complex but C µ = C µ . We saw in subsection 3.4.3 that the constants C µ represent the total charges. To solve equation (5.17), let us write χ 0 0 = h α e A αB for some functions h α with spin weight one. Then (5.17) takes the simple form Thus C µ ν is a constant dim g × dim g real or complex matrix. Expanding h µ in terms of the s = 1 spin weighted spherical harmonics with the constant coefficients h µ jm as and recalling that the action of the edth operators on spin-s spherical harmonics is Thus the expansion coefficients h µ jm must be eigenvectors of C µ ν with the real non-positive eigenvalues − 1 3 (j − 1)(j + 2). By the definition of the charge matrix C µ ν C ν = 0 holds, i.e. λ = 0 is always an eigenvalue with eigenvector C µ . Thus, in particular, h µ 1m can always be non-zero, and if C µ is the only eigenvector of C µ ν with zero eigenvalue then h µ 1m = C µ h m for some constants h m . If the dimension of the kernel space of C µ ν is k > 1, then h µ 1m will be the linear combination of the vectors of a basis of this kernel with arbitrary constants h m ,ĥ m , ... etc. However, since C µ ν is a finite matrix, it may have only finitely many eigenvalues, and hence only finitely many coefficients h µ jm can be non-zero. Nevertheless, since the total (electric) charges C µ can be specified freely, in general no eigenvalues of C µ ν has the form − 1 3 (j − 1)(j + 2) for some j ≥ 2. Therefore, apart from the exceptional cases in which the charges have this structure, the expansion coefficient functions χ 0 0 and χ 0 0 are combinations only of the spherical harmonics 1 Y 1m and −1 Y 1m , respectively. Similarly, if we write χ 1 0 = k α e A αB , then equation (5.18) reduces to the linear, but inhomogeneous equation Following the strategy of the previous paragraph and expanding k µ as for the expansion coefficients k µ jm of the solution of the homogeneous equation we obtain the condition Thus the general solution of the homogeneous equation contains non-trivial k µ jm for j = 2 only if the matrix C µ ν is chosen to be very special. This solution depends only on finitely many parameters. A particular solution of the inhomogeneous equation (5.27) can also be found by using the spin weighted spherical harmonics and the expansion of their product, s Y jm s ′ Y j ′ m ′ , by the appropriate Clebsch-Gordan coefficients and the spherical harmonics Thus, to summarize, in the present special stationary case the asymptotic field equations can be integrated completely and these solutions (with the second order accuracy, i.e. keeping only χ 0 0 , χ 1 0 and χ 0 1 ) can be indexed by the freely specifiable total (electric) charges C µ and the expansion coefficients h µ 1m and k µ 2m (and, in the complex case, bỹ h µ 1m andk µ 2m , too). Other parameters may occur in the solutions (viz. in the expansion coefficients χ 0 0 , χ 1 0 , etc) only when the charges are chosen in very specific ways. Special stationary solutions: Examples First consider the generic case, i.e. when no non-vanishing eigenvalue of C µ ν has the form ± 1 3 (j − 1)(j + 2) for some j ∈ N. Then by (5.26) we have that h µ = 1 m=−1 h µ 1m 1 Y 1m and h µ = 1 m=−1h µ 1m −1 Y 1m , and then, using the expressions how the edth operators act on the spin weighted spherical harmonics, (5.27) reduces to Its second line can be written as 1 where H µ jm , j = 1, 2, are explicitly given, homogeneous quadratic expressions of h µ 1m andh µ 1m , and are built also from the structure constants and the Clebsch-Gordan coefficients. With this substitution we obtain that Moreover, if C µ ν has an eigenvalue 1 5 ((J − 1)(J + 2) − 4) for some J ≥ 3 then k µ Jm are its eigenvectors while k µ jm = 0 for all j = 3, ..., J − 1, J + 1, ...; otherwise the coefficients k µ jm are vanishing for all j ≥ 3. The first of (5.31) can be inverted to find the unique solution k µ 1m precisely when − 4 5 is not an eigenvalue of C µ ν , otherwise the corresponding eigenvector can be freely added to the particular solution. Similarly, k µ 2m is determined only up to the general solution of the homogeneous equation C µ ν k ν 2m = 0. If the only eigenvector of C µ ν with zero eigenvalue is the charge vector C µ , then h µ 1m = C µ h m andh µ 1m = C µh m , which imply that H µ jm = 0. (For the sake of simplicity we assume that the structure constants are real.) Hence k µ 1m is either an arbitrary combination of the eigenvectors of C µ ν with eigenvalue − 4 5 or zero, while k µ 2m is a solution of C µ ν k ν 2m = 0. If, in addition to C µ ,Ĉ µ is another eigenvector of C µ ν with zero eigenvalue, then h µ = 1 m=−1 (C µ h m +Ĉ µĥ m ) 1 Y 1m , implying that H µ jm are proportional to c µ αβ C αĈ β = C µ βĈ β = 0, i.e. H µ jm = 0 again. In the other extreme special case when the charge matrix is vanishing (e.g. if C µ = 0) k µ 1m = − 1 2 H µ 1m holds, k µ 2m are arbitrary, but now H µ 2m = 0 must hold. This yields restrictions on the coefficients h µ 1m andh µ 1m . The evaluation of the explicit formulae show that then H µ 10 = 0, but H µ 1±1 are still non-zero. To illustrate the above possibilities, let us specify the Lie algebra g. First it is chosen to be the abstract Lie algebra sl(2, R), so(3) (i.e. su(2)) or sl(2, C); and let us choose the basis {e 1 , e 2 , e 3 } such that [e 2 , e 3 ] = ∓e 1 , [e 3 , e 1 ] = e 2 and [e 1 , e 2 ] = e 3 , where the upper sign corresponds to sl(2, R) and the lower to so(3) and sl (2, C). In this basis the components of the Killing-Cartan metric are g αβ = 2diag(−1, ±1, ±1). The eigenvalues of C µ ν are λ = 0, in which case the corresponding eigenvector is proportional to the total charge vector C µ , and λ = ± 1 √ 2 g αβ C α C β . The non-zero eigenvalues can be real for g = sl(2, R) if g αβ C α C β > 0 and for g = sl(2, C) also in special cases (e.g. for purely imaginary C µ ), but never for g = so(3). Thus the configurations for g = so(3) are always generic.	2011-05-30T14:28:14.000Z	2010-12-21T00:00:00.000	{ "year": 2010, "sha1": "56fecf2d5a3845799979985ba356ae6243fb92bc", "oa_license": null, "oa_url": "http://arxiv.org/pdf/1012.4662", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "56fecf2d5a3845799979985ba356ae6243fb92bc", "s2fieldsofstudy": [ "Mathematics", "Physics" ], "extfieldsofstudy": [ "Physics" ] }
890206	pes2o/s2orc	v3-fos-license	Adiabatic Markovian Dynamics We propose a theory of adiabaticity in quantum Markovian dynamics based on a decomposition of the Hilbert space induced by the asymptotic behavior of the Lindblad semigroup. A central idea of our approach is that the natural generalization of the concept of eigenspace of the Hamiltonian in the case of Markovian dynamics is a noiseless subsystem with a minimal noisy cofactor. Unlike previous attempts to define adiabaticity for open systems, our approach deals exclusively with physical entities and provides a simple, intuitive picture at the underlying Hilbert-space level, linking the notion of adiabaticity to the theory of noiseless subsystems. As an application of our theory, we propose a framework for decoherence-assisted computation in noiseless codes under general Markovian noise. We also formulate a dissipation-driven approach to holonomic computation based on adiabatic dragging of subsystems that is generally not achievable by non-dissipative means. Introduction.-The adiabatic theorem is a simple and powerful result that has been known since the early days of quantum mechanics [1,2]. It states roughly that a closed system in an eigenstate of a continuously perturbed Hamiltonian remains in an instantaneous eigenstate in the limit of slow perturbations if the corresponding eigenvalue is separated from the rest of the spectrum by a gap. Adiabaticity in quantum mechanics has applications in a wide range of areas, including quantum chemistry [3], geometric phases [4,5], quantum Hall effect [6], STIRAP [7], and quantum phase transitions [8]. More recently, the adiabatic theorem has been the subject of increased interest in relation to quantum information processing, where it has served as a basis for a variety of schemes, including holonomic quantum computation [9] and adiabatic quantum algorithms [10]. Given the importance of the concept of adiabaticity in closed quantum systems, it is natural to ask how this concept extends to the dynamics of systems interacting with an environment. This question is of particular interest from the point of view of quantum information processing where decoherence is a major obstacle to the construction of reliable quantum devices, and at the same time non-unitary processes are an important tool for quantum control [11]. In Ref. [12], Sarandy and Lidar proposed an approach to the adiabatic dynamics of open quantum systems, defining adiabaticity as the regime in which the operator subspaces corresponding to the instantaneous Jordan blocks of the generator of the dynamics evolve independently (for adiabaticity in weakly open systems, see Ref. [13]). This definition is motivated by the formal analogy between the Schrödinger equation and the timedependent Markovian master equation written in a coherence basis, both being first-order linear vector differential equations with the difference that the generator of the master equation is generally not diagonalizable (hence the Jordan decomposition). But while in closed systems the phenomenon of adiabaticity concerns the decou-pled evolution of eigenspaces of the Hamiltonian which themselves are Hilbert spaces containing physical states, the Jordan blocks correspond to generally nonorthogonal subspaces of the space of linear operators that need not contain density matrices or even observables and may decay to zero even when mutually decoupled. In the present paper, we propose a different approach, based primarily on physical considerations, which yields an inequivalent picture of open-system adiabaticity that links adiabatic dynamics to the theory of noiseless subsystems [14]. Taking as a ground the basic physical characteristic of adiabatic closed-system evolutions-namely, that these are quasi-static evolutions where under sufficiently slow changes of the Hamiltonian a system in a stationary state evolves so as to remain in a stationary state with respect to the changed Hamiltonian-we look for a generalization of this phenomenon to the case of Markovian dynamics. The key insight of our approach is that the natural generalization of the eigenspaces of the Hamiltonian corresponding to distinct eigenvalues are noiseless subsystems whose noiseful cofactors support unique fixed states. A decomposition of the Hilbert space into such subsystems arises naturally from the asymptotic behavior of the Lindblad semigroup [15]. We define adiabaticity as the regime in which the stationary states over such a noiseless subsystem and its cofactor remain stationary with respect to the Lindbladian as it changes. We derive an adiabatic theorem based on this definition. To illustrate the utility of our formalism, we propose two applications. One is a framework for decoherenceassisted computation in noiseless codes which generalizes the approach of Beige et al. [16] to subsystems and general noise models. The other is a dissipation-driven approach to holonomic quantum computation based on adiabatic "dragging" of subsystems [17] along paths that are generally not achievable by non-dissipative means. Generalization of eigenspaces.-Our starting point is the observation that the eigenstates of a Hamiltonian H are the stationary state vectors of its dynamics. In particular, all stationary density matrices under the evolution dρ/dt = −i[H, ρ] (we seth = 1) have the direct-sum form ρ = i p i ρ i , i p i = 1, p i ≥ 0, where ρ i are density matrices over the eigenspaces H i of H corresponding to distinct eigenvalues. In more general quantum processes, the stationary states are organized as operators over noiseless subsystems tensored with a fixed density matrix over the corresponding noiseful co-subsystem [18]. Consider a time-homogenous finite-dimensional Markovian dynamics described by the Lindblad equation [19] dρ dt where L i are Lindblad operators. As shown in Ref. [15], Eq. (1) induces a decomposition of the Hilbert space where H A ij are noiseless subsystems [14], H B j are noiseful subsystems that support unique fixed states, and K is a decaying subspace. More particularly, it was shown that for any initial state ρ(0), the solution of Eq. (1) satisfies where ρ A j are density matrices on the unitarily noiseless subsystems H A j = i H A ij evolving under the Hamiltonians H A j , ̺ B j are fixed full-support states on H B j , and k p k = 1, p k ≥ 0. The noiseless subsystems H A ij are the eigenspaces of H A j . The stationary states have the form ij . This suggests that the subsystems H A ij whose cofactors H B j support unique fixed states ̺ B j can be thought of as the generalization of eigenspaces corresponding to distinct eigenvalues. How do we find the decomposition (2) for a given Lindbladian L? An algorithm for finding the noiseless subsystems of a completely positive trace-preserving (CPTP) map that runs in time O[(dimH) 6 ] was described in Ref. [18] (see also Ref. [20]). It is based on finding the left and right operator eigenspaces corresponding to the eigenvalue 1 of the CPTP map. Since Eq. (1) is equivalent to the continuous application of an infinitesimal CPTP map, the same algorithm can be used here (the eigenvalue 1 of the map translates to eigenvalue 0 of L). Before we introduce adiabaticity for Markovian dynamics, let us briefly review the closed-system case. Adiabaticity in closed systems.-Consider a timedependent Hamiltonian H(t/T ) changing along a differentiable curve H(s), s ∈ [0, 1]. Let ǫ i (s) be an eigenvalue of H(s) with multiplicity m, and P i (s) be the (twicedifferentiable) projector on the corresponding eigenspace H i (s) = P i (s)H. [Note that m = const(s) implies that ǫ i (s) is separated from the rest of the spectrum by a gap. The adiabatic theorem has been extended to cases without a gap [21], but in this paper we restrict to the standard formulation.] The eigenspace H i (t/T ) is said to evolve adiabatically under Equivalently, if we change the basis via a unitary U (s) so that P i becomes fixed, in the new basis the dynamics is driven by the effective Adiabaticity then refers to the regime in which any state initially in H i remains in H i despite the action of 1 T V (t/T ). The adiabatic theorem states [2] that in the limit of large T , one approaches perfect adiabaticity where the states in H i evolve via the , where ∆ > 0 is a fixed energy scale (e.g., the energy gap). Note that unlike the "folk" adiabatic condition which is known to be insufficient [22], this theorem (similarly to the one derived below) is concerned with the scaling of the error as a function of T for a fixed curve H(s). Similarly to the closed-system case, the assumption that dimH A kl (s) and dimH B l (s) are constant implies an analogue of the gap condition (see Appendix A). Definition. The noiseless subsystem H A kl (t/T ) and its noisy cofactor H B l (t/T ) evolve adiabatically under As in the case of closed systems, it is convenient to consider a basis rotated by a unitary U (s), in which H A kl and H B l are fixed. In this basis, the master equation is where L(s) is the Lindbladian with H(s) replaced by U (s)H(s)U (s) † and L i (s) by U (s)L i (s)U (s) † , and V (s) = i dU(s) ds U (s) † . (We will not use a different notation for ρ in this basis but will keep in mind the basis we are working in.) Adiabaticity then means that any state Consider Markovian dynamics satisfying the above assumptions. In the limit of large T , perfect adiabaticity is approached with an error that scales as O( 1 T ∆ ), where ∆ > 0 is some fixed energy scale. In the adiabatic limit, the Proof. Let us divide the total time T into N steps, each of length δt, T = N δt. We will take δt = N/∆ (hence, T = N 2 /∆) such that when N → ∞, δt is short on the time scale of change of the Lindbladian but long on the time scale for reaching the asymptotic regime of the instantaneous Lindbladian. The differentiability assumptions about L(s) and P kl (s) imply that we can The evolution of the density matrix during a single time step can then be written Assume that the state at time t has the form Then the first term on the right-hand side of Eq. (5) is ]. Using noiseless-subsystem properties of the Lindbladian [23,24], in Appendix A we show that this term is equal to −i the last inequality can be verified by a simple algebra). We therefore see that if the initial state is of the form (6), it will remain of this form for all times, up 2 ), which in the limit N → ∞ yields the differential equation Note. Our theorem includes an adiabatic theorem for closed systems as a special case. However, the convergence rate stated in our theorem is weaker than the standard one [the error is O( 1 ∆T ) as opposed O( 1 ∆T )] since our proof captures dissipative cases as well. (In Appendix A, we describe a natural energy scale ∆ associated with the curve L(s), which can be regarded as a generalization of the minimum energy gap.) Decoherence-assisted computation in noiseless codes.-Computation in noiseless subsystems requires operations that keep the information inside the code [25]. However, the Hamiltonians that preserve the code in general may be rather complicated and may not be naturally available in a particular experimental setup. Thus strategies for achieving encoded universality [26] by other means are of particular interest [27]. An immediate implication of the above theorem is that for the common case of time-homogenous Markovian noise with Lindbladian L [to play the role of L(t/T ) in Eq. (4)], any Hamiltonian perturbation 1 T V (t/T ) acting during t ∈ [0, T ] would give rise to (possibly non-trivial) unitary evolutions inside the noiseless subsystems H A ij of L within an arbitrary precision for sufficiently large T . Thus given a set of available interactions {V µ } that can be turned on with variable strength, for a given subsystem H A kl one can produce the set of effective interactions (Note that preparation of the states on H B l is not needed as they quickly decay to the fixed points.) Encoded universality is achieved if the set {V eff µ } spans the Lie algebra su(m) over H A kl . Remarkably this is possible even if the Hamiltonians {V µ } commute (see example below). Such an approach was first proposed in Ref. [16] for noiseless subspaces (dimH B l = 1) under certain noise models that can be interpreted as continuous Zeno measurements projecting onto the subspace. Equation (36) provides a generalization of this idea to noiseless subsystems (that may exist even when no noiseless subspaces exist) and arbitrary time-homogenous Markovian models. As an example, in Appendix B we study a two-level noiseless subsystem of three spin-1 2 particles under collective decoherence [14]. The noiseless subsystem involves highly entangled states, and non-local interactions are in principle required to perform operations on the encoded qubit. However, we find that the decoherence process itself can be used to induce an effective universal set of gates on the code by acting with local Hamiltonians. Holonomic quantum computation via dissipation.-In the previous method, we assumed that the perturbation 1 T V (s) is applied by the experimenter. However, the conclusions are valid also if we assume that the description is with respect to an instantaneous basis of a time-dependent noiseless subsystem H A kl (s) of L(s), where the perturbation now arises from the time dependence of the basis. As L(s) acts trivially on H A kl (s), the effective transformation in H A kl (s) is not of dynamical origin. Indeed, in the adiabatic limit, an initial state ρ AB (0) over H A kl (0) ⊗ H B l (0) transforms via the superoperator lim δs→0 P kl (1)P kl (1 − δs)...P kl (δs)P kl (0) which is an intrinsically geometric quantity defined via the projectors P kl (s (1), so that the final basis is the same as the initial one, the resultant transformation is a gaugeinvariant quantity that generalizes the standard holonomy associated with parallel transport of Hamiltonian eigenspaces [5]. We note that the idea of adiabatically "dragging" a subsystem (rather than a subspace) along suitable paths in order to perform geometric gates inside it has been proposed for the case of Hamiltonian dynamics as a powerful tool for robust computation [17]. However, a subsystem cannot be dragged along an arbitrary path H A (s) by a Hamiltonian since some paths necessarily give rise to correlations between H A (s) and H B (s). This problem does not exist here since the Lindbladian acting on H B (s) severs any such correlations. (For dissipation-driven holonomies in subspaces, see Ref. [28].) The mathematical foundations of these geometric transformations will be studied elsewhere. Here we show that the method can be used for universal quantum computation. Consider a two-qubit system H = H A ⊗ H B and a depolarizing Markovian channel acting locally on The Hamiltonian in the exponent can be easily diagonalized and one sees that U (1) = U (0) = I. Hence, if we change the Lindbladian via the unitary U (s), we will take the noiseless subsystem H A around a loop H A (s) ⊗ H B (s) = U (s)H A ⊗ H B with a single-valued basis. According to Eq. (36) (here ̺ B = I B 2 ), the subsystem will experience the effective Hamiltonian bσ A x which gives rise to the transformation e −ibσ A x at the closing of the loop. Similarly, by exchanging σ z and σ x , we can generate the unitary e −ibσ A z . To perform an entangling gate between two qubits, A and A ′ , we can start with the same Lindbladian acting on B and rotate it via the uni- x . This set of gates is universal. Conclusion.-We introduced a theory of adiabatic Markovian dynamics that relates the notion of adiabaticity to the theory of noiseless subsystems. We proved an adiabatic theorem for such dynamics and proposed two novel methods of quantum information processing based on it-decoherence-assisted computation in noiseless subsystems and dissipation-driven holonomic computation-that add to the developing picture of dissipation as a powerful quantum computation primitive [29]. A natural problem for future research would be to find exact bounds on the adiabatic error in Markovian dynamics similar to those obtained for closed systems, e.g., in Ref. [30]. Acknowledgments.-We thank Lorenza Viola for helpful comments. This work was supported by the Spanish MICINN via the Ramón y Cajal program (JC), contract FIS2008-01236/FIS, and project QOIT (CONSOLIDER2006-00019), and by the Generalitat de Catalunya via CIRIT 2005SGR-00994. OO was also supported by the Foundational Questions Institute (FQXi). Before we go in detail through the main steps of the proof, it is convenient to introduce an energy scale ∆ associated with the curve L(s). This quantity can be regarded as a generalization of the minimal spectral gap of the Hamiltonian from the case of closed systems, and is a suitable choice in view of certain later calculations. Although it is not the purpose here, this energy scale could be useful for deriving exact bounds on the error and not just its scaling with T . As shown in Ref. [23], the subsystem H A kl is noiseless under the evolution driven by L(s), if and only if for every s the Hamiltonian and the Lindblad operators have the block forms where the upper-left block corresponds to H A kl ⊗ H B l , and Then it is not difficult to verify (see also Ref. [24]) that L(s) preserves the subspace B 2 of operators with vanish-ing lower right block, where B(H) denotes the space of linear operators over H. It further preserves the subspace of operators with vanishing lower right and offdiagonal blocks, where it acts as where L B (s) is a local Lindbladian with Hamiltonian H B 1 (s) and Lindblad operators L B 1j (s). Note that L B (s) has a non-degenerate eigenvalue 0 with a corresponding right eigenoperator ̺ B l (s), and all its other eigenvalues have negative real parts, since ̺ B l (s) is an attractive fixed point. By continuity, the magnitudes of the real parts of the non-zero eigenvalues of L B (s) have a minimum value in the interval s ∈ [0, 1]. Denote that value by ∆ 1 > 0. We will also need another quantity, ∆ 2 , which is the minimum of the magnitudes of the non-zero eigenvalues of L(s)P 2 in the interval s ∈ [0, 1], where P 2 is the projector on B 2 (this minimum also exist by the assumption of continuity in a closed interval). Then we can define which we will serve as a natural energy scale in our analysis. If H A kl is a noiseless subspace (dim H B l = 1), ∆ 1 does not exist and ∆ = ∆2. We note that in the case of closed systems, where H A k1 is an eigenspace of the Hamiltonian, ∆ is exactly the minimum gap that separates this eigenspace from the rest of the spectrum. This is because the non-zero eigenvalues of L(s)P 2 are ±i(E k (s) − E m (s)), m = k, where E n are the energies of the eigenspaces H A n1 . Remark. The existence of ∆ > 0 follows naturally from continuity and the assumption that dim H A kl and dim H B l are fixed during the closed interval s ∈ [0, 1]. The condition dim H A kl = const(s) can be thought of as an analogue of the closed-system requirement that the eigenspace does not cross other energy levels. However, it may be possible to relax the condition dim H B l = const(s) as long as we require ∆ > 0 for the open subintervals of s ∈ [0, 1] during which dim H B l = const(s). Before we proceed, we will need another observation. By the definition of H A kl ⊗ H B l , the only right eigenoperators of L(s) with eigenvalue 0 inside the subspace B 2 are those with τ 2 = 0 and τ AB 1 = τ A kl ⊗ ̺ B l (s). Denote the subspace of these operators by B 0 (s) [B 0 (s) ⊂ B 1 ⊂ B 2 ]: Let P ′ (s) be the projector on the subspace B 2 ⊖ B 0 (s). Then the superoperator L ′ (s) ≡ L(s)P ′ (s) over B 2 ⊖ B 0 (s) has only non-zero eigenvalues and is therefore invertible. Its inverse, L ′−1 ( t T ), has magnitude which is bounded by the inverse of ∆, The boundedness of this operator will be used at a certain stage of the proof, and the energy scale ∆ was chosen as described above since it provides the bound (16). We are now ready to go through the steps of the proof. Main proof Let us divide the total time T into N time steps, each of length δt, T = N δt. We will take δt = N/∆ (hence, T = N 2 /∆) such that when N → ∞, δt is short on the time scale of change of the Lidbladian but long on the time scale for reaching the asymptotic regime of the instantaneous Lindbladian. The evolution of the density matrix of the system during one time step can be written Let us now assume that the state at time t has the form Then the first term on the right-hand side of Eq. (18) is for some τ B l (t), because H A kl is noiseless under L(s). We will now show that for sufficiently large δt, First of all, we have T e δt 0 dt ′ L( t+t ′ T ) = e δt L( t T ) + O( 1 N ). Also, under the action of L( t T ) (for fixed t) any state ρ AB on H A kl ⊗ H B l decays towards ρ A ⊗ ̺ B l ( t T ), where ρ A = Tr B ρ AB , with a rate at least ∆. Since δt = N/∆, we have e δt L( t T ) ρ(t) = ρ A kl (t)⊗ [̺ B l ( t T )+ O( 1 N )]. (In fact, the error O( 1 N ) in the latter expression is an overestimate, but for our purposes this precision suffices.) Therefore, for the first term on the right-hand side of Eq. (18) we obtain	2010-07-30T17:45:35.000Z	2010-02-10T00:00:00.000	{ "year": 2010, "sha1": "1be0d2f9d113695c220ef83b215a5cfe837de03b", "oa_license": null, "oa_url": "http://arxiv.org/pdf/1002.2219", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "5be45eab64dd605569892c6066d9211bbe07b87d", "s2fieldsofstudy": [ "Mathematics" ], "extfieldsofstudy": [ "Physics", "Medicine" ] }
