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==== Front Front PhysiolFront PhysiolFront. Physiol.Frontiers in Physiology1664-042XFrontiers Media S.A. 10.3389/fphys.2016.00364PhysiologyOriginal ResearchTheoretical Considerations and a Mathematical Model for the Analysis of the Biomechanical Response of Human Keratinized Oral Mucosa Tsaira Aikaterini 1Karagiannidis Panagiotis 23Sidira Margarita 1Kassavetis Spyros 2Kugiumtzis Dimitris 4Logothetidis Stergios 2Naka Olga 1Pissiotis Argirios 1Michalakis Konstantinos 15*1Department of Prosthodontics, Faculty of Health Sciences, School of Dentistry, Aristotle UniversityThessaloniki, Greece2Laboratory of Thin Films-Nanosystems and Nanometrology, School of Physics, Aristotle UniversityThessaloniki, Greece3Department of Engineering, Cambridge Graphene Centre, Cambridge UniversityCambridge, UK4Department of Electrical and Computer Engineering, School of Engineering, Aristotle UniversityThessaloniki, Greece5Division of Graduate and Postgraduate Prosthodontics, Tufts University School of Dental MedicineBoston, MA, USAEdited by: Thimios Mitsiadis, University of Zurich, Switzerland Reviewed by: Jean-Christophe Farges, Claude Bernard University Lyon 1, France; Christian Hellmich, Vienna University of Technology, Austria; Nenad Filipovic, University of Kragujevac, Serbia *Correspondence: Konstantinos Michalakis kmichalakis@hotmail.com; konstantinos.michalakis@tufts.edu; kmichalakis@dent.auth.grThis article was submitted to Craniofacial Biology, a section of the journal Frontiers in Physiology 29 8 2016 2016 7 36428 5 2016 09 8 2016 Copyright © 2016 Tsaira, Karagiannidis, Sidira, Kassavetis, Kugiumtzis, Logothetidis, Naka, Pissiotis and Michalakis.2016Tsaira, Karagiannidis, Sidira, Kassavetis, Kugiumtzis, Logothetidis, Naka, Pissiotis and MichalakisThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Removable complete and partial dentures are supported by the residual alveolar ridges consisting of mucosa, submucosa, periosteum, and bone. An understanding of the biomechanical behavior of the oral mucosa is essential in order to improve the denture-bearing foundations for complete and partially edentulous patients. The purpose of this paper was to examine the biomechanical behavior of the soft tissues supporting a removable denture and develop a model for that reason. Keratinized oral mucosa blocks with their underlying bone were harvested from the maxillary palatal area adjacent to the edentulous ridges of a cadaver. The compressive response of the oral mucosa was tested by using atomic force microscopy. The specimens were first scanned in order their topography to be obtained. The mechanical properties of the specimens were tested using a single crystal silicon pyramidal tip, which traversed toward the keratinized oral mucosa specimens. Loading-unloading cycles were registered and four mathematical models were tested using MATLAB to note which one approximates the force-displacement curve as close as possible: a. spherical, b. conical, c. third order polynomial, d. Murphy (fourth order polynomial, non-linear Hertzian based). The third order polynomial model showed the best accuracy in representing the force-displacement data of the tested specimens. A model was developed in order to analyze the biomechanical behavior of the human oral keratinized mucosa and obtain information about its mechanical properties. human oral keratinized mucosaatomic force microscopyoral tissue mechanicscontact mechanicspolynomial modelmathematical modelcurve fitting ==== Body Introduction Removable complete and partial dentures are supported by the residual alveolar ridges consisting of mucosa, submucosa, periosteum, and bone. This supporting area has been calculated to be 23 cm2 for the maxilla, and about 12.25 cm2 for the edentulous mandible (Hobkirk and Zarb, 2013). It should be mentioned however that these figures vary, depending on the size of the maxilla or mandible and the amount of resorption after teeth extraction. Nevertheless, these numbers are substantially lower than the supporting mechanism of the teeth—i.e., the periodontal ligament—which is about 45 cm2, for each dental arch (Hobkirk and Zarb, 2013). Besides the smaller area of the denture bearing surface in comparison with that of the teeth, there are some other distinct differences too. These include the involved sensory mechanisms and the anatomical features of each structure. The periodontal ligament is connective tissue with a thickness of 0.15–0.35 mm, consisting of collagen, oxytalan and eulanin fibers, glycosaminoglycans and blood vessels ranging between 4 and 47% of the total tissue volume (Jonas and Riede, 1980; Blaushild et al., 1992; Johnson and Pylypas, 1992; Embery et al., 1995; Sloan and Carter, 1995; Michalakis et al., 2012). The periodontal ligament is organized into six different groups of fibers which are not unidirectionally distributed: 1. transeptal, 2. alveolar crest, 3. horizontal, 4. oblique, 5. apical, and 6. interradicular, which are present only between the roots of multirooted teeth (Carranza, 1990; Lindhe et al., 2003). The existence of many blood vessels into the periodontal ligament and the hemodynamic pressure that these vessels exert affects its biomechanical response (Kristiansen and Heyreaas, 1989; Sims, 1995; Ioi et al., 2002a,b). The biomechanical response of the periodontal ligament to occlusal loads is not clear (Caputo and Standlee, 1987). Three focal hypotheses have been made in the past, in order to describe the way in which the periodontal ligament supports the tooth: (i) the tensional mechanism model, supporting the idea that the fibers have a wavy configuration and consequently load transmission from the tooth to the neighboring alveolar bone occurs through a gradual unfolding of these fibers (Mühlemann, 1951; Picton, 1965, 1969), (ii) the viscoelastic model, considering that tooth movement within the socket is controlled more by the vascular elements than by the fibers (Bien, 1966; Fung, 1973; Natali et al., 2004), (iii) the collagenous thixotropic model, supporting the notion that tooth support is possible because of the periodontal ligament's thixotropic gel properties (Kardos and Simpson, 1979, 1980). The oral mucosa covering the hard palate and the attached gingiva is termed masticatory mucosa and consists of the stratified squamous epithelium at the surface and the lamina propria which lies deeper. The stratified squamous epithelium consists of four layers, which—from most superficial to deepest—are: (a) stratum corneum, (b) stratum granulosum, (c) stratum spinosum, (d) stratum basale (Nanci, 2013). Lamina propria is connective tissue, which is composed of cells, mainly fibroblasts, and an extracellular matrix, consisting of a ground substance and fibers. Fibroblasts are responsible for the secretion of collagen and other elements of the extracellular matrix. The ground substance of the matrix is composed of glycoproteins, glysosaminoglycans and proteoglycans, while the fibers are mainly collagen, providing tensile strength and flexibility to the tissue and elastic fibers, contributing resiliency. Below the oral mucosa of the attached gingiva and the hard palate lies the mucoperiosteum with dense collagenous connective tissue attaching directly to the periosteum. The mucoperiosteum contains fat and salivary glands (Slavkin and Bavetta, 1972; Newcomb, 1981; Clausen et al., 1983; Meyer et al., 1984; Dahllöf et al., 1986; Bourke et al., 2000). An understanding of the biomechanical behavior of the oral mucosa is essential in order to improve the denture-bearing foundations for complete and partially edentulous patients, by better managing traumatized tissues and giving instructions to patients regarding the time which is required for tissues to recover, after applying occlusal loads during daytime. Furthermore, finite element analysis models studying the deformation of oral mucosa under occlusal loading require use of an equation, which unfortunately is not supplied by the bibliography. Nevertheless, finite element analyses of the oral mucosa have been performed in the past and several material models have been adopted in order mucosal behavior to be interpreted. These include linear elastic, biphasic, multi-phasic elastic, and hyperelastic models (Chen et al., 2015). Additionally, knowledge of oral mucosa biomechanics can be helpful in fabricating dental materials with similar or complementary behavior to that of oral tissues (Saitoh et al., 2010; Hong et al., 2012). Although, numerous articles have been published on the biomechanics of the periodontal ligament (Kurashima, 1965; Komatsu and Viidik, 1966; Daly et al., 1974; Wills et al., 1976; Atkinson and Ralph, 1977; Wills and Picton, 1978; Dorow et al., 2003; Natali et al., 2003; Bergomi et al., 2010, 2011), the research on the biomechanics of keratinized oral mucosa is scarce (Keilig et al., 2009; Goktas et al., 2011). The purpose of this study was to examine the biomechanical behavior of the soft tissues supporting a removable complete denture and develop a model for that reason. Materials and methods Tissue preparation This in vitro study was performed in accordance with the guidelines of the Declaration of Helsinki and the research protocol was approved by the Ethics Committee of the Aristotle University (256/06-07-2011), prior to initiation. Eight 12 × 8 × 8 mm keratinized oral mucosa blocks with their underlying bone were provided by the Laboratory of Anatomy of the Medical Faculty of the Health Sciences School of the Aristotle University of Thessaloniki (Goktas et al., 2011; Herris et al., 2013). The specimens were harvested from maxillary edentulous areas, by using a low speed 0.2 mm thickness diamond disc (Thin Flex X929-7 TP; Abrasive Technology Inc, Lewis Center, OH, USA) under continuous saline irrigation (Figure 1). To prevent dehydration, the specimens were then stored in a 10% neutral buffered formalin solution (water 91.9–92%, formaldehyde 4%, methyl alcohol 1–2%, sodium phosphate dibasic 0.65%, sodium phosphate monobasic, monohydrate 0.4%) until the mechanical analysis testing, which took place 1 h after. Figure 1 Human keratinized oral mucosa specimen with the underlying bone, tested with AFM. The compressive response of the oral mucosa was the only biomechanical characteristic tested, by using an Atomic Force Microscope (Solver P47H; NT-MDT Co., Moscow, Russia). A standard square based pyramidal single crystal silicon (Si) tip (NSG 10; NT-MDT Co., Moscow, Russia) with a < 10 nm typical curvature tip radius and a lateral surface of 500 μm was used. A nominal spring constant of 12.9 0.06 N/m was used after calculating it by using the Sader method (Sader et al., 1999). The length of the cantilever was 95 ± 5 μm, the width was 30 ± 3 μm, while the thickness was 2 ± 0.5 μm. Before initiation of the experimental procedures a calibration of the cantilever took place. First the deflection was converted to force using the Hooke's law and then the response of the cantilever was subtracted from the measurement. For this purpose a force-displacement curve in a hard surface was acquired, followed by a force-displacement curve on the sample. The difference of these measurements supplied the indentation depth. The specimens were then fixed on glass slabs with Histoacryl topical tissue adhesive (B. Braun Corp., Melsungen, Germany), which was used according to manufacturer's instructions. The fixing luting agent was left to set for a period of 1 min before initiation of the measurements. For each specimen a time-period of 3 min was required for the preparation and testing procedures. Specimens topography The specimens were first scanned in order their topography to be obtained. This can be achieved in two ways: (1) the contact mode and, (2) the tapping (semicontact) mode (Ethier and Simmons, 2008). In the contact mode the probe exerts a constant force to the specimens, which has as a result the application of large lateral forces and therefore a possible deformation of the specimen (Ethier and Simmons, 2008). Thus, the tapping mode, in which the cantilever is either magnetically or acoustically driven, was selected. (Figure 2) During the scanning period the oscillating tip of the cantilever was moving in very close proximity to the surface of the specimen, touching it regularly. Figure 2 The tapping mode selected for the purposes of this study. The oscillations of the cantilever are due to repulsive and attracting forces, and they have been discussed previously in the literature (Goodman and Garcia, 1991; García and San Paulo, 1999). The first ones are short range forces with an exponential decaying and can be considered as Pauli's exclusion principle interaction, electron-electron Coulomb interaction and hard sphere repulsion. The second ones are long range forces, including van der Waals interactions, electrostatic and chemical forces. For the interaction between the tip of the cantilever and the specimen's surface, the van der Waals forces and van der Waals potential obey to the following (Hamaker, 1937): (1) FvdW=−(AR6d2) (2) VvdW=−(AR6d) Where, A is the Hamaker constant related to the material, R is the sphere radius and d is the separation between the tip and the half-space surface (Argento and French, 1996). The scanning rate was set at ~5,6 μm/s step 11 nm Hz, and the specimens' surface roughness was determined by the two following equations: Mean absolute deviation surface roughness (Ra) (3) Ra=1NxNyΣi=1NxΣj=1Ny|Z(i,j)−Zmean| Where Zmean represents the mean height, as this was calculated over the entire area of the biologic specimen, discretized in the grid of Z(i,j), i = 1,…,Nx and j = 1,…,Ny The Root Mean Square (RMS) surface roughness (4) RRMS=1NxNyΣi=1NxΣj=1Ny(Z(i,j)−Zmean)2 which represents the average deviation between the height and the mean surface. Additionally, Ry (peak to valley) measurements were recorded. Two- and three-dimensional images of the specimens' topography were obtained (Figure 3). Figure 3 Three-dimensional AFM image of keratinized oral mucosa. Indentation test The mechanical properties of the specimens were then tested. The Si pyramidal tip traversed vertically toward the keratinized oral mucosa specimens and the deflection of the cantilever was measured. As the rigid tip moved toward the soft biologic specimen the latter deflected around the probe. This problem of contact mechanics is based on the Hertz theory and the deflection of the cantilever arm is given by the following (Haga et al., 2002; Ethier and Simmons, 2008): (5) z−zo=Fkc+π2F(1−ν)Etanα Where z is the vertical deflection, zo denotes the height of the probe where the force F applied to the biologic specimen becomes non-zero, kc is the stiffness of the cantilever, E is the elastic modulus of the biologic material in N/m−2, v is the Poisson's ratio of the material (due to high water content Poisson's ratio for most biological specimens is considered to be 0.5), and α is the face angle for the silicon-nitride cantilever (Dimitriadis et al., 2002; Ethier and Simmons, 2008; Figure 4). Figure 4 Indentation of the human keratinized oral mucosa by the Si3N4 four-sided pyramid tip. The force applied by a four sided pyramidal tip is given by: (6) F=E(δ)1−v2tan a2δ2 (7) while,a=tan a2δ A vertical oscillating frequency of ~331 kHz was used and data were recorded at multiple sites of the tested specimens (Weisenhorn et al., 1992, 1993a,b; Stolz et al., 2004; Rahmat and Hubert, 2010; Figure 5). Each loading-unloading cycle lasted 0.3 s. Figure 5 Load-unload curve. Force calibration and displacement data Identification of the first contact between the tip of the cantilever and the material tested presents a challenge in the indentation tests of soft biologic tissues, when using atomic force microscopy (Stolz et al., 2004; Herris et al., 2013). The abrupt change in the force-development curve, due to the generation of repulsive forces between the two interacting bodies, was taken as the initial contact point (z0), and the corresponding force (F0) at that point was zero. The indentation depth was given by the following (Rahmat and Hubert, 2010; Herris et al., 2013): (8) h=(z−z0)−(d−d0) where, z denotes the displacement of the cantilever base, d0 represents the deflection of the cantilever at initial contact and d is the deflection of the cantilever. It should be pointed out that the cantilever's deflection was given by (9) d=d0+(Fk) where, F represents the force and k denotes the stiffness of the cantilever. Room temperature (21 ± 1°C) and relative humidity (50 ± 10%) were recorded throughout the experiments. Results The indentation tests were performed only on the coronal surface of the specimens. Indentation tests on sagittal planes were not performed due to their low clinical application. Data from the indentation tests of the tested specimens was collected and force-displacement diagrams have been obtained (Figure 5). It should be mentioned that the negative values in the curve correspond to the initial phase of the testing procedure, when the tip lands on the surface of specimen. Five loading-unloading cycles were registered. The simplest fitting approach was selected. That consisted of a visual inspection of the force-displacement curve and identification of the initial contact point (zo, do), as already presented earlier (Lin et al., 2007). The area of the curve representing the noncontact region was ignored and for the contact region of the curve four mathematical models were tested using MATLAB (Mathworks Inc; Natick, MA, USA) to note which one approximates the force-displacement curve, as close as possible: a. spherical, b. conical, c. third order polynomial, d. Murphy (fourth order polynomial, non-linear Hertzian based), which is a fourth order polynomial model (Murphy et al., 2013; Table 1, Figure 6). Table 1 Comparison of the numerical fit results including the goodness-of-fit statistic of the normalized mean square error, where the normalization is by the sample variance. Fit name No.1 No.2 No.3 No.4 No.5 exp = 3/2 0.126403 0.009939 0.021102 0.003426 0.004355 exp = 2 0.047443 0.085310 0.117485 0.060889 0.066963 Murphy 0.000380 0.002366 0.004634 0.002658 0.01629 Polynomial 3 0.000396 0.000071 0.000142 0.000189 0.000023 Figure 6 Oral mucosa force-displacement curve (loading) with different models fitting. The goodness-of-fit measure presented in Table 1 is the normalized mean square error (NMSE), and was defined as follows: (10) NMSE=∑i=1n(xi−x^i)2∑i=1n(xi−x¯)2 where xi is the i-th observation of variable X, x^i is the corresponding estimated value by the fitted model, x¯ is the sample mean of the n observations of X. The spherical and the conical models did not approximate the force-displacement curves The model presented by Murphy et al. (2013), with the form (11) F=Γ1δ4+Γ2δ3+Γ3δ2 performed well for only one specimen. The third order polynomial model of the form (12) f(δ)=P1δ3+P2δ2+P3δ+P4 approximated the curve very closely for all tested specimens. Therefore, a proposition of a model which fits the experimental data better than the aforementioned models and the Hertz model (Herz, 1881), which is fully elastic, is attempted: Murphy et al. (2013) have speculated that the Young's modulus of biological materials varies with displacement and is given by the following second order polynomial: (13) E(δ)=k1δ2+k2δ+Eb The authors hypothesized that the triad set k1, k2, and Eb governs the modulus of elasticity of the material they tested. Specifically k1 and k2 represent the non-linear region, while Eb represents the contact stiffness, which is in the elastic region of the force-displacement curve. The coefficients Γ1, Γ2, and Γ3 of their fourth order polynomial (11) are: (14) Γ1=2πk1(tan a)(1−ν2), (15) Γ2=2πk2(tan a)(1−ν2), and (16) Γ3=2πEb(tan a)(1−ν2) Accordingly, in the present study E(δ) is given by the following equation: (17) E(δ)=k1δ+k2+Eb where k1, k2, and Eb describe the oral mucosa's and mucoperiosteum's properties as a triad set, and (18) Eb=k3δ Eb represents the mucoperiosteum's contact stiffness and δ is the indentation depth. A similar approach has been adopted by Murphy et al. (2013). Other researchers have come independently to the same conclusion (Herris et al., 2013). The constant term P4 of (12) is omitted, as when F = 0, δ = 0. Then the general polynomial model (12) becomes: (19) F=P1δ3+P2δ2+P3δ Therefore, by substituting E(δ) from (17) and (18) to (6), the latter becomes: (20) F=k1tan a(1−ν2)2δ3+k2tan a(1−ν2)2δ2+k3tan a(1−ν2)2δ where F is the applied force, δ is the indentation depth, α is the face angle for the silicon-nitride cantilever, v is the Poisson's ratio of the mucosa, and k1, k2, and k3 are the moduli of elasticity for the stratified squamous epithelium, lamina propria and mucoperiosteum respectively. Discussion In the present ex vivo study AFM was used to test the mechanical properties of human keratinized oral mucosa. The specimens were harvested from the edentulous areas which support the maxillary complete denture (Hobkirk and Zarb, 2013). The force-development curves were analyzed and a third order polynomial model different than the classic elastic Hertz model (Herz, 1881) was developed in order to describe its biomechanical response. In the past 25 years AFM has been used to study the mechanical properties of materials. This concept has also been used in the early 90's for “force-displacement” data collection from biological materials, including tendons, ligaments, muscle tissues and menisci (Burnham and Colton, 1989; Radmacher et al., 1992; Tao et al., 1992; Weiss et al., 2002; Sweigart et al., 2004; Yin and Elliot, 2004; Van Loocke et al., 2006, 2008; Villegas et al., 2007; Cheng and Gan, 2008; Ciarletta et al., 2008). A nano-indenter could have been used in this study, as well. However, nano-indenters have a resolution of approximately 100 nN, while the forces applied by the AFM can range from pico-Newtons to several hundreds of micro-Newtons by changing the stiffness of the cantilever. Thus, the sensitivity and the versatility of the AFM makes it an ideal tool for mechanical properties testing of biological materials (Stolz et al., 2009; Notbohm et al., 2012). Additionally, use of the tapping mode of the AFM prevents the distortion of the biologic specimen, as it is presented later in the discussion. In most of the published research, standard manufacturers' cantilevers and pyramidal tips have been employed, while modifications with attachment of silica microspheres have also been reported (Mahaffy et al., 2000; Dimitriadis et al., 2002). The solution for the microsphere tips is supplied by the original Hertz model which deals with the shallow contact between two spherical bodies (Herz, 1881). The original Hertzian theory has been used by many researchers who studied contact deformation and many modifications have been made in order to account for large deformations (Gao and Gao, 2000), viscoelasticity (Gillies et al., 2002; Yang et al., 2004; Attard, 2007; Chen et al., 2013), anisotropy (Batra and Jiang, 2008), multi-layered structures (Ai et al., 2002), and adhesive interactions (Cao et al., 2005; Yang, 2006; Ebenstein, 2011; Chen et al., 2013; Kohn and Ebenstein, 2013). In the present study a sharp pyramidal tip was employed. It should be mentioned that the Bilodeau solution applies for this case (Bilodeau, 1992). The model developed in the present study is probably valid only when the forces are applied to the coronal part of the oral keratinized mucosa, as it has been proven that tissues are both inhomogeneous and anisotropic. Biological specimens' anisotropic properties have been demonstrated with nonlinear laser scanning microscopy, which has been used for elastin and collagen distribution—within the specimens—imaging (Herris et al., 2013). Additionally their mechanical properties are site and direction-specific dependent (Stolz et al., 2004). It has been shown in the past that the indentation elastic moduli of biological specimens differ depending on the depth, increasing from the superficial to deep layers (Herris et al., 2013). Furthermore, it has been demonstrated that both the Young's modulus and Poisson's ratio change during different development stages of the cell (Zhang et al., 2009). Three different moduli of elasticity were incorporated to the model developed in this study, with k1 being the modulus of elasticity of stratified squamous epithelium, k2 of lamina propria and k3 of mucoperiosteum. This is in accordance with the present experimental results and previous research findings, which have demonstrated that the elastic moduli increase from the superficial to the deep layer (Herris et al., 2013; Murphy et al., 2013). Furthermore, the cells and the extracellular matrix present different elastic modulus and may contribute to the observed inhomogeneity (Trickey et al., 2006; Han et al., 2011). Third-degree polynomial models have also proven to be valid for mechanical forces acting on cell biomembranes, as well as in other anatomical structures as the descending aorta (Stefanadis et al., 2000; Zhang et al., 2009). The third degree polynomial model without a constant term which was tested in the present study not only approximated better the force-displacement curve of the keratinized oral mucosa, but it also provided a simpler model than the fourth order polynomial presented by Murphy et al. (2013). It should be mentioned however that different biological tissues have been examined in these two studies. As already mentioned, before testing the specimens were kept in a buffered 10% formalin solution until testing, which occurred within an hour. The 10% formalin solution is actually a 4% formaldehyde, which is by definition 1.3 molar. A totally unbuffered formaldehyde solution exerts an osmotic pressure of about 1300 mO. Isotonic salt solutions present osmolarities of 250–350 mO. Therefore, it is expected that formalin diffuses into tissues faster. Formaldehyde has a molecular weight of 30 and it is expected to penetrate the tissues fast. Nevertheless, fixation actually takes a relatively long time. It has been estimated that a time period of 6–16 h, depending on the specimen, is needed. Furthermore, since all specimens received the same treatment simultaneously it can be assumed that formalin penetration was uniform in all specimens. Macroscopically, no swelling was noticed within the 1-h period between specimen harvesting and testing (Bono et al., 2001; Thavarajah et al., 2012). The human oral keratinized tissue tested in the present study was bonded with cyanoacrylate cement to a glass slab, since there is scientific evidence that force-displacement results depend strongly on whether or not the specimens are attached to the substrate (Yang, 1998). A glass substrate was used as its modulus of elasticity is 50 GPa, which is much higher than that of biologic specimens, while the modulus of elasticity of the pyramidal tip employed was 150 GPa (Grafström et al., 1993; Weisenhorn et al., 1993a). Therefore, the deformation of both the tip and the substrate can be considered as negligible. The loading-unloading curves of the specimens tested did not coincide, suggesting a viscoelastic behavior (Lakes, 1999). Furthermore, the hysteresis of the loading-unloading curves, indicates dissipation of energy. During loading, the area beneath represents the energy stored, while during unloading, this area represents the energy recovered. Quantification of the hysteresis can be performed by introduction of the plasticity index η. In experiments involving solid objects this parameter characterizes the elastic/plastic behavior of the material, when external forces are applied. The value of plasticity index can range between 0, indicating a fully elastic material, and 1, when the material displays a fully plastic behavior (Briscoe et al., 1998; Klymenko et al., 2009). The tapping mode was selected for the present study, as it presents more advantages than the contact and the non-contact imaging methods. The tapping mode eliminates the lateral, frictional forces transmitted from the tip of the cantilever to the specimen's surface. In this mode the probe oscillates with sufficient amplitude to prevent it being trapped by adhesive meniscus forces from the contaminant layer (e.g., water), and it makes intermittent contact with the surface of the specimen (Manning et al., 2003; Rogers et al., 2004). As a result, tissue damage is minimal, if any, and recovery is guaranteed. The oscillation frequency usually ranges between 50,000 and 500,000 cycles per second. An oscillation frequency of 311,000 cycles was used in the present study. With this frequency the tip-specimen adhesion forces are overcome and only vertical forces are applied. The high frequency oscillations of the tip may be regarded as a disadvantage of the method, as they are not comparable to the human chewing frequency. It should be mentioned however that, neither the forces applied by the tip to the specimen are comparable to mastication forces. Atomic force microscopy, like all laboratory techniques, has some limitations. It should be pointed out however that, in studies like the present one, a fundamental assumption is made: the mechanical response of biological materials relies on contribution of different structures which act in sequence. In this way interpretation of the mechanical properties of materials which present a hierarchy can be made (Bonilla et al., 2015). Development of a model describing the behavior of oral mucosa under mechanical forces is instrumental for the knowledge of its mechanical properties, such as the Young's modulus, and an understanding of how masticatory function is affected by mechanical interactions. Moreover, this modeling assists in fabricating biomaterials (e.g., tissue conditioners) which will act in a similar way with, or in a complimentary way to the oral keratinized mucosa. It should be mentioned that ex-vivo studies performed with AFM present certain drawbacks, including the identification of the most appropriate area for force application, the high frequency oscillation of the cantilever, the possible minor dehydration of the tissue, the molecular roughness of the pyramidal tip and the uncertainty of the first contact between the tip and the biological specimen. These have also been acknowledged by other authors and possibly affect measurement's accuracy (Vinckier and Semenza, 1998). Further studies are needed to determine the contribution of each layer, as well as, that of the cells and the extracellular matrix in the biomechanical behavior of the oral mucosa. Conclusion Within the limitations of the present study, the following conclusions can be made regarding the force-displacement data of the human oral keratinized mucosa: The third order polynomial model examined in the present study showed a very good accuracy. The Murphy model (fourth order) performed well for only one specimen. The spherical and the conical models did not approximate the force-displacement curves. A mathematical model for the analysis of the biomechanical response of human keratinized oral mucosa was developed. Author note The results of this paper were presented at the IADR General Session, March 20–23, 2013, Seattle, WA, USA, and at the European Society of Biomechanics, August 25–28, 2013, Patras, Greece. Author contributions All authors (AT, PK, MS, SK, DK, SL, ON, AP, and KM) have made substantial contributions to the conception and/or design of the work; or the acquisition, analysis, or interpretation of data for the work; and greatly assisted in drafting the work or revising it critically for important intellectual content; and they approved the submitted version; and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. ==== Refs References Ai Z. Y. Yue Z. Q. Tham L. G. Yang M. (2002 ). Extended Sneddon and Muki solutions for multilayered elastic materials . Int. J. Eng. Sci. 40 , 1453 –1483 . 10.1016/S0020-7225(02)00022-8 Argento C. French R. H. 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==== Front Front ImmunolFront ImmunolFront. Immunol.Frontiers in Immunology1664-3224Frontiers Media S.A. 10.3389/fimmu.2016.00323ImmunologyReviewNK Cell Influence on the Outcome of Primary Epstein–Barr Virus Infection Chijioke Obinna 12Landtwing Vanessa 2Münz Christian 2*1Institute of Surgical Pathology, University Hospital Zürich, Zürich, Switzerland2Viral Immunobiology, Institute of Experimental Immunology, University of Zürich, Zürich, SwitzerlandEdited by: Eric Vivier, Centre d’Immunologie de Marseille-Luminy, France Reviewed by: Bojan Polić, University of Rijeka, Croatia; Nicholas D. Huntington, Walter and Eliza Hall Institute of Medical Research, Australia *Correspondence: Christian Münz, christian.muenz@uzh.chSpecialty section: This article was submitted to NK Cell Biology, a section of the journal Frontiers in Immunology 29 8 2016 2016 7 32330 6 2016 15 8 2016 Copyright © 2016 Chijioke, Landtwing and Münz.2016Chijioke, Landtwing and MünzThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.The herpesvirus Epstein–Barr virus (EBV) was discovered as the first human candidate tumor virus in Burkitt’s lymphoma more than 50 years ago. Despite its strong growth transforming capacity, more than 90% of the human adult population carries this virus asymptomatically under near perfect immune control. The mode of primary EBV infection is in part responsible for EBV-associated diseases, including Hodgkin’s lymphoma. It is, therefore, important to understand which circumstances lead to symptomatic primary EBV infection, called infectious mononucleosis (IM). Innate immune control of lytic viral replication by early-differentiated natural killer (NK) cells was found to attenuate IM symptoms and continuous loss of the respective NK cell subset during the first decade of life might predispose for IM during adolescence. In this review, we discuss the evidence that NK cells are involved in the immune control of EBV, mechanisms by which they might detect and control lytic EBV replication, and compare NK cell subpopulations that expand during different human herpesvirus infections. lytic EBV infectionNKG2DDNAM-1infectious mononucleosishumanized miceKrebsliga Schweiz10.13039/501100004361KFS-3234-08-2013Universität Zürich10.13039/501100006447KFSP MS, KFSP HHLDWorldwide Cancer Research10.13039/50110000728714-1033Sparks10.13039/50110000086115UOZ01Seventh Framework Programme10.13039/501100004963COST Mye-EUNITERSchweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung10.13039/501100001711310030_162560, CRSII3_160708 ==== Body Epstein–Barr Virus Infection and Predisposing Factors for EBV Disease The herpesvirus Epstein–Barr virus (EBV) was discovered in 1964 by electron microscopy in Burkitt’s lymphoma, the most common childhood tumor in sub-Saharan Africa (1). It is arguably the most potent human tumor virus, because it readily transforms primary human B cells into immortalized lymphoblastoid cell lines (LCLs) in culture (2). This strong growth transforming capacity is due to the latent EBV proteins, six nuclear antigens (EBNAs) and two latent membrane proteins (LMPs), which are expressed as the default infection program in B cells (3). Lytic EBV replication occurs in LCLs only at low levels and triggers the expression of around 80 gene products under the guidance of the immediate early lytic transactivator BZLF-1 for the production of infectious DNA virus particles (4). In addition to Burkitt’s lymphoma, EBV is associated with numerous malignancies, mostly of B and epithelial cell origin, such as Hodgkin’s lymphoma and nasopharyngeal carcinoma (3). Despite this strong growth transforming capacity, EBV is carried by more than 90% of the human adult population as an asymptomatic persistent infection. Epstein–Barr virus infection remains asymptomatic in most persistently infected individuals despite transforming latent EBV protein expression (5). In healthy EBV carriers, the expression of all six EBNAs and the two LMPs can be found in naïve B cells of secondary lymphoid organs like the tonsils (6) (Figure 1). In germinal center B cells, only the subset of viral proteins that is also present in Hodgkin’s lymphoma is expressed (EBNA1, LMP1 and 2). Finally, in homeostatically proliferating memory B cells, the latency pattern of Burkitt’s lymphoma is present with EBNA1 as the only expressed protein (7). Reactivation from this persistent EBV reservoir of memory B cells into lytic replication seems to occur after B cell activation and plasma cell differentiation (8). These findings, however, indicated that healthy EBV carriers are continuously challenged with transforming latent EBV expression programs, which could result in tumor formation without immune control. Figure 1 Role of NK cells in the immune control of the EBV life cycle. Epstein–Barr virus (EBV) is transmitted via saliva exchange and infects submucosal B cells. In infected naïve B cells, the latency III EBV program can be found (EBNA1, 2, 3A–C, LP, and LMP1 and 2). Activation via EBV infection drives infected B cells into differentiation. In resulting germinal center B cells, latency II EBV infection can be found (EBNA1, LMP1 and 2). These EBV proteins allow EBV-infected B cells to survive and enter the memory B cell pool. In memory B cells, all EBV proteins expression is switched off (latency 0). Upon B cell receptor cross-linking, the lytic EBV cycle is activated due to plasma cell differentiation, which allows epithelial cell infection for further amplification of infectious virus before shedding into saliva. NK cells target lytically EBV replicating cells via their activating NKG2D and DNAM-1 receptors. It remains unclear if also lytically EBV replicating epithelial cells can be recognized by NK cells. Indeed, immune suppression after organ transplantation or due to human immunodeficiency virus (HIV) co-infection causes the occurrence of EBV-associated malignancies, such as post-transplant lymphoproliferative disease (PTLD) and immunoblastic lymphoma (3). Some of these lymphoproliferations can be treated by the adoptive transfer of EBV-specific T cell lines (9). Moreover, some individuals remain EBV seronegative despite carrying the virus, and seem to control persistent EBV infection entirely by cell-mediated immunity (10). Thus, cell-mediated immunity by T cells seems to be sufficient to control persistent EBV infection (11). In addition to direct immune suppression of cell-mediated immunity, the conditions under which this immune response is primed, seems to be decisive for an asymptomatic immune control of EBV infection. Indeed, if primary infection with EBV is delayed into adulthood, the virus is more frequently acquired with symptomatic primary infection, called infectious mononucleosis (IM) (12). This immunopathology by massive CD8+ T cell expansion and activation seems to result from an uncontrolled lytic EBV replication, because most of the expanding CD8+ T cells are directed against lytic EBV antigens (13). This massive lymphocytosis seems to transiently compromise EBV-specific immune control with an increased susceptibility to some EBV-associated malignancies, such as Hodgkin’s lymphoma up to 5–10 years after IM (14). In this review, we discuss the innate arm of cell-mediated immune control of EBV, which could explain the different outcomes of primary infection with this tumor virus and might be required to limit initial viral titers so that long-lasting adaptive cell-mediated immune control can be efficiently primed. Primary Immunodeficiencies That Affect NK Cell Function and Predispose for EBV Disease Evidence that loss of cytotoxic cell-mediated immune control predisposes for EBV-associated diseases comes from primary immunodeficiencies that sensitize for EBV-associated malignancies (15, 16). A subset of these affect, in addition to T cell responses, natural killer (NK) cell responses and hint toward an important function of cell-mediated innate immunity in EBV-specific immune control. The underlying genetic lesions affect gene products that are involved in NK cell differentiation, stimulation, and cytotoxic effector function. Natural killer cell differentiation is disrupted by mutations in GATA-binding protein 2 (GATA2) and minichromosome maintenance complex component 4 (MCM4). Accordingly, a GATA2 mutation was identified later in the first indicator patient with susceptibility to herpesvirus induced diseases (17, 18). GATA2 is a hematopoietic transcription factor that is required for the development of several immune cell lineages, including B cells, CD4+ T cells, dendritic cells, neutrophils, and monocytes in addition to NK cells (19). With respect to EBV-associated diseases, patients with GATA2 mutations have been diagnosed with chronic active EBV infection (CAEBV) and virus-positive smooth muscle tumors (20, 21). In contrast to this multilineage deficiency in patients with GATA2 mutations, partial deficiency of the DNA helicase MCM4 blocked differentiation of the human CD56dim NK cell subset, while other hematopoietic lineages seemed to be unaffected (22). One of the afflicted patients suffered from an EBV-associated lymphoma (23). Thus, compromised NK cell differentiation is associated with uncontrolled EBV infection. In addition to NK cell differentiation, some mutations that impact NK cell stimulation are associated with EBV disease. These include deficiencies in SLAM-associated protein (SAP) of X-linked lymphoproliferative disease type 1 (XLP1), in magnesium transporter 1 (MAGT1) of X-linked immunodeficiency with magnesium defect, EBV infection, and neoplasia (XMEN), in CD27, in phosphatidylinositol 3 kinase (PI3K) 110δ and in FcγR3A (CD16). XLP1, also known as Duncan’s disease, primarily manifests in boys (24). Primary infection with EBV often leads to fatal IM in the affected patients, if they cannot be identified early enough and treated with bone marrow transplantation (25). The underlying mutations in SAP were identified in 1998 (26–28) and affect the adaptor protein of SLAM receptors that mediate their co-activating function in T and NK cells. Two of these SLAM receptors, 2B4 and NTB-A, increase NK cell cytotoxicity (29, 30), but XLP1-associated SAP mutations might primarily compromise EBV-specific CD8+ T cell immune control (31–33). Furthermore, deficiency in the magnesium transporter MAGT1 results in diminished free magnesium levels within cells, which is associated with downregulation of the activating NKG2D receptor on cytotoxic lymphocytes, T, and NK cells (34). Supplementation of magnesium results in decreased EBV loads in the affected XMEN patients. Another activating co-receptor on T and NK cells is CD27. Mutations in this CD70 engaging co-receptor predispose for EBV-associated lymphoproliferations (35, 36). Also, loss-of-function mutations in the signaling molecule PI3K 110δ of activating receptors are associated with persistent EBV viremia (37). Finally, the activating FcγR on NK cells, CD16, seems to be required for EBV-specific immune control. Mutations in CD16 were reported to be associated with persisting IM symptoms (38, 39). These primary immunodeficiencies identify 2B4, NKG2D, CD27, and FcγR as important receptors in EBV-specific cell-mediated immune control. Apart from these activating receptors, the cytotoxic effector machinery also seems to be important in EBV-specific immune control. Accordingly, mutations in perforin, Munc13-4, and Munc18-2 have been identified in patients with EBV-associated diseases. Mutations in perforin are responsible for type 2 familial hemophagocytic lymphohistiocytosis (FHL2). Persistent IM has been described in one FHL2 patient (40). Munc18-2 and 13-4 mediate docking and activation of syntaxin 11 for cytotoxic granule fusion with the cell membrane, respectively. Mutations in these two components of the cytotoxic machinery were found in patients with CAEBV (41). These genetic lesions point toward a role of cytotoxic lymphocytes in EBV-specific immune control. Primarily, prolonged IM resulting in CAEBV seems to be associated with primary immunodeficiencies that affect NK cell function. NK Cell Expansion During Primary EBV Infection Natural killer cell expansion during primary EBV infection has first been reported in a study by Tomkinson et al. (42), in which peripheral NK cells (identified as CD16+ lymphocytes) were described to be significantly increased in both frequency (1.5-fold) and absolute number (4-fold) in – by these measures – a similar manner to CD8+ T cells in a cohort of IM patients. However, since the authors had to use a strategy for gating lymphocytes that included activated and, thus, blasted cells, CD16+ monocytes could not be excluded from the analysis and might account for some of the quantitative changes ascribed to the NK cell compartment. Still, threefold to sixfold increases in the number of bulk NK cells in IM patients were found by other groups as well (43, 44) and these increases were found to be inversely correlated with viral load in blood (43). Likewise, higher NK cell counts tended to be associated with less severe disease (43). On the contrary, a large and, notably, prospective study of primary EBV infection (45), while also reporting expansions of NK cells during the acute phase, positively correlated NK cell numbers with blood viral load and also positively correlated blood viral load with disease severity (45). Similarly, the increase in NK cells in IM patients was related to greater disease severity by another group, although the small number of subjects in that study precluded statistical significance (44). A study by Azzi et al. (46) detailed the phenotype of NK cells during IM and convalescence in pediatric patients and demonstrated the lack of influence of primary EBV infection on the expression of killer cell immunoglobulin-like receptors (KIRs) but instead noted an up to fivefold expansion of an early-differentiated NK cell subset (Figure 2). This accumulated NKG2A+KIR−CD57− NK cell subset was the only identifiable subset within the NK cell compartment that proliferated in the acute phase and importantly, this proliferating early-differentiated NK cell subset also correlated with viral load in PBMCs (46). Although overall NK cell numbers and frequencies contract early after the onset of symptoms (43–46), these early-differentiated NK cells remain elevated in frequency up to 6 months after the acute symptomatic phase (46–48), but over time accumulate signs of differentiation (46, 47). Asymptomatic primary EBV infection is mostly found in young children (49, 50) compared to a symptomatic outcome, i.e., IM, in up to 75% of cases of primary EBV infection in adolescents (45). While asymptomatic infection was associated with high viral load, phenotype, and frequencies of antigen-specific CD8+ T cells similar to IM, the massive expansion of CD8+ T cell numbers typically seen in IM was absent (49). It might be speculated that the confinement of CD8+ T cell expansion is exerted by the EBV-responsive early-differentiated NKG2A+KIR− NK cell subset, especially since this subset is highest in both frequency and numbers in newborns and young children but decreases with age (46). Whether the loss of early-differentiated NK cells during adolescence is associated with a specific molecular imprint that affects NK cell homeostasis, e.g., the result of changes in the expression of transcription factors, has not yet been explored in the current literature. One explanation for such an age-dependent effect, however, is an increased burden and accumulation of various infectious challenges with advancing years that can likely be expected to have an impact on the differentiation of NK cells. One of these challenges, namely infection with the human cytomegalovirus (HCMV), that seems to drive NK cell differentiation via IL-12 and IL-15 production, is discussed below. Thus, dynamics within the NK cell compartment over time might in part explain the age-dependent occurrence of symptomatic primary EBV infection. Figure 2 EBV and HCMV expand different NK cell populations. Epstein–Barr virus (EBV) expands early-differentiated NKG2A/CD94, NTB-A, 2B4, CD27, CD16, NKG2D, and DNAM-1-positive NK cells, which after expansion upregulate the senescence marker CD57. Human cytomegalovirus (HCMV) in contrast expands late-differentiated CD94/NKG2C, CD16, CD2, KIR, and CD57-positive NK cells. NK Cell Reactivity Against Lytic EBV Infection Indeed, the trigger of peripheral NK cell accumulation in primary EBV infection does not seem to be caused by the inflammatory status of IM itself, e.g., increased levels of pro-inflammatory cytokines, since patients with equally inflammatory conditions but lacking evidence of EBV seroconversion do not show any expansions in their NK cell compartment (46). Instead, there is evidence that the state of the infectious cycle of EBV, either latent or lytic, drives the expansion of NK cells during infection, specifically changes that are inherent to lytic replication. In mice with reconstituted human immune system components (HIS mice), NK cell expansion only occurs during infection with wild-type EBV, but not with recombinant EBV engineered to only establish latent infection (EBV BZLF-1 knockout or BZ1KO EBV) (51). Furthermore, proliferation of NKG2A+KIR− NK cells was only seen after in vitro infection of cord blood with wild-type EBV but not with BZ1KO EBV (46). It is, therefore, conceivable that the expansion of the cytotoxic lymphocyte populations, namely NK and CD8+ T cells, during EBV infection is driven by the amount of available antigen (45, 46, 51), since the expansion of total CD8+ T cell and NK cell numbers as well as viral load correlate (45). Actually, lytic replication might not only be responsible for the expansion of the early-differentiated NK cell subset, but seems to also be a target of NK cells itself (Figure 1). In EBV-infected HIS mice, NK cells protect from high viral load, elevated cytokine levels, splenomegaly, weight loss, and occurrence of lymphoproliferative tumors, as well as limit the expansion of CD8+ T cells (51). Most of the protective effects of NK cells are lost in HIS mice only latently infected with EBV, but regained when these mice are infected with a recombinant virus reverted to allow for lytic replication (51). Also, in EBV-infected HIS mice depleted of NK cells, there is an increased abundance of lytic proteins and cell-free viral DNA indicative of ongoing uncontrolled lytic replication (51). In vitro, NK cells respond to and kill an EBV-positive B cell line more efficiently when these cells are in the lytic as compared to the latent phase of infection (51–53), in particular NK cells with the NKG2A+KIR− phenotype (46). The preferential killing of lytic cells was sensitive to blocking of CD112 and ULBP-1, ligands of the activating NK cell receptors DNAM-1 and NKG2D, respectively (52) as well as directly blocking DNAM-1 (53). Therefore, the identification of activating receptors or combinations thereof crucial in NK cell-mediated protection in vivo holds promise to further our understanding of the intricate interplay between EBV with the host’s immune system and HIS mice might constitute a feasible model to answer such questions (51). Differences Between EBV-Driven NK Cell Expansion and Other Human Herpesvirus Infections In contrast to EBV infection, other herpesviruses either do not change the NK cell composition, such as recurrent α-herpesvirus infection by herpes simplex virus 2 (HSV2) (54), or expand terminally differentiated NKG2C+KIR+CD16+ NK cells, such as the β-herpesvirus HCMV (55–57) or the γ-herpesvirus Kaposi sarcoma-associated herpesvirus (KSHV) in HIV-infected individuals (58). Accumulation of terminally differentiated NK cells is primarily connected to HCMV infection (Figure 2) and it has been argued that in other viral infections, for which such terminal NK cell differentiation can be observed, such as with Hantavirus (59), Chikungunya virus (60), HIV (61), and hepatitis virus (62), mainly HCMV-positive individuals are affected by this alteration in NK cell repertoire composition (63–65). This HCMV-driven terminal NK cell expansion has been linked to NK cell stimulation by cells that produce the NKG2C ligand HLA-E plus the NK cell proliferation stimulating cytokine IL-15 on their surface (59, 66). Expansion of NKG2C-positive NK cells could be obtained with HCMV infected fibroblasts plus IL-15 (67) and bystander monocytes were able to provide NK cell stimulating cytokines, including IL-12 (68). However, HCMV-infected individuals with NKG2C deficiency also develop NK cell populations that more vigorously secrete IFN-γ upon stimulation, the so-called adaptive NK cell populations (69), and the NKG2C genotype does not affect the outcome of congenital HCMV infection (57). Therefore, HLA-E-mediated NK cell stimulation might not be essential for the expansion and anti-viral function of NKG2C-positive NK cell populations, but IL-15 and IL-12 might be more important (65). Accordingly, one patient with IL-12Rβ1 deficiency did not carry adaptive NK cell populations (70). Therefore, cytokines might be one of the main drivers of adaptive NK cell expansion, as originally proposed in mice (71). These adaptive NK cells are terminally differentiated NKG2C-positive NK cells during HCMV infection, while for the early-differentiated NK cells that expand and persist for 6 months during acute EBV infection adaptive features have still to be investigated. In contrast to direct immune control of lytic EBV replication by early-differentiated NK cells (51), the role of terminally differentiated NK cells is less clear during HCMV infection. Only for decidual NKG2C-positive NK cells it was shown that they directly kill HCMV-infected autologous decidual fibroblasts in an HLA-E dependent fashion (72). Most studies, however, implicate the NKG2C-positive NK cell subset that expands during HCMV infection in mediating superior antibody-dependent cellular cytotoxicity (ADCC) against antibody opsonized HCMV-infected macrophages or fibroblasts (73, 74). In these studies, both IFN-γ production and degranulation of NKG2C-positive NK cells of HCMV-infected donors were superior upon opsonized target recognition compared to NKG2C-negative NK cell populations. These superior effector functions most likely result from epigenetic modifications, as has been shown for the IFN-γ gene locus in NKG2C-positive NK cells of HCMV infected individuals (75–78). Thus, early-differentiated NK cells that expand during EBV infection might directly recognize lytically EBV replicating targets, while the terminally differentiated NK cells in HCMV-infected individuals mainly promote ADCC. Author Contributions All authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding Research in our laboratory is supported by grants from Cancer Research Switzerland (KFS-3234-08-2013), Cancer Research Zurich, Worldwide Cancer Research (14-1033), KFSPMS and KFSPHHLD of the University of Zurich, the Sobek Foundation, the Swiss Vaccine Research Institute, the SPARKS Foundation (15UOZ01), COST Mye-EUNITER of EU FP7, and the Swiss National Science Foundation (310030_162560 and CRSII3_160708). ==== Refs References 1 Epstein MA Henle G Achong BG Barr YM Morphological and biological studies on a virus in cultured lymphoblasts from Burkitt’s lymphoma . J Exp Med (1964 ) 121 :761 –70 .10.1084/jem.121.5.761 14278230 2 Nilsson K Klein G Henle W Henle G The establishment of lymphoblastoid lines from adult and fetal human lymphoid tissue and its dependence on EBV . Int J Cancer (1971 ) 8 :443 –50 .10.1002/ijc.2910080312 4332899 3 Cesarman E . Gammaherpesviruses and lymphoproliferative disorders . 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==== Front Front Mol BiosciFront Mol BiosciFront. Mol. Biosci.Frontiers in Molecular Biosciences2296-889XFrontiers Media S.A. 10.3389/fmolb.2016.00044Molecular BiosciencesMini ReviewParB Partition Proteins: Complex Formation and Spreading at Bacterial and Plasmid Centromeres Funnell Barbara E. *Department of Molecular Genetics, University of TorontoToronto, ON, CanadaEdited by: Manuel Espinosa, Spanish National Research Council - Centro de Investigaciones Biológicas, Spain Reviewed by: Christopher Morton Thomas, University of Birmingham, UK; Jean-Yves Bouet, Centre National de la Recherche Scientifique, France *Correspondence: Barbara Funnell b.funnell@utoronto.caThis article was submitted to Molecular Recognition, a section of the journal Frontiers in Molecular Biosciences 29 8 2016 2016 3 4427 6 2016 15 8 2016 Copyright © 2016 Funnell.2016FunnellThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.In bacteria, active partition systems contribute to the faithful segregation of both chromosomes and low-copy-number plasmids. Each system depends on a site-specific DNA binding protein to recognize and assemble a partition complex at a centromere-like site, commonly called parS. Many plasmid, and all chromosomal centromere-binding proteins are dimeric helix-turn-helix DNA binding proteins, which are commonly named ParB. Although the overall sequence conservation among ParBs is not high, the proteins share similar domain and functional organization, and they assemble into similar higher-order complexes. In vivo, ParBs “spread,” that is, DNA binding extends away from the parS site into the surrounding non-specific DNA, a feature that reflects higher-order complex assembly. ParBs bridge and pair DNA at parS and non-specific DNA sites. ParB dimers interact with each other via flexible conformations of an N-terminal region. This review will focus on the properties of the HTH centromere-binding protein, in light of recent experimental evidence and models that are adding to our understanding of how these proteins assemble into large and dynamic partition complexes at and around their specific DNA sites. chromosome dynamicssegregationParABSDNA-bindingbridgingCanadian Institutes of Health Research10.13039/50110000002437997 ==== Body In bacteria, the segregation, or partition, of low-copy-number plasmids and cellular chromosomes depends on the activity of site-specific DNA binding proteins to recognize one or more copies of a centromere-like DNA site. These “centromere-binding proteins” generally work in concert with an ATPase or GTPase, resulting in dynamic movement and positioning of plasmids or chromosomal domains during the cell cycle (reviewed in Wang et al., 2013; Baxter and Funnell, 2014; Bouet et al., 2014). Centromere-binding proteins fall into one of two structural classes, as helix-turn-helix (HTH), or ribbon-helix-helix site-specific DNA binding proteins. In bacteria, the proteins of all chromosomal, and many plasmid partition systems are members of the HTH class. They share similar properties in vivo and in vitro, including similar domain organization and DNA-binding properties, although there are also interesting differences. They all form large partition complexes in vivo that can be visualized as foci using fluorescence approaches. This review will focus on the properties of the HTH centromere-binding proteins, and in particular, how they assemble into large partition complexes. I will discuss the contribution of protein domains, the “spreading” phenomenon that has been reported as a general property of HTH centromere-binding proteins, and how flexibility in the protein allows multiple conformations and binding modes in complex assembly. The components of partition systems are commonly named ParA (the partition ATPase), ParB (the centromere-binding protein), and parS (the centromere or partition site). Plasmids typically contain one parS located near the parA and parB genes, although some have multiple parS sites. Bacterial chromosomes contain several parS sites, which are primarily located in the chromosomal domain that contains the replication origin. Bacterial ParA and ParB are also called Soj and Spo0J, respectively, because their genes were first defined by roles in sporulation of Bacillus subtilis (Ireton et al., 1994). For simplicity, I will use the ParABS nomenclature, with specific names for some of the discussion to be consistent with published literature. HTH ParBs share a similar domain organization although the primary sequence conservation is not high among members of this family. In general, the protein is divided into three regions: A central HTH DNA binding domain is flanked by a C-terminal dimer domain and an N-terminal region necessary for protein oligomerization (Figure 1A). Flexible linkers connect the domains, and flexibility in domain organization, orientation, and folding have been observed in biochemical experiments and crystal structures. The most highly conserved sequence among plasmid and chromosomal ParBs is a short arginine-rich motif in the N-terminus, which is often called an arginine patch (Yamaichi and Niki, 2000). ParA interactions are often specified by residues near the N-terminus of ParB (Radnedge et al., 1998; Figge et al., 2003; Leonard et al., 2005; Ah-Seng et al., 2009), although there are exceptions. For example, the ParA interactions for RK2 KorB and Pseudomonas aeruginosa ParB map to the center and dimer domain, respectively (Lukaszewicz et al., 2002; Bartosik et al., 2004). There are also added complexities to the general arrangement. For example, P1 ParB and its relatives contain an additional site-specific DNA binding activity within the dimer domain. These ParBs recognize both an inverted repeat and a second DNA motif in their parS sites, via their HTH domain and dimer domains, respectively (Schumacher and Funnell, 2005). Figure 1 Assembly of ParB partition complexes. (A) Cartoon of the conserved domain structure of HTH ParBs, shown as a dimer. The black rectangles represent the regions of the protein for which there is some structural information: the C-terminal dimer domain, the HTH DNA binding domain, and the N-terminal domain. The three regions are connected by flexible linker sequences (arrows). The linker length here is represented as short, as in the HpSpo0J and P1 ParB published structures (Schumacher and Funnell, 2005; Chen et al., 2015), but may be longer in other ParBs. The wavy line represents the region that interacts with ParA in many, although not all, ParBs. The position of the HTH motif (blue) and the conserved arginine patch motif (RR, red) are indicated in one monomer. (B) Diagrams of 1D + 3D and caging models for higher-order ParB binding and partition complex assembly. ParB dimers bound to parS (in red) nucleate complex assembly and interact with other ParBs in green. Arrows in the caging architecture illustrate that dynamic associations maintain the cluster of ParB. Partition complex assembly, spreading, and bridging Partition complex assembly begins with the recognition of parS by a dimer of ParB, followed by loading of multiple ParB dimers to form a very large protein-DNA complex (Baxter and Funnell, 2014). These higher-order complexes are necessary as both the substrates and the activators of the mechanisms of partition. The number of ParB protein foci observed inside cells is usually lower than the number of parS sites, leading to the idea that inter and intra-molecular pairing of parS sites occurs in plasmids and chromosomal domains. Spreading is an unusual feature for site-specific DNA binding proteins that is common to HTH ParBs, and it reflects how ParB assembles into higher-order complexes (Rodionov et al., 1999; Murray et al., 2006; Breier and Grossman, 2007; Sanchez et al., 2015). Measured by ChIP approaches, in vivo ParB binding extends beyond parS into the surrounding non-specific DNA, often many kb away from the site. Binding is maximal at parS, and diminishes non-linearly as a function of distance from parS. Spreading can be impeded by “roadblocks,” which are strong binding sites for other proteins (Rodionov et al., 1999; Murray et al., 2006). Spreading has also been inferred from the ability of some ParBs, especially when overexpressed, to silence expression of nearby genes (Lynch and Wang, 1995; Rodionov et al., 1999; Bartosik et al., 2004; Bingle et al., 2005; Kusiak et al., 2011). Silencing is likely a consequence of protein overexpression and is not necessary for partition (Rodionov and Yarmolinsky, 2004). Spreading ability is required however, as ParB mutants that do not spread are defective in partition (Rodionov et al., 1999; Autret et al., 2001; Breier and Grossman, 2007; Kusiak et al., 2011; Graham et al., 2014). In particular, the arginine patch in ParB is essential for spreading, focus formation, and partition activity in vivo. The first and simplest model described spreading as lateral protein-protein association along the DNA as a one-dimensional filament (Rodionov et al., 1999). However some properties of ParBs lead investigators to question this idea. Studies with plasmid KorB and with B. subtilis Spo0J (BsSpo0J) argued that the intracellular concentration of ParB was insufficient to account for the amount of spreading observed in vivo if arranged as a one-dimensional filament (Bingle et al., 2005; Graham et al., 2014). It was also difficult to demonstrate biochemically that ParB binding to parS increased the affinity of ParB for adjacent non-specific DNA. Two other models have emerged recently, based on sophisticated microscope and ChIP-seq technologies as well as computer modeling and traditional biochemistry (Broedersz et al., 2014; Graham et al., 2014; Sanchez et al., 2015). The first proposes that limited lateral ParB-ParB interactions (1D) in combination with inter and intra-molecular looping and bridging (3D) act to build large complexes and coalesce many ParB molecules into foci (Broedersz et al., 2014; Graham et al., 2014; Figure 1B). Computer modeling was used to argue that neither 1D nor 3D interactions alone could generate ParB foci; that the 1D + 3D arrangement allows focus formation because it creates a surface tension on the ParB cluster that counteracts the tendency for entropy to disperse the protein on DNA (Broedersz et al., 2014). Elegant in vitro TIRF microscopy experiments provided experimental support for the bridging activity: flow-stretched DNA was condensed by BsSpo0J in a manner that is most consistent with ParB bridging across loops within the same or across different DNA molecules (Graham et al., 2014). The experiments were however unable to demonstrate any sequence-specificity for parS, leading to the suggestion that experiments on flow-stretched DNA were not recapitulating an undefined aspect of critical nucleation properties of ParB bound to parS. For example, one factor missing from these experiments is DNA supercoiling, which affects chromosome compaction and may strongly influence the DNA binding properties of ParB in higher-order complexes. Mutations in conserved arginine residues of the arginine patch motif eliminated bridging in the TIRF assay, consistent with the requirement for this motif for spreading and partition in vivo (Graham et al., 2014). Interestingly, BsSpo0J mutated at one residue in the arginine patch (G77S), which is unable to form foci or spread in vivo, was still able to bridge DNA, and with slightly higher stability than that of wild-type BsSpo0J. Therefore the bridging activity of ParB is necessary but not sufficient for complex formation. These observations lead to the suggestion that the G77S mutation may promote inappropriate bridging and/or alter the dynamics of bridging necessary for proper complex assembly in vivo. Modeling the spreading/bridging behavior also predicted that roadblocks would decrease the probability of loops forming in their vicinity, interfering with complex assembly beyond the roadblock (Broedersz et al., 2014). In contrast, a second model proposes that a network of stochastic binding of ParB explains the clustering of ParB molecules around parS (Sanchez et al., 2015). In essence, the nucleation of ParB by parS creates, and maintains a very high localized concentration of ParB in a “cage” by many weak but dynamic interactions with itself (dimer-dimer interactions via the N-terminal domains), as well as with non-specific DNA around parS (Figure 1B). In caging, these interactions do not need to occur simultaneously or to bridge DNA (Figure 1B). Computer modeling of the patterns of ParB occupancy around parS measured by ChIP was used to argue that they are not consistent with either 1D lateral spreading or a combination of 1D spreading and 3D bridging. Biochemical examination showed no evidence that binding of one ParB could stabilize binding of an adjacent ParB. The caging model neither requires nor excludes bridging, although bridging interactions are intuitively attractive as part of the dynamic glue. The model does depend on other properties of the DNA chromosome, such as topology or organization by other nucleoid-binding proteins in vivo to help restrict the DNA within the cage. Roadblocks could alter local DNA organization and reduce the proximity of parS to the rest of the DNA in three-dimensional space; that is, place this DNA outside the cage. Both models agree that ParB binding to parS must nucleate the formation of higher-order complexes to explain ParB clustering and foci in vivo (Broedersz et al., 2014; Sanchez et al., 2015). This is most simple to envision in plasmid systems such as P1, in which ParB's affinity for parS is at least 10,000-fold higher than that for non-specific DNA (Funnell, 1991). However this affinity difference is small for some ParBs (Broedersz et al., 2014; Taylor et al., 2015), and it was suggested that a conformational change is induced in ParB by parS-specific binding to effectively anchor the focus at the site. Both models agree that multiple ParB-ParB interaction interfaces must be involved in assembly of higher-order partition complexes, and that the dynamics of these interactions are critical for proper assembly and function in partition. How bridging activities detected in vitro contribute to ParB activity in vivo remains to be resolved. Further refinement of these models will depend on the ability to completely reconstitute the parS-dependent complex assembly in vitro, which will in turn depend on identifying the other factors necessary for caging or bridging, and on the nature of ParB-ParB and ParB-DNA interactions at the molecular level. The influence of ParA on complex architecture and dynamics has also yet to be defined (see below). ParB-ParB and ParB-DNA interactions in higher-order-complex assembly Site-specific DNA binding of ParBs to cognate parS sites has been examined directly and in detail in many different partition systems, but the parS-dependent formation of higher order, large partition complexes has been difficult to reconstitute in vitro. However, there are insights arising from structural biology of several plasmid and chromosomal ParBs, which are leading to a preliminary, albeit incomplete, picture of partition complex assembly. Although there are no structures of a full length ParB, those of individual domains or combinations of domains, with and without parS DNA, have provided clues concerning the three dimensional organization of the protein with respect to DNA and to itself. There are structures of the HTH domains of three plasmid ParBs (P1 ParB, F SopB, and RP4 KorB) in complex with their specific DNA sites, and of their dimer domains (SopB and KorB dimer domains solved separately from the HTH; Delbrück et al., 2002; Khare et al., 2004; Schumacher and Funnell, 2005; Schumacher et al., 2010). Structures of two chromosomal ParB fragments, each containing the N-terminal region and adjacent HTH domain, have visualized the oligomerization interactions of the proteins. These ParBs are Thermus thermophilus Spo0J (TtSpo0J) and Helicobacter pylori Spo0J (HpSpo0J); structure of the latter was solved bound to parS (Leonard et al., 2004; Chen et al., 2015). One of the first themes to highlight is that of flexibility. Taken together, the structures indicate that these three regions of the protein are connected by flexible linkers, and that their orientation with respect to each other can vary. The conformation of the N-terminus is particularly flexible (Chen et al., 2015). Second, ParBs are bona-fide HTH site-specific DNA binding proteins, but with a twist. As expected, the HTH domains contact inverted repeat sequences within parS via helix insertion into the major groove of DNA. However, unexpected features emerged from the structures. First, residues outside of the recognition helix also contribute to specificity for parS in SopB, KorB, and HpSpo0J (Khare et al., 2004; Schumacher et al., 2010; Sanchez et al., 2013; Chen et al., 2015). Second, both the P1 ParB and HpSpo0J structures demonstrated bridging across parS sites mediated by the dimer and N-terminal domains, respectively (Schumacher and Funnell, 2005; Chen et al., 2015). Each monomer of a P1 ParB dimer interacts with a half-site on a different DNA molecule, effectively pairing two parS sites. HpSpo0J-parS bridging interactions are mediated by the N-terminal oligomerization regions of the protein (Figure 2). Four monomers of HpSpo0J (monomeric because it lacks the C-terminal dimer domain) interact with two parS oligos and with each other in a cross-bridge arrangement across the DNA molecules (molecules A–D in Figure 2). The monomers share a common HTH domain, but show different conformations in the extended N-terminal regions as well as different interactions with each other. There are two adjacent (AB and CD) and one transverse (AC) sets of protein-protein interactions, which are distinct. For example, the conserved arginine patch motifs are close to each other at the AC interface, but not at the AB or CD interfaces (Figure 2). In the structure, there is no BD interaction, leaving these surfaces available, perhaps for interactions with different conformations of ParB or with different partners. Figure 2 Structure of HpSpo0J monomers (lacking C-terminal dimer domain) bound to and across parS DNA (PDB 4UMK, Chen et al., 2015). Four monomers (A to D) make adjacent (AB and CD) and transverse (AC) interactions. The arginine patch motif is illustrated by two red arginines from each monomer. The arrangement on the left is rotated approximately 90° and magnified on the right to illustrate the environments of these arginines in the different interactions. The images were generated using the PyMOL Molecular Graphics System, Version 1.8.2.1 Schrödinger, LLC. The overall fold of HpSpo0J is similar to that of TtSpo0J, except for a bend in the linker between the N-terminal and HTH domains (Leonard et al., 2004; Chen et al., 2015). It was suggested that the TtSpo0J structure may represent a closed conformation that opens up following DNA binding to the HpSpo0J architecture. How do these structures inform us of higher-order complex assembly, particularly when ParB binds, bridges, and spreads on non-specific DNA adjacent to and away from parS? The simplest model is that the HTH is responsible for both specific and non-specific DNA interactions, which is supported by the observation that a triple substitution in the HTH domain of F SopB impairs both DNA binding activities (Ah-Seng et al., 2009). In this case the HpSpo0J-DNA structure may represent ParB-ParB and ParB-DNA interactions during spreading at any DNA site. The requirement for the N-terminus in higher-order complex formation in vivo is also consistent with this picture. The flexibility of and variation in the cross-monomer interactions and interfaces seen in the HpSpo0J structure make it an attractive model for the ability of ParB to make multiple and flexible interactions during complex assembly and maintenance. However, one recent study suggests that the specific and non-specific DNA binding activities may be distinct (Taylor et al., 2015). The specific and non-specific DNA binding activities of BsSpo0J showed different properties in vitro, including different abilities to protect the HTH domain from proteolysis. The results lead to the proposal that the N-terminus contains a DNA binding region that is distinct from the HTH. These observations may also reflect differences between plasmid and chromosomal ParBs. We must await identification of the protein-DNA contacts at non-specific sites before we can confirm the organization of ParB during higher-order partition complex assembly. The role of ParA in ParB-DNA complexes During partition, ParAs form patterns on the surface of the bacterial nucleoid due to dynamic interactions with ParB bound to parS (Hatano et al., 2007; Ringgaard et al., 2009; Hatano and Niki, 2010; Ah-Seng et al., 2013). The patterning is necessary for the segregation of plasmids and chromosomal domains, and the molecular mechanisms involved are still being defined. ParA is not necessary to form the large ParB-DNA complexes seen in vivo and in vitro, but ParA can influence or modulate these complexes. For example, the behavior of several parA and parB mutants supports a proposal that ParA is necessary to separate pairs or groups of plasmids during segregation (Fung et al., 2001; Ah-Seng et al., 2013). For the ParBs that interact with ParA via N-terminal regions that are adjacent to the flexible ParB-ParB oligomerization interface, one attractive idea is that ParA-ParB interactions at the N-terminus may influence the available conformations for ParB-ParB interactions next door. For example, specific interference with the transverse ParB-ParB interactions in the HpSpo0J structure might favor intramolecular associations over intermolecular ones (Figure 2). Flexibility and order in partition complex assembly Taken together, the biochemistry and structural biology of the N-terminal regions of ParBs imply that the folding and structures are flexible, dynamic, and fluid. Recent experiments using magnetic tweezers support the idea that the complexes are not highly ordered (Taylor et al., 2015). ParB-ParB (dimer-dimer) interactions via the N-terminal domain must contribute to higher-order complex assembly and function. It is attractive to consider that the flexibility of the N-terminus, in folding and conformation, is important for the dynamics and architecture of the large higher-order partition complex in vivo. This conformational flexibility resembles the properties of so-called “intrinsically disordered” proteins and domains whose unstructured properties allow proteins to sample and bind to multiple targets and in multiple ways (Wright and Dyson, 1999; Uversky, 2016). The same region in ParB could be involved, directly or indirectly, in different binding scenarios with different partners, including ParA, and potentially non-specific DNA. Why are flexibility, dynamics, and size important for these partition complexes? ParBs are engaging in multiple and constantly changing interactions during partition. Clustering creates a condensed, organized DNA substrate and provides a high density of ParBs available to ParA. Defining the molecular nature of these interactions continues to be an essential step toward the understanding of these intriguing DNA binding proteins. Author contributions The author confirms being the sole contributor of this work and approved it for publication. Funding This work was supported by Canadian Institutes of Health Research grant 133613. Conflict of interest statement The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. ==== Refs References Ah-Seng Y. Lopez F. Pasta F. Lane D. Bouet J.-Y. (2009 ). Dual role of DNA in regulating ATP hydrolysis by the SopA partition protein . J. Biol. Chem. 284 , 30067 –30075 . 10.1074/jbc.M109.044800 19740757 Ah-Seng Y. Rech J. Lane D. Bouet J. Y. (2013 ). Defining the role of ATP hydrolysis in mitotic segregation of bacterial plasmids . PLoS Genet 9 :e1003956 . 10.1371/journal.pgen.1003956 24367270 Autret S. Nair R. 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==== Front Front MicrobiolFront MicrobiolFront. Microbiol.Frontiers in Microbiology1664-302XFrontiers Media S.A. 10.3389/fmicb.2016.01347MicrobiologyOriginal ResearchMycobacterium tuberculosis Zinc Metalloprotease-1 Assists Mycobacterial Dissemination in Zebrafish Vemula Mani H. 1Medisetti Raghavender 2†Ganji Rakesh 1†Jakkala Kiran 1Sankati Swetha 1Chatti Kiranam 2Banerjee Sharmistha 1*1Department of Biochemistry, School of Life Sciences, University of HyderabadHyderabad, India2Biology Department, Dr. Reddy’s Institute of Life SciencesHyderabad, IndiaEdited by: Leonard Peruski, Centers for Disease Control and Prevention, USA Reviewed by: Mattias Collin, Lund University, Sweden; Yusuf Akhter, Central University of Himachal Pradesh, India *Correspondence: Sharmistha Banerjee, sbsl@uohyd.ac.in†These authors have contributed equally to this work. This article was submitted to Infectious Diseases, a section of the journal Frontiers in Microbiology 29 8 2016 2016 7 134708 5 2016 15 8 2016 Copyright © 2016 Vemula, Medisetti, Ganji, Jakkala, Sankati, Chatti and Banerjee.2016Vemula, Medisetti, Ganji, Jakkala, Sankati, Chatti and BanerjeeThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Zinc metalloprotease-1 (Zmp1) from Mycobacterium tuberculosis (M.tb), the tuberculosis (TB) causing bacillus, is a virulence factor involved in inflammasome inactivation and phagosome maturation arrest. We earlier reported that Zmp1 was secreted under granuloma-like stress conditions, induced Th2 cytokine microenvironment and was highly immunogenic in TB patients as evident from high anti-Zmp1 antibody titers in their sera. In this study, we deciphered a new physiological role of Zmp1 in mycobacterial dissemination. Exogenous treatment of THP-1 cells with 500 nM and 1 μM of recombinant Zmp1 (rZmp1) resulted in necrotic cell death. Apart from inducing secretion of necrotic cytokines, TNFα, IL-6, and IL-1β, it also induced the release of chemotactic chemokines, MCP-1, MIP-1β, and IL-8, suggesting its likely function in cell migration and mycobacterial dissemination. This was confirmed by Gap closure and Boyden chamber assays, where Zmp1 treated CHO or THP-1 cells showed ∼2 fold increased cell migration compared to the untreated cells. Additionally, Zebrafish-M. marinum based host–pathogen model was used to study mycobacterial dissemination in vivo. Td-Tomato labeled M. marinum (TdM. marinum) when injected with rZmp1 showed increased dissemination to tail region from the site of injection as compared to the untreated control fish in a dose-dependent manner. Summing up these observations along with the earlier reports, we propose that Zmp1, a multi-faceted protein, when released by mycobacteria in granuloma, may lead to necrotic cell damage and release of chemotactic chemokines by surrounding infected macrophages, attracting new immune cells, which in turn may lead to fresh cellular infections, thus assisting mycobacterial dissemination. tuberculosisMycobacterium tuberculosisZinc metalloprotease-1zebrafishmycobacterial disseminationDepartment of Biotechnology, Ministry of Science and Technology10.13039/501100001407BT/IN/New Indigo/05/SB/2013, BT/PR3260/BRB/10/967/2011 ==== Body Introduction Tuberculosis (TB) is a highly contagious, chronic, airborne infection caused by bacilli belonging to genus mycobacteria, primarily, Mycobacterium tuberculosis (M.tb) (O’Garra et al., 2013). TB continues to pose a major health challenge worldwide, especially as a co-epidemic with HIV (Global Tuberculosis Report, 2015). India is one of the worst hit countries accounting for 23% of total global TB cases resulting in 2.2 lakhs mortality annually as estimated by World Health Organization (WHO) (Global Tuberculosis Report, 2015). The TB-causing bacilli, M.tb enter the host through aerosol route and are engulfed by alveolar macrophages, polymorphonuclear neutrophils and type 2 pneumocytes by phagocytosis (Smith, 2003). In most of the cases, the infection does not result in disease as the bacillus has evolved evasion strategies to live in balance with the immune response, thus remaining latent for decades (Babalola, 2015). A hallmark of immune reaction to TB bacilli is the formation of granuloma, by which host attempts to contain the infection (Guirado and Schlesinger, 2013). The granuloma is an aggregate of various immune cells, such as, macrophages, dendritic cells, and lymphocytes whose function depends on the cytokine environment generated due to TB infection (Ordway et al., 2006). Some of the infected cells undergo necrosis and create an acellular central zone where TB bacilli persist within granuloma. This necrotic zone eventually disintegrates in certain immunocompromised hosts, triggered by a mechanism still unknown, causing reactivation (Silva Miranda et al., 2012; Guirado and Schlesinger, 2013). A significant aspect of the pathogenesis of virulent mycobacteria, like M.tb, is the ability to modulate cell death pathways where apoptotic cell death is considered bactericidal and necrotic cell death possibly assists bacterial dissemination and transmission (Chen et al., 2008; Gan et al., 2008; Divangahi et al., 2009; Behar et al., 2010). In this regard, several secretory proteins of M.tb have been implicated in either initial establishment of lung infections or extrapulmonary dissemination. To exemplify, ESAT-6 limits macrophage responses by inhibiting signaling from Toll-like receptor-2 (TLR-2) and causes phagosomal membrane lysis, thus helping establishment of infection, while HbhA, a glycoprotein, found on M.tb surface and also in culture filtrates, is not required for initial infection, but has possible role in dissemination to extrapulmonary regions (Pethe et al., 2001; de Jonge et al., 2007; Pathak et al., 2007). Yet other proteins, unique to mycobacteria genus, like PE25/PPE41 protein complex, has been shown to induce necrosis in macrophages and speculated to have a role in dissemination and disease reactivation (Tundup et al., 2014). An interesting group of secreted proteins is extracellular Zinc-metalloproteases. These have been documented to contribute to the virulence of pathogenic bacteria by a variety of mechanisms. Several of these are established exotoxins and virulence factors, such as metalloprotease from V. cholerae O1 serotype (Finkelstein and Hanne, 1982; Hase and Finkelstein, 1993) or enterotoxin from Bacteroides fragilis (Obiso et al., 1995). In local bacterial infections, such as by Staphylococcus or Pseudomonas (keratitis, dermatitis) or by Streptococcus (pneumonia), the secreted metalloproteases cause necrotic or hemorrhagic tissue damage through digestion of structural components of the ground substance, enhancing vascular permeability permitting bacterial dissemination (Miyoshi and Shinoda, 2000). Similarly, clostridial neurotoxins are Zinc-metalloproteases that act by specifically cleaving a synaptic vesicle membrane or the presynaptic plasma membrane protein (Hayashi et al., 1994). So far, there are evidences of three Zinc-metalloproteases from M.tb in the culture filtrate, namely, Rv2869 (Rip), Rv2467 (pepN) and Rv0198c (Zmp1). Rv2869 (Rip), a secretory metalloprotease has been shown to regulate intramembrane proteolysis and proteolytic degradation of anti-sigma substrates like RsdA controlling the SigD mediated transcriptional regulation in mycobacteria during stationary phase and hypoxia (Raman et al., 2004; Calamita et al., 2005) and thereby playing a role in mycobacterial virulence (Makinoshima and Glickman, 2005; Sklar et al., 2010). Rv0198c (Zmp1), supported by deletion mutant studies, was implicated in suppression of inflammasome activation by inhibiting caspase-1 activity and phagosome maturation, leading to decreased pathogen clearance suggesting a key role of Zmp1 during M.tb pathogenicity (Master et al., 2008; Johansen et al., 2011). In a recent study from our lab, we identified and characterized purified Zmp1 as a mycobacterial antigen that is secreted during granuloma-like stress conditions and generated Th2 cytokine microenvironment upon exogenous treatment of PBMCs, which was supported by recording specific and robust humoral response in a large cohort of TB patients (Vemula et al., 2016). Interestingly, the purified Zmp1 protein was earlier shown to cleave synthetically generated neuropeptides (Petrera et al., 2012). With M.tb Zmp1 reported as a virulence factor holding the properties of immunomodulation, high immunogenicity and proteolysis of synthetic neuropeptides, we further extended the study on the other possible roles of Zmp1 in the pathogenesis of M.tb. In this study, we observed that exogenous treatment by high concentration of purified recombinant Zmp1 possibly induced necrotic cell death of THP1 cells, while sub-toxic levels promoted cell migration and release of chemotactic chemokines. In agreement with the reports that necrotic cell death during TB infection is an imperative precondition for bacterial dissemination, we further established, using zebrafish infected with fluorescently labeled Mycobacterium marinum that exogenous (secretory) Zmp1 helped in dissemination of mycobacteria. With this study, along with the earlier observations from our laboratory and others, M.tb Zmp1 has emerged as a multi-faceted protein which can be further explored as either a vaccine candidate, biomarker or anti-mycobacterial target. Materials and Methods The cell lines used were Chinese Hamster Ovary (CHO) cell line and THP-1 Monocyte leukemia cell line (Cat#TIB-202, ATCC). Bacterial strains used were M.tb H37Rv and Mycobacterium marinum. Cell culture media and fetal bovine serum were purchased from Himedia, India. Casein was obtained from Sigma-Aldrich, USA. BD OptEIATM capture ELISA sets and eBioscience Human cytokine ELISA Ready-Set-Go kits were used. MTT, DMSO, and other chemicals were purchased from HiMedia, India. All plasticware and glassware were obtained from Tarsons, India; Eppendorf, Germany, and Corning, USA. All procedures for zebrafish experimentation were as per guidelines published by the National Institutes of Health USA for care and use of zebrafish (Wilson et al., 2009). Zebrafish husbandry and all experimental procedures were part of a protocol approved by an Institutional Biosafety Committee (IBSC Approval No. DRILS/IBSC/2013/06). Indigenous wildtype male and female adult zebrafish were used for this study (obtained from Vikrant Aquaculture, Mumbai, India and further generations bred in our laboratory). Fish were maintained in a custom-built recirculation system with polysulphone housing tanks containing purified water (Millipore ELIX system grade) with 200 mg/L sea salt at 28°C under a 14:10 h light and dark cycle (Westerfield, 2000). Fish were fed three times daily with live hatched brine shrimp and dry food. Zebrafish were spawned in a Mini Mass Embryo Production System (MEPS® – Aquatic Habitats). Fertilized eggs were collected and maintained in either tank water or in embryo medium. Larval stages were raised in static tanks and fed with paramecia or spirulina from 5 dpf (day post-fertilization) to 15 dpf and then weaned onto Artemia as food. Maintenance of Cell Lines The THP-1 cell lines were maintained in RPMI 1640 media supplemented with 10% FBS (South American origin, Gibco) and incubated at 37°C with 5% CO2. The media was changed when the confluency of the cells reached 90%. The CHO cell lines were maintained in RPMI media supplemented with 10% FBS and incubated at 37°C with 5% CO2. The media was changed when the cells reached 90% confluency, the cells were trypsinized with the Trypsin-EDTA (Sigma) and washed with phosphate buffered saline pH 7.4 (PBS) (Ganji et al., 2016b). Growth and Maintenance of Mycobacterial Culture Mycobacterium marinum transformed with pTEC27 harboring Td-Tomato (TdM. marinum) was plated on 7H10 agar media (Hi-media, India) supplemented with 10% Oleic acid, Albumin, Dextrose and Catalase (OADC, Hi-Media, India) in presence of Hygromycin (50 μg/mL). The plate was incubated at 30°C until colonies appeared. The colonies were picked into the 7H9 broth supplemented with 10% OADC, Hygromycin (50 μg/mL) and the broth culture was incubated at 30°C at 180 rpm until the OD600 nm reached 0.8–1. The culture was checked for any contamination and single suspension of bacteria was obtained as described earlier (Ganji et al., 2016a). Growth and Maintenance of Bacterial Cultures pET28a (+) plasmid carrying zmp1 (Rv0198c) (Vemula et al., 2016) or Rv0047c genes were transformed into Escherichia coli BL21-DE3 cells. A single bacterial colony carrying plasmid were inoculated into LB medium containing kanamycin (34 μg/mL) and were incubated overnight at 37°C with vigorous shaking (180 rpm). The next day, secondary cultures were set up in LB media with 2% inoculum of overnight grown primary cultures and were incubated at 37°C with vigorous shaking (180 rpm). When OD at 600 nm reached to 0.6, the cultures were induced with 0.5 mM β-D-Isopropyl thiogalactoside (IPTG) kept for 4 h at 37°C. The bacterial pellets were harvested and rZmp1 and rRv0047c proteins were purified using similar methodology as described previously (Vemula et al., 2016). The proteins were made endotoxin-free using polymyxin B agarose beads (Sigma Aldrich). The residual endotoxin levels were quantified using PierceTM LAL Chromogenic Endotoxin Quantitation Kit. The residual endotoxin (LPS) levels in the purified rZmp1 and rRv0047c were observed to be 0.046 ± 0.001 EU/mL and 0.043 ± 0.001 EU/mL, respectively. The proteolytic activity of rZmp1 using casein as substrate was performed as described earlier (Vemula et al., 2016). Heat-denaturation of rZmp1 or rRv0047c was performed by heating the purified proteins at 100°C for 15 min followed by snap-freezing on ice. Native and heat denatured rZmp1 were later checked for caseinase activity (Supplementary Figure S1A). MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide) Assay 0.2 × 105 cells (THP-1 and CHO cell lines) per well were seeded in 200 μL media into a 96 well plate and incubated overnight at 37°C and 5% CO2. The cells were incubated exogenously with varying concentrations of purified rZmp1 protein ranging from 5 nM to 2 μM for 24 h. Similarly, THP-1 cells were exogenously treated with rRv0047c at concentrations 50 and 100 nM. The cell viability was assessed using MTT assay (Supplementary Figures S1B and S2). Twenty microliter of 5 mg/mL MTT solution was added to each well and incubated the plate at 37°C, 5% CO2 for 4 h. The plate was centrifuged at 3000 rpm for 5 min and excess media (150 μL) was removed. The formazan crystals were dissolved in 150 μL of DMSO. Absorbance was measured at 540 nm using multi-well plate reader. Gap Closure Assay Chinese Hamster Ovary cells were allowed to reach >90% confluence in 35 mm cell culture dish. For the assay, prepared 10 ml of base media with 50 nM of rZmp1 protein and filter sterilized. Meanwhile, a line was drawn with a marker on the bottom of the dish. Using a sterile tip, three separate wounds were scratched through the cells moving perpendicular to the line drawn before. Cells were rinsed gently with PBS and replaced with 1.5 mL of media either containing rZmp1 protein or only buffer (control set). Pictures were taken using phase contrast at 10× magnification at 0, 16, 24, and 48 h. The same was repeated with the control set without rZmp1. The gap closure was measured in both control set and rZmp1 containing set using ImageJ software (Liang et al., 2007). Boyden Chamber Assay 24-well tissue culture plate containing cell culture inserts with polycarbonate membrane of 8 μm pore size were taken. 200 μL of media with 0.4 × 105 cells without rZmp1 was added to upper chamber of cell insert and 750 μL of media without cells containing only 50 nM of endotoxin-free rZmp1 protein or rRv0047c protein was added to lower chamber. Corresponding heat-denatured protein controls were also used. The plate was then placed at 37°C with 5% CO2 for 24 h. The cells in the upper and lower chamber were collected separately by centrifuging at 200–300 g for 5 min. The cell numbers were then determined by Trypan blue exclusion assay using hemocytometer. Capture ELISA Protocol Capture ELISA for cytokine measurements was performed as per the manufacturer’s instructions. Briefly, 50 μL of diluted capture antibody was added to the wells of ELISA/RIA compatible 96-well plate and kept overnight at 4°C. Followed by three PBS-T (PBS containing 0.05% Tween-20) and one PBS washes. The coated plate was blocked using 3% bovine serum albumin in PBS at 37°C for 1 h. After blocking, washes were performed with PBS-T and PBS as mentioned before. Fifty microliter sample was added and incubated at 37°C for 2 h which was followed by five PBS-T and one PBS washes. Then 50 μL of diluted detection antibody along with streptavidin-HRP conjugate was added and incubated at 37°C for 1 h, followed by 7 PBS-T and one PBS washes. 3,3′,5,5′-Tetramethylbenzidine (TMB) was used as substrate according to manufacturer’s directions. Dispensed 100 μl into each well in dark and incubated at RT (5–30 min) for color development. The reaction was stopped using 100 μl of 2N H2SO4. Read the optical density (OD) for each well with a microplate reader set to 450 nm. Lactate Dehydrogenase (LDH) Assay The lactate dehydrogenase activity was determined by measuring the decrease in the absorbance at 340 nm resulting from the oxidation of NADH. The reaction mix consisted of 0.66 mM NADH and 3 mM Sodium pyruvate in 0.2M Tris-HCl, pH 7.3. To 100 μL of reaction mix 20 μL of culture supernatant of THP-1 cells treated with or without rZmp1 was added and monitored the change in NADH levels at 340 nm. Propidium Iodide Staining for Flow Cytometry THP-1 cells were treated with or without rZmp1 (500 nM and 1 μM) for 24 h. After incubation, the media was removed, added prewarmed RPMI complete media containing 40 μg/mL of propidium iodide (PI) for 15 min at room temperature (RT) in dark. After staining, the cells were washed twice in PBS and then fixed with 3% paraformaldehyde at RT for 20 min. Then the cells were washed twice with PBS followed by RNase (50 μg/mL) treatment at 37°C for 15 min. The cells were then washed with PBS and resuspended in PBS for flow cytometry analyses. TdM. Marinum Dissemination Assay in Zebrafish Fluorescently labeled TdM. marinum was injected at 200 CFU (colony forming unit) with or without rZmp1 [25 to 100 nM native or 100 nM heat-denatured protein (HD-rZmp1)] or non-specific, recombinant mycobacterial protein, rRv0047c (100 nM native or 100 nM heat-denatured protein (HD-rRv0047c)) into yolk sac of 1 dpf (day post fertilization) Zebrafish. Images were taken at the 0 day of injection and on the 5th day after injection of TdM. marinum using fluorescent microscopy. Fishes were scanned for the presence of TdM. marinum in the whole organism and images were taken accordingly. Fluorescence intensities of TdM. marinum in the fish were quantified using ImageJ software. Total fluorescence was calculated by taking count of all the regions in a whole fish (Benard et al., 2012). Dissemination of TdM. marinum toward tail region was calculated by two parameters and plotted: (1) Relative percentage of fish with TdM. marinum disseminated to tail region = (Number of fish with TdM. marinum disseminated to tail region / total number of fish injected with TdM. marinum in the category) × 100; (2) Dissemination to tail region = (Fluorescence intensity in tail region/total fluorescence intensity in whole fish) × 100. Survival of Zebrafish for the rZmp1was evaluated by injecting the fish with rZmp1 (ranging from 25 to 100 nM or 100 nM heat-denatured rZmp1) alone without any TdM. marinum and monitored the fish over the course of 5 days. Graphs and Statistical Analyses SigmaPlot software version 11.0.0.77 (Systat Software, Inc., USA) was used for different statistical analyses. For cytokine data and cell migration data, One-way ANOVA was performed with Holm–Sidak multiple pair-wise comparison method and the threshold for significance was set at p < 0.05. The error bars represent the ± standard deviation (SD) from the mean of at least three independent experiments. For statistical analyses of mycobacterial dissemination data, One-way ANOVA on ranks was performed with Dunn’s method for pair-wise comparison method for comparing more than two groups or Students t-test with Mann–Whitney Rank sum test was performed for comparing two groups. The threshold for significance was set at p < 0.05. They were represented as box plots using SigmaPlot. Within the plots, the upper quartile of the box represents the 75th percentile and the lower quartile for the 25th percentile. The line inside the box represents the median. The whiskers arising from either side of the upper half and the lower half of the box correspond to 1.5 times the interquartile range (IQR) (Benjamin et al., 2013; Vemula et al., 2016). Any datum to the further extreme of the whiskers is termed as outlier. Results Higher Concentrations of Exogenous rZmp1 Treatment Caused Necrotic Cell Death Secreted Zinc metalloproteases across pathogenic bacteria are known to act as toxins causing a range of pathological effects by virtue of their proteolytic activities, quite often causing necrotic tissue damage (Hase and Finkelstein, 1993). To begin with, we checked for the toxicity of the recombinant Zmp1 (rZmp1) on human monocyte leukemia cell line THP-1 and Chinese Hamster Ovary (CHO) cell lines (Figure 1; Supplementary Figure S1B). THP-1 monocytes or CHO cells were treated with varying concentrations of endotoxin-free rZmp1 and assayed for cell death using MTT assay. It was observed that though rZmp1 protein was not toxic at lower concentrations of 50 nM but was toxic at high concentrations at 24 h (Figure 1A). The IC50 calculated for Zmp1 cytotoxicity on THP-1cells was ∼550 nM (Figure 1B). We further observed that the toxicity at higher concentrations was due to necrotic mode of cell death as determined by flow cytometry using Propidium Iodide (PI) staining of the rZmp1 treated cells and Lactate Dehydrogenase (LDH) assay of the supernatants of the rZmp1 treated cells (Figures 2A–C). It was observed that rZmp1 treated cells at both 500 nM and 1 μM concentrations showed increased PI stained population suggesting cell death due to necrotic damage (Figures 2A,B). The 500 nM and 1 μM rZmp1 treated cells showed 66.6 and 75.51% PI positive population, respectively (Figures 2A,B). Upon LDH assay of the culture supernatants of the rZmp1 treated and untreated THP-1 cells, it was observed that culture supernatants of rZmp1 (both 500 nM and 1 μM) treated cells showed increased LDH activity (500 nM rZmp1: 0.548 ± 0.03 Abs340; 1 μM rZmp1: 0.558 ± 0.02 Abs340) as compared to untreated controls (0.388 ± 0.02 Abs340) suggesting the mode of cell death to be necrotic in nature (Figure 2C). We further checked the levels of cytokines, TNFα, IL-6, and IL-1β, which are implicated in necrosis (Kaczmarek et al., 2013; Moriwaki et al., 2015), in the culture supernatants of the THP-1 cells treated with rZmp1 (500 nM and 1 μM) (Figures 2D–F). The levels of cytokines as measured by ELISA were TNFα (Untreated: 54.82 ± 14.75 pg/mL; 1 μM rZmp1 treated: 239.46 ± 35.31 pg/mL; p < 0.05) (Figure 2D), IL-6 (Untreated: 91.93 ± 15.92 pg/mL; 1 μM rZmp1 treated: 1318.47 ± 74.23 pg/mL; p < 0.05) (Figure 2E) and IL-1β (Untreated: 328.19 ± 168.89 pg/mL; 1 μM rZmp1 treated: 2665.31 ± 1366.98 pg/mL; p < 0.05) (Figure 2F), respectively. As anticipated there was significant increase in the secretions of these cytokines by THP-1 into the culture supernatants upon rZmp1 exposure as compared to untreated cells further pointing that exogenous treatment of rZmp1 at higher concentrations, possibly, induced necrosis. FIGURE 1 Exogenous treatment with rZmp1 at higher concentrations caused cell death with IC50 of ∼550 nM in the conditions studied. (A) Bar graph represents the percentage THP-1 cell viability upon exogenous treatment with varying concentrations of rZmp1. (B) Scatter plot represents the percentage THP-1 cell death upon exogenous treatment with varying concentrations of rZmp1. The dotted lines depict the concentration of rZmp1 (∼550 nM) at which there was 50% cell death (IC50). All the experiments were performed more than three times. The error bar represents standard deviation from mean. FIGURE 2 Exogenous treatment of THP-1 with rZmp1 induced necrotic cell death. (A) The dot plots show the propidium iodide staining for the conditions, Untreated unstained, Untreated stained, 500 nM rZmp1 and 1 μM rZmp1 treated cells. The THP-1 cells were either only buffer treated or rZmp1 treated (500 nM and 1 μM) for 24 h. Propidium iodide staining followed by flow cytometry were used to evaluate necrosis of THP-1 cells. (B) The histogram plot depicts the extent of PI staining upon rZmp1 treatment. (C) Bar graphs represent lactate dehydrogenase activity of the culture supernatants of THP-1 cells treated either with buffer or rZmp1. (D–F) Bar graph represents the levels of cytokines, (D) TNFα, (E) IL-6 and (F) IL-1β determined in the culture supernatants of rZmp1 treated cells, using capture ELISA method. All the experiments were performed at the least three times. The error bars represent standard deviation from mean. ∗represents statistical significance with p < 0.05. rZmp1 Promoted Cell Migration in THP-1 Cells and Induced Secretion of Chemotactic Chemokines Necrotic cell death of the M.tb infected cells has been linked to mycobacterial dissemination (Molloy et al., 1994; Lee et al., 2011; Butler et al., 2012; Roca and Ramakrishnan, 2013). With high concentration of rZmp1 causing necrotic cell death, we proceeded to assess for the ability of rZmp1 to promote cell migration. For these assays, a sub-toxic level of rZmp1 concentration was used, so that excessive cell deaths can be avoided. The preliminary experiments were performed on CHO cells using the gap closure assays, which were further supported by Boyden chamber assays using THP-1 cells (Figures 3 and 4). Gap closure assays were performed using untreated or CHO cells treated with 50 nM rZmp1 (Figure 3A). Images were taken at different time points (0, 16, 24, and 48 h) and gap width was calculated and plotted (Figure 3B). Exogenous treatment of rZmp1 resulted in increased gap closure as compared to the untreated cells by 2.05 ± 0.47 folds at 48 hr (Figure 3B). FIGURE 3 rZmp1 treatment helped CHO cell migration as observed by Gap closure assay. (A) Representative microscopic image from experiment 1 showing CHO cell migration as function of time upon 50 nM rZmp1 exogenous treatment. Monolayer of CHO cells were grown in 35 mm cell culture dish. A gap was introduced using a sharp tip and the cells were either only buffer treated (Upper panel) or 50 nM rZmp1 treated (lower panel). The extent of gap closure was monitored microscopically at regular intervals (0, 16, 24, and 48 h). (B) Bar graphs represents the distance of gap closure in μm (micrometers) relative to 0 h calculated using ImageJ software at regular intervals from three independent experiments. The error bars represent the standard deviation from mean. FIGURE 4 Boyden chamber migration assays with rZmp1 treated and untreated THP-1 cells. (A) Representative microscopic image showing THP-1 cells migrated to the lower chamber upon 50 nM rZmp1 treatment. THP-1 cells were seeded into the upper chamber of the Boyden chamber and the lower chamber contains media along with buffer control or 50 nM rZmp1 and incubated for 24 hr. 50 nM rZmp1 containing lower chamber showed more THP-1 cells migrated from upper chamber. (B,C) Bar graph represents the fold change in THP-1 migration in untreated, rZmp1 (50, 100 nM native or 50 nM HD-rZmp1) or rRv0047c (50 nM native or 50 nM HD-rRv0047c) treated cells. The number of cells migrated to the lower chamber were counted using Trypan Blue live cell staining assay and plotted the increase in fold change in THP-1 cells migrated from upper chamber to lower chamber. (D-F) The culture supernatants of rZmp1 (50 and 100 nM) treated cells were assayed for chemokines implicated in cell migration or chemotaxis, (D) MIP-1β, (E) MCP-1 and (F) IL-8 using capture ELISA method. All the experiments were performed at the least three times. The error bars represent standard deviation from mean. ∗represents statistical significance with p < 0.05. The ability of rZmp1 to promote cell migration was further confirmed by Boyden chamber assays using THP-1 monocyte cell line. In this assay, we preloaded the cells in the upper chamber with complete media and the lower chamber with media containing 50 and 100 nM endotoxin-free rZmp1, expecting that if rZmp1 indeed influenced migration of cells, the cells would move from the upper chamber to the lower chamber. It was observed that there was increased migration of THP-1 cells from the upper chamber to the lower chamber containing 50 and 100 nM rZmp1 as compared to control without rZmp1 (Figures 4A,B). The fold changes in the migration of THP-1 cell line in the presence of 50 and 100 nM rZmp1 were 1.55 ± 0.20 and 1.81 ± 0.21 fold, respectively as compared to untreated, exhibiting a dose dependent increase in the cell migration (Figure 4B). To further confirm that this property is specific to rZmp1 and not due to the residual endotoxin present in the recombinant protein preparations, we performed the same experiment with 50 nM heat-denatured rZmp1 (HD-rZmp1) and a randomly selected non-specific recombinant mycobacterial protein, rRv0047c [50 nM native or 50 nM heat-denatured protein (HD-rRv0047c)], which was purified and made endotoxin-free under conditions similar to that of rZmp1 purifications from recombinant protein expressing E. coli. It was observed that the migration of THP-1 cell line was specific to rZmp1 treatments and is not induced by rRv0047c or heat-denatured rZmp1 (Figure 4C). Taken together the gap closure assays and Boyden chamber assays confirmed involvement of rZmp1 in migration of cells. We further checked if rZmp1 treatment of THP-1 cells resulted in release of chemotactic chemokines, such as MCP-1, MIP-1β and IL-8 that are known to recruit monocytes, neutrophils, memory T cells, and dendritic cells to the sites of infection (Taub et al., 1995; Gerszten et al., 1999; Bystry et al., 2001; Deshmane et al., 2009). THP-1 cells upon incubation with rZmp1 at 50 and 100 nM showed increased levels of all the three cytokines compared to the untreated cells (Figures 4D–F). The cytokine levels in the culture supernatants of THP-1 cells untreated or rZmp1 treated were, MCP-1 (Untreated: 57.97 ± 11.2 pg/mL; 50 nM rZmp1: 654.82 ± 118.97 pg/mL; 100 nM rZmp1: 1473.54 ± 443.12 pg/mL; p < 0.05) (Figure 4D), MIP-1β (Untreated: 38.51 ± 15.71 pg/ml; 50 nM rZmp1: 478.28 ± 42.06 pg/mL; 100 nM rZmp1: 548.58 ± 149.59 pg/mL; p < 0.05) (Figure 4E) and IL-8 (Untreated: 78.42 ± 79.08 pg/mL; 50 nM rZmp1: 1495.44 ± 25.88 pg/mL; 100 nM rZmp1: 1626.99 ± 130.99 pg/mL; p < 0.05) (Figure 4F). Summing up the results so far, rZmp1 that induced necrotic cell death at higher concentrations, promoted cell migration and secretion of chemotactic chemokines by THP1 cells upon exogenous treatments at sub-toxic levels. One can thus speculate that upon causing necrosis, M.tb Zmp1 induces secretion of chemotactic chemokines that would assist migration of monocytes and dendritic cells to the necrotic site, causing fresh rounds of infections of these cells and thereby help dissemination of mycobacteria. rZmp1 Helped in Dissemination of Mycobacteria To examine our hypothesis that Zmp1 indeed helped in mycobacterial dissemination, we used Zebrafish as a model organism which was infected with fluorescently labeled M. marinum (TdM. marinum) which harbored pTEC27 plasmid having Td-Tomato gene. Although Zmp1 of M. marinum is 84% similar to M.tb Zmp1, unlike M.tb Zmp1, M. marinum Zmp1 is predicted to be a cytosolic protein (Kapopoulou et al., 2011). Therefore, to simulate the presence of secreted Zmp1 in the vicinity of mycobacteria, M. marinum was either mixed with rZmp1or with buffer alone before injections into Zebrafish. We injected 200 CFU of TdM. marinum either mixed with buffer or with various concentrations of endotoxin-free rZmp1 (25, 50, 100, and 100 nM of heat-denatured rZmp1) into the yolk sac of 1 dpf (days post fertilization) Zebrafish larva (refer materials and methods) (Figure 5A). Initially toxicity of injected rZmp1 protein on Zebrafish was checked and found that rZmp1 was not toxic at any of the concentrations of 25, 50, and 100 nM used (Figure 5B). After injections with TdM. marinum with or without rZmp1, the fishes were monitored for 5 days and images were taken using fluorescence microscope on 0 day and fifth day (Figure 5A). Then we determined the spread of TdM. marinum from the site of injection to the tail region using two parameters (Figure 5C). Firstly, we calculated the percentage of fish in a category with fluorescence in the tail region and secondly, we measured the percentage of fluorescence intensity in tail region with respect to whole fish fluorescence intensity for each fish (Figure 5C). Using these parameters, we observed that there was an increase in dissemination of fluorescently labeled M. marinum to the tail regions from the site of injection in the infected Zebrafish with rZmp1 than in infected Zebrafish without rZmp1 in a dose dependent manner ranging from 25 to 100 nM (Figure 6A). It was observed that 54.84% of control fish showed fluorescent TdM. marinum in tail region (n = 31), while 76.67% of fishes injected with TdM. Marinum + 25 nM rZmp1 (n = 30), 90% of fishes injected with TdM. Marinum + 50 nM rZmp1 (n = 20) and 93.1% of fishes injected with TdM. Marinum + 100 nM rZmp1 (n = 29) showed fluorescent TdM. marinum in the tail region (Figure 6B). This indicated that rZmp1 helped in the dissemination of TdM. marinum from the site of injection to the tail region in a dose-dependent manner (Figure 6B). In order to confirm that the impact of rZmp1 on mycobacterial dissemination is specific, we heat inactivated rZmp1 by denaturing the protein at 100°C for 15 min. The 100 nM of heat-denatured rZmp1 was mixed with TdM. marinum and injected into Zebrafishes similar to other groups. Only 45.45% of fishes (n = 22) showed fluorescent TdM. marinum in the tail region, which was similar to untreated control group (Figure 6B). FIGURE 5 Schematic representation of M. marinum dissemination assay in Zebrafish. (A) 1 day post fertilization fish were taken for the experiments and injected with 200 CFU of Td-Tomato labeled M. marinum (TdM. marinum) along with either only buffer control or rZmp1 (25, 50, and 100 nM) into the yolk sac. The fish were then incubated in E3 medium for 5 days, followed by visualization of TdM. marinum under fluorescent microscope on 5th day to evaluate for bacterial dissemination to tail region. (B) Table represents the survival of Zebrafish for rZmp1 which was evaluated by injecting the fish with rZmp1 (ranging from 25 to 100 nM) alone without any TdM. marinum and monitored the fish over the course of 5 days. (C) To determine the bacterial dissemination to tail region, firstly each fish was divided into three regions, head, mid-region and tail-region as depicted in the figure. Fish were then scanned under fluorescence microscopy for the presence of TdM. marinum in the whole organism and images were taken accordingly. Fluorescence intensity of TdM. marinum present in the fish was quantified using ImageJ software. Total fluorescence was calculated by taking count of all the regions in a whole fish. Dissemination of TdM. marinum toward tail region was calculated by two parameters and plotted: (1) Relative percentage of fish with TdM. marinum disseminated to tail region and (2) Dissemination to tail region. FIGURE 6 Zmp1 helped in the dissemination of M. marinum in Zebrafish model. (A) Representative images showing the presence of fluorescently labeled M. marinum (TdM. marinum; Red) in the tail region of the Zebrafish. One day post fertilization Zebrafish were injected with TdM. marinum either with buffer control (Untreated) or rZmp1 (25–100 nM) or heat-denatured rZmp1 (100 nM). The dissemination of TdM. marinum to the tail region was then observed under fluorescence microscope on 5th day post infection (refer materials and methods). (B) Bar graph representing the relative percentage of fish with TdM. marinum disseminated to tail region. (C) The box plot represents the percentage of dissemination of TdM. marinum to the tail region. All the experiments were performed more than three times. n represents the number of fish used per category. ∗represent statistical significance with p < 0.05. We then measured the fluorescence intensity of TdM. marinum per fish and calculated the ratio of fluorescence intensity in the tail region with respect to the whole fish intensities (Figure 5C). With this, we observed that the control group infected with TdM. marinum without rZmp1 (Median: 2.66%; IQR: 0-16.55) showed significantly lesser percentage of fluorescence intensity in tail region compared to Zebrafish category infected with TdM. marinum+100nM rZmp1 (Median: 23.91%; IQR: 12.1 – 30.59) further confirming that rZmp1 helped in the enhanced dissemination of TdM. marinum from the site of injection to the tail region (Figure 6C). Further, Zebrafish group injected with TdM. Marinum + 100 nM heat-denatured rZmp1 (Median: 0%, IQR: 0 – 19.31) showed dissemination similar to the rZmp1 untreated control group (Median: 2.66%; IQR: 0-16.55) (Figure 6C). The assays were also performed using rRv0047c (100 nM native or 100 nM heat-denatured), which was also used in Boyden chamber assay as a negative control. Dissemination of TdM. marinum in rRv0047 (n = 21) injected zebrafish groups were similar to that of control group (n = 17) as assessed by calculating the ratio of fluorescence intensity in the tail region with respect to the whole fish intensities (Supplementary Figure S3). These analyses confirmed the role of Zmp1 in mycobacterial dissemination in Zebrafish model. Discussion With this study we added a new physiological function to the multi-faceted M.tb Zmp1. We observed that, exogenous treatment of macrophages with high concentrations resulted in necrotic cell death, at the same time, sub-toxic levels stimulated cells to migrate and release chemotactic chemokines. Together, these properties perhaps helped rZmp1 facilitating dissemination of mycobacteria, as observed in zebrafish infected with fluorescence labeled M. marinum. Previous reports involving Zmp1 deletion mutants of M.tb advocated that Zmp1 is implicated in suppression of inflammasome activation. In these studies it was observed that deletion of Zmp1 resulted in improved pro-inflammatory response and antigen presentation (Master et al., 2008). Complementing these inferences, we also observed that exogenous treatment of PBMCs with rZmp1 biased the immune response toward Th2 (Vemula et al., 2016). It is well established that when Th2 wing of immune response gets activated it tends to suppress the pro-inflammatory Th1 response. In this study, we observed that rZmp1 treatment induced secretions of cytokines TNFα, IL-6, and IL-1β (Figures 2D–F) which are well-documented to be associated in inducing necrotic cell death (Vanden Berghe et al., 2006; Kaczmarek et al., 2013; Moriwaki et al., 2015). In our conditions, we observed that rZmp1 exogenous treatment of THP-1 cells, induced a pro-inflammatory response showing necrotic cell damage. A possible explanation may be that in our earlier experiments (Vemula et al., 2016), we had observed that rZmp1 treatment of PBMCs and THP-1 cells resulted in release of high titers of IL-4, IL-6, and IL-1β, along with high TNFα. Concurrent high levels of TNFα with IL-4 increases the toxic potentiality of TNFα, causing necrotic tissue damage. This may result in liquefaction of TB granuloma, hence dissemination and relapse (Fenhalls et al., 2000; Rook, 2007). Also, TNFα at higher levels, in an autocrine or paracrine mode induces necrosis through RIP1/RIP3 kinase pathway (Roca and Ramakrishnan, 2013). In general, necrosis of the M.tb infected cells has been associated with the dissemination of bacilli and further infection of fresh cells. Careful evaluations with complementary experiments are further required to understand the exact mechanism of necrotic death caused by rZmp1. Zinc containing metalloproteases are widely distributed among prokaryotes and eukaryotes. They are known to play a critical role in bacterial pathogenesis and in various physiological processes such as mammalian cells adhesion, migration, extracellular matrix remodeling and cell–cell interactions (Kearns et al., 2002). For example, Zmp1 protein of Clostridium difficile has been shown to degrade the fibrin network of fibroblasts and thus help in bacterial infection and dissemination (Cafardi et al., 2013). Pap6 of Vibrio harveyi and Hemagglutinin/protease from Vibrio cholerae have been shown to digest various ECM components such as fibronectin, collagen and gelatin (Booth et al., 1983; Finkelstein et al., 1983; Teo et al., 2003). Digestion of fibronectin network by proteases results in fibroblasts losing adherence property and thus may lead to cell migration (Cafardi et al., 2013). StcE, a secreted protease of E. coli, expressed during infection, contributes to virulence by affecting crucial neutrophil recruitment, migration and also oxidative burst production (Szabady et al., 2009). SslE, another extracellular zinc metalloprotease of E. coli through its mucinase activity help bacterial translocation through mucin-matrix allowing its penetration through the mucosal surface and reach host epithelium to make a niche inside the host (Nesta et al., 2014; Valeri et al., 2015). Based on the evidences that Zinc metalloproteases help in cell adhesion and migration, we sought to check the effect of rZmp1 on the cell migration. We employed both Gap closure assays and Boyden chamber assays on CHO cells and THP-1 cells, respectively and observed that rZmp1 treatments showed improved cell migration compared to control treatments (Figures 3 and 4). Though Gap closure assays using CHO cells confirmed the effect of rZmp1 on migration, Boyden chamber assays using THP-1 cells, which creates a concentration gradient between upper and lower chamber, could be inferred in the context of either cell migration and/or chemotaxis (Chen, 2005; Liang et al., 2007). We also learnt that rZmp1 treatment induced secretion of chemotactic chemokines, MCP-1, MIP-1β and IL-8 in the extracellular milieu (Figures 4D–F). Hypothesizing that together with induction of necrosis, release of chemotactic chemokines and induction of cell migration, Zmp1 may influence dissemination of mycobacteria from the site of infection, we used Zebrafish to confirm the same. Zebrafish-M. marinum as a host-pathogen model has been used extensively for understanding tuberculosis pathogenesis, primarily due to the ease of genetic tractability and the visible transparency of Zebrafish larva during the early developmental stages (Ramakrishnan, 2013). Zebrafish, as a model, has been, so far successfully used to study the dissemination of various pathogens like Mycobacterium, Salmonella, Burkholderia, Staphylococcus, Shigella, and Candida (Torraca et al., 2014). M. marinum, the close relative of M.tb, can infect Zebrafish and is known to cause formation of granuloma structures similar to M.tb granuloma in humans. It can also cause latent infections in adult Zebrafish that can be reactivated upon immunosuppressive treatment (Ramakrishnan, 2013). The model has provided several insights into the understanding of tuberculosis infections. For example, understanding the role of granuloma in pathogenesis (Swaim et al., 2006; Davis and Ramakrishnan, 2009; Ramakrishnan, 2013), role of bacterial efflux pumps in drug resistance (Adams et al., 2011), role of LTA4H in mediating inflammation against mycobacteria (Tobin et al., 2010), induction of necrotic death of infected cells mediated by TNFα (Roca and Ramakrishnan, 2013) and to study the manipulation of macrophage recruitment by mycobacteria (Cambier et al., 2014). Several therapeutic strategies against tuberculosis were employed or underway based on the insights from Zebrafish model (Torraca et al., 2014). In the current study, we used Zebrafish model to elucidate the role of rZmp1 in the dissemination of M. marinum and we have observed that upon injection of TdM. marinum mixed with rZmp1 resulted in dissemination of TdM. marinum to the tail region compared to the TdM. marinum injected alone or with heat-denatured rZmp1 (Figures 6A–C). Based on our quantitative analyses, we could infer that the presence of rZmp1 not only helped dissemination of TdM. marinum to the tail region per fish but also the number of fishes showing increased dissemination to the tail region were high (Figure 6C). Overall, the observations confirmed the possible role of rZmp1 in dissemination of M. marinum in Zebrafish. We had earlier shown that Zmp1 is secreted by the virulent M.tb strain H37Rv in granuloma-like stress conditions (Vemula et al., 2016). With the present observations on rZmp1 inducing necrosis and the release of chemotactic chemokines from macrophages, we propose the following hypothesis that Zmp1 when released by mycobacteria in granuloma, may lead to both necrotic damage of the cells and release of chemotactic factors from surrounding infected macrophages. This would attract the uninfected immune cells like monocytes, dendritic cells and lymphocytes toward the site of necrosis. These cells may then get freshly infected by M.tb released from necrotic cells, thus assisting mycobacterial dissemination. The study has added new dimension to the understanding of molecular basis of mycobacterial pathogenesis. One may further explore how the concentration gradient of Zmp1 is decisive in necrosis on one hand and also lead a possible systemic dissemination on the other or does secretion of Zmp1 into acellular milieu of granuloma causes granuloma dissolution owing to proteolytic activity of Zmp1 and hence may have possible role in reactivation. In-depth studies in these lines would help in designing new anti-mycobacterial strategies to confront the emerging problem of drug resistance in M.tb. Author Contributions Conceived and designed the experiments: MHV, RG, RM, SB, and KC. Performed the experiments: MHV, RG, RM, SS, and KJ. Analyzed the data: MHV, RG, RM, SB, and KC. Contributed reagents/materials/analysis tools: SB and KC. Contributed to the writing of the manuscript: MHV, RG, RM, SB, and KC. We declare that all the authors have approved the article for submission, its contents, order of authorship. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We thank for financial assistance BT/IN/New Indigo/05/SB/2013 and BT/PR3260/BRB/10/967/2011 from DBT, India to SB. MHV, RG and KJ are funded by CSIR. Infrastructure support by DBT-CREBB, DST-FIST to the Department of Biochemistry and School of Life Sciences, UH is acknowledged. We thank Dr. Lalita Ramakrishnan and Dr. Deepak Saini for pTEC27 plasmid. Supplementary Material The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb.2016.01347 FIGURE S1 Caseinase activity and cytotoxicity on CHO cells using purified, endotoxin-free rZmp1. (A) Purified rZmp1 protein or heat-denatured rZmp1 (+ represents ∼ 4.8 μg and HD represents heat-denatured rZmp1) was incubated with casein (+ represents 10 μg) at 37°C in 100 mM Tris pH8.0. Small black arrowheads show the Casein (lane 2) and cleaved product of Casein (lanes 3 and 4). (B) Bar graph represents the cytotoxicity of CHO cells exogenously treated with endotoxin-free rZmp1 as determined using MTT assay. Click here for additional data file. Click here for additional data file. FIGURE S2 Exogenous treatment of THP-1 with rRv0047c does not cause cytotoxicity. Bar graph represents the cytotoxicity of THP-1 cells exogenously treated with endotoxin-free rRv0047c [50, 100 nM or Heat-denatured (HD)] as determined using MTT assay. rRv0047c did not show any cytotoxicity on THP-1 cells at the above mentioned concentrations. These concentrations were used for Boyden chamber assays. All the experiments were performed more than three times. Click here for additional data file. Click here for additional data file. FIGURE S3 Dissemination of M. marinum mixed with non-specific protein, rRv0047c, or residual concentrations of LPS in Zebrafish. The box plot represents the percentage of dissemination of TdM. marinum to the tail region. One day post fertilization Zebrafish were injected with TdM. marinum either with buffer control (Untreated) or non-specific rRv0047c (100 nM or 100 nM HD-rRv0047c) or LPS (1.25 pg). The dissemination of TdM. marinum to the tail region was then observed under fluorescence microscope on 5th day post infection and fluorescence intensities were calculated using ImageJ software (refer materials and methods). All the experiments were performed more than three times. ‘n’ represents the number of fish used per category. Click here for additional data file. Click here for additional data file. ==== Refs References Adams K. N. Takaki K. Connolly L. E. Wiedenhoft H. Winglee K. Humbert O. (2011 ). Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux mechanism. Cell 145 39 –53 . 10.1016/j.cell.2011.02.022 21376383 Babalola M. O. (2015 ). 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==== Front Front PsycholFront PsycholFront. Psychol.Frontiers in Psychology1664-1078Frontiers Media S.A. 10.3389/fpsyg.2016.01284PsychologyOriginal ResearchSalience in Second Language Acquisition: Physical Form, Learner Attention, and Instructional Focus Cintrón-Valentín Myrna C. 1*Ellis Nick C. 1231Department of Psychology, University of Michigan, Ann ArborMI, USA2Department of Linguistics, University of Michigan, Ann ArborMI, USA3English Language Institute, University of Michigan, Ann ArborMI, USAEdited by: Adriana Hanulikova, University of Freiburg, Germany Reviewed by: Patrick Rebuschat, Lancaster University, UK; Karin Madlener, University of Basel, Switzerland *Correspondence: Myrna C. Cintrón-Valentín, mcintron@umich.eduThis article was submitted to Language Sciences, a section of the journal Frontiers in Psychology 29 8 2016 2016 7 128429 2 2016 11 8 2016 Copyright © 2016 Cintrón-Valentín and Ellis.2016Cintrón-Valentín and EllisThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.We consider the role of physical form, prior experience, and form focused instruction (FFI) in adult language learning. (1) When presented with competing cues to interpretation, learners are more likely to attend to physically more salient cues in the input. (2) Learned attention is an associative learning phenomenon where prior-learned cues block those that are experienced later. (3) The low salience of morphosyntactic cues can be overcome by FFI, which leads learners to attend cues which might otherwise be ignored. Experiment 1 used eye-tracking to investigate how language background influences learners’ attention to morphological cues, as well as the attentional processes whereby different types of FFI overcome low cue salience, learned attention and blocking. Chinese native speakers (no L1 verb-tense morphology) viewed Latin utterances combining lexical and morphological cues to temporality under control conditions (CCs) and three types of explicit FFI: verb grammar instruction (VG), verb salience with textual enhancement (VS), and verb pretraining (VP), and their use of these cues was assessed in a subsequent comprehension test. CC participants were significantly more sensitive to the adverbs than verb morphology. Instructed participants showed greater sensitivity to the verbs. These results reveal attentional processes whereby learners’ prior linguistic experience can shape their attention toward cues in the input, and whereby FFI helps learners overcome the long-term blocking of verb-tense morphology. Experiment 2 examined the role of modality of input presentation – aural or visual – in L1 English learners’ attentional focus on morphological cues and the effectiveness of different FFI manipulations. CC participants showed greater sensitivity toward the adverb cue. FFI was effective in increasing attention to verb-tense morphology, however, the processing of morphological cues was considerably more difficult under aural presentation. From visual exposure, the FFI conditions were broadly equivalent at tuning attention to the morphology, although VP resulted in balanced attention to both cues. The effectiveness of morphological salience-raising varied across modality: VS was effective under visual exposure, but not under aural exposure. From aural exposure, only VG was effective. These results demonstrate how salience in physical form, learner attention, and instructional focus all variously affect the success of L2 acquisition. second language acquisitionmorphologytenselearned attentionfocus on formgrammar instructionform-focused instructionperceptual linguistic salience ==== Body Introduction Psychological Aspects of Salience Psychological research uses the term salience to refer to the property of a stimulus to stand out from the rest. Salient items or features are more likely to be perceived, to be attended to, and are more likely to enter into subsequent cognitive processing and learning. Salience can be independently determined by physics and the environment, and by our knowledge of the world. It is useful to think of three aspects of salience, one relating to psychophysics, the other two to what we have learned. (1) The physical world, our embodiment, and our sensory systems come together to cause certain sensations to be more intense (louder, brighter, heavier, etc.) than others. (2) As we experience the world, we learn from it, and our resultant knowledge values some associations higher than others. We know that some stimulus cues have affordances: they are associated with outcomes or possibilities that are important to us, while others are negligible (Gibson, 1977; James, 1890a, chap. 11). (3) We also have expectations about what is going to happen next in known contexts, we are surprised when our expectations are violated, and we pay more attention as a result. Each of the three phenomena is explained in more detail below. Psychophysical Salience Loud noises, bright lights, and moving stimuli capture our attention. Salience arises in sensory data from contrasts between items and their context. Stimuli with unique features compared to their neighbors (e.g., Os in a field of Ts, a red poppy in a field of yellow) “pop out” from the scene but in a shared feature context will not (Os among Qs; Treisman and Gelade, 1980). These are aspects of bottom-up processing (Shiffrin and Schneider, 1977). Salient Associations Attention can also be driven by top–down, memory dependent, expectation-driven processing. Emotional, cognitive, and motivational factors affect the salience of stimuli. These associations make a stimulus cue “dear.” A loved one stands out from the crowd, as does a stimulus with weighty associations ($500000.0 versus $0.000005, however, similar the amount of pixels, characters, or ink in their sensation). The units of perception are influenced by prior association: “The chief cerebral conditions of perception are the paths of association irradiating from the sense-impression, which may have been already formed” (James, 1890b, p. 82). Psychological salience is experience-dependent: hotdog, sushi, and mean different things to people of different cultural and linguistic experience. This is why, contra sensation, the units of perception cannot be measured in physical terms. They are subjective. Hence George Miller’s definition of the units of short-term memory as “chunks”: “We are dealing here with a process of organizing or grouping the input into familiar units or chunks, and a great deal of learning has gone into the formation of these familiar units” (Miller, 1956, p. 91). Context and Surprisal The evolutionary role of cognition is to predict what is going to happen next, given that anticipation affords survival value. We find structure in time (Elman, 1990). The brain is a prediction machine (Clark, 2013). One consequence is that it is surprisal – when prediction goes wrong – that maximally drives learning from a single trial. Otherwise, the regularities of the usual course of our experiences sum little by little, trial after trial, to drive our expectations. Cognition is probabilistic, its expectations a conspiracy tuned from statistical learning over our experiences (Ellis, 2002). Salience and Learning Rescorla and Wagner (1972) presented a formal model of conditioning which expresses the capacity of any cue [conditioned stimulus (CS), for example a bell in Pavlovian conditioning] to become associated with an outcome [unconditioned stimulus (US), for example food in Pavlovian conditioning] on any given experience of their pairing. This formula summarized over 80 years of research in associative learning, and it elegantly encapsulates the three factors of psychophysical salience, psychological salience, and surprisal. The role of US surprise and of CS and US salience in the process of conditioning can be summarized as follows: dV=ab(L−V). The associative strength of the US to the CS is referred to by the letter V and the change in this strength which occurs on each trial of conditioning is called dV. On the right hand side, a is the salience of the US, b is the salience of the CS, and L is the amount of processing given to a completely unpredicted US. Thus both the salience of the cue (a) and the psychological importance of the outcome (b) are essential factors in any associative learning. As for (L–V), the more a CS is associated with a US, the less additional association the US can induce. As Beckett (1954) put it: “habit is a great deadener.” Alternatively, with novel associations where V is close to zero, there is much surprisal, and consequently much learning: first impressions, first love, first time... This is arguably the most influential formula in the history of learning theory. Physical salience, psychological salience, and surprisal interactively affect what we learn from our experiences of the world. Salience in Second Language Acquisition (SLA) Naturalistic second language (L2) learners tend to focus more in their language processing upon open-class words (nouns, verbs, adjectives, and adverbs) than on grammatical cues. Their language attainment often stabilizes at a “Basic Variety” of interlanguage that predominantly comprises open-class words; closed-class items—in particular, grammatical morphemes and prepositions—are rare if present at all (Noyau et al., 1995). Although naturalistic second language learners are surrounded by language input, the available target language, not all of it becomes intake, that subset of input that actually gets in and which the learner utilizes in some way (Corder, 1967). A classic case study is that of the naturalistic language learner, Wes, who was described as being very fluent, with high levels of strategic competence, but low levels of grammatical accuracy: “using 90% correct in obligatory contexts as the criterion for acquisition, none of the grammatical morphemes counted has changed from unacquired to acquired status over a 5 years period” (Schmidt, 1984, p. 5). Although the Basic Variety is sufficient for everyday communicative purposes, grammatical morphemes and closed-class words tend not to be put to full use (e.g., Van Patten, 1996, 2006; Clahsen and Felser, 2006). Many untutored L2 learners initially make temporal references mostly by use of temporal adverbs, prepositional phrases, serialization, and calendric reference, with the grammatical expression of tense and aspect emerging only slowly thereafter, if at all (Meisel, 1987; Bardovi-Harlig, 1992, 2000; Noyau et al., 1995). Psychophysical Salience One factor determining the learning of cues is psychophysical salience: prepositional phrases, temporal adverbs, and lexical linguistic cues are salient and stressed in the speech stream. Verb inflections are not. In his landmark study of first language acquisition, Brown (1973, p. 343) breaks down the measurement of perceptual salience, or “clarity of acoustical marking,” into “such variables as amount of phonetic substance, stress level, usual serial position in a sentence, and so on” Brown (1973, p. 463). Many grammatical form-function relationships in English, like grammatical particles and inflections such as the third person singular -s, are of low salience in the language stream. This is a result of the well-documented effect of frequency and automatization in the evolution of language. The basic principles of automatization that apply to all kinds of motor skills (like playing a sport or a musical instrument) are that through repetition, sequences of units that were previously independent come to be processed as a single unit or chunk (Ellis, 1996). The more frequently speakers use a form, the more they abbreviate it: this is a law-like relationship across languages. Zipf (1949) summarized this in the principle of least effort – speakers want to minimize articulatory effort so they tend to choose the most frequent words, and the more they use them, automatization of production causes their shortening. Frequently used words become shorter with use. Grammatical functors are the most frequent elements of a language, thus they lose their emphasis and tend to become abbreviated and phonologically fused with surrounding material (Bybee, 2000; Jurafsky et al., 2001; Zuraw, 2003). Thus grammatical function words and bound inflections tend to be short and low in stress, with the result that these cues are difficult to perceive. In a corpus study by Cutler and Carter (1987), 86% of strong syllables occurred in open class words and only 14% in closed-class words; for weak syllables, 72% occurred in closed-class words and 28% in open-class words. When grammatical function words (by, for, no, you, etc.) are clipped out of connected speech and presented in isolation at levels where their open-class equivalents (buy, four, know, ewe, etc.) are perceived 90–100% correctly, adult native speakers can recognize them only 40–50% of the time (Herron and Bates, 1997). Clitics, accent-less words or particles that depend accentually on an adjacent accented word and form a prosodic unit together with it, are the extreme examples of this: the /s/ of ‘he’s’, /l/ of ‘I’ll’, and /v/ of ‘I’ve’ can never be pronounced in isolation. In sum, grammatical functors are difficult to perceive from bottom-up auditory evidence alone. Fluent language processors can perceive these elements in continuous speech because their language knowledge provides top–down support. But this is exactly the knowledge that learners lack. Thus the low psychophysical salience of grammatical functors contributes to L2 learners’ difficulty in learning them (Goldschneider and DeKeyser, 2001; Ellis, 2006b). Salience as Modulated by Modality Spoken and written language are very different media, with spoken language being fleeting while writing provides more permanent visual substance on the page, allowing the reader to attend linguistic form at their discretion. Attention to language form may therefore pose different challenges in written and spoken modalities. VanPatten (1990) showed that L2 learners of Spanish have difficulty simultaneously attending to meaning and form of aural input. He had them process spoken Spanish passages for meaning while simultaneously monitoring the input for either lexical content words like inflacíon or for grammatico-morphological forms like the definite article la or the verb morpheme –n. Monitoring grammatico-morphological forms negatively affected comprehension, whereas attention to lexical items did not. Wong (2001) replicated this study while also adding conditions in the written modality. She showed that comprehension was worse from aural language than from written language. Furthermore, while the results from the aural conditions replicated the patterns found by VanPatten, the number of idea units recalled by readers who had to pay attention to the definite article in the written input was not significantly less than those who read the passage for content only or for those who had to attend to the lexical item inflacíon. Thus modality can differentially affect the salience of forms and their input processing: written language can make grammatical forms more salient and more easily processed. Learned Attention In addition to psychophysical factors, there are attentional factors which affect the salience of grammatical functors. The first relates to their redundancy. Grammatical morphemes often appear in redundant contexts where their interpretation is not essential for correct interpretation of the sentence (Terrell, 1991; Van Patten, 1996; Schmidt, 2001). Tense markers often appear in contexts where other cues have already established the temporal reference (e.g., “yesterday he walked”), plural markers are accompanied by quantifiers or numerals (“27 cats”), etc. Hence their neglect does not result in communicative breakdown, they carry little psychological importance of the outcome (term b in the Rescorla-Wagner equation), and the Basic Variety satisfices (Simon, 1957) for everyday communicative purposes. Still again, there are attentional biases that result from L2 learners’ history of learning – from their knowledge of a prior language. Ellis (2006a,b) attributes L2 difficulties in acquiring inflectional morphology to an effect of learned attention known as blocking (Kamin, 1969; Mackintosh, 1975; Kruschke and Blair, 2000; Kruschke, 2006). Blocking is an associative learning phenomenon, occurring in animals and humans alike, that shifts learners’ attention to input as a result of prior experience (Rescorla and Wagner, 1972; Shanks, 1995; Wills, 2005). Knowing that a particular stimulus is associated with a particular outcome makes it harder to learn that another cue, subsequently paired with that same outcome, is also a good predictor of it. The prior association “blocks” further associations. All languages have lexical and phrasal means of expressing temporality. So anyone with knowledge of any first language is aware that that there are reliable and frequently used lexical cues to temporal reference (words like German gestern, French hier, Spanish ayer, English yesterday). Such are cues to look out for in an L2 because of their frequency, their reliability of interpretation, and their salience. Learned attention theory holds that, once known, such cues block the acquisition of less salient and less reliable verb tense morphology from analysis of redundant utterances such as Yesterday I walked. The Input Processing (IP) theory of SLA (Van Patten, 1996) includes a Lexical Preference Principle: “Learners will process lexical items for meaning before grammatical forms when both encode the same semantic information” (Van Patten, 2006, p. 118), and a Preference for Non-redundancy Principle: “Learners are more likely to process non-redundant meaningful grammatical markers before they process redundant meaningful markers” (Van Patten, 2006, p. 119). Summing up, grammatical functors abound in the input, but, as a result of their low salience, their redundancy, the low contingency of their form-function mappings, and adult acquirers’ learned attentional biases and L1-tuned automatized processing of language, they are simply not implicitly learned by many naturalistic learners whose attentional focus is on communication. Prior Experiments on Learned Attention and Blocking in SLA Ellis and Sagarra (2010, 2011) and Ellis et al. (2014) report a series of experimental investigations of learned attention in SLA involving the learning of a small number of Latin expressions and their English translations. We sketch them in some detail here because they introduce key concepts and because we build on their design in the present study. In Ellis and Sagarra (2011) there were three groups: Adverb Pretraining, Verb Pretraining, and Control. In Phase 1, Adverb Pretraining participants learned two adverbs and their temporal reference – hodie today and heri yesterday; Verb Pretraining participants learned verbs (shown in either first, second, or third person) and their temporal reference – e.g., cogito present or cogitavisti past; the Control group had no such pretraining. During Phase 2, Sentence Exposure, all participants were shown sentences which appropriately combined an adverb and a verb (e.g., heri cogitavi, hodie cogitas, cras cogitabis) and learned whether these sentences referred to the past, the present, or the future. In Phase 3, the Reception test, all combinations of adverb and verb tense marking were presented individually and participants were asked to judge whether each sentence referred to the past, present, or future. The logic of the design was that in Phase 2 every utterance contained two temporal references – an adverb and a verb inflection. If participants paid equal attention to these two cues, then in Phase 3 their judgments should be equally affected by them. If, however, they paid more attention to adverb (/verb) cues, then their judgments would be swayed toward them in Phase 3. The Control Group illustrate the normal state of affairs when learners are exposed to utterance with both cues and learn from their combination. Multiple regression analysis, where the dependent variable was the mean temporal interpretation for each of the Phase 3 strings and the independent variables were the information conveyed by the adverbial and verbal inflection cues showed in standardized ß coefficients, Control Group Time = 0.93 Adverb + 0.17 Verb. The adverb cues far outweighed the verbal inflections in terms of learnability. We believe this is a result of two factors (i) the greater salience of the adverbial cues, and (ii) learned attention to adverbial cues which blocks the acquisition of verbal morphology. The two other groups reacted to the cues in quite different ways – the Adverb pretraining group followed the adverb cue, the Verb pretraining group tended to follow the verb cue: Adverb Group Time = 0.99 Adverb – 0.01 Verb; Verb Group Time = 0.76 Adverb + 0.60 Verb. Pretraining on the verb in non-redundant contexts did allow acquisition of this cue when its processing was task-essential, but still, the adverb predominated. Ellis and Sagarra (2010, Experiment 2) and Ellis and Sagarra (2011, Experiments 2 and 3) also illustrated long-term language transfer effects whereby the nature of learners’ first language (+/- verb tense morphology) biased the acquisition of morphological versus lexical cues to temporal reference in the same subset of Latin. First language speakers of Chinese (no tense morphology) were less able than first language speakers of Spanish or Russian (rich morphology) to acquire inflectional cues from the same language experience where adverbial and verbal cues were equally available, with learned attention to tense morphology being in standardized ß coefficients: Chinese (-0.02) < English (0.17) < Russian (0.22) < Spanish (0.41) (Ellis and Sagarra, 2011, p. 612). These findings suggest that there is a long-term attention to language, a processing bias affecting subsequent cue learning that comes from a lifetime of prior L1 usage. Enhancing Attention to Non-salient Forms: The Role of Form-Focused Instruction Several theories of SLA (e.g., Schmidt, 2001; Ellis, 2005) emphasize the centrality of attention. Schmidt’s (2001) Noticing Hypothesis holds that conscious attention to linguistic forms in the input is an important precondition to learning: “people learn about the things they attend to and do not learn much about the things they do not attend to” (Schmidt, 2001, p. 30). Form focused instruction (FFI) attempts to encourage noticing, drawing learners’ attention to linguistic forms that might otherwise be ignored (Spada, 1997; Spada and Tomita, 2010; Ellis, 2012). Variants of FFI vary in the degree and manner in which they recruit learner consciousness and in the role of the learner’s metalinguistic awareness of the target forms (Ellis, 1994; Rebuschat, 2015). Explicit instruction traditionally centers upon “some sort of rule being thought about during the learning process” (DeKeyser, 1995). This type of instruction can be deductive, when learners are presented with grammar rule explanation, or inductive, when they are asked to attend to a particular set of forms with the purpose of inferring the rules on their own. This may include explicit metalinguistic feedback, which provides “comments, information, or questions, related to the well-formedness of the learner’s utterance” (Lyster and Ranta, 1997, p. 47). Conversely, through more implicit instruction, learners are expected to infer regularities of form-meaning patterns without awareness. Having laid out the bare contrast like this, we emphasize that there is no simple binary divide between explicit and implicit instruction, that implicit and explicit knowledge interact, and that this is still an area of considerable research inquiry (e.g., Ellis, 1994, 2005; Rebuschat, 2015). Long (1991) and Doughty and Long (2003) describe how a focus on meaning can be improved upon by periodic attention to language as object: during otherwise meaning-focused lessons, learners’ attention is briefly shifted to linguistic code features, in context, to induce noticing. This is known as focus-on-form. Doughty and Williams (1998) give the following examples of focus-on-form techniques, ranging from less to more explicit: input flood, where texts are saturated with L2 models; input elaboration; input enhancement, where learner attention is drawn to the target through visual highlighting or auditory stress; corrective feedback on error, such as recasting; and input processing, where learners are given practice in using L2 rather than L1 cues. Norris and Ortega’s (2000) meta-analysis comparing the outcomes from studies that employed differing levels of explicitness of L2 input demonstrated that FFI instruction results in substantial target-oriented L2 gains, that explicit types of instruction are more effective than implicit types, and that the effectiveness of L2 instruction is durable. More recent meta-analyses of effects of type of instruction by Spada and Tomita (2010) and Goo et al. (2015) likewise report large advantages of explicit instruction in L2 acquisition. However, the studies gathered in these meta-analyses used a wide variety of types of instruction, learner, targeted feature, and method of assessment. There is need to compare FFI methods upon the processing of the same target feature in similar populations of learners. This is one of the aims of our current study, which employs a series of explicit and implicit FFI techniques to contrast and illuminate the processes by which these different methods help learners refocus their attention to non-salient forms in the input. In the following sections we will discuss and operationalize the different types of FFI included in our design: (1) Verb grammar (VG), (2) Textual enhancement (TE), and (3) Verb pretraining (VP) in isolation in task-essential rather than redundant contexts. Verb Grammar (VG) One method that has been widely investigated both in SLA research and practice is that of explicit grammar instruction (EGI) which Terrell (1991, p. 53) defines as “the use of instructional strategies to draw the students’ attention to, or focus on, form and/or structure,” with instruction targeted at increasing the salience of inflections and other commonly ignored features by, first, pointing them out and explaining their structure and, second, providing meaningful input that contains many instances of the same grammatical meaning-form relationship. Ellis (2006) reviews studies of EGI demonstrating that learning through explicit means alone, that is, without the provision of tasks requiring the learner to practice the target features before being tested on their knowledge of these forms, seems to be ineffective (e.g., Ellis, 1993; VanPatten and Oikennon, 1996). We therefore operationalized VG as short metalinguistic description of simple regular tense morphology in Latin which was followed by a sentence exposure phase where leaners were presented with phrases combining adverbs and verb cues to temporality and were asked to determine the appropriate tense before proceeding to comprehension. Textual Enhancement Another common FFI technique is the use of Textual Enhancement such as color-coding, boldfacing and underlining, to increase learners’ awareness of non-salient forms in the input (Sharwood-Smith, 1993; Doughty and Williams, 1998). Han et al. (2008) and Lee and Huang (2008) review studies of TE and conclude that there are conflicting findings regarding its effectiveness. They suggest that these discrepancies may be explained by differences between studies in such factors as learners’ target and native languages, the type, complexity and communicative value of target forms, learner proficiency, treatment intensity, and the measures used to assess noticing and processing of these forms. In the present study, we used boldfacing and color to make verb-tense inflections more salient. This condition is therefore called verbal salience (VS). Contrary to the VG condition, we did not explicitly direct learners to attend to the enhanced verb-inflections. Nevertheless, given that we did provide VS participants with explicit feedback on their correctness during the exposure phase, we consider VS an explicit FFI technique designed to promote induction of the target form. Verb Pretraining The effect of blocking is particularly potent whenever the cue to be processed is met in a redundant context where other cues have the same interpretation and have been learned previously or are more salient. One way to counteract this type of blocking is to ensure that early in L2 experience, the cue is experienced on its own in situations in which it must be processed for successful interpretation (VanPatten and Oikennon, 1996). Ellis and Sagarra’s (2010, 2011) VP conditions tested the effects of this and demonstrated that once the cue has been consolidated into the processing system, it continues to contribute to processing in subsequent situations of potential cue competition. For continuity, replication, and comparison, we include VP here to compare its efficiency and operation with VG and VS conditions. VP does not explicitly provide learners with a metalinguistic description of verb-tense morphology, but rather gives them opportunity to infer how verb tense morphology works by processing Latin verb forms for temporality and providing feedback on their correctness. Eye-Tracking as a Measure of Attention Second Language Acquisition research is increasingly recognizing eye tracking as a research tool (Winke et al., 2013) because it “allows for the study of moment-by-moment processing decisions during natural, uninterrupted comprehension, and critically, without the need to rely on participants’ strategic or metalinguistic responses” (Roberts and Siyanova-Chanturia, 2013, p. 214). Ellis et al. (2014) used eye movement recordings to measure participants’ overt attention to adverb and verb cues and found that pretraining on different cue dimensions (adverb pretraining versus verb pretraining) led to differences in learners’ overt attention to these cues during processing, and that these in turn led to differences in their covert attention to these cues during the comprehension and production tasks. Aims The current studies extend previous research on salience and learned attention in SLA by (i) exploring and comparing the degree to which VG, VS, and VP methods of FFI might serve to counteract learned attention effects whereby learners’ prior experience with adverbial cues in their L1 block their processing of verb inflections in the L2, and (ii) comparing their effects in aural and visual modalities of language. In Experiment 1 we use eye-tracking to measure Chinese L1 speakers’ visual attention to form in these various FFI conditions of visual language exposure. The control condition (CC) and VP conditions allow us to replicate Ellis and Sagarra (2011), as well as to extend the findings in Ellis et al. (2014) using a more complex verbal system. The inclusion of VG and VS, additionally allow us to further compare the effects of these manipulations to VP. Experiment 1 focuses upon several research questions: Research Question 1 (RQ1): do the effects of physical salience and learned attentional biases toward adverbial cues, under normal conditions of exposure (CC), prejudice the acquisition of verbal tense morphology, as indexed in participants’ relative reliance on these cues in subsequent language comprehension? Research Question 2 (RQ2): does early experience of morphological cues to temporal reference, through each of the FFI treatments VG, VS, VP, counteract the effects of physical salience and learned attentional biases, as indexed by participants’ relative reliance on these cues in subsequent language comprehension? Research Question 3 (RQ3): does early experience of morphological cues to temporal reference lead to biases in subsequent overt perceptual attention (as indexed by number of fixations) during Sentence Exposure, where there are both adverbial and morphological cues to the same interpretation? Research Question 4 (RQ4): does any bias in overt attention to input cues in turn lead to subsequent attentional biases to the adverbial or morphological cues in subsequent language comprehension? In Experiment 2 we compare the processing of auditory and visual input to assess effects of modality on salience, and again we contrast the effectiveness of VG, VS and VP methods of FFI in counteracting learned attention effects. The research questions of Experiment 2 are: Research Question 5 (RQ5): as in Experiment 1, does early experience of morphological cues to temporal reference, through each of the FFI treatments VG, VS, and VP, counteract the effects of physical salience and learned attentional biases, as indexed by participants’ relative reliance on these cues in subsequent language comprehension. Research Question 6 (RQ6): are each of the FFI treatments VG, VS, and VP equally effective in reattuning learners’ attention to the non-salient morphological cues through visual and auditory modalities of exposure? Experiment 1 Introduction Cintrón-Valentín and Ellis (2015) used eye-tracking to investigate the attentional processes whereby different types of FFI instruction overcome learned attention and blocking effects in learners’ online processing of L2 input. English native speakers viewed Latin utterances combining lexical and morphological cues to temporality under control conditions (CC) and three types of explicit FFI: verb grammar instruction (VG), verb salience with textual enhancement (VS), and verb pretraining (VP). All groups participated in three phases: exposure, comprehension test, and production test. VG participants viewed a short lesson on Latin tense morphology prior to exposure. VS participants saw the verb inflections highlighted in bold and red during exposure. VP participants had an additional introductory phase where they were presented with solitary verb forms and trained on their English translations. Instructed participants showed greater sensitivity to morphological cues in comprehension and production testing. Eye-tracking measures revealed how FFI affects learners’ attention during online processing and thus modulates long-term blocking of verb morphology. This experiment aims to replicate these findings in another population of learners, L1 Chinese speakers, whose L1 does not exhibit verb-tense morphology. In Chinese languages, “gender, plurality and tense are either indicated by lexical choice or not indicated at all” (Li and Thompson, 1987, p. 825). As a result, L1 speakers of Chinese languages are particularly prone to long-term attentional blocking of verb tense morphology (Ellis and Sagarra, 2011). Participants Chinese native speakers who had not learned Latin or Italian previously were recruited from a major university in the USA (n = 58) or its local community (n = 9). They were volunteers and either participated as part of an undergraduate Psychology course requirement (n = 3) or they were compensated with 10 dollars for their time (n = 64). All were bilingual with high-level English language proficiency sufficient to admit them to study in English. However, all had learned English as a L2 after the age of 5 years. They were randomly assigned to one of four conditions: CC, n = 19 (12 females and 7 males), age range 19–35 years (M = 24.58); VG, n = 18 (13 females and 5 males), age range 20–26 years (M = 22.50); VS, n = 14 (11 females and 4 males), age range 19–30 years (M = 23.13) and; VP, n = 15 (10 females and 4 males), age range 20–34 years (M = 24.80). Of these participants, seven (CC = 4; VG = 2; VS = 1) were excluded from the eye-tracking analyses due to poor data quality. All participants received oral instructions in their native language prior to the start of the experiment, with the exception of three participants in the Chinese CC group and four participants in the Chinese VG group. Although it was originally intended that all participants would receive these additional instructions in their native language, to ensure that they were indeed bilingual, the research assistants were not all fluent in Chinese. Procedure The experiment was programmed in E-Prime (Schneider et al., 2002). It took less than 1 h to complete. There were three phases: Pretraining, Sentence Exposure, and Comprehension testing. The procedure of these phases is shown in Table 1. Table 1 The design of Phases 1–3 of Experiment 1. Pretraining (Phase 1) (+ feedback) Sentence Exposure (Phase 2) (+ feedback) 36 (18 × 2) randomized trials Comprehension Test (Phase 3) (- feedback) 66 randomized trials Stimulus Semidiem Control group Present No pretraining Verb Pretraining group (36 randomized trials) cogito “I think” cogitas “you think” cogitat “X thinks” cogitavi “I thought” cogitavisti “you thought” cogitavit “X thought” Verb Salience group No pretraining Verb Grammar group Brief Grammar Lesson See Figure 1 hodie cogito hodie cogitas hodie cogitat cogito hodie cogitas hodie cogitat hodie Past heri cogitavi heri cogitavisti heri cogitavit cogitavi heri cogitavisti heri cogitavit heri Future cras cogitabo cras cogitabis cras cogitabit cogitabo cras cogitabis cras cogitabit cras hodie heri cras cogito/as/at cogitavi/visti/vit cogitabo/bis/bit hodie cogito/as/at hodie cogitavi/visti/vit hodie cogitabo/bis/bit heri cogito/as/at heri cogitavi/visti/vit heri cogitabo/bis/bit cras cogito/as/at cras cogitavi/visti/vit cras cogitabo/bis/bit cogito/as/at hodie cogitavi/visti/vit hodie cogitabo/bis/bit hodie cogito/as/at heri cogitavi/visti/vit heri cogitabo/bis/bit heri cogito/as/at cras cogitavi/visti/vit cras cogitabo/bis/bit cras 3 1 5 3 1 5 3 2 4 2 1 3 4 3 5 3 2 4 2 1 3 4 3 5 The rating scale for the Comprehension Test ranged from 1 (past) to 5 (future). The correct answer for each trial is shown in the semidiem column.Pretraining Verb pretraining participants engaged in a phase that involved training on verb inflections. On each trial they saw one of the past (cogitavi, cogitavisti, cogitavit) or present (cogito, cogitas, cogitat) inflected verbs and learned that each corresponded to either X think(s) or X thought by clicking the appropriate alternative with the mouse. A correct choice returned the feedback “Correct” or “Incorrect – the meaning of [Latin word] is [English word].” The 36 trials thus involved each of the three persons singular of present and past tense being presented six times in random order. Keeping the same number of trials of pretraining for all participants allows evaluation of what is gained from that amount of experience. This permits comparison across contents and conditions of pretraining, for example auditory versus visual modality as in Experiment 2 which follows here, and those of Ellis and Sagarra (2010, 2011) which vary with regard to the different levels of grammatical number and person. We report performance levels at the end of training in 2.4.1. Pretraining for the VG participants involved a brief lesson on Latin verb-tense morphology using the three slides shown in Figure 1. Although they could view each of the slides for an undetermined amount of time, they were not allowed to take notes and could not regress to previous slides. FIGURE 1 Grammar Lesson Slides for Grammar Instruction Condition. Sentence Exposure During Sentence Exposure, participants were exposed to 18 sentences (see Table 1) that appropriately combined the adverb with a verb (half in adverb-verb word order and half in verb-adverb order) and had to choose whether these sentences referred to the present, the past, or the future. Both word orders were used to counterbalance which cue was experienced first across sentences. Each of the 18 sentences was presented twice during this phase of the experiment. Feedback was given for both correct and incorrect choices. For correct answers, the word “correct” would appear on the screen, whereas for incorrect answers, participants would see the word “wrong” accompanied by the correct answer (e.g., “Wrong – [heri cogitavisti] is [past]”). The Sentence Exposure procedure was identical for the CC, VG, and VP groups. For VS participants only, the stimuli were textually enhanced so that the verbal inflections were highlighted in bold and red to increase the salience of these items (see Figure 2). Participants were not made aware of this beforehand and were given the same instructions for this task as were the other groups. FIGURE 2 An example trial from the Verb Salience Condition, where the verb inflections were highlighted in bold and red during Sentence Exposure. Comprehension Test In this phase, participants were presented with all single-word items (verbs and adverbs) and all possible combinations of adverbs and verb tenses for a total of 12 single-word items and 54 two-word items (comprised of 27 unique combinations), respectively (a grand total of 66 trials; see Table 1). The two-word items were presented in two different word orders, counterbalancing the cue participants would experience first. The presentation of all possible combinations meant that participants experienced sentences that were familiar to them from the previous task and also combinations in which the verb and adverb were incongruent in their time reference. Before the start of the task participants were told that there would be both congruent and incongruent sentences. They were asked to judge their temporal reference on a five-point scale by using their mouse to select the appropriate answer. The possible scale points were labeled (1) “past,” (2) “both past and present,” (3) “present,” (4) “both present and future,” and (5) “future.” Participants were told they could also choose 3 if they encountered an incongruent sentence with both past and future cues. For example, the participant could be presented with an incongruent sentence such as heri cogitabo “Yesterday I will think,” for which the correct answer was 3 and understood as the average of the items’ tenses (past [1] + future [5]/2 = 3). The correct answer for each trial, which Ellis and Sagarra (2011) referred to as the semidiem, is shown in Table 1. This task separately assessed the degree to which participants attended the adverb and verbal cues by determining the relative weight that learners put on adverbial and inflectional cues to time reference. For this reason, feedback was not provided. The logic behind the experiment follows that of previous studies of learned attention and blocking (Ellis and Sagarra, 2010, 2011; Ellis et al., 2014). During Sentence Exposure, regardless of condition, all participants experience both the adverb and verbal cue together. If they pay equal attention to both cues during this phase then their judgment during the Comprehension test should be equally affected by both cues. However, if they are biased toward one cue or the other, it is expected that their judgment in the Comprehension test will be swayed toward the corresponding cue. Because the CC participants only saw the two cues together, their performance was expected to mirror how learners typically weigh these cues, which in the native speakers of English studied in Ellis and Sagarra (2010, 2011) was characterized by the overshadowing of morphological cues by the more salient and reliable adverbial cues. Eye-Tracking Eye-movement recordings were gathered using an ISCAN-ETL 400 eye-imaging system with a sampling rate of 60 Hz. The eye-tracking cameras were mounted on headgear. Before the start of Pretraining (or Sentence Exposure for the CC and VS participants), the participants’ gaze was calibrated using a six-point calibration sequence. This sequence was again repeated for all participants before starting Comprehension testing. Stimuli were presented in E-Prime and were positioned within a screen area of 640 × 480 pixels. In the Sentence Exposure phase, the left stimulus (STIML) was centered at coordinates (x, y) 94, 99, and the right stimulus (STIMR) was positioned at coordinates 454, 99. For Comprehension testing, STIML and STIMR were positioned at 109, 108 and 505, 108, respectively. Participants’ fixations were analyzed using ILAB (Version 3.6.4), an open-source program developed for the analysis of eye-movement recordings (Gitelman, 2002) through the MATLAB software platform (Version 7.12.0.635) (MathWorks Inc, 2011). For each condition, fixations were analyzed from 600 ms after the start of Sentence Exposure and Comprehension testing trials (coinciding with the end of the presentation of a fixation cross at the center of the screen) until the end of each trial (coinciding with participant response). Region of interest (ROI) analyses were calculated using two positions (left and right) at the upper-most part of the screen. Both ROIs had a height of 200 pixels and a width of 250 pixels; the ROI for STIML was centralized at 175, 103 pixels and the ROI for STIMR, at 465, 103 pixels. These relatively large ROIs reflect our simple setup, which involved merely a chin rest and forehead bar to stabilize the participant’s head position. In some cases, for individual subjects it was necessary to edit coordinates for both ROIs to adjust for drift. Fixation analyses were run using the default ILAB fixation velocity/distance calculation parameters, with fixations determined according to degree of movement (horizontal 1.02°; vertical 1.09°) and a minimum duration of 100 ms. Eye-movement analysis was done blind to stimulus content: the random order of stimulus presentation for each participant entailed that right and left fixation durations were assigned as verb and adverb fixation durations only in subsequent statistical analysis on the basis of trial number. Results Behavioral Data Verb Pretraining Data Mean performance in the first quarter of Verb Pretraining was 79%. By the fourth quarter, mean performance was 93% (nine participants attained 100%, five 89%, and one 56%) demonstrating that the amount of training in Phase 1 was at an appropriate level. Sentence Exposure Data Mean performance in the first quarter of Sentence Exposure was 60% for the CC group, 62% for the VG group, 49% for the VS group, and 74% for the VP group: the prior experience of VP participants gave them an advantage in the first quarter compared to the other groups. However, performance evened out for all groups by the end of the phase. Mean performance in the final quarter was 82% for the CC group, 84% for the VG group, 73% for the VS group, and 89% for the VP group. A one-way ANOVA on these final quarter scores did not reveal a significant group effect, F(3,63) = 1.55, p = 0.21. Comprehension Data For each participant, we computed the Pearson correlation between the temporal ratings they provided for each of the 54 two item stimuli in the comprehension phase and the information given in each sentence by the corresponding adverb and verb cues. This correlation thus shows the degree to which each participant is biased by the verb and adverb cues. Figure 3 illustrates the group mean correlations. Following Corey et al. (1998), when averaging or performing inferential statistics on the correlation coefficients, we first transformed the r values to z values, then performed the statistics, and then reverse transformed to report the values. Participants in the four groups differed in their cue use. Chinese CC participants were more influenced by the adverb, M = 0.51, 95% CI = [0.34, 0.69] than the verb, M = 0.03, 95% CI = [-0.06, 0.11]. Chinese Verb grammar participants were more influenced by the verb, M = 0.53, 95% CI = [0.35, 0.71], than by the adverb, M = 0.13, 95% CI = [-0.04, 0.29]. Chinese VS participants were more influenced by the verb, M = 0.54, 95% CI = [0.39, 0.69], than by the adverb, M = 0.13, 95% CI = [0.04, 0.22]. Likewise, Chinese VP participants were more influenced by the verb, M = 0.61, 95% CI = [0.45, 0.77], but relative to the other FFI groups, maintained some sensitivity toward the adverb cue, M = 0.47, 95% CI = [0.30, 0.64]. The one VP participant who attained less than 88% in Phase 1 pretraining showed little influence of verb bias in later comprehension (r = 0.11) compared to adverb bias (r = 0.88). FIGURE 3 Group mean correlations between individual participants’ Comprehension sentence ratings and the information given by the corresponding adverb and verb cues. Error bars are 2 standard errors long. Chi = Chinese. An ANOVA (4 Groups × 2 Cues, with subjects nested within groups) revealed an overall effect of group, F(3,63) = 3.83, p = 0.01; and a significant group by cue interaction, F(3,63) = 7.80, p < 0.001. Individual ANOVAs (2 Groups × 2 Cues) of each FFI group against the CC were conducted using Bonferroni adjusted alpha levels of 0.017 per test (0.05/3). The results yielded a significant interaction of group and cue for the CC group versus the VG group, F(1,35) = 18.73, p < 0.001; for the CC group versus the VS group, F(1,32) = 25.84, p < 0.001; and for the CC group versus the VP group, F(1,32) = 8.51, p = 0.006. All FFI treatments therefore increased sensitivity to the verb cue. Figure 4 shows the reliability of these patterns across individual group members. Most CC individuals were predominantly influenced by the adverb cue, whereas most VS and VG participants were more influenced by the verb cue. Verb pretraining participants were more scattered: most showed greater sensitivity to the verb, though there were some who lay close to the 45° diagonal, suggesting that they were more evenly affected by both cues. FIGURE 4 Sensitivity to adverbial and verbal inflectional cues to temporal reference in each participant. Chi = Chinese. Eye-Tracking Data Sentence Exposure Figure 5 and Table 2 show the group mean fixation duration of these participants as they were studying the adverb and verb cues during exposure to the Latin sentences. Figure 5A shows the total fixation duration on these cues. Figure 5B shows these data as the proportion of the total fixations on each trial. The pattern in these Figures is clear, all groups looked at the verb more than the adverb, but it was the three FFI groups that did so to a greater extent. Individual ANOVAs on the total fixations (2 Groups × 2 Cues, with subjects nested within groups) were conducted using Bonferroni adjusted alpha levels of 0.017 per test (0.05/3). The results revealed a significant group by cue interactions for the VG group versus the CC group, F(1,29) = 6.86, p = 0.014; for the VS group versus the CC group, F(1,28) = 17.71, p < 0.001; but the interaction marginally failed to reach significance for the VP group versus the CC group, F(1,28) = 4.06, p = 0.05. VG and VS therefore paid more attention than the CC group to the verb cue during processing. FIGURE 5 Mean Group Fixation Duration on the Adverb and Verb cues in Sentence Exposure. (A) Participants’ total fixation duration during Exposure. (B) Participants’ proportion fixation duration during exposure. Error bars are 2 standard errors long. Chi = Chinese. Table 2 Mean participant fixations on the adverb and verb cues by the four groups of Experiment 1. Group Cue Mean 95% CI Mean total fixation duration (ms.) Control Adverb 819.2 [402, 1236] Verb 1141.6 [628, 1656] Verb grammar Adverb 958.7 [340, 1577] Verb 1959.2 [662, 3256] Verb salience Adverb 300.3 [23, 578] Verb 1262.7 [631, 1894] Verb pretraining Adverb 708.8 [375, 1041] Verb 1581.1 [681, 2480] Mean proportion fixation time Control Adverb 0.41 [0.33, 0.50] Verb 0.59 [0.50, 0.67] Verb grammar Adverb 0.34 [0.25, 0.43] Verb 0.66 [0.58, 0.75] Verb salience Adverb 0.17 [0.07, 0.27] Verb 0.83 [0.73, 0.93] Verb pretraining Adverb 0.34 [0.20, 0.48] Verb 0.66 [0.52, 0.80] Correlations between Attention to Cue in Sentence Exposure and Subsequent Cue Comprehension Pearson correlations investigating the relations between attention in the Sentence Exposure phase and comprehension ability in the Comprehension Phase across all the participants and groups of Experiment 1 show that the proportion of fixation time spent on the adverb during Sentence Exposure correlates significantly with later adverbial bias in Comprehension (r = 0.50, p < 0.001). Likewise, proportion of fixation time spent on the verb during Sentence Exposure correlates significantly with later verb bias in Comprehension (r = 0.45, p < 0.001). Sentence Exposure Eye-Tracking Over Trials Although the random order of stimuli was different for each participant, we can determine the degree to which the participants attended to the verb and adverb cues over trials. Figure 6A shows the total fixation on each cue by trial of experience in all L1 Chinese groups. It can be seen that CC participants initially spent more time looking at the verb, but interest in this cue waned over trials and more attention was paid to the adverbial cue. Participants in the three FFI conditions, however, maintained a steady attentional preference for the verb cue. These patterns are clearer in Figure 6B, which plots the proportion of fixation time on each trial spent on the adverb and verb cues, respectively. FIGURE 6 Mean Group Fixation Duration on the Adverb and Verb cues Over Trials (solid lines and circles = verb, and dotted lines and triangles = adverb). (A) Participants’ total fixation duration by each trial of exposure. (B) Participants’ proportion fixation by each trial of exposure. Discussion The behavioral results of Experiment 1 show that under CC, adverbs were better attended than verb inflections. This finding replicates that of Ellis and Sagarra (2010, 2011) and Cintrón-Valentín and Ellis (2015). In the linguistic input, adverbial cues are more salient, simple and reliable cues compared to the verb-tense inflections. Furthermore, the adult language learners’ prior use of adverb temporal reference in their Chinese L1 could have resulted in long-term blocking. In contrast to the CC treatment, training on the isolated verb cue under the VP condition reversed this bias, resulting in a better use of the verb cue during comprehension. This finding also replicates that of Ellis and Sagarra (2010, 2011) and Cintrón-Valentín and Ellis (2015), showing short-term learned attention effects, where prior learning of an isolated cue during pretraining shifts learners’ attention to that cue in subsequent testing. In the two other FFI conditions, VG, where learners were first exposed to a short instructional sequence on how Latin verb-tense morphology works in Latin, and VS, where the verb inflections were made more salient by means of textual enhancement manipulations during exposure, participants were better able to use the verb cues in comprehension relative to the adverb cue than those in the CC condition. Of the three FFI conditions, VP resulted in more balanced acquisition of both verbal and adverbial cues. The eye-tracking data show how these FFI treatments affected attention to cues in the input processing. All participants looked at the verbs more than they did the adverb during sentence exposure. However, participants in the VG and VS conditions fixated upon the verbs significantly more during input processing than did Control participants. The VP group, however, did not differ significantly from the control group, although the same numerical trend was evident. The correlation analyses suggest that the relative amount participants spent processing the verb/adverb cues during exposure determined cue usage in subsequent comprehension testing. The trial-by-trial analyses illustrated in Figure 6, show that CC participants initially spend more time looking at the verb, however, participants rapidly lose interest in the verb cue across trials and more attention is paid to the adverbial cue. One possible interpretation is that learners initially first fixate more on the verb + inflection because it is the longer word form, however, over trials they come to realize that the adverb is the simpler and more reliable cue, and as a result they shift their attention to it. The FFI participants on the other hand – for whom the verb forms or their functions were made more salient – pay more attention to verb from the start of language exposure, and this focus persists, leading to subsequent attention and use of this cue. Experiment 2 Introduction Our previous studies examining the effects of learned attention and blocking and the effects of FFI in overcoming learned attentional biases in L2 acquisition Ellis and Sagarra (2010, 2011) and Ellis et al. (2014) have focused on the learning of Latin only through the visual modality. As described in the introduction, spoken and written language are very different mediums. Whereas readers have the advantage of being able to control the amount and speed at which they process visual input, the fleeting nature of spoken language does not afford listeners the same advantage. These differences could well-affect the degree to which different language forms are salient in the input and thus control the degree to which they are attended, perceived, processed, and learned. Indeed Leow (2015, p. 122), in reviewing the relevance for instruction of this work on learned attention, explicitly asks for a potential replication study which addresses the issue of whether the findings can be extrapolated to the aural mode. This experiment therefore aims to replicate and extend previous work by comparing the attentional processes of L1 speakers of English in control (CC), VG, VS, and VP conditions who learn from aural input with those whose input experience is visual. Participants Participants were 200 individuals recruited from a major