46224921	pes2o/s2orc	v3-fos-license	Association between readiness for behavior change and complaints of vocal problems in teachers Purpose: To verify the association between readiness to behavioral change and self-reported vocal problems of teachers in schools from the municipal education network of Montes Claros, Minas Gerais, Brazil. Methods: The URICA-VOICE scale was used to measure the motivational stages of 138 teachers. Readiness to change was the variable outcome, whereas the independent variables referred to sociodemographic, economic, occupational, lifestyle, general health, and teacher’s own voice factors. Results: The majority (59.4%) of teachers were in the pre-contemplation stage of the URICA-VOICE scale. The variables use of medication, perception of voice failure, and demand for speech and language therapy were retained for the final model. Conclusion: The low readiness to change showed the need for increased awareness of the risks and benefits related to the voice and general health. The results can provide public health intervention strategies that deal with individuals at various stages in the decision-making process. DOI: 10.1590/2317-1782/20152013088 171 Prontidão para mudança comportamental pela URICA-VOZ CoDAS 2015;27(2):170-7 INTRODUÇÃO Promoção da saúde é o processo que visa aumentar a capacidade dos indivíduos e das comunidades para controle e melhoria da própria saúde, sendo imprescindível para a construção de uma cultura de valorização do bem-estar, do autocuidado e da qualidade de vida. Existem diversas teorias de promoção de saúde e a maioria vem das ciências comportamentais e das sociais, porque a prática tem o foco no comportamento dos indivíduos e na forma como é organizada a sociedade. As teorias mais utilizadas, entre 2000 e 2005, em ordem decrescente, foram o Modelo Transteórico, a Teoria Social Cognitiva e o Modelo de Crenças em Saúde. O Modelo Transteórico, ou Modelo de Fases de Mudança, surgiu no final da década de 1970 e trabalha com o conceito de estágios de mudança para integrar processos e princípios de mudança provenientes das principais teorias de intervenção. O Modelo Transteórico pode ser considerado um importante instrumento de auxílio para compreender a mudança comportamental relacionada à saúde. Utilizando o construto do Modelo Transteórico, foram criadas escalas para a mensuração dos estágios motivacionais, dentre elas a University Rhode Island Change Assessment (URICA). Trata-se de uma medida de autorrelato do tipo escalar empregada de forma genérica para todos os tipos de problemas e está baseada nos estágios de mudança contendo quatro subescalas: pré-contemplação, contemplação, ação e manutenção (Quadro 1). As duas primeiras etapas descrevem o desenvolvimento da intenção de agir, enquanto as duas últimas etapas descrevem o processo de colocar em prática a intenção de mudar. É importante ter em mente que não se pode analisar esse modelo como linear e estático, porque as pessoas não passam pelas fases de forma sequencial, tanto podem saltá-las como também ter recaídas, entrando e saindo dos estágios, o que leva a uma evolução dinâmica, delineando uma espiral. A recaída é normal e prevista quando se busca uma mudança de comportamento por longo prazo. No Brasil, já existe uma versão adaptada da URICA para pessoas com disfonia cujo questionário contém 32 itens. A utilidade clínica e em pesquisa da URICA é inquestionável, pois auxilia na compreensão da motivação do indivíduo sobre sua capacidade de mudança comportamental relacionada à saúde. Entre os profissionais da voz, a categoria dos professores é a mais vulnerável para o aparecimento de disfonia. Portanto, a percepção que o professor tem sobre o seu problema de voz pode ser uma ferramenta importante para a detecção precoce de problemas vocais. Além do mais, o fonoaudiólogo deve valorizar as necessidades do paciente considerando sua própria perspectiva, para isso é essencial ter conhecimento dos dados sociodemográficos, características individuais, crenças e valores. Estudos relacionando o Modelo Transteórico aos distúrbios fonoaudiológicos ainda são escassos, o que demonstra a importância de novas pesquisas. Neste sentido, o objetivo deste estudo foi verificar a prontidão para mudança comportamental das professoras da rede municipal de ensino de Montes Claros, Minas Gerais, com queixa de disfonia e a associação com as variáveis sociodemográficas, econômicas, ocupacionais, estilo de vida, saúde geral e demais fatores relacionados à voz. INTRODUCTION Health promotion is a process that aims to increase the capacity of individuals and communities to control and improve their own health, which is essential for building a culture of wellness, self-care, and quality of life (1) .There are several health promotion theories, and the majority come from behavioral and social sciences because of the practice of focusing on the behavior of individuals and how society is organized (2) .The most commonly used theories between 2000 and 2005 were Transtheoretical Model, Social Cognitive Theory and the Belief Model in Health (3) . The Transtheoretical Model, or Model of Change Phases, came up in the late 1970s and is based on the concept of stages of change to integrate processes and change principles resulting from the main intervention theories (4)(5)(6) .The Transtheoretical Model can be considered an important tool to help understand the health-related behavior change (4) . On the basis of the Transtheoretical Model, scales were created to measure motivational stages, including University Rhode Island Change Assessment (URICA).This is a selfreport scale measure used generally for all types of problems (5,7) , and it is based on the stages of change containing four subscales: pre-contemplation, contemplation, action, and maintenance (Chart 1).The first two steps describe the development of intention to act, and the last two steps describe the process of putting the intention to change into practice (8) . Important to remember that one cannot consider this model as linear and static because people do not go through stages sequentially; they can skip them or relapse, going in and out of stages (9) , which leads to a dynamic evolution in spiral.Relapse is normal and expected when one is seeking a long-term behavior change (5) . There is already an adapted version of URICA for people with dysphonia in Brazil, which is a questionnaire containing 32 items (7) .Clinical and research utility of URICA is unquestionable (10) as it helps understand the motivation of individuals regarding their capacity of changing health-related behavior (4) . Among voice professionals, the category of teachers is the most vulnerable to the onset of dysphonia (11) .Therefore, the perception that a teacher has on his voice problem may be an important tool for the early detection of vocal problems (12) .Moreover, the speech therapist must value the patient's needs considering their own perspective, and it is essential to know about their sociodemographic status, individual characteristics, beliefs, and values (13) . The literature lacks studies relating the Transtheoretical Model to speech disorders, which shows the importance of further research.So, the objective of this study was to assess the readiness for behavior change among teachers in the municipal school Montes Claros, Minas Gerais, who had complaints of dysphonia, and the association with sociodemographic, economic, occupational, lifestyle, general health, and other variables related to voice. METHODS This epidemiological, cross-sectional, analytical study was conducted in Montes Claros, northern Minas Gerais, Brazil.The city has about 370,000 inhabitants and is the main regional urban center in the State.The target population consisted of teachers of the first 5 years of primary education in municipal schools.According to data from the Municipal Department of Education, the network is composed of 640 teachers. First, the sample calculation was performed by simple random sampling to estimate the prevalence of voice disorders.Calculation was set with a confidence level of 99%, accuracy of 5%, and estimated prevalence of self-reported voice alteration of 11.6% (11) , so it was concluded that 196 teachers from municipal schools should be allocated in the study, considering possible losses.Male teachers were excluded due to their small number in the municipal network and anatomical differences of the larynx.Teachers not currently acting in the field and those of physical education were also excluded. The data were collected in visits to schools.During recess time, the teachers were informed about the research Source: Adapted from the National Institutes of Health (9) and the voluntary and anonymous character of participation.The self-administered questionnaire was structured and delivered in individual envelopes to teachers randomly chosen, and a date was set to collect it and clarify any doubts.Those who had vocal complaints were instructed to fill the URICA-VOICE questionnaire. Application of URICA-VOICE questionnaire We used the adapted version of the URICA-VOICE questionnaire with 32 items, whose questions concerning stages (pre-contemplation, contemplation, action, and maintenance) have possibilities of answers in scales such as five-point Likert scale, as oriented by the Health and Addictive Behaviors: Investigating Transtheoretical Solutions (HABITS) at the University of Maryland, Baltimore (14) .As recommended, the Yesple sum of statements was applied corresponding to each stage of change divided by the number of questions; later on the results of contemplation stages, action, and maintenance were summed up and then subtracted from the result of the precontemplation stage.Scores lower than or equal to 8.0 indicates that the individual is in pre-contemplation stage; 8.1-11.0, in contemplation stage; 11.1-14.0, in action stage; and equal to or greater than 14.1, in maintenance stage (7,14) . Data analysis The result of URICA-VOICE questionnaire was considered as dependent variable, which as dichotomized into precontemplation and contemplation stages. Independent variables were divided into blocks: Statistical analysis was made with the Predictive Analytics SoftWare application (PASW® STATISTIC), version 18.0.Afterward, the bivariate analysis was done using the χ 2 -test of all dichotomized independent variables and the outcome variable.Those presenting descriptive level p≤0.20 were selected for the logistic regression, which identified the variables associated with readiness to change. This research was approved by the Research Ethics Committee of Universidade Estadual de Montes Claros (Unimontes) under Number 2889/11.All participants signed the informed consent before answering the questionnaire. RESULTS At first, a prevalence of self-reported voice problems was verified.In total, 226 teachers answered the questionnaire and 61.1% reported having voice problems.Of the 138 who provided affirmative answers, 58.0% had a change starting in up to 3 weeks before acute problem and 42.0%, a change over 3 weeks before chronic problem.As stated in the guideline of the American Academy of Otolaryngology-Head and Neck Surgery Foundation (15) , acute changes are those lasting less than 3 weeks. Age of all 138 participants ranged from 26 to 54 years, with mean age of 41.4 years and standard deviation (SD) of 5.6.Number of children varied from 0 to 5, with mean of 1.9 (SD±1.1).The number of family members varied from 1 to 10, with mean of 4.0 (SD±1.3).Teaching experience varied from 1 year and 6 months to 29 years, with mean of 16 years and 5 months (SD±6 years and 4 months).The minimum number of students per classroom was 17 and the maximum was 40, with mean of 25.9 (SD±4.0).Main characteristics of the sample are shown in Tables 1 and 2. With respect to teachers' distribution according to readiness to change, based on URICA-VOICE scale, 59.4% were in pre-contemplation stage; 37.0% in contemplation stage; 3.6% in action stage; and no one was in the maintenance stage.The level of readiness to change was in the score ranging from 1.4 to 12.5, with mean of 7.4 points (SD±2.1). Considering that only five teachers were in the action stage, they were excluded from other statistical analyses.They reported voice problems for over a month, and three of them said they had sought speech therapy.One of them mentioned not having sought a speech therapist because the only symptom was sore throat and she believed it was a result of hypothyroidism. The variable outcome was considered in two categories: pre-contemplation and contemplation.65.0% of teachers with acute voice problems were in the pre-contemplation stage and 35.0% were in the contemplation stage; among those with chronic voice problem, 56.6% were in the precontemplation stage and 43.4% in the contemplation stage (p=0.330). The bivariate analysis between the main group characteristics and the two stages for behavioral change by URICA-VOICE scale is presented in Table 3. Table 4 shows the variables in the final model of statistical multivariate analysis.Those who reported using medication, failed to realize voice problems, or did/were doing speech therapy are more likely to present readiness for behavior change. DISCUSSION The prevalence of self-reported voice disorders was similar to those found in other studies (60.0 and 64.8%) (16,17) , but above the prevalences of 11.6% to the national extent (11) and 47.6% in Florianopolis, Santa Catarina (18) , and below a study conducted in Maceio, Alagoas, whose prevalence of dysphonia was 87.3% (19) .This variation is probably due to differences in methodology between these studies. In this study, the readiness for behavior change among teachers of municipal schools who reported dysphonia was proved low in relation to voice care.At first, a rating was not found that quantifies how much the observed result in Transtheoretical Model studies is satisfactory in the literature.However, a larger number of people in more advanced stages in URICA-VOICE stage were expected due to the high prevalence of dysphonia among teachers.Low readiness to change may result from two factors: 1. participants are aware of the existence of a problem to be faced, but are not well oriented to the consequences of this behavior; and 2. participants are trying to change behavior, but have no ability or support to do so (20) . Individuals in the pre-contemplation stage are often characterized as resistant to change, unmotivated, and not ready to attend therapeutic or health-promotion programs.A possible explanation for this attitude is that traditional health-promotion programs are probably not good motivators to meet their needs (3) .Some teachers were in the contemplation stage, which means that they intended to change behavior, perhaps because they recognize the positive aspects of such action.Five teachers with chronic dysphonia were in the action stage, that is, they were developing new behaviors. Study conducted with 66 patients under dysphonia treatment in University hospitals of voice clinics at two educational institutions of Speech Therapy found 30.3% of patients in the pre-contemplation stage, 57.6% in the contemplation stage, 12.1% in stage action, and none in the maintenance stage (7) .This difference related to this study, or higher percentage in the stages of contemplation and action, can be attributed to the fact that individuals were under speech therapy; therefore, they presented higher levels of motivation. In the analysis of this study, comparing the two stages, the variables associated with a greater predisposition/awareness (contemplation stage) for behavior change as to the voice were: use of medication, perception of voice failures, and demand for speech therapy.Conceivably, such factors may contribute to the vocal problem awareness, interfering in readiness to behavior change, reaching the contemplation stage. The search for treatment was associated with voice complaint, although most participants were in the pre-contemplation stage and search for assistance usually occurs in more advanced readiness stages, when the individual considered solving his vocal problem already.Therefore, further studies are needed for a better understanding of these results. Information about the patients' perception of their own voice help guide the therapy and can help increase the awareness of behavior change necessity.A possible strategy for individuals in the pre-contemplation stage would be to customize information about the negative impacts of dysphonia in quality of life and the benefits of speech therapy.Educational programs are basic conditions whose main purpose should be the empowerment of the individual and the community. For those who are in the contemplation stage, they should motivate them, encouraging them to make specific plans (9) .Note that many can remain in this stage for long periods of time, the so-called chronic contemplation (20) .An activity for patients with vocal problems who are undergoing treatment would be voice recording during speech therapy process, which could be a valuable resource to encourage the patient, because this would motivate them to realize the progressive improvement of their voice.Due to lack of resources in outpatient public services, such recordings are limited to the beginning or end of treatment (7) . The transition from a behavioral change stage to another depends on appropriate strategies and interventions in each specific moment.The Transtheoretical Model has ten change processes applicable to specific stages, and focuses on these activities so that progress can occur (3) . It must be noted that the results of this study should be interpreted based on some of their limitations.Some of these limitations refer to the target audience, which were restricted to teachers in municipal public education, teaching from first to fifth grade.Others refer to be highlighted as concerned to the detection of voice problems, for they were measured by self-reported vocal complaints.It is important to note that the group was not made up of individuals enrolled in therapy programs, and the Transtheoretical Model has been mainly used to measure the dimensions of readiness to change in individuals under treatment. Despite limitations, the importance of the topic and the lack of literature on the subject highlight the magnitude of the problem and the need for studies in the area.Results should motivate health-promotion activities to improve quality of life as to voice care and general health, which is a commitment of the speech therapist (21) .The Transtheoretical Model is an opportunity for the professional to work with health-promotion habits and to verify that each stage requires specific actions for adherence to vocal care. CONCLUSION Most teachers with vocal complaints are in the precontemplation stage in URICA-VOICE scale.This shows the need for an education process aimed to the risks of voice misuse and abuse, and to presenting the benefits of general and vocal health.Variables associated with readiness for behavior change are related to medication use, perceived voice failure, and the search for speech therapy.These results may support public health interventions involving people at different stages of the decision-making process as to behavioral change for vocal health. Chart 1 . Determination to change behavior or develop new ones.Help with feedback, solving problems, social support, and reinforcement In process of solving the problem 4. Maintenance Maintain behavior change over time; prevention to relapse; consolidate the gains Help with coping, reminders, find alternative avoiding slips, and relapse Trying to get rid of the problem Stages of change, according to the Transtheoretical Model • Block 5 -voice: voice changes (hoarseness, dry throat, vocal fatigue, throat clearing, voice fails, vocal effort, pain when speaking, sore throat, etc.), voice use in daily life, sick leave for vocal problems, and default due to voice problems; and • Block 6 -health care: medical consultations due to voice problems and speech therapy. • Block 1 -demographic and economic data: age, education, marital status, number of children, and income; • Block 2 -occupational data: teaching experience, work shifts, average number of students per class, perception of noise in classroom, school, out of school, and ventilation; • Block 3 -lifestyle: hydration during lessons, water intake per day, fruit juice intake, amount of juice per day, physical activity, frequency of alcohol intake, alcoholic doses when drinking, and smoking; • Block 4 -health: treatment for gastroesophageal reflux, medical diagnosis of respiratory allergy, perception of breathing problem, and use of medication for diseases (hypertension, diabetes, depression or anxiety, sleep disturbances, rheumatism, etc.); Table 1 . Readiness for behavior change by URICA-VOICE scale Sociodemographic, economic, occupational, and lifestyle data of 138 teachers with voice problems allocated in public schools, Montes Table 2 . Health and vocal problems of 138 teachers with vocal disorders from the municipal network, Montes Claros, Minas Gerais, 2013 Table 3 . Association between stages of pre-contemplation and contemplation according to URICA-VOICE scale and independent variables Readiness for behavior change by URICA-VOICE scale χ 2 -test Table 4 . Multivariate analysis for factor associated with readiness to change behavior among teachers with complaint of dysphonia, Montes Claros, Minas Gerais, 2013 Backward logistic regression: LR Caption: OR = odds ratio; 95%CI = 95% confidence interval	2018-04-03T06:17:17.126Z	2015-03-01T00:00:00.000	{ "year": 2015, "sha1": "c4ef0cfd99b6e460da86cfa06b47899badbc8f98", "oa_license": "CCBY", "oa_url": "http://www.scielo.br/pdf/codas/v27n2/2317-1782-codas-27-02-00170.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "9cfd7839eaa5576951da0b5b35d1f5f0b187c4e6", "s2fieldsofstudy": [ "Education", "Psychology" ], "extfieldsofstudy": [ "Psychology", "Medicine" ] }
3394486	pes2o/s2orc	v3-fos-license	Antibacterial activity of 3,3′,4′-Trihydroxyflavone from Justicia wynaadensis against diabetic wound and urinary tract infection The present investigation was designed to study the effect of an active compound isolated from Justicia wynaadensis against multi drug resistant organisms (MDRO's) associated with diabetic patients. The drug resistant pathogens implicated in wound and urinary tract infection of diabetic patients were isolated and identified by molecular sequencing. Solvent–solvent fractionation of crude methanol extract produced hexane, chloroform, ethyl acetate and methanol–water fraction, among which chloroform fraction was found to be potent when compared with other three fractions. Further, chloroform fraction was subjected to preparatory HPLC (High-Performance Liquid Chromatography), that produced four sub-fractions; chloroform HPLC fraction 1 (CHF1) through CHF4. Among the sub-fractions, CHF1 inhibited the pathogens effectively in comparison to other three sub-fractions. The purity of CHF1 was found to be >95%. Therefore, CHF1 was further characterized by NMR and FTIR analysis and based on the structure elucidated, the compound was found to be 3,3′,4′-Trihydroxyflavone. The effective dose of this bioactive compound ranged from 32 μg/mL to 1.2 mg/mL. Thus, the present study shows that 3,3′,4′-Trihydroxyflavone isolated from J. wynaadensis is an interesting biopharmaceutical agent and could be considered as a source of antimicrobial agent for the treatment of various infections and used as a template molecule for future drug development. Introduction Infectious diseases are among the leading causes of death globally and are more frequent in patients with diabetes mellitus. The recurrence of microbial complications in diabetic vascular disease, foot ulceration and gangrene that lead to limb amputation. 2 While, UTI is allied with acute pyelonephritis (upper UTI) and emphysematous pyelonephritis necessitating hospital admission. 3 The first line of treatment in these patients is the usage of antibiotics. The choice of antibiotic depends on several factors such as severity of infection, antibiotics used previously and the resistance to antibiotics by the infecting microorganism. However, excessive use of antibiotics is of concern as their overall effect on the patient is unclear. The increasing incidence of antibiotic resistance among human pathogens against conventional antibiotics demands search for alternate modes of treatment. 4 Medicinal plants have been found to be effective in treating microbial infections associated with diabetes. Bioassay with activity-guided fractionation has provided directions to isolate new bioactive compounds with novel targets to overcome drug resistance among the pathogenic microorganisms. 5 The search for more potent and safer biomolecules has continued to be an important area of active research. Therefore, in this study, we selected Justicia wynaadensis, which is known traditionally to possess vital bioactive molecules having various medicinal properties. 6 Justicia, the largest genus of Acanthaceae, has approximately 600 species that are found in pantropical and tropical regions. 7 J. wynaadensis is endemic to the rainforest region of the Western Ghats. 8 To the best of our knowledge, there are no reports available, that describes the isolation and characterization of any antimicrobial compound from this plant. In the present study, an attempt has been made to isolate a bioactive compound with potent antibacterial activity against drug resistant microorganisms isolated from wound and UTI of patients with diabetes. Multidrug resistance organisms Isolation and identification of MDRO's from diabetic wound and UTI were performed as follows: briefly, one hundred wound and urine samples each were collected from diabetic patients with random blood glucose levels >140 mg/dl and HbA1c >7.5 percent adopting aseptic conditions. The isolates were cultured on various selective media and identified by biochemical methods. 9 Further, the isolates were cultured on Mueller-Hinton medium, and tested against known antibiotics (Hi Media Laboratories, Pvt Ltd., Mumbai). Penicillin-G, oxacillin, erythromycin, linezolid, co-trimoxazole, vancomycin, cefotaxime, ciprofloxacin, tetracycline, imipenem, amoxyclav, ampicillin, gentamycin, cefuroxime, levofloxacin, norfloxacin, doxycycline HCl, amikacin chloramphenicol, cefoxitin, amphotericin-B, and clotrimazole. The organisms that offered resistance to three or more classes of antibiotics were selected and identified by molecular sequencing. Bacteria Genomic DNA was isolated by employing the conventional phenol-chloroform method. 10 Amplification of 16S rDNA was performed using the universal primers 27f (5 -AGAGTTTGATCCTGGCTCA-3 ) and 1492r (5 -TACGGCTACCTTGTTACGACTT-3 ). 11 The optimal cycling conditions were the following: 95 • C for 5 min; 35 cycles of 94 • C for 15 s, 56 • C for 30 s, and 72 • C for 90 s; and a final extension at 72 • C for 7 min. Each PCR mixture of 50 L contained 1× Taq buffer (Fermentas, USA), 200 M each dNTPs, 1.5 mM MgCl 2 , 0.4 M of primers, template DNA (100-250 ng) and 1.25 U of Taq polymerase (Fermentas, USA). The PCR products were loaded in 1.5% agarose gel in 1× TAE (Tris acetate EDTA) buffer and were allowed to run at 50 V for 45 min. The gel was stained with ethidium bromide bath (10 g/mL) and the gel image was captured in a gel doc. DNA sequencing was carried out at Amnion Biosciences Pvt. Ltd., Bangalore. Collection of plant material J. wynaadensis was collected in the month of August 2014 from Western Ghats of Karnataka, India and authenticated by a taxonomist. A voucher specimen of this plant material is deposited at the herbarium library, JSS college of Pharmacology, Mysore, India (Accession No.: Jsscp-Pcog-16). Extract preparation The leaves were separated from the plant material, washed thoroughly under running tap water and shade-dried on sterile blotters. The ground material (100 g) was soaked in 500 mL of methanol and was placed on a water bath in a sealed container for proper steam effect at 50 • C for 4 h with frequent stirring. The extract was filtered through Whattman filter paper and the filtrate was evaporated to dryness under reduced pressure using a rotavapor (Buchi, Rotavapour R-3, Switzerland). Fractionation of crude methanol extract The methanol extract was weighed and reconstituted in methanol: water 1:1 (v/v), subjected for solvent-solvent partitioning with sequential addition of hexane, chloroform and ethyl acetate in the ratio 1:1 (v/v), the extraction was repeated thrice. The solvent fractions were concentrated to dryness in rotavapor to afford four solvent fractions: hexane (HF), chloroform (CF), ethyl acetate (EF) and methanol-water (MF). Antibacterial activity of four solvent fractions The pathogens isolated from diabetic wound and UTI were inoculated into 10 mL of sterile nutrient broth and incubated at 37 • C for 24 h. The cultures were aseptically swabbed on sterile Mueller-Hinton agar plates using sterile cotton swab. Twenty-five microliter of each of the four fractions (0.5 mg) was loaded onto the sterile disc placed on the microbial lawn and antibacterial activity was evaluated by measuring the zone of inhibition against the test organisms. Analytical HPLC analysis Analytical RPHPLC was carried out in a Shimadzu HPLC system using a C18 column (100Å, 5 m, 4.6 mm × 250 mm) containing LC-10AT vp pump, SCL-10A vp system controller, and SPD-10AV vp UV/vis detector. Chromatographic separation was achieved using methanol, acetic acid, and water in the ratio 18:2:80 (v/v) as the mobile phase under isocratic condition. The column was washed with methanol, equilibrated with the filtered and degassed mobile phase for 30 min, and 20 L of 1 mg/mL standard mixture or 2 mg/mL chloroform fraction (CF) was injected. The sample or standard run was carried out for 1 h at 1 mL/min flow rate and the detection system was set at 280 nm. Preparative HPLC analysis Preparative HPLC analysis of the chloroform fraction (CF) was carried using Shimadzu (LC-8A) series HPLC system, DI diode array detector (SP-M10AVP), reverse phase C18 column (20 mm × 250 mm). Samples (2 mL, 30 mg/mL) were injected into the column repeatedly and the reference molecule, quercetin was used as a standard (1 mL, 1 mg/mL). An Isocratic elution method was employed using a mobile phase of methanol, acetic acid and water in the ratio 18:2:80. The flow rate was 10 mL/min and the absorption was measured at a wavelength of 280 and 320 nm. From the gradient elution, four major peaks were obtained at retention time 20.5, 30.1, 37.5 and 57.1 min and were collected, and labelled CHF1 through CHF4. The collected isolated fractions were dried by removing the solvent using rotavapour at 40 • C. The samples were kept in a vacuum desiccator for removal of traces of water and then stored at 4 • C until further analysis. Antibacterial activity of four fractions obtained from preparatory HPLC Twenty-five microliter of each of the four fractions CHF1-CHF4 (0.25 mg) obtained from preparative HPLC were loaded onto the sterile disc and placed on the microbial lawn and antibacterial activity was evaluated by measuring the zone of inhibition against the test organisms. Each organism was tested in triplicates. Characterization of isolated active principle Among the isolated fractions, CHF1 eluting at 20.5 min was again re-chromatographed under same condition to verify its purity and then characterized by spectroscopic methods as follows. LC/MS The analytical LC/MS experiment was performed in Waters Acquity UPLC system coupled with Synapt G2 Si HDMS mass spectrometer. Waters MassLynx and TargetLynx softwares were used for data acquisition and data processing respectively. LC conditions Two microlitre of 2 mg/mL CHF1 was injected into Acquity BEH C18 RP column (130Å, 1.7 m, 10 mm × 50 mm). Binary gradient containing mobile phase A (0.1% formic acid in water, v/v) and mobile phase B (acetonitrile) was used for chromatographic separation at 15,000 psi and 0.3 mL/min flow rate. The gradient used was as follows: 98% A and 2% B from 0 to 4 min, 2% A and 98% B from 4 to 6 min, and 98% A and 2% B from 6 to 8 min. Column and sample temperatures were maintained at 50 • C and 24 • C respectively; and the run time was set for 8 min. MS conditions Time of flight MS (TOF-MS) was used in negative electron spray ionization mode. Capillary, sampling cone and extraction cone voltages were set at 1.8 kV, 40 kV and 4 kV respectively, with a source temperature of 100 • C and pressure of 3.5 mbar. Nitrogen was used as the desolvation gas at 200 • C with a flow rate of 500 L/h and pressure of 1.4 mbar. Helium (damping gas, 180 mL/min, 2 mbar), Argon (trap gas, 2 mL/min, 7.6 mbar) and nitrogen (IMS gas, 90 mL/min, 2.5 mbar) were the gases used in Tri-wave. Collision induced dissociation was used as the fragmentation method at a trap collision energy of 4 eV. Ion mobility separation (IMS) was set on a linear ramp with IMS gas velocity ascent from 300 to 600 m/s, and wave height (V) ascent from 8 to 20. Transfer wave velocity was set at 247 m/s with a wave height of 0.2. TOF was operated between 50 and 1500 m/z with low mass resolution of 4.7 and high mass resolution of 15. Data was obtained in continuum mode with full scan acquisition time of 8 min, scan time of 1 s and interscan delay of 0.024 s. Minimum inhibitory concentration of the active principle Minimum inhibitory concentration of the active compound was determined by micro broth dilution technique as per CLSI guidelines. Briefly, the cell suspensions were prepared from bacterial cultures grown on Trypticose soya broth and was adjusted to 1-2 × 10 5 cells/mL as per McFarland's standards. The standard reference drug, chloramphenicol (4-246 g/mL) and the bioactive molecule, 3,3 ,4 -Trihydroxyflavone (16-1024 g/mL) were prepared in Mueller-Hinton broth. Each individual drug concentrations (90 L) were mixed with 10 L inoculum in 96 well plate. Chloramphenicol was used as a positive control, while the broth without active compound served as negative control. The above treated bacterial plates were incubated at 37 • C and observed after 24-48 h and optical density was measured at 600 nm in Tecan plate reader. All the tests were conducted in triplicates. Statistical analyses Statistical analysis was performed with Graph Pad Prism (Graph Pad Software, Inc.). One-way ANOVA followed by student's 't' test was used for testing potent fraction with other fractions. p < 0.05; p < 0.01; **p < 0.001 were considered statistically significant. Data are expressed as mean ± SEM. Species identification of the isolates The clinical isolates used in this study were identified by 16S rDNA sequence analysis. Fig. 1 shows the single sharp 1500 bp amplified region of the 16S rDNA ( Fig. 1A and B). The size of the amplified DNA was confirmed by using a standard marker DNA ladder. The partial 16S rDNA sequences thus obtained were retrieved in FASTA format and subjected to BLAST search and deposited in Gen bank under the accession numbers as listed in (Table 1). In total, 16 organisms were identified with 8 organisms each from wound and urine samples. Fractionation of methanol crude extract and phytochemical analysis The total yield of crude methanol extract was found to be 5.25 g/100 g of the dry powder. Fractionation of the crude methanol extract (Fig. 2) by solvent-solvent partitioning gave four different fractions: hexane (yield = 47.23%), chloroform (yield = 24.57%), ethyl acetate (yield = 15.23%) and methanol-water (yield = 7.04%). Phytochemical analysis of these four fractions revealed the presence and absence of medicinally important phytoconstituents such as alkaloids, tannins, saponins, terpenoids, flavonoids, glycosides, phlobatannins and steroids. Chloroform fraction had alkaoids, tannins, saponins, flavonoids, glycosides and steroids whereas, methanol-water fraction had alkaloids, tannins and glycosides. However, the hexane fraction and ethyl acetate fraction were rich in at least one of phlobatannins, terpenoids, glycosides, saponins and steroids. Antibacterial activity The four solvent fractions (HF, CF, EF and MF) were tested for their efficacy against the isolated MDROs. The antibacterial activity of the four fractions against a total of 16 isolates is shown in Fig. 3A when compared with other two fractions and methanol-water fraction had the least activity against organisms tested from wound samples. Interestingly, in case of UTI isolates, chloroform fraction showed maximum antibacterial activity. Maximum activity was observed against S. aureus, P. aeruginosa, K. pneumoniae, E. faecalis and E. coli. Hexane fraction was effective against E. aerogenes, Proteus mirabilis and S. epidermidis but showed least activity against K. pneumoniae and E. coli. Ethyl acetate fraction was found to be effective against E. aerogenes and E. faecalis but a moderate activity was observed for P. aeruginosa, P. mirabilis and S. aureus. However, the methanol-water fraction was not found to be effective against any organisms. Since chloroform fraction showed potential antibacterial effect, it was sub-fractionated using preparative HPLC which resulted in four Sub-fractions (Fig. 4). The total percentage Sub-fractionation of chloroform fraction Among the four sub-fractions screened for potential antibacterial activity against organisms isolated from diabetic wound (Fig. 5A), CHF1 showed maximum activity against E. coli, K. pneumoniae and E. aerogenes among gram negative isolates and also for E. faecalis and S. aureus among gram positive isolates. A moderate inhibition was observed for S. epidermidis and S. haemolyticus. CHF2 was effective against E. faecalis, E. aerogenes and E. coli. However, CHF3 and CHF4 were less effective when compared with CHF 1 and CHF2. We found that, the results obtained from chloroform fraction against microorganism of diabetic wound were similar to that of UTI samples (Fig. 5B). Consistently, CHF1 showed maximum inhibitory activity for P. aeruginosa, E. faecalis, S. aureus, K. pneumonaie, and E. coli. While, minimum inhibitory effect was observed for E. aerogenes, P. mirabilis and S. epidermidis. CHF2 was effective against E. faecalis, K. pneumoniae and S. aureus and a moderate zone of inhibition was observed for P. mirabilis, S. epidermidis and E. coli but the activity was less when compared to CHF1. Chloroform HPLC fraction (CHF3) was found to be more effective against Gram negative isolates than Gram positive isolates. Chloroform HPLC fraction 4 (CHF4) had least antibacterial effect when compared with other three sub-fractions. It was further subjected to check the purity of the CHF1 through analytical HPLC (Fig. 6A) and the structure of the active compound in CHF1 was identified as 3,3 ,4 -Trihydroxyflavone (Fig. 6B) based on the NMR and other spectral data (Fig. 7). Minimal inhibitory concentration of 3,3 ,4 -Trihydroxyflavone The active compound, 3,3 ,4 -Trihydroxyflavone was significantly effective against diabetic wound isolates compared to UTI isolates. It showed an MIC value of 32 g/mL against E. faecalis and S. aureus, and 64 and 128 g/mL K. pneumoniae, E. aerogenes and E. coli, respectively. While, the concentration of reference antibiotic for the corresponding organisms ranged between 8 and 32 g/mL. However, we found the MIC value against S. epidermidis and S. haemolyticus to be 1024 g/mL. In comparison to pathogens from wound samples, the MIC value of 3,3 ,4 -Trihydroxyflavone against K. pneumoniae, S. aureus, E. faecalis and E. coli was also found to be 64 and 128 g/mL, respectively. The MIC of the reference antibiotic for these isolates ranged between 4 and 32 g/mL. However, the MIC of P. aeruginosa was found to be 32 g/mL, and appeared to be relatively greater than that of reference antibiotic. Discussion Opportunistic organisms take advantage of low resistance of the diabetic host to infection. The commensals of the body become pathogenic in conditions such as diabetic wound and UTI. Diabetics have severe neutropenia and impaired humoral as well as cellular immunity which may be the major contributing factor to the cause of infection. 12 Although opportunistic fungi are rare compared with bacteria, they can be the cause of life threatening infections in immune compromised individuals. Thus, the major infective organisms may depend on several factors such as age, gender and severity of infection. We identified eight different bacterial isolates in wound and UTI of patients with diabetes. Polymicrobial infections were also observed with as many as five isolates in a single individual. While, S. aureus was the most common organisms in wound; E. coli was most common in UTI. Gram negative bacteria were predominant in both wound and UTI when compared to Gram positive bacteria. This observation is in agreement with studies reported from several investigators. [13][14][15][16] The presence of E. coli as the most predominant pathogen in diabetic UTI is supported by several studies. [17][18][19][20] The major mechanism of drug resistance in UTI among patients with diabetes is the ESBL production by the infecting organisms. In our study, 30.8% of E. coli and 44.4% of K. pnemoniae were ESBL producers. This was higher than 26.6% reported by Khurana. 21 The present study also confirms the colonization of MDRO notably by MRSA predominance with over 22% of S. aureus being methicillin resistant. This observation agrees with the study of Caputo,22 who reported 20% MRSA in diabetic wound infections. Inhibition zone among associated bacteria are the great interest to search for antibacterial substances. Isolation and screening for secondary metabolite-producing bacteria have been strongly investigated. There are numerous reports on the antimicrobial activity of crude plant extracts and its bioactive compounds. 23 In the present study, we have shown the potential of J. wynaadensis as a source of natural antimicrobials. The chloroform fraction obtained through solvent-solvent fractionation showed strong antimicrobial activity against most of the tested drug resistant organisms. While the other fractions showed moderate antimicrobial activity. This is likely due to the presence of bioactive molecule in chloroform fraction. The antibacterial activity of the methanolic extract of J. wynaadensis against K. pneumoniae has also been reported by Ponnamma and Manjunath. 24 In a recent study reported by Subbu, 25 the methanol extract of Tragia involucrata was found to be effective against E. aerogenes, P. aeruginosa, S. aureus, Proteus vulgaris and E. coli of which were isolated from diabetic foot ulcers and UTI, which is in agreement with our results. We performed further fractionation of the chloroform fraction to obtain four sub-fractions and upon screening for antibacterial properties, we found CHF1 to be highly potent when compared with other three sub-fractions (CHF2, CHF3 and CHF4). This led us to know that CHF1 indeed possesses the bioactive molecule responsible for its potent effects. The maximum inhibitory activity against E. coli, K. pneumonaie, P. aeruginosa, S. aureus and E. faecalis could be due to a common mechanism of action. However, the minimum inhibitory effect of all the sub-fractions observed against P. mirabilis, E. aerogenes and S. haemolyticus suggests that these pathogens might adopt a different kind of resistant pattern in order to multiply in great number. Identifying such resistant pattern will provide new insights to combat such MDROs. Therefore, we subjected CHF1 for its purity through HPLC and found >95% pure. Once the purity was confirmed, characterization and structure elucidation of the isolated molecule was carried out by using different spectroscopic techniques: 1 H (400 MHz) and 13 C NMR (100 MHz). Comparison of the 1 H and 13 C NMR along with the FTIR spectrum indicated the presence of hydroxyl group and presence of hydrogen and carbon atoms. The identified compound, 3,3 ,4 -Trihydroxyflavone is a flavonoid. The antimicrobial activity of flavonoids has been reported earlier. 26 We speculate that the antimicrobial activity observed in this study is due to the presence of flavonoid. A flavonoid rich plant extracts from different species have been reported to possess antibacterial activity. 27,28 Several flavonoids including apigenin, galangin, flavone and flavonol glycosides, isoflavones, flavanones, and chalcones have been shown to possess potent antibacterial activity. 29 Antibacterial flavonoids may have various cell targets, instead of one particular site of activity. One of their molecular activity is to form complex with proteins through nonspecific strengths such as hydrogen bonding and hydrophobic impacts, and also by covalent bond formation. Hence, their method of antimicrobial activity might be identified with their capacity to inactivate microbial adhesins, compounds and cell envelope transport proteins. 30,31 The antimicrobial activity of 3,3 ,4 -Trihydroxyflavone might be due to one or more of the mechanisms of action as mentioned above. The minimal inhibitory concentration (MIC) of 3,3 ,4 -Trihydroxyflavone, the isolated compound from J. wynaadensis extract against the tested bacteria are promising. According to Rios and Recio, 32 MIC value ranging less than 1 mg/mL for crude extracts or 0.1 mg/mL for isolated compounds should be considered effective and also proposed that bioactive compounds that show activity in the range between 0.1 mg/mL (extract) and 0.01 mg/mL (isolated compound) would be of very interesting. While, Gibbons 33 suggests that isolated phytochemicals should have MIC values <1 mg/mL. The MIC of this compound ranging from 32 to 1024 g/mL suggests that the 3,3 ,4 -Trihydroxyflavone hold a lot of promise since the organisms tested were multi drug resistant. And hence, can be derivatized to increase its specificity and potency. The 3,3 ,4 -Trihydroxyflavone structure can serve as a useful template for the synthesis of novel anti-microbial agents. From our study and from other studies, it is evident that emergence of drug resistance is becoming a major obstacle to effective treatment of infections in diabetic individuals. The lack of effective and safe antibiotics to treat serious bacterial infections and lack of new antibiotic drug development by pharma industry is alarming. Consequently, botanicals as well as complementary and alternative medicine are seriously considered to address the issue of emerging drug resistance. The use of traditional medicinal plants for primary health care has steadily increased worldwide. Plants are a rich source of phytochemicals that could be used as antimicrobials and developed as new drugs. Conclusion Prevention and cure of infectious diseases using phytochemicals especially flavonoids are well known. The present study shows that 3,3 ,4 -Trihydroxyflavone isolated from J. wynaadensis is an interesting biopharmaceutical agent against the multi drug resistant organisms isolated from diabetic wound and UTI infections. It could be considered as a source of antimicrobial agent for the treatment of various infections and used as a template molecule for future drug development.	2018-01-17T07:02:47.186Z	2017-08-10T00:00:00.000	{ "year": 2017, "sha1": "59230b88fd5156692e37913858874e8b30590f38", "oa_license": "CCBYNCND", "oa_url": "https://doi.org/10.1016/j.bjm.2017.05.002", "oa_status": "HYBRID", "pdf_src": "PubMedCentral", "pdf_hash": "59230b88fd5156692e37913858874e8b30590f38", "s2fieldsofstudy": [ "Chemistry" ], "extfieldsofstudy": [ "Chemistry", "Medicine" ] }
209748013	pes2o/s2orc	v3-fos-license	Prokineticin 2 relieves hypoxia/reoxygenation-induced injury through activation of Akt/mTOR pathway in H9c2 cardiomyocytes Abstract Prokineticin 2 (PK2) was reported to be decreased in the hearts of end-state heart failure patients. Our study aimed to explore the effects of PK2 on hypoxia/reoxygenation (H/R) injury and the underlying mechanism. H9c2 cardiomyocytes were treated with 5 nM PK2 in the presence or absence of 5 mM dual phosphatidylinositol 3-kinase (PI3K)/the mammalian target of rapamycin (mTOR) inhibitor (BEZ235) for 24 h and then subjected to H/R treatment. Cell viability and lactate dehydrogenase (LDH) release were evaluated by CCK-8 and LDH release assays, respectively. Apoptosis was determined by flow cytometry analysis. Oxidative stress was assessed by measuring superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities and malondialdehyde (MDA) content. Results showed that H/R treatment decreased PK2 expression and inactivated the Akt/mTOR pathway in H9c2 cardiomyocytes. PK2 treatment activated the Akt/mTOR pathway in H/R-exposed H9c2 cardiomyocytes. H/R stimulation suppressed cell viability, increased LDH release, induced apoptosis and oxidative stress in H9c2 cardiomyocytes, while these effects were neutralised by treatment with PK2. However, the inhibitory effects of PK2 on H/R-induced injury in H9c2 cardiomyocytes were abolished by the addition of BEZ235. In conclusion, PK2 relieved H/R-induced injury in H9c2 cardiomyocytes by activation of the Akt/mTOR pathway. Introduction Acute myocardial infarction (AMI), the most severe manifestation of coronary artery disease, has long been a leading cause of morbidity and mortality of cardiovascular disease worldwide [1]. It is estimated that there will be approximately 23 million patients with AMI in China by 2030, which seriously threatens people's health [2]. It is well established that ischaemia/reperfusion (I/R)-induced myocardial injury remains the principal cause of AMI worldwide, which is characterized by the interruption of the myocardial blood supply and subsequent irreversible damage to heart muscle [3]. Several lines of evidence have demonstrated that I/R-induced myocardial injury causes mitochondrial dysfunction with excess production of reactive oxygen species (ROS) and oxidative stress, and if prolonged, leads to the aggravation of cardiomyocyte apoptosis and ultimately cardiac failure [4]. Thus, the exploration of effective therapeutic approaches will open new avenues to protect myocardial cells from I/R injury and improve the clinical outcomes in patients with AMI. Prokineticin 2 (PK2), also known as Bv8, is a small chemokine-like secreted protein that exerts its biological activities via activation of two cognate G protein-linked receptors, namely prokineticin receptor 1 (PKR1) and 2 (PKR2) [5]. PK2 is well-documented to be highly expressed in activated immune cells and inflamed murine tissues with infiltrating neutrophils, showing proinflammatory activities via PKR1 [6]. Recent literatures report that PK2 is also implicated in regulating diverse essential physiological and/or pathological processes, including circadian rhythms, neuroprotection, angiogenesis and pain perception [7][8][9][10]. More importantly, it was reported that the expression of PK2 and its receptors were decreased in the hearts of end-state heart failure patients [11]. PK2 activated the protein kinase B (Akt) pathway to prevent cardiomyocytes from apoptosis against hypoxic insult [11]. Still, little is known about the effects of PK2 on I/R injury in AMI. In the present study, we aimed to investigate the effects of PK2 on hypoxia/reoxygenation (H/R)-induced injury, apoptosis and oxidative stress in H9c2 cardiomyocytes and further explored the underlying mechanism. Cell culture and treatment The H9c2 cardiomyocytes derived from rat embryonic heart were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and routinely maintained in complete Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/L glucose, 10% heat-inactivated foetal bovine serum (FBS; Hyclone, Logan, UT, USA) and antibiotics (100 U/ ml penicillin and 100 lg/ml streptomycin) at 37 C in a humidified incubator containing 95% air and 5% CO 2 . To establish H/R injury model, H9c2 cardiomyocytes were transferred to FBS-and glucose-free DMEM and incubated under hypoxic conditions in an incubator supplied with a gas mixture containing 1% O 2 , 94% N 2 and 5% CO 2 for 12 h at 37 C, followed by reoxygenation (95% air and 5% CO 2 ) in complete medium in a normoxic chamber for another 12 h at 37 C. H9c2 cardiomyocytes in the normal control were cultured under normoxic conditions. PK2 and BEZ235 (a dual phosphatidylinositol 3-kinase (PI3K)/the mammalian target of rapamycin (mTOR) inhibitor) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulphoxide (DMSO) to a final concentration of 5 nM and 5 mM, respectively. In some experiments, H9c2 cardiomyocytes were incubated in FBS-and glucose-free DMEM supplemented with 5 nM PK2 in the presence or absence of 5 mM BEZ235 and then subjected to H/R treatment. Cell viability assay Cell viability was assessed using the cell counting kit-8 (CCK-8) assay. In brief, H9c2 cardiomyocytes were inoculated in 96well plates at an optimal density of 5 Â 10 4 cells/well. Subsequent to the aforementioned treatments, cells were incubated with 10 ll CCK-8 reagent (Beyotime, Shanghai, China) at 37 C for another 2 h. The optical density at a wavelength of 450 nm was detected using a microplate reader (MQX 200, BioTek Instruments, Winooski, VT, USA). Lactate dehydrogenase (LDH) release assay Cell necrosis induced by the H/R injury was evaluated by measuring the release of LDH, a biochemical indicator of cellular damage. Following experimental treatments, H9c2 cardiomyocytes were harvested and lysed, and the supernatants were then centrifuged for 15 min. LDH release in the supernatant was measured using a commercial LDH kit (Takara, Dalian, China) according to the manufacturer's protocols. Analysis of oxidative stress The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in the supernatant were measured using a SOD Activity Assay Kit (BioVision, Milpitas, CA, USA), CAT and GSH-Px assay kits (Nanjing Jiancheng Bioengineer Company, Nanjing, China), respectively. The content of malondialdehyde (MDA), an index of lipid peroxidation, was determined by a Lipid Peroxidation MDA Assay Kit (Beyotime) according to the manufacturer's instructions. Flow cytometry analysis of apoptosis Apoptosis of H9c2 cardiomyocytes was analysed using an Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (BD Biosciences, San Jose, CA, USA). Briefly, H9c2 cardiomyocytes were harvested by trypsinization following treatments and washed twice with PBS. After being resuspended in 100 lL of 1Â binding buffer, H9c2 cardiomyocytes were incubated with 5 lL annexin V-FITC and 5 lL PI in the dark for 15 min. The apoptotic cells were detected by a FACScan flow cytometry (BD Biosciences, San Jose, CA, USA) equipped with CellQuest TM software (BD Biosciences). Caspase-3 and caspase-9 activity assay The activities of caspase-3 and caspase-9 in the supernatant were detected using the colorimetric caspase-3 and caspase-9 activity assay kits (Beyotime), respectively. Absorbance at 405 nm was measured with a microplate reader (MQX 200, BioTek Instruments). Statistical analysis All experimental results were shown as mean ± standard deviation (SD). All statistical analysis was evaluated through Student's t-test or one-way analysis of variance using GraphPad Prism software version 6.0 (GraphPad Software Inc., San Diego, CA, USA). p values less than .05 were considered to be statistically significant. H/R treatment decreased PK2 expression and inactivated the Akt/mTOR pathway in H9c2 cardiomyocytes As compared with the control group, H/R treatment resulted in a significant suppression of cell viability in H9c2 cardiomyocytes (Figure 1(A)). In addition, western blot analysis showed that PK2 protein level was reduced in response to H/R exposure with respect to control group (Figure 1(B)). It has been reported that the Akt/mTOR pathway is involved in cardiovascular diseases including AMI [12]. The effect of H/ R treatment on the Akt/mTOR pathway in H9c2 cardiomyocytes was explored. As displayed in Figure 1(C), we found that the protein levels of p-Akt (Ser473) and p-mTOR (Ser2448) were decreased, whereas the expression of total Akt and mTOR were not changed in the H/R treatment group relative to control group, suggesting that H/R treatment inhibited the Akt/mTOR pathway in H9c2 cardiomyocytes. PK2 activated the Akt/mTOR pathway in H/R-treated H9c2 cardiomyocytes Next, we examined the effect of PK2 on the Akt/mTOR pathway in H/R-treated H9c2 cardiomyocytes. Western blot analysis indicated that administration with PK2 increased the phosphorylation of Akt and mTOR in H9c2 cardiomyocytes versus control group. Notably, treatment with PK2 evidently reversed the reduction of p-Akt and p-mTOR expression induced by H/R in H9c2 cardiomyocytes, while these effects were abrogated following the addition of BEZ235, a dual PI3K/mTOR inhibitor (Figure 2). These results suggested that PK2 activated the Akt/mTOR pathway in H/R-treated H9c2 cardiomyocytes. PK2 suppressed H/R-induced injury by activation of the Akt/mTOR pathway in H9c2 cardiomyocytes To explore the effects of PK2 on H/R-induced injury in H9c2 cardiomyocytes, cell viability and LDH release were evaluated by CCK-8 assay and LDH release assay, respectively. The results demonstrated that PK2 treatment alone showed no effect on cell viability and LDH release in H9c2 cardiomyocytes ( Figure 3(A,B)). Besides, H/R-induced decrease of cell viability and increase of LDH release were restored by treatment with PK2, which were offset following exposure to a combination of PK2 and BEZ235 (Figure 3(A,B)). Together, these data indicated that PK2 impeded H/R-induced injury by activation of the Akt/mTOR pathway in H9c2 cardiomyocytes. PK2 inhibited H/R-induced apoptosis by activation of the Akt/mTOR pathway in H9c2 cardiomyocytes To clarify the effect of PK2 on apoptosis in H/R-treated H9c2 cardiomyocytes, flow cytometry analysis was applied to detect cell apoptosis. As presented in Figure 4, no significant difference in apoptotic rate was observed between PK2treated group and control group. H/R exposure led to a higher ratio of apoptosis in H9c2 cardiomyocytes, while this effect was significantly attenuated by PK2 treatment. However, inhibition of the Akt/mTOR pathway by BEZ235 successfully abolished the suppressive effect of PK2 on H/Rinduced apoptosis in H9c2 cardiomyocytes. Thus, we concluded that PK2 inhibited