university in the USA. They were volunteers who participated as part of an undergraduate psychology course requirement (n = 182) or were paid $10 for their participation (n = 18). Inclusion criteria required participants to be native English speakers who had not learned Latin or Italian previously. They could know Spanish but could not have been raised bilingually before the age of 6 years. They were randomly assigned to one of eight conditions regarding instruction and modality of presentation. Those who received Aural presentation only were split into CCA (Control Condition Aural), n = 25 (15 females and 10 males), age range 17–45 years (M = 21.56); VGA (Verb Grammar Aural), n = 25 (16 females and 9 males), age range 18–22 years (M = 18.84); VSA (Verb Salience Aural), n = 25 (22 females and 3 males), age range 17–20 years (M = 18.36); and VPA (Verb Pretraining Aural), n = 25 (15 females and 10 males), age range 18–20 years (M = 18.44). Participants who received instruction in the Visual modality only were split into CCV, n = 25 (18 females and 7 males), age range 18–21 years (M = 18.40); VGV (Verb Grammar Visual), n = 25 (8 females and 17 males), age range 17–22 years (M = 18.68); VSV (Verb Salience Visual), n = 25 (9 females and 16 males), age range 18–20 years (M = 18.68); and VPV (Verb Pretraining Visual), n = 25 (14 females and 11 males), age range 18–21 years (M = 18.52). Procedure The experiment was programmed in PsychoPy (Peirce, 2007) and consisted of the same phases as presented in Experiment 1 (see Table 3 for detailed procedure). However, the stimulus set for Experiment 2 was more complex than that of Experiment 1. In Experiment 1 participants were presented with one verb stem, cogit-, which was combined with all appropriate past, present and future inflections, whereas in Experiment 2, participants were presented with four different verb stems and their appropriate past, present and future inflections. Table 3 The design of Phases 1–3 of Experiment 2. Pretraining (Phase 1) (+ feedback) Sentence Exposure (Phase 2) (+ feedback) 48 (24 × 2) randomized trials Comprehension Test (Phase 3) (- feedback) 48 randomized trials Stimulus Semidiem Control group Present No pretraining Verb Pretraining group (36 randomized trials) nato “I swim” cantas “You sing” pugnas “You fight” fleat “He/She cries” cantavi “I sang” natavisti “You swam” pugnavit “He/She fought” fleavit “He/She cried” Verb Salience group No pretraining Verb Grammar group Brief Grammar Lesson See Figure 1 hodie cantas hodie fleat hodie pugnas hodie nato cantas hodie fleat hodie pugnas hodie nato hodie Past heri cantavi heri fleavi heri fleavit heri pugnavit heri natavisti cantavi heri fleavi heri fleavit heri pugnavit heri natavisti heri Future cras cantabit cras pugnabis cras natabo cantabit cras pugnabis cras natabo cras verb-o/as/at cras verb-avi/visti/vit cras verb-abo/ bis/ bit cras hodie verb–o/as/at hodie verb-avi/visti/vit hodie verb-abo/bis/bit heri verb-o/as/at heri verb-avi/visti/vit heri verb-abo/bis/bit cras verb-o/as/at cras verb-avi/visti/vit cras verb-abo/bis/bit verb-o/as/at hodie verb-avi/visti/vit hodie verb-abo/bis/bit hodie verb-o/as/at heri verb-avi/visti/vit heri verb-abo/bis/ bit heri 3 1 5 3 2 4 2 1 3 4 3 5 3 2 4 2 1 3 The rating scale for the Comprehension Test ranged from 1 (past) to 5 (future). The correct answer for each trial is shown in the semidiem column.Pretraining Participants in the VP group were first pretrained on verb inflections and determined that each made reference to either present or past time. On each trial, Visual participants saw, or Aural participants heard, one of the four verb stems (cant-, flea-, nat-, pugn-) combining an inflection referencing the past (-avi -avisti, -avit) or present (-o, -as, -at). Participants were additionally presented with a picture of a stick figure that represented the action of the verb (see Figure 7). They were asked to select the Latin verb’s temporal (past/present) reference from an on-screen menu. Feedback was provided on their responses. In this phase they were not asked about the verb meaning, thus their understanding was focused upon the morphological tense reference. FIGURE 7 Pictures presented during Verb Pretraining, Exposure and Comprehension testing. During Pretraining and Exposure each picture was presented in the presence of the corresponding Latin verb (either aurally or visually, depending on the condition), where (A) corresponded to any form of the verb pugno (representing the verb to fight in English); (B) corresponded to any form of the verb canto (representing the verb to sing in English); (C) corresponded to any form of the verb nato (representing the verb to swim in English); and (D) corresponded to any form of fleo (representing the verb to cry in English). During the Comprehension testing phase, a pane where all four pictures were present was shown in each trial, again in the presence of the corresponding Latin verb. Pretraining for the VG participants involved a brief lesson on Latin verb-tense morphology using similar slides to those shown in Figure 1, except that the Latin verb amare was used as an example in slides 2 and 3. Regardless of modality of language exposure these slides were presented visually. Sentence Exposure In Sentence Exposure, Visual participants saw, Aural participants heard, 24 different sentence combinations, which appropriately combined the adverb with a verb stem (see Table 3). While the sentence was exposed, participants saw onscreen a picture of a stick figure which appropriately represented the action the verb was referencing. Again, in this phase they were not asked to make any judgments regarding the picture they were shown. After each sentence, participants were asked to identify whether the sentences referred to the past, present, or future, responding via a visual menu presented on the computer screen. The sample of stimuli selected for presentation in Sentence Exposure ensured that each verb root was (1) presented once in each tense, and (2) appropriately combined one of the agreement markers for each tense. The Sentence Exposure procedure was identical for the CC, VG, and VP groups. For VS participants only, the stimuli were either textually or aurally enhanced to increase their salience, so for Visual presentation the verbal inflections were highlighted in bold and red, and for Aural presentation the verb inflections were spoken emphatically. Feedback was given for both correct and incorrect choices. For correct answers, the word “correct” would appear on the screen, whereas for incorrect answers, participants would see the word “wrong” accompanied by the correct answer (e.g., “Wrong – [heri cantavit] is [past]”). Comprehension Test Here, participants were presented with randomized verb-adverb/adverb-verb combinations as well as a selection of single word items. The single word items were verbs (canto, fleat, natat, pugnas, cantavi, fleavit, natavit, pugnavisti, cantabit, fleabis, natabo, pugnabit), half of which had been previously presented in the same inflection during Sentence Exposure. For the randomized verb-adverb/adverb-verb combinations, similar to Experiment 1, participants experienced sentences that were familiar to them from the previous task, but also combinations in which the verb and adverb were incongruent in their time reference. Here, participants saw six congruent combinations they had previously experienced during Sentence Exposure (heri fleavi, heri pugnavit, heri natavisti, hodie cantas, hodie nato, cras pugnabis) as well as six new congruent combinations they had not seen before (heri natavi, heri cantavit, hodie fleo, hodie fleas, cras cantabis, cras pugnabo). For the trials involving incongruent combinations, each of the verbs used for the congruent combinations were combined with all possible adverb forms. Overall this led to a total of 12 single-word items and 36 two-word combinations. In each of the trials, participants were additionally presented with a four-picture pane menu, where they saw the four pictures they had been previously presented with during Sentence Exposure. The position of the pictures was counterbalanced in the pane. On each trial, participants were asked to make two judgments. The first judgment was whether the word string referred to the past, present, or future on a five-point scale. The possible scale points were the same as in Experiment 1. For the second judgment, participants were asked to select the picture that best represented the word or phrase they were presented with. This judgment tested how well they had processed the meaning of the verbs to which they had been exposed. Feedback was not provided. Results Visual Modality Verb Pretraining Data Mean performance in the first quarter of Verb Pretraining was 63%. By the fourth quarter, mean performance was 86%, demonstrating acceptable completion of Phase 1. Sentence Exposure Data Mean performance in the first quarter of Sentence Exposure was 61% for the CCV group, 84% for the VGV group, 68% for the VSV group, and 82% for the VPV group. Both the VGV and the VPV groups were at an advantage in the first quarter compared to the other groups. However, performance evened out for all groups by the end of the phase. Mean performance in the final quarter was 96% for the CCV group, 98% for the VGV group, 93% for the VSV group, and 95% for the VPV group. A one-way ANOVA on these final quarter scores did not reveal a significant group effect, F(3,96) = 1.71, p = 0.17. Comprehension Data Perception of Time Cues Each participant’s temporal rating responses for the strings in Comprehension testing were correlated with the information provided by the verb cue and the information separately provided by the adverb cue to determine the degree to which each participant was biased by each cue type. Pearson correlations between each participant’s temporal rating responses and the information provided by the verb and adverb cues separately are illustrated in Figure 8A. CCV participants were more influenced by the adverb, M = 0.89, 95% CI = [0.82, 0.96] than the verb, M = 0.02, 95% CI = [-0.03, 0.07]. VGV participants were more influenced by the verb, M = 0.78, 95% CI = [0.59, 0.97], than by the adverb, M = 0.03, 95% CI = [-0.07, 0.12]. VSV participants were more influenced by the verb, M = 0.73, 95% CI = [0.59, 0.86], than by the adverb, M = 0.17, 95% CI = [0.06, 0.27]. Contrary to the other FFI conditions VPV participants were more influenced by the adverb, M = 0.81, 95% CI = [0.60, 0.91], but showed some sensitivity toward the verb cue, M = 0.43, 95% CI = [0.29, 0.57]. FIGURE 8 Group mean correlations between individual participants’ Comprehension sentence ratings and the information given by the corresponding adverb and verb cues. (A) Group mean correlations for the visual modality treatments (B) Group mean correlations for the aural modality treatments. Error bars are 2 standard errors long. If we compare the comprehension data for the VPV participants with that of the Chinese VP participants in Experiment 1 (verb: M = 0.61, 95% CI = [0.45, 0.77]; adverb: M = 0.47, 95% CI = [0.30, 0.64], the pattern is quite different, with the VPV participants showing a greater degree of sensitivity toward the adverb cue relative to the verb cue. Although the VPV participants showed an increase in their sensitivity toward the verb cue when, compared to the CCV group (as confirmed by our analysis of variance below), it seems that the greater complexity of the stimulus set in Experiment 2 had an impact on the learners’ attentional focus, and thus on the degree of sensitivity they showed toward the verbal morphological cues during comprehension. An ANOVA (4 Groups × 2 Cues, with subjects nested within groups) revealed a significant group by cue interaction, F(3,96) = 7.80, p < 0.001. As in Experiment 1, individual ANOVAs (2 Groups × 2 Cues) of each FFI group against the CC were conducted using Bonferroni adjusted alpha levels of 0.017 per test (0.05/3). The results yielded a significant interaction of group and cue for the CCV group versus the VGV group, F(1,48) = 82.19, p < 0.001; for the CCV group versus the VSV group, F(1,48) = 73.72, p < 0.001; and for the CCV group versus the VPV group, F(1,48) = 8.68, p = 0.004. These results replicate those of Experiment 1, where the FFI conditions increased sensitivity to the verb cue. Figure 9A shows the reliability of these patterns across individual group members. For the groups in the visual modality, most CCV participants were influenced by the adverb cue, whereas most VSV and VGV participants were more influenced by the verb cue. VPV participants were more scattered: most showed greater sensitivity to the adverb, though there were some who showed greater sensitivity toward the verb, and one participant who lay close to the 45° diagonal, suggesting that they were more evenly affected by both cues. FIGURE 9 Sensitivity to adverbial and verbal inflectional cues to temporal reference in each participant. (A) The groups in the visual modality. (B) The groups in the aural modality. To determine if the effects of FFI on cue use during comprehension testing differed based upon the nature of the items, that is, whether they were trained (i.e., previously presented during Sentence Exposure) or generalization items (i.e., only presented during Comprehension Testing) we ran a three-way ANOVA (4 Groups × 2 item type × 2 cues). The analyses revealed a non-significant effect of item type F(1,96) = 0.19, n.s., a statistically significant group by cue interaction, F(3,96) = 28.56, p < 0.001, but no significant three-way interaction between item type (Trained or Generalization), group, and cue use, F(3,96) = 0.69, p = 0.56. Thus, participants performed at a similar level, regardless of item type (see Figure 10). FIGURE 10 Group mean correlations between individual participants’ Comprehension sentence ratings and the information given by the corresponding adverb and verb cues for the Visual Modality. (A) Group mean correlations for the trained items (B) Group mean correlations for the generalization items. Error bars are 2 standard errors long. Perception of Verb Meaning Mean accuracy scores for verb meaning was 0.82 for the CCV group, 0.80 for the VGV group, 0.76 for the VSV group, and 0.91 for the VPV group. A one-way ANOVA on each of the conditions’ mean accuracy scores for the picture ratings showed a non-significant effect of group, F(3,96) = 2.59, p = 0.06. Post hoc Tukey HSD tests demonstrated just one significant pairwise group difference: between the VPV group, and the VSV group, p = 0.04, 95% CI = [0.004, 0.30]. Aural Modality Verb Pretraining Data Mean performance in the first quarter of Verb Pretraining was 66%. By the fourth quarter, mean performance was 80%, demonstrating acceptable completion of Phase 1. Sentence Exposure Data Mean performance in the first quarter of Sentence Exposure was 49% for the CCA group, 50% for the VGA group, 54% for the VSA group, and 67% for the VPA group. The pretraining on the verb allowed the VPA participants to be at an advantage in the first quarter compared to the other groups, and this advantage also persisted. Mean performance in the final quarter was 78% for the CCA group, 68% for the VGA group, 77% for the VSA group, and 87% for the VPA group. A one-way ANOVA on these final quarter scores revealed a significant group effect, F(3,96) = 3.12, p = 0.03. Post hoc Tukey HSD tests demonstrated one significant pairwise group difference: between the VPA group, and the VGA group, p = 0.02, 95% CI = [0.02, 0.35]. Comprehension Data Perception of Time Cues Pearson correlations between each participant’s temporal rating responses and the information provided by the verb and adverb cues separately are illustrated in Figure 8B. CCA participants were more influenced by the adverb, M = 0.78, 95% CI = [0.62, 0.94] than the verb, M = 0.07, 95% CI = [-0.001, 0.15]. VGA participants were more influenced by the verb, M = 0.47, 95% CI = [0.32, 0.62], than by the adverb, M = 0.33, 95% CI = [0.19, 0.48]. Contrary to the VGA condition, VSA participants were more influenced by the adverb, M = 0.71, 95% CI = [0.56, 0.86], than by the verb, M = 0.14, 95% CI = [0.05, 0.23]. Likewise, VPA participants were more influenced by the adverb, M = 0.71, 95% CI = [0.55, 0.99], but showed some sensitivity toward the verb cue, M = 0.21, 95% CI = [0.09, 0.33]. The general patterns observed here were reliable across individual group members. Figure 9B shows the aural modality data. Again, most CCA individuals were predominantly influenced by the adverb cue. VGA participants showed greater sensitivity toward the verb, whereas VSA participants were predominantly influenced by the adverb. Similar to those in the visual modality, VPA participants were more scattered: most showed greater sensitivity toward the adverb, a small group of participants showed greater sensitivity toward the verb, and one participant lay close to the 45° diagonal, suggesting that they were more evenly affected by both cues. An ANOVA (4 Groups × 2 Cues, with subjects nested within groups) revealed a significant group by cue interaction, F(3,96) = 5.53, p = 0.002. Individual ANOVAs (2 Groups × 2 Cues) of each FFI group against the CCA group yielded a significant interaction of group and cue for the CCA group versus the VGA group, F(1,48) = 13.09, p < 0.001; a significant main effect of cue for the CCA group versus the VSA group, F(1,48) = 35.32, p < 0.001, but no significant group by cue interaction, F(1,48) = 0.63, p = 0.43; and a significant main effect of cue for the CCA group versus the VPA group, F(1,48) = 30.15, p < 0.001, but no significant group by cue interaction, F(1,48) = 1.07, p = 0.31. Contrary to the results of Experiment 1 and those for Visual presentation described in 3.4.1, it seems only the VGA group increased sensitivity to the verb cue. As for the visual modality, to determine if the effects of FFI on cue use during comprehension testing differed based upon the nature of the items (i.e., Trained or Generalization) we ran a three-way ANOVA (4 Groups × 2 item type × 2 cues). The analyses revealed a non-significant effect of item type F(1,96) = 0.0002, n.s., a statistically significant item type by cue interaction, F(1,96) = 8.04, p = 0.006, but no significant three-way interaction between item type (Trained or Generalization), group, and cue use, F(3,96) = 0.70, p = 0.55. Thus, similar to the visual modality, participants in the aural modality performed at a similar level, regardless of item type (see Figure 11). FIGURE 11 Group mean correlations between individual participants’ Comprehension sentence ratings and the information given by the corresponding adverb and verb cues for the Aural Modality. (A) Group mean correlations for the trained items (B) Group mean correlations for the generalization items. Error bars are 2 standard errors long. Perception of Verb Meaning Mean accuracy scores for verb meaning was 0.59 for the CCA group, 0.56 for the VGA group, 0.63 for the VSA group, and 0.80 for the VPA group. A one-way ANOVA on each of the conditions’ mean accuracy scores for the picture ratings showed a significant effect of group, F(3,96) = 5.35, p = 0.002. Post hoc Tukey HSD tests demonstrated three significant pairwise group differences: between the VPA group and the CCA group, p = 0.01; between the VPA group and the VGA group, p = 0.002; and between the VPA group and the VSA group, M = 0.62, p = 0.05. Modality by FFI Interactions To determine if the effects of FFI on cue use during comprehension testing differed across modality of presentation, we ran a three-way ANOVA (4 Groups × 2 modalities × 2 cues). The analyses revealed a statistically significant three-way interaction between modality of input presentation (Aural or Visual), group, and cue use, F(3,384) = 9.85, p < 0.001. Inspection of Figure 8 shows the major loci of this interaction. Under CC, participants process the adverb and pay little or no heed to the verb. This is so for CCA and CCV. Pretraining on the verb in VP allows them a little better use of this cue, especially in VPV, but still it is overshadowed by the adverb. Making the verbal inflections salient during sentence exposure with visual presentation VSV allows learners to attend and learn to use verb morphology. But this is absolutely not so with auditory presentation VSA. Grammar instruction, however, does allow learners to make use of the morphological cues, both from auditory presentation, and particularly from visual exposure. Discussion The findings for the Visual conditions follow that of the prior learned attention studies. The CCV group showed greater sensitivity toward the adverb than the verb cue. The VPV treatment allows participants to show sensitivity toward the verb cue, while also showing sensitivity to use of adverbs. As in Experiment 1, and in Cintrón-Valentín and Ellis (2015), both VG and VS treatments in the visual modality shifted learners’ attention to the verb cue in subsequent testing. The general behavioral data for the Aural conditions show consistency in that learners in the CCA group focus more on the adverb than the verb cues. However, the other findings are in contrast to these patterns following Visual exposure. Of the three FFI conditions, only VGA produced a shift in their attention toward the verb cue. VSA participants’ performance was similar to that of the CCA participants, showing more sensitivity toward the adverb relative to the verb cue. Although VPA participants showed an increase in their verb sensitivity, when compared to that of the CCA group, their attention was greater toward the adverb cue than to the verb cue. We will discuss these disparities below. General Discussion Experiment 1 tested whether under normal conditions of exposure (CC), the effects of physical salience and learned attentional biases toward adverbial cues, prejudice the acquisition of verbal tense morphology, as indexed in participants’ relative reliance on these cues in subsequent language comprehension (RQ1). The results of Experiment 1 lend support to the idea that the limited attainment of adult second and foreign language learners follows general principles of associative learning and cognition wherein salience and attention are key factors (Ellis, 2006a,b). Under normal conditions of language exposure (CC), adverbs were better processed than verb inflections. We interpret this phenomenon, a standard finding in SLA research (e.g., Schmidt, 1984; Bardovi-Harlig, 1992, 2000; Noyau et al., 1995; Van Patten, 1996, 2006; Clahsen and Felser, 2006), as relating firstly to the relative salience, simplicity and reliability of adverb cues which render them more learnable when compared to verb-tense morphology, and secondly, to adult language learners’ prior knowledge of the use of adverb temporal reference in their L1 which results in the long-term blocking of these forms. This is apparent in adult language learners’ difficulty in learning morphology compared to child learners, and in studies such as Ellis and Sagarra (2010, Experiment 2) and Ellis and Sagarra (2011, Experiments 2 and 3) which demonstrate long-term language transfer effects whereby the nature of learners’ first language (+/- verb tense morphology) biased the acquisition of morphological versus lexical cues to temporal reference in Latin. First language speakers of Chinese (no tense morphology) were less able than first language speakers of Spanish or Russian (rich morphology) to acquire inflectional cues from the same language experience. Experiment 1 also tested whether early experience of morpho-logical cues to temporal reference, through each of the FFI treatments VG, VS, and VP, counteract the effects of physical salience and learned attentional biases, as indexed by participants’ relative reliance on these cues in subsequent language comprehension (RQ2). The behavioral data demonstrated that all FFI interventions resulted in better attention to and use of the verbal inflectional cues. Participants in the VG group were initially provided with declarative statements about morphological function but still had to put this knowledge to use in subsequent phases. VS learners were introduced to the verbal cues during the exposure phase but still had to determine their function. Both of these treatments resulted in participants’ attending these cues and using them over adverbial cues. In contrast, the verb pretraining in VP, where learners had to process the Latin verb forms for meaning in English, resulted in a more balanced acquisition of both verbal and adverbial cues. We believe that this is because, having learned to some extent how to use the morphology, they were next able to consider the role of adverbs too. This interpretation is consonant with other findings in the literature, where in the early stages of learning, when learners are confronted with multiple cues to interpretation, they typically focus upon one cue at a time. As they reduce errors of estimation regarding the outcome or interpretation of the cue, they then consider the role of the other cues (MacWhinney, 2001). The results of the VG condition are consonant with prior findings in the literature on FFI (see, for instance, the meta-analyses of effects of type of instruction by Norris and Ortega, 2000; Spada and Tomita, 2010; Goo et al., 2015), which suggest that instructional conditions involving a focus on the rules underlying specific L2 structures generally lead to large advantages in the acquisition of target forms. In terms of the results obtained for the VS condition, as we explained in the introduction, reviews of the effectiveness of textual enhancement (TE) have yielded conflicting findings (Han et al., 2008; Lee and Huang, 2008), largely due to a wide variety of methodological differences. However, one pattern that seems to apply is that the provision of compound enhancement, that is, “TE in combination with attention-getting strategies such as corrective feedback” (Han et al., 2008, p. 609) tends to be more effective in encouraging noticing and subsequent processing than simple enhancement. The sentence exposure phase for our VS participants involved exactly this – visual salience and corrective feedback. Two additional research questions in Experiment 1 concerned (i) whether early experience of morphological cues to temporal reference lead to biases in subsequent overt perceptual attention (as indexed by number of fixations) during Sentence Exposure, where there are both adverbial and morphological cues to the same interpretation (RQ3), and (ii) whether any bias in overt attention to input cues in turn lead to subsequent attentional biases to the adverbial or morphological cues in subsequent language comprehension (RQ4). The eye-tracking data from Experiments 1 show how the FFI treatments affected attention to cues during input processing. All participants fixated upon the verbs significantly more than they did the adverb. As shown by the group by cue interactions, participants in the VG and VS conditions attended the inflections more than the control participants, as did the VP participants although in the latter case the interaction failed to reach significance. Additionally, the correlation analyses showed that the relative amount participants spent processing the verb/adverb cues during language exposure determined their cue usage in subsequent comprehension. The trial-by-trial analyses of Figure 6 show that although control participants initially spent more time looking at the verb, their interest in this cue waned over trials, with more attention being paid to the adverbial cue. One interpretation is that CC learners initially first fixated more on the verb + inflection because it is the longer more salient form. Initially, over the first 20 trials or so, they tried to induce the system of how the inflections signal temporality, but realizing that the adverb is the simpler and more reliable cue, they eventually shifted their attention to it. The FFI groups on the other hand – who were initially made aware of the verb forms or their functions, (1) by having the verb-inflections explained during pretraining (VG), (2) by having the inflections made more salient by textual enhancement during exposure (VS), (3) or by being pretrained on the verbal cues in non-redundant situations (VP) – paid more attention to the verbs from the start of the exposure phase, and this persisted through the end of the trials. Overall, the eye movement findings in the current study replicate with Chinese L1 speakers those of Cintrón-Valentín and Ellis (2015) with English L1 learners. Experiment 2 investigated the effects of modality of language exposure upon the salience and consequent processing of linguistic form, and on the ways in which different types of FFI interact with these very different mediums. The fleeting nature of spoken language does not afford listeners the control of scrutiny of input as does visual presentation, and these differences could well-affect the degree to which forms are salient in the input. This experiment therefore compared the attentional processes of L1 speakers of English in control (CC), VG, VS, and VP conditions who were exposed to aural input with those whose input experience was visual. Consonant with prior studies, it showed that participants under control conditions (either CCV or CCA) showed greater sensitivity toward the adverb than the verb cue. Two specific research questions in Experiment 2 concerned (i) whether each of the FFI treatments VG, VS, and VP, counteract the effects of physical salience and learned attentional biases, as indexed by participants’ relative reliance on these cues in subsequent language comprehension (RQ5), and (ii) whether each of the FFI treatments VG, VS, and VP are equally effective in reattuning learners’ attention to the non-salient morphological cues through visual and auditory modalities of exposure (RQ6). Regarding RQ5, all forms of FFI were effective in increasing attention to verbal morphology in the visual modality, although VP resulted in balanced attention to both cues. However, it was generally the case that attending to the morphological cues was considerably more difficult under aural than under visual presentation. Only grammar instruction (VG) was successful in reattuning learners’ attention to the non-salient morphological cues in both modalities. This instruction allowed learners to become aware of forms and their patterning of function prior to sentence exposure, and their subsequent processing of these cues in the input promoted their use during comprehension testing. Also relating to RQ6, a major difference was seen in the effectiveness of morphological salience-raising in the two modalities. Increasing the salience of the verb morphology was very effective with visual exposure. Textually enhancing the morphology promoted attention to and analysis of these cues during sentence exposure, and this in turn resulted in subsequent use of these cues even when no longer made salient. In contrast, although emphasized pronunciation of these cues led to their use during sentence exposure in the aural group, this did not result in sustained use of these cues once the emphasis was removed. With aural presentation, the emphasized inflectional forms were attended but not analyzed, so that abrupt removal of the emphasis removed the cues themselves. There are many limitations to this study. Concerns include the small range of constructions being taught, the short-term nature of the experiment, the experimental environment and lack of ecological validity, the lack of long-term delayed testing, and the small range of outcome measures. The latter is a particular worry. As pointed out by one reviewer, attention/processing in this experiment is assessed through a considered comprehension temporal rating task, which likely taps predominantly explicit knowledge. Future research could well-incorporate a battery of measures ranging in their implicitness/explicitness. Meta-analyses of effects of instruction demonstrate that effectiveness varies as a result of explicitness of measure. Much also remains to be done particularly with regard to assessing the transfer of visual language experience to aural competence and vice-versa. We are currently comparing the effects of instruction under aural, visual, and bimodal conditions. The findings in this study reinforce and extend prior studies in second language instruction. Specifically, in the absence of instruction, learners tend to ignore non-salient features in the input, such as verb morphology. FFI can increase the salience of inflections and other commonly ignored features by (i) explicitly identifying the forms and their functions, as in VG, (ii) by having the inflections made more salient by textual enhancement, as in VS, or (iii) by introducing the verb alone in a non-redundant context, as in VP. These are the type of techniques that help learners attend to verb morphology, and broadly, they do so to the same extent in the visual modality. However, our results demonstrate that the effectiveness of different types of FFI techniques in enhancing the salience and processing of these forms can vary as a function of modality of input presentation (i.e., aural or visual). Here, VG was effective across modalities, but VS was only advantageous in the visual modality. These findings should be considered in the design of optimal L2 instruction programs. Here, we only examined one specific type of construction, that of verb-tense morphology. We do not believe that the findings in this study will necessarily be true for all linguistic constructions. As the literature on FFI shows, different forms will require different levels of explicitness and explanation (Long, 2006, chap. 5; Spada and Tomita, 2010; Tolentino and Tokowicz, 2014). Taken together, these findings demonstrate a range of effects of salience in L2 acquisition. Morphological forms are less well-attended than lexical forms. We believe that this reflects a combination of their relative psychophysical slightness in comparison to lexical cues, as well as effects of learned attention and blocking. There are effects of modality – attention to morphological cues is more effective from visual than from aural input. There are effects of instruction – form-focusing techniques such as grammar instruction, verb pretraining, or enhancing the salience of forms through typological or prosodic enhancement, can increase attention to these forms and increase processing. There are interactions between the effectiveness of FFI and modality such that typological salience enhancement from visual input is effective, while prosodic enhancement from aural input is not. Finally, brief EGI prior to language exposure is an effective means of raising the salience of otherwise ignored cues and turning input into intake. In learning a second language, some attention to form is necessary, and the forms that need to be attended are often the least salient in the input. Successful L2 acquisition rests on attention-focusing manipulations which raise the significance of otherwise non-salient cues. Author Contributions The Abstract, Introduction and Discussion Sections were all contributed to equally by both authors. The methods section was written up by MC-V and edited by NE. The results were analyzed/written up by MC-V and edited by NE. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE 1256260. 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==== Front Front PsycholFront PsycholFront. Psychol.Frontiers in Psychology1664-1078Frontiers Media S.A. 10.3389/fpsyg.2016.01278PsychologyOriginal ResearchAn fMRI Study of the Impact of Block Building and Board Games on Spatial Ability Newman Sharlene D. *Hansen Mitchell T. Gutierrez Arianna Department of Psychological and Brain Sciences, Indiana UniversityBloomington, IN, USAEdited by: Natasha Kirkham, Cornell University, USA Reviewed by: Vanessa R. Simmering, University of Wisconsin-Madison, USA; Amy S. Joh, Seton Hall University, USA *Correspondence: Sharlene D. Newman sdnewman@indiana.eduThis article was submitted to Developmental Psychology, a section of the journal Frontiers in Psychology 29 8 2016 2016 7 127828 4 2016 10 8 2016 Copyright © 2016 Newman, Hansen and Gutierrez.2016Newman, Hansen and GutierrezThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Previous studies have found that block play, board games, and puzzles result in better spatial ability. This study focused on examining the differential impact of structured block play and board games on spatial processing. Two groups of 8-year-old children were studied. One group participated in a five session block play training paradigm and the second group had a similar training protocol but played a word/spelling board game. A mental rotation task was assessed before and after training. The mental rotation task was performed during fMRI to observe the neural changes associated with the two play protocols. Only the block play group showed effects of training for both behavioral measures and fMRI measured brain activation. Behaviorally, the block play group showed improvements in both reaction time and accuracy. Additionally, the block play group showed increased involvement of regions that have been linked to spatial working memory and spatial processing after training. The board game group showed non-significant improvements in mental rotation performance, likely related to practice effects, and no training related brain activation differences. While the current study is preliminary, it does suggest that different “spatial” play activities have differential impacts on spatial processing with structured block play but not board games showing a significant impact on mental rotation performance. spatial processinggame playfMRIblock buildingmental rotation ==== Body Introduction Play is an important way that young children learn (Singer et al., 2006). Playing with spatial toys and engaging in spatial activities may prove to be an essential part of the development of spatial thinking. There are a number of studies that have related spatial play with spatial skill (Levine et al., 2012; Jirout and Newcombe, 2015) and number processing (Cheng and Mix, 2014; Verdine et al., 2014; Casey et al., 2015). For example, in a recent study by Jirout and Newcombe (2015) a large group of 4- to 7-year-old children were studied. There it was found that those who frequently participated in block play, puzzles, and board games had higher spatial ability than those who participated more in other activities like drawing, playing with sound-producing toys, trucks, and riding bikes. While studies seem to suggest a relationship between games like block building, board games, and puzzles to spatial processing, there are few studies that have explored the differential impact of these spatial games on spatial processing. There is some indication that they are not equivalent. One of the very few studies directly comparing spatial games examined their impact on mathematics. Cheng and Mix (2014) examined the effect of two types of spatial training (mental rotation and puzzles) in 7-year-old children. Both mental rotation and puzzle play have been suggested to impact spatial skill. They found that only mental rotation training resulted in improvements in performance on missing-term mathematics problems (e.g., 2 + __ = 7); but mental rotation training failed to improve place-value understanding. The differential effect of puzzles and mental rotation was observed on mathematics and not directly on spatial thinking. However, the results observed by Cheng and Mix demonstrate that different types of spatial training have different consequences and further that different types of spatial play may have a different impact on spatial processing. The current study is a preliminary examination of the differential neural and behavioral impact of playing a structured block building and board game on spatial processing. The effect of a 5 day, 30 min per day training, with 8-year-old children was examined using both behavioral and functional magnetic resonance imaging (fMRI). Play was used here as it is an activity that children engage in regularly. We hypothesized that while both block play and board games may result in improvements in some aspects of spatial thinking, the impact of these two games will vary, as shown by Cheng and Mix (2014). Both games examined are commercially available games—Blocks Rock! and Scrabble. Block play Block play has garnered a great deal of attention in terms of its potential link to spatial thinking (Casey et al., 2008). There are at least two key types of spatial skills closely related to block building—spatial visualization and mental rotation. Spatial visualization involves mentally combining objects to produce designs. As an individual is working with blocks, he or she is mentally visualizing how blocks will fit and interact with one another. Also the blocks are three-dimensional objects. Understanding how these complex objects fit together and relate to each other in designs that can be built either flat on the table or up off of the table provides additional spatial perspective to improve visualization. The second skill, mental rotation, involves mentally visualizing what an object will look like after it is rotated (Casey et al., 2008). Piaget and Inhelder (1971) proposed that children under seven were unable to perform mental rotation or dynamic imagery. However, more recent work has found that this proposal is incorrect. Mental rotation develops in infancy (Quinn and Liben, 2008; Moore and Johnson, 2011). Additionally it has been shown that manually interacting with objects improves a child's ability to mentally rotate it (Möhring and Frick, 2013; Frick and Wang, 2014) with infants who are mobile having better mental rotation ability. This suggests that physically interacting with the blocks during block play may also be an important aspect of the game. Although, many preschool and elementary programs as well as homes have block toys, how these toys are played with has an impact on whether and how spatial skills are developed. Two types of block play have been considered, free play where children are provided blocks and they create designs, and structured block play in which children build a model of a structure (Verdine et al., 2014). It is structured block play that has been suggested to require the analysis of a spatial representation and that may result in more significant improvements in spatial ability. By spatial analysis we mean the ability to specify the parts and the overall configuration of an object and to understand how the parts are related to form a whole. It includes the ability to segment an object into parts and to integrate those parts into a coherent whole (e.g., Delis et al., 1986, 1988; Stiles and Stern, 2009). As mentioned above, the blocks are 3D and the structures built are 3D. During structured block play a 2D picture of a structure is copied. In other words, a 2D-3D spatial transformation is performed. Training in 2D-3D spatial transformations has been found to improve mental rotation performance in girls (Tzuriel and Egozi, 2007, 2010). This suggests another mechanism by which structured block play may improve spatial processing. Again, while classrooms may have block building activities, there is not enough structured play for children to greatly enhance spatial learning (Casey et al., 2015). Casey et al. (2015) suggests that “if this skill were taught in a more systematic way in the early childhood classroom, it might have the potential to further develop spatial reasoning.” The primary goal of the current study was to further explore the impact of structured block play on spatial ability in young elementary school children. Board games In addition to block play, board games, and puzzles have also been linked to improved spatial processing (Ramani and Siegler, 2008; Siegler and Ramani, 2009; Jirout and Newcombe, 2015). For example, Siegler and Ramani have shown that number knowledge is improved in preschoolers who played a linear number-based board game like Chutes and Ladders. They also reported that the gains in number knowledge persisted. There are a number of different types of board games with some focusing on counting while others, like Scrabble, focusing on spelling. Board games that focus on spelling are an interesting category because Jirout and Newcombe (2015) found that playing word and spelling games with parents had a marginally significant effect on Block Design score. A possible explanation for such a relationship is that the spatial relationships between letters are extremely important for spelling. For example, “biek” is not a word while “bike” is because the spatial relationship between the “k” and “e” is important. Spelling games like Scrabble also use spatial language (e.g., building words up or down) which draws the child's attention to spatial relationships. Additionally, Scrabble, like structured block play provides a similar sensory-motor experience due to the hand-eye movements necessary to manipulate pieces (letters) and place them on the board. This sensory-motor experience has been found to be important to spatial processing (Ballard et al., 1992). While there are some similarities between structured block play and board games like Scrabble, there are also differences. For example, structured block play requires the building of complex spatial configurations and more explicitly focuses on spatial analysis and spatial working memory. Scrabble, on the other hand, focuses on word creation from a jumbled set of letters. Therefore, while Scrabble has spatial components (e.g., words are spatially organized letters and the use of spatial language), it does not require spatial working memory processes related to holding a non-verbal spatial configuration in short-term memory. This difference predicts that different brain regions will be recruited during structured block play and Scrabble. One such region is the parahippocampus which has been linked to spatial memory (Johnsrude et al., 1999; Bohbot et al., 2000; Burgess et al., 2001). By recruiting spatial processing regions like the parahippocampus during structured block play these regions begin to develop their processing strategy which may result in differential involvement during mental rotation before and after structured block play training. Current study Based on previous research the hypothesis tested here is that structured block play will result in greater spatial processing gains than board games and will therefore have different behavioral and neural consequences. Greater gains in spatial processing for block play are thought to be due to its emphasis on spatial analysis; this includes determining the spatial relationships between parts and its emphasis on spatial working memory. To test this hypothesis we employed a combination of behavioral and neuroimaging methods. A 2D letter mental rotation task was used to test spatial processing. Mental rotation is a test of spatial visualization and analysis which has long been used as a measure of spatial processing ability. Because of the age of our population (8-years) the complexity (2D) and familiarity (letters) of the objects to be rotated were simplified (Kosslyn et al., 1990; Lütke and Lange-Küttner, 2015). By simplifying the stimuli the objects to be rotated were easier to encode and identify (Bialystok, 1989). It was also important to use stimuli different from the blocks used during block play. There has been some debate regarding whether spatial training transfers to other tasks and stimuli (Kail, 1986; Kail and Park, 1990). Scrabble may be expected to have an advantage during mental rotation here due to the use of letter stimuli; therefore a greater increase in performance by the block play group will demonstrate that the training not only transfers to another task but also to other object stimuli. Methods Participants Thirty-six (male = 21) 8-year-old children participated in this study (see Table 1 for demographic information). All were typically developing children with no history of neurological disorders. Six participants were excluded due to excessive motion in the scanner (>5 mm) and three were lost to attrition, leaving 28 (male = 15) total usable imaging participants. Of the 28, 14 (male = 8) were placed in the block play group and 14 (male-7) were placed in the board game group. Parents completed a short survey regarding their child's play behavior and parental education level (see Appendix). Participants had a variety of block building experiences (e.g., playing with Legos) prior to this study (information was obtained from parental survey); as such the two groups were equated on spatial play. Therefore, the groups were balanced on gender, age, mathematics test score, parental education and the amount of previous spatial play. Parental consent and child assent were both obtained prior to the first experimental session, in accordance with the Indiana University Institutional Review Board. Table 1 Demographic information. Block play Board game t-value Age (years) 8 years; 3 months ± 0.29 8 years; 2 months ± 0.17 <1 % Female 46% 43% Math test 53% ± 25% 47% ± 24% <1 Parent education Bachelor's degree Bachelor's degree Age, the age of the children; % Female, the proportion of subjects in each group who were girls; Math test, score on an abbreviated test of mathematics ability; Parent education, average level of education of the participant's parents ranging from high school to graduate/professional school. Experimental design The participants took part in seven sessions, all on separate days. The first and last sessions were pre- and post-training evaluations. The middle five sessions were the training sessions. The mean number of days between the first and final session was similar for both groups (blocks play: 12.7 ± 4.4; Scrabble: 12 ± 6.5; p = 0.36). MRI scanning was performed during the pre- and post-training sessions. All sessions took place in the Department of Psychological and Brain Sciences at Indiana University. Pre-training Parents completed a survey to obtain information regarding the child's prior play activities, musical training, and number/mathematics activities as well as demographic information and history of learning disorders (see Appendix for survey details). The groups were constructed in an attempt to equate them on these measures. Musical training, mathematics skill, gender as well as socio-economic status, for which parental education is a proxy, have all been linked to spatial processing. Mathematics competency was assessed by using a subset of questions from the Grade 2 Mathematics California Standards Tests from 2003 to 2007. Questions from the Number Sense and Algebra and Functions sections were used. Participants were given 15 min to complete as many of the 24 questions as possible. Parental education was averaged within each group. The scanner task was a mental rotation task. A pair of letters was presented. The letter on the left was oriented in a normal upright position, and the letter on the right was either rotated and non-mirrored or rotated and mirrored. The angle of rotation varied from 30 to 180° (see Figure 1). Easy problems had an angle of rotation that was 90° or less. If the right letter was only rotated, the letter pair was considered the same and the participant pressed a button with their right index finger. If the right letter was rotated and mirrored then the pair was considered different and they pressed a button with their left index finger. Half of the trials were same and half were different. A block design was employed. Each block contained six letter pairs, and the blocks were separated by 12 s fixation periods. The blocks contained a mixture of same and different pairs and angles of rotation. If the participant did not answer a trial within 10 s, the program moved to the next trial. There were 24 s fixation periods to start and end the mental rotation task. The duration of the mental rotation task was 360 s. Accuracy and reaction time were recorded. Figure 1 Example stimuli. Both upper and lower case letters were presented. The difficulty was manipulated by varying the angle of rotation (left = easy, low degree of rotation and right = hard, large angle of rotation). The pre-training mental rotation performance was also used to match groups. A more lenient one-tailed t-test was used to compare performance across groups. Prior to training the groups showed no differences in RT for easy (p = 0.2), hard (p = 0.28), or when they were combined (p = 0.47) on the mental rotation task. There were also no differences in accuracy for easy (p = 0.37), hard (p = 0.11), or when they were combined (p = 0.14). Training Participants were separated into one of two groups—block play with the game Blocks Rock! and a board game, Scrabble. Both games are commercially available and both games are competitive games in which two players interact. The Blocks Rock! game has a set of cards, two identical sets of blocks of varying shape, size, and color and a bell. Each player has a set of blocks and one player turns over a card during play that has a particular structure, point value, and how to build the structure (e.g., up or flat on the table). The complexity of the structure increases during play. Each player attempts to build the structure as fast as possible with the player who does so correctly first and rings the bell being awarded the points displayed on the card. The score is kept and once all cards have been played the winner is the player with the most points. Scrabble is a popular competitive word game and the standard rules were used during play. During each training session, participants played either Blocks Rock! or Scrabble for 30 min with either another participant matched for skill level or a research assistant who adjusted their play to match the subject. Score was kept for each training session for motivational purposes. Post-training Participants played the same game from their training sessions for 15 min before completing the MRI portion of the session. The MRI protocol for the post-training session was identical to that of the pre-training session. Imaging parameters Participants underwent MRI scanning using a 12-channel head coil and a Siemens 3T Tim Trio MRI scanner. The first scan was an anatomical T1-weighted scan used to co-register functional images. An MPRAGE sequence (192 sagittal slices; FOV = 256 mm, matrix = 256 × 256, TR = 1800 ms, TE = 2.67 ms, TI = 900 ms, flip angle = 9°, slice thickness = 1 mm, resulting in 1 × 1 × 1-mm voxels) was used. The experimental functional scan was a multiband EPI scan (33 axial slices using the following protocol: field of view = 192 mm, matrix = 128 × 128, iPAT factor = 2, TR = 2000 ms, TE = 30 ms, flip angle = 60°, slice thickness = 3.8 mm, 0 gap). Data analysis fMRI data were analyzed with SPM8 (Wellcome Trust Centre for Neuroimaging; http://www.fil.ion.ucl.ac.uk/spm). fMRI data were preprocessed in several steps including slice timing correction, motion correction by realignment, co-registration between functional and anatomical scans, spatial normalization, and smoothing. All functional data were resampled to 2 mm3 isomorphic voxels normalized to the Montreal Neurological Institute (MNI) template. For spatial smoothing an 8 mm FWHM Gaussian kernel was applied. On the preprocessed fMRI data of individual subjects, a canonical statistical analysis based on the general linear model (GLM) and Gaussian random field theory was performed (Friston et al., 1995). The hemodynamic response for the stimuli blocks were modeled with a canonical HRF built on the onsets of the blocks with the block duration included in the analysis. For each individual data analysis, regressors were built for the mental rotation blocks, fixation blocks, and six regressors from the realignment step were included in the model to remove unexpected effects from noise from head movement. In order to examine the activation related to mental rotation at each time point and for each group contrast images for mental rotation compared to fixation were computed. This was performed to allow for inspection of the results prior to group comparisons to ensure analysis quality. Next the effect of training was examined for each group separately. The mental rotation minus fixation contrasts for each timepoint were analyzed using a paired t-test to compare the pre- and post-training activation for each group. Additionally, to examine activation differences between groups at each timepoint one-sample t-tests were performed (e.g., block play groups minus board game group for the post-training scan). For the contrasts examined we applied a Monte Carlo simulation of the brain volume to establish an appropriate voxel contiguity threshold. The threshold obtained from the simulation has the advantage of higher sensitivity to smaller effect sizes (Slotnick and Schacter, 2004). The result of the Monte Carlo simulation indicated that a cluster size of 20 contiguous resampled voxels using an uncorrected threshold of p < 0.005 would be appropriate to control type I error, at p < 0.05 corrected for the multiple comparisons in the whole brain volume analysis. Results Behavioral (reaction time) A 2 (block play vs. board game) × 2 (pre vs. post) × 2 (easy vs. difficult) between subjects ANOVA was performed on the RT data using SAS 9.4. The results showed a significant effect of training [F(1, 108) = 3.78; p = 0.054; η2 = 0.034]; and difficulty [F(1, 108) = 10.5; p = 0.002; η2 = 0.089]. Although none of the interactions were found to be significant, because our a priori hypothesis was that the block play group would show an effect of training but not the board game group and because there was a trend of an interaction between group and training for RT (see Figure 2), we examined each group separately using a within-subjects ANOVA. The block play group showed a significant effect of training [F(1, 14) = 8.92; p = 0.0098; η2 = 0.083] and a significant effect for difficulty [F(1, 14) = 771.76; p = 0.023; η2 = 0.067]. The interaction between training and difficulty was not significant. The board game group failed to show an effect of training [F < 1]. An effect of difficulty was also not observed [F(1, 13) = 5.12; p = 0.26; η2 = 0.11]. Also, the board game group showed an interaction between training and difficulty [F(1, 13) = 5.75; p = 0.032; η2 = 0.024]. See Supplemental Data for complete behavioral statistics. Figure 2 Mental rotation behavioral results. There is a main effect of difficulty and training. While both groups showed some improvements in performance post training, only the block play group showed significant RT and accuracy improvements after training. Error bars depict standard error. Behavioral (accuracy) The same 2 (block play vs. board game) × 2 (pre vs. post) × 2 (easy vs. difficult) between subjects ANOVA was performed on the accuracy data. The results showed a significant effect of training [F(1, 108) = 5.06; p = 0.027; η2 = 0.045], difficulty [F(1, 108) = 15; p = 0.0002; η2 = 0.12]; and a trending effect of group [F(1, 108) = 3.36; p = 0.069; η2 = 0.03]. Although none of the interactions were found to be significant, because our a priori hypothesis was that the block play group would show an effect of training but not the board game, we examined each group separately using a within-subjects ANOVA. The block play group showed a significant effect of training [F(1, 14) = 5.75; p = 0.031; η2 = 0.0422] and a trending effect of difficulty for accuracy [F(1, 14) = 76.71; p = 0.072; η2 = 0.16]. The interaction between training and difficulty was not significant. The board game group failed to show an effect of training [F(1, 13) = 3.51; p = 0.084; η2 = 0.048] while the effect was trending. An effect of difficulty was observed for accuracy [F(1, 13) = 89,401; p = 0.0021; η2 = 0.085]. fMRI To examine the effect of training in each group, the post-training minus pre-training contrast was examined. It showed that the block play group had increased activation in the anterior lobe of the cerebellum extending into the right parahippocampus and the bilateral fusiform gyrus (see Figure 3; Table 2) after block play training than prior to training. The board game group failed to show any significant activation when comparing the pre- and post-scans, analogous to the behavioral finding of no effects of training. Figure 3 The post- minus pre-training contrast for the block play group. Increased activation is observed in the anterior lobe of the cerebellum, the parahippocampus and the fusiform gyrus after training for the block play group. Table 2 Activation details for significant activation clusters. Region BA k t MNI coordinates x y z BLOCK PLAY (POST- MINUS PRE-TRAINING) Right cerebellum anterior lobe 590 4.92 16 −32 −10 Right cerebellum anterior lobe 4.81 20 −30 −20 Left cerebellum anterior lobe 4.26 0 −38 −8 Left fusiform gyrus 37 34 3.53 −36 −54 −10 Left fusiform gyrus 37 3.4 −36 −46 −12 Right fusiform gyrus 37 20 3.36 36 −42 −8 POST-TRAINING (BLOCK PLAY GROUP MINUS BOARD GAME GROUP) Left medial frontal gyrus 9 37 3.73 −8 50 32 Left precentral gyrus 6 13 3.14 −44 −6 24 BA, Broadman's area; k, cluster extent; t, t-test; x, y, z are coordinates in standardized space. The direct comparison of the block play and board game group was also examined for the post-training scan (Figure 4). The results showed that the block play group elicited greater activation in the medial prefrontal cortex and the left precentral gyrus (at a lower extent threshold) than did the board game group after training. There was no difference pre-training difference between groups. Figure 4 The increased activation for the block play group compared to the board game group after training. The increased activation is observed in the medial prefrontal cortex and the precentral gyrus. Discussion The goal of this preliminary study was to examine the impact of two very different games that have both been suggested to impact spatial reasoning skills—block play and board games. As predicted and as suggested by previous work, both block play and board games were found to result in performance improvements, although in different ways. It should be noted that some improvement may be due to practice effects (performing the same task twice). The same practice effects would be expected to be observed for both groups. While, on average both groups did show faster reaction times and increased accuracy after training, only the block play group showed significant training effects. The neuroimaging results mirrored the behavioral data in that only the block play group showed significant changes in brain activation after training. Together these results provide some support for a differential effect of board games and block play on spatial processing. Block play has been shown to impact spatial ability in children. In a recent study that examined 847 4- to 7-year-old children it was found that spatial play, including block building and playing with puzzles and board games, was associated with increased spatial ability (Jirout and Newcombe, 2015). Here we found that 5, 30-min structured block play sessions resulted in changes to the neural network responsible for mental rotation as well as increased the speed and accuracy of mental rotation performance. Structured block play in which children build a given structure requires the ability to analyze a spatial representation. It is thought to develop skills in estimation, measurement, patterning, part-whole relations, visualization, symmetry, transformation, and balance (Casey and Bobb, 2003; Stiles and Stern, 2009; Verdine et al., 2014). Blocks Rock! is precisely this type of structured block play game as it requires players to construct a specified structure as accurately and fast as possible. Therefore, it not only encourages accuracy but speed in analyzing and then building the structures. It was predicted that block play would impact spatial processing; however the specific aspects of spatial processing impacted was not known. The imaging data may provide some hints. Block play training resulted in increased activation in the parahippocampal gyri, cerebellum, and the fusiform gyri. These regions have all been implicated in different aspects of spatial processing (Aguirre et al., 1996; Johnsrude et al., 1999; Aminoff et al., 2007; Stoodley et al., 2010). The parahippocampus has been linked to spatial memory encoding (Johnsrude et al., 1999; Bohbot et al., 2000; Burgess et al., 2001); and spatial navigation (Aguirre et al., 1996; Mellet et al., 2000), particularly the posterior aspect of the region as found here (Aminoff et al., 2007). There cerebellum has also been linked to both spatial and motor processing (Shen et al., 1999; Stoodley et al., 2012) and sensory motor integration (Bastian, 2006; Wiestler et al., 2011), while the fusiform gyrus has been linked to visual object recognition (Chao et al., 1999). All of these processes are involved in structured block play. For example, structured block play is analogous to block copy tasks that have a long history of use in neuropsychology research. Ballard et al. (1992) detailed the intricate hand-eye coordination and spatial memory strategy required to perform a block copy task. In that study it is was found that participants memorized sequences of moves then executed those moves in an iterative pattern moving back and forth between fixating and memorizing and fixating and executing the movement. This requires spatial working memory, sensory-motor processing, and visual object processing. In fact, a similar process is thought to be involved in the solution of a visuospatial problem solving task, the Tower of London (Owen, 2005) with neuroimaging studies of the task showing the involvement of these same brain regions (Dagher et al., 2001; Rowe et al., 2001; Newman et al., 2003). There are at least two mental rotation strategies that have been identified. One is the holistic strategy in which the image is mentally rotated as a whole, while the other is a piecemeal or viewpoint independent strategy that involves the analysis of the internal relations of image parts (Khooshabeh et al., 2013). Motor simulation is more strongly linked to the holistic strategy. Interestingly, the holistic strategy has been found to be faster and one used by individuals with better visuospatial ability (Cooper, 1975). This suggests that the block play training likely reinforced the holistic strategy resulting in increased motor simulation, as suggested by increased activation of the cerebellum. Further support for this hypothesis comes from the finding that the block play group showed increased activation in the precentral gyrus after training compared to the board game group. The role of the precentral cortex in mental rotation has largely been suggested to be related to motor simulation due to the region's link to motor planning (Cohen and Bookheimer, 1994; Zacks, 2008). In sum, the block play group shows a change in activation in regions linked to both motor and spatial processing while the board game group showed no changes in brain activation. This result raises the possibility that the block play group changed how they were performing the mental rotation task after training. However, it is unclear whether there was a strategy change or whether block play resulted in refining an existing strategy. Further research is required to explore this question. The board game used here was Scrabble. Previous research has shown that, like block play, board games including word/spelling games may result in improvements in spatial processing (Verdine et al., 2014; Jirout and Newcombe, 2015). This may be particularly true for Scrabble because, as mentioned earlier, spelling is spatial in that the spatial relationships between the letters are important. Additionally, spatial language is used during game play (e.g., up and down). Also, Scrabble may be expected to have an advantage here given that the game involves viewing letters (the same stimuli used in the mental rotation task) in words where the alignment of the letters vary on a different axis on a board, or 2D plane. Lange-Küttner (2009) found that 7-year-old children were sensitive to the spatial axis of the frame in a drawing task; therefore, the practice that Scrabble provides in viewing and rotating words along the x-y axis may be expected to impact spatial processing. However, the board game group here failed to show brain activation changes as a result of training and the improvements in behavioral performance were just under the significance threshold. One possible explanation for the failure to show an effect is the absence of 2D-3D transformation and this absence of letter rotation made the Scrabble game the less powerful training game despite its multiple spatial attributes. While Scrabble failed to show significant improvements in mental rotation, the hypothesis that board games improve spatial processing cannot be ruled out here. It could be that board game play does benefit spatial processing, but not the processes recruited during mental rotation. Scrabble also has a numerical component with an emphasis on counting. For example, available places must be counted once the game has advanced and many places are occupied. Also free places must be counted to be able to fit the intended word into the empty space. This emphasis on counting and getting objects (words) to fit within a given context may facilitate different spatial processes than block play. Additionally, counting may also recruit verbal working memory resources instead of spatial working memory. Further research that uses a longer training, a larger sample, and/or different spatial tasks are required to determine the precise character of the spatial processing impacted. Future directions and limitations The results from this preliminary study are very promising and suggest that structured block play may be an important tool to help improve spatial processing. However, the results are preliminary in that the number of subjects is rather small. With a larger sample size the behavioral results may become more robust. Additionally, the training period was relatively short. This short training period may have caused the behavioral effects to be small, particularly for the board game group. Also, Blocks Rock! rewards players who complete the structure fastest; therefore the speed of the block building game may have resulted in the children being more attentive. This increase in attentiveness may have made the effects more salient for block play. Further research is necessary with a larger sample and a more extensive training protocol to confirm the results. The age of the children examined here was chosen based on the literature regarding the development of mental rotation and spatial processing ability (Piaget and Inhelder, 1971; Bialystok, 1989; Kosslyn et al., 1990). It seems that differences in mental rotation appear around 7-years. Also, younger children have difficulty with the types of mental rotation tasks that are typically used in MRI environments. However, it would be important to examine how structured block play may differentially impact mental rotation performance at different developmental stages. Here we found increased activation due to training instead of a decrease in activation that has been previously linked to greater efficiency. We hypothesize that this increase may be related to the age of the participants. In this study the children are still developing mental rotation skills. Therefore, they start with very inefficient strategies that must be improved or even developed. Once the strategy becomes more entrenched and more efficient decreased levels of activation are expected. A longitudinal study in which cognitive ability including spatial ability and spatial working memory is assessed to determine the developmental consequences of block play is necessary to fully characterize the impact block play on spatial ability. Finally, we failed here to obtain a comprehensive assessment of spatial processing ability in our sample prior to training. Obtaining a measure that explores different tasks and objects with varying complexity including 2D and 3D objects (e.g., the rotated color cube test, Lütke and Lange-Küttner, 2015) is necessary. In addition, using unfamiliar objects may also be important as the use of letters may strongly activate the what visual system and interfere with the rotation decision (Lange-Küttner and Küttner, 2015). Some support for this idea comes from a previous mental rotation practice study by Kail and Park (1990). There participants completed 3360 trials of mental rotation on letters and found that rotation experience did not transfer to other objects. It was suggested that the benefits of letter rotation training failed to transfer to other objects because a memory/instance based strategy was used instead of changes to the mental rotation process; therefore, activating the what visual system. Some evidence may also be observed here in that post-training activation for the block play group showed increased activation of the fusiform gyrus—part of the what processing system—as well as spatial processing regions even though letter stimuli were not included in the block play training. In any case, it may be important in future studies to examine both familiar and unfamiliar objects in the mental rotation task. Conclusions There has been some debate in the literature regarding whether training on one visuospatial task transfers to other tasks. The results presented here suggest that they can. Here training on a speeded, structured block building game, Blocks Rock!, resulted in transfer to mental rotation performance. While it appears that both the block play and board games resulted in some improvements in mental rotation performance the improvements were greater for the block play group. The findings have important implications on child play activities. Given the importance of spatial thinking to success in science, technology, engineering, and mathematics (Newcombe, 2010), using games like structured block building may prove to be important for helping to set a solid foundation. Ethics statement All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Author contributions SN is the PI and was responsible for most of the writing. MH and AG were responsible for the data collection and analyses and contributed to the writing. Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. SN has received research grants from the LaCrosse Family Business Trust. This work supported by the LaCrosse Family Business Trust. The authors would also like to thank Morgan Newman, Adrian Paneto, David Raymond, Amanda Tinkey, Shreya Hurli, and Marissa Malaret. Supplementary material The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpsyg.2016.01278 Click here for additional data file. Click here for additional data file. ==== Refs References Aguirre G. K. Detre J. A. Alsop D. C. D'Esposito M. (1996 ). The parahippocampus subserves topographical learning in man . Cereb. Cortex 6 , 823 –829 . 8922339 Aminoff E. Gronau N. 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==== Front Front Plant SciFront Plant SciFront. Plant Sci.Frontiers in Plant Science1664-462XFrontiers Media S.A. 10.3389/fpls.2016.01283Plant ScienceOriginal ResearchCan Adverse Effects of Acidity and Aluminum Toxicity Be Alleviated by Appropriate Rootstock Selection in Cucumber? Rouphael Youssef 1Rea Elvira 2†Cardarelli Mariateresa 2Bitterlich Michael 3Schwarz Dietmar 3Colla Giuseppe 4*1Department of Agricultural Sciences, University of Naples Federico IINaples, Italy2Centro di Ricerca per lo Studio delle Relazioni tra Pianta e Suolo, Consiglio per la Ricerca in Agricoltura e l'Analisi dell'Economia AgrariaRome, Italy3Leibniz Institute of Vegetable and Ornamental CropsGroßbeeren, Germany4Department of Agricultural and Forestry Sciences, University of TusciaViterbo, ItalyEdited by: Elena Prats, Spanish National Research Council, Spain Reviewed by: Paolo Sambo, University of Padua, Italy; Spyridon Alexandros Petropoulos, University of Thessaly, Greece *Correspondence: Giuseppe Colla giucolla@unitus.itThis article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science †In Memoriam: This article is dedicated to our deceased friend and colleague Dr. Elvira Rea, scientist in plant physiology and plant nutrition. She was a close and helpful collaborator over many years and made a significant contribution to this research. 29 8 2016 2016 7 128329 5 2016 11 8 2016 Copyright © 2016 Rouphael, Rea, Cardarelli, Bitterlich, Schwarz and Colla.2016Rouphael, Rea, Cardarelli, Bitterlich, Schwarz and CollaThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Low-pH and aluminum (Al) stresses are the major constraints that limit crop yield in acidic soils. Grafting vegetable elite cultivars onto appropriate rootstocks may represent an effective tool to improve crop tolerance to acidity and Al toxicity. Two greenhouse hydroponic experiments were performed to evaluate growth, yield, biomass production, chlorophyll index, electrolyte leakage, mineral composition, and assimilate partitioning in plant tissues of cucumber plants (Cucumis sativus L. “Ekron”) either non-grafted or grafted onto “P360” (Cucurbita maxima Duchesne × Cucurbita moschata Duchesne; E/C) or figleaf gourd (Cucurbita ficifolia Bouché; E/F). Cucumber plants were cultured in pots and supplied with nutrient solutions having different pH and Al concentrations: pH 6, pH 3.5, pH 3.5 + 1.5 mM Al, and pH 3.5 + 3 mM Al (Experiment 1, 14 days) and pH 6, pH 3.5, and pH 3.5 + 0.75 mM Al (Experiment 2, 67 days). Significant depression in shoot and root biomass was observed in response to acidity and Al concentrations, with Al-stress being more phytotoxic than low pH treatment. Significant decrease in yield, shoot, and root biomass, leaf area, SPAD index, N, K, Ca, Mg, Mn, and B concentration in aerial parts (leaves and stems) in response to low pH with more detrimental effects at pH 3.5 + Al. Grafted E/C plants grown under low pH and Al had higher yield, shoot, and root biomass compared to E/F and non-grafted plants. This better crop performance of E/C plants in response to Al stress was related to (i) a reduced translocation of Al from roots to the shoot, (ii) a better shoot and root nutritional status in K, Ca, Mg, Mn, and Zn concentration, (iii) a higher chlorophyll synthesis, as well as (iv) the ability to maintain cell membrane stability and integrity (lower electrolyte leakage). Data provide insight into the role of grafting on Al stress tolerance in cucumber. Al tolerancechlorophyll indexCucumis sativus L.graft combinationmembrane stabilitymineral compositionpH level ==== Body Introduction Acidic soils represent 50% of the earth's arable land (Inostroza-Blancheteau et al., 2012), and in those soils aluminum (Al) toxicity is the main factor restricting crop productivity (Kochian, 1995). In acidic soils with pH lower than 5, Al-containing minerals (e.g., aluminosilicates) are solubilized in the phytotoxic form Al3+ (Kochian, 1995; Seguel et al., 2013). The root is the first organ feeling Al3+ toxicity, thus conditioning stress sensitivity and hampering crop productivity (Delhaize and Ryan, 1995; Rengel et al., 2015). For many plant species, Al concentration in the range of 1–2 mg L−1 can inhibit root elongation by damaging the cell structure of the root apex and thus influencing water and nutrient uptake (Kochian et al., 2015; Rengel et al., 2015). Al also interferes with cell membrane stability, enzyme function, and the synthesis of chlorophyll (Simon et al., 1994; Rouphael et al., 2015). Several authors reported that exposure to Al3+ in the rooting medium strongly inhibit Ca2+ and Mg2+ fluxes across the plasma membrane of root cells (Rengel and Elliott, 1992; Horst et al., 2010; Bose et al., 2011). Similarly, Al toxicity can decease or even block the uptake of K+, since it interacts with various different plasma-membrane channel proteins (Kochian et al., 2005). This may decrease cations uptake leading to nutrient deficiencies, thus affecting metabolism and productivity of crops. However, plant species have different degrees of adaptation to Al; these differences are often related to the specific mechanisms developed for mitigating stress (Panda and Matsumoto, 2007). The mechanisms conferring tolerance to Al have been classified into mechanisms of exclusion and those of intracellular tolerance (Kochian, 1995). Exclusion mechanisms not allowing Al entering the symplast can take place by exudation of Al-chelating compounds (e.g., organic acids), raising the pH in the rhizospheric environment and by binding of Al in the cell wall itself (Inostroza-Blancheteau et al., 2012; Brunner and Sperisen, 2013; Kochian et al., 2015). Internal tolerance mechanisms include those that bind the Al entering the root cells as well as the sequestration of Al in subcellular compartments (e.g., vacuoles). To overcome the limitations of Al toxicity several solutions have been proposed. Amendments such as gypsum, lime, and phosphate fertilizers are common agricultural practices used to raise the soil pH causing the Al to become insoluble (Nawrot et al., 2001). Liming is only efficient in the topsoil, while it does not remedy the subsoil acidity (>1 m), since deep incorporation of lime is difficult to realize and very costly (Choudhary and Singh, 2011; Yang et al., 2013). Another possible solution to alleviate the negative effects of acid soils would be the use Al-tolerant cultivars obtained by breeding and/or biotechnological approaches (Choudhary and Singh, 2011). However, the long time needed for the breeding of Al-tolerant cultivars as well as the complexity of the Al-trait make this task extremely difficult. In order to overcome this situation more rapidly, grafting elite vegetable cultivars onto rootstocks tolerating higher Al concentrations and low pH could be a key tool in alleviating the effects of acidity and Al toxicity on crop productivity. By selecting suitable rootstocks, grafting can control scion morphology (Albacete et al., 2015), increase productivity and fruit quality (Colla et al., 2008; Proietti et al., 2008; Kyriacou et al., 2016), improve nitrogen use efficiency (Colla et al., 2010a, 2011), and also induce tolerance against several abiotic stresses, among them nutrient toxicity, heavy metals, and alkalinity (Edelstein et al., 2005, 2007; Rouphael et al., 2008a,b; Colla et al., 2010b; Savvas et al., 2010, 2013; Kumar et al., 2015a). Therefore, we hypothesized that the effectiveness of root genotypes to enhance the uptake of nutrients and to limit the Al accumulation in aerial parts may be improved by grafting onto suitable rootstocks. To verify the above hypothesis, two hydroponic greenhouse experiments were conducted (1) to assess the effects of grafting combinations on morphological traits of cucumber at early development stage in response to different nutrient solution pH and Al concentrations; and (2) to elucidate the agronomical, physiological, and mineral composition changes of cucumber mediated by grafting under acidity and Al toxicity conditions using long-term treatments. Materials and methods Plant material selection and growth conditions A short- and a long-term experiment were performed to characterize the response to acidity and Al toxicity in cucumber (Cucumis sativus L.) either non-grafted or grafted onto vigorous rootstocks. Both experiments were conducted in a polyethylene greenhouse at the experimental station of the University of Tuscia, located in Viterbo (latitude 42°25′N, longitude 12°08′E, altitude 310 m). Inside the greenhouse the mean air temperatures amounted to 25°C varying between 18 and 30°C, while the mean day/night relative humiditiy was 55/80%. In the short-term experiment (Experiment 1), C. sativus L. “Ekron” (E; Enza Zaden, Verona, Italy) was grafted onto the commercial rootstock “P360” (C; Cucurbita maxima × Cucurbita moschata; Società Agricola Italiana Sementi, Cesena, Italy), whereas in the long-term experiment (Experiment 2) cucumber “Ekron” was grafted onto the “P360” and also onto the figleaf gourd (F) (Cucurbita ficifolia Bouché; Società Agricola Italiana Sementi, Cesena, Italy) using the procedure of insertion grafting (Lee et al., 2010). In both experiments the non-grafted “Ekron” was used as control. The Cucurbita hybrid “P360” and the figleaf gourd were selected as the most representative commercial rootstocks used in the Mediterranean basin and Asia, respectively (Lee et al., 2010). In Experiment 1, grafted and non-grafted seedlings were transplanted 20 days after sowing, at the two true-leaf stages into plastic pots of 2 L volume (one plant per pot) filled with quartziferous sand and placed over 16 cm wide and 5 m long benches at a plant density of 11 plants m−2. In Experiment 2, cucumber seedlings were transplanted on 22 May into pots containing 17.7 L of quartziferous sand. Pots were disposed in double rows at a plant density of 2.5 plants m−2, as used commercially for cucumber under greenhouse conditions. Cucumber plants were grown as vertical cordons and trained to the umbrella system as described by Rouphael et al. (2010). Experimental design, treatments, and nutrient solution management Experiment 1 contained eight treatments, derived by the factorial combination of two grafting combinations (grafted E/C and non-grafted E plants) and four nutrient solutions (pH 6.0, pH 3.5, pH 3.5 + 1.5 mM Al, and pH 3.5 + 3.0 mM Al). Any treatment had four replications, amounting to a total of 32 experimental unit plots with eight plants each (n = 256 plants). In Experiment 2, nine treatments, derived by the factorial combination of three grafting combinations (non-grafted E, grafted E/C and E/F) and three nutrient solutions (pH 6.0, pH 3.5, and pH 3.5 + 0.75 mM Al) were compared with four replications, amounting to a total of 36 experimental unit plots (n = 180 plants). Each experimental unit consisted of five plants. Plants were drip-irrigated automatically 2-4 times per day in Experiment 1 and 3–10 times in Experiment 2, to ensure adequate substrate moisture (Colla et al., 2012, 2013). The basic nutrient solution (pH 6.0) was a modified Hoagland and Arnon formulation containing the following macro- and micro-nutrients: 13.0 mM N–NO3−, 1.6 mM S, 0.3 mM P, 4.3 mM K, 4.0 mM Ca, 1.3 mM Mg, 20 μM Fe, 9 μM Mn, 0.3 μM Cu, 1.6 μM Zn, 20 μM B, and 0.3 μM Mo. The low pH (3.5) treatments had the same basic nutrient composition plus HCl which was added to decrease the nutrient solution pH, thus simulating the effects of acidity. The Al treatments were generated by adding AlCl3·6 H2O to the basic nutrient solution. The stressed treatments (pH 3.5 and pH 3.5 + Al) started at transplanting in Experiment 1, and 7 days after transplanting in Experiment 2. Yield assessment and growth measurements In Experiment 2, fruits of all plants were harvested manually 2–3 times per week from 16 June to 25 July. The number of fruits per plant, the mean fruit weight, and the marketable yield were recorded. At final harvest, all plants were separated into leaves, stems and roots, and dried in a forced-air oven at 80°C for 72 h for biomass determination. Root-to-shoot ratio was calculated by dividing root dry weight by the sum of leaf and stem dry weights. The final leaf area was also measured with an electronic leaf area meter (Delta-T Devices Ltd., Cambridge, UK). Fruit quality analysis On 1 July (43 days after transplanting), five fruits were sampled from each plot for quality analysis. Fruit shape index (SI) was defined by the ratio of equatorial and longitudinal lengths. Fruit firmness was measured using a penetrometer (Bertuzzi FT 011; Brugherio, Milan, Italy), fitted with an 8 mm-diameter round-head probe. A part of the homogenate prepared under low speed and filtered under cheesecloth was used for determining the total soluble solid content at 20°C using an Atago N1 refractometer (Atago Co. Ltd., Japan). The pH of the juice was measured with a pH electrode (HI-9023, Hanna Instruments, Padova, Italy). The titratable acidity of the juice, expressed as % w/v malic acid content, was determined by titration of an aliquot of 25 mL with 0.1 M NaOH to a pH endpoint of 8.1 on an automatic titrator. Fruit dry matter percentage was also determined by drying a part of the homogenate in a forced air oven at 8°C for 72 h. Analysis of aluminum and mineral nutrient concentrations in plant tissues The dried plant tissues: leaf, stem, fruit, and root tissues were ground in a Wiley mill to pass through a 20-mesh screen, then 0.5 g samples were analyzed for the following macro- micro-nutrients and toxic element: N, P, K, Ca, Mg, Fe, Mn, Zn, B, and Al. Nitrogen (total N) concentration in the four plant tissues was determined after mineralization with sulfuric acid (H2SO4, 96%, Carlo Erba Reagents, Cornaredo, Milan, Italy) in the presence of potassium sulfate (K2SO4) and a low concentration of copper (Cu) according to the Kjeldahl method (Bremner, 1965). Phosphorus, K, Ca, Mg, Fe, Mn, Zn, B, and Al were determined by dry ashing at 400°C for 24 h, dissolving the ash in HNO3 (1:20 w/v) and assaying the solution obtained using an inductively coupled plasma emission spectrophotometer (ICP Iris, Thermo Optek, Milan, Italy; Karla, 1998). SPAD index measurement One day before the end of Experiment 2 the Soil Plant Analysis Development (SPAD index), a non-destructive and indirect measurement of leaf chlorophyll content, was measured on the fully expanded leaves by means of a portable chlorophyll meter SPAD-502 (Konica-Minolta corporation, Ltd., Osaka, Japan). Measurements were made at the central point of the leaflet between the midrib and the leaf margin. Fifteen leaves were randomly measured and averaged to a single SPAD value for each treatment. Determination of electrolyte leakage At the same date of the SPAD index measurement, the membrane integrity in leaves was measured in terms of electrolyte leakage as described by Lutts et al. (1995). Briefly, 10 pieces of equal-sized leaves (10 × 10 mm) collected from four plants per plot were placed in individual vials containing 10 ml of distilled water. The vials were incubated at room temperature (25°C) for 24 h with continuously shaking and the initial electrical conductivity (EC1) of the bathing solution was measured using a conductivity meter (HI991301; Hanna Instruments, Padova, Italy). To measure the total electrolytes released from leaf tissues, vials were then autoclaved at 120°C for 20 min and cooled at 25°C to obtain the final electrical conductivity (EC2). The EL was calculated using the following formula: EL (%) = (EC1/EC2) × 100. Statistical analysis All experimental data were subjected to a two-way ANOVA using the SPSS software package (SPSS, 2001). When ANOVA indicated that either nutrient solution or graft combination or their interaction was significant, mean separation was performed using the Duncan's multiple range test at p = 0.05 on each of the significant variables measured. Results Growth response and leaf symptoms In Experiment 1, shoot and root dry mass of cucumber plants were significantly (p < 0.01) affected by nutrient solution and grafting combination interaction (data not shown). Shoot dry mass at pH 3.5 in non-grafted and grafted plants was decreased by 26 and 25% over the pH 6.0 treatment (Figure 1A). Moreover, shoot dry mass was reduced by 67 and 58% over the pH 6.0 treatment at pH 3.5 + 1.5 mM Al in non-grafted and rootstock-grafted plants, and further decreased by 84 and 76% at pH 3.5 + 3.0 mM Al (Figure 1A). Similarly, root dry mass declined at pH 3.5 + 1.5 mM Al and especially at pH 3.5 + 3.0 mM Al, however, less in rootstock-grafted plants (Figure 1B). Plant growth traits in particular plant height, leaf area and number were significantly (p < 0.01) affected by nutrient solution and grafting combination interaction (data not shown). The biometric plant traits decreased under acidity and Al concentration especially at pH 3.5 + 3.0 mM Al (data not shown), however, the reduction in plant growth was less pronounced in rootstock-grafted plants (Figure 2). The most evident symptom of adverse low pH conditions (i.e., acidity) is a yellowing between leaf veins giving the leaves a “marbled appearance,” indicating magnesium deficiency. These symptoms recorded after 14 days in plants grafted onto pumpkin rootstock were less pronounced than those observed in the non-grafted plants (Figure 3). Figure 1 Effects of grafting combination, nutrient solution pH and aluminum concentration on shoot (A) and root (B) dry mass of cucumber plants grown in experiment 1 (14 days after transplanting). Different letters indicate significant differences according to Duncan's test (P = 0.05). Values are the means of four replicate samples. Figure 2 Effects of nutrient solution pH and aluminum concentration on non-grafted (A) and grafted (B) cucumber plants in experiment 1 (14 days after transplanting). Figure 3 Fully expanded leaves of non-grafted (A) and grafted (B) cucumber plants grown in experiment 1 (14 days after transplanting) and treated with a nutrient solution at pH 3.5. In Experiment 2, shoot and root dry mass at the end of the growing season were significantly affected by nutrient solution, grafting combination, and their interactions, whereas the root-to-shoot ratio (R/S) was significantly influenced by both main factors (Table 1). For instance, the shoot dry mass was decreased by 30, 25, and 20% over control at low adverse pH (3.5) treatment in non-grafted E, E/F, and E/C, and strongly decreased by 77, 73, and 66% at pH 3.5 + Al (Table 1). Consistent with the shoot dry mass, the root dry weight of cucumber plants decreased by 16, 11, and 8% at pH 3.5 in E, E/F, and E/C, respectively and decreased substantially by 65, 48, and 36% at pH 3.5 + Al (Table 1). The highest R/S ratio was recorded at pH 3.5 + Al compared to pH 6.0 and pH 3.5 treatments, whereas among grafting combination the E/C combination exhibited the highest R/S ratios (Table 1). Table 1 Effects of grafting combination, nutrient solution pH, and aluminum concentration on shoot and root biomass dry weight, and on the root-to-shoot ratio (R/S) of cucumber plants grown in experiment 2. Solution Graft combination Shoot (g plant−1) Root (g plant−1) R/S pH 6.0 E 101.1a 6.3a 0.06 E/F 96.0a 5.6ab 0.06 E/C 97.8a 6.4a 0.07 Mean 98.3a 6.1a 0.06b pH 3.5 E 70.7c (30) 5.3b 0.07 E/F 72.3c (25) 5.0bc 0.07 E/C 77.8b (20) 5.9ab 0.08 Mean 73.6b 5.4b 0.07b pH 3.5 + Al E 23.2e (77) 2.2d 0.09 E/F 24.7e (73) 2.9d 0.12 E/C 33.6d (66) 4.1c 0.12 Mean 27.2c 3.1c 0.11a SIGNIFICANCE Solution (S) *** *** *** Graft combination (G) ** * * S × G * ** ns E, cucumber hybrid “Ekron” (Cucumis sativus L.); E/F, “Ekron” grafted onto figleaf gourd rootstock (Cucurbita ficifolia Bouché); E/C, “Ekron” grafted onto squash hybrid rootstock “P360” (Cucurbita maxima Duch. × Cucurbita moschata Duch.). Values are the means of four replicate samples. The percentage of reduction in solution treatments having pH 3.5 and pH 3.5 + Al with respect to the solution treatment having pH 6.0 (control) are reported in parenthesis. Means within columns separated using Duncan's multiple range test, P = 0.05. ns., *, **, *** Nonsignificant or significant at P < 0.05, 0.01, or 0.001, respectively. Marketable yield and yield components In grafted and non-grafted plants, the marketable yield diminished in response to a decrease of the nutrient solution pH with more adverse effects at pH 3.5 + Al (Table 2). The reduced marketable yield was mostly caused by a reduced number of fruits per plant and mean weight (Table 2). Moreover, at pH 3.5 and pH 3.5 + Al, the percentage of yield reduction in comparison to the pH 6.0 treatment was lower in E/C (25 and 71%, respectively) than in E/F (27 and 79%, respectively) and non-grafted plants (32 and 81%, respectively; Table 2). Under Al toxicity conditions, plants of E/C had the highest marketable fruit yield (+50%) compared to those at E and E/F combinations (Table 2). The relatively low yield of non-grafted and E/F plants at pH 3.5 + Al was attributed to a reduction in the fruit mean weight and not to a change in the number of fruit per plant (Table 2). Table 2 Effects of grafting combination, nutrient solution pH, and aluminum concentration on marketable fruit yield, marketable fruit mean weight, and number of cucumber plants grown in experiment 2. Solution Graft combination Yield (kg plant−1) Fruit Number (n. plant−1) Mean weight (g fruit−1) pH 6.0 E 4.04a 12.5a 323.2 E/F 3.91a 12.4a 315.3 E/C 3.90a 12.1a 322.3 Mean 3.95a 12.3a 320.3a pH 3.5 E 2.73c (32) 8.6b 317.4 E/F 2.85bc (27) 9.0b 316.7 E/C 2.94b (25) 8.5b 345.9 Mean 2.84b 8.7b 326.7a pH 3.5 + Al E 0.77e (81) 3.0d 256.7 E/F 0.80e (79) 3.8c 210.5 E/C 1.17d (71) 4.1c 285.4 Mean 0.91c 3.6c 250.9b SIGNIFICANCE Solution (S) *** *** *** Graft combination (G) ns ns * S × G ** * ns E, cucumber hybrid “Ekron” (Cucumis sativus L.); E/F, “Ekron” grafted onto figleaf gourd rootstock (Cucurbita ficifolia Bouché); E/C, “Ekron” grafted onto squash hybrid rootstock “P360” (Cucurbita maxima Duch. × Cucurbita moschata Duch.). Values are the means of four replicate samples. The percentage of reduction in solution treatments having pH 3.5 and pH 3.5 + Al with respect to the solution treatment having pH 6.0 (control) are reported in parenthesis. Means within columns separated using Duncan's multiple range test P = 0.05. ns., *, **, *** Nonsignificant or significant at P < 0.05, 0.01, or 0.001, respectively. Fruit quality No significant changes were observed for titratable acidity (Table 3). The fruit SI was only influenced by nutrient solution pH and Al concentration with the highest values observed in cucumbers treated with pH 3.5 + Al compared to pH 3.5 and pH 6.0 treatments (Table 3). Under pH 3.5 and Al toxicity, the fruit firmness, dry matter, and total soluble solid contents decreased, but they were not significantly affected by the grafting onto figleaf gourd or pumpkin (Table 3). Moreover, when averaged over nutrient solution pH and Al concentration the highest fruit dry matter content was recorded with E/F, followed by E/C combination, whereas the lowest values were observed with non-grafted plants. Table 3 Effects of grafting combination, nutrient solution pH, and aluminum concentration on fruit shape index (SI), firmness, dry matter content (DMC), total soluble solids (TSS) content, juice pH, and titratable acidity (TA) of cucumber fruits grown in experiment 2. Solution Graft combination SI Firmness (N cm−2) DMC (%) TSS (°brix) pH TA (%) pH 6.0 E 0.24 1.53 4.41 3.87a 6.12 0.11 E/F 0.23 1.56 4.34 3.63a 6.12 0.09 E/C 0.23 1.52 4.24 3.70a 6.18 0.09 Mean 0.23b 1.54a 4.33a 3.74a 6.14a 0.10 pH 3.5 E 0.23 1.32 3.43 2.92d 5.81 0.08 E/F 0.23 1.37 3.70 3.14c 5.90 0.08 E/C 0.23 1.60 3.77 3.17c 5.97 0.11 Mean 0.23b 1.43ab 3.63b 3.08b 5.89b 0.09 pH 3.5 + Al E 0.27 1.29 3.34 3.22c 5.52 0.09 E/F 0.28 1.23 4.34 3.53bc 5.28 0.10 E/C 0.24 1.43 3.78 3.09cd 5.61 0.08 Mean 0.26a 1.31b 3.82b 3.28b 5.47c 0.09 SIGNIFICANCE Solution (S) *** * *** *** *** ns Graft combination (G) ns ns * ns ns ns S × G ns ns ns ** ns ns E, cucumber hybrid “Ekron” (Cucumis sativus L.); E/F, “Ekron” grafted onto figleaf gourd rootstock (Cucurbita ficifolia Bouché); E/C, “Ekron” grafted onto squash hybrid rootstock “P360” (Cucurbita maxima Duch. × Cucurbita moschata Duch.). Values are the means of four replicate samples. Means within columns separated using Duncan's multiple range test, P = 0.05. ns., *, **, *** Nonsignificant or significant at P < 0.05, 0.01, or 0.001, respectively. Aluminum uptake and distribution Al treatment increased Al concentration in leaves, stems, and roots but did not affect it in fruits (Table 4). The partitioning of Al within the plant tissues was in the following order: roots > leaves > stems > fruits. The highest shoot (leaf and stem) and root Al concentrations were observed in cucumber plants treated with pH 3.5 + Al (Table 4). There was a marked influence of grafting combination on the accumulation of Al in aerial vegetative plant parts, whereas no statistical effects were observed in fruits and roots. Particularly, the accumulation of Al in leaf tissue at pH 3.5 + Al, with respect to pH 6.0, was significantly lower in E/C plants (22%), in comparison to E/F and non-grafted plants (72 and 87%, respectively). Table 4 Effects of grafting combination, nutrient solution pH, and aluminum concentration on Al distribution in leaf, stem, fruit, and root tissues of cucumber plants grown in experiment 2. Solution Graft combination Al (mg kg−1 DW) Leaves Stems Fruits Roots pH 6.0 E 85.7c 10.6 6.2 263.7 E/F 80.5c 13.7 6.2 271.1 E/C 87.2c 7.8 6.0 236.0 Mean 84.5b 10.7b 6.1 257.0b pH 3.5 E 90.5c 10.1 6.1 303.4 E/F 88.0c 10.6 7.3 468.7 E/C 93.0c 9.6 7.5 734.0 Mean 90.5b 10.1b 6.9 502.1b pH 3.5 + Al E 160.0a 22.5 7.7 7697.0 E/F 138.3b 19.8 10.0 6575.7 E/C 106.4c 16.6 7.1 5395.8 Mean 134.9a 19.6a 8.9 6556.1a SIGNIFICANCE Solution (S) *** *** ns *** Graft combination (G) * * ns ns S × G ** ns ns ns E, cucumber hybrid “Ekron” (Cucumis sativus L.); E/F, “Ekron” grafted onto figleaf gourd rootstock (Cucurbita ficifolia Bouché); E/C, “Ekron” grafted onto squash hybrid rootstock “P360” (Cucurbita maxima Duch. × Cucurbita moschata Duch.). Values are the means of four replicate samples. Means within columns separated using Duncan's multiple range test, P = 0.05. ns., *, **, *** Nonsignificant or significant at P < 0.05, 0.01, or 0.001, respectively. Mineral composition and partitioning A different pattern of macronutrient accumulation and distribution in plant tissues was detected as a function of nutrient solution and grafting combination (Table 5). The concentration of N in leaf and fruit tissues were negatively affected by decreasing pH and the addition of 0.75 mM Al in the nutrient solution, while it remained unaffected in stems, fruits, and roots as compared with pH 6.0 (Table 5). Under Al stress conditions E/C plants markedly had higher N in their leaves than E/F and non-grafted plants (Table 5). Decreasing the pH from 6.0 to 3.5 in the nutrient solution and the further addition of Al significantly decreased P concentration in stems, fruits and roots (Table 5). The concentration of P in roots was lower in non-grafted than grafted plants in both treatments mentioned before (Table 5). Table 5 Effects of grafting combination, nutrient solution pH, and aluminum concentration on macronutrient composition of leaves, stems, fruits, and roots of cucumber plants grown in experiment 2. Solution Graft combination Macronutrients (g kg−1 DW) N P K Ca Mg Leaves Stems Fruits Roots Leaves Stems Fruits Roots Leaves Stems Fruits Roots Leaves Stems Fruits Roots Leaves Stems Fruits Roots pH 6.0 E 44.9a 35.2 40.3 33.2 7.3 9.2 5.9 26.3a 33.2ab 40.5 36.0 10.8 42.4 10.1 3.7 6.3 7.4ab 4.9a 2.5 0.9 E/F 43.6ab 28.7 37.5 32.9 5.7 8.6 6.8 25.9a 29.9b 35.9 35.3 13.8 42.6 8.0 3.5 6.3 7.0b 4.6ab 2.7 1.1 E/C 45.2a 33.5 41.0 30.3 6.3 9.2 6.8 23.2a 39.4a 37.7 35.5 15.4 46.3 10.6 5.2 6.6 8.1a 5.3a 2.4 1.4 Mean 44.6a 32.5 39.6a 32.1 6.5 9.0a 6.5a 25.1a 34.2a 38.0a 35.6a 13.3 43.8a 9.6 4.1a 6.4 7.5a 4.9a 2.6a 1.1 pH 3.5 E 42.5ab 30.3 35.8 27.0 6.4 9.1 6.1 10.8c 34.4ab 40.0 35.7 11.8 37.6 9.6 3.8 4.7 7.2ab 4.1b 2.4 0.8 E/F 40.7b 28.5 32.0 28.9 5.1 8.7 5.8 17.1bc 29.2b 34.9 32.2 20.1 33.9 8.6 3.2 6.0 6.0b 4.2b 2.4 1.3 E/C 42.1ab 29.3 35.7 30.0 5.6 9.4 6.0 20.0b 34.3ab 36.1 33.1 18.7 38.1 10.0 4.7 6.4 7.3ab 4.5b 2.2 1.5 Mean 41.8b 29.4 34.5b 28.6 5.7 9.1a 6.0a 16.0b 32.6a 37.0a 33.6ab 16.9 36.5b 9.4 3.9a 5.7 6.8b 4.3b 2.3b 1.2 pH 3.5 + Al E 33.0d 27.7 33.9 29.7 5.0 6.0 3.4 11.3c 32.5ab 39.4 31.2 15.5 26.0 10.6 2.8 5.5 4.9c 3.9b 2.1 1.2 E/F 32.4d 25.8 32.0 31.1 5.7 6.9 4.2 16.5bc 27.8b 31.9 28.9 17.5 20.1 8.2 2.7 4.4 4.3c 3.8b 2.0 0.9 E/C 37.9c 27.5 33.7 30.5 5.7 6.8 5.1 17.5b 29.1b 36.5 32.8 13.1 28.1 11.6 3.1 7.2 6.6b 4.1b 2.1 1.9 Mean 34.4c 27.0 33.2b 30.4 5.4 6.5b 4.3b 15.1b 29.8b 35.9b 31.0b 15.3 24.7c 10.2 2.9b 5.7 5.3c 3.9c 2.0c 1.3 SIGNIFICANCE Solution (S) *** ns ** ns ns *** *** *** ** *** ** ns *** ns *** ns *** ** *** ns Graft combination (G) ns ns ns ns ns ns ns * *** ns ns ns * *** *** * *** * ns ** S × G * ns ns ns ns ns ns * * ns ns ns ns ns ns ns ** ** ns ns E, cucumber hybrid “Ekron” (Cucumis sativus L.); E/F, “Ekron” grafted onto figleaf gourd rootstock (Cucurbita ficifolia Bouché); E/C, “Ekron” grafted onto squash hybrid rootstock “P360” (Cucurbita maxima Duch. × Cucurbita moschata Duch.). Values are the means of four replicate samples. Means within columns separated using Duncan's multiple range test, P = 0.05. ns., *, **, *** Nonsignificant or significant at P < 0.05. 0.01, or 0.001, respectively. The mean K concentration of the grafting combinations in stems and fruits decreased in both treatments (pH and pH + Al), more detrimental when Al was supplied compared with only pH 3.5 (Table 5). Comparing the grafting treatments, the lowest K concentration was found in E/F (Table 5). The Ca concentration was also significantly affected by the low pH in the nutrient solution and the addition of Al but compared with K not in the fruits but in the roots. At pH 3.5 + Al the leaves and fruits had significantly lower Ca concentration in comparison to those observed at pH 3.5 and control treatment (Table 5). Moreover, the concentration of Ca in all plant tissues (leaf, stem, fruit, and root) was higher in E/C than in E/F and in non-grafted plants. Decreasing the pH and the addition of Al in the nutrient solution decreased significantly the Mg concentration in leaves, stems, and roots especially under Al stress. The concentration of Mg in leaves of Al-treated plants was significantly affected by the grafting combination with values recorded for E/C plants being higher than E/F and non-grafted plants. Similarly to leaves, the Mg concentration in roots was significantly higher in E/C than in other grafting combinations (Table 5). The effect of low pH and Al-supply on tissue micronutrient concentrations was highly significant (Table 6). The concentration of Fe in leaf tissue decreased in both stressed plant treatments with the lowest values recorded in non-grafted plants supplied with pH 3.5 + Al. Concerning the grafting combination, the lowest concentration of Fe in the roots was observed in E/F plants (Table 6). Moreover, the concentrations of Mn (in all plant tissues), Zn (fruits and roots), and B (leaves) declined with increasing acidity and Al concentration in the nutrient solution. Finally, when averaged over nutrient solution, the Mn and Zn concentrations in all plant tissues were significantly higher in E/C as compared with other grafting combinations (Table 6). Table 6 Effects of grafting combination, nutrient solution pH and aluminum concentration on micronutrient composition of leaves, stems, fruits, and roots of cucumber plants grown in experiment 2. Solution Graft combination Micronutrients (mg kg−1 DW) Fe Mn Zn B Leaves Stems Fruits Roots Leaves Stems Fruits Roots Leaves Stems Fruits Roots Leaves Stems Fruits Roots pH 6.0 E 56.5bc 30.6c 27.0 202.6 254.4 54.6ab 23.4c 64.2 48.0bc 40.9 32.5 28.0 67.6 15.8 13.2 7.8 E/F 73.3ab 27.6c 30.6 123.5 269.8 41.1bc 23.7c 64.0 32.9d 49.1 30.0 77.4 82.5 16.3 13.9 10.5 E/C 62.3b 43.9a 24.8 202.6 302.3 63.9a 35.6a 82.6 65.8a 61.5 30.8 56.8 68.1 14.7 13.3 10.7 Mean 64.1 34.0ab 27.4a 176.2 262.1a 53.2a 27.6a 70.3a 48.9 50.5 31.1a 54.1b 72.7a 15.6 13.4 9.6 pH 3.5 E 59.8bc 35.9b 31.2 192.4 205.5 43.3bc 26.0bc 62.2 50.4b 53.3 30.3 29.9 58.3 14.8 12.7 11.7 E/F 80.7a 39.0a 34.6 170.1 219.3 48.1b 22.2c 66.1 30.9d 49.7 27.6 63.4 57.9 16.3 13.4 13.7 E/C 72.9ab 38.8a 29.2 176.7 248.5 48.1b 30.5b 71.4 55.5b 81.8 32.7 72.1 59.1 15.4 11.9 9.3 Mean 71.1 37.9a 31.6a 179.7 212.4b 46.5b 26.2a 66.6a 45.6 61.6 30.2a 55.1b 58.4b 15.5 12.7 11.5 pH 3.5 + Al E 48.0c 31.2bc 19.0 205.5 141.0 37.5b 15.9d 37.9 45.4c 55.2 23.3 64.3 52.3 16.9 13.1 7.7 E/F 65.8ab 30.8c 19.6 130.2 132.4 42.6bc 17.4d 32.1 47.3c 62.5 22.2 86.8 61.9 18.2 13.6 10.4 E/C 82.2a 30.3c 21.1 208.6 163.3 47.6b 18.7d 75.2 50.7bc 82.9 30.2 72.6 47.5 13.3 12.8 11.6 Mean 65.3 30.8b 19.9b 181.4 136.7c 42.6b 17.4b 48.4b 47.8 66.9 25.2b 74.6a 53.9b 16.1 13.1 9.9 SIGNIFICANCE Solution (S) ns *** *** ns *** *** *** * ns ns *** * *** ns ns ns Graft combination (G) *** * ns ** ** ** *** * *** *** *** *** ns ns ns ns S × G * * ns ns ns * * ns * ns ns ns ns ns ns ns E, cucumber hybrid “Ekron” (Cucumis sativus L.); E/F, “Ekron” grafted onto fig-leaf gourd rootstock (Cucurbita ficifolia Bouché); E/C, “Ekron” grafted onto squash hybrid rootstock “P360” (Cucurbita maxima Duch. × Cucurbita moschata Duch.). Values are the means of four replicate samples. Means within columns separated using Duncan's multiple range test, P = 0.05. ns., *, **, *** Nonsignificant or significant at P < 0.05. 0.01, or 0.001, respectively. Leaf area, SPAD index, and electrolyte leakage The total leaf area and electrolyte leakage were significantly affected by grafting combination, nutrient solution and their interaction, whereas the SPAD index was significantly influenced by grafting combination and nutrient solution with no interaction of these two factors (Table 7). The total leaf area decreased in response to a decrease of nutrient solution pH, with more detrimental at pH 3.5 + Al (Table 7). However, comparing pH 3.5 + Al with pH 6, the leaf area was less reduced in E/C (46%) than in E/F (61%) and non-grafted plants (68%; Table 7). The highest mean SPAD index of the grafting treatments was recorded at pH 6.0, while it was significantly lower at pH 3.5 + Al. The chlorophyll content recorded in grafted plants (avg. 40.8) was higher by 7% compared with ungrafted plants (38.3). Finally, the highest electrolyte leakage values were recorded in E/C and non-grafted plants supplied with pH 3.5 + Al (Table 7). Table 7 Effects of grafting combination, nutrient solution pH, and aluminum concentration on final leaf area, SPAD index, and leaf electrolyte leakage of cucumber plants grown in experiment 2. Solution Graft combination Leaf area (m2 plant−1) SPAD index Electrolyte leakage (%) pH 6.0 E 1.24a 47.3 33.4e E/F 1.11a 49.1 35.0e E/C 1.05ab 48.3 38.6de Mean 1.13a 48.2a 35.7c pH 3.5 E 0.73c 36.6 51.2c E/F 0.79bc 37.4 48.4d E/C 0.85b 41.0 42.8d Mean 0.79b 38.3ab 47.5b pH 3.5 + Al E 0.39e 31.1 68.3a E/F 0.43e 33.8 64.9a E/C 0.57d 35.2 61.1b Mean 0.46c 33.4b 64.8a SIGNIFICANCE Solution (S) *** *** *** Graft combination (G) ns ** ns S × G * ns ** E, cucumber hybrid “Ekron” (Cucumis sativus L.); E/F, “Ekron” grafted onto figleaf gourd rootstock (Cucurbita ficifolia Bouché); E/C, “Ekron” grafted onto squash hybrid rootstock “P360” (Cucurbita maxima Duch. × Cucurbita moschata Duch.). Values are the means of four replicate samples. Means within columns separated using Duncan's multiple range test, P = 0.05. ns., *, **, *** Nonsignificant or significant at P < 0.05, 0.01, or 0.001, respectively. Discussion Under acidic soils, reduced plant growth and consequently productivity are induced by different morphological, biochemical, and physiological alterations (Kochian et al., 2015; Rengel et al., 2015). Rapid damage and growth inhibition of the root system is the primary symptom of Al toxicity at concentrations in the μM range, which may be associated with interference in the elongation and division of meristematic cells (Rengel and Zhang, 2003; Kochian et al., 2004; Sivaguru et al., 2013). This interference reduces the absorption of water and nutrients, thus conditioning stress sensitivity and limiting plant biomass production (Inostroza-Blancheteau et al., 2012). In agreement, in both experiments a significant reduction in root, shoot biomass, and yield was observed at low pH (3.5) and even more, when Al was added (Figure 1; Tables 1, 2), suggesting that these two stresses differ in their inhibition on plant growth (Rangel et al., 2005; Bose et al., 2010). Moreover, the reduction in plant biomass production depended on the Al concentration. The constrained crop growth and fruit yield observed under pH 3.5 and under pH 3.5 + Al has been reported in previous open field and greenhouse studies on tomato (Nogueirol et al., 2015) and zucchini squash (Rouphael et al., 2015). Nevertheless, decreasing pH and increasing Al concentration in the nutrient solution improved fruit quality characteristics such as fruit DM and TSS contents (Table 3). These phenomena have been found before for zucchini and explained by a decrease in water accumulation by the fruit without any effect on the synthesis and accumulation of organic solutes (Rouphael et al., 2012, 2015). When cucumber cv. Ekron were grafted onto pumpkin rootstock “P360” compared with non-grafted and grafted onto figleaf gourd, the suppression of growth and production suppression under Al-supply was mitigated indicating a genetic diversity in Al-tolerance among cucumber rootstocks. The Al-tolerance of cucumber grafted onto pumpkin “P360” may be attributed to a greater uptake and translocation of K, Ca, Mg, Mn, and Zn to the aerial parts (leaves and stems). The enhanced nutritional status and abiotic stress-tolerance of grafted vegetables has often been associated with an enlarged and more vigorous root system (Colla et al., 2010c; Savvas et al., 2010). The root is the first organ sensing abiotic stresses in soil or substrates, such as acidity and Al toxicity. Therefore, the enhanced root growth (Table 1), length, and density (data not shown) of a pumpkin compared with a cucumber rootstock are important aspects for cucumbers cultivated in acidic conditions with or without Al toxicity. Al accumulates in the epidermis and in the outer cortex of the root (Delhaize et al., 1993; Ryan et al., 2001). Many studies demonstrated that the major fraction of absorbed Al (30–90% of total Al) is localized in the apoplast (Yang et al., 2000; Ryan et al., 2001; Pereira et al., 2010). In Experiment 2, Al concentration in the root tissues was 50 times higher than in the cucumber leaves and even higher than in stems or fruits (Table 4). This indicates that the endodermis of roots represents a barrier to the transport of Al to the shoot (Dogan et al., 2014). Interestingly, this ratio was independent of the type of rootstock used although the absolute Al concentration differed between grafted and non-grafted root systems. The different Al accumulation in plant tissues highly depends on the mechanism (exclusion or internal tolerance) used by plants to confer tolerance to Al toxicity (Delhaize and Ryan, 1995; Kochian, 1995). Our results showed that Al concentration in cucumber roots was similar between grafted (E/C and E/F) and non-grafted plants grown under Al-stress (Table 4). This indicates that neither the figleaf gourd nor the pumpkin rootstocks were able to exclude Al from the root apex by exudation of organic acids (citrate, malate, and oxalate) into the rhizosphere (Brunner and Sperisen, 2013). On the other hand, the lowest Al accumulation in leaves with pH 3.5 + Al was recorded in the E/C combination compared to the other grafting combinations (Table 4) indicating that another mechanism was involved in mitigating the Al toxicity effect (Barceló and Poscheinrieder, 2002; Brunner and Sperisen, 2013). It appears that grafting cucumber onto pumpkin rootstock can restrict the Al root-to-shoot translocation (Table 4), throughout “the sequestration of this toxic element into less sensitive parts of the plant and cell compartments (e.g., vacuoles)” (Inostroza-Blancheteau et al., 2012; Brunner and Sperisen, 2013; Kochian et al., 2015). This suppression of Al uptake and translocation to shoots of grafted plants is consistent with previous reports indicating that appropriate rootstocks of cucurbitaceous and solanaceous species may limit the uptake of toxic elements and heavy metals (Edelstein et al., 2005; Rouphael et al., 2008a; Kumar et al., 2015b). Authors show that grafting annual fruit crops, such as melon, cucumber, and tomato onto vigorous rootstocks (i.e., pumpkin for cucurbits and “Maxifort” for tomato) may significantly restrict trace elements' (B and Cu) and heavy metal (Ni) concentrations in leaf and fruit tissue, thereby alleviating their detrimental effects on crop productivity and human health via the food chain (Savvas et al., 2010). Nutrient uptake is a crucial aspect for the maintenance of homeostasis and plant growth under unfavorable soil conditions (i.e., edaphic stress; Seguel et al., 2013). Tolerance of Al is mostly reflected in limited alterations of macro and micronutrient's acquisition and translocation (Andrade et al., 2009). In the present study, decreasing pH level and increasing Al concentration in the nutrient solution depressed concentrations of macro- (N, P, K, Ca, and Mg) and micro-elements (Mn and Zn) in shoots (leaves and/or stems) and to a lesser degree in roots in both grafted and non-grafted plants (Tables 5, 6). Therefore, excessive Al accumulation affected uptake, translocation and accumulation of these nutrients in plant tissues, and hence was responsible for mineral imbalances and deficiencies as well as for the depression of plant growth and yield (Rouphael et al., 2015). Studies have demonstrated that Al interferes directly with several different plasma membrane channel proteins, thus reducing the uptake of mono- and divalent cations such as K and Ca (Gassmann and Schroeder, 1994; Piñeros and Tester, 1995). Al-stress has been reported to reduce Ca through three mechanisms: “(1) inhibition of Ca transport via symplasm by Al, (2) disruption of Ca homeostasis in cytoplasm by Al, and (3) Ca displacement by Al from apoplasm” (Delhaize and Ryan, 1995; Kochian, 1995). In addition to the reduction in K and Ca uptake, Al can also decrease Mg uptake in aerial parts (Rengel and Robinson, 1989), as we can confirm with our current data. The competition between these two cations (Al and Mg) was demonstrated for membrane transporters and metal binding sites in enzymatic reactions (Rengel and Robinson, 1989; Pécsváradi et al., 2009). However, grafted and non-grafted plants diverged in their tissue nutrient concentration in relation to acidity and Al toxicity. Grafting cucumber plants onto the pumpkin rootstock (E/C) increased K, Ca, Mg, Mn, and Zn in shoot and/or root (Tables 5, 6) suggesting that selecting appropriate rootstocks can maintain nutrient homeostasis under Al-stress. The lower reduction in cation uptake observed in the E/C combination under Al-stress, could be associated to the lower inhibition of root growth and elongation since cation (especially K) accumulation contributes to the expansion of cell elongation (Cristancho et al., 2011). Similarly, the shoot and root Mg concentrations were higher in E/C compared to the other grafting combinations. It remains to be elucidated whether higher Mg uptake in E/C combination is a consequence of a greater root growth and/or greater Al tolerance of Mg-uptake systems (Rengel et al., 2015). From above we could suggest that an efficient metabolism system exists in E/C combination under Al toxicity, indicating that Al tolerance is correlated with enhanced mineral nutrient concentrations in plant tissues (Rouphael et al., 2015). These results are consistent with several studies (Rouphael et al., 2008a; Kumar et al., 2015a,b) reporting that rootstocks used in grafting of annual fruit crops were able to enhance the uptake of some macro- and micro-elements under heavy metal (e.g., Cd, Ni, and Cu) toxicity. High Al concentrations in soil or substrates hamper plant development at a physiological and biochemical level, affect its photosynthetic rate, total chlorophyll content, and also inhibit electron transport in PSII (Chen, 2006; Inostroza-Blancheteau et al., 2012). This was confirmed with our measurements of the SPAD index, widely used as a non-destructive estimate of chlorophyll content. It dropped sharply in leaves of Al stressed non-grafted plants compared to both E/C and E/F plants (Table 7). This suggests the occurrence of chlorophyll degradation and early senescence, likely due to the harmful effects of reactive oxygen species on chloroplasts (Chen, 2006; Rouphael et al., 2015). Contrarily, the grafted cucumber plants in particular E/C combination were able to maintain a higher chlorophyll content in both stress treatments, thus exhibiting the highest yield and biomass. In addition to a reduced chlorophyll content, reductions in total leaf area at low pH and especially at Al-supply could be caused by a premature leaf senescence and ion toxicity (Seguel et al., 2013). Maintenance of a large leaf area upon Al stress in E/C combination (Table 7) may be crucial to guarantee production and translocation of photosynthates to the fruits, thus increasing the final yield in cucumber. Adverse pH conditions and Al-stress induced impairment of membrane integrity and affect all physiological activities linked to membrane functioning (Rouphael et al., 2015). This is related to the fact that Al-enhanced oxidative stress caused by an increased production of reactive oxygen species leads to lipid and protein oxidation (Boscolo et al., 2003; Jones et al., 2006). The results of Experiment 2 indicate that in cucumber the electrolyte leakage percentage (i.e., degree of cell membrane injury) was significantly increased, and depended on the nutrient solution pH and the Al concentration (Table 7). The degree of cell membrane injury induced by Al-stress has often been related to calcium concentration, an important factor in increasing structural stability of cell membrane (Borer et al., 2005). Increasing Al concentration in the nutrient solution reduced leaf and root calcium uptake resulting in a reduction of cell membrane integrity (Pereira et al., 2010). However, E/C plants reduced the amount of ion leakage in Al-stress treatment by improving the Ca uptake in shoot and root tissues indicating that grafting cucumber onto pumpkin rootstocks has facilitated the membrane functions (i.e., semipermeability). Conclusions Our findings indicate that in both grafted and non-grafted plants, agronomical, physiological, and mineral composition responses were negatively affected by acidity and Al concentration, with Al-stress being more phytotoxic than low pH treatment. The results of both experiments were able to verify our hypothesis that grafting onto suitable rootstocks (i.e., pumpkin) may limit the Al accumulation in the aerial parts, improve the uptake of nutrients (K, Ca, Mg, Mn, and Zn), enhance chlorophyll synthesis as well as the root genotypes to control the cell membrane stability; thus alleviating the impacts of adverse pH level and Al toxicity on crop productivity. These results might be useful to assist the selection of tolerant rootstocks in breeding programs particularly in acidic soils, where Al-toxicity is a major agronomic constraint. Author contributions YR performed the long term experiment with agronomical and physiological analysis and he was involved in writing the manuscript (Experiment 2); ER and MC performed the mineral analysis in both experiments and they collaborated in manuscript preparation; MB and DS were involved in the short term experiment (Experiment 1) and in manuscript preparation; GC defined the scientific hypothesis, set up the experimental protocols, made the statistical analysis of experimental data, and he was involved in the manuscript preparation. Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This work was partially supported by the German–Italian Cooperation VIGONI Program in the frame of the project “The effectiveness of grafting to improve the vegetable crop tolerance to adverse pH conditions in the root zone” (2011–2013). 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Frontiers in Neuroscience 1662-4548 1662-453X Frontiers Media S.A. 27621700 10.3389/fnins.2016.00373 Neuroscience Hypothesis and Theory Language Impairments in ASD Resulting from a Failed Domestication of the Human Brain Benítez-Burraco Antonio 1 Lattanzi Wanda 2 † Murphy Elliot 3 * † 1 Department of Philology, University of Huelva Huelva, Spain 2 Institute of Anatomy and Cell Biology, Università Cattolica del Sacro Cuore Rome, Italy 3 Division of Psychology and Language Sciences, University College London London, UK Edited by: Yuri Bozzi, University of Trento, Italy Reviewed by: Suhash Chakraborty, Hindustan Aeronautics Limited Hospital, India; Roberto Canitano, University Hospital of Siena, Italy *Correspondence: Elliot Murphy elliotmurphy91@gmail.com This article was submitted to Child and Adolescent Psychiatry, a section of the journal Frontiers in Neuroscience †These authors have contributed equally to this work. 29 8 2016 2016 10 37301 4 2016 02 8 2016 Copyright © 2016 Benítez-Burraco, Lattanzi and Murphy. 