H/R-induced apoptosis by activation of the Akt/mTOR pathway in H9c2 cardiomyocytes. PK2 regulated caspase-3/9 activities and apoptosisrelated protein expression by activation of the Akt/ mTOR pathway To further confirm whether PK2 could inhibit H/R-induced apoptosis in H9c2 cardiomyocytes, caspase-3 and caspase-9 activities were measured by caspase-3 and caspase-9 activity assays. The results revealed that PK2 alone did not affect caspase-3 and caspase-9 activities in H9c2 cardiomyocytes ( Figure 5(A,B)). H/R stimulation enhanced the activities of caspase-3 and caspase-9 in H9c2 cardiomyocytes, while PK2 treatment apparently undermined H/R-induced increase of caspase-3 and caspase-9 activities ( Figure 5(A,B)). However, the presence of BEZ235 nullified the inhibitory effects of PK2 on H/R-induced increase of caspase-3 and caspase-9 activities in H9c2 cardiomyocytes ( Figure 5(A,B)). Meanwhile, western blot analysis demonstrated that the protein levels of Bax and Bcl-2 were unchanged after PK2 treatment in H9c2 cardiomyocytes ( Figure 5(C)). H/R exposure resulted in a significant increase of Bax and an evident decrease of Bcl-2 in H9c2 cardiomyocytes ( Figure 5(C)). PK2 treatment effectively reduced Bax level and elevated Bcl-2 level in H9c2 cardiomyocytes in response to H/R, which was overturned following treatment with BEZ235 ( Figure 5(C)). Collectively, these results proved that PK2 regulated caspase-3/9 activities and apoptosisrelated protein expression by activation of the Akt/mTOR pathway in H9c2 cardiomyocytes. PK2 suppressed H/R-induced oxidative stress by activation of the Akt/mTOR pathway in H9c2 cardiomyocytes To investigate the effect of PK2 on oxidative stress in H/Rexposed H9c2 cardiomyocytes, the level of oxidative stress indicator MDA and the activities of anti-oxidant enzymes including SOD, CAT, and GSH-Px were measured. As shown in Figure 6(A-D), PK2 treatment alone showed no significant effect on MDA content and SOD, CAT and GSH-Px activities, while H/R stimulation notably augmented MDA content and blocked the activities of SOD, CAT and GSH-Px in H9c2 cardiomyocytes. Interestingly, treatment with PK2 recuperated H/ R-induced enhancement of MDA content and inhibition of SOD, CAT and GSH-Px activities in H9c2 cardiomyocytes, while these effects were abolished by the addition of BEZ235. Therefore, we concluded that PK2 retarded H/Rinduced oxidative stress by activation of the Akt/mTOR pathway in H9c2 cardiomyocytes. Discussion AMI is a complex event responsible for cardiac fibrosis, malignant arrhythmia and heart failure [13], and searching for novel therapeutic targets to treat AMI has become a research focus in the cardiovascular field. Increasing experimental data have shown that H/R-induced myocardial injury in AMI is closely implicated in ROS-induced oxidative stress, calpain activation and cardiomyocyte apoptosis [14]. Wide range of evidence proves that cellular hypoxia disrupts microenvironment homeostasis, resulting in cardiomyocyte necrosis and apoptosis [15]. Cardiomyocyte apoptosis is an important pathological process of H/R-induced myocardial injury and strongly associated with heart dysfunction [16]. In addition, it is extensively believed that oxidative stress is a major cause of cardiomyocyte apoptosis in the development of H/Rinduced myocardial injury [17]. Therefore, inhibition of cardiomyocyte apoptosis and oxidative stress might be an effective therapeutic strategy for preventing H/R-induced myocardial injury in AMI. Herein, we demonstrated that H/R stimulation suppressed cell viability, increased LDH release, induced apoptosis and oxidative stress in H9c2 cardiomyocytes, indicating that H/R induced myocardial injury. Prokineticins (PKs), a newly identified peptide family, has been shown to participate in cardiac repair possibly by inducing angiogenesis or regenerating cardiomyocytes [18]. Dysregulation of PKs and their receptors has been reportedly associated with heart failure [19]. PK2, a member of PK family that located at chromosome 3p21.1, is a powerful angiogenic factor. Recent studies have documented that PK2/PKR1 signalling acts as a new linker between development of obesity, diabetes and cardiovascular diseases [20]. Previously, it was reported that PK2 or overexpressing PKR1 protected cardiomyocytes against hypoxia-mediated apoptosis by activating Akt pathway [11]. In addition to its role in cardiomyocyte protection against hypoxia-induced injury, PK2 was also reported to inhibit serum starvation-induced apoptosis in adrenal cortical capillary endothelial cells and luteal endothelial cells [21,22]. A previous study indicated that the expression profile of PKR1 and PKR2 might manage the effects of PK2 on coronary endothelial cells, favouring endothelial cell barrier dysfunction or compensatory angiogenesis during myocardial infarction [23]. However, whether PK2 has a protective effect on H/R-induced injury in endothelial cells is unknown and deserves further research. It is worth noting that PK2 has been demonstrated to be activated by hypoxiaischemia in primary cortical cultures, and blocking PK2 decreases infarct volume, suppresses central inflammation, and improves behavioural functional outcome in an in vivo stroke model [24]. In the present study, we demonstrated that PK2 expression was decreased in response to H/R stimulation. PK2 treatment activated the Akt/mTOR pathway in H/ R-exposed H9c2 cardiomyocytes. We further manifested that PK2 treatment suppressed H/R-induced reduction of viability, increase of LDH release, apoptosis and oxidative stress in H9c2 cardiomyocytes, which were abolished following the addition of BEZ235, a dual PI3K/mTOR inhibitor. These results suggested that PK2 protected cardiomyocytes from H/Rinduced injury by activation of the Akt/mTOR pathway. The Akt/mTOR signalling pathway is a well-recognized signal transduction pathway that participates in the regulation of cell proliferation, adhesion, apoptosis and survival under pathological conditions [25]. Accumulating evidence shows that the Akt/mTOR pathway is involved in cardiomyocyte protection against hypoxia-induced injury [26]. It was documented that ginsenoside Rg1 protected cardiomyocytes from hypoxia-induced cell injury through activation of the PI3K/ Akt/mTOR pathway [27]. Moreover, a previous study reported that miR-21 inhibited H/R-induced autophagy and apoptosis in H9c2 cells by the activation of the Akt/mTOR pathway [28]. In addition, it was demonstrated that dragon's blood extracts exerted cardioprotective efficacy against myocardial injury in AMI mouse model through activating the PI3K/Akt/ mTOR pathway [12]. In our study, we showed that H/R exposure suppressed the activation of Akt/mTOR pathway in H9c2 cardiomyocytes, consistent with the previous study [29]. PK2 activated the Akt/mTOR pathway in H/R-exposed H9c2 cardiomyocytes, which was in accordance with the previous report [11]. We also found that PK2 treatment recuperated H/Rinduced enhancement of MDA content and inhibition of SOD, CAT and GSH-Px activities by activation of the Akt/ mTOR pathway in H9c2 cardiomyocytes. It has been demonstrated that NF-E2-related factor (Nrf2) is an important factor Figure 2. Effect of PK2 on the Akt/mTOR pathway in H/R-treated H9c2 cardiomyocytes. H9c2 cardiomyocytes were treated with 5 nM PK2 in the presence or absence of 5 mM BEZ235, followed by H/R treatment for 12/12 h, or H9c2 cardiomyocytes were only treated with 5 nM PK2 for 24 h. Western blot was performed to measure the protein levels of p-Akt, Akt, p-mTOR, and mTOR. Ã p < .05. . Effects of PK2 on the apoptosis-related protein activities and expression in H9c2 cardiomyocytes. H9c2 cardiomyocytes were treated with 5 nM PK2 in the presence or absence of 5 mM BEZ235, followed by H/R treatment for 12/12 h, or H9c2 cardiomyocytes were only treated with 5 nM PK2 for 24 h. (A and B) The activities of caspase-3 and caspase-9 in treated H9c2 cardiomyocytes were examined by caspase-3 and caspase-9 activity assays. (C) The protein levels of Bax and Bcl-2 in treated H9c2 cardiomyocytes were determined by western blot. Ã p < .05. NS, not significant. associated with the expression of cytoprotective genes in response to oxidative stress [30]. Nrf2 knockdown inhibited the expression of Nrf2 downstream target genes, including SOD, CAT, and GSH-Px, at the mRNA and protein levels in H9c2 cells [31]. Nrf2 expression was mediated by the Akt/ mTOR pathway [31,32]. Therefore, we inferred that PK2 could increase protein levels of SOD, CAT, and GSH-Px in H/Rexposed H9c2 cardiomyocytes. Conclusion In summary, our study provided the evidence that PK2 relieved H/R-induced injury in H9c2 cardiomyocytes by attenuating apoptosis and oxidative stress through activation of Akt/mTOR pathway, heightening our understanding of the roles and molecular mechanisms of PK2 in H/R-induced injury in AMI. Therefore, our study suggested that PK2 might be a potential therapeutic target to protect against H/Rinduced injury. Disclosure statement No potential conflict of interest was reported by the authors.	2020-01-05T14:03:13.326Z	2020-01-01T00:00:00.000	{ "year": 2020, "sha1": "9caeedcb7d961f14ecba42088297d47f631b768a", "oa_license": "CCBY", "oa_url": "https://www.tandfonline.com/doi/pdf/10.1080/21691401.2019.1709850?needAccess=true", "oa_status": "GOLD", "pdf_src": "TaylorAndFrancis", "pdf_hash": "6a75394aec54873eb5b105b4e05b716f447bb272", "s2fieldsofstudy": [ "Biology", "Chemistry", "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
33605647	pes2o/s2orc	v3-fos-license	Prosthetic Functional Rehabilitation Following Resection of an Oral Malignoma – A Case Report Citation: Zupancic-Cepic L, Eder J, Schmid-Schwap M, Piehslinger E (2017) Prosthetic Functional Rehabilitation Following Resection of an Oral Malignoma – A Case Report. J Nov Physiother Phys Rehabil 4(1): 009-013. DOI: http://doi.org/10.17352/2455-5487.000039 Abstract Tumor surgery in the orofacial region frequently requires resection of major parts of the jawbone and the adjacent facial and pharyngeal soft tissue resulting in large-scale hard and soft tissue defects. Consequences of such defects may include masticatory dysfunction, speech disturbances and swallowing problems as well as signifi cant aesthetic impairment for the patients concerned. Thus, comprehensive therapy with reconstruction of the missing tissue and subsequent prosthetic rehabilitation is of major importance for restoring and improving the tumor patient’s quality of life. The case reported illustrates the stepwise prosthetic rehabilitation of masticatory function in a patient after radiochemotherapy and surgical treatment of a squamous cell carcinoma in the right retromolar trigone (T4N2bM0) with neck dissection, hemimandibulectomy and mandibular reconstruction with titanium plate and pectoralis major fl ap. Case report Full and complete rehabilitation of masticatory function may only be achieved by a fi nal prosthetic restoration. Selection of the prosthetic restoration will be depend on the intraoral environment, in particular the number of residual teeth or the teeth worth preserving, and the condition of the potential denture support. Implant-borne restorations will also be used quite commonly, e.g. in cases of edentulousness. However, for irradiated tumor patients it must be considered, that the risk of osteoradionecrosis will be increased [3]. Case Report At the age of 52 years, patient F.K., heavy smoker under medication for high blood pressure (Enac Hexal 20mg), underwent treatment for a squamous cell carcinoma in the right retromolar trigone (T4N2bM0) at the University Clinic of Oral and Maxillofacial Surgery of the Vienna General Hospital. Introduction Treatment of orofacial tumors commonly requires partial resection of the mandibular bone potentially affecting mandibular functionality [1]. For such interventions, soft tissue and muscle tissue may also need to be sacrifi ced frequently involving signifi cant impairment: facial asymmetry, mandibular deviation, masticatory dysfunction, speech disturbances and swallowing diffi culties as well as considerable aesthetic impairment [2,3]. The extent of the remaining deformity will essentially depend on the type of surgery having been used, the number of residual teeth, the extent of the impairment of lingual function and the extent of the damage and injury of motor and sensory nerve endings [2]. The tractive force of the contralateral muscles will cause a slanted deviation of the mandible with loss of regular occlusion [4]. Mandibular reconstruction and subsequent prosthetic rehabilitation is intended to provide for a stable intermaxillary relation with improvement of oral functions as well as aesthetics [4,5]. Principally, mandibular defects may be reconstructed using alloplastic material (e.g. titanium plate), non-vascularized or vascularized autologous tissue or a combination of the same [4]. Microvascular free osteocutaneous fl aps have been described as gold standard in literature with the free fi bular transplant being considered as the clear favorite choice [6,7]. Upon comprehensive dental assessment and evaluation the precise and detailed course of treatment was planned. For restoring a stable intermaxillary relation and occlusion fully prosthetic rehabilitation using removable dentures in both jaws was planned. In the maxilla, the anterior tooth gap (#9) was restored with a bridge supported by teeth #8 and #10 made of gold alloy with ceramic veneering (Porta Geo Ti, Wieland) and the two terminal gaps were restored with a clasp-tooth-crownsupported metal frame denture of a chrome-cobalt alloy ( Figure 7). In the mandible, a telescope metal frame denture with shortened row of teeth on the resection side was manufactured ( Figure 8). This was to ensure a stable intermaxillary relation and occlusion for the patient (Figures 9,10). The detailed course of treatment has been illustrated in Discussion Surgical treatment of tumor lesions in the oral cavity (tongue, fl oor of the mouth, alveolar ridge, oropharynx) will frequently be associated with an unfavorable setting for the anchorage of prosthetic restorations [3]. The primary problems mostly involve disturbed function of tongue and facial muscles, cicatricial pull, restricted mouth opening, mandibular deviation and facial asymmetry as well as major reduction of the socalled neutral zone. The term neutral zone defi nes the space in the oral cavity where the outward force of the lingual musculature is equal and balanced with the inward forces exerted by the buccinator and lip muscles. Removable partial prostheses manufactured under adequate consideration of the neutral zone (neutral zone technique) will ensure suffi cient prosthetic stability and retention [8]. Case reports show that this technique will also be well suitable for the prosthetic rehabilitation in patients with partial mandibulectomy [8,9]. However, it must be noted that such patients are frequently reconstructed using muscle fl aps, although these show a poor bearing capacity and will be unsuitable as denture support. In such cases, use of endosseous Radiotherapy will result in obliteration of small blood vessels and disorders of bone vitality. The bone will become increasingly susceptible to bacterial infections and, as a consequence, may develop osteoradionecrosis [4]. Under this setting, the risks and benefi ts of implantation should be carefully weighed. However, survival rates seen with implants In the case reported, the right lateral mandible resected was prosthetically restored using a telescopic hybrid denture providing for good prosthetic retention as a result of the friction of the telescope system [11]. For avoiding direct support of the denture on the pectoralis transplant and thus minimizing the risk of dehiscence, a shortened row of teeth was used on the resection side (to premolars) while dentition to the fi rst molar on the contralateral side allowed regular articular support and the occlusal concept of canine guidance. Conclusion Loss of jaw segments and of normal tissue anatomy as a result of the surgical treatment of oral tumors frequently represents a particular challenge in the functional masticatory rehabilitation of the patient. In some of the cases certain compromises may also need to be accepted. Presence of residual dentition worth preserving in the resected mandible may provide for adequate denture support and potentially replace or at least delay a more intricate and time-consuming implant-supported rehabilitation.	2019-03-16T13:12:40.435Z	2017-09-01T00:00:00.000	{ "year": 2017, "sha1": "a3cbc5c81619e67dac08dae566a8509bd82c65ae", "oa_license": "CCBYNC", "oa_url": "https://www.peertechz.com/articles/JNPPR-4-139.pdf", "oa_status": "HYBRID", "pdf_src": "MergedPDFExtraction", "pdf_hash": "c9ed1fe9f1275b0bd6ab07e156af5de6b732e2cc", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
119633230	pes2o/s2orc	v3-fos-license	A bulk-surface reaction-diffusion system for cell polarization We propose a model for cell polarization as a response to an external signal which results in a system of PDEs for different variants of a protein on the cell surface and interior respectively. We study stationary states of this model in certain parameter regimes in which several reaction rates on the membrane as well as the diffusion coefficient within the cell are large. It turns out that in suitable scaling limits steady states converge to solutions of some obstacle type problems. For these limiting problems we prove the onset of polarization if the total mass of protein is sufficiently small. For some variants we can even characterize precisely the critical mass for which polarization occurs. that corresponding models must address is the mechanism by which relatively weak chemical gradients yield large spatial changes of the concentrations of chemicals at the cellular level. A significant number of the models used to describe cell polarization rely on the local excitation, global inhibition (LEGI) mechanism which was suggested in the seminal paper about cell polarization [20]. Different forms in which the chemical pathways might be arranged in a way consistent with this mechanism, and yield the specific chemical patterns associated to cell polarization, are described in [28]. Typically polarization is achieved by the combination of an internal pattern forming system, a response to an external signal that imposes some directional preference to the pattern, and the amplification of small concentration differences. In this paper we study a minimal model for this amplification step and examine when polarization patterns appear. These are characterized by the onset of clearly distinct regions in which the concentrations of some chemicals have different orders of magnitude. The most remarkable feature of the proposed model is that for a suitable choice of parameters it is possible to approximate the model by a generalized obstacle problem. This asymptotic reduction yields a clean and analytically tractable characterization of polarized states and allows for a rigorous investigation of the onset of polarization patterns. The bio-chemical model that we study in this paper is a system of PDEs that is motivated by the GTPase cycle model presented in [25,26]. We consider two versions of one protein on the cell surface which is either in an active or in an inactive state. We denote the first surface concentration as u and the second as v. Furthermore the inactive proteins can move to the interior of the cell and vice versa. We denote the volume concentration in the cytosol by w. Our model contains three types of activation processes of the proteins which all take place on the cell membrane. First there is an intrinsic activation with rate a 1 . Second there is an activation by a positive feedback mechanism and a rate law given by a Michaelis-Menten law. Third, there is an activation induced by an external (or internal) chemical signal. We assume here that this signal has already been processed and has lead to a concentration field c on the membrane of a chemical that catalyzes the activation (the function c could be also interpreted as the surface concentration of some activated receptors). For the deactivation we again prescribe a Michaelis-Menten rate law. The use of Michaelis-Menten laws stems from the fact that the corresponding processes require some catalyzation, as the intrinsic activation and deactivation of GTPase proteins is typically very slow [2]. To introduce our mathematical model, let Ω ⊂ R 3 denote the cell and Γ := ∂Ω the cell membrane. The assumptions described above give rise to the following bulk-surface reaction diffusion system. Here c : Γ × (0, T ) → R is the (processed) external stimulus, with some abuse of notation ∆u and ∆v denote the Laplace-Beltrami operator on the surface Γ, while ∆w is the usual Laplacian. We complement the system with initial conditions w(·, 0) = w 0 in Ω, u(·, 0) = u 0 on Γ, v(·, 0) = v 0 on Γ , where w 0 : Ω → R and u 0 , v 0 : Γ → R are given nonnegative data. Solutions of (1)-(5) satisfy the mass conservation propertŷ Ω w(x, t) dx +ˆΓ(u(y, t) + v(y, t)) dH 2 (y) =ˆΩ w 0 dx +ˆΓ(u 0 + v 0 ) dH 2 for all t ≥ 0. (6) In this paper we will mainly study for given c = c(x) the stationary solutions of (1)- (4). Polarization patterns arise here under the assumption that several of the reaction rate parameters a 1 , a 2 , ..., a 7 , the diffusion coefficient D and the total mass of proteins are large. This will be parametrized by a large parameter ε −1 > 0. The most remarkable feature of the patterns is that a suitably rescaled version of u converges as ε → 0 to the solution of a variational inequality which is closely related to the classical obstacle problem [17]. Responsible for this is the presence of the inhibitory Michaelis-Menten reaction term u 1+u , whereas the intrinsic activation term a 1 v and the positive feedback activation term a 2 uv a 3 +u do not contribute to the limit. Let us give a simple heuristic explanation for the impact of the Michaelis-Menten reaction term R(u) = u 1+u . In the parameter regimes that we consider it will be convenient to introduce the rescaled concentration U = εu in order to account for the large values of u in some regions. Then, the function R U ε converges in a suitable sense to the maximal monotone graph ξ(U ) such that ξ(0) = [0, 1], ξ(U ) = 1 if U > 0. It is well known that several variational inequalities, for instance the one associated to the obstacle problem, can be reformulated in terms of maximal monotone graphs [5], [7,Sec.2.2], compare the Remark 3.4 below. Actually, in this paper we will consider two different types of scaling limits for stationary solutions of (1)- (4). In the first one we will assume that D = ∞ before taking the limit ε → 0, which is motivated by the fact that the cytosolic diffusion is typically much larger than lateral diffusion over the membrane [16]. Then the stationary version of equation (3) becomes just the formula w = M −´Γ(u + v), where M is the total amount of protein, and the system reduces to two coupled surface PDEs that include, as a remnant of the bulk-surface coupling, a nonlocal term. Taking then the limit ε → 0 we obtain that the limit satisfies a variational inequality for a suitable PDE. In the second scaling limit we will take D large but finite (finite cytosolic diffusion case). The limit ε → 0 will then yield a variational inequality for an operator which contains, in addition to partial derivatives, the concatenation of the Neumann to Dirichlet map (cf. Appendix 4.4) and the Laplace-Beltrami operator. One particular advantage of the limiting obstacle-type problems is that they allow for an easy characterization of polarized states: these are described by the property that the rescaled concentration of the active proteins takes the value zero on a set of positive measure and it takes a positive value on the complement, which has also positive measure. We will prove in this paper the onset of polarization patterns, for both cases, if the total (rescaled) mass of protein m is sufficiently small. In addition, we show that patterns are localized in regions, where the signal c is large. We will also prove that in the case D = ∞, for given concentration c, there exists a critical mass of protein m * such that polarization takes place for m < m * and it does not for m > m * . In the case D < ∞ due to the presence of the nonlocal contributions, it is not clear if the critical mass m * exists with the same degree of generality concerning the domain Ω and the concentration c. However, in the case of spherical domains Ω the nonlocal operator reduces to the Dirichlet to Neumann operator (cf. Appendix 4.4) and we will be able to prove the existence of the critical mass m * if D < ∞ for these particular domains and general concentrations c. The model (1)-(4) is different from LEGI-type models and rather describes the signal amplification following a first polarization of the cell, expressed by a heterogeneous distribution c. In the scaling limits under consideration solutions of (1)-(4) do not exhibit spontaneous patterns if c is constant unlike the classical Gierer-Meinhardt models (cf. [13]). The system (1)-(5) is closely related to models for spontaneous cell polarization (in absence of an external signal) considered in [26,15]. There, more general reaction kinetics but spatially homogeneous rate constants are assumed. The model (1)-(5) belongs to the class of bulk-surface partial differential equations that appears in a variety of different applications and has attracted quite some attention over the last years, see for example [18,21,24,8] and the references therein for applications to cell biology and [27,3,9,11,15] for recent well-posedness results and rigorous asymptotic limits. In [9], for a different bulk-surface system where the nonlinearity is contained in the Robin boundary condition, a fast-reaction limit was derived that also takes the form of an obstacle-type problem involving the Dirichlet to Neumann map. However, compared to our contribution the asymptotic analysis and the limiting obstacle-type problems are different. The convergence of elliptic PDEs with suitably rescaled Michaelis-Menten reactions to a classical obstacle model has already been observed in [4]. There the motivation was an approximation of the obstacle problem by a bounded penalty method suitable for numerical simulations. No investigation of pattern forming properties in the sense of our analysis has been done in [4,9]. For an investigation of axially symmetric cap-like patterns in a related but different model see [8]. The plan of the paper is the following. In Section 2 we will briefly review the well-posedness of the initial value problem (1)-(5) and establish the existence of stationary states (cf. (17)- (21)). In Section 3 we will first investigate the limit of infinite cytosolic diffusion D → ∞ for stationary solutions. In this limit w converges to a constant and the model (1)-(5) becomes a nonlocal elliptic system (cf. (23)-(25)) containing the nonlocal term´Γ (u + v). Next, we prove that in some suitable scaling limit the solution u of the rescaled system (27)- (29) converges to the unique solution of an obstacle problem for the Laplace operator (cf. Theorem 3.2). In Section 3.3 we derive precise conditions for the onset of polarization and characterize the positivity set of u for small mass in Section 3.4. In Section 4 we consider the analogous problems for finite cytosolic diffusion coefficients D. We derive in a scaling limit analogous to the case D = ∞ a generalized obstacle problem in Theorem 4.2 containing the Dirichlet to Neumann operator. We can prove also prove polarization for small mass for the resulting model (cf. Subsection 4.2). We obtain a more precise description of the localization property, as well as the existence of a critical mass in the case of spherical domains Ω in Subsections 4.3 and 4.4. Well-posedness and existence of steady states Let us state the main assumptions that we impose in the following. Assumption 2.1. Let Ω ⊂ R 3 be an open, bounded, connected set with C 3 -regular boundary Γ = ∂Ω. Assume a 1 , a 2 ≥ 0, a 3 , a 4 , a 5 , a 6 , a 7 > 0 and D ≥ 1. Furthermore we assume that c : Γ → R + is continuous and strictly positive. This in particular implies that there exists c 0 > 0 such that Remark 2.2. Throughout the paper we will denote by W k,p (Ω) the usual Sobolev spaces over Ω and by W k,p (Γ) the corresponding Sobolev spaces on the manifold Γ. For T > 0 we set Ω T := Ω × (0, T ) and Γ T := Γ × (0, T ). For a Banach space X we consider the Bochner spaces L 2 (0, T ; X) and the spaces H 1 (0, T ; X) of functions in L 2 (0, T ; X) with weak time derivative in L 2 (0, T ; X). The relevant diffusion operator on Γ is the corresponding Laplace-Beltrami operator, see for example [23]. For simplicity we just write ∆ if there is no reason for confusion. We remark that classical elliptic L p -and Schauder-regularity results [14, Sec. 6.1, Sec. 9.5] and a partition of unity argument yield the corresponding L p -regularity properties for elliptic equations involving the Laplace-Beltrami operator on Γ. In fact, in local coordinates the Laplace-Beltrami operator corresponds to an elliptic operator in divergence form (with C 2 -regular coefficients in our case). Similarly one deduces parabolic L 2 -regularity in analogy to [10, Theorem 7.1.5] for evolution problems on Γ involving the Laplace-Beltrami operator. For simplicity we will often neglect dx and dH 2 in the corresponding integrals if there is no reason for confusion. For a function ϕ : Γ → R we let ϕ : We first quote an existence and uniqueness result for the full evolution problem that was shown in [15], Proposition 3.3 (there uniformly bounded initial data were assumed and only the case of constant c is covered, but inspecting the proof we see that the result remains valid under the present assumptions). The following a-priori estimate will be useful to prove the existence of steady states as well as to pass to the limit D → ∞. We next prove the existence of stationary states using Theorem 2.3 and the a-priori estimates from Proposition 2.4. Moreover, Proof. Denote by X the set of all with C 1 from Proposition 2.4. Fix T ≥ 3. By Theorem 2.3 for any (u 0 , v 0 , w 0 ) ∈ X there exists a unique weak solution (u, v, w) to the evolution problem in (0, T ). By Proposition 2.4 and by the mass conservation property (6) we deduce that (u(t), v(t), w(t)) ∈ X for all t ∈ (0, T ). Moreover, the bounds (9), (10) are satisfied for any σ > 0. In particular (see e.g. [10], Theorem 3, Section 5. with bounds that depend only on σ and the data. In particular, for any τ ∈ (0, 1) the map is well-defined, and by Sobolev embedding compact. Considering differences of two solutions with initial data (w 0 , u 0 , v 0 ) and (w 0 ,ũ 0 ,ṽ 0 ), respectively, we obtain by similar calculations as in Proposition 2.4 that Therefore S(τ ) is continuous for any 0 < τ < T . By Schauders fixed point theorem for any τ ∈ (0, 1), τ = 1/N , there exists a fixed point (u τ , v τ , w τ ) ∈ X of S(τ ). Since the system (1)-(4) is autonomous and solutions are unique we obtain a solution (u τ , v τ , w τ ) of this system on (0, T ) with the property that By Proposition 2.4 we obtain for any N ∈ N uniform bounds Therefore there exists a subsequence N → ∞ (not relabeled), hence τ = τ (N ) → 0, such that It is easy to show that (u, v, w) is a solution to (1)-(4) on the time interval (1,2). Moreover, by the Aubin-Lions lemma, we have strong Next, since by [10], Chapter 5.9, we have uniform bounds in We then compute that in L 2 (Γ) where we have used that u τ is uniformly bounded in H 1 ((1, 2); L 2 (Ω)) ֒→ C [1,2]. We therefore have obtained a time-independent solution of (1)-(4) on (1, 2) that satisfies the mass constraint. This gives the required solution of the stationary system. Starting from the H 1 regularity of the solution and the Sobolev embeddings [14,Theorem 6.31] that w ∈ C 2, 1 2 (Ω). Standard Schauder regularity then implies higher regularity for v, u. The estimate (22) is obtained from (u, v, w) ∈ X and (9). 3 The limit of infinite cytosolic diffusivity: D = ∞ Passage to the limit D → ∞ In biological cells the diffusion in the cytosol is typically much faster than the lateral diffusion on the membrane. This means in our rescaling that D ≫ 1 and motivates to consider an asymptotic reduction D → ∞. We obtain a system of two surface PDEs for u, v, whereas the variable w becomes spatially constant and is determined by the prescribed total amount of protein in the cell. This introduces a nonlocal term in the PDE system for u, v. Theorem 3.1. For any M > 0 there exist u, v ∈ C 2 (Γ), and a nonnegative constant w, such that Proof. Consider a sequence (D k ) k with D k → ∞ as k → ∞ and let (u k , v k , w k ) be a solution to (17)- (21) with D replaced by D k . By the bound (22) and standard compactness arguments we deduce the existence of a subsequence (not relabeled) . Moreover, by the Rellich theorem and the compactness of the trace mapping H 1 (Ω) → L 2 (Γ) we have strong convergence in L 2 (Γ) 2 ×L 2 (Ω). This allows to pass to the limit in the equations (17), (18) and (21) and to deduce (23)- (25). The higher regularity of the solution is deduced from standard regularity results as above. Derivation of the obstacle problem Our goal in this section is to consider a suitable scaling limit of the system (23)- (25). More precisely, we introduce the following rescalings and assume that the coefficients a 1 , a 2 , a 3 are kept of order one and that a 4 , a 5 , a 6 , a 7 > 0. Upon redefining c, without loss of generality we can assume that a 7 = 1. Moreover, in order to obtain a nontrivial limit we also need to assume that the total number of proteins M rescales like 1 ε , more precisely M = m ε for some fixed m > 0. Denoting by (u ε , v ε , w ε ) the solution of the rescaled system (23)- (25) an asymptotic expansion shows that we should expect that U ε := εu ε , v ε , w ε are of order one. We therefore rewrite the rescaled system as We continue to assume that w ε ≥ 0, hence´Γ U ε + εv ε ≤ m. For convenience we state our results mainly in terms of a function g : Γ → (0, 1) instead of c, is a family of nonnegative solutions of (27)- (29) and fix an arbitrary sequence (ε j ) j∈N with lim j→∞ ε j = 0. Moreover, there exists α ∈ R such that the functions u and ξ satisfy almost everywhere on Γ and we haveˆΓ u = m. Taking the limit in (38) we obtain where ϕ is an arbitrary test function in C 2 (Γ). In particular this implies that (c + a 5 )v is an absolutely continuous measure with can be written in terms of a bounded density, more precisely We now consider the limit of (27). Multiplying this equation by ϕ where ϕ ∈ C 2 (Γ) and using (39) we obtain the limit equation which is satisfied in the sense of distributions. Using (41) we obtain Due to the boundedness of g and ξ classical regularity theory implies that u is bounded in W 2,p (Γ) for any p < ∞. This gives (31). On the other hand, taking the limit of (29), using (39) and the uniform boundedness of w ε we obtain (32). It remains to prove ξu = u. Using (27), (35) and applying classical regularity arguments [29,Proposition 4.3], (see also [12]), we obtain uniform estimates for U ε j in W 1,q (Γ) for all 1 < q < 2. Then U ε j → u in L q (Γ) and for any test function ϕ ∈ C 0 (Γ) This implies ξu = u. We remark that by an integration of (42) first over Γ and then over {u > 0} (and using the Moreover, by Stampacchia's lemma [12,Proposition 3.23] and the W 2,p (Γ)-regularity of u one obtains ∆u = 0 almost everywhere in {u = 0}, which yields the representation formula In particular, from the second equality in (43) and (44) we deduce that ξ, α are determined by u. Critical mass for polarization. Without loss of generality, rescaling u and α accordingly, we set in the following a 4 = 1. Thus, we consider a solution (u, ξ, α) of the problem Here g is as in (30) and we also recall the compatibility condition (43) for α, which by (46) in particular yields We have a variational principle for the problem (45), (46). Proposition 3.3. Fix 0 ≤ α ≤ α * and consider the minimization problem Then the following properties hold. We use the direct method of the calculus of variations. For any If 0 ≤ α < α * this shows the coercivity of I and then also the existence of a minimizer. (We remark that for α > α * we can take a sequence v n = n such that the functional converges to −∞.) In the case α = α * we have I(v) = I(v + γ) for any γ ∈ R. We then can minimize I in the class {v ∈ H 1 (Γ) :´Γ v = 0} and obtain a minimizer v * for this constrained minimization problem, which in addition satisfies, since Moreover, v * solves the Euler-Lagrange equation −∆v * = −(1 − g) + α * g + λ, for some Lagrange multiplier λ (we even find λ = 0 by integration over Γ). By elliptic regularity, v * is bounded. But then u := v * − min Γ v * is nonnegative and satisfies (49). 3. (50) is the weak form of the Euler-Lagrange inequality and holds for any solution of (49). Vice versa, for a nonnegative solution u ∈ H 1 (Γ) of (50) and any other v ∈ H 1 (Γ), v ≥ 0 we deduce from (50) that Therefore, u is a minimizer in (49). We have not yet shown that the assumption u ∈ W 2,p (Γ), 1 ≤ p < ∞ for a minimizer u of (49) that appears in item 6 of the previous proposition is always satisfied. This however will follow from Proposition 3.3 together with Theorem 3.7 below. We will say that a solution to (45), (46) exhibits polarization if the measure of both of the two regions {u > 0} and {u = 0} is positive. It turns out that it is possible to give a necessary and sufficient condition for polarization in this model. To this end we define an auxiliary function u * . Equation (55) has infinitely many solutions due to the definition of α * , which implieś Γ [−(1 − g) + α * g] = 0. The solutions differ by a constant and they are continuous in Γ. Then, if u is any solution of (55) we obtain that u * = u − min Γ u and u * is unique. We define In the following we denote g max = max Γ g and α 0 = (1−gmax) gmax . Notice that α 0 ≤ α * and strict inequality holds if g is not constant. In case that g is constant no polarization occurs. More precisely we have the following equivalence. Proof. Let u ≥ 0 be constant. Since m > 0 we have u > 0 and ξ = 1. Then (45) implies that g is constant. This yields u * = 0 and α = α 0 = α * . Vice versa assume that g is constant. By m > 0 the set {u > 0} has positive measure and (43) yields that α = 1−g g and ξ = 1. But then (45) implies ∆u = 0 and u is constant. Our main results concerning the onset of polarized states is contained in the following theorem, see also the remark below. Theorem 3.7. Let g be as in (30) Proof. Solvability of the variational inequality and monotonicity. The variational inequality (50) can be solved for each α ≤ α * , see Proposition 3.3, and yields a unique solution for α < α * and a solution that is unique up to additive constants if α = α * . We next show that solutions for 0 ≤ α ≤ α 0 are trivial, unless α = α 0 and g is constant. Consider α = α 0 and the unique solution u of the variational inequality (50). Assume that u ≡ 0. We note that Using which gives a contradiction, unless g ≡ g max and u is a constant. Now for all 0 ≤ α < α 0 by the monotonicity property and comparing u with the trivial solution for α = α 0 we deduce that the solution of (50) vanishes identically, also in the case that g is constant. By Proposition 3.3 this proves the claim. This in particular shows that for any m > 0 the solution (u, ξ, α) of (45)-(47) is unique (here we use that ξ is determined by u, α through the representation formula (44)). Furthermore, consider the map α → m that assigns to α the total mass m of the solution of the variational inequality (50). By the monotonicity and uniqueness properties proved above α → m is strictly increasing on [α 0 , α * ]. This equivalence together with the monotonicity property shown above proves that m → (α, u) is strictly monotone. Finally, by (44) then also the map m → ξ is monotone. If m ≥ m * we must have necessarily α = α * . Since α =´Γ we obtain that ξ = 1 for a.e. x ∈ Γ, whence u solves (55). Thus u and u * only differ by a constant A and using (56) we obtain A = m − m * , whence item c) in Theorem 3.7 follows. Localization of {u By the results of the previous section, for non-constant g and sufficiently small mass polarization occurs. We can say a bit more about the patterns that arise if we consider the limit of vanishing mass: in a well-defined sense the polarized region concentrates on the set where g (and hence also c) takes its maximal value. To give a precise statement, let S = {x ∈ Γ \| g(x) = g max } and α 0 = 1−gmax gmax as above. 2. Assume that u n attains a maximum in x n . Then g(x n ) → g max . Finite cytosolic diffusion D < ∞ In this section we now deal with the case of finite cytosolic diffusion D < ∞ and the fully coupled system. Using a similar rescaling as in the case D = ∞ we derive an asymptotic limit in the form of a bulk-surface obstacle problem. In the case of spherical cell geometry we characterize polarized states in terms of this limit problem and in terms of the total amount of proteins in the cell. Derivation of a nonlocal obstacle problem For finite D we use a similar rescaling of the steady-state equation (17)- (21) as in the previous section but consider in addition to (26) that D becomes large with ε → 0, more precisely D 1 ε D with D of order one. This yields the following bulk-surface system. andˆΓ By Theorem 2.5 for given m > 0 there exists a nonnegative solution of this system. We first prove some uniform bounds. With these uniform estimates we can pass to the limit ε → 0. We then obtain a generalized obstacle type problem. For p > 2 we test (70) with (κ p w) p−1 , κ p := a 6 a 5 as well as (69) with v p−1 . This yields and hence v is bounded in L p (Γ) for any 1 ≤ p < ∞. By [22,Theorem 3.14] we then obtain that w ∈ C 0,γ (Ω) for some γ > 0, with which in particular proves the desired maximum bound for w. As a harmonic function, w is smooth in the open set Ω. The maximum bound for w and (69) yield v ∈ L ∞ (Γ), with a corresponding bound. By standard L p -regularity for (68) we finally deduce that u ∈ W 2,p (Γ) for any 1 ≤ p < ∞ and u W 2,p (Ω) ≤ C(a 4 , a 5 , a 6 , m 0 , c, Ω, p). We can express the system above as an equation for u only, but involving a non-local (pseudodifferential) operator given by the Neumann to Dirichlet operator T applied to the Laplace-Beltrami operator on Γ. For properties of the Neumann to Dirichlet (and the Dirichlet to Neumann) operators see the appendix. and v is given by v = 1 − g a 5 (a 6 w + a 4 ξ). Localization of {u For simplicity we set a 4 = 1 in the following. As above let S = {x ∈ Γ \| g(x) = g max } and α 0 = 1−gmax gmax . Proposition 4.4. Consider a sequence m k → 0 of positive numbers and for k ∈ N any nonnegative solution (u k , v k , w k , ξ k ) of (68)-(71) with´Γ u k = m k . Then the following holds 1. u k W 2,p (Γ) → 0 for any 1 ≤ p < ∞. 2. We multiply (75) by a positive test function ϕ and integrate by parts to obtain Using m k → 0 and the properties from the first item, we deduce that lim sup k→∞ w k ≤ Γ (1−g)ϕ a 6´Γ gϕ . We now take a sequence (ϕ ℓ ) ℓ of test functions such that spt(ϕ ℓ ) ⊂ {g > g max − 1 ℓ } and obtain lim sup 3. Since u k is locally C 2 -regular in {u k > 0} we have −∆u k (x k ) ≥ 0 and ξ(x k ) = 1 and hence It follows together with item 2 of this proof that It follows that w k → 1 a 6 α 0 and that g(x k ) → g max . 4. Integrate (75) over Ω k and recall that ξ k = 1 in Ω k . Since u k ∈ W 2,p (Γ) we have´Ω k ∆u k = 0. This yields and the claim follows by item 2 and since w k − w k → 0 in C 0 (Γ). Moreover, by item 1, (98) and the Sobolev embedding T ∆u k → 0 in W 1,p (Γ) for all 1 ≤ p < ∞ and uniformly on Γ. Then, possibly after increasing k 0 , we deduce On the other hand, since D(x 0 , r) ⊂ {dist(·, S) > r} In addition 0 ≤ u k < (α 0 +1)θ 32 r 2 for k ≥ k 0 for k 0 ∈ N sufficiently large, only depending on δ, since u k → 0 uniformly by item 3. Then as in Proposition 3.9 the weak maximum principle yields a contradiction. Refined analysis for the case of spherical cell shapes In the following we restrict ourselves to the case of a spherical cell and let Ω = B(0, 1) ⊂ R 3 . This allows us to characterize the system (68)-(71) as a generalized obstacle problem that involves the Dirichlet to Neumann operator N (see the appendix for a definition). In order to reduce the number of parameters we define ℓ = a 6 D . and if in addition w is given by a 6 w = α − ℓg N (u) + (u − u) and v by (77). The number α is then determined by Proof. By Proposition 4.3 (see in particular (78)) it is sufficient to prove that for Ω = B(0, 1) and any u ∈ H 2 (Γ) we have hence ∂ n F = N u on S 2 , and let G denote the solution of We now consider the position vector field, η(x) = x for x ∈ B(0, 1). We observe that and that on Γ = S 2 ∂ n G + η · ∇F + F = ∂ ∂n G + n · D 2 F n + 2n · ∇F where in the last step we have used that u = F on Γ and that ∆ = ∂ 2 r + 2 r ∂ r + 1 r 2 ∆ S 2 in spherical coordinates. Hence G + η · ∇F + F is constant and since´Γ G =´Γ η · ∇F = 0 we deduce which proves the claim. We assume that the maximum of U is positive and argue by a maximum principle. By [1] (see also [19]) for any maximum point x 0 of U there exists a sequence (x k ) k in Γ with x k → x 0 and lim k→∞ ∆U (x k ) ≤ 0. By assumption and continuity of U we can assume without loss of generality that U (x k ) > 0 for all k ∈ N. Hence ξ 1 (x k ) = 1 ≥ ξ 2 (x k ) and the second term in (85) as well as the third term are nonpositive in x k . We next consider the term involving the Dirichlet to Neumann operator: Let F be the solution of hence N (U ) = ∇F · ν. By assumption and the weak maximum principle F attains a positive maximum in x 0 and by the Hopf boundary point lemma and the strong maximum principle we have N (U )(x 0 ) = (∇F · ν)(x 0 ) > 0 unless U is constant. By (95) the Dirichlet to Neumann map N : is continuous for any 1 < p < ∞. By Sobolev embedding we deduce that N (U ) is continuous and, unless U is constant, Finally, in x 0 the term U −U is also nonnegative. Therefore, from (85) we deduce a contradiction unless U is a positive constant. Hence, u 1 ≤ u 2 or u 1 = u 2 + γ for some positive constant γ. 2. Next consider α 1 = α 2 . If U is not constant then u 1 ≤ u 2 , but interchanging the roles of u 1 , u 2 also gives u 2 ≤ u 1 and hence equality. We draw some first conclusions on the polarization properties of the model. Corollary 4.9. Let u ∈ W 2,p (Γ) for any 1 ≤ p < ∞ and ξ ∈ L ∞ (Γ) with 0 ≤ ξ ≤ 1, ξu = u on Γ be given such that (81), (82) are satisfied. Assume m :=´Γ u > 0. Then u is constant if and only if g is constant. In that case we have ξ = 1 on Γ. In order to characterize the critical value of α, below we need that´ψg = 0. In the following let us fix any nontrivial nonnegative solution ψ of (90). By the argument from above a solution of (86) exists if and only if (Note that α * is well-defined by Lemma 4.10.) Then all solutions (u, α) of (86) have α = α * and can be written as u = u * + c, where c is constant and u * is the unique solution with min Γ u * = 0. We now argue exactly as in the case ℓ = 0. We define the critical mass as and obtain the following characterization for the onset of polarized states. c) By part a) for any solution (u, ξ, α) with m < m * we have α ≤ α * . If α = α * then we would have u = u * + c with c < 0, a contradiction to u ≥ 0. This shows α < α * . Finally, if u > 0 almost everywhere then ξ = 1 almost everywhere and we deduce that u is a solution of (86). But this requires α = α * .	2019-02-15T15:21:08.000Z	2019-02-15T00:00:00.000	{ "year": 2020, "sha1": "3c5e194f2ac54b02417e589cf07efe29ec751f91", "oa_license": null, "oa_url": "http://arxiv.org/pdf/1902.05842", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "3c5e194f2ac54b02417e589cf07efe29ec751f91", "s2fieldsofstudy": [ "Mathematics" ], "extfieldsofstudy": [ "Materials Science", "Mathematics", "Physics" ] }
17674305	pes2o/s2orc	v3-fos-license	The Proposed CLEO-c Program and R Measurement Prospects The proposed experimental program (CLEO-c) for a charm factory based on a modification of the Cornell Electron Storage Ring is summarized. The prospects for R measurements over the range 3 GeV to 7 GeV are examined in detail. Adapted from"CLEO-c and CESR-c: A New Frontier of Weak and Strong Interactions", the CLEO-c project description, by the CLEO Collaboration. CLEO-C PROGRAM OVERVIEW The CLEO collaboration is proposing a focused three-year program of charm and QCD physics with the CLEO detector operating in the range √ s = 3 − 5 GeV. The CLEO-c physics program includes a set of measurements that will substantially advance our understanding of important Standard Model processes and set the stage for understanding the larger theory in which we imagine the Standard Model to be embedded. Much of this program revolves around the strong interactions and the pressing need to develop sufficiently powerful tools to deal with an intrinsically non-perturbative theory. At the present time, and for the last twenty years, progress in weak interaction physics and the study of heavy flavor physics has been achieved primarily by seeking those few probes of weak-scale physics that successfully evade or minimize the role of strong interaction physics. The preëminence of the mode B → J/ψK S in measuring sin 2β stems almost entirely from the absence of complications due to the strong interactions. Similarly, the discovery of a previously unrecognized symmetry in QCD, which led to Heavy Quark Effective Theory (HQET), created an opportunity in heavy-to-heavy quark decays where strong interaction effects are minimized. HQET's identification of the zero-recoil limit as the optimal kinematic point at which to measure b → cℓν decays now dominates the extractions of \|V cb \| in B physics -not because it is experimentally optimal (quite the opposite!) but because it offers a way to minimize the complications of strong interactions. If we had similar strategies that would allow us to extract \|V ub \| from b → uℓν measurements without form factor uncertainties, \|V td \| from B d mixing measurements without decay constant and bag parameter uncertainties, or \|V ts \| from B s mixing measurements (or limits) without its corresponding decay constant and bag parameter uncertain- * Adapted from "CLEO-c and CESR-c: A New Frontier of Weak and Strong Interactions", the CLEO-c project description, by the CLEO Collaboration ties, we would be well on our way to understanding the CKM matrix at the few percent level. In the current state of the field this is an unrealized dream. Across the spectrum of heavy flavor physics the study of weak-scale phenomena and the extraction of quark mixing matrix (CKM) parameters remain fundamentally limited by our restricted capacity to deal with the strong interaction dynamics. Moreover, as we look to the future beyond the Standard Model, and beyond the realm of today's heavy flavor physics, we anticipate that the larger theory in which the Standard Model lives will certainly be either strongly coupled or will have strongly coupled sectors. Both Technicolor, which is modeled on QCD and is ab initio strongly coupled, and Supersymmetry, which needs strongly coupled sectors to break the supersymmetry, are prime examples of candidates for physics beyond the Standard Model. Strong coupling is a phenomenon to be expected: weak coupling is the exception in field theory, not the norm. Nevertheless our ability to compute reliably in a strongly coupled theory is far from developed, as evidenced by the careful identification and exploitation of golden modes in heavy quark physics. Techniques such as lattice gauge theory that deal squarely with strongly coupled theories will eventually determine our progress on all fronts of particle physics. At the present time absence of adequate theoretical tools significantly limits the physics we can obtain from heavy quark experiments. Recent advances in Lattice QCD (LQCD), however, may offer hope. Algorithmic advances, and, to a lesser extent, improved computing hardware have produced a wide variety of nonperturbative results with accuracies of order 10-20%. This is particularly true for analyses of systems involving heavy quarks, such as B and D mesons or the Υ and ψ quarkonia. First-generation unquenched calculations have been completed for decay constants and semileptonic form factors, for mixing and for spectra. There is strong interest within the LQCD community in pursuing much higher precision, and the techniques needed to reduce errors to a few percent exist. A small number of calculations have achieved errors of 5% or less, including calculations of heavy-quark masses and the strong coupling constant; much more is possible within the next few years. But the push towards high precision is hampered by a lack of sufficiently accurate data against which to test and calibrate the new theoretical techniques. CLEO-c proposes to address this challenge by confronting it in the charm system at threshold where the experimental conditions are optimal. With high statistics data obtained from the decays of charmed mesons and charmonium, we will provide unprecedentedly precise data to confront theory. We will supplement the charmonium data with ∼ 4 fb −1 of bottomonium data to be taken by CLEO III in the year prior to conversion to CLEO-c. Decay constants, form factors, spectroscopy of open and hidden charm and hidden bottom, and an immense variety of absolute branching ratio determinations will be provided with accuracies at the level of 1-2%. Precision measurements will demand precision theory. The measurements proposed below are therefore an essential and integral part of the global program in heavy flavor physics of this decade and the larger program of the as yet unknown physics of the next decade. By exploiting capabilities which are unique to the charm sector and the charm energy region, and programmatic opportunities that are unique to CESR and CLEO, our measurements will explore a large set of critical weak and strong interaction phenomena. These in turn will drive theoretical advances that will both extend and enable the full program of flavor physics targeted by Babar, Belle, CDF, D0, BTeV, LHCb, ATLAS, and CMS, and will lay the foundation for strong interaction theory to meet the requirements of future physics beyond the Standard Model. Run Plan and Datasets The CESR accelerator will be operated at center-of-mass energies corresponding to √ s ∼ 4140, √ s ∼ 3770(ψ ′′ ), and √ s ∼ 3100(J/ψ) for approximately one calendar year each. Taking into account the anticipated luminosity which will range from 5×10 32 cm −2 s −1 down to about 1×10 32 cm −2 s −1 over this energy range, the proposed run plan will yield 3 fb −1 each at the ψ ′′ and at √ s ∼ 4140 above D s D s threshold, and 1 fb −1 at the J/ψ. These integrated luminosities correspond to samples of 1.5 million D s D s pairs, 30 million DD pairs, and one billion ψ decays. As a point of reference, note that these datasets will exceed those of the Mark III experiment by factors of 480, 310, and 170, respectively. If time and luminosity allow, modest additional data samples will be obtained at the Λ c Λ c threshold region, the τ + τ − threshold region, the ψ(3684), and over a set of scan points for an R vs. √ s determination In addition, prior to the conversion to low energy operation, we plan to take ∼ 4 fb −1 spread over the Υ(1S), Υ(2S), and Υ(3S) resonances to launch the QCD part of the program. These datasets will increase the available bb bound state data by more than an order of magnitude. Hardware Requirements The conversion of the CESR accelerator for low energy operation will require the addition of 18 meters of wiggler magnets to enhance transverse cooling of the beam at low energies. In the CLEO III detector the solenoidal field will be reduced to 1.0 T, and the silicon vertex detector may be replaced with a small, low mass inner drift chamber. No other changes are needed to carry out the proposed program. Measurements The principal measurement targets include: From the muonic decays alone the decay constants f D and f Ds can be determined to a precision of about 2%. The decay constants measure the nonperturbative wave function of the meson at zero inter-quark separation and appear in all processes where constituent quarks must approach each other at distances small compared to the meson size. Note that while f π and f K are known to 0.3% and 0.9% respectively, f Ds and f D are only known to about 35% and 100% respectively, and f B and f Bs are unlikely to be measured to any useful precision in this decade. Semileptonic charm decays: Absolute branching ratios in critically interesting modes like D → πℓν and D → Kℓν will be measured to ∼ 1%, and form factor slopes to ∼ 4%. Form factors in all modes can be measured across the full range of q 2 with excellent resolution. Semileptonic decays are the primary source of data for the CKM elements \|V ub \|, \|V cb \|, \|V cd \|, and \|V cs \|, but these CKM elements cannot be extracted without accurate knowledge of form factors. Currently, semileptonic branching ratios are known with uncertainties that range from 5% to 73% -in the cases where they are known at all -and form factor measurements are limited by resolution and background. Inclusive semileptonic decays such as D → eX, D s → eX, and Λ c → eX will also be examined and branching ratios will be measured to a precisions of 1 − 5%. Currently, such quantities are known with uncertainties that range from 4% to 63%. Hadronic decays of charmed mesons. The rate for the critical normalizing modes D → Kπ, D + → Kππ, and D s → φπ will be established to a precision of order 1 − 2%. Currently these are known with uncertainties that range up to 25% and are even larger for other hadronic decays of interest. Many important B meson branching ratios are normalized with respect to these subsidiary charm modes. Rare decays, DD mixing, and CP violating decays. CLEO-c can search for rare decays with a typical sensitivity of 10 −6 , study mixing with a sensitivity to x = ∆M/M and y = ∆Γ/2Γ of under 1%, and detect any CP violating asymmetries that may be present with a sensitivity of better than 1%. CLEO-c will also search for evidence of new physics within τ decays. Quarkonia and QCD. With approximately one billion J/ψ's produced, CLEO-c will exploit the natural glue factory, ψ → γgg → γX, to search for "glueballs" and other gluerich states. The region of 1 < M X < 3 GeV/c 2 will be explored with partial wave analyses for evidence of scalar or tensor glueballs, glueball-qq mixtures, exotic quantum numbers, quark-glue hybrids, and other evidence for new forms of matter predicted by QCD but not yet cleanly observed. (a) Masses, widths, spin-parity quantum numbers, decay modes, and production mechanisms will be established for any states that are identified. (b) Reported glueball candidates such as the tensor candidate f J (2220), and the scalar states f 0 (1710), f 0 (1500), and f 0 (1370) will be explored in detail and spin-parity assignments clarified. (c) The inclusive photon spectrum in J/ψ → γX will be examined with < 20 MeV photon energy resolution. States with up to 100 MeV width and inclusive branching ratios above 1×10 −4 will be identified. The ∼ 4 fb −1 of CLEO bb resonance data (to be taken prior to conversion to low energy operation) will also be exploited to survey the physics of the Υ(1S), Υ(2S), and Υ(3S), resonances. We will measure leptonic widths (related to decay constants of mesons with open flavor) and photonic transition matrix elements (related to form factors in semileptonic decays of open flavor mesons). Comparing experimental results with LQCD predictions for the Υ (and ψ) spectra, leptonic widths and form factors test both the heavy-quark action that is used for LQCD simulations of B's and D's, and the specific techniques used to analyze B and D decay constants and form factors in LQCD. CLEO-c will also make spectroscopic searches for new states of the bb system and for exotic hybrid states such as cgc and perhaps bgb. Analysis of Υ(1S) → γX will play an important role in establishing or debunking any glueball candidates found in the J/ψ data. 6. Spot checks of R. The ratio R = σ(e + e − → hadrons)/σ(e + e − → µ + µ − ) will be measured at various values of √ s with a precision of 2% per point. The R measurements are critical to interpretation of precision electroweak data and the g − 2 experiment. Unique Features of the CLEO-c Program Many of the measurements described above have been done or attempted by other experiments such as Mark III and BES, and many are accessible to the B-factory experiments operating at the Υ(4S). What makes CLEO-c unique? Compared to the Mark III and BES experiments which have taken data on the same ψ resonances as we propose here, CLEO-c will have: 1. Vastly more data. As noted above the CLEO-c data sample will be ∼ 200−500 times larger than the corresponding Mark III datasets. Compared to BES, CLEO-c will have 270 times as much D and D s data, and 20 times as many ψ(3100) decays. One order of magnitude opens new vistas; two orders of magnitude can change a field. A modern detector. Mark III was built twenty years ago, and BES was modeled on Mark III. Detectors have gone through several generations of development since then. In every resolution and performance parameter -hit resolution, momentum and energy resolution, mass resolution, particle ID capability, solid angle coverage -CLEO III is superior to these other detectors by substantial margins. Photon energy resolution, for example, is factors of 10-20 times better (depending on E γ ); charged particle momentum resolution is 2-3 times better (depending on p T ). Particle identification with the RICH detector, augmented by energy loss (dE/dx) measurements in the drift chamber, give tens to hundreds of sigma Kπ separation across the full kinematic range. A 25% increase of solid angle coverage relative to BES gives CLEO-c a huge advantage in analyses such as double-tag measurements that require every particle to be reconstructed. The gains go as 1.25 n where n is the total track and photon multiplicity of the event. This implies a typical effective luminosity gain of 8 in such analyses. For studies that involve partial wave analysis, the increase in solid angle coverage means angular distributions can be measured across the full angular range without large variations in efficiency. This translates to substantial gains in the reliability and precision with which J P C can be measured for a given state. On the other hand, CLEO-c will not have any advantage in statistics or in detector performance when compared to Babar and Belle. The three detectors are all similar, and with an anticipated 400 fb −1 of Υ(4S) data, Babar and Belle will each have about 500 million continuum e + e − → cc events. Yet the data CLEO-c takes at charm threshold has distinct and powerful advantages over continuum charm production data taken at the B-factories, which we list here: 1. Charm events produced at threshold are extremely clean. The charged and neutral multiplicities in ψ(3770) events are 5.0 and 2.4, compared with 11.0 and 5.6 in Υ(4S) events. This alone substantially reduces combinatorics, but in addition the ψ(3770) decays are spherical, distributing decay products uniformly in the detector solid angle. Low multiplicity in CLEO-c translates to high efficiency and low systematic error. 2. Charm events produced at threshold are pure DD. No additional fragmentation particles are produced. The same is true for ψ(4140) decaying to DD * , D sDs and D sDs * , and also for threshold production of Λ cΛc . This allows the use of kinematic constraints such as total candidate energy and beam constrained mass, and also permits effective use of missing mass methods and neutrino reconstruction. The crisp definition of the initial state is a uniquely powerful advantage of threshold charm production that is absent in continuum charm production. Double-tag studies are pristine. The pure production of DD states, together with the low multiplicity and high branching ratios characteristic of typical D decays permits effective use of double-tag studies in which one D meson is fully reconstructed and the rest of the event is examined without bias but with substantial kinematic knowledge. These techniques, pioneered by Mark III many years ago allow one to make absolute branching ratio determinations. Backgrounds under these conditions are heavily suppressed. Very low background conditions minimize both statistical and systematic errors. Signal/Background is optimum at threshold. The cross section for the signal ψ(3770) → DD is equal to the cross section for the underlying continuum e + e − → hadrons background. By contrast, for cc production at √ s = 10.6 GeV the signal is only 1/4 of the total hadronic cross section. In addition, the cc fragmentation distributes the final states among many charm hadron species. Neutrino reconstruction is clean. For leptonic and semileptonic decays the lost neutrino can be treated as a missing mass problem and in the double tagged mode these measurements have low backgrounds. The missing mass resolution is under a pion mass. For semileptonic decays this also means that the resolution on q 2 is excellent, about 3 times better than is available in continuum charm reconstruction at √ s = 10.6 GeV. For D mixing and some CP violation studies, the fact that the D andD are produced in a coherent quantum state in ψ(3770) decay is of central importance for the subsequent evolution and decay of these particles. The same is true for the CP = +1 mode ψ(4140) → γDD. The coherence of the two initialstate particles allows simple methods to measure DD mixing parameters and check for direct CP violation. In addition to the advantages of studying open-charm decays at threshold, the CLEO-c program includes the opportunity to use a huge charmonium data sample in searches for glueballs, hybrids, and exotic states. If found -or if not found -these states will present a powerful challenge to QCD calculations. Furthermore, CLEO will have the unique ability to compare results between both high statistics J/ψ and Υ data sets, and further cross check with the 25 fb −1 of existing two-photon data. These corroboratory measurements will be used to eliminate spurious glueball candidates. Theory, moreover, will be forced to confront precision data in both open-and hidden-flavor charm and bottom mesons simultaneously. Taken together, these technical and programmatic features constitute formidable advantages for the CLEO-c proposal. THE IMPACT OF CLEO-c The measurements of leptonic decay constants and semileptonic form factors, together with the study of QCD spectroscopy both in the cc and bb quarkonium sectors will yield an extensive set of 1 − 2% precision results that will rigorously constrain theoretical calculations. The calculations which survive these tests will be validated for use in a wide variety of areas where the interesting physics cannot be extracted without theoretical input. This broader impact of CLEO-c results extends beyond the borders of CLEO-c measurements and affects most of the core issues in heavy flavor physics. We list here some of the areas that will be most notable: Currently limited by form factor calculations to an estimated 25% accuracy, and unlikely to improve beyond 10% in present-day extrapolations. Pinning down form factor technology in the closely related charm decays such as D → πℓν and with D → ρℓν, CLEO-c data will open the door to 5% or better precision in \|V ub \|. Extraction of \|V td \| and \|V ts \|. Currently limited by ignorance of f B B B d and f Bs B Bs , our only prospect for separately extracting \|V td \| and \|V ts \| from B mixing measurements is through improving decay constant calculations to the percent level. Determinations of the charmed decay constants f D and f Ds will underwrite the required theoretical advances and open the door to ∼ 5% determinations of \|V td \| and \|V ts \|. Currently known only at the ∼ 10% level by direct measurement, CLEO-c will provide absolute branching ratio measurements of leptonic and semileptonic decays from which \|V cd \| and \|V cs \| can be extracted to ∼1% accuracy. Here as elsewhere, form factor and decay constant calculations must be advanced to a comparable level of precision and validated by the entire range of CLEO-c measurements for CKM determinations at the percent level to be valid. Extraction of \|V cb \|. Currently limited by a variety of both experimental and theoretical inputs. Prominent among these are the theoretical control of the form factors and the experimental determination of B(D → Kπ). CLEO-c will drive form factor technology, and measure the normalizing branching ratios at the sub-percent level. Unitarity tests of CKM. Currently poorly satisfied by the first two rows of the CKM matrix, which fail both orthogonality and orthonormality conditions at the 2 − 3σ level. Unitarity conditions can be probed with 1% precision when \|V cd \| and \|V cs \| are provided at this level by CLEO-c. Over-constraining the Standard Unitarity Triangle. Provided \|V ub \| and \|V td \| have been determined at the 5% level, as discussed in items 1 and 2 above, the triangle sides will have been measured with precision comparable to the phase quantity sin 2β -thereby allowing for the first time meaningful comparison of the sides of the unitarity triangle with one of the angles. New forms of matter. In the quarkonium studies new forms of gluonic matter may be identified. Current results in this field are murky and contradictory. The high statistics data sample, high resolution detector, and clean initial state will be an unprecedented combination in this field, and offer the best hope for incisive experimental results. MEASUREMENTS OF R CLEO-c can approach measurement of R in two ways. The first involves an explicit scan over √ s to measure R directly at different energies. The second involves the use of radiative returns to determine an average R over a range of energies below those accessible directly by CESR-c. R scanning A scan of the energy range between 3 and 5 GeV will clarify the energy dependence of R just below the open charm threshold, from J/ψ to ψ(3770), and above it where several relatively broad cc resonances exist. A possible scenario is to scan this energy range with a steps of 100 and 20 MeV, below and above ψ(3770) respectively, collecting about 10 4 hadronic events per point. Such a scan will require an integrated luminosity of about 100 pb −1 . It will also serve as an introduction to a more detailed potential study of the cc resonances in this energy range as well as D * and D s mesons copiously produced in their decays The previous experience of CLEO (which measured R with an accuracy of 2% in the vicinity of the Υ(4S) resonance [1]) and recent progress in the calculations of radiative corrections together with the hermeticity of the CLEO-III detector allows one to expect a systematic uncertainty of about 2%. After that a scan of the energy range from 5 to 7 GeV will be needed to solve the dramatic contradiction between the old measurement of MARK-I [2] and the unpublished results of Crystal Ball [3]. Here a scan in steps of 100 MeV with 10 4 events per point will be adequate, requiring an integrated luminosity of 50 pb −1 . At an average luminosity of 3 × 10 32 cm −2 s −1 it will take one week of collection time to scan the entire energy range between 3 and 7 GeV. Radiative returns The second approach to measuring R at CLEO-c will be to use radiative return events [4,5]. Such events would allow CLEO-c to measure R in the 1-3 GeV energy range which is of crucial importance to reduce the overall uncertainty in α(M Z ) and (g − 2) µ . Recent experience of BABAR confirms statistical feasibility of such R measurements [6]. While running at the ψ(3770) one can expect ∼ 10 4 fully contained radiative return events in the range 1-3 GeV per 1 fb −1 of data. However, it is still unclear whether systematic errors can be controlled well enough to make the radiative return measurements meaningful, i.e., to reach an uncertainty of less than 5%. Various dedicated experiments have recently been proposed to improve our knowledge of R in the crucial 1-3 GeV energy range [7]. If they are approved, the CLEO-c measurements would be most valuable as an independent check of the dedicated experiments. A 2% measurement of R between 3 and 7 GeV will significantly reduce the uncertainty of the hadronic contribution to (g − 2) µ and especially α(M Z ). If radiative return events can be utilized to reach the 5% accuracy between 1 and 3 GeV, remarkable improvement is expected. For example, the α(M Z ) uncertainty would be a factor of 2 smaller than it is at present. 'Modern' R Measurements for Charm Spectroscopy Studies Although cc states below strong decay threshold (3.7 GeV) were studied at e + e − machines some time ago, higher mass states are very poorly defined. Most quark model analyses [8] find 3 poorly defined L=2 states above a mass of 4 GeV based on on low statistics R data from 30 years ago. A detailed study of the properties of these states will test the long-range qq potential in a controlled way. In addition, the mass region from 4-5 GeV is where lattice calculations have predicted charmed hybrids to be, as detailed in other sections of this document. Thus, a means to study this spectroscopy with more detail than the previous experiments is required. CLEO-c proposes a study that will provide a direct look at the states through a scan of the mass region 3.7-5 GeV. Because very little is known about the states above DD threshold, a broad look is appropriate. However, the complexities of extraction above the inelastic threshold require more detailed information about the final state. The proposed study will be similar to the R measurement described above. However it will measure the final state whenever possible by reconstructing D mesons in that the dominant decay modes will be DD, D * D , D * D * , and D * * D . Previous measurements of these decay rates are strongly in disagreement with quark model calculations [8], but the data is of poor quality. Data would be collected with a loose trigger in the region 3.7-5 GeV. Similar to an R measurement, runs will be at closely spaced energies (steps of 20 MeV). However, the runs need to be much longer (∼100,000 events) than is typical [9]. Although the BES data have higher statistics than the older SLAC experiments, no attempts have been made to analyze the composition of the final state because of detector limitations. With the larger event samples and the acceptance of CLEO, one would be able to reconstruct at least one D meson in about 10% of the events. This would be sufficient to characterize the final state and measure the angular distribution. The overall efficiency (summed decay branching ratio times detection efficiency) is about 16% for D 0 mesons and 6% for D + mesons for prominent decay modes. At a luminosity of 3 × 10 32 cm −2 s −1 , each step would require about 2 days of data taking. Existing quark model calculations for charmed hybrid mesons [10] assume the quarks to have orbital angular momentum 1. For decay mechanisms normally used in quark models, decays to DD mesons will be suppressed. If this symmetry is correct, a state with very low decay rate to DD would signal the possibility of a hybrid. Total widths are about 10-30 MeV in these models, similar to the conventional cc states at the same mass. This measurement is related to other CLEO-c measurements outlined in this document. It is closely related to the R measurement discussed in the previous section, but will require much larger statistics. SUMMARY The high-precision charm and quarkonium data we propose to take will permit a broad suite of studies of weak and strong interaction physics. In the threshold charm sector measurements are uniquely clean and make possible the unambiguous determinations of physical quantities discussed briefly above, and at greater length in the chapters that follow. The advances in strong interaction calculations that we expect to drive will in turn underwrite advances in weak interaction physics not only in CLEO-c, but in all heavy quark endeavors and in future explorations of physics beyond the Standard Model. CLEO-c stands to make a significant impact upon the R determination in 3-7 GeV range, with direct 2% measurements appearing feasible with a fairly detailed scan. In addition, the use of radiative returns may provide a useful crosscheck for direct low energy measurements at other facilities if the systematic uncertainties can be controlled at the 5% level.	2019-04-14T02:26:22.031Z	2001-07-27T00:00:00.000	{ "year": 2001, "sha1": "8c3a554059c19c01776ec9439207362b43918b14", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "8c3a554059c19c01776ec9439207362b43918b14", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
258872403	pes2o/s2orc	v3-fos-license	Synthesis, In Vitro Evaluation and Molecular Docking Studies of Novel Thiophenyl Thiazolyl-Pyridine Hybrids as Potential Anticancer Agents Many literature reports revealed the anticancer activity of pyridine and thiazole derivatives, especially in lung cancer. Therefore, a new series of thiazolyl pyridines linked with thiophene moiety via hydrazone group was prepared by one-pot multi-component reaction of (E)-1-(4-methyl-2-(2-(1-(thiophen-2-yl)ethylidene)hydrazinyl)thiazol-5-yl)ethanone with benzaldehyde derivatives and malononitrile in a good yield. Then, compound 5 and the thiazolyl pyridines were investigated for their in vitro anticancer activity against lung cancer (A549) cell line using MTT assay compared to doxorubicin as a reference drug. The structure of all the newly synthesized compounds was established based on spectroscopic data and elemental analyses. For better insight to investigate their mechanism of action on A549 cell line, docking studies were performed, targeting epidermal growth factor receptor (EGFR) tyrosine kinase. The results obtained revealed that the tested compounds displayed excellent anticancer activities against lung cancer cell line except 8c and 8f compared to reference drug. Based on the data obtained, it can be inferred that the novel compounds, as well as their key intermediate, compound 5, demonstrated potent anticancer activity against lung carcinoma by inhibiting EGFR. Introduction The world's most serious affliction, cancer, is the second leading reason for death worldwide. It is one of the life-threatening diseases that impair human health [1]. Globally, in 2020, 19.3 million new cancer cases and 9.9 million cancer-related deaths were recorded, according to the International Agency for Research on Cancer (IARC). By the end of 2030, these numbers are predicted to increase, reaching more than 24.1 million and 13.0 million [2]. Lung cancer, one of the most common types of cancer, is responsible for about 19% of cancer deaths globally, as compared to other types such as stomach, colorectal, hepatic and mammary gland cancers [3]. According to Hanahan and Weinberg, cancer is a diverse disease that displays characteristics associated with uncontrolled growth, proliferation and invasion into other organs and tissues. Cancer cells undergo significant biological changes that give rise to new cellular properties, which are established markers of cancer, including sustained proliferative signaling, growth suppressor inactivation, resistance to apoptosis, increased replicative capacity, promotion of angiogenesis, invasion of neighboring tissues and metastasis [4]. Although chemotherapy is commonly used to treat cancer, it can have significant adverse effects on healthy tissues, resulting in long-term damage to organs such as the heart, lungs, kidneys and reproductive system [5,6]. Numerous investigations have explored the use of diverse sulfur and nitrogen heterocycles, such as thiophene, thiazole, and pyridine, for the treatment of various diseases. Literature suggests that compounds featuring a thiophene core have generated significant attention in the field of drug discovery owing to their potential as anticancer agents, and they operate through multiple pathways implicated in cancer [7][8][9][10][11]. Thiazoles are important heterocyclic compounds due to their diversity of pharmacological properties and significant medicinal utility [12], including anti-bacterial, antimalarial, antiviral, antidiabetic and anticancer activities [13]. Additionally, drugs containing thiazole have been involved in several commercially available remedies for cancer, such as dasatinib [14], patellamide A [15], tiazofurin [16], ixabepilone [17], dabrafenib [18] and epothilone [19]. Moreover, anti-cancer drugs comprising a pyridine ring are streptonigrin, streptonigrone, and lavendamycin. As an attempt to develop a new anti-cancer drug, cytotoxicity and topoisomerase-inhibitory investigations were performed on some pyridine derivatives. Moreover, a large number of pyridine compounds have been recorded for their cardiotonic, antituberculosis, antibacterial and anti-hepatitis B virus activities [20,21]. Molecular hybridization of two or more heterocyclic rings enhances biological activity, such as antimalarial, tubulin inhibition, antibacterial, antidiabetic and anticancer activities [22][23][24][25][26][27]. Several studies have reported that thiazole-pyridine hybrids exhibit cytotoxicity towards cancer cells and can induce apoptosis in cancer cells ( Figure 1) [28][29][30][31][32]. Ivasechko et al. reported the anticancer activity of newly synthesized thiazole-pyridine hybrids ( Figure 1, I and II) against different cancer cells, with compound I being the most reactive and sensitive to lung, melanoma, colon and CNS cancer cell lines. Both compounds had shown their ability to affect DNA and cause nuclear morphology alteration that can induce genetic instability in tumor cells. Moreover, the anti-tumor activity of new series of pyridone-thiazole hybrid compounds (Figure 1, III) was estimated against colon, gastric and hepatocellular cancer, revealing the most potent one with chlorine atom in ortho position against gastric and hepatic cancer. Moreover, Vasu et al. screened nine imidazo[1,2-a]pyridine-thiazole derivatives for their anticancer activity and showed that hybrid (Figure 1, IV) was the most potent inhibitor of NF-κB activity. Combining pyridine with five-membered heterocycle containing nitrogen and sulfur (thiazole core) in a single hybrid structure via linker promotes interesting structural and biological characteristics, including anticancer effect on breast, liver and lung cancer (Figure 1, V). The pyridinethiazole hybrid (shown in Figure 1, VI) was tested for its antiproliferative activity against various human carcinoma cell lines. It demonstrated notable anti-proliferative activity, specifically against the hepatocyte carcinoma (HEPG2) cell line. Cancer is associated with the upregulation of epidermal growth factor receptors (EGFRs), potent mediators of normal cell growth and development, along with cell proliferation, apoptosis, angiogenesis and invasion for cancer cells [33]. In normal cells, low EGFR levels were recorded; on the contrary, higher levels were detected in many cancer types as lung, breast, esophagus and colon cancers. Since the amino thiazole derivatives have shown potent EGFR kinase inhibition property, the compounds containing this moiety could be potential EGFR inhibitors for anti-cancer therapy [34]. On the basis of these findings, and in continuation of our previous work on the synthesis of anticancer agents [9,20,[35][36][37][38][39][40][41][42] from cheap laboratory-available starting materials with anticipated biological activities, here, hoping to obtain promising molecular candidates as anticancer agents (Figure 1), the aim of this study is to synthesize a novel series of heterocyclic compounds which are hybrids of all the mentioned bioactive heterocyclic moieties. On the basis of these findings, and in continuation of our previous work on the synthesis of anticancer agents [9,20,[35][36][37][38][39][40][41][42] from cheap laboratory-available starting materials with anticipated biological activities, here, hoping to obtain promising molecular candidates as anticancer agents (Figure 1), the aim of this study is to synthesize a novel series of heterocyclic compounds which are hybrids of all the mentioned bioactive heterocyclic moieties. Chemistry The required starting compound 5, namely, (E)-1-(4-methyl-2-(2-(1-(thiophen-2-yl) ethylidene)hydrazinyl)thiazol-5-yl)ethenone, was prepared as previously reported [43][44][45][46] (Scheme 1) and was used as a building block for preparing a new series of thiazolyl pyridines incorporating thiophene moiety 8a-f via one-pot three-component reaction. Thus, the reaction of compound 5 with benzaldehyde (or its derivatives) 6a-f and malononitrile 7 in the presence of ammonium acetate in glacial acetic acid under reflux led to the formation of products 8a-f (Scheme 2). The structure of the latter compounds, 8a-f, was confirmed by elemental analyses and spectral data. For example, the IR spectra revealed, in each case, two stretching absorption bands in the region ῡ 3148-3204 and 3353-3448 cm −1 attributed to the NH, NH2 groups, in addition to a sharp band in the region ῡ 2207-2222 cm −1 due to the nitrile group. The 1 H-NMR spectra revealed three singlet signals in the region δ 6.87-6.94, 8.05-8.19 and 8.59-8.81 ppm assigned for the NH2, pyridyl-H and Chemistry The required starting compound 5, namely, (E)-1-(4-methyl-2-(2-(1-(thiophen-2-yl) ethylidene)hydrazinyl)thiazol-5-yl)ethenone, was prepared as previously reported [43][44][45][46] (Scheme 1) and was used as a building block for preparing a new series of thiazolyl pyridines incorporating thiophene moiety 8a-f via one-pot three-component reaction. Thus, the reaction of compound 5 with benzaldehyde (or its derivatives) 6a-f and malononitrile 7 in the presence of ammonium acetate in glacial acetic acid under reflux led to the formation of products 8a-f (Scheme 2). The structure of the latter compounds, 8a-f, was confirmed by elemental analyses and spectral data. For example, the IR spectra revealed, in each case, two stretching absorption bands in the region On the basis of these findings, and in continuation of our previous work on the synthesis of anticancer agents [9,20,[35][36][37][38][39][40][41][42] from cheap laboratory-available starting materials with anticipated biological activities, here, hoping to obtain promising molecular candidates as anticancer agents (Figure 1), the aim of this study is to synthesize a novel series of heterocyclic compounds which are hybrids of all the mentioned bioactive heterocyclic moieties. Chemistry The required starting compound 5, namely, (E)-1-(4-methyl-2-(2-(1-(thiophen-2-yl) ethylidene)hydrazinyl)thiazol-5-yl)ethenone, was prepared as previously reported [43][44][45][46] (Scheme 1) and was used as a building block for preparing a new series of thiazolyl pyridines incorporating thiophene moiety 8a-f via one-pot three-component reaction. Thus, the reaction of compound 5 with benzaldehyde (or its derivatives) 6a-f and malononitrile 7 in the presence of ammonium acetate in glacial acetic acid under reflux led to the formation of products 8a-f (Scheme 2). The structure of the latter compounds, 8a-f, was confirmed by elemental analyses and spectral data. For example, the IR spectra revealed, in each case, two stretching absorption bands in the region ῡ 3148-3204 and 3353-3448 cm −1 attributed to the NH, NH2 groups, in addition to a sharp band in the region ῡ 2207-2222 cm −1 due to the nitrile group. The 1 H-NMR spectra revealed three singlet signals in the region δ 6.87-6.94, 8.05-8.19 and 8.59-8.81 ppm assigned for the NH2, pyridyl-H and NH protons, respectively, in addition to the multiplet peaks in the aromatic region due to 3148-3204 and 3353-3448 cm −1 attributed to the NH, NH 2 groups, in addition to a sharp band in the region 3 of 16 hybrids used as potent anticancer agents and the targeted ngs, and in continuation of our previous work on the [9,20,[35][36][37][38][39][40][41][42] from cheap laboratory-available starting logical activities, here, hoping to obtain promising er agents (Figure 1), the aim of this study is to synthesize pounds which are hybrids of all the mentioned bioactive pound 5, namely, (E)-1-(4-methyl-2-(2-(1-(thiophen-2-yl) )ethenone, was prepared as previously reported [43][44][45][46] ing block for preparing a new series of thiazolyl pyridines 8a-f via one-pot three-component reaction. Thus, the nzaldehyde (or its derivatives) 6a-f and malononitrile 7 acetate in glacial acetic acid under reflux led to the me 2). The structure of the latter compounds, 8a-f, was and spectral data. For example, the IR spectra revealed, rption bands in the region ῡ 3148-3204 and 3353-3448 roups, in addition to a sharp band in the region ῡ 2207p. The 1 H-NMR spectra revealed three singlet signals in and 8.59-8.81 ppm assigned for the NH2, pyridyl-H and ition to the multiplet peaks in the aromatic region due to 2207-2222 cm −1 due to the nitrile group. The 1 H-NMR spectra revealed three singlet signals in the region δ 6.87-6.94, 8.05-8.19 and 8.59-8.81 ppm assigned for the NH 2, pyridyl-H and NH protons, respectively, in addition to the multiplet peaks in the aromatic region due to the aromatic protons. The mass spectra for all the new compounds 8a-f revealed, in each case, a molecular ion peak which is in accordance with the respective molecular weight. the aromatic protons. The mass spectra for all the new compounds 8a-f revealed, in each case, a molecular ion peak which is in accordance with the respective molecular weight . The synthetic pathway of one-pot synthesis of amino cyanopyridine derivatives 8af [20,47] starts with the Claisen-Schimdt condensation reaction between the acetylthiazole derivative and substituted benzaldehyde derivatives 6 to form the α, β-unsaturated ketone intermediate. Then, the latter intermediate was reacted with malononitrile through the Michael addition reaction in the presence of ammonium acetate, followed by cyclization, auto-oxidation and finally tautomerization to afford the corresponding pyridine derivatives, 8a-f (Scheme 3). The synthetic pathway of one-pot synthesis of amino cyanopyridine derivatives 8af [20,47] starts with the Claisen-Schimdt condensation reaction between the acetylthiazole derivative and substituted benzaldehyde derivatives 6 to form the α, β-unsaturated ketone intermediate. Then, the latter intermediate was reacted with malononitrile through the Michael addition reaction in the presence of ammonium acetate, followed by cyclization, auto-oxidation and finally tautomerization to afford the corresponding pyridine derivatives, 8a-f (Scheme 3). The synthetic pathway of one-pot synthesis of amino cyanopyridine derivatives 8a-f [20,47] starts with the Claisen-Schimdt condensation reaction between the acetylthiazole derivative and substituted benzaldehyde derivatives 6 to form the α, β-unsaturated ketone intermediate. Then, the latter intermediate was reacted with malononitrile through the Michael addition reaction in the presence of ammonium acetate, followed by cyclization, auto-oxidation and finally tautomerization to afford the corresponding pyridine derivatives, 8a-f (Scheme 3). The mass spectrum fragmentation of the newly synthesized compound 8b, taken as a representative example of series 8a-f, was investigated. The mass spectrum of 8b showed a molecular ion peak at m/z = 444 (1.76%), corresponding to its molecular formula C 23 H 20 N 6 S 2 . This molecular ion underwent fragmentation via homolytic and heterolytic α-cleavage, as shown in Scheme 4, to give the following fragment ions (molecular formula, m/z (%)): C 17 Cytotoxic Activity The acetylthiazole derivative 5 and the newly synthesized compounds 8a-f were evaluated for their in vitro anticancer screening against human lung cancer (A549) cell line using MTT colorimetric assay with doxorubicin as a reference drug. Data obtained from MTT assay are depicted in Table 1 and show that generally, the tested compounds exert promising cytotoxic activities against A549 cell line except 8c and 8f when compared to doxorubicin that has IC 50 of 0.460 µM. It was noticed that compound 5 has IC 50 value of 0.452 µM. This potency may be due to the presence of both thiophene and thiazole rings in one hybrid molecule through hydrazone linkage exerting this action on A549 cell line in a good manner, which is reflected as an efficient anticancer agent. This current result is in agreement with Rodríguez et al. [48] who reported an anticancer activity against the A549 cell line for a compound containing thiophene moiety after 24 h treatment. Similarly, a series of compounds comprising thiazole ring was noted to have anticancer activity after treatment of A549 cell line for 24 h [49]. Regarding compounds 8a-f, compounds 8e and 8f were identified as the most and least effective ones against the A549 cell line with IC 50 of 0.302 and 0.788 µM, respectively, while the rest showed an intermediate activity. The anticancer activity of the newly synthesized products 8a-f can be attributed to the addition of a substituted pyridine ring to structure 5, elucidating that this addition has an influence on the cytotoxicity against human lung cancer cell line. This finding is in line with Suma et al. [50] who recorded an anticancer activity for a series of compounds containing both pyridine and thiazole rings with IC 50 ranging from 0.66 to 16.03 µM with the most effective one having IC 50 of 0.66 µM after 24 h treatment of A549 cell line. Molecular Docking Studies To investigate the binding mode of all the tested compounds, 5 and 8a-f, towards EGFR tyrosine kinase domain, molecular docking was conducted, where the results were compared with a known EGFR inhibitor as erlotinib, with binding energy S = −21.9889 Kcal/mol in addition to their corresponding binding energies, as shown in Table 2. It was found that compound 5, having energy E = 41.2253 Kcal/mol, interacts by the nitrogen atom of hydrazone linkage through hydrogen bond with key amino acid threonine (Thr830) with binding energy S = −20.1495 Kcal/mol and bond length of 2.16 Å, suggesting that threonine is the most reactive amino acid in the protein structure with compound 5 as compared to the series. Based on the interaction of compounds 8a-f with EGFR, as shown in Figure 2, they can be divided into two groups: group A and group B. In group A, which comprises 8a,c,d and has mean energy E = 82.7210 Kcal/mol, it was observed that all of them interact via hydrogen bonding (via the hydrogen atom of the amine moiety attached to the pyridine ring) with amino acid Asp831 residue with mean binding energy S = −24.2234 Kcal/mol and mean bond length of 1.989 Å. Meanwhile, group B including 8b,e,f, with mean energy E = 75.8467 Kcal/mol and mean binding energy S = −24.4538 Kcal/mol, showed two arene-cation interactions, one between the substituted benzene ring attached to pyridine ring and lysine (Lys721) residue in three compounds and the other between thiophene ring and arginine (Arg817) residue in the case of 8b and 8e; except for 8f, the arene-cation interaction was between pyridine ring and amino acid arginine (Arg817). This change in the interaction may be due to the variance in the molecular weight since 8f has the highest molecular weight within this group. Concurrently, it was noticed that group B exhibits a slightly more stable interaction with the EGFR active site, which may be due to the difference in molecular weight among all the tested compounds. Chemistry Melting points were measured using an Electrothermal Gallenkamp digital melting point apparatus and were reported uncorrected. IR spectra were recorded in potassium bromide discs using PyeUnicam SP-1000 spectrometer. 1 H-NMR spectra were recorded using deuterated dimethyl sulfoxide (DMSO-d 6 ) solution on a Varian Mercury VX-300 MHz spectrometer, and 13 C-NMR spectra were recorded at 75.46 MHz. Chemical shifts are quoted in δ and were reported related to that of solvents. Mass spectra were recorded using a Shimadzu GCMS-Qp-2010 Plus mass spectrometer (Tokyo, Japan) operating at 70 eV. Elemental analyses were carried out by the Microanalytical Centre of Cairo University, Giza, Egypt. General procedure for synthesis of products 8a-f A mixture of compound 5, benzaldehyde or its derivatives 6a-f (2 mmol each) and malononitrile 7 in glacial acetic acid (5 mL) containing 0.5 g of ammonium acetate was refluxed for 4 h and then cooled. The solid formed was filtered and crystallized from acetic acid to give products 8a-f (see Supporting Information File). ( Maintenance of Cell Line Human lung adenocarcinoma (A549) cell line was obtained from Egyptian holding company for vaccines and sera (VACSERA) (Cell Culture lab., Cairo, Egypt). The cell line was maintained according to Esmail et al. [51]. Cytotoxicity Assay MTT assay is a sensitive, quantitative and reliable colorimetric method that measures cells ' viability. It is based on the fact that the water-soluble substrate 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), utilized as 5 mg/mL, can be converted by mitochondrial lactate dehydrogenase enzymes (LDH) into a water-insoluble dark blue formazan in living cells. The insoluble purple formazan product is dissolved into a colored solution by the addition of a solubilization solution as dimethyl sulfoxide (DMSO). This colored solution's absorbance can be measured using a spectrophotometer at a wavelength between 500 and 600 nm [52]. The cytotoxic effect of the synthesized compounds and the reference medication (doxorubicin) was evaluated using MTT assay. The tested compounds 8a-f were dissolved in DMSO and used at different concentrations to perform the assay. Precultured cell lines were exposed to the tested materials at 2-fold serial dilutions for 24 h at 37 • C following the decantation of growing media. Microscopically, the treated cell lines were inspected for morphological alterations and detached cells. Phosphate buffer saline (PBS), pH 7.2 ± 0.2, was used to remove dead cells. Residual live cells were treated with 0.5 % MTT stain as 25 µL/well. The plate was incubated for 3-4 h at 37 • C. A total of 0.05 mL of DMSO was used to dissolve formed intra-cytoplasmic MTT formazan crystals for 30 min on a plate shaker. Optical densities were determined using ELISA plate reader (Biotek-8000, USA). Data were reported for three independent experiments and represented as means [53]. The viability percentage was computed as follows: Cell viability percentage = (optical density of treated cells/optical density of untreated cells) × 100 [54]. IC 50 of the tested compounds were determined using Master -plex-2010 program. Morphological changes were observed 24 h post-treatment using an inverted phase contrast microscope according to Zhang et al. [55]. Molecular Docking Study Docking studies for the compounds were performed using Molecular Operating Environment (MOE) program, 2009.10 version, to examine ligand-protein interactions at the protein active site. The Protein Data Bank (PDB) was used to download the EGFR protein structure (www.rcsb.org (accessed on 8 March 2023), with PDB code: 4HJO). The grid box patterns were 30.077, 5.987 and 1.141. For preparation of the protein, the standard ligand molecules were removed from the protein's active site; then, hydrogen atoms were added while maintaining all the heavy atoms in place. Partial charges were calculated using the MMFF94x force field. The program's final structure was saved as a Pdb file and visualized using BIOVIA Discovery Studio V6.1.0.15350 program, where the target molecule seemed to fit into the protein's active domain in 3D form. Conclusions Overall result, compound 5 and both groups A and B have nearly the same and higher binding energies than previously reported inhibitors as erlotinib, respectively, indicating that synthesized moieties have a greater binding ability with EGFR protein, and resulting in more effective blockage. Based on these findings, compound 5 and amino cyanopyridine derivatives 8a-f were observed to have promising chemotherapeutic effect on lung cancer cells. Compound 5 has the smallest molecular weight, while compounds 8a-f have higher molecular weight. The observed interactions may be accountable for the EGFR inhibition potency of all tested compounds that can vary according to the molecular weight. Also, the tested compounds record better anticancer activity except 8c and 8f as compared to the reference drug. As a result, the current investigation indicated that the described thiazolyl pyridines are promising EGFR inhibitors and pave the way for the synthesis of other libraries based on the reported scaffold, which may eventually result in the creation of an effective therapy for lung cancer.	2023-05-25T15:03:52.346Z	2023-05-23T00:00:00.000	{ "year": 2023, "sha1": "d58f031972444cf39544bd86c8e9a4d2d1c87175", "oa_license": "CCBY", "oa_url": null, "oa_status": null, "pdf_src": "PubMedCentral", "pdf_hash": "9f8554cb3f3e3beb2052bd65f611ecfc94574ceb", "s2fieldsofstudy": [ "Chemistry", "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
28193622	pes2o/s2orc	v3-fos-license	LRP6 is identified as a potential prognostic marker for oral squamous cell carcinoma via MALDI-IMS Oral squamous cell carcinoma (OSCC) is a leading cause of cancer-related deaths worldwide, with 500 000 new cases each year. However, the mechanisms underlying OSCC development are relatively unknown. In this study, matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS)-based proteomic strategy was used to profile the differentially expressed peptides/proteins between OSCC tissues and their adjacent noncancerous tissues. Sixty-seven unique peptide peaks and five distinct proteins were identified with changed expression levels. Among them, LRP6 expression was found to be upregulated in OSCC tissues, and correlated with a cluster of clinicopathologic parameters, including smoking, drinking, tumor differentiation status, lymph node metastasis and survival time. Notably, knockdown of LRP6 inhibited the proliferation ability of OSCC cells. Furthermore, we demonstrated that the expression of LRP6 in OSCC cells is positively correlated with its downstream oncogene, FGF8. The present study suggests that LRP6 could be a potential biomarker for OSCC patients, and might further assist in the therapeutic decisions in OSCC treatment. Oral cancer is one of the most common cancers worldwide, and over 90% of oral cancers are oral squamous cell carcinomas (OSCCs). 1 Despite the advanced therapeutic strategies applied in treating OSCC patients during recent years, 2,3 only limited improvement was achieved in the overall prognosis of this disease, and the 5-year survival rate is still below 50%. 4,5 Therefore, a better understanding of OSCC etiopathogenesis, especially the molecular determinants in OSCC prognosis and valuable biomarkers, is still needed. Proteomics stands for a concept of an entire set of proteins expressed by a whole genome, which fills the gap between cell function and the information encoded by genome. Proteomics approaches are powerful tools in screening molecules in either clinical or experimental samples, and have been widely applied in cancer research. Matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) is an emerging technique that allows profiling up to hundreds of molecules directly from a tissue section or tissue array. 6 One of the major advantages of this technology lies in that it is capable of measuring both the abundance and distribution of the entire proteome throughout a tissue section, without any artificial labeling or modification. 7 Although possessing 'profiling' property, other proteomic approaches, such as twodimensional gel electrophoresis or liquid chromatography LC-MS/MS, are incapable of analyzing the spatial distribution of peptides/proteins in the histological context. 8 MALDI-IMS has been used in many proteomics-related researches. 9,10 Thus far, a small but increasing number of studies have used MALDI-IMS in screening the abnormally expressed peptides/ proteins in diverse cancers, including brain cancer, 11 prostate cancer, 12 lung cancer 13 and head and neck cancer, 14 and a cluster of identified peptides/proteins were shown as potential biomarkers. In this study, the MALDI-IMS-based proteomic strategy is used to compare protein expression patterns between OSCC tissues and adjacent noncancerous counterparts. LRP6 is found to be highly expressed in OSCC areas, and is associated with an index of histopathological parameters. Further, we demonstrate that a combination of LRP6 and its downstream protein, FGF8, could be a potential prognostic factor for OSCC outcome. MALDI imaging. To determine OSCC-specific peptide/protein expression patterns, 10 OSCC clinical tissues, containing both cancer and adjacent noncancerous region, were subjected to MALDI-IMS analyses. The schematic flow diagram of MALDI-IMS analyses was shown in Figure 1a. The representative H&E staining images indicating the areas for MALDI-IMS analyses, and the tumor or non-tumor areas for comparing peak intensities, was shown in Figure 2a. Representative MS spectrums from OSCC (red) and adjacent noncancerous (green) areas were shown in Figure 1b. By principal component analysis, each peak was calculated and converted into a unique point in a two-or three-dimensional coordinate system. As shown in Figure 1c, the points representing the peaks from OSCC areas gathered in a separated cluster, in contrast to those points representing the peaks from adjacent noncancerous areas, suggesting histological heterogeneity of the identified peaks. Further, normalization of each peak was performed according to the average ionic intensity, and the changed peaks between OSCC and control areas were subsequently calculated by using the ClinprotTools 3.0 Software (Bruker Daltonics, Ettlingen, Germany). As listed in Supplementary Table S1, 45 peaks were upregulated in OSCC areas (Po0.01), whereas 22 peaks were downregulated in cancerous areas (Po0.01). MALDI-IMS images of 18 peaks with most discriminative expression (9 upregulated and 9 downregulated in OSCC areas) were shown in Figure 2b, respectively. Additionally, altered levels of 67 peaks were shown in Figure 2c. Protein identification. Out of the 67 identified peaks, 5 peaks with relative higher signal/noise value and intensity were manually selected for protein identification. The original MS/MS results were submitted to and analyzed by Mascot service and/or ExPASy protein sequence database. As a result, five distinct proteins were positively identified, including S27A3, LRP6, MKKS, DOCK9 and HXA2, corresponding to peak 808.69, 823.27, 924.83, 1538.99 and 3409.33, respectively. Detailed information of these identified proteins, including Uniprot access number, peptide sequence and protein description, were provided in Table 1. Bioinformatics analysis. To investigate the potential roles of the five identified proteins in OSCC development, proteinprotein interaction (PPI) network with functional annotations was further established ( Figure 3a). As shown in Figure 3b, a total of 1069 paired PPIs were extracted from the pre-PPI network, which was built up based on the predicted proteins that correlated with the five identified proteins. The PPI network was further processed by GO annotation functional cluster analysis. Strikingly, a significant proportion of LRP6associated proteins were found in the regulation of cell proliferation (Figures 3c-e). By gene ontology (GO) annotation, the P-value for genes in the regulation of cell proliferation is 5.2E − 19. Particularly, the P-value for genes in a positive regulation of cell proliferation is 1.2E − 67, and the P-value for genes in a negative regulation of cell proliferation is 8.5E − 50. Considering that the unlimited cell proliferation is an important feature of cancer cells, thus LRP6 was chosen for further studies. LRP6 expression is correlated with OSCC development. To extend our findings in MALDI-IMS and bioinformatics analysis, the expression of LRP6 in OSCC and normal oral mucous tissues was examined by immunostaining. Clinicopathologic information of clinical samples was summarized in Supplementary Table S2. As shown in Figure 4a, LRP6 signal was positively detected in both the cytoplasm and membrane, which were consistent with previous studies. 15 Notably, strong LRP6 immunoreactivity was found in most tumor cases, whereas most of the cases of normal mucous exhibited only weak staining of LRP6 (t-test; OSCC N = 51, normal N = 28; Po0.001; Figure 4b). Next, we sought to evaluate the relevance between LRP6 expression and a series of clinicopathologic factors in OSCC samples. We found that the level of LRP6 expression was positively associated with smoking (t-test; with smoking N = 8, without smoking N = 20; P = 0.0047; Supplementary Table S2 and Figure 4d) and drinking (t-test; with drinking N = 13, without drinking N = 15; P = 0.0095; Supplementary Table S2 and Figure 4l). Further, LRP6 immunoreactivity was more intense in tumor with lymph node metastasis (t-test; with node metastasis N = 9, without node metastasis N = 19; P = 0.0206; Supplementary Table S2 and Figure 4h). In contrast to welldifferentiated tumor, a higher level of LRP6 was found to be in the poorly and moderately differentiated (ANOVA; well differentiated 18, moderately differentiated 5, poorly differentiated 5; P = 0.0056; Supplementary Table S2 and Figure 4c). However, no apparent correlation was observed between LRP6 expression and patient gender, age, tumor size, tumor location or clinical stage (Supplementary Table S2). To evaluate the relations between LRP6 expression and the HPV status, HPV p16 IHC staining was performed. HPV p16 expression was defined based on IHC staining score: score = 0, negative; score 40, positive; score ⩽ 8, low; score 49, high. As shown in Figures 4e and f, no obvious difference in LRP6 expression was found between p16-positive and -negative samples, or between the sample with high or low p16 expression. Further, statistical analyses showed that the expression of HPV p16 is not correlated with LRP6 (P = 0.1559, R 2 = 0.04968; Figure 4g). These data suggest that LRP6 expression is not associated with HPV infection status in OSCCs. Further, the Kaplan-Meier method and log-rank test were used to estimate the regulatory role of LRP6 expression on the survival rates of OSCC patients. As a result, those patients with high LRP6 expression level showed significantly decreased average survival time after surgery than those patients with low LRP6 expression (Figure 4i). Further, a shortened metastasis-free survival time is more likely to be associated with patients with high LRP6 expression ( Figure 4j). The relationship between LRP6 expression and the outcome of tongue cancer patients was also examined, as tongue cancer was ranked as the most common subtype of OSCCs. 16 As shown in Figure 4k, LRP6 expression was negatively associated with survival time of tongue cancer patients. These results suggested that LRP6 was upregulated in OSCCs, and is negatively correlated with patient outcome. Modulation of LRP6 regulates OSCC cell proliferation. It has been demonstrated that LRP6 is involved in regulating proliferation in cancer cells. 17 As a pilot test, LRP6 expressions in one normal oral squamous cell line (HOK) and six human OSCC cell lines (HSC-4, HSC-3, Cal-27, Um1, Um2, SCC9) were examined. As shown in Figures 5a and b, LRP6 was highly expressed in HSC-3 and Cal-27 cell lines both at protein and RNA levels, and the LRP6 expression was relatively low in HOK, HSC-4, Um1, Um2 and SCC9 cell lines. Therefore, HSC-3 and Cal-27 cell lines were selected as in vitro cell models. To avoid off-target effects, two siRNAs targeting different sites of LRP6 were designed. As shown in Figures 5c and d, each siRNA could efficiently reduce the LRP6 expression levels in both two cell lines. Notably, knockdown of LRP6 by either siLRP6 substantially reduced the proliferation rate of HSC-3 and Cal-27 cells (Figure 5e), revealed by the colony formation assay. Similar proliferation inhibitory effects were observed from the CCK8 assay (Figure 5f). These results suggested that LRP6 possessed a proproliferative property in OSCC cell lines. LRP6 expression is positively correlated with protooncogenic protein FGF8. Previously, we reported that FGF8 promoted cell proliferation in colorectal cancer cells. 18 Considering that LRP6 is an essential Wnt coreceptor for activating the canonical Wnt/β-catenin signaling pathway, 19 and FGF8, which was found in the LRP6associated PPI network (Figure 3e), was a potential downstream gene of the Wnt pathway, 20 the regulatory role of LRP6 on FGF8 expression in OSCC tissues was of particular interest. Then, the expression pattern of LRP6 and FGF8 was compared in OSCC samples. FGF8 immunostaining was performed using the sister tissue slides corresponding to those used for LRP6 immunostaining. As shown in Figure 6a, the immunostaining signals of FGF8 was well paralleled with LRP6 in OSCC tissues. To further confirm the concurrent expression of LRP6 and FGF8 in OSCC tissues, co-immunofluorescent staining was conducted. As shown in Figure 6b, strong FGF8 signal (green) was frequently found in those cells with strong LRP6 signal (red), suggesting that expression of FGF8 and LRP6 was positively related. Next, we examined whether concurrent expression of LRP6 and FGF8 could be a better prognostic factor for OSCC patients than LRP6 or FGF8 expression alone. As shown in Figures 6c and e, the patients with high expression of both LRP6 and FGF8 showed even shorter overall survival time compared with those patients with high LRP6 or FGF8 expression alone. Likewise, the concurrently low LRP6 and FGF8 expressions are associated with a better survival rate compared with low LRP6 or FGF8 expression alone. Similar relationship between concurrent expression of LRP6/FGF8 and patient outcome was also observed in tongue cancer (Figures 6d and f). FGF8 is required for LRP6-induced proliferation in OSCC cell lines. Next, we sought to determine whether FGF8 is required for LRP6-induced proliferation. As expected, overexpression of LRP6 triggered FGF8 transcription in both HSC-3 and HSC-4 cell lines (Figure 7a). Two distinct siRNAs targeting FGF8 was designed, and treatment with either siRNA markedly inhibited endogenous FGF8 expression Figure 7b). Notably, knockdown of FGF8 by siRNAs substantially abolished LRP6-induced cell proliferation in both HSC-3 and HSC-4 cell lines, revealed by the CCK8 (Figure 7c) and colony formation assay (Figure 7d). These results suggest that FGF8 played an important role in LRP6induced proliferation in OSCC cell lines. Discussion OSCCs possess poor prognosis and strong potential metastasis, and the mortality rate of this disease is nearly 50% within 5 years. In spite of a large body of studies, the molecular mechanisms responsible for OSCC development remain unclear. Proteomic strategies have been widely applied as vital tools to screen potential diagnostic and prognostic biomarkers in human cancers. Compared with conventional proteomic technologies, MALDI image-based proteomics provide the spatial information of each detected signal across the tissue context. 21 Further, disease-specific areas on the sample tissue slide can be precisely defined, according to an H&E-stained sister slide. These results have indicated that tumor or noncancerous areas were selected for comparing peak intensities, and these areas were defined according to H&E staining. Sixty-seven specific peptide peaks were detected with changed expression level in this study, including 45 peaks upregulated in OSCC areas and 22 peaks upregulated in noncancerous areas. MALDI-IMS allows unbiased analysis of intact tissue sections, avoiding homogenization and separation steps and protecting the anatomical feathers in situ. 22 MALDI-IMS has been applied in previous OSCC study; however, these studies failed in identifying proteins. In the present data, five proteins with altered expression levels, including S27A3, LRP6, MKKS, DOCK9 and HXA2, were successfully identified. The potential protein interaction network of each protein was addressed by bioinformatics analyses. PPI network with functional annotations was established and further processed by GO annotation cluster analysis. The bioinformatics analysis revealed that LRP6 was closely associated with the regulation of cell proliferation and cell cycle. Our data, together with previous reports, 23,24 suggest that MALDI-IMS combined with bioinformatics analyses is a powerful tool in identifying new cancer biomarkers. It is documented that aberrantly increased LRP6 expression is involved in the development of several human cancer types, including breast cancer and prostate cancer. 25,26 Engineered overexpression of LRP6 was found to promote either cell proliferation or invasion in multiple in vitro or in vivo models. 27 LRP6 expression level was validated via immunostaining, as the above results show that LRP6 was overexpressed in OSCC tissues. It has been reported that LRP6 was associated with diverse physiologic processes, including lipoprotein metabolism, protease regulation, glucose homeostasis, cell differentiation and cell migration. 28 Our results show that LRP6 expression was strongly associated with lymph node metastasis and tumor differentiation status, as well as the habit of smoking and drinking. Notably, our results showed that the increased expression of LRP6 is associated with a shortened survival time, for either overall OSCC patients or tongue cancer patient subset. LRP6 was previously involved in the regulation of cancer cells. It has been shown that the expression of LRP6 promote the proliferation rate of human fibrosarcoma HT1080 cells by altering the subcellular distribution of β-catenin. 29 Another report revealed that the Wnt signaling was significantly activated in LRP6 transgenic mice, which contributed to the development of breast cancer. 30 In this study, we showed that the proliferation rate of HSC-3 and Cal-27 OSCC cell lines were markedly inhibited after LRP6 knockdown, suggesting that LRP6 is probably a proproliferative factor in OSCCs. It is widely accepted that LRP6 functions as essential coactivators for the canonical Wnt/β-catenin signaling. 19 Wnt ligands interact with both frizzled and LRP6 to initiate canonical Wnt signaling. 31 The binding of Wnt leads to phosphorylation of LRP6 coreceptor at cytoplasmic residues by glycogen synthase kinase-3 and casein kinase-1. 32 Activated LRP6 recruits the scaffold protein axin to the membrane and prevents it from participation in the degradation of β-catenin, thereby enhancing translocation of β-catenin to the nucleus where it interacts with the LEF-1/TCF family of transcription factors to regulate transcription of Wnt target genes. 33 Studies in chick and zebrafish have suggested that ectopic expression of Wnt1 can alter FGF8 expression through a pathway involving Lmx1b, 34 Pax2 and En2. 20 Additionally, Wnt/β-catenin signaling was reported to regulate isthmic FGF8 expression in mouse. 35 These results suggested that FGF8 was a potential downstream gene of Wnt signaling, and our results indicated that LRP6 promote the expression of FGF8 in OSCC cells. In a previous study, we reported that activation of FGF8 contributed to metastasis and poor prognosis in patients with colorectal cancer. FGF8 can accelerate the growth rate, increase the clonogenic capability and induce an invasive phenotype in colorectal cancer cells, suggesting the proto-oncogenic property of FGF8. 18 Here, by bioinformatics analyses, we found that FGF8 was present in LRP6-related PPI network. Indeed, overexpression of LRP6 triggered FGF8 expression in OSCC cell lines, and knockdown of FGF8 largely abolished LRP6-induced proliferation in OSCC cell lines. Interestingly, FGF8 expression is positively associated with LRP6 expression in OSCC clinical samples by immunostaining. More importantly, in contrast to LRP6 expression alone, our data show that concurrent expression of LRP6 and FGF8 could serve as a better factor to predict OSCC patient outcome. Materials and Methods Clinical samples. Ten OSCC specimens containing adjacent noncancerous areas for MALDI-IMS analysis and 20 normal oral mucous tissues for IHC analysis were collected from the Department of Oral and Maxillofacial Surgery, Hospital of Stomatology, Sichuan University. All the samples were obtained with informed consent of patients. This study was approved by the Institutional Ethics Committee of Sichuan University. The cancerous or noncancerous areas were identified by two pathologists independently, according to the HE staining. The pathologists were blinded to patient outcomes and other clinical information. If the evaluations did not agree, the sample were re-evaluated and then classified according to the assessment given most frequently by the pathologists.	2018-04-03T01:06:46.011Z	2017-09-01T00:00:00.000	{ "year": 2017, "sha1": "0e8b38c0fe63ec176f6ff491a379f4950c19bbde", "oa_license": "CCBY", "oa_url": "https://www.nature.com/articles/cddis2017433.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "0e8b38c0fe63ec176f6ff491a379f4950c19bbde", "s2fieldsofstudy": [ "Biology", "Medicine" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
265205311	pes2o/s2orc	v3-fos-license	A NEW q -HERMITE-HADAMARD’S INEQUALITY AND ESTIMATES FOR MIDPOINT TYPE INEQUALITIES FOR CONVEX FUNCTIONS . This paper proves a new q -Hermite-Hadamard inequality for convex functions using quantum integrals. We also prove some new midpoint-type inequalities for q -differentiable convex functions. Moreover, we present some examples to illustrate our established results, supplemented with graphs. It is also well known that ϕ is convex if and only if it satisfies the Hermite-Hadamard inequality, stated below (see, [6]): where ϕ : I → R is a convex function and α 1 , α 2 ∈ I with α 1 < α 2 .In [7,11], authors proved some bounds for the right and left sides of the inequality (1.1) which are called trapezoid and midpoint inequalities, respectively. On the other hand, in [2], Alp et al. proved the following version of quantum Hermite-Hadamard type for convex functions using the left quantum integrals: Recently, Bermudo et al. [3] used the right quantum integrals and proved the following variant of Hermite-Hadamard type inequality for convex functions: Several papers are devoted to finding bounds for the left and right sides of inequalities (1.2) and (1.3).In [12], Noor et al. proved several bounds for the right-hand side of the inequality of (1.2).Alp et al., by using convex functions, established some new results for the left side of the inequality (1.2) in [2].In [1,4], quantum Simpson's and Newton's type inequalities for convex and co-ordinated convex functions were proved.For more recent results, one can consult [5,10]. Inspired by the ongoing studies, we prove a new version of quantum Hermite-Hadamard inequalities for convex functions.We also prove some new corresponding quantum midpoint inequalities for q-differentiable convex functions.We present many examples to illustrate our results, supplemented with graphs. BASICS OF q-CALCULUS In this section, we recall some basics of quantum calculus, and throughout this paper, let 0 < q < 1 be a constant. The q-number or q-analogue of n ∈ N is given by (see [9]) The q-Jackson integral for the function ϕ over [0, α 1 ] is defined as (see, [8]): and q-Jackson integral for a function ϕ over [α 1 , α 2 ] is as follows (see, [8]): (2.4) The function ϕ is said to be q-differentiable function on (2.5) The function ϕ is said to be q-integrable function on On the other hand, Bermudo et al. defined new quantum derivatives and quantum integral which are called right q-derivative and right q-integral: ).The right q-derivative of mapping ϕ : [α 1 , α 2 ] → R is defined as: if it exists and it is finite. q-HERMITE-HADAMARD INEQUALITY In this section, we prove the new quantum Hermite-Hadamard inequality and give an example to illustrate the obtained inequality. → R be a convex mapping, then we have the following inequality: Proof.Since ϕ is a convex function, we have q-integrating the inequality (3.2) over [0, 1], we have It is obvious from Definition 2 and Definition 4 that This completes the proof. Remark 1.In Theorem 1, if we set the limit as q → 1 − , then we obtain the classical Hermite-Hadamard inequality (1.1). Example 1.The function ϕ (x) = e x is convex on R so that it also convex on [0, 1] .For this convex function, we have q n e q n 2 and q n e − q n 2 . FIGURE 1. Example 1 We also have It is obvious from Figure 1 that we have the inequality for q ∈ (0, 1) .On the other hand Figure 1 shows that ϒ mid → 1 0 e x dx = e − 1 for q → 1. MIDPOINT INEQUALITIES In this section, we prove some left-estimates of the newly proved Hermite-Hadamard inequality (3.1) using the q-differentiability of the function. Let us start with the following lemma. If the functions α 1 D q ϕ and α 2 D q ϕ are continuous and integrable over [α 1 , α 2 ], then we have the following new equality: By applying Lemma 1 to calculate the integral I 1 , we have Similarly, by using Lemma 2 to calculate the integral I 2 , we obtain Thus, we establish the desired equality by putting (4.3) and (4.4) in (4.2).The proof is completed. Theorem 2. We assume that the conditions Lemma 3 hold.If the functions α 1 D q ϕ and α 2 D q ϕ are convex, then the following inequality holds: Proof.By taking modulus in (4.1), we have 1 Since the functions α 1 D q ϕ and α 2 D q ϕ are convex, we have 1 Thus, the proof is completed. Remark 2. In Theorem 2, if we set the limit as q → 1 − , then we obtain [11, Theorem 2.2].Theorem 3. We assume that the conditions Lemma 3 hold.If the functions α 1 D q ϕ s and α 2 D q ϕ s , s ≥ 1 are convex, then the following inequality holds: Proof.By using the power mean inequality in (4.6), we have 1 . By using the convexity of the functions α 1 D q ϕ s and α 2 D q ϕ s , we have 1 Thus, the proof is completed. Theorem 4. We assume that the conditions Lemma 3 hold.If the functions α 1 D q ϕ s and α 2 D q ϕ s , s > 1 are convex, then the following inequality holds: Proof.By applying Hölder's inequality in (4.6), we have 1 . Since the functions α 1 D q ϕ s and α 2 D q ϕ s are convex, we have 1 Hence, the proof is completed. EXAMPLE In this section, we give an example to support one of the newly established inequalities. CONCLUDING REMARKS In this paper, we have established some new Hermite-Hadamard inequality for convex functions in the context of quantum calculus.Moreover, we have proved some quantum estimates for midpoint-type inequalities for q -differentiable convex functions.It is also has proven by some mathematical examples that the newly established inequalities are valid for any convex function.It is an interesting and new problem that the upcoming researchers can obtain similar inequalities for coordinated convex functions.	2023-11-15T16:18:47.637Z	2023-01-01T00:00:00.000	{ "year": 2023, "sha1": "e33a82c606f1cd9acaed108b7057122b0711937b", "oa_license": null, "oa_url": "http://mat76.mat.uni-miskolc.hu/mnotes/download_article/4200.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "09c25a704860ed92f307e777690da10655d4a89d", "s2fieldsofstudy": [ "Mathematics" ], "extfieldsofstudy": [] }
213461754	pes2o/s2orc	v3-fos-license	Fabrication and Properties of 3D Mullite Fiber-Reinforced Al2O3 Matrix Composites Prepared by Sol-Gel Process Fiber toughening is an effective method to improve the brittleness of ceramic materials. In this paper, three-dimensional mullite fiber reinforced alumina ceramic matrix (3D mullitef/Al2O3) composites were prepared by sol-gel process. Alumina sol was prepared by two ways in the inorganic aluminium salt method, and the alumina sol with good fluidity and high solid content was used as a precursor. The preparation process of alumina sol was studied by XRD and TGA-DSC. The results show that the sol is transformed into Al2O3 at about 1100°C, and the crystal phase is α-Al2O3 phase. The mechanical properties of the sample were analyzed by three-point bending test. The microstructure of the fractures was observed by scanning electron microscopy. The results show that with the increase of the immersion cycles, the density of composites increases continuously, and the toughening mechanism of continuous fiber extraction, interfacial debonding and crack deflection is fully utilized during fracture, which is characterized by ductile fracture behavior and achieved the purpose of toughening. Introduction With the development of high-performance aviation turbine engines, turbine inlet temperatures continue to increase, the temperature of the hot-end parts can reach above 1600C, and the working temperature has far exceeded the working limit of superalloys [1,2]. Continuous non-oxide fiber reinforced non-oxide ceramic matrix composites are prone to oxidative failure and even catastrophic failure in service environments with high temperatures, oxygen and moisture [3,4]. Oxide ceramic matrix composites are resistant to high temperatures and oxidation, and they don't form catastrophic fractures due to oxidative failure. This type of composite is relatively low cost and is one of the most promising candidates for aero-engine hot end components. Countries such as the United States and Japan began to focus on the field of oxide/oxide ceramic matrix composites at the end of the last century, and gradually realized their application in the field of hot end components such as aeroengines [5,6]. Oxide reinforcing fibers generally used are continuous mullite fibers or alumina fibers, but the surface activity of the oxide fiber is high, and it is easy to react with the matrix to form a strong interface during the preparation of the composite, or the fiber is eroded during the preparation process, resulting in a decrease in performance. There are usually two ways to avoid the strong interface [7] used the chemical vapor infiltration method (CVI) to deposit PyC and PyC/SiC interfaces on Nextel 720 fibers, and the flexural strength of the composites was measured to be 56 MPa and 267 MPa, respectively. (2) Preparation of porous matrix: cracks can undergo continuous nonlinear deflection in the matrix during the fracture process, but the substrate has poor thermal creep properties, the material is prone to creep damage in high temperature environment [8], and the porous matrix has lower thermal conductivity, so commonly used in the field of non-structural materials such as heat insulation [9]. Therefore, it has become a top priority to study the preparation of an interface layer of a suitable oxide fiber/oxide ceramic matrix composite. The preparation methods of oxide ceramic matrix composites mainly include hot pressing sintering method, precursor impregnation cracking method, sol-gel method and electrophoretic deposition method [10][11][12][13]. Among them, the sol-gel method is especially suitable for the preparation and molding of continuous fiber toughened ceramic matrix composite due to its low densification temperature, convenient process and the advantages of near-net size processing [14]. In this paper, the alumina matrix was prepared by sol-gel method, and the pyrolysis carbon (PyC) interfacial layer was prepared by chemical vapor infiltration process. And the effect of interface layer on the mechanical properties and microstructure of the composite was studied. Sample Preparation Formulation of alumina sol: using AlCl 3 ·6H 2 O as precursor, preparing one solution with deionized water and absolute ethanol in a certain ratio, and stirring well to dissolve the aluminum salt. After forming the colorless transparent solution and standing for more than 2 hours, a pure, uniform and stable alumina sol is obtained. Preparation of PyC interface phase: the mullite fiber preform was placed in a chemical vapor deposition furnace with argon gas as a kind of shielding gas at a flow rate of 1.0 L/min, butane as a kind of PyC precursor gas at a flow rate of 4.0 L/min, deposition temperature was 1000C and deposited for 3 h to deposit the surface of the mullite fiber preform to obtain the PyC interface phase. After the deposition was completed, it was taken out and cooled by the furnace. The cycle impregnation densification step: the sample by preparing the pyrolytic carbon interface was impregnated with the aluminum sol prepared by a vacuum impregnation process, the immersion time was 0.5 h, the gel treatment temperature was 120C, and the time was 5 h. Then the sample was vacuum pyrolyzed at the temperature of 1100C, heating rate of 5C/min, constant temperature of 1 h. The above procedure was repeated 16 times to obtain a kind of densified composite material called L1. The following preparation processes were the same as the above, but the deposition time of the pyrolytic carbon interface on the L2 sample was 12 h, the deposition of the pyrolytic carbon interface time on the L3 sample was 18 h, and the deposition of the pyrolytic carbon interface on the L4 sample was 24 h. The rest of the densification process parameters were the same. Testing and characterization Phase analysis of the material was carried out using D8 ADVANCE X-ray diffractometer (XRD) from Bruker, Germany. The change of mullite xerogel during heat treatment was analyzed by STA 449C synchronous thermal analyzer (TGA-DSC) from Netzsch, Germany. Sample density was tested using the Archimedes drainage method. The micro-morphology of the carbon fiber-reinforced mullite- The bending strength and flexural modulus of the composite were tested by three-point bending method. The equipment used was the Landmark dynamic and static fatigue testing system of MTS Company of USA. The sample size was 3 mm  4 mm  40 mm, the span was 30 mm, and the loading was performed. The rate was 0.5 mm/min. The fracture toughness of the composite was tested by the single-edge slit beam method. The equipment used was the AG-IC100KN electronic universal testing machine of Shimadzu Corporation of Japan. The sample size was 3 mm  4 mm  40 mm and the span was 30 mm. The side slot had a width of 0.2 mm, a groove depth of 1.5 mm and a loading rate of 0.05 mm/min. Alumina sol performance analysis The Al 2 O 3 sol in this paper is close to a completely transparent liquid, and there is a kind of distinct Tyndall phenomenon when irradiated with a laser beam. After being left at room temperature for one month, the sol didn't change significantly. The physical properties of the sol are shown in Table 1. The viscosity of the sol is 8.5 mPas, the solid content after drying at 120C is 29.6%, and the yield of the ceramic after heat treatment at 1100C is 20.4%. It can be seen that the sol has the characteristics of high solid content, low viscosity and high ceramic yield. The high solid content and ceramic yield are beneficial to improve the densification efficiency of the composite material, and the low viscosity facilitates the impregnation of the sol into the interior of the mullite fiber preform. The gel powder obtained by drying the sol at 120C is subjected to TGA-DSC analysis, and the results are shown in Figure 1. It can be seen from the TGA curve that it is divided into three stages. The first stage: from room temperature to 150C, the weight loss is relatively gentle, the weight loss is about 5%, and the DSC curve has an endothermic peak at 140C at this stage. It is judged that this is mainly caused by the residual free water and crystallized in the matrix after drying. The second stage: from 150C to 350C, the weight loss in this stage shows a linear downward trend, about 60%. This means that the system undergoes a relatively severe chemical reaction during this phase, which can be confirmed by the obvious endothermic peak on the DSC curve. There is an obvious endothermic peak at 200C on the DSC curve, which should be caused by decomposition of the base hydroxyl group and Cldecomposition. The third stage: from 350C to1300C, the weight loss is very small at this stage, and there are two corresponding pairs on the DSC curve. The thermal peak is considered to be mainly caused by the formation of Al 2 O 3 crystal phase, and the matrix changes from amorphous to crystalline, AlOOH  -Al 2 O 3 . The XRD analysis of the gel powder after heat treatment at different temperatures is carried out, and the results are shown in Figure 2. It can be seen from Figure 2 that the XRD patterns of the samples respectively prepared at 1100C, 1200C, 1300C and 1400C have Al 2 O 3 crystal plane diffraction peaks. Its crystal phase is -Al 2 O 3 , which has a hexagonal crystal structure that makes the crystal structure of the sample denser, so it will cause the sample to shrink to a certain volume. However, obvious -Al 2 O 3 diffraction peaks are not showed at 900C and 1000C, which indicates that -Al 2 O 3 phase is not formed at this temperature. In addition, the higher the sintering temperature is, the sharper the diffraction peak becomes, and the amorphous structure gradually becomes crystalline, which indicates that the degree of crystallization of Al 2 O 3 is better. It can be seen from Figure 2 that the diffraction peak of the sample sintered at 1100C to 1400C doesn't change much, so the sintering temperature is set to 1100C, which can ensure that the amorphous form in the matrix is completely converted into the crystalline -Al 2 O 3 that can reduce the sintering cost and speed up the experiment. Effect of pyrolytic carbon interface on mechanical properties of materials The properties of the four composite materials prepared are shown in Table 2. It can be seen that the PyC (pyrolysis carbon) interface attached to the fiber significantly improved the flexural strength of the material. The room temperature flexural strength of L2 composites reached 140.9 MPa, and the room temperature flexural strength of L3 composites is 167.8 MPa, while the room temperature flexural strength of L4 composites is 146.3 MPa. At the same time, it can be seen from Table 2 that the composite material deposited with PyC interface exhibits obvious ductile fracture behavior, mullite fiber effectively toughens and strengthens the alumina matrix, and the room temperature of the Mullite f /Al 2 O 3 composite without PyC interface is deposited. The bending strength is only 110.6 MPa, and the strength is low, which shows obvious brittle fracture behavior. However, the porosity and density of the three are not much different, which indicates that the deposition of PyC interface has little effect on the same aluminum sol and impregnation process. In addition, the pyrolytic carbon interface layer greatly improves the fracture toughness of the material. Since the pyrolytic carbon is has loose layered structure and low density, its presence can prevent the diffusion reaction of three-dimensional mullite fiber preforms and alumina matrix at high temperatures, and the three-dimensional mullite fiber preform caused by the direct precipitation of the alumina matrix on the mullite fiber cannot function as a reinforcement. Therefore, the presence of the PyC interface phase ensures that the material has suitable interfacial strength after the high temperature preparation process and enhances the fracture toughness of the material. At the same time, the PyC interface phase has mechanical compatibility, which causes the micro-cracks of the matrix to deflect and maintain good load transfer between the fiber preform and the matrix. In the composite material containing the PyC interface, due to the barrier effect of the PyC interface, the corrosive gas generated during the gel ceramization process does not directly contact the fiber surface, so that the fiber strength is largely retained. Due to the existence of the loose layered interface of PyC, the diffusion of the fiber and the matrix is hindered, and the fiber/matrix achieves weak interfacial bonding, which greatly improves the toughness and strength of the composite. Figure 3 is a microscopic scan of the PyC/Mullite f fracture at different times of deposition. The graph shows that the thickness of the interface layer is about 0.3 μm (L2), the thickness of the interface layer of b is about 0.5 μm (L3), and the thickness of the interfacial layer of c is about 0.7 μm (L4). The microstructure exhibits a distinct loose layered structure. There is a significant interfacial debonding between the pyrolytic carbon interface prepared in the bending fracture test and the fiber. Figure 5 is the Mullite f /Al 2 O 3 composite containing a PyC interface phase. It can be seen that a large amount of fiber is withdrawn during the fracture process, absorbing most of the energy during the fracture process. Cracks in the axial propagation of the fiber occur during the fracture process, and the crack propagation of the fiber is prevented to cause brittle fracture of the material. Figure 4 is a microstructural view of the fracture of the Mullite f /Al 2 O 3 composite. It can be clearly seen from the figure that the fracture is flat and there is no fiber-out phenomenon. During the fracture process, the crack directly passes through the mullite fiber due to the stress concentration effect, and the toughening mechanism of the fiber is not exerted. Simultaneously, the matrix and the fiber have already exhibited a very strong bonding state during the pyrolysis process. It can be seen from the figure that there are pores which aren't densified. This is the material in the late stage of impregnationpyrolysis. As the number of impregnation-pyrolysis increases, the pores in the fiber bundle gradually close and block, which causes the sol not to infiltrate into the pores left in the fiber bundle. Conclusions The mullite fiber composite alumina ceramic matrix composites with different thicknesses of pyrolytic carbon interfacial layer are prepared by sol-gel method and CVI method. Studies have shown that the alumina sol prepared by the inorganic aluminum salt method is completely converted to -Al 2 O 3 at about 1100C, after repeated cycles of impregnation and cleavage, the material of the composite pyrolytic carbon interface layer is greatly improved than that of the uncomposited material, which is characterized by typical ductile fracture behavior and the fracture toughness reached 8.6 Mpam 1/2 , the bending strength reached 167.8Mpa, and the mechanical properties of the sample are the best when the thickness of the pyrolytic carbon interface layer is 0.5μm.	2019-12-05T09:43:36.756Z	2019-11-27T00:00:00.000	{ "year": 2017, "sha1": "5d83f818cc6ef4c67d3e7a6d3aeed29def88fcaf", "oa_license": null, "oa_url": "https://doi.org/10.1088/1757-899x/678/1/012032", "oa_status": "GOLD", "pdf_src": "IOP", "pdf_hash": "96b8cdf484088debca803f84a5bb75602987b7e4", "s2fieldsofstudy": [ "Materials Science", "Engineering" ], "extfieldsofstudy": [ "Physics", "Materials Science" ] }

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