2016 Benítez-Burraco, Lattanzi and Murphy https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Autism spectrum disorders (ASD) are pervasive neurodevelopmental disorders entailing social and cognitive deficits, including marked problems with language. Numerous genes have been associated with ASD, but it is unclear how language deficits arise from gene mutation or dysregulation. It is also unclear why ASD shows such high prevalence within human populations. Interestingly, the emergence of a modern faculty of language has been hypothesized to be linked to changes in the human brain/skull, but also to the process of self-domestication of the human species. It is our intention to show that people with ASD exhibit less marked domesticated traits at the morphological, physiological, and behavioral levels. We also discuss many ASD candidates represented among the genes known to be involved in the “domestication syndrome” (the constellation of traits exhibited by domesticated mammals, which seemingly results from the hypofunction of the neural crest) and among the set of genes involved in language function closely connected to them. Moreover, many of these genes show altered expression profiles in the brain of autists. In addition, some candidates for domestication and language-readiness show the same expression profile in people with ASD and chimps in different brain areas involved in language processing. Similarities regarding the brain oscillatory behavior of these areas can be expected too. We conclude that ASD may represent an abnormal ontogenetic itinerary for the human faculty of language resulting in part from changes in genes important for the “domestication syndrome” and, ultimately, from the normal functioning of the neural crest. autism domestication language evolution neural oscillations language deficits ==== Body pmcIntroduction Autism spectrum disorders (ASD) are pervasive neurodevelopmental conditions characterized by several and severe cognitive and social deficits, including language and communication problems, repetitive and stereotypical behavior, and problems with social interaction (Bailey et al., 1996). In DSM-V, language deficits are no longer explicitly postulated as a central feature of ASD because they are subsumed in its distinctive communication problems. Nevertheless, it is clear that ASD entails a typical language profile and language developmental path (reviewed in Benítez-Burraco and Murphy, 2016; see also Tager-Flusberg et al., 2005; Tager-Flusberg, 2006; Eigsti et al., 2007; Bourguignon et al., 2012). Because of the masking effect of a variable IQ, and the variable degree of functionality exhibited by ASD patients, it is difficult to hypothesize a core language deficit in this condition. The impairment of the oromotor function has been claimed to account for expressive language problems in some autistic subjects (Belmonte et al., 2013). Comprehension problems seemingly result from other underlying deficit(s), including a reduced effect of semantic priming (Preissler, 2008), problems with phonological processing (Lindgren et al., 2009), or impairment of procedural memory (Walenski et al., 2006). At the neural level, ASD entails atypical development, wiring and interconnection of areas involved in language processing (Stefanatos and Baron, 2011; Bourguignon et al., 2012). Not surprisingly, functional differences in language processing tasks of ASD compared with unaffected subjects have been attested as well (Courchesne and Pierce, 2005; Scott-Van Zeeland et al., 2010a,b). For instance, microstructural anomalies and reduced lateralization patterns have been observed in the arcuate fasciculus of ASD patients (Fletcher et al., 2010), suggesting that a constraint on the integrative processes during development may contribute to language impairment in this condition (Schipul et al., 2011). We also wish to highlight both increased and decreased intra- and inter-hemispheric connectivity (Hahamy et al., 2015), and abnormal responses to linguistic stimuli (reviewed in Stefanatos and Baron, 2011, pp 259–262). Intriguingly, the ASD phenotype is characterized by increased intrinsic functional connectivity during the first years of life (the time window where language is acquired) and reduced connectivity in adolescent and adult states (Uddin et al., 2013). In spite of this growing body of neurobiological data, a comprehensive view of language processing in the ASD brain is still lacking. Specifically, ASD studies need to move beyond simplistic models of language processing and focus instead on how collections of brain areas jointly engaged in specific, impaired cognitive operations (see Fedorenko and Thompson-Schill, 2014, for a general discussion). This is a real challenge, provided that abnormal brain profiles are not expected to easily map on to anomalous categories or computations of linguistic theories (see Poeppel, 2012; Murphy, 2016a, for discussion). We have recently proposed a translational theory of language deficits in ASD as amounting to abnormal patterns of brain rhythms (Benítez-Burraco and Murphy, 2016); although a clarification and empirical validation of this hypothesis is still pending. Finally, we wish to emphasize that ASD has been associated with sequence variants, copy number variation (CNVs), and/or changes in the expression patterns of an extensive number of genes (Geschwind and State, 2015). Despite the remarkable genetic heterogeneity, it is noteworthy that all these genes tend to converge on specific pathways and neural mechanisms, functionally relevant in this condition and expected to account for its associated deficits (Willsey and State, 2015). Specifically, several candidates for language impairment in ASD have been proposed, including MET, CTTNBP2, EN2, NBEA, HRAS, and PTEN (Comings et al., 1996; Naqvi et al., 2000; Cheung et al., 2001; Castermans et al., 2003; Benayed et al., 2005; Campbell et al., 2006). Nonetheless, the gap between genes and language deficits in ASD still remains open (see Jeste and Geschwind, 2014, for a general discussion, and Benítez-Burraco and Murphy, 2016, for a specific discussion on candidates for language dysfunction in ASD). The aim of this paper is to contribute to the bridging of the gap between the genetic backdrop and language deficits observed in ASD. To this end, we will primarily focus on language evolution. There exists a strong, deep link between evolution and (abnormal) development. Recently-evolved neural networks seem to be more sensitive to damage because of their lower levels of resilience (Toro et al., 2010). As a consequence, aspects of brain development and function that are preferably impaired in modern populations are expected to be involved in recently evolved, human-specific cognitive abilities. Some comprehensive accounts of the human condition set against the cognitive profiles of other primates have been recently put forth (Seed and Tomasello, 2010; Platt et al., 2016). Comparative genomics also provides valuable information about the sources of the observed differences and similarities in the human genome (Rogers and Gibbs, 2014; Franchini and Pollard, 2015). Likewise, we are beginning to achieve an advanced understanding of the genetic changes that occurred after our split from extinct hominins (Pääbo, 2014; Zhou et al., 2015). We expect that the same factors that prompted the transition from an ape-like cognition to our specific mode of cognition are involved in the etiology of cognitive disorders involving language deficits and, particularly, of ASD (see Benítez-Burraco, 2016a, for a general discussion). In what follows, the focus is placed on one aspect of this evolutionary process: the self-domestication of the human species. At present, we have a decent understanding of how our language-readiness (that is, our species-specific ability to learn and use language) may have evolved. Accordingly, among the changes brought about by human evolution, one very relevant aspect is the ability to transcend (better than other species) the signature limits of core knowledge systems and thus go beyond modular boundaries (Mithen, 1996; Spelke, 2003; Carruthers, 2006; Hauser, 2009; Boeckx, 2011; Wynn and Coolidge, 2011). As hypothesized in Boeckx and Benítez-Burraco (2014a), our language-readiness boils down to this enhanced cognitive ability, but also to its embedding inside cognitive systems responsible for interpretation (thought) and externalization (speech). This language-readiness was seemingly brought about by specific changes in the skull/brain developmental path (resulting in a more globular brain), which entailed new patterns of long-distance connections among distributed neurons and, ultimately, new patterns of brain rhythmicity, including an adequate degree and pattern of cortical inhibition. Interestingly, brain rhythms are heritable components of brain function (Linkenkaer-Hansen et al., 2007; Hall et al., 2011) and have been linked to computational primitives of language (Murphy, 2015a,b, 2016a), allowing for a good explanation (and not just a description) of linguistic computation (and of language deficits) at the brain level, and specifically, for a satisfactory mapping of language deficits to neural dysfunction and its genetic basis in ASD (Benítez-Burraco and Murphy, 2016). We have found many candidates for ASD among the genes known to be involved in the emergence of language-readiness (Benítez-Burraco and Boeckx, 2015). At the same time, the emergence of modern-like languages (and perhaps of core aspects of language too) was seemingly favored by changes in the cultural niche of our ancestors. The archeological record shows that cognitive modernity (encompassing language-readiness) did not automatically entail behavioral modernity (seemingly resulting from using fully-fledged languages), which only appeared long after the emergence of anatomically-modern humans (AMHs) together with changes in human cultural dynamics. Current linguistic research has shown that aspects of linguistic complexity (including core aspects of grammatical knowledge) correlate with aspects of social complexity (Wray and Grace, 2007; Lupyan and Dale, 2010). Moreover, core properties of human languages (like duality of patterning) can develop in response to environmental pressure, as research into emergent sign languages has nicely illustrated, implying that they cannot be regarded as part of the biological endowment (see Benítez-Burraco, 2016b, for discussion). Importantly, language acquisition by the child demands a prolonged socialization window that enables her to receive the proper amount of triggering stimuli and to interact with other conspecifics. All this means that the intrinsic cognitive machinery may be not enough for granting the acquisition of a successful tool for linguistic cognition and that the environment has to be of the right kind too (see Sterelny, 2011 on behavioral modernity set against cognitive modernity). It has been hypothesized that the social conditions (or the cultural niche) that facilitated the enhancement of linguistic structure through a cultural mechanism were brought about by a process of human self-domestication (see Thomas, 2014, for details, and Hare and Tomasello, 2005; Deacon, 2009, on relaxed selective pressures resulting from self-domestication as explanations of the emergence of key aspects of behavioral modernity). Different factors may have contributed to human self-domestication, from adaptation to the human-made environment to selection against aggression to sexual selection. We have hypothesized (Benítez-Burraco et al., in press) that the very changes that brought about our globular skull/brain and our language-readiness may have also fuelled the emergence of a (self-domesticated) phenotype in the human species. Accordingly, we have found numerous links between the candidates for globularization and language-readiness, and genes important for the development and function of neural crest cells (NCC). Indeed, the hypofunction of the neural crest (NC) has been claimed to account for the constellation of distinctive traits observed in domestic mammals (the “domestication syndrome”) (Wilkins et al., 2014). Because of the deep link between evolution and development, we expect that examining the signatures of the domesticated phenotype in people with ASD contributes to a better understanding of etiology of ASD, and specifically, of language deficits in this condition. In a recent paper Reser (2014) found similarities between autism and species of solitary mammals. Although the focus was put on behavior, the author suggests that future research will benefit from investigating the neurobiological, genetic and epigenetic causes of these similarities. Here we try to push research in this direction. The paper is structured as follows. First, we provide a general account of the domesticated traits that are absent or attenuated in ASD. Then we move to the genes and focus on candidates for ASD that are found among the set of genes involved in the domestication syndrome and the evolution of language-readiness, as characterized in Benítez-Burraco et al. (in press), showing that they exhibit a distinctive expression profile in the brain of autists. Finally, we compare the ASD phenotype with wild primates, focusing on the expression profile of these genes, but also on oscillatory signatures of areas important for language processing, considering that language impairment in ASD can be interpreted as an “oscillopathic” condition (see Benítez-Burraco and Murphy, 2016). We will conclude that ASD (and language deficits in ASD) can be viewed as an abnormal ontogenetic itinerary for the human faculty of language, resulting in part from changes in genes important for the domestication syndrome and seemingly from changes in the normal functioning of the NC. Domestic traits in the ASD phenotype Wilkins et al. (2014) provide a comprehensive summary of traits known to be modified in domesticated mammals, many of them concerning the cranial region. These include changes in ear size and shape, changes in the orofacial area (including shorter snouts and smaller jaws), changes in dentition (particularly, smaller teeth), and a reduced brain capacity (specifically, of components of the forebrain such as the amygdala or parts of the limbic system). Other distinctive traits commonly found in domesticated strains are depigmentation, neoteny, shorter reproductive cycles, and increased docility, which is thought to result from adrenal size reduction and adrenal hypofunction as well as from reduced levels of stress hormones (including adrenocorticoids, adrenocorticotropic hormone, cortisol, and corticosterone). This delayed adrenal maturation also involves a hypofunction of the sympathetic nervous system and an increase of the duration of the immaturity of the hypothalamic-pituitary-adrenal system (the HPA axis), which provides the animal with a longer socialization window. According to Wilkins et al. (2014), the multiple phenotypic traits that characterize the domestication syndrome emerge as unselected by-products from a developmental reduction in NCC inputs, resulting from selection for tameness. Interestingly, compared to extinct hominins, AMHs exhibit a number of domesticated traits, including reduced brains (at least during the last 50,000 years), changes in dentition, reduction of aggressiveness, and retention of juvenile characteristics (see Thomas, 2014, for details). Intriguingly, most of these features are generally attenuated in ASD (Figure 1). Figure 1 Anomalous presentation of domesticated traits in people with ASD. Main clinical features observed in ASD patients and concerning the domestication syndrome are shown. The child diagram was gathered from Iconfinder output (available at http://www.iconfinder.com/icons/525448/boy_child_kid_male_man_person_white_icon). To begin with, ASD subjects show significant differences with healthy controls regarding minor physical anomalies, particularly in the craniofacial region (assumed to result from deviations during fetal development and suggested to constitute external markers of atypical brain growth) (Tripi et al., 2008; Manouilenko et al., 2014). Specifically, in adults the abnormal shape of the ears is robustly associated with autistic traits, with higher scores correlating with poorer functioning (Manouilenko et al., 2014). Regarding the changes in the orofacial region, prepubertal boys with ASD show significant differences in facial morphology compared to typically developing (TD) boys (Aldridge et al., 2011). This distinctive facial phenotype is more pronounced in subjects with severe symptoms, significant cognitive impairment, and language regression (Obafemi-Ajayi et al., 2015). Concerning tooth peculiarities, children with ASD show greater abnormalities in dentition, including missing teeth, diastemas, or reverse overjets (Luppanapornlarp et al., 2010). With respect to brain size, head circumference is significantly larger in people with ASD, with nearly 15% suffering from macrocephaly. Higher brain volumes correlate with lower functioning abilities; indeed nearly 9% ASD individuals exhibit brain overgrowth (Sacco et al., 2015). It is worth noticing that higher head circumference and brain size values are observed only during early childhood (Fukumoto et al., 2008; Courchesne et al., 2011, although see Raznahan et al., 2013), particularly when ASD is presented with regression (Nordahl et al., 2011). Typically, early brain overgrowth is followed by a decrease in structural volumes (Courchesne et al., 2011). Although brain overgrowth may result from a dysregulation of the overall systemic growth (see below), it is thought to impact on cognition. This is believed to occur as a result of the reduced networking efficiency among widespread regions of the cortex, due to the increased long-distance connections (Lewis et al., 2013). Specifically, people with ASD show increased volumes of the amygdala (Mosconi et al., 2009; Murphy et al., 2012), which correlate with the severity of their social and communication impairments (Schumann et al., 2009). In the TD population, higher amygdala volumes are associated with poorer language abilities in infancy (Ortiz-Mantilla et al., 2010). Regarding the behavioral traits associated with the domestication syndrome, we wish to highlight that aggressive behaviors are frequent in children with ASD (with about 25% of them having scores in the clinical range), and correlate with lower cognitive outcomes (Hill et al., 2014). Children with ASD display more reactive than proactive aggression attitudes (Farmer et al., 2015). Likewise, irritability is also commonly observed in affected individuals (Mikita et al., 2015). Additionally, ASD is commonly found to be comorbid with generalized anxiety disorder (Hollocks et al., 2014; Bitsika et al., 2015). Several studies have been carried out to learn more about the physiological basis of this anomalous response to the social environment. Interestingly, higher serum cortisol responses are usually found in children with ASD, particularly after stressor stimulation, when prolonged duration and recovery of cortisol elevation is also observed (Spratt et al., 2012). Moreover, children with ASD show a distinctive diurnal rhythm of cortisol compared to their TD peers; this involves elevated cortisol levels at the end of the day and dampened linear decline across the day in some children (Tomarken et al., 2015). Dysregulation of the diurnal rhythm as a whole has been found in low functioning ASD (Taylor and Corbett, 2014). Also, anxiety symptoms correlate with high cortisol levels in ASD pediatric patients (Bitsika et al., 2015). Plasma levels of adrenocorticotropic hormone are also significantly higher in children with ASD, and correlate positively with the severity of the symptoms (Hamza et al., 2010). The HPA axis in ASD responds in a more sluggish way to physiological or physical manipulation. Accordingly, Taylor and Corbett (2014) found hyper-responsiveness of the HPA axis when unpleasant stimuli or relatively benign social situations are involved, whereas they observed hypo-responsiveness in conditions involving social evaluative threat. On the whole, the HPA axis may be more reactive to stress in social anxiety disorder and ASD (Spratt et al., 2012; Jacobson, 2014). Because children with autism and anxiety disorders show a blunted cortisol response to psychosocial stress, and given that reduced cortisol responsiveness is significantly related to increased anxiety symptoms, Hollocks et al. (2014) suggested that a non-adaptive physiological response to psychosocial stress may exist in ASD. Finally, it is worth considering some other traits commonly observed in domesticated mammals: neoteny, alterations of reproductive cycles, and pigmentation changes. Regarding neotenic features, it is noteworthy that children with ASD exhibit an early generalized overgrowth (van Daalen et al., 2007; Fukumoto et al., 2008; Chawarska et al., 2011). Typically, boys with ASD show increased body size at birth and during infancy, with postnatal overgrowth correlating with lower adaptive functioning, greater severity of social deficits, and poorer verbal skills (Chawarska et al., 2011; Campbell et al., 2014). Interestingly, higher levels of androgens are found in children and adolescents with ASD. This correlates with the severity of autistic traits and might account for the precocious puberty also reported in this condition (El-Baz et al., 2014). These findings emphasize the role of elevated pre- and postnatal testosterone levels in the liability for ASD (see Hauth et al., 2014). Testosterone significantly affects brain development, particularly targeting the hypothalamus, the amygdala and the hippocampus, impacting on aspects of memory consolidation (Filová et al., 2013). High perinatal testosterone concentration negatively correlates with early vocabulary development in TD boys (Hollier et al., 2013). Interestingly, children with elevated androgen levels due to congenital adrenal hyperplasia show atypical patterns of brain asymmetry in the perisylvian areas, and language/learning disabilities (Plante et al., 1996). Less data on reproductive functions in females is available, due to the lower prevalence of ASD among women. Nevertheless, women with ASD reported significantly more irregular menstrual cycles and dysmenorrhea (Ingudomnukul et al., 2007; Hamilton et al., 2011). Likewise, an increase in premenstrual syndrome has been observed in women with ASD (Obaydi and Puri, 2008; Hamilton et al., 2011), who are more likely to exhibit behavioral issues related to the onset of periods (Burke et al., 2009). In addition, delayed age of menarche seems to correlate with the severity of autistic traits (Hergüner and Hergüner, 2016). These findings lend support to the androgen theory of ASD, according to which elevated levels of testosterone during fetal development may contribute to the development of ASD. Finally, concerning changes in pigmentation, it is of interest that hypomelanotic diseases usually entail autistic symptoms, as is commonly observed in hypomelanosis of Ito (OMIM#300337; Akefeldt and Gillberg, 1991; von Aster et al., 1997; Gómez-Lado et al., 2004). It has been hypothesized that the comorbidity between hypomelanosis and ASD may result from a deficiency in vitamin D (Eyles, 2010; Bakare et al., 2011). In fact, core symptoms of ASD improve after vitamin D supplementation (Jia et al., 2015). Interestingly, core candidates for the globularization of the AMH skull/brain and the evolution of language-readiness are involved in vitamin D homeostasis and function (see Benítez-Burraco and Boeckx, 2015, for details). As noted above, regardless of the different selectionist scenarios that may account for the traits commonly found in domesticated mammals, a role for NC hypofunction during embryonic development has been proposed (see Wilkins et al., 2014, for details). No comprehensive view of the role (if any) of the NC in the aetiopathogenesis of ASD has been provided to date. Still, it is important to note that neurocristopathies (that is, conditions resulting from NC defects) commonly involve autistic features. For instance, in CHARGE syndrome (OMIM#214800) autistic traits coexist with developmental abnormalities affecting endocrine, reproductive, urinary and digestive systems, along with skeletal and craniofacial features (Fernell et al., 1999). Given this background, we will now examine whether candidates for ASD are overrepresented among the genes believed to play a central role in NC development and function, with a special emphasis on those that interact with genes important for the globularization of the AMH skull/brain. ASD and the genetics of the domestication syndrome In order to improve our characterization of the domesticated traits in ASD, it is of interest to assess whether candidate genes for this condition (with a particular emphasis on language disabilities) are overrepresented among, or are functionally related to, candidates for domestication. We have relied on an extended list of candidates, which includes the core set of genes proposed by Wilkins et al. (2014), plus a subset of the genes involved in the globularization of the AMH skull/brain and the emergence of language-readiness that are functionally related to them, through direct interaction, and/or that play a role in the development and function of the NC (see Benítez-Burraco et al., in press, for details). Our list also comprises NC-related genes known to play a key role in craniofacial development and/or disorders. As noted above, most of the domesticated traits result from the modification of the cranial region and many of the ASD distinctive features concern the skull, face and brain. Moreover, as reasoned in Boeckx and Benítez-Burraco (2014a,b), we expect that our language-readiness resulted from changes in the development of the skull/brain, but also from the refinement of the externalization devices, specifically, the orofacial region: As also noted above, the impairment of oromotor function has been hypothesized to account for some language deficits in ASD. Table 1 provides a full list and a schematic characterization of these genes. Table 1 List of putative candidate genes for domestication and ASD. Gene symbol Gene name Domesticationa Language-readinessb NCCc Craniofaciald Brain rhythmicitye ASD Candidatef Differentially expressedg ALX1 Aristaless-like homeobox protein 1 + + ALX3 Aristaless-like homeobox protein 3 + + ALX4 Aristaless-like homeobox protein 4 + + AXIN2 Axin 2 + + + BAZ1B Bromodomain adjacent to zinc finger domain 1B + + BMP2 Bone morphogenetic protein 2 + + + + BMP7 Bone morphogenetic protein 7 + + + + CDC42 Cell division cycle 42 + + + CHD7 Chromodomain helicase DNA binding protein 7 + + + + CITED2 Cbp/p300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 2 + + + CTNNB1 Catenin Beta 1 + + + + DLX1 Distal-less homeobox 1 + + + DLX2 Distal-less homeobox 2 + + + DLX5 Distal-less homeobox 5 + + + + + + DLX6 Distal-less homeobox 6 + + + + + EDN1 Endothelin 1 + + + EDN3 Endothelin 3 + + + EDNRA Endothelin receptor type A + + + EDNRB Endothelin receptor type B + + + ERF ETS2 repressor factor + + + FGF7 Fibroblast growth factor 7 + + FGF8 Fibroblast growth factor 8 + + + + FGFR1 Fibroblast growth factor receptor 1 + + + + FGFR2 Fibroblast growth factor receptor 2 + + + FOXD3 Forkhead box D3 + + + FOXP2 Forkhead box P2 + + FREM1 FRAS1 related extracellular matrix 1 + + GDNF Glial-derived neurotrophic factor + + GLI3 GLI family zinc finger 3 + + + + GRHL3 Grainyhead like transcription factor 3 + + GSC Goosecoid homeobox + + HES1 Hes family bHLH transcription factor 1 + + + + HOXA2 Homeobox A2 + + + HSH2D Hematopoietic SH2 domain containing + + KIT KIT proto-oncogene receptor tyrosine kinase + + + + MAGOH Mago homolog, exon junction complex core component + + + MITF Microphthalmia-associated transcription factor + + + MSX1 Msh homeobox 1 + + + + MSX2 Msh homeobox 2 + + + NCAM1 Neural cell adhesion molecule 1 + + + + NODAL Nodal growth differentiation factor + + + NOG Noggin + + NTN1 Netrin 1 + + + PAX3 Paired box 3 + + + PAX6 Paired box 6 + + + + + PAX7 Paired box 7 + + POLR1A Polymerase (RNA) I subunit A + + POU3F2 POU class 3 homeobox 2 + + + + PQBP1 Polyglutamine binding protein 1 + + + PTCH1 Patched 1 + + + + RET Ret proto-oncogene + + + ROBO1 Roundabout guidance receptor 1 + + + ROBO2 Roundabout guidance receptor 2 + + + RUNX2 Runt related transcription factor 2 + + + + SATB2 Special AT-rich sequence binding- homeobox 2 + + + SHH Sonic hedgehog + + + + SIX2 Sine oculis-related homeobox 2 + + SLIT1 Slit guidance ligand 1 + + SLIT2 Slit guidance ligand 2 + + + SOX2 Sex determining region Y-box 2 + + + + SOX9 Sex determining region Y-box 9 + + + + + + SOX10 Sex determining region Y-box 10 + + + + SPECC1L Sperm antigen with calponin homology and coiled-coil domains 1-like + + + TCF12 Trascription factor 12 + + + TCOF1 Treacle ribosome biogenesis factor 1 + + VCAN Versican + + + ZIC1 Zinc finger protein family member 1 + + + a Core candidates for the “domestication syndrome” according to Wilkins et al. (2014) (bold italicized tags) plus language-readiness genes functionally interacting with them according to Benítez-Burraco et al. (in press) (regular tags). b Genes highlighted as candidates for globularization of the AMH skull/brain and the emergence of language-readiness according to Boeckx and Benítez-Burraco (2014a,b) and Benítez-Burraco and Boeckx (2015). c Involved in neural crest (NC) development and function. d Involved in craniofacial development and/or found mutated in craniofacial syndromes. e Involved in brain oscillation and rhythmicity. f Candidate for ASD as resulting from genomic studies (pathogenic SNPs, association studies, CNVs, functional studies, etc.). g Differentially expressed in postmortem brain tissues of ASD-vs.-control individuals (see text for details). When we tried to identify ASD-candidates among this extended list of genes via PubMed (http://www.ncbi.nlm.nih.gov/pubmed), we found out that nearly 25% of them have been suggested to play a role in the aetiopathogenesis of ASD. If we also consider genes that we found differentially expressed in postmortem brain tissues isolated from patients (as discussed in the subsequent section), the percentage rises above 50%. Interestingly, some of these genes are thought to be involved in brain rhythmicity (see Table 1), plausibly contributing to the oscillopathic signature of the ASD brain during language processing. We expect that the genes we highlight here are functionally interconnected and map on to specific pathways, signaling cascades, or aspects of brain development and function, of interest for language processing and the aetiopathology of ASD. In silico analyses offer promising insights. Accordingly, String 10 (http://www.string-db.org) predicts quite robust links between most of these genes (Figure 2). Likewise, ontology analyses by Panther (http://www.pantherdb.org) suggest that they might play biological functions important for ASD and be part of signaling pathways known to be impaired in this condition (Table 2). Figure 2 Functional links among candidates for domestication and ASD. The chart (drawn using String 10.0 license-free software, http://string-db.org/) shows the network of known and predicted interactions among all genes listed in Table 1. The colored nodes symbolize proteins: small nodes are proteins with unknown 3D structure, while large nodes are those with known 3D structures. The color of the edges represent different kind of known protein-protein associations. Green: activation, red: inhibition, dark blue: binding, light blue: phenotype, dark purple: catalysis, light purple: posttranslational modification, black: reaction, yellow: transcriptional regulation. Edges ending in an arrow symbolize positive effects, edges ending in a bar symbolize negative effects, whereas edges ending in a circle symbolize unspecified effects. Gray edges symbolize predicted links based on literature search ((co-mention in PubMed abstracts). Stronger associations between proteins are represented by thicker lines. The medium confidence value was 0.0400 (a 40% probability that a predicted link exists between two enzymes in the same metabolic map in the KEGG database: http://www.genome.jp/kegg/pathway.html). String 10 predicts associations between proteins that derive from a limited set of databases: genomic context, high-throughput experiments, conserved coexpression, and the knowledge previously gained from text mining (Szklarczyk et al., 2015). This is why the figure does not represent a fully connected graph (evidence for additional links are provided in the main text). Importantly, the diagram only represents the potential connectivity between the involved proteins, which has to be mapped onto particular biochemical networks, signaling pathways, cellular properties, aspects of neuronal function, or cell-types of interest that can be confidently related to aspects of language development and function. Table 2 GO classifications of candidates for domestication and ASD. Biological process %a Pathway Metabolic process (GO:0008152) 21.60% Axon guidance mediated by Slit/Robo (P00008) 12.20% Biological regulation (GO:0065007) 18.80% TGF-beta signaling pathway (P00052) 10.20% Developmental process (GO:0032502) 17.00% Endothelin signaling pathway (P00019) 8.20% Cellular process (GO:0009987) 13.80% Gonadotropin releasing hormone receptor pathway (P06664) 8.20% Multicellular organismal process (GO:0032501) 11.50% Wnt signaling pathway (P00057) 8.20% Immune system process (GO:0002376) 4.10% FGF signaling pathway (P00021) 8.20% Apoptotic process (GO:0006915) 3.20% Angiogenesis (P00005) 6.10% Response to stimulus (GO:0050896) 3.20% Hedgehog signaling pathway (P00025) 6.10% Biological adhesion (GO:0022610) 3.20% Axon guidance mediated by netrin (P00009) 4.10% Localization (GO:0051179) 1.80% CCKR signaling map (P06959) 4.10% a Numbers refer to percent of gene hit against total of process or pathway hits. Only the top 10 functions, filted after Bonferroni post-hoc correction, have been included. Candidate genes: a functional characterization Some of Wilkins et al.'s (2014) original candidates for the domestication syndrome are candidates for ASD. KIT mutations have been found in patients featuring ASD symptoms (Kilsby et al., 2013). KIT is a tyrosine kinase receptor (Kasamatsu et al., 2008), which acts as a key developmental regulator in the NC-derived processes of hematopoiesis, melanogenesis, and gametogenesis (Rothschild et al., 2003). In rats mutations of Kit impair hippocampal synaptic potentiation and spatial learning and memory (Katafuchi et al., 2000). Likewise, whole-genome sequencing analyses have identified deleterious variants of CHD7 in ASD probands (Jiang et al., 2013). CHD7 is known to be the main candidate for CHARGE syndrome (Vissers et al., 2004; Lalani et al., 2006), mentioned above. Interestingly, CHARGE syndrome also involves microcephaly, face asymmetry, cleft lip/palate, along with variable degrees of intellectual disability (Pisano et al., 2014; Hale et al., 2016, for review). Changes in the expression pattern of CDH7 can also result in behavioral anomalies resembling the autistic phenotype. Accordingly, in utero exposure to heavy metals in mice increases autism-like behavioral phenotypes in adult animals through inducing the hypomethylation of Chd7 (Hill et al., 2015). FOXD3, encoding a transcription factor, is downregulated by DISC1 (Drerup et al., 2009), a robust candidate for schizophrenia that has been also associated to ASD (Williams et al., 2009; Zheng et al., 2011; Kanduri et al., 2016). FOXD3 maps within one of the present-day human-specific differentially-methylated genomic regions (DMRs) (Gokhman et al., 2014). Interestingly, loss of Disc1 results in abnormal NCC migration and differentiation (Drerup et al., 2009). Also, DISC1 downregulates SOX10, another NC gene, involved in the maintenance of precursor NCC pools, in the timing of NCC migration onset, and in the induction of their differentiation; it is also implicated in oligodendrocyte differentiation (Hattori et al., 2014). In turn, SOX10 interacts with PAX3, another core candidate proposed by Wilkins et al. (2014), and with POU3F2 (Smit et al., 2000). Sequence and CNVs affecting POU3F2 have been found in subjects with ASD, and in individuals with different developmental and language delays (Huang et al., 2005; Lin et al., 2011). POU3F2 is a known interactor of FOXP2, the renowned “language gene” (Maricic et al., 2013). AMHs bear a derived allele of the binding site which is less efficient in activating transcription than the Neanderthal/Denisovan counterpart (Maricic et al., 2013). Likewise, POU3F2 has been associated with human accelerated conserved non-coding sequences (haCNSs) (Miller et al., 2014). Also, it interacts with PQBP1, which has been linked to intellectual disability (Wang et al., 2013) and developmental delay and microcephaly (Li et al., 2013). Also SOX9, considered a master regulator of craniofacial development and related to several congenital skeletal malformations (Mansour et al., 2002; Gordon et al., 2009; Lee and Saint-Jeannet, 2011), is found among the candidates for ASD. Accordingly, gene and miRNA expression profiling using cell-line derived total RNA has revealed SOX9 as one of the genes dysregulated in ASD (Ghahramani Seno et al., 2011). As discussed in detail by Benítez-Burraco et al. (in press) SOX9 interacts with BMP2, BMP7, DLX2, and HES1. All of them are core components of the network believed important for globularization and language-readiness (reviewed in Boeckx and Benítez-Burraco, 2014a). In addition, all of them are involved in NCC development and migration, and in the patterning of NC-derived tissues (Mallo, 2001; Gajavelli et al., 2004; Correia et al., 2007; Glejzer et al., 2011; Ishii et al., 2012). BMP2 is a key osteogenic regulator, which has been associated to craniosynostosis (Justice et al., 2012; Lattanzi et al., 2013). BMP2, BMP7, and DLX2 act upstream SOX9 (Sperber et al., 2008; Li et al., 2013). In turn, SOX9 mediates the retinoic acid-induced expression of HES1, known also to be involved in language function, craniofacial development, and neuron growth and interconnection (reviewed in Boeckx and Benítez-Burraco, 2014b). Importantly, retinoic acid also regulates the expression of other genes that are relevant for language, like FOXP2 (Devanna et al., 2014), or for globularization, like ASCL1 (see Benítez-Burraco and Boeckx, 2015, for details). Retinoic acid has proven to be important for brain plasticity (Luo et al., 2009), and memory and learning processes (Etchamendy et al., 2003; Jiang et al., 2012). Recent whole-exome sequencing analyses have linked retinoic acid regulation pathways to ASD (Moreno-Ramos et al., 2015). In neuronal cells reduced levels of RORA downregulate multiple transcriptional targets that are significantly enriched in biological functions negatively impacted in ASD and which include known ASD-associated genes, like A2BP1, CYP19A1, ITPR1, NLGN1, and NTRK2 (Sarachana and Hu, 2013a). RORA itself is downregulated in postmortem prefrontal cortex and cerebellum of subjects with ASD (Nguyen et al., 2010). RORA is differentially regulated in them by masculine and feminine hormones: Whereas it is under negative feedback regulation by androgens, it is under positive regulation by estrogens (Sarachana et al., 2011; Sarachana and Hu, 2013b). In certain regions of the brain this sexually dimorphic expression is also found in several of RORA's targets and this correlation is much higher in the cortex of males (Hu et al., 2015). Perhaps not surprisingly, synthetic RORα/γ agonist improve autistic symptoms in animal models of the disease, particularly, repetitive behavior (Wang et al., 2016). We wish to highlight two other genes thought to be involved in the changes that brought about modern language that are also candidates for ASD and interact with core candidates for the domestication syndrome as posited by Wilkins et al. The first one is DLX5, involved in crucial aspects of NC development (McLarren et al., 2003; Ruest et al., 2003), but also of skull and brain development (Kraus and Lufkin, 2006; Wang et al., 2010). Accordingly, it plays a role in thalamic development (Jones and Rubenstein, 2004) and contributes to regulate the migration and differentiation of precursors of GABA-expressing neurons in the forebrain (Cobos et al., 2006). DLX5 is a candidate for ASD (Nakashima et al., 2010), due to an ultraconserved cis-regulatory element (Poitras et al., 2010), which is bound by GTF2I, encoded by one of the genes commonly deleted in Williams-Beuren syndrome (OMIM#194050) and a candidate for ASD too (Malenfant et al., 2012). Additionally, DLX5 is regulated by MECP2 (Miyano et al., 2008), encoded by the main candidate for Rett syndrome (OMIM#312750), a condition entailing problems for motor coordination, autistic behavior, and language regression (Uchino et al., 2001; Veenstra-VanderWeele and Cook, 2004). Interestingly, Dlx5/6(±) mice exhibit abnormal pattern of γ rhythms resulting from alterations in GABAergic interneurons, particularly in fast-spiking interneurons (Cho et al., 2015). In addition, DLX5 interacts with key candidates for language evolution, in particular, with RUNX2 and FOXP2 (see Boeckx and Benítez-Burraco, 2014a, for details). The second one is NCAM1, which is also a target of both RUNX2 (Kuhlwilm et al., 2013) and FOXP2 (Konopka et al., 2009). In mice mutations in the gene affect working/episodic-like memory (Bisaz et al., 2013), whereas overexpression of the Ncam1 extracellular proteolytic cleavage fragment impacts on GABAergic innervation, affecting long- and short-term potentiation in the prefrontal cortex (Brennaman et al., 2011). NCAM1 encodes a cell adhesion protein involved in axonal and dendritic growth and synaptic plasticity (Rønn et al., 2000; Hansen et al., 2008). It interacts with VCAM1 which is involved in cell adhesion and the control of neurogenesis (Kokovay et al., 2012), and which bears a fixed (D414G) change in AMHs compared to Neanderthals/Denisovans (Pääbo, 2014). VCAM1 is upregulated by CLOCK, which plays a key role in the modulation of circadian rhythm (Gao et al., 2014). Together with other circadian-relevant genes CLOCK seems to be involved in the psychopathology of ASD cases entailing sleep disturbances (Yang et al., 2016). The circadian modulation of synaptic function has been hypothesized to contribute decisively to ASD (Bourgeron, 2007). In turn, CLOCK interacts with RUNX2 and with several other candidates for language-readiness, like DUSP1, involved in vocal learning (Doi et al., 2007), and USF1, which regulates synaptic plasticity, neuronal survival and differentiation (Tabuchi et al., 2002; Steiger et al., 2004). USF1 binds the promoter of FMR1 (Kumari and Usdin, 2001), a strong candidate for Fragile-X syndrome (OMIM#300624), which presents with language problems and ASD features (Kaufmann et al., 2004; Smith et al., 2012). The regulatory region of USF1 shows many fixed or high frequency changes compared to Denisovans (Meyer et al., 2012). As shown in Table 1, several of the genes important for globularization and language-readiness are involved in NC development and function and some of them are also candidates for ASD. Accordingly, we expect them to contribute to the abnormal domesticated features observed in patients with ASD, and also to their distinctive language profile. Among them we wish mention CTNNB1, DLX1, DLX6, PAX6, and ROBO2. CTNNB1 is a component of the Wnt/β-catenin signaling pathway, known to be impaired in ASD (Cao et al., 2012; Zhang et al., 2012; Martin et al., 2013). CTNNB1 controls aspects of NC development, from NC induction, lineage decisions, to differentiation (Hari et al., 2012). As noted in Boeckx and Benítez-Burraco (2014b), CTNNB1 is expected to interact with many of the genes highlighted as important for the evolution of language-readiness, specifically with RUNX2 and SLIT2/ROBO1 signals. Regarding DLX1, it is a robust NC marker (Ishii et al., 2012), involved in patterning and morphogenetic processes in NC-derived tissues (Mallo, 2001). It also regulates the development of the skull and the brain (Andrews et al., 2003; Jones and Rubenstein, 2004). In mice Dlx1 downregulation results in reduced glutamatergic input to the hippocampus (Jones et al., 2011), as well as in changes in interneuron subtypes and migration patterns in the cortex (Ghanem et al., 2008). DLX1 is found to be downregulated in ASD (Voineagu et al., 2011; McKinsey et al., 2013). ROBO2 is one of the DLX1 interactors. Slit/Robo signaling regulates early NCC migration (Jia et al., 2005) ROBO2 is also involved in thalamocortical axons (TCA) development, known to be important for the modulation of cognitive functions (López-Bendito et al., 2007; Marcos-Mondéjar et al., 2012). ROBO2 is a candidate for ASD (Suda et al., 2011), but also for different types of language disorders, like dyslexia (Fisher et al., 2002) and speech-sound disorder and reading (Stein et al., 2004). It has been related as well to expressive vocabulary growth in the normal population (St Pourcain et al., 2014). Finally, PAX6 controls the migration of NCCs from the anterior midbrain (Matsuo et al., 1993). PAX6 is involved as well in the development of the brain (Valverde et al., 2000; Tyas et al., 2003; Caballero et al., 2014). Mutations on PAX6 have been reported in some forms of ASD (Maekawa et al., 2009), although they also impact in working memory (Bamiou et al., 2007). Alterations of PAX6 expression in the brain of people with ASD may account for the observed imbalance in excitatory/inhibitory neuronal activity (Kim et al., 2014). And like many of the genes reviewed above, PAX6 is functionally related to both FOXP2 and RUNX2, and it also targets POU3F2 (see Benítez-Burraco and Boeckx, 2015, for details). Most of the NCC-genes mentioned here are known to play a key role in the development and patterning of the craniofacial complex, and to be associated to congenital craniofacial defects (Table 1) (see Twigg and Wilkie, 2015 for review). Many of these genes are known candidates for ASD, including DLX5 and DLX6 (reviewed above), FGFR2, MSX1, POLR1A, and PTCH1. Both DLX5 and DLX6 are indeed required for NC-derived facial morphogenesis (Gitton et al., 2011) FGFRs are among the main craniosynostosis-associated genes. In particular, gain-of-function mutations in FGFR2 are typically associated to Apert (OMIM#101200) and Crouzon (OMIM#123500) syndromes, while both FGFR1 and FGFR2 are found mutated in Pfeiffer syndrome (OMIM#101600) (Lattanzi et al., 2012). All these syndromic craniosynostoses occasionally present with variable degree of ASD-like mental retardation (Morey-Canellas et al., 2003). MSX1 encodes a transcriptional repressor involved in craniofacial development and shaping (particularly in odontogenesis) (Alappat et al., 2003; Lattanzi, forthcoming). It is expressed in the NC (Khadka et al., 2006), where it acts as a master regulator of gene expression (Attanasio et al., 2013). Although it has not been associated to ASD, MSX1 is a direct downstream target of DLX5 during early inner ear formation (Sajan et al., 2011). The gene is also a critical intrinsic dopaminergic neuron determinant (Andersson et al., 2006) and is found mutated in some patients with Wolf-Hirschhorn syndrome (OMIM#194190), a clinical condition entailing profound mental retardation and craniofacial dysmorphism (Campbell et al., 1989). POLR1A, found mutated in acrofacial dysostosis (Cincinnati type, OMIM#616462) involving microcephaly, plays a role in the regulation of NC-derived skeletal precursor cells (Weaver et al., 2015). In some ASD subjects CNVs result in fusion transcripts involving POLR1A, although no fusion transcripts have been detected to date (Holt et al., 2012). Finally, it is worth mentioning that genes encoding primary cilium signaling molecules, such as SHH, GLI3, and PTCH1, are all primarily involved in congenital malformations affecting the midline craniofacial compartment (Brugmann et al., 2010; Rice et al., 2010). Specifically, PTCH1 is required in the NC-dependent orofacial development and gives rise to orofacial clefting, when mutated (Metzis et al., 2013). Heterozygous mutations of either SHH or PTCH1 are typically found in holoprosencephaly (OMIM#610828, and #236100), a genetically heterogeneous, highly prevalent congenital forebrain anomaly in humans, associated with mental retardation and craniofacial malformations (Ming et al., 2002; Mercier et al., 2011). In addition, a 22-bp deletion in this gene has been found in a girl with ASD and Gorlin syndrome, a complex condition involving macrocrania and hypertelorism (Delbroek et al., 2011). Candidate genes: expression profiles in the ASD brain If our hypothesis is on the right track, we expect that the genes we highlight here are dysregulated in the brain of people with ASD, particularly in areas important for language processing. Accordingly, we surveyed the Gene Expression Omnibus (GEO) repository (https://www.ncbi.nlm.nih.gov/gds) searching for their expression profiles in the cerebellum and the temporal cortex (but also in the frontal and occipital cortices) in patients with ASD. This should help identify new candidates for ASD in the context of domestication and language-readiness (Table 1). Overall, we could find significant expression values for some of our candidates and learnt that they are up- or downregulated in the brain of autists (Figure 3). Figure 3 Expression profiles of candidate genes in the ASD brain. Data were gathered from the following microarray expression datasets available on the Gene Expression Omnibus database (GEO datasets, http://www.ncbi.nlm.nih.gov/gds): GSE28521 (Voineagu et al., 2011) for the temporal and frontal cortices, GSE38322 (Ginsberg et al., 2012) for the cerebellum and the occipital cortex. Data are shown as log transformation of fold changes (logFC) between patients and corresponding controls. Only genes showing statistically significant (p < 0.05) differential expression were considered. Additional details may be found in the Supplemental information file. Among the genes that are significantly downregulated in the cerebellum we found AXIN2, EDNRB, SOX2, SPECC1L, TCF12, and VCAN, whereas CDC42, and PQBP1 are found upregulated in this region (Figure 3). Although none of them has been associated to ASD, they stroke us as promising candidates for the atypical presentation of the domestication syndrome in ASD. AXIN2 is expressed in the cranial NC and is needed for NC-derived frontal bone osteogenesis (Yu et al., 2005; Li et al., 2015a). This gene is also expressed as a specific marker for suture stem cells (Maruyama et al., 2016), but also acts as a negative regulator of canonical Wnt pathway, contributing to the stability of CTNNB1 (Li et al., 2015a). Speech alterations are also observed in people with AXIN2 mutations causing non-syndromic oligodontia (Liu et al., 2015). EDNRB encodes a receptor for endothelins, known to be potent vasoactive peptides. Mutations in this gene are associated to increased susceptibility to Hirschsprung disease (OMIM#600155), a neurocristopathy characterized by congenital absence of intrinsic ganglion cells in the enteric nervous plexa (Amiel et al., 2008). Waardenburg syndrome (OMIM#277580), a genetically heterogeneous condition which may involve developmental delay subsidiary to sensorineural hearing loss, has also been associated with mutations in EDNRB (Read and Newton, 1997). SOX2, one of core candidates for domestication (Wilkins et al., 2014), encodes an interactors of the GLI factors as part of the SHH-GLI signaling pathway involved in NCC fate (Oosterveen et al., 2012, 2013; Peterson et al., 2012), but also in the globularization of the AMH skull/brain (see Boeckx et al., submitted for details). SOX2 interacts as well with the BMP signaling (Li et al., 2015b). Interestingly, SOX2 regulates PQBP1, highlighted above as one of POU3F2 interactors. SPECC1L is found mutated in Opitz G/BBB syndrome (OMIM# #145410) and in facial clefting (Kruszka et al., 2015). This gene functions in NC development (Wilson et al., 2016) and is specifically involved in facial morphogenesis (Saadi et al., 2011). TCF12 is highly expressed in embryonic precursors of skull/brain structures, including NC-derived head mesenchyme (Uittenbogaard and Chiaramello, 2002). TCF12 directly interacts with TWIST1, mutated in Saethre-Chotzen syndrome (OMIM#601622), which features complex craniosynostosys with variable degrees of intellectual disability, including ASD traits (Maliepaard et al., 2014). Indeed, loss-of-function mutations of TCF12 have been identified in patients with coronal synostosis, which sometimes involves intellectual disability (Sharma et al., 2013; di Rocco et al., 2014; Paumard-Hernández et al., 2015; Piard et al., 2015). VCAN encodes versican-1, a protein that guides migratory NCCs (Dutt et al., 2006) and which shows a fixed N3042D change in AMHs (Pääbo, 2014). Finally, CDC42 controls NC stem cell proliferation (Fuchs et al., 2009). Inactivation of Cdc42 in NCCs causes craniofacial and cardiovascular morphogenesis defects (Liu et al., 2013). As discussed in detail in Boeckx and Benítez-Burraco (2014b) CDC42 is an important gene regarding language evolution, because of its functional connections with core candidates for globularization and the externalization of language (including FOXP2, RUNX2, SLIT2, and ROBO1), with genes related to language disorders (like CMIP), and with genes known to have changed after our split from Neanderthals and Denisovans (like ITGB4, ARHGAP32, ANAPC10, and CDC42EP4). With regards to the temporal cortex, we found that EDNRB, POU3F2, SOX2, VCAN, and TCF12 are downregulated in subjects with ASD, whereas KIT, PQBP1, and SLIT2 are upregulated in them (Figure 3). As noted above, POU3F2, SOX2, KIT are known candidates for ASD. We have already reviewed all these genes. Concerning the frontal cortex we found that BMP7, EDNRB, PAX6, ERF, and MSX1 are significantly downregulated, whereas CITED2 and DLX1 are significantly upregulated (Figure 3). As noted in Table 1, DLX1, PAX6, and MSX1 have been previously associated to ASD. Most of these genes have been already reviewed. BMP7 is a NCC gene involved in regulation of osteogenesis (Cheng et al., 2003; Anderson et al., 2006) and in skull and brain development (Yuge et al., 2011; Segklia et al., 2012). BMP7 is also closely related to some of the core candidates for globularization and language- readiness, like BMP2, DLX1, DLX2, and RUNX2. BMP7 is predicted (according to String 10) to interact with SOX2 via NOG, involved in dopamine neuron production and an inhibitor of BMP signaling (Chiba et al., 2008). Developmental delay and learning disabilities are commonly observed in people with mutations in BMP7 (Wyatt et al., 2010). ERF encodes a member of the ETS family of transcription factors, expressed in migratory cells, including NCCs (Paratore et al., 2002). ERF haploinsufficiency gives rise to either coronal or multisuture synostosis, midface hypoplasia, often associated with behavioral and learning difficulties (Twigg et al., 2013). Concerning CITED2, this is a functional partner of both FOXP2 and RUNX2 (Luo et al., 2005; Vernes et al., 2011; Nelson et al., 2013), two important genes for the emergence of modern language. CITED2 is also involved in craniofacial development (Bhattacherjee et al., 2009) and in the establishment of left-right axis through interactions with the BMP signaling and Nodal (Preis et al., 2006; Lopes Floro et al., 2011). Interestingly, 99% of AMHs bear a highly disruptive intergenic change near CITED2 compared to Altai Neanderthals and Denisovans (Prüfer et al., 2014). Finally, regarding the occipital cortex, we found that EDN3, CDC42, and PQBP1 are significantly upregulated in ASD, whereas SOX2, SOX9, and VCAN are downregulated. We have already considered all these genes, with the exception of EDN3. This gene encodes an endothelium-derived vasoactive peptide which binds the product of EDNRB, playing a key role in the development of neural crest-derived cell lineages, such as melanocytes and enteric neurons. Although EDN3 is a candidate for Waardenburg syndrome and Hirschsprung disease, the gene has been found significantly dysregulated in children with ASD (Glatt et al., 2011). As shown in Figure 3, genes that exhibit significant changes in their expression levels in the ASD brain are consistently down- or upregulated across all the regions under analysis. Considering their functions and the phenotypes resulting from their mutation, the most promising of these genes are CDC42 and PQBP1 (which are upregulated) and EDNRB and SOX2 (which are found downregulated). As noted above, to date none of them have been associated to ASD, but they emerge as reasonably involved in the pathogenesis of this condition. Noteworthy age-related differences in the intrinsic functional connectivity of the brain are observed in ASD: adult patients show reduced connectivity while children tend to exhibit an increased connectivity (Uddin et al., 2013). Therefore, we have further analyzed ASD brain expression data in an age-matched fashion. Due to the available sample characteristics, only expression data obtained from the cerebellum could be analyzed (see Supplemental file for further details). The reduction of the age-related bias in the patients-vs.-controls comparison, enabled finding a higher number of statistically significant dysregulated genes in the ASD brain. Accordingly, we found that in the cerebellum of ASD children (below 11 years old), CDC42, MSX1, MSX2, NODAL, PQBP1, and SLIT2 were downregulated, whereas, AXIN2, CHD7, CITED2, EDNRB, FGF8, NCAM1, PAX7, PTCH1, RET, ROBO1, SOX2, SPECC1L, TCF12, VCAN, and ZIC1 were upregulated. In turn, in the cerebellum of adult patients (aged 22–60 years) we found that CDC42, CTNNB1, DLX1, EDNRA, EDNRB, HES1, KIT, MAGOH, MITF, NCAM1, NTN1, POU3F2, PQBP1, PTCH1, RET, ROBO1, SATB2, SOX2, SPECC1L, TCF12, VCAN, and ZIC1 are downregulated, whereas only AXIN2, CTNNB1, DLX1, EDN1, and MSX1 are upregulated. Overall, we concluded that nearly one third of the candidates for domestication are dysregulated in the cerebellum of people with ASD, and that more than a half are specifically dysregulated in the cerebellum of either children and/or adults with this condition. Genes that are dysregulated in both children and adults with ASD can be regarded as significant contributors to the atypical presentation of the domestication syndrome in this condition. This list encompasses 14 genes: AXIN2, CDC42, EDNRB, MSX1, NCAM1, PQBP1, PTCH1, RET, ROBO1, SOX2, SPECC1L, TCF12, VCAN, and ZIC1. Interestingly, they exhibit opposite expression profiles in children and adults with ASD (Figure 4). Most of these genes have been already discussed here. In addition, RET, encoding a cadherin that plays a crucial role in NC development, is a candidate for Hirschsprung disease (OMIM# 142623; Edery et al., 1994) and is found to be differentially expressed after RUNX2 transfection in neuroblastoma cells (Kuhlwilm et al., 2013). RET is downstream ASCL1 (another candidate for Hirschsprung disease) in noradrenergic brain stem neurons important for respiratory rhythm modulation (Dauger et al., 2001). Likewise, ZIC1 is needed for NC development (along with PAX3) and plays a key role in craniofacial development (Milet et al., 2013; Plouhinec et al., 2014). Mutations in ZIC1 result in severe coronal synostosis associated with learning difficulties (Twigg et al., 2015). Figure 4 Expression profiles of candidates genes in the cerebellum of children and adults with ASD. Expression data were obtained from the microarray expression dataset GSE38322 (Ginsberg et al., 2012) available on the Gene Expression Omnibus database (GEO datasets, http://www.ncbi.nlm.nih.gov/gds). Data are shown as log transformation of fold changes (logFC) between patients and corresponding controls. Only genes showing statistically significant (p < 0.05) differential expression were considered. Additional details may be found in the Supplemental information file. In the last section of the paper we attempt refining our characterization of ASD as an atypically-domesticated phenotype. In doing so, we will focus on brain function, with a special emphasis on brain oscillations. Accordingly, we will compare the oscillopathic profile of people with ASD during language processing with the oscillatory signature of the TD population and that of non-domesticated primates. Additionally, we will examine the expression profile in the primate brain of the candidate genes we highlight here. ASD and wild primates: from brain oscillation to gene expression patterns ASD and primate oscillomes As we have discussed in detail in Benítez-Burraco and Murphy (2016) language impairment in ASD (as ASD itself) can be satisfactorily characterized as an oscillopathic condition. With cognitive disorders exhibiting disorder-specific abnormal oscillatory profiles, it is also noteworthy that species-specific oscillatory patterns seemingly emerge as slight variations within the network constellation that constitutes a universal brain syntax (Buzsáki and Watson, 2012; Buzsáki et al., 2013). The differences in brain capacity between domesticated and non-domesticated animals (Wilkins et al., 2014) would be predicted to give rise to a corresponding alteration in oscillatory properties (i.e., features of neural oscillations which form part of an individual's “oscillome,” as it is termed in Murphy and Benítez-Burraco, in press; Murphy, 2016a,b). Although we feel that current knowledge is too scarce to permit any reasonable linking hypotheses between the primate and ASD oscillomes, we would like to briefly sketch out a possible route to increasing our understanding of the neural signature of domestication (and failed domestication itineraries). Call vocalizations have been found not to be impaired when the homolog of Broca's region in the monkey brain is lesioned, which suggests that other area (like the limbic system and brainstem) are involved (Sage et al., 2006). However, macaques appear to share similar call comprehension substrates with human language comprehension in the left posterior temporal gyrus (Heffner and Heffner, 1986). It would be of interest, for instance, to compare the rhythmic properties of this region of the TD brain with those of the primate brain to see if any particular activity (e.g., coupling and synchronization) is marked in humans. This would also yield insights into how the primate call comprehension system “interfaces” with other cognitive systems (given the appropriate experimental environment), and would also permit the exploration of similar interface properties of human language comprehension, which requires the transfer of information to two interfaces (Figure 5). Figure 5 A schematic view of language representing the systems and interfaces of interest and the levels of analysis. “Cognome” refers to the operations available to the human nervous system (Poeppel, 2012) and “dynome” refers to brain dynamics (Kopell et al., 2014; Murphy and Benítez-Burraco, in press). See text for details. Discounting work on evoked potentials (which is itself fairly modest), there are currently only a handful of empirical studies of the monkey oscillome. Brincat and Miller (2015), for instance, discovered functional differences and frequency-specific interactions between the Rhesus hippocampus (HPC) and prefrontal cortex (PFC) during object pair association learning. θ synchrony was found to be greater after errors and decreased after learning; correct associations increased β-α synchrony, which was also greater in the HPC-PFC direction. Esghaei et al. (2015) also suggested that the macaque visual cortex employs phase-amplitude coupling to regulate inter-neuronal correlations, and so the potential for generic oscillomic processes to yield insights into cognitive (dys)function seems apparent. During the internally monitored continuation phase of a synchronization-continuation task, β also appears to increase in Rhesus monkeys (Bartolo and Merchant, 2015), suggesting that—as in humans (Murphy, 2015a)—β is responsible for maintaining the existing cognitive set in memory. β is also involved in “the attentive state and external cues as opposed to detailed muscle activities” in Japanese monkeys (Macaca fuscata) (Watanabe et al., 2015). Finally, pulvinar γ is involved in feedforward processing for snake images, and also in cortico-pulvinar-cortical integration for face images (Le et al., 2016), while Ramirez-Villegas et al.'s (2015) study of the macaque hippocampal CA3-CA1 network pointed to the role of γ in memory reactivation, transfer and consolidation. Currently, there are no studies comparing the oscillome of domesticated and non-domesticated primates, but our prediction would be that non-domesticated primates display a degree of oscillomic difference with domesticated primates comparable to the difference between TD individuals and people with ASD. Table 3 summarizes existing knowledge of the ASD and primate oscillome during a range of cognitive tasks, and it is hard to find any correlations or connections between the two. However, we feel that comparatively exploring these oscillomes will permit a greater understanding of the atavistic neural oscillations of the non-domesticated human and primate brains. Future research should also seek to compare the oscillomes of domesticated and non-domesticated primates in an effort to investigate neural signatures of domestication. Table 3 Summary of the patterns of rhythmicity observed in wild primates and the observed oscillomic differences in ASD compared to TD subjects. Frequency band Oscillomic monkey profile Oscillopathic profile of autism spectrum disorder Delta (~0.5–4 Hz) Decreased phase-amplitude coupling with γ yields increased visual attention, suggesting that cross-frequency coupling suppression modulates attention. Increased in eyes-closed resting state exam; predicted to be disrupted in processing phrases involving raising and passives. Theta (~4–10 Hz) Decreased phase-amplitude coupling with γ yields increased visual attention; greater HPC-PFC synchrony after object pair association errors. Reduced cross-frequency coupling with γ; does not synergistically engage with γ during speech; predicted to be disrupted in certain memory retrieval processes. Alpha (~8–12 Hz) Increased synchrony with β during correct object pair associations. Reduced cross-cortically; reduced resting-state α-γ phase amplitude coupling; increased in resting state; predicted to be disrupted during certain lexicalizations. Beta (~10–30 Hz) Increased synchrony with α during object pair associations; increases during continuation phase of a synchronization-continuation task. Reduced in picture-naming tasks; predicted to be disrupted in the maintenance of syntactic objects in raising, passives and wh-questions. Gamma (~30–100 Hz) Involved in processing snake and face images increases during action sequence updating and memory consolidation, reactivation, and transfer. Over-connectivity gives rise to increased γ; reduced in rSTG and lIFG during picture naming; predicted to be disrupted quite generally in linguistic cognition. Candidate genes: expression profiles in the primate brain If our hypothesis turns to be on the right track, we further expect that the genes that we have found dysregulated in the brain of people with ASD show similar expression profiles in conditions where normal socialization failed to occur. Because feral children are scarce and not easily available we examined the expression profiles of these genes in wild primates (chimps). In particular, we selected available gene expression profiles obtained from chimp brain areas that are known to be involved in language processing in humans (the cerebellum, the temporal cortex, and the frontal cortex), as we did for people with ASD. We learnt that most of the genes that we had previously found differentially expressed in the ASD brain data exhibit the same expression pattern in the chimp brain, including EDNRB (in the cerebellum), BMP7, DLX1, EDNRB, MSX1, and PAX6 (in the frontal cortex), and VCAN (in the temporal cortex) (Figure 6). Figure 6 Comparative expression profiles in chimpanzees and subjects with ASD of candidate genes. Data were obtained from microarray expression datasets available on the Gene Expression Omnibus database (GEO datasets, http://www.ncbi.nlm.nih.gov/gds): GSE28521 (Voineagu et al., 2011) for the temporal and frontal cortices, and GSE38322 (Ginsberg et al., 2012) for the cerebellum of subjects with ASD; GSE22569 (Somel et al., 2011; Liu et al., 2012) for the cerebellum, GSE18142 (Konopka et al., 2009) for the frontal cortex, and GSE7540 (Cáceres et al., 2003) for the temporal cortex of chimps. Data are shown as log transformation of fold changes (logFC) between patients and corresponding controls. Only genes showing statistically significant (p < 0.05) differential expression were considered. Additional details may be found in the Supplemental information file. Note that the plot is intended to display the overall trend of gene expression, given that the relative expression values (i.e., logFC) were obtained from comparative analyses performed on different datasets (based on different designs, samples, and batches). Conclusions Socialization is a crucial step needed for the achievement of many cognitive abilities that are a signature of the human condition. Language is one of the most prominent of such abilities. Several high prevalent pathological conditions impact on human-specific cognitive abilities, including schizophrenia and ASD. In ASD, social abilities are seriously compromised, but other core cognitive skills, including language, also exhibit differences with the non-affected population. ASD is a multifactorial condition, with wide clinical and genetic heterogeneity. It is still not clear how ASD features emerge from genomic and/or environmental cues during development. In this paper we have focused on language deficits in ASD, although we expect that the lessons we draw here contribute to shedding light on the whole profile of this cognitive condition. In doing so we have adopted and evolutionary perspective, because of the robust link that exists between (abnormal) development and evolution. As we have shown, domesticated traits are absent or are attenuated in people with ASD, and genes that we believe important for the (self)domestication of our species and the evolution of our distinctive cognitive abilities (including language) show abnormal expression patterns in the brains of people with autism. What is more: abnormalities can be traced to the time window when crucial brain rewiring occurs during language acquisition and when changes in the normal configuration of the brain occurs in children with ASD. Additionally, some features of the ASD phenotype can be found (or expected to be found) in wild primates. On the whole, we think that our approach can help illuminate the etiology of ASD primarily because provides robust links between the genome and the environment, and between development and evolution, in line with the current evo-devo approaches to cognitive diseases (see Benítez-Burraco, 2016a, for review). In this sense, the putative involvement of the neural crest in the aetiopathogenesis of ASD emerges as a promising avenue for future research. At the same time, we expect that our approach with help illuminate the evolutionary history of our language-readiness: Our results support the view that language evolution benefitted from a favorable social context that may have resulted from our (self)-domestication. Author contributions AB contributed to all Sections and drafted the manuscript, WL contributed to Sections “Domestic Traits in the ASD Phenotype” and “ASD and the Genetics of the Domestication Syndrome” and revised the manuscript, EM contributed to Sections “Introduction” and “ASD and Wild Primates: from Brain Oscillation to Gene Expression Patterns” and revised the manuscript. Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Preparation of this work was supported in part by funds from the Spanish Ministry of Economy and Competitiveness (grant numbers FFI2014-61888-EXP and FFI-2013-43823-P to ABB), in part by “Linea D1- 2015” intramural funds from Università Cattolica S. Cuore (to WL), and in part by an Economic and Social Research Council scholarship (1474910) (to EM). ==== Refs References Akefeldt A. Gillberg C. (1991). 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==== Front Front ImmunolFront ImmunolFront. Immunol.Frontiers in Immunology1664-3224Frontiers Media S.A. 10.3389/fimmu.2016.00327ImmunologyOriginal ResearchCombination of In Silico Methods in the Search for Potential CD4+ and CD8+ T Cell Epitopes in the Proteome of Leishmania braziliensis e Silva Rafael de Freitas 12Ferreira Luiz Felipe Gomes Rebello 3Hernandes Marcelo Zaldini 3de Brito Maria Edileuza Felinto 2de Oliveira Beatriz Coutinho 2da Silva Ailton Alvaro 2de-Melo-Neto Osvaldo Pompílio 4Rezende Antônio Mauro 4*†Pereira Valéria Rêgo Alves 2*†1Department of Natural Sciences, Universidade de Pernambuco, Garanhuns, Pernambuco, Brazil2Department of Immunology, Fundação Oswaldo Cruz, Recife, Pernambuco, Brazil3Department of Pharmaceutical Sciences, Universidade Federal de Pernambuco, Recife, Pernambuco, Brazil4Department of Microbiology, Fundação Oswaldo Cruz, Recife, Pernambuco, BrazilEdited by: José Mordoh, Fundación Instituto Leloir, Argentina Reviewed by: Yvonne Paterson, University of Pennsylvania, USA; Vijay Panchanathan, Perdana University, Malaysia *Correspondence: Antônio Mauro Rezende, antonio.rezende@cpqam.fiocruz.br; Valéria Rêgo Alves Pereira, valeria@cpqam.fiocruz.br†Antônio Mauro Rezende and Valéria Rêgo Alves Pereira contributed equally to this work. Specialty section: This article was submitted to Immunotherapies and Vaccines, a section of the journal Frontiers in Immunology 29 8 2016 2016 7 32728 6 2016 16 8 2016 Copyright © 2016 e Silva, Ferreira, Hernandes, de Brito, de Oliveira, da Silva, de-Melo-Neto, Rezende and Pereira.2016e Silva, Ferreira, Hernandes, de Brito, de Oliveira, da Silva, de-Melo-Neto, Rezende and PereiraThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.The leishmaniases are neglected tropical diseases widespread throughout the globe, which are caused by protozoans from the genus Leishmania and are transmitted by infected phlebotomine flies. The development of a safe and effective vaccine against these diseases has been seen as the best alternative to control and reduce the number of cases. To support vaccine development, this work has applied an in silico approach to search for high potential peptide epitopes able to bind to different major histocompatibility complex Class I and Class II (MHC I and MHC II) molecules from different human populations. First, the predicted proteome of Leishmania braziliensis was compared and analyzed by modern linear programs to find epitopes with the capacity to trigger an immune response. This approach resulted in thousands of epitopes derived from 8,000 proteins conserved among different Leishmania species. Epitopes from proteins similar to those found in host species were excluded, and epitopes from proteins conserved between different Leishmania species and belonging to surface proteins were preferentially selected. The resulting epitopes were then clustered, to avoid redundancies, resulting in a total of 230 individual epitopes for MHC I and 2,319 for MHC II. These were used for molecular modeling and docking with MHC structures retrieved from the Protein Data Bank. Molecular docking then ranked epitopes based on their predicted binding affinity to both MHC I and II. Peptides corresponding to the top 10 ranked epitopes were synthesized and evaluated in vitro for their capacity to stimulate peripheral blood mononuclear cells (PBMC) from post-treated cutaneous leishmaniasis patients, with PBMC from healthy donors used as control. From the 10 peptides tested, 50% showed to be immunogenic and capable to stimulate the proliferation of lymphocytes from recovered individuals. neglected tropical diseasescutaneous leishmaniasisLeishmania braziliensisvaccine developmentCD4+ CD8+ T cell epitopesConselho Nacional de Desenvolvimento Científico e Tecnológico10.13039/501100003593404259/2012 ==== Body Introduction The leishmaniases constitute an important group of neglected tropical diseases (1), which affect and impact “the bottom billion” of people living in poverty by inducing disfiguration, loss of productivity, and a burden of 3.3 million disability-adjusted life years (DALY) (2–4). It is estimated that one-quarter of the world’s population, 1.7 billion people, are living in risk areas for leishmaniasis (5). Until recently, 98 countries have reported cases of leishmaniasis with 0.7–1.2 and 0.2–0.4 million cases reported annually of cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL), respectively (6). The leishmaniases are caused by protozoans from the genus Leishmania, transmitted to humans and other mammals by phlebotomine sand fly bites (7). These diseases have multiple clinical forms and the main ones are CL, widely distributed among targeted populations and which affects the skin and mucous; VL, the more lethal form if not treated, which affects mainly the reticuloendothelial system of the liver and the spleen; and mucocutaenous leishmaniasis (MCL), which affects the mucous and has a poor prognosis. In Brazil, most of CL cases are caused by Leishmania braziliensis. The disease is found in different regions (8), but many patients experience spontaneous cure that is associated with IFN-γ production (9). Diverse strategies are being used to control the leishmaniasis, namely, the control of infected animals and vectors and the chemotherapy of affected individuals. These approaches, however, have a high cost and can also induce resistance in parasites and vectors (10). Therefore, a safe and effective vaccine against human leishmaniasis is urgent to address these issues. Vaccines against Leishmania spp. have the major task of correctly activating the immune system to develop a protective response composed mainly by CD4+ and CD8+ T cells, producing and secreting IFN-γ. This response has been associated with disease control, macrophage activation, and parasite elimination from the host (11–13). Our previous data show that cells from patients infected with L. braziliensis produce high amounts of IFN-γ after stimulation with whole lysed parasite (14, 15). To initiate cellular response, dendritic cells (DCs), which are specialized antigen-presenting cells (APCs), have the unique capacity of priming naive T cells by presenting peptide antigens bound to major histocompatibility proteins (MHCs), co-stimulating and secreting cytokines, and thus mounting a T cell response against Leishmania spp. Some Leishmania species, e.g., Leishmania amazonensis and Leishmania mexicana, may fail to activate DCs, and consequently, no effective T cell response is mounted (16, 17). L. braziliensis is capable of activating DCs and inducing a protective immune response (18). It is estimated that each mature DC expresses 106–107 MHC Class II (MHC II) and 105 MHC Class I (MHC I) molecules (19). The activation of CD8+ T cells is a result of the specific engagement of 9-mer-peptide to MHC I proteins (9-mer-p-MHC Class I), while CD4+ T cells are activated by 15-mer-peptides bound to MHC II (15-mer-p-MHC Class II). There is no such thing as an ideal antigen, and the search for antigens that could generate immunogenic epitopes for a potential vaccine against Leishmania spp. is thus critical. In this sense, reverse vaccinology has been constantly increasing its value, and now diverse in silico approaches are available for the identification of potential antigens and epitopes for vaccines. Since experimental methods are difficult and time consuming, reverse vaccinology using in silico methods has narrowed the vast amount of molecules to be tested, increasing the odds of finding better candidates (20). In addition, many pathogen genomes and proteomes are currently available in public data banks and can be assessed regarding their potential antigen diversity and variability. Thus, sequence- and structure-based methods investigating the binding affinity of peptides to MHC I and MHC II molecules and other parameters may aid in the search for new antigens in order to support vaccine development (21). John et al. (22) have used only sequence-based methods to search for different epitopes in the predicted proteome of Leishmania spp. Agallou et al. (23) have recently reported the construction of a multi-epitope peptide vaccine against leishmaniasis by analyzing four known proteins from Leishmania infantum. In this context, we hypothesized that a combination of modern sequence and protein structure algorithms would help the search, within the whole predicted proteome from L. braziliensis, for potential immunogenic epitopes with high affinity for both human MHC I and MHC II. Thus, the aim of this work was to combine robust in silico approaches in the search for potential immunogenic T cell epitopes, based on the proteome of L. braziliensis, for the development of an anti-Leishmania vaccine. Results Linear Epitope Prediction With the goal of developing a peptide vaccine based on in silico approaches, many studies have shown its feasibility, and different attempts have been carried out in order to find good epitopes capable of stimulating the immune system and its memory arm (21, 24–26), but these mainly rely on computational tools, which focus on epitope prediction. Here, we have developed an in silico pipeline combining the linear prediction of epitopes with a sequence of structural refinements to confirm the potential of some epitopes to bind to MHC molecules and thus stimulate the immune system. Several computational tools were applied with the goal of minimizing the number of candidate epitopes identified as well as maximizing their potential as inducers of protective immunity. The whole strategy used can be seen in the workflow described in Figure 1. Figure 1 Methodology flowchart used in this work. TriTrypDB was used to retrieve the predicted proteome of Leishmania spp. (A). Predicted proteome sequences were analysed by different methods (B). EpitopeDB relational database was created, and managed using MySQL as database management system. Parsers and algorithms in PERL and SQL languages were developed in order to access and integrate the results (C). Data was clustered (D). Clustered data was used for molecular modeling (E). The initial epitope prediction tools used here (NETMHC and NETCTL) were selected based on two criteria, namely, predictors of MHC I and MHC II binding affinity and predictors in which their accuracy and performance applied to trypanosomatid protein sequences have already been assessed by Resende et al. (27). NETMHC and NETCTL were then used to predict MHC I-binding epitopes, and NETMHC Class II was used for a MHC II prediction. For each allele supertype, the epitopes predicted were those classified by the tools as strong binders. Simultaneously, different sets of sequence analysis were performed in order to exclude all epitopes that belonged to proteins conserved in humans and mice, so as to avoid potential autoimmune epitopes; select epitopes belonging to proteins conserved between different Leishmania species, potentially able to induce an immune response against multiple species; select epitopes that came from proteins, which were predicted as extracellular or secreted and having a maximum of one trans-membrane domain, therefore selecting epitopes from proteins easier to express and which should be generally exposed to the host immune system. The number of predicted epitopes for both MHC I and MHC II derived from these predictions, and the allele supertypes, are summarized in Table 1. If all predictions for different MHC alleles are considered, the total number of epitopes found in this stage is 657, 6,710, and 64,553, for NETMHC, NETCTL, and NETMHCII, respectively. Next, as the total epitope prediction includes some degree of redundancy, a clustering step was performed, considering the sequence similarity among the predicted epitopes, and this analysis resulted in 168 groups for MHC I and 2,138 groups for MHC II. Subsequently, each group was dismembered to reveal the exact sequence of each epitope, resulting in 230 individual epitopes for MHC I and 2,319 epitopes for MHC II. Table 1 Number of predicted epitopes by bioinformatics tools. Allele supertype MHC Class I prediction NETMHC NETCTL Number of predicted epitopes HLA-A1 17 556 HLA-A2 181 685 HLA-A3 86 579 HLA-A24 18 444 HLA-A26 61 515 HLA-B7 20 1,620 HLA-B27 229 1,092 HLA-B44 31 466 HLA-B58 14 753 Allele supertype MHC Class II prediction NETMHC Class II Number of predicted epitopes HLA-DPA 7,021 HLA-DPB 1,558 HLA-DQA 14,762 HLA-DRB 41,212 Fitting Linear Epitopes into MHC Structures As the main objective of this work was to select predicted epitopes with a good binding affinity to a large number of MHC receptor alleles, a molecular modeling approach was used aiming to find the most in silico stable epitope + allele complexes. Structures from 33 different alleles of MHC I (21) and MHC II (12) were downloaded and processed as described in the Section “Materials and Methods,” prior to modeling their interaction with the individual predicted epitopes. The sequence of steps followed can be found in Figure 2. The first step consists of the replacement of the co-crystallized peptide (present at each downloaded structure) for each one of the predicted epitopes. The total number of complexes is composed of 4,830 complexes for MHC I (21 MHC I alleles times 230 predicted epitopes) and 27,828 complexes for MHC II receptor (12 MHC II alleles times 2,319 predicted epitopes). As the overall combinations of all the MHC structures and predicted epitopes achieved an impressive number of 32,658 complexes, a distributed computing strategy was adopted to process this large-scale problem in a feasible time. Figure 2 The sequence on which the Rosetta’s protocols were used. The total number of complexes generated and the average Interface score (Isc) of it are shown. The computational demand of each protocol compared to the FixBB protocol (clock picture), for a single complex (epitope + allele), can also be found. Every complex image consists of an example containing a MHC II allele (PDB: 3LQZ) and the epitope #1,677, after each protocol. The MHC II allele is in green, while the predicted epitope color range from blue (FixBB) to black (Relax), red (FlexPepDock with 100 solutions), and yellow (FlexPepDock with 500 solutions). With all the 32,658 complexes (receptor + epitope) generated and their respective epitopes energetically relaxed, the molecular docking could be started. This was carried out using the Rosetta’s FlexPepDock protocol. However, during the development of this molecular modeling protocol, several preliminary evaluations were made in order to find a good trade-off between precision and computational demand. First of all, it has been noticed that the use of Rosetta’s FixBB protocol to replace the co-crystallized peptide by the predicted epitopes generates typical unstable structures, with high positive Interface scores (Isc). Therefore, three complexes (predicted epitope + allele), that had the best, a regular and the worst Isc values, based solely on the structures obtained by FixBB, were selected and submitted to the Rosetta’s Relax protocol. This step was performed to verify if the Relax protocol could stabilize the selected prediction in more favorable conformations. The Relax protocol results can be found at Figure S1 in Supplementary Material. It is possible to notice an increase in affinity of the complexes (predicted epitope + MHC receptor) after the relaxing step. Filtering Epitope–MHC Complexes After the analyses performed previously, there were still an impracticable number of complexes (32,658) to be used as input for Rosetta’s FlexPepDock protocol. This universe of complexes demands a computational effort that is not feasible even using a computational grid environment. Thus, a filtering strategy had to be adopted in order to select the most promising predicted epitopes to the largest possible number of MHC receptor alleles. First, knowing that epitopes containing 9 residues (MHC I) can be windows of 15-residue epitopes (MHC II), the epitopes from the Class I prediction were matched with the Class II epitopes in order to find windows of Class I and Class II epitopes. A total of 385 pairs of 15-residue epitopes and their respective 9-residue windows were found. The filtering strategy required the Isc scores derived from the calculation of the whole set of epitope + allele complexes, computed using the Rosetta’s Relax protocol (called rescore procedure, as detailed in the Section “Materials and Methods”). These Isc scores were used to estimate the frequency that each predicted epitope appears on the list of top 30% ranked candidates for all available MHC structures (Figure 3). If a 15-residue epitope has a good affinity for a MHC II allele, at the top 30% best scored epitopes for each target structure, and its 9-residue window also has a good affinity for a MHC I allele, this “pair” of predicted epitopes (MHC II and MHC I) might be a good candidate for better immunogenic properties. The 385 pairs of 15-residue epitopes and their respective 9-residue windows were then sorted according to the sum of their frequency within the 30% best scored epitopes. After the exclusion of repetitions, the 30% cutoff recovered 81 and 285 predicted epitopes of MHC I and MHC II, respectively, varying from 12 (at 10 and 15%) to 18 (at 25%), within the total 33 alleles (21 for MHC Class I and 12 for MHC Class II). Choosing a cutoff larger than 30% would not increase significantly the number of occurrences. Figure S2 in Supplementary Material shows the plot of percentage cutoff for the top ranked candidates, ranging from 10 to 50%, and Figure S3 in Supplementary Material presents the filtering algorithm. Figure 3 The filtering approach used in order to reduce the number of complexes calculated at FlexPepDock step. Details can be found in text. Based on such filtering approach, the 100 best-ranked pairs (or 1,764 predicted epitope + allele complexes) were used at the next molecular docking step, with Rosetta’s FlexPepDock protocol, because this number of calculations required a viable computational demand, about 18-fold lower than the initial set of complexes (32,658). Molecular Docking The 100 best-ranked pairs after the filtering strategy were then used at the molecular docking step with 100 docked solutions each. From this set, the top 10 pairs of predicted epitopes, with the best average Isc among the alleles, were selected for an enhanced run of Rosetta’s FlexPepDock protocol with 500 docked solutions, hence increasing the chance of finding new docking solutions with higher affinities for these 10 pairs of predicted epitopes. The final results obtained through the molecular docking of these 10 pairs of predicted epitopes defined a total of 4 unique 9-residue epitopes predicted for MHC I and 10 unique 15-residue epitopes predicted for MHC II. Figure 4 shows the superposition of the best-docked solutions derived from the alleles with the highest binding affinity (lowest Isc) to their respective MHC targets [alleles with Protein Data Bank (PDB) IDs 4NQV and 3LQZ for MHC I and MHC II, respectively]. One can see that the best docking solutions are quite similar in position, displaying a homogeneous result. The way these solutions are positioned are consistent with the known binding mechanism for these MHCs, as each predicted epitope bound to the correct key anchor residues localized within the binding groove of its corresponding MHC target (28). The MHC I (Figure 4A) predicted epitopes were anchored by residues located at the epitope extremities, while the MHC II predicted epitopes (Figure 4B) were anchored by residues positioned in the middle. The final selected 14 predicted epitopes are those that displayed the best binding affinity among the 2,549 candidate epitopes (230 targeting MHC I + 2,319 targeting MHC II). Figure 4 Superimposition of the best solutions for the (A) 4 predicted MHC I and (B) 10 MHC II epitopes. Epitope–MHC Interaction Features Detailed analyses were then conducted in order to identify important intermolecular aspects responsible for the affinity of these final predicted epitopes to the MHC targets, as follows. First, the correlation between the Interface-buried surface area (Ibsa), commonly used to measure the size of the macromolecule interface (29), and the Isc was evaluated. Figure 5 presents the average Ibsa and Isc values for all the complexes (epitope + allele) formed by each one of the 33 MHC structures. A strong correlation between Ibsa and Isc was seen, with bigger Ibsa values being accompanied by lower (more stable) values for Isc. This was to be expected, since when the contact area between the ligand (predicted epitope) and the receptor (allele structure) is larger, there are more intermolecular interactions between them stabilizing the complex, with lower (more negative) Isc values. Thus, an increase on Ibsa contributes in a favorable way to the binding affinity of the complexes, by typically lowering the Isc. Figure 5 Correlation between the average Interface-buried surface area (Ibsa, in square angstroms) and the average Interface score (Isc). This was performed for all the complexes generated with the 33 MHC receptors, after the FlexPepDock protocol (with 500 solutions). In a similar analysis to the one presented in Figure 5, Figure 6 shows the correlation between the average Interface hydrogen bonds (Ihb) and the average Isc values for all the complexes (apitope + allele) formed by each one of the 33 MHC structures. As more hydrogen bonds are formed between the receptor and the predicted epitope, the Isc value is lower, i.e., the complex has a higher binding affinity, emphasizing the fact that the peptides bind to the MHC alleles (particularly MHC II) via an “extensive hydrogen bond network” (28). To further investigate this correlation, three complexes included in the final results (FPD500) were selected and analyzed (Figure 6). It is important to notice that the larger Class II epitopes (with 15 residues) have a greater natural probability to form hydrogen bonds, because of the higher number of residues (two-thirds more). It is also important to emphasize that the average Isc, highlighted in Figure 2 as “Avrg. score,” can be observed as progressively more negative with each successive step of the methodology applied, meaning more stable epitope + allele complexes identified during the in silico procedures. Figure 6 Correlation between the average Interface score (Isc) and the average Interface hydrogen bonds (Ihb), for the complexes obtained by the FlexPepDock protocol with 500 solutions (FPD500). The blue dots correspond to the MHC II alleles (total of 12), while the red dots correspond to the MHC I alleles (total of 21). (A) The best docking solution of the final set of results, showing the predicted epitope that formed the highest number of hydrogen bonds and generated the lowest (most stable) Isc. (B) The worst overall solution within the final results, having a low number of hydrogen bonds and the highest (less stable) Isc value. (C) One of the worst solutions for a MHC II complex, having a reduced affinity between the allele and the predicted epitope. Only the side chain groups with hydrogen bonds are shown. The epitope’s alpha-carbons are highlighted in dark blue. Epitope Tracing Next, the identity of the 10 highest ranking epitopes was investigated, and they were seen to derive from a total of 4 L. braziliensis proteins. The first of these, a conserved hypothetical protein, is encoded by a CDS localized to chromosome 34 and encompasses at least 4 potential epitopes. Interestingly, the protein segment encompassing the 4 15-mer epitopes is 19 aa in linear length. Therefore, the four potential epitopes with high affinity for MHC I and MHC II molecules are found within this particular segment, with minor differences between each of these four epitopes, but all producing high scores when analyzed by the approaches described above. Another conserved hypothetical protein located on chromosome 1 encodes at least three epitopes. Here, the three epitopes were found in a window 18 aa in length. The third protein, found in chromosome 14, encodes two 15-mer epitopes located in a window of 16 aa. Finally, the last and largest protein, also hypothetical, encodes only one 15-mer epitope. All the genes encoding for those proteins are syntenic with other genes from trypanosomatids. This indicates that these proteins have evolved in the same genetic loci from diverse trypanosomatids, and the potential epitopes may induce cross-protection against other pathogenic species from the same family. Validation of Peptide Epitopes In order to have an experimental validation of the results derived from the bioinformatic approach, peptides corresponding to the 10 highest ranking 15-mer epitopes described above were commercially synthesized. These were then used for an evaluation of their ability to induce proliferation of peripheral blood mononuclear cells (PBMC) derived from human patients cured after treatment. These PBMC were capable of proliferating when stimulated with total antigen from L. braziliensis (7.6 ± 6.1), and non-stimulated cells had minimal levels of proliferation (data not shown). The results from the assays carried out with the synthetic peptides are presented as the mean percentage of proliferation, with its SD, calculated with the data from the proliferation of PBMC derived from 10 patients, when exposed to the individual peptides. Peptides 2, 4, 8, 9, and 10 were capable of stimulating proliferation of many of the PBMC derived from the afflicted patients. However, no significant difference was observed when the mean proliferation values obtained for these peptides with the PBMC from the 10 patients was compared with the mean values derived from the data with PBMC from the control group, consisting of five healthy volunteers. In contrast, significant statistical differences were observed for the mean PBMC proliferation results between samples from treated patients and control volunteers for the assays carried out with peptides 1, 3, 5, 6, and 7 (Figure 7). These five peptides are derived from three of the four L. braziliensis proteins described above, confirming that the positive response is not associated with one specific protein. The possibility that the improved results from some peptides might also be associated with a stronger response from a reduced number of individuals was also investigated. The PBMC from two patients did respond better to a greater number of peptides, four in all, but these include peptides included among both groups described above. In all, the PBMC proliferation data highlight the potential of the computational approaches used for epitope selection and indicate that some of the synthetic peptides tested would be recognized by the immune system to mount a protective immune response. Figure 7 Comparison of the proliferation data from PBMC derived from the cutaneous leishmaniasis post treatment (PT) patients with the PBMC proliferation data from the control group, in response to different peptides. The asterisk indicates significant differences (p < 0.05) between patients and control group. The horizontal bars represent the mean values for each group. The median percentage levels and the corresponding SD for each group tested are described below. Discussion So far, different approaches have tried to address the lack of an anti-Leishmania vaccine capable of being effectively used against the leishmaniasis. However, most of the vaccines under development have failed in very early assays, due to factors such as poor antigen response, absence of good animal models, and lack of standardization (30). An ideal vaccine should also be capable of stimulating a promiscuous response against different Leishmania species, but generally only one species has been considered at a time. Moreover, only a few papers have described predictions of T cell epitopes from Leishmania spp. proteomes (31–35). Most of these have focused on epitopes, which bind to MHC I, with a focus on CD8+ T cell response and not considering the induction of a response mediated by CD4+ T cells (31, 32, 35, 36). CD8+ T cells have a major role in protecting against CL, but evidence has been provided that they may also exacerbate and compromise the disease outcome (37). In this context, we have searched for natural epitopes that would stimulate both CD4+ and CD8+ T cells, in order to establish a more balanced response that could favor the prevention of disease progression. However, development of CD4+ T cell predicted epitopes is still a challenge, since we do not fully understand the epitope motifs that bind to the MHC II groove, most of the peptides may not be naturally processed by APCs, and the positional alignment is critical for the prediction (38, 39). To overcome these issues, the epitopes predicted here were tested through different criteria in order to define a final set of peptides. One of these criteria was the capacity to bind with high affinity to both MHC I and MHC II, as demonstrated by the high scores of the complexes and the elevated number of hydrogen bonds, for example. Thus, it is expected that, during natural processing by APCs, these peptides could bind either to MHC I or MHC II or be able to bind both molecules. We hypothesized that the differences observed in the cell proliferation are a consequence of the number of T cells present in the peripheral blood of recovered patients. In addition, these peptides may differ in how they are presented and activate T cells since they have different linear sequences. In terms of target MHC molecules, this work has crucially focused in simulating peptide epitopes with different allele supertypes expressed by different human populations. Allele selection considered their distribution across the globe and other important parameters, such as the promiscuity of the selected peptides to which they bind and PDB crystallized structures. To do so, most of the work was based on human MHC alleles and in order to reduce bias due to MHC multiplicity, allele supertypes were used (40–42). Supertypes share specific residues at some anchor positions, thus, they are capable of binding to overlapping groups of peptides (43). It is important to consider the human MHC molecules, since the purpose of the work is to develop a vaccine that would be applied to humans. Previous works have already helped the search for potential epitopes that could bind to murine MHC, since it is the most used experimental model for preclinical assays, testing potential vaccines against leishmaniasis (23, 36). Most of the in silico developed peptides based on the murine model, however, may fail to translate good results to humans, due to the huge differences between human and mouse immune responses. All the computational efforts performed here were therefore dedicated to simulate peptides with human MHC alleles. One of the major problems of bioinformatics these days is the huge amount of data generated. Linear epitope predictions using predicted proteomes provide a large number of potential epitopes, which cannot possibly be tested. Therefore, this large number must be reduced into a feasible number of epitopes, which can be experimentally tested for their immunogenicity. Thus, the option here to solve this issue was for molecular modeling protocols, mainly through the application of molecular docking approaches, adding another layer of strength to the data. Here, it has been shown that the best-ranked peptide epitopes are clearly those that establish the highest number of molecular interactions (like hydrogen bonds and hydrophobic contacts, for example) with the chemical groups in the MHC groove. Nevertheless, the in silico methods employed and some decisions taken during the process may have imposed some limitations. One of these is that, the tools available to predict linear epitopes use learning machine techniques, which have a huge dependency on training dataset. However, those tools are not currently trained with experimental data from trypanosomatids, such as L. braziliensis (27). Additionally, other factors have also guided the decisions, such as the exclusion of proteins with more than one transmembrane helix that might be difficult for further expression, and exclusion of proteins conserved but with high similarity with human sequences, which is not desired for a vaccine. Moreover, one limitation related to structural approach is the limited diversity of HLA structure alleles available on the PDB bank. Here, just 21 and 12 alleles of MHCI and MHCII, respectively, were tested. This aspect could affect the performance of the method used here. Based on the results presented so far, it seems that the proposed combination of approaches is consistent enough to be applied in cases of reverse vaccinology, when there is a large quantity of candidate epitopes to be tested. The strategy of distributed computing (computational grid) alongside the filtering algorithms has turned an unpractical problem, into a feasible task, done in weeks. Moreover, in the context of leishmaniasis, the results of this research identified peptide epitopes with high potential to stimulate the immune system to develop a protective response. Materials and Methods Linear Epitope Prediction Proteome Retrieval and Conservancy The available proteomes (from L. braziliensis, Leishmania major, and L. infantum) were downloaded from TriTrypDB (44) and used to perform, in parallel, different bioinformatics analyses. Only protein sequences from L. braziliensis with more than 60% conservancy with other Leishmania species verified through BLAST protein alignment were considered for epitope analysis. This parameter was taken into account since an ideal vaccine should be capable to induce protection in individuals against as many species as possible. MHC Class I and MHC Class II Prediction Tools and Binding Affinity Prediction NetMHC and NetCTL tools were used for a MHC I predictions, while MHC II predictions were made using the NetMHCII tool for the most prevalent allele supertypes (45–47). Both NetMHC and NetCTL are epitope predictors; however, NETMHC just predicts the epitope, while NETCLC also considers other predictions, such as the transport efficiency prediction mediated by the transporter associated with antigen processing (TAP) protein and the C-terminal proteasomal cleavage prediction. The cutoff score defined to select peptides with high affinity for those tools was ≥1 in order to maximize the number of true positive predictions. Similarity and Biological Features of Protein Candidates In order to exclude protein candidates with high degree of similarity with proteins of humans and mice, the BLAST sequence alignment tool (48) was used to compare parasite protein sequences against host protein sequences. Proteins with degree of similarity equal or higher than 40% with human or mice proteins were excluded from the next steps. Moreover, transmembrane helix and subcellular localization predictions were performed using the TMHMM tool (49) and WoLF PSORT (50), respectively. All the data obtained after running the methods described above were deposited in a relational database, which is managed using MySQL as a database management system (DBMS). Parsers and algorithms in PERL and SQL languages were developed in order to access and integrate the results deposited in the databank. Clusterization An in-house algorithm based on BLAST alignment results was developed in order to group the data with high similarity. A threshold of 60% of identity and 100% of coverage between any two epitopes were used in order to cluster them in the same group. In addition, group selection was performed based on selective criteria: peptide epitopes with high affinity predicted for at least three different allele of either MHC Class I or MHC Class II, or peptide epitopes derived from at least three different proteins. Molecular Modeling Approach Preparing MHC Structures from PDB The structures from the 33 different alleles of MHC I (21 PDB structures: 2HJL, 3C9N, 3HCV, 3KPP, 3L3D, 3RL1, 3VCL, 3VFS, 3 × 11, 4F7M, 4G8G, 4HWZ, 4JQX, 4MJ5, 4MJI, 4NQV, 4O2C, 4QRR, 4QRU, 4WU5, and 4XXC) and MHC II (12 PDB structures: 1A6A, 1BX2, 1H15, 1S9V, 1UVQ, 1YMM, 2NNA, 2Q6W, 3C5J, 3LQZ, 3PL6, and 3WEX) were downloaded from RCSB PDB (51). These structures were then prepared by removing the water molecules, ligands, and duplicated residues or alleles. Furthermore, using the PyMol (52) software in build mode, the co-crystallized small peptide chain, for each structure, was modified to have the same length as the predicted peptides used in this work (9 residues for MHC I and 15 residues for Class II). In addition, each non-canonical amino acid found in the co-crystallized peptide was manually mutated to alanine. An in-house developed software named GriDoMol was then used to prepare and submit into a computational grid environment all the in silico procedures required to combine the MHC structures to the predicted epitopes and to compile the results obtained at each step into formatted datasheets. This computational grid environment was assembled, in our laboratory, by using eight computers, each containing 2× Intel Xeon quadcore (total of eight cores per computer) chipset and 16 GB RAM memory. Producing MHC–Epitope Complexes The sequence of steps on which the Rosetta framework (53) protocols were used can be found in Figure 2. For the replacement of the co-crystallized peptides for each one of the predicted epitopes, the Rosetta’s FixBB protocol, available within the Rosetta framework, was used. However, the Rosetta’s FixBB protocol does not move the backbone atoms, and thus the replacement of the co-crystallized peptide for a new one may produce unstable final conformations. Therefore, the Rosetta’s Relax protocol was used, right after the FixBB protocol, in order to energetically stabilize each one of the new epitopes. Moreover, all the MHC receptor’s residues have been locked unmovable to prevent conformational changes on the receptor side, while only the epitope’s residues were allowed to move and rotate toward a more stable conformation in the chemical neighborhood. In order to quantify the binding affinity between receptors and predicted epitopes, at this particular step, the score function named Isc was chosen from the Rosetta’s FlexPepDock protocol (54), which is the sum over the energetic contributions of the interface residues on both the receptor and the predicted epitope. Hence, all the 32,658 structures obtained by the application of the Rosetta’s Relax protocol were only rescored using the Rosetta’s FlexPepDock protocol, in order to obtain the Isc values, but keeping the same structure obtained by Relax protocol (i.e., without any change on the atomic coordinates). In other words, the rescore procedure just recomputed the energy (with a better scoring function) at the same geometry, without perturbing the chemical system. Scoring Potential Epitopes through Molecular Docking While each run of the FixBB or Relax protocols takes a few seconds to generate the result, each run of the FlexPepDock protocol, using the default setup (100 docked solutions obtained as result), takes hours to complete. Thus, a filtering strategy had to be adopted in order to select the most promising predicted epitopes, based on their Isc scores (obtained by rescoring, as mentioned above) with receptors and the frequency of affinity observed along different MHC receptor structures. The Rosetta’s FlexPepDock protocol, using the refinement approach, was applied to perform the molecular docking, allowing the full flexibility for the predicted epitope and the side chain flexibility for the residues at the receptor’s interface. This procedure searched for the predicted epitopes with the best binding affinities for MHC’s alleles. For each predicted epitope, the best docking solutions were selected according to the Isc. At the end, the predicted epitopes were ranked by the average Isc of their solutions along the MHC’s alleles, granting an overall view of each epitope’s average affinity. From the list of 100 pairs previously described, the first 10 pairs of predicted epitopes with the best average Isc among the alleles have been selected for an enhanced run of Rosetta’s FlexPepDock protocol, increasing the number of generated structures from 100 to 500. The computational cost of such calculation (500 docked solutions) was about 5 times greater than the default 100 docked solutions. Validation of Peptide Epitopes Synthetic Peptides and Storage Peptides corresponding to the top 10 ranked 15-mer peptides were synthesized (Genome Biotechnology, Brazil). Linear peptides were purified through a high performance liquid chromatography (HPLC) approach with a final purity greater than 95%. All the synthetic peptides were individually resuspended in DMSO and stored at −80°C until use. Sampling and Isolation of Peripheral Blood Mononuclear Cells All individuals included in this research signed a written informed consent before blood collection, following recommendations of the Ethics Committee from the Centro de Pesquisas Aggeu Magalhães (CPqAM-FIOCRUZ, Project: 522.964). From each individual, a total of 30 mL of peripheral blood was collected by venipuncture in sodium-heparin tubes (Vacuette, USA). The blood was diluted (1:1 v/v) with phosphate-buffered saline (PBS, pH 7.2) and deposited onto the Ficoll–Paque PLUS density (1.077 g/mL) gradient (GE Healthcare, USA) and centrifuged. Subsequently, the PBMC layer was individually removed and washed twice with PBS. CFDA-SE Labeling and Cell Culture About 4 × 106 cells were resuspended in 1 mL of PBS containing 2 μM of carboxyfluorescein diacetate succinimidyl ester (CFDA-SE, Invitrogen, USA) and incubated at 37°C for 10 min. The CFDA-SE concentration was previously titrated in order to prevent inhibition of cell proliferation or cell death. After incubation, cell labeling was quenched with 1 mL of ice-cold (4°C) RPMI 1,640 containing 2 mM of l-glutamine, 50 mg/L of gentamicin sulfate, and 2 mg/L of amphotericin B, supplemented with 10% fetal bovine serum (both Cultilab reagents, Brazil). The cells were pelleted and washed with PBS followed by resuspension in 1 mL of RPMI 1,640 supplemented at a density of 2 × 106 cells/mL. The PBMC were plated in 96-well U bottom plates (BD Falcon) at a density of 2 × 105 cells/well with 20 μg/mL of each peptide. These cells were then incubated at 37°C with 5% CO2 for 96 h. For each patient or control volunteer tested, non-stimulated and phytohemagglutinin (PHA)-stimulated cells were evaluated as intra-experimental controls. Flow Cytometry Analysis The analyses were performed on a FACScalibur flow cytometer (Becton Dickinson Company, USA) equipped with an argon laser (wavelength 488 nm). Fluorescence of 20,000 lymphocyte gated events, based on scatter parameters of size and granulosity, was acquired. The data were analyzed and treated with FlowJo v10.1 (Tree Star Inc., USA). Non-stimulated cells were used during the analysis for setting quadrant parameters and to set the basal level of lymphocyte proliferation. For the statistical analysis, the data were analyzed with non-parametric Mann–Whitney U-test. Differences were considered statistically significant when p < 0.05. Author Contributions Conceived and designed the methods: RS, MH, AR, and VP. Performed the in silico approaches: RS, LF, MH, and AR. Performed the epitope validation: RS, VP, MB, BO, and AS. Analyzed the data: RS, LF, MH, Od-M-N, AR, VP, MB, BO, and AS. Wrote the paper: RS, LF, MH, Od-M-N, AR, and VP. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. LF is the recipient of a doctoral fellowship received from the “Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco” (FACEPE). MH, Od-M-N, and VP acknowledge research fellowships received from the “Conselho Nacional de Desenvolvimento Científico e Tecnológico” (CNPq). Funding This study was supported by Chamada MCTI/CNPq/MS-SCTIE – Decit No. 40/2012 – Pesquisa em Doenças Negligenciadas. Processo No.: 404259/2012. Supplementary Material The Supplementary Material for this article can be found online at http://journal.frontiersin.org/article/10.3389/fimmu.2016.00327 Click here for additional data file. ==== Refs References 1 WHO . Neglected Tropical Diseases, Hidden Successes, Emerging Opportunities . Geneva : World Health Organization (2009 ). 59 p. 2 Hotez PJ Ferris MT . 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==== Front Front PsycholFront PsycholFront. Psychol.Frontiers in Psychology1664-1078Frontiers Media S.A. 10.3389/fpsyg.2016.01296PsychologyOriginal ResearchA Qualitative Analysis of Emotional Facilitators in Exercise Wienke Benjamin 1*Jekauc Darko 121Department of Sport and Exercise Psychology, Humboldt University of Berlin, BerlinGermany2Department for Sport Science, University of Konstanz, KonstanzGermanyEdited by: Duarte Araújo, University of Lisbon, Portugal Reviewed by: Jonathon Headrick, Queensland University of Technology, Australia; Rui Sofia, University of Minho, Portugal *Correspondence: Benjamin Wienke, bw@yourpersonaltrainer.deThis article was submitted to Movement Science and Sport Psychology, a section of the journal Frontiers in Psychology 29 8 2016 2016 7 129630 4 2016 12 8 2016 Copyright © 2016 Wienke and Jekauc.2016Wienke and JekaucThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Although previous research has shown that emotions are consistently associated with sport and exercise behavior, the working mechanisms are not understood to the extent of creating an intervention. The aim of this study is to identify situations and aspects of recreational sport and exercise, which lead to positive emotional reactions in people taking part in regular and long-term exercise. In this study, 24 adults (12 female, 12 male) distributed over three age groups (young, middle, and late adulthood), took part in recreational sports (individual or team sport) for at least 5 years. Semi-structured in depth interviews with questions about sport and exercise habits, long term participation and emotional response in a sporting environment were conducted in order to ascertain those situations and aspects of the exercise program triggering positive emotions. Interviews were transcribed verbatim and followed Grounded Theory principles. Emerging concepts were grouped and merged into different categories representing the key aspects of sport and exercise. Four factors were identified which are associated with the emergence of positive emotions in recreational sport and exercise. Firstly, perceived competence is one of the major factors influencing emotions during exercise and can represent individual and collective success and progress, competition and challenge. Secondly, perceived social interaction is another factor comprising of all sorts of peer-related aspects such as communication with others, being part of a group and creating close relationships or friendships. Thirdly, novelty experience in contrast to other none-sporting activities such as work, family or other leisure activities was another factor. The last factor found was the perceived physical exertion comprising of the degree of exhaustion, a possibly delayed turnaround in the emotional response and the aspect of sport being a physical compensation for everyday sedentary life. The results of this study provide the starting point for the development of interventions to enhance positive emotions in sports in order to increase maintenance and adherence to recreational sport and exercise. emotionsgrounded theoryexercise maintenanceperceived competencebelongingnessnovelty experienceexercise intensity ==== Body Introduction Evidence shows a wide range of health benefits from exercise and physical activity (Paffenbarger et al., 1993; Mensink, 2003; Rütten et al., 2005; Krug et al., 2013; Reiner et al., 2013), but also poor compliance and adherence to exercise programs (Mensink, 2003; Ekkekakis and Lind, 2006; World Health Organization [WHO], 2010; Krug et al., 2013; Eurobarometer, 2014). These health improvements only persist due to regular participation in physical activity and exercise whereas a significant decline or drop out causes a decrease or complete loss of initially gained benefits (Mujika and Padilla, 2000a,b). Regardless of people knowing this necessity to continue exercising, the majority of the population remains physically inactive even those who are younger (Jekauc et al., 2012). Furthermore, empirical evidence has identified high dropout rates of up to 50% in exercise programs, within the first 6 months (Dishman and Buckworth, 1996; Annesi, 2003) and a common relapse to lower or no levels of activity after the exercise program has finished (Amireault et al., 2013). Effort has been made to encourage behavior change (Biddle et al., 2012) with marginal success (Rhodes and Pfaeffli, 2010). Even though insights about the motivational process of initiation of physical activity and exercise is advanced, there is a paucity of research which investigates the maintenance of such activities (McAuley et al., 2007; van Stralen et al., 2009; Kwasnicka et al., 2016) as well as a lack of effective practice (Amireault et al., 2013). Previous research indicates that the initiation of physical activity and exercise has different influences (e.g., strategic planning and recovery self-efficacy; Schwarzer et al., 2007) in comparison to the maintenance (habit, environmental, and social influences; Kwasnicka et al., 2016) of it (see also Biddle and Mutrie, 2008). Therefore there is a high interest in identifying key factors which affect physical activity and exercise maintenance such as specific facilitators (Amireault et al., 2013). Although recent findings support the notion of facilitators which foster behavior maintenance (previous physical activity habits, positive attitude, expectations; Amireault et al., 2013; perception of competence, social affiliation, environmental conditions, sense of autonomy, specific physical activity; Rhodes and Kates, 2015), the capability of established theoretical frameworks is fairly limited (Rhodes and Pfaeffli, 2010; Amireault et al., 2013). Indeed, most factors are based on cognition, but established cognitive theories and models can only explain parts of people’s behavior (Jekauc et al., 2015; Kwasnicka et al., 2016) revealing a lack of theoretical foundation. Perhaps diverting attention to other domains such as facilitators of emotions in the context of sport and exercise is more promising (Williams, 2008; Rhodes and Kates, 2015) and may lead to a better understanding, which despite years of research is still limited (Rhodes et al., 2009). To succeed using this approach a clear definition is crucial to distinguish between different terms and constructs used (Tuson and Sinyor, 1993). According to Caspersen et al. (1985) “physical activity is defined as any bodily movement produced by skeletal muscles that results in energy expenditure” […] which “can be categorized into occupational, sports, conditioning, household, or other activities” (p. 126). Instead, sport and exercise are subcategories of physical activity, which are often planned, structured, and repetitive with the purpose of improving or maintaining physical capability (Caspersen et al., 1985). Since sport and exercise are subsets of physical activity, evidence drawn from sport or exercise might be valid for each other and broadly for physical activity but not the other way round. Following the approach from Buckworth et al. (2013), this article is mostly focused on exercise because the majority of researchers used methods that fit the definition of exercise mentioned above, to measure physical activity. Since classic competitive sports are expected to have a specific emotional pattern (e.g., influenced by a higher goal orientation or different expectations such as external rewards), a look at recreational sports might result in a broader insight. The same procedure is required for the differentiation of affects and emotions. According to Russell and Feldman Barrett (2009) the core affect (because it is considered to be basic or irreducible; Ekkekakis, 2003) is characterized as a “neurophysiological state consciously accessible as a simple primitive non-reflective feeling most evident in […] emotion but always available to consciousness” (p. 104) such as pleasure and displeasure, tension and relaxation, or energy and tiredness (Ekkekakis, 2012). Core affect can occur on its own or as a part of emotion, e.g., like pride which is feeling good about oneself whereas ‘feeling good’ represents the core affect and ‘about oneself’ the other, cognitive, component (Russell, 2003). Thus emotions are a kind of affective state, but they do not apply vice versa. Making reference to Russell and Barrett (1999) and Ekkekakis (2012) simplifies the definition of emotions that are elicited by and are a reaction to and about a certain stimulus, with its cognitive appraisal involved as an elemental characteristic and reveals a more complex phenomenon in comparison to basic affect (Ekkekakis and Petruzzello, 2000). The previously mentioned example of ‘pride’ represents an emotion which includes all these criteria mentioned before and demonstrates the close relation between the two concepts of affect and emotion. In this article, the focus is primarily on emotions and not affects not only due to the qualitative design of the study. The aim of the present study is to identify facilitators which affect the emotional response to recreational sport and exercise and thus the maintenance of both. Research about the role of emotions in relation to sport and exercise or facilitators is sparse (Rhodes and Kates, 2015) and the few models that exist focus on leisure time activities that seek pleasure, enjoyment, and fun (Rhodes et al., 2009). Positive affects are one of the fundamental basics in order to understand the nature of enjoyment (Scanlan and Simons, 1992) which in turn reinforce behavior (Rhodes and Kates, 2015; Kwasnicka et al., 2016) and enhance participation and maintenance in sport and exercise (Stenseng et al., 2015). Thus facilitators for positive emotion during exercise must be identified to prompt the development of interventions charged with stimuli which affect one or more facilitators to ensure that each individual experiences positive emotion (for details see the results section below; Scanlan and Simons, 1992). Materials and Methods A qualitative study was conducted to identify facilitators that affect the emotional response to recreational sport and exercise. Participants In order to ensure heterogeneity of the sample the participants were evenly recruited across three factors: age, sex, and type of exercise. Age was divided in three age categories: 18–34 years (AG1), 35–59 years (AG2) and older than 60 years (AG3). Sex (male vs. female) and type of exercise (group vs. individual) consisted of two categories. Combining these three factors (3 × 2 × 2) 12 cells emerged. For each cell, two participants were recruited. Thus, the sample consisted of 24 participants. All participants took voluntarily part in this study and were recruited in Berlin as native German speakers. Ethical approval for the study was provided by the Humboldt University and all participants signed an informed consent form at the beginning of the study. Participants were involved in formal recreational sports such as soccer, volleyball, or swimming and informal recreational sports like climbing, martial arts, or paddling; organized in sport clubs, training groups, or exercising on their own. None of the participants were a high-level-competitive or professional athlete or worked in a job that involved sport. All participants described their actual level of engagement in popular sports as more or less ambitious. The average training frequency was three times a week (mean: 2,98; SD = 1,84) and the duration was around 90 min (mean: 94,04; SD = 34,42). Compared to their age, participants reported an involvement in recreational sports of around 70% of their lifespan (e.g., age of 28 and 22 years of sport participation results in 78, 57% of engagement; mean: 69,89; SD = 22,77) with a minimum of 6 years engagement. Of the 24 participants, there were even 12 that took part in competitions (e.g., league game or small tournament), five participated in competitions previously and seven had never participated in any competitions. Data Collection Students of Sport Science of the Humboldt University of Berlin through acquaintances or friends recruited suitable participants without any deeper relationship to the interviewer. At the start of each interview, relevant information about the study, the procedure and the data usage were given to the participants which had agreed to be interviewed and recorded. Data collection was made via voluntary, non-directive, face to face interviews, which where standardized using a semi-structure interview manual according to Tong et al. (2007) which refers to a gradual three level approach (three types of questions from the general to the specific) in a quiet environment. Participants were asked open-ended-questions about their experiences, habits, and emotions in the context of sport and exercise. Firstly, questions remained general in order to be able to get to know the participant, e.g., “How did you get into sport and exercise?” “What is your first memory in the context of sport and exercise?” “Which of these kind of sports/or what kind of exercise do you do regularly?” The second step was to ask about their habits and how they emerged. “What significance does regular exercise have in your everyday life?” “How much do you have to think about deciding whether to exercise?” “If you decide not to exercise, could you describe the inner monolog with yourself any further?” “Would you miss something after quitting your sport/exercise?” The final part of the interview contained questions especially about emotions within exercise and sport: “What kind of feelings do you experience whilst you exercise?” ”In which kind of situation do you experience the most enjoyment?” “How significant is the experience of pleasure to you, to be able to keep on exercising?” In addition to the participant and the interviewer, a third person attended to write minutes of the interview, which lasted approximately 20–40 min. Referring to GAT (“Conversation Analytic Transcription System”; Selting et al., 2009) the interviews where transcribed verbatim. Data Analysis Verbatim transcripts of the audio recordings were used in content analysis related to the Grounded Theory (Strauss and Corbin, 1996). The first step was the open encoding of the full text of the whole 24 interviews. Therefore the interviews were read completely by three different independent researchers and thoughts were written down in memorandum. Relevant information was grouped into different concepts. This working phase was done independently by two researchers, who met for a weekly meeting to compare and to discuss their findings and stayed as close as possible to the script in order to avoid bias. All concepts were grouped within categories (e.g., performance, process, achievement, competition, and challenge in the context of positive emotions were summarized in the categoryperceived competence (PC; Figure 1) and were checked for connections between each other in the axial encoding process. In that second step categories that seemed synonymous were merged into one so that only the varying categories remained [e.g., categories such as closeness to nature and curiosity and otherness were merged into novelty experience (NE)]. The last working step was selective coding to ensure the integrity of the four categories by searching for appropriate concepts and by analyzing the context in which they were mentioned. The final data was organized accordingly into categories and subcategories in order to be able to formulate the theory that is the focus of this study. FIGURE 1 Concepts which form each of the four categories. Results The majority of participants (22 out of 24) with their long lasting engagement in recreational sports and exercise referred to positive emotions related to this activity. For a better understanding, it is crucial to discover facilitators for these positive emotions to increase sport and exercise involvement and its maintenance. Four different factors with a high to moderate impact on the emotional experience could be revealed (Figure 2). The strongest and the most frequently mentioned concepts refer to the category PC which was mentioned in 22 out of 24 interviews. The second conceptual group was related to perceived social interaction (PSI), which was mentioned in 19 out of 24 interviews. The third group was related to NE and was mentioned in 18 out of 24 interviews. Lastly, the physical dimension perceived physical exertion (PPE) was found in 14 out of 24 interviews. Whereas three of these facilitators are psychological, one is also affected by interoceptive signals. According to the three independent factors (sex, age, and sport type) some differences have occurred as discussed below (Table 1). FIGURE 2 Facilitators of positive emotions in a sporting environment. Table 1 Occurrence of concepts related to positive emotions corresponding to one of the four categories. Groups (sample size) Concepts (24) Male (12) Female (12) Individuals (12) Team sports (12) AG1 18–34y (8) AG2 35–59y (8) AG3 +60y (8) Perceived competence 22 11 11 11 11 8 7 7 Perceived social interaction 19 9 10 9 10 7 6 6 Novelty experience 18 8 10 9 9 5 6 7 Perceived physical exertion 14 10 4 6 8 7 5 2 Perceived Competence Firstly, PC was found to be the most influential factor in the emotional experience in recreational sport and exercise, -feeling competent and capable was often combined with positive emotions. PC represents the own individual sense of how well one is performing in a particular situation in relation to exercise, in the present, retrospectively or in the future. Whilst performing well during a task, being successful or winning a game, mastering a challenge, achieving a goal or making progress created positive emotions in all participants such as pleasure and enjoyment; there was also often excitement or pride: “When I think back to how we won the championship I remember being happy for weeks”; “It is simply awesome when you are allowed to shoot the free kick and are able to fool the wall–I always really look forward to doing that”; “If something really cool worked out, then you are very happy about it. Sometimes you are also proud when you are making progress. After our tournament I was proud of my good results.” Perceived competence is not only binding on the individual performance; collective accomplishments were also perceived and evaluated: “If you manage to score or prevent a good move being successful or if you see a good move which you saw 30 years ago done by somebody and he replicates that and you comment “hey you didn’t forget that.” These sorts of things makes you happy or happy for the others”; “I am also happy for my teammates when they are doing well and sometimes even when the opponents do something great.” Only two participants did not refer to any concepts concerning PC as a facilitator for enjoyment and pleasure in recreational sport and exercise. Furthermore there were no cues among the interviews for a negative emotional response to PC or a positive response to a lack of PC. Also sex did not have an impact on PC among the interviews. Men and women mentioned PC in the context of positive emotions equally (both 11 out of 12 interviews). The same result was shown in the individual and team-sport participants (both 11 out of 12). Only the age factor showed a very small variation. In AG1 everybody mentioned concepts related to PC. In both other groups (AG2 and AG3) only seven out of eight mentioned PC as a factor related to pleasure and enjoyment. Perceived Social Interaction Secondly, PSI is another important factor that influences emotional experience in a sport and exercise-related environment and was mentioned in 19 out of 24 interviews. Many participants appreciate the company of others in the context of recreational sport or during exercise. To have social bonds with others or exercise with them triggers positive emotions, whereas being on your own does not (in category 3, NE, being alone triggered positive emotions due to cognitive rest or relaxation). The PSI category comprises of all sorts of peer-related aspects like communication with others, to be part of a group and to create close and warm relationships with others such as companionship or friendship. Meeting friends and teammates, spending time together or exercising together, communicating, and socializing made participants experience positive emotions. In other words, accompanied participants described themselves and especially their emotional state more positively and enthusiastically than in situations where they exercised alone without friends or peers: “Because it is so incredibly fun and (despite finding that every now and then I think about quitting) I realize that it is so much fun and such a pleasurable experience and I experience amazing things with my teammates and that’s the thing that keeps me going–not so much the success. It is the community, great experiences, and good feelings.” “Swimming was enjoyable, but if you are part of a team, it is a lot more fun” ”Climbing in a group is like basketball–I really look forward to these amazing days, its similar to snowboarding with several friends a few days–I really enjoy that“ “The fun and pleasure is enormous and we all stayed together because we functioned very well as a group” “What one wants to have regularly is to meet the people who are also there and with whom you have fun with and love to train with and everything that goes with it.” Perceived social interaction as a facilitator of enjoyment and pleasure has reasonable homogeneity among the cells sex, age, or sport-type. Almost the same frequency was found in both sport-types (individual or team): 9 out of 12 individuals compared to 10 out of 12 team sports interviews mentioned PSI related concepts. However, one difference must be considered: the dissimilar description pattern of PSI from the different types of recreational sport. Both, individual and team-sport groups described PSI in a sports-related environment, e.g., meeting people before or after exercise as well as for other non-sporting purposes: “The aim is to have fun with the other girls, and they organize a lot of things to do outside of handball. For them, it is like a replacement for family.” “Well I have to say that the camaraderie and the team spirit is quite strong. You travel together to tournaments or drink a beer or go to eat together after training. Some of them also came to my birthday. We also sometimes met at our trainers place to watch a game on his television. That’s pretty cool.” However, participants from team sports also described PSI during exercise or at least directly related to it: “A smile on your face and cheering on your teammates, having fun together and a good time”; “It’s really the team spirit which entices me, this kind of sport is fascinating to me.” On account of this, PSI triggers positive emotions directly through the involvement with others such as teammates or indirectly through the company of others. However, it was the environmental concepts that were the most commonly described. In five interviews participants did not mention any concepts concerning PSI as a facilitator for enjoyment and pleasure in sport and exercise. However, the majority of the participants described positive emotions in situations, when they were in the company of significant others. None of the participants stated negative emotions whilst they were exercising with others (the only exception was at the start of a marathon among hundreds of other people which hindered the performance). The difference between male and female participants was very weak (male 9 out of 12 and female 10 out of 12 participants); this also applied to the three age groups (AG1: seven out of eight, AG2 and AG3: six out of eight). Only two elderly participants explicitly mentioned being fine to exercise on their own, but didn’t exclude company per se. “Well I also like to go (exercise) alone and enjoy the time I have for myself.” This is a reference to the third category that describes relief and relaxation when people are able to ‘switch off their heads’ and let their thoughts go. Novelty Experience Thirdly, NE in contrast to the daily life routine was another factor influencing some of the emotional experiences in recreational sport and exercise mentioned in 18 out of 24 interviews. Whilst typically most kinds of exercise are dissimilar to most work-, family-, or other none-sporting commitments and activities, the divergent character of NE lies in the nature of sport and exercise itself. The difference is not limited to activities per se and can be found in many dimensions, e.g., the diversity of surroundings, the character of certain people involved in sports or the alternating demand of exercise–sometimes playful or challenging. Concepts such as enjoying the landscape and countryside, to be outside and do outdoor activities- in contrast to most indoor activities, to relax on a cognitive level, to gain new experiences, or to do something non-productive just for fun; curiosity or variety were all grouped into NE. “And when it was lightly raining and you were completely alone out on the water and you are paddling slowly and there is hardly any noise… you really hear the rain on the water, like singing. It’s wonderful–at least that’s what I think.” “On the one hand I find the snow and the mountains amazing, as well as the landscape which is thrilling and the sun–which is enormous,” “the otherness of the game compared to daily life, most emotions are triggered by that.” “It is when you are on high up in the mountains on a slope and you get this feeling of free movement whilst descending, the swinging to the left and right, and then moving to adapt to the course of the landscape. It is then I get the feeling of pleasure and satisfaction.” Another part of NE is the cognitive relaxation component. To switch off the brain and let thoughts go, triggers positive emotions in and concentrates the mind on something other than stress and is a contrast to the focus on daily life. Amongst the interviews exercise was often mentioned as an important tool to compensate other influences. NE is only one element of this method, representing the cognitive aspect and must be distinguished from PPE (fourth category), which is related to a physical dimension: [When exercising] “Then I’m not able to think about my job anymore. When I was on the water at the weekend and returned to study on Monday, my head was totally free. I only realized that much later. The mental concentration required while you are sailing- you have to focus on the route, the rules, the environment, and adapting to the elements–had a tremendous recuperative effect on me and I was filled with inner satisfaction and peace.” “That moment of diving into the water I find really off-putting but afterward it is pure relaxation. Nothing to think about, nothing else to do. Because of that, I often miscount. Only when I leave the water and have to dry myself, does it turn back to being bad”; “it’s more like turning off from your job. That’s my goal, switching off from the office job and daily routine and to have a bit of fun doing something you want to do.” “You can turn off your head on the track and run because you don’t have to do anything in particular and you can simply let your thoughts go.” In comparison to the male participants (8 out of 12), female participants referred slightly more to concepts related to NE (10 out of 12). However, there were no differences between the individuals and team-sport groups (individuals- 9 out of 12; team sport- 9 out of 12). Among the three age groups a slight increase in the amount of statements about the concepts was found (AG1: five out of eight; AG2: six out of eight; AG3: seven out of eight). Perceived Physical Exertion The last factor found was PPE. Concepts related to this category were found in 14 out of 24 interviews. In addition to its psychological component, this category exclusively contains a physical dimension of the body, while the other three are on a pure psychological level. PPE is the phenomenon that sport and exercise per se but also fatigue and exhaustion caused by such activities generates a specific and desired emotional response. This effect was described whilst exercising and especially when pushing oneself to the limit but was mentioned more after exercising. Similarly to NE, this category is also a contrast to the daily life routine with its typical lack of movement and physical demand. Therefore PPE represents an inner drive for movement, the pleasure after exercise or the physical balance desired after long periods of sitting or leading sedentary lifestyle per se. Participants referred to positive emotions like pleasure and enjoyment in situations where they could satisfy this drive for movement or do other things that were different to the daily routine such as sport. Even the perception of exhaustion due to strenuous exercise, the opportunity to burn off energy or the growing muscle soreness triggered positive emotions: “The feeling of pleasure during sport is when you are exercising and push yourself, you get feelings of happiness. For me, when I know that I can work out and when I am finished, I pushed myself and afterward I am actually more satisfied and happy.” “Well running sometimes feels a bit like torture but nevertheless I feel happy during the run and afterward too.” “And if something went wrong you are able to really push yourself to the limit when doing sport and mostly you always feel better afterward. Because of that it is a really good way to balance out my job.” Another component of PPE is the inner need for movement. People described an impulse to exercise but not necessarily for a certain purpose or a specific goal. They responded with positive emotions whilst fulfilling the desire to exercise: “It has an enormous impact and if I’m not exercising one day I notice that I don’t feel as well as I normally do. I need that very much at least once a day, if not more often.” “When I arrive at home I feel well balanced, if I wouldn’t exercise I would sit at home with the same incomplete work and be frustrated and nervous and in a bad mood. I would then start to concentrate more on the smaller things that aren’t so important.” “Well the regularity is very important for me, I’m angry if my training is canceled and then I realize that I miss it because I’m not balanced.” “I’m used to it. If I was completely without exercise …well there are some periods in between where you are forced to interrupt exercise but then I miss it quite a lot.” To underline this point a common theme about missing exercise was very strong among the interviewees. All participants mentioned, that they would miss their familiar sports (24 out of 24). One crucial part of PPE is the follow-up or rebound effect. In these interviews all participants stated positive emotions after exercising (but only if those emotions were related to a physical response or effect such as exhaustion and not due to evaluation, for example due to a lost match): “When I’m not exercising, I recognize after a while that I’m not feeling physically well. I certainly need to push myself physically and afterward the endorphins are released. You always feel better after having exercised.” “I am simply more relaxed and less tense when I’m physically exhausted and you have a total different feeling about life. You sleep very well because you have pushed yourself physically and because it cleared your head from thinking about your job you have a better feeling about life. ” “Well emotionally balanced and after exercise a happy exhaustion. Therefore, satisfied, happy, and physically exhausted.” “It is like a happy exhaustion after a training session. Frequently, I recognized then that I am finished with the training session due to these feelings. It’s a kind of very physical happiness.” “When you are finished you think about what you have done and that you managed to overcome your doubts and do it, which is probably like a reward system in itself. Well after exercising you have, 99% of the time, positive feelings afterward.” There was a strong difference between male and female participants. 10 out of 12 men reported situations or concepts related to PPE while only 4 out of 12 women did. The need to move, push oneself to their limits, be physically exhausted, or to get the positive emotions afterward was substantially stronger among male participants. The factor sport type, only slightly differed. Individual-participants mentioned 6 out of 12 times concepts related to PPE whilst in 8 out of 12 interviews team participants mentioned it. According to the age factor a strong decline occurred from AG1 (seven out of eight) via AG2 (five out of eight) to AG3 (two out of eight). Discussion To change sport and exercise experiences and thus behavior, it is crucial to identify the major influential factors. As described above there is no doubt that positive emotions are major reasons to adopt and maintain corresponding activities (Jekauc, 2015; Rhodes and Kates, 2015; Stenseng et al., 2015), while a lack of positive emotions leads to dropping out (Scanlan and Simons, 1992). Furthermore perceived enjoyment is a key factor for commitment to sport (Scanlan and Lewthwaite, 1986; Scanlan et al., 1993) and reflects a positive emotional response like general feelings (e.g., pleasure) to the sporting experience (Scanlan and Simons, 1992). This study also provides strong evidence for the assumption, that positive emotions such as fun and enjoyment encourage maintenance of recreational sport and exercise in the long term. Participants even referred to positive emotions in a sporting context occasionally without being directly asked (aside from the specific questions). To enhance participation in recreational sport and exercise, commitment and adherence, a better understanding of factors which makes this experience more enjoyable is crucial, starting with the general to the specific (Scanlan and Simons, 1992; Ekkekakis and Petruzzello, 1999). According to Weiss and Raedeke (2004) the factors PC, enjoyment, and the social environment impact adherence and maintenance of physical activity among young people. Similarities were described by Scanlan and Simons (1992) with PC and challenge, social interactions, elements of activity itself, and extrinsic rewards as mediators which often arise in research. In this article, categories similar to the factors competence, elements of activity and social environment and, respectively, social interactions were found. Challenge is included in the first category: PC. Concepts concerning extrinsic rewards were not found during the interviews. Similarly to the factors mentioned above, the four categories found in these interviews with an impact on emotional experience are PC, PSI, NE, and PPE. Concepts referring to these facilitators occurred frequently among all twenty four interviews suggesting also a dynamic interplay. Perceived Competence Perceived competence is one category which is deeply anchored in the character of sport and exercise not only because of the Olympic motto, “Citius–Altius–Fortius” but because in despite of progression, many appropriate activities require special skills to perform them. Positive emotions like fun enhance habits or channel positive behavior change (Rhodes and Kates, 2015; Kwasnicka et al., 2016) while negative emotions strengthen actions to avoid something. PC therefore is a facilitator to evolve toward a certain skill level (Deci and Ryan, 2000; Ekkekakis et al., 2005; Cohn and Fredrickson, 2006) as well as helping to sustain this ability as a result of the fun and enjoyment experienced during sport and exercise. The importance of PC as a facilitator for positive emotions is underlined due to it commonness and robustness among all interviews regardless of the independent factors. Furthermore it might reinforce itself since the perception of competence result in positive emotions which in turn support optimal performance (if perceived as convenient; McCarthy, 2011). White (1959) was the first, who described the interaction between competence and enjoyment. He proposed an intrinsic drive of an individual to deal effectively with the surrounding environment and the positive emotions after doing so (Harter, 1978). In his opinion effectance motivation just aims for the feeling of efficacy, not for any consequences (White, 1959). As described in his basic model, this type of motivation leads to mastery attempts in a certain domain. After a successful attempt and PC, a feeling of efficacy arises, accompanied by pleasure, which in turn increases or at least maintains effectance motivation (White, 1959; Harter, 1978). Based on White’s (1959) concept of effectance motivation, Harter (1978) developed the competence motivation theory which describes the intrinsic striving of a child to develop competence in certain domains and to achieve mastery level. Related to the amount of competence motivation the child will search for correspondence between challenge and expected success. If the person succeeds in those mastery attempts with an optimal amount of challenge, they will experience PC and control as well as a positive affect response. Significant others may also enhance these perceptions giving social support in different ways and indirectly influence the emotional response. In sum these processes increase the desire “to continue being effective” (Weiss and Raedeke, 2004; S.227). It is reasonable to assume that this process applies for the most part equally in older individuals–with its usual characteristic structure but with different intensities of influential behavior and habits. In summary, in a recreational sport or exercise -related environment, PC is undoubtedly a major facilitator on emotions with a robust effect on the emotional response and furthermore essential for any type of motivation (Deci and Ryan, 2000). Connections to PSI, NE and PPE were found (e.g., collective success, flow, and push to the limit). Perceived Social Interaction Humans do have a natural need to establish and sustain close and warm relationships (Maslow, 1968). Forming these bonds as well as amplifying them is ensued by positive emotions such as pleasure and happiness. To trigger these emotions people need frequent social interactions with others, ideally experienced as pleasant and enjoyable. However, more importantly is the perception of belonging and the social tie itself rather than mere social contact (Baumeister and Leary, 1995). Furthermore, a consistent and long-term interaction with familiar people is experienced as more satisfactory than changing partners or strangers. This effect increases due to mutuality, which is supportive but not necessarily essential (Clark, 1984; Baumeister and Leary, 1995). The facilitator PSI may promote positive emotions during sport and exercise and afterward. Most kind of sports provide an appropriate environment for social interactions because many people tend to exercise with others. According to Coon (1946), affiliating in groups is inherently instinctive rather than practical (e.g., cost-effective, same time, interests, etc.). In addition, it seems that behavior change from individuals within a group is more promising than changing them alone (Lewin, 1951; Kwasnicka et al., 2016). Perhaps positive motivation from others who enjoy the physical activity might enhance the attitude toward it and the effect is cumulative. Although this collective reinforcement might also apply to negative emotions, it is reasonable, that the majority of emotions whilst exercising are positive. As the participants stated, –‘otherwise people won’t continue with an unpleasant activity.’ However, PSI as mentioned by the majority of the participants enhances their emotional experience in the context of sport and exercise. This indicates that the need for attachment and belonging is affected which can be considered to be fundamental (Baumeister and Leary, 1995; Lavigne et al., 2011). People experience pleasure and enjoyment when creating new relationships such as entering a new sport, team, or club, but this was even more so when reinforcing or sustaining existing ties (Baumeister and Leary, 1995). On the other hand, it seems that positive emotions also influence belongingness (Stenseng et al., 2015) and due to their reciprocal relationship are self-energizing. Many participants reported, that either their parents or their friends were the initial cause of starting their engagement in sports and exercise–both figures in which the participants have a bond and attachment. PSI might convert recreational sport and exercise into something more attractive. Participants also underlined positive emotions while exercising together with friends or being accompanied by them in an exercise context. These emotions might arise due to the exercise itself, due to the satisfaction of the need to belong or due to a combination of both. Although participants thought that their sporting-activities were enjoyable, they explained that the intensity of positive emotions was weaker when exercising alone. Similarly lower levels of enjoyment were reported from participants who spent time together without recreational sport and exercise. The highest degree of positive emotions occurred when exercising along with others or directly together. Recent findings suggest, that a basic function of emotion is to encourage the formation and sustenance of relationships (Baumeister and Leary, 1995). Because people are happy and also resist the dissolution of relationships, they participate more often and are more likely to continue with recreational sport and exercise much longer than they normally would without these social ties. Along with recent evidence this study confirms the connection between belongingness and positive emotions in recreational sport and exercise (Stenseng et al., 2015). Furthermore relationships with NE and PPE were found (interaction with teammates, reduced negativity of high intensity). Novelty Experience According to the Latin proverb, ‘variatio delectat,’ variation can be seen as a source of positive emotions. Furthermore, the distraction hypothesis may provide an explanation for the ‘feel better’ effect normally associated with exercise. It assumes, that distraction from stressful stimuli rather than the physical exercise itself enhances positive emotions (Morgan, 1985). Participants often reported, that they enjoyed situations in which they could ‘turn off their head’ and let their thoughts go. However, research could not verify a stronger impact of exercise in terms of distraction in comparison to equivalent activities such as rest or relaxation on emotional states (Yeung, 1996). It seems that the distraction hypothesis is not able to explain why participants choose exercise rather than rest or relaxation. Other distractions and influences due to cognitive factors such as non-associative thoughts about external surroundings, etc. might affect mood states during exercise (Goode and Roth, 1993), however, the intensity of this cognitive task must still match (Fillingim et al., 1989). Yeung (1996), concludes that the effect of exercise on negative emotions is not superior compared to equivalent activities for distraction but in fact is superior when it comes to improving positive mood. If the attitude toward exercise is positive, distraction might reinforce that. Cognitive processes might attenuate negative signals from the body and as a result the overall sensation is primarily positive (this mechanism is discussed further in category 4, PPE). Another approach might be the theory of sensation seeking, a concept which describes the need for various, novel and complex sensations and the acceptance of accompanied risks (Zuckerman, 1979). Although the four scales (thrill and adventure seeking, experience seeking, disinhibition, and boredom susceptibility) suit NE on first impression, the differences found among the three independent factors did not match with recent evidence. While sensation seeking is stronger amongst younger people and men (Zuckerman et al., 1978; Steinberg et al., 2008; Cross et al., 2013), the participants reported the opposite in terms of age, sex, and readiness to assume risk. Since sensory deprivation reinforces inter alia the urge to move and extrinsic sensory stimuli (Solomon et al., 1957), people might desire similar needs without such need for extremes. The seeming paradox of this category is that new experiences and impressions, curiosity and excitement are completely opposite to relaxation and cognitive balance or rest which are also comprised in NE. However, both dimensions share in common a positive valence and the contrast to the daily life routine even though they are opposites. The first form of NE describes the positive state of ‘feelings’ in such a situation- high arousal and activation of a person. Seeking sensation, adventure, challenge, or just the desire not only to move but also to gain other experiences in comparison to everyday life might be one explanation for such an emotional response and resulting behavior. The second appearance of NE describes the lower but fair arousal and activation of a person seeking some relaxation and rest. In this particular situation people also described positive emotions in contrast to a stressful daily routine, which meant switching off and letting their thoughts go. In both forms of NE the outcome is nearly the same: positive emotions, enjoyment, and satisfaction. However, the trigger is a different one. Therefore none of the participants described a relaxation effect following a state of excitement or curiosity accompanied by rest. Positive emotions were intended to promote curiosity and novelty to explore the surroundings for resources (Ekkekakis et al., 2005; Cohn and Fredrickson, 2006) rather than responding to a stimulus demonstrating a strong affinity between NE and positive emotions. Phenomena such as the ‘runners high’ underline the interplay between NE and PPE. Perceived Physical Exertion In relation to exercise-intensity, the emotional response of individuals differs in terms of magnitude, direction, or accountable sources (Ekkekakis and Petruzzello, 1999; Ekkekakis et al., 2000, 2005; van Landuyt et al., 2000; Hall et al., 2002; Ekkekakis, 2003). It is reasonable to assume, that individuals always seek the best emotional outcome and choose the appropriate intensity accordingly, whereas a deviation might lead to less pleasure and enjoyment (Ekkekakis, 2003; Ekkekakis et al., 2005; Ekkekakis and Lind, 2006; Vazou-Ekkekakis and Ekkekakis, 2009). However, some people experience positive emotions at lower levels of intensity and duration of exercise (Ekkekakis and Petruzzello, 1999; Ekkekakis et al., 2000; Reed and Ones, 2006), while some feel comfortable with moderate to high exercise intensity, others will not (Yeung, 1996; van Landuyt et al., 2000; Ekkekakis, 2003). As intensity approaches functional limits, divergent sensations vanished as well as positive valence (Ekkekakis and Petruzzello, 1999; Hall et al., 2002; Ekkekakis, 2003, 2009; Ekkekakis et al., 2005, 2011). Despite these negative emotions some participants (especially those with a higher fitness level) chose demanding intensities and pushed themselves to their limits possibly to generate a higher physical contrast to that of the daily life demand on the body via exhaustion or pain [“hurt so good”(Ekkekakis, 2003)]. Perhaps other influences attenuate the unpleasant sensations associated with strenuous exercise and generate the desire to exercise. Although the main impact on emotions in this domain is physical and increases proportionally with exercise intensity whilst cognitive effects diminish (Ekkekakis, 2003, 2009). However, it still has a cognitive component (e.g., conscience, pride, guilt, etc.; connection to the first category PC). Another explanation, why people choose intensities that have minimal positive emotions might be due to a follow-up or rebound effect after the physical activity is finished. In general, exercise is consistently ensued by positive emotions (Tuson and Sinyor, 1993; Yeung, 1996; Ekkekakis, 2003), even if it is at a demanding intensity (Hall et al., 2002). This is despite recent emotional bias during exercise (Ekkekakis and Petruzzello, 1999) which might exceed pre-exercise states independently of intensity (Ekkekakis et al., 2008). This affective contrast (Solomon, 1980, 1991) might be due to an interference of two affective dynamic processes with different patterns in terms of effect direction, emergence, and duration. In contrast to each other, the a-process has a negative valence, which is stimulated beyond a certain threshold, then matches the intensity and disappears along with it. Whereas the b-process has a positive valence, is triggered by the first, has a completely opposite bias and appears to be more consistent with slower emergence and decline (Solomon, 1980, 1991). The sensation during exercise is the result of subtraction of both whereas in the recovery-phase the b-process solely determines the emotional outcome. This leads to the conclusion that during high intensity exercise individuals might experience negative emotions, due to a high salient influence of the a-process, but afterward the emotional response is always positive because only the b-process remains. The concept of flow (Csikszentmihalyi, 1975) or phenomena such as the ‘runners high’ might be reflected through the b-process during exercise and mark the threshold where positive emotions occur. However, the a-process might include interoceptive signals which rise as the intensity increases whereas the b-process is charged with cognitive signals which diminish with growing intensity in favor of the first process, which is similar to the dual mode concept (Ekkekakis, 2003, 2009). Another explanation for positive emotions due to the influence of the b-process might be a feeling of relief and alleviation accompanied by biochemical processes inducing regeneration (Hatfield and Landers, 1986). Due to this relationship, positive emotions vanish whilst exercise intensity increases and will return immediately upon exercise cessation explaining the ‘feel-better-effect’ commonly mentioned by participants and people in general (Morgan, 1985). In other words, some individuals may not initially enjoy the activity itself but seek the positive emotions afterward. Statements among those interviewed supported these notions. However, in their review, Rhodes and Kates (2015) did not find any evidence, that post-exercise emotional experience influenced future exercise behavior. Either a lack of sensitivity and power of the method, different mechanics between emotional experience during and after exercise, aforementioned relationships with other facilitators or habitual issues such as the overall exercise experience which might attenuate current emotional influences cause this contrariness which must be clarified through future research. Relating to the strong difference in terms of sex, references to a higher intensity and especially to exceeding physical limits and pushing oneself hard were noticeably common amongst men. According to the opponent-process model (Solomon, 1980, 1991), men might have a higher resistance to the a-process or potent factors like PC (category 1) boosting the b-process, whilst women do not. Reinforced by a weaker fitness level, they perceive exercise as more strenuous and less pleasant and may avoid higher intensity exercise (Ekkekakis and Petruzzello, 1999; Ekkekakis and Lind, 2006). The difference in terms of sport type is small. While participants in individual recreational sports could decide autonomously, those people in a team or group must adapt to the collective intensity expected–this normally results in a decrease of positive valence (Dishman et al., 1994; Ekkekakis and Petruzzello, 1999; Vazou-Ekkekakis and Ekkekakis, 2009) as well as it being due to supra-threshold intensities (Ekkekakis et al., 2011). However, the slightly higher frequency in positive emotion-related statements of team-sports participants suggests that team-dynamics as well as providing a distraction attenuate the interoceptive signals that improve the resistance to the a-process, raise the threshold and reinforce the b-process during exercise and later on as well. The age groups show a strong decline in positive emotions related to exercise intensity. A transition from interoceptive dominance at a younger age to cognitive dominance can be expected. According to the opponent-process model (R. L. Solomon, 1980, 1991), a higher resistance to the a-process or a higher sensitivity to the b-process and lower self-awareness at a younger age might reflect the urge to move and the need for vigorous exercise. An inverted relationship at an older age with higher maturity induces avoidance to strenuous exercise not only because of health issues, restrictions, and ailments but actually increased rationalizing results in the motivation to do exercise for other reasons. As one ages the threshold for desired high intensity exercise seems to weaken which is reinforced by a decreasing fitness level influencing the overall exercise experience (Ekkekakis et al., 2005). Self Determination Theory According to the self determination theory (Deci and Ryan, 2010) humans have three psychological needs which are essential for intrinsic motivation to adopt and adhere behavior (Kwasnicka et al., 2016) or an activity in particular: the need for competence, relatedness, and autonomy. Although the theory is focused on intrinsic and extrinsic motivation and this study is focused on facilitators of positive emotions, the similarities are striking. The perception of competence and affiliation plays an important role to elicit positive emotions through satisfaction of these basic needs. This leads to the assumption, that positive emotions and intrinsic motivation are related constructs. Furthermore it is not surprising that the need for autonomy was not as strong as the other two among the interviews since all participants were voluntary involved in recreational sports in their leisure time and autonomy was naturally present. Nevertheless there are some characteristics of autonomy in common with NE, such as self-control or the ability to choose, e.g., skiing or sailing a self-chosen course or running at one’s own desired pace. Additionally NE is described as contrast to the repetition of daily routine which contains commitments and limited autonomy. However, when this is restricted due to a forced interruption (e.g., such as injuries or appointments) this negate any positive emotions. The selective coding process revealed that even a high level of perception of competence or relatedness seemed to both facilitate positive emotions even more if this perception was further increased, e.g., one is performing well and later on is victorious or teammates become close friends. In contrast references to autonomy were more common when it was threatened which indicates, that in this case it is a much stronger facilitator for negative emotions. However, as an avenue for future work the different function of negative emotions (e.g., survival, avoid harm) and positive emotions (e.g., adaptation, extend resources) require separate attention since negative emotions influence behavior in a different way (Cohn and Fredrickson, 2006). Implications This study is a novel and perhaps promising approach to illuminate facilitators of positive emotions to explain exercise maintenance. Firstly, these findings encourage continuation of practical application studies to advance the knowledge about potential facilitators of emotional responses to exercise. Secondly, based on these results, interventions could be developed which enhance the emotional response during exercise by manipulating these four facilitators (e.g., due to a corresponding design or instructor behavior). Thirdly, recommendations for the design and evaluation of exercise programs can be made in so far as to reduce the drop-out rate and to increase commitment and adherence to sport. In an experimental study, Jekauc (2015) could show that emotions could be manipulated to increase adherence in exercise. However, further quantitative studies are needed to explicitly test the relevance of the factors discovered here. Strength and Limitations This study has some strengths and limitations. For the best approach to the emotional dimension, Dishman (1994) and Rhodes et al. (2009) proposed a qualitative and therefore inductive design because qualitative methods meant having a higher capability to illuminate the different sources of enjoyment by including a broad spectrum of influences as expected in the sporting environment (Scanlan and Simons, 1992). Also the value of information is much higher if participants are able to express their feelings with their own words. They might reveal factors which might otherwise be missed. The semi structure interview guide guaranteed a uniform approach. For a qualitative study, it has quite a large sample of 24 participants which is heterogeneous as sex, age, and type of sport are systematically varied. Furthermore the analysis via Grounded Theory ensured systematical categorization. However, the sample is not large enough to be a representative sample. Despite a progressive but sometimes deficient differentiation of constructs and convergence of several theories, terms in the literature are still used inconsistently which hinders the ability to compare. Presented findings were drawn from recreational sport and exercise and might not count for specific aspects such as competitive sport or for a broader remit such as physical activity. Conclusion In this study, four facilitators of positive emotions during exercise and recreational sport were identified: PC, PSI, NE, and PPE. These results could provide the starting point for the development of interventions aiming at the promotion of positive emotional states in sport in order to increase maintenance and adherence to sport and exercise. Future steps will be to design intervention studies according to the findings of this study and to test their effectiveness in experimental studies. Author Contributions BW is the main author of the submitted article and responsible for the overall conception and design of this manuscript. He is also responsible for the data collection, analysis, and interpretation. DJ is the second author and contributed to the design of the study, he was involved in the interpretation and provided edits to the paper. Both authors read and approved the final manuscript. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. ==== Refs References Amireault S. Godin G. Vézina-Im L.-A. (2013 ). Determinants of physical activity maintenance: a systematic review and meta-analyses. Health Psychol. Rev. 7 55 –91 . 10.1080/17437199.2012.701060 Annesi J. (2003 ). Effects of a cognitive behavioral treatment package on exercise attendance and drop out in fitness centers. Eur. J. 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==== Front Front PharmacolFront PharmacolFront. Pharmacol.Frontiers in Pharmacology1663-9812Frontiers Media S.A. 10.3389/fphar.2016.00254PharmacologyOriginal ResearchFrom Traditional Resource to Global Commodities:—A Comparison of Rhodiola Species Using NMR Spectroscopy—Metabolomics and HPTLC Booker Anthony 12Zhai Lixiang 13Gkouva Christina 1Li Shuyuan 3Heinrich Michael 1*1Research Cluster Biodiversity and Medicines/Centre for Pharmacognosy and Phytotherapy, UCL School of Pharmacy, University of LondonLondon, UK2Division of Herbal and East Asian Medicine, Department of Life Sciences, University of WestminsterLondon, UK3Department of Traditional Chinese Medicine, School of Traditional Chinese Medicine, Guangdong Pharmaceutical UniversityGuangzhou, ChinaEdited by: Judith Maria Rollinger, University of Vienna, Austria Reviewed by: Pierluigi Caboni, University of Cagliari, Italy; Maria Halabalaki, Athens State University, Greece *Correspondence: Michael Heinrich m.heinrich@ucl.ac.ukThis article was submitted to Ethnopharmacology, a section of the journal Frontiers in Pharmacology 29 8 2016 2016 7 25413 5 2016 02 8 2016 Copyright © 2016 Booker, Zhai, Gkouva, Li and Heinrich.2016Booker, Zhai, Gkouva, Li and HeinrichThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.The fast developing international trade of products based on traditional knowledge and their value chains has become an important aspect of the ethnopharmacological debate. The structure and diversity of value chains and their impact on the phytochemical composition of herbal medicinal products, as well as the underlying government policies and regulations, have been overlooked in the debate about quality problems in transnational trade. Rhodiola species, including Rhodiola rosea L. and Rhodiola crenulata (Hook. f. & Thomson) H. Ohba, are used as traditional herbal medicines. Faced with resource depletion and environment destruction, R. rosea and R. crenulata are becoming endangered, making them more economically valuable to collectors and middlemen, and also increasing the risk of adulteration and low quality. Rhodiola products have been subject to adulteration and we recently assessed 39 commercial products for their composition and quality. However, the range of Rhodiola species potentially implicated has not been assessed. Also, the ability of selected analytical techniques in differentiating these species is not known yet. Using a strategy previously developed by our group, we compare the phytochemical differences among Rhodiola raw materials available on the market to provide a practical method for the identification of different Rhodiola species from Europe and Asia and the detection of potential adulterants. Nuclear magnetic resonance spectroscopy coupled with multivariate analysis software and high performance thin layer chromatography techniques were used to analyse the samples. Rosavin and rosarin were mainly present in R. rosea but also in Rosea sachalinensis Borris. 30% of the Rhodiola samples purchased from the Chinese market were adulterated by other Rhodiola spp. The utilization of a combined platform based on 1H-NMR and HPTLC methods resulted in an integrated analysis of different Rhodiola species. We identified adulteration at the earliest stage of the value chains, i.e., during collection as a key problem involving several species. This project also highlights the need to further study the links between producers and consumers in national and trans-national trade. Rhodiolametabolomicsherb qualityadulterationHPTLCNMR ==== Body Introduction While medicinal plants and spices have been traded for centuries on a global scale, the fast developing international trade of products now includes a large number of species which are used based on local and traditional knowledge and practice. The value chains of such products are starting to become an important topic in the ethnopharmacological debate. The structure and diversity of value chains, as well as their impact on the phytochemical composition of herbal medicinal products (HMPs) has been overlooked in quality issues in transnational trade. Different government policies and regulations governing trade in herbal medicinal products impact on such value chains. Medicinal Rhodiola species, including Rhodiola rosea L. and Rhodiola crenulata (Hook. f. & Thomson) H. Ohba (Figure 1), have been used widely in Europe and Asia as traditional herbal medicines with numerous claims for their therapeutic effects. Faced with resource depletion and environment destruction, R. rosea and R. crenulata are becoming endangered, making them more economically valuable to collectors and middlemen, and also increasing the risk of adulteration and low quality. Poor quality and adulterated R. rosea products have been previously reported (Booker et al., 2015; Xin et al., 2015) and this paper investigates some aspects of the value chains that leads to the production of such products. Figure 1 Rhodiola species. (A) R. rosea; (B) R. crenulata. Photos taken by A. Booker, Sichuan-Tibet border, June, 2015. Adulteration of R. rosea products with R. crenulata has been previously reported but our fieldwork investigations suggested that other species may be implicated, and particularly Rhodiola sachalinensis, another species that appears to contain rosavins (the main marker compounds used for the identification of R. rosea). The genus Rhodiola (Crassulaceae) comprises ~90 species of succulent and herbaceous perennial plants, which mainly show a circumpolar distribution across the northern hemisphere (Xia et al., 2005; Lu and Lan, 2013). Rhodiola species usually grow in mountainous areas such as rock ledges, precipices, tundra, brooks, and river banks (Zhu and Lou, 2010). Ethnopharmacological importance of key Rhodiola species In Europe and North America, Sedum roseum (L.) Scop. (commonly named under its synonym R. rosea L.) is the most well-known and widely used among the different species. It is also known as golden root, or artic root which reputedly demonstrates the economic importance and the geographical distribution of the plant. It has a rich history of traditional use in Russia, Europe and Asia with various uses according to the region (e.g., as shown in Table 1). Table 1 Traditional uses of R. rosea in different regions. Region Use References Russia Escalation of physical endurance Remedy against fatigue and high altitude sickness Aphrodisiac Shikov et al., 2014; Alm, 2004 Norway Astringent Cure for scurvy Remedy against hair-loss and urinary tract disorders Alm, 2004 Iceland and Denmark Alleviation of headaches Alm, 2004 France Stimulant Astringent Panossian et al., 2010 Alaska Cure for sores Remedy against tuberculosis Alm, 2004 Mongolia Remedy against tuberculosis Anticancer Escalation of physical endurance Treatment for lung inflammation Brown et al., 2002; World Health Organization, 2013 In Europe, the first documented medicinal use of R. rosea can be traced back to Dioscorides in 77 A.D. (Brown et al., 2002). In C. v. Linne's Materia Medica, the root of R. rosea was recommended for several conditions such as headaches, “hysteria,” hernias and discharges (C. v. Linne, 1749 in Panossian et al., 2010). Throughout the years, it has appeared in many pharmacopeias and medicinal books of different countries such as Sweden, France, Norway, Germany, Iceland, Estonia, and Russia (Brown et al., 2002; Alm, 2004; Panossian et al., 2010; Shikov et al., 2014). In China, 73 different Rhodiola species have been reported, mainly in the northwest and southwest regions such as Tibet and the Sichuan province. The adaptogenic and tonic properties of the Rhodiola plants have been widely used in traditional Chinese and Tibetan medicine (Li and Zhang, 2008). They are generally referred to with the Pinyin name Hong Jing Tian [red (or glorious) view of heaven] with slight alterations for each species (Table 2). Table 2 Examples of the similar Pin Yin names of different Rhodiola species in China. Scientific name Pin Yin name R. rosea L. Qiang Wei (rose smell) Hong Jing Tian R. sachalinensis Borris. Gao Shan (high mountain) Hong Jing Tian R. quadrifida (Pall.) Fisch. & C.A.Mey Si Lie (four split) Hong Jing Tian R. crenulata (Hook. f. & Thomson) H. Ohba Da Hua (big flower) Hong Jing Tian R. yunnanensis (Franch.) S.H. Fu Yunnan (From Yunnan) Hong Jing Tian R. kirilowii (Regel) Maxim. Xia Ye (narrow leaf) Hong Jing Tian R. fastigiata (Hook. f. & Thomson) S.H. Fu Chang Bian (clustered) Hong Jing Tian R. crenulata can be traced back to Tibetan medicine books including “The Four Medical Tantras” (rgyud-bzhi in Tibetan, Si Bu Yi Dian in Chinese), Yue Wang's Classical Medicinal Book (Somaratsa in Tibetan, Yue Wang Yao Zhen in Chinese), and Jing Zhu Materia Medica [Shel Gong Shel Phreng in Tibetan, Jing Zhu Ben Cao in Chinese (Lu and Lan, 2013)]. It is used for treatment of cough, hemoptysis, pneumonia, and abnormal vaginal discharge. In Traditional Chinese Medicine (TCM), it has effects of nourishing qi as well as promoting blood circulation and is mainly prescribed for qi deficiency and blood stasis (QDBS), stroke, hemiplegia, and fatigue. It is commonly used in China and Tibet for treating altitude sickness. Phytochemical and pharmacological research Research on the phytochemistry and pharmacology of Rhodiola spp. was initiated in the 1960s in the Soviet Union and Scandinavia, mainly focusing on R. rosea (Brown et al., 2002). After the turn of the century the interest in this plant spread globally. Intensive phytochemical research led to the detection of known and novel compounds in R. rosea and related species (Ma et al., 2006; Yousef et al., 2006). Between 2000 and 2015 an increased number of publications stemming from Asian research groups have focused on the detection of novel compounds from Rhodiola species, usually in combination with their respective pharmacological assessments (Fan et al., 2001; Nakamura et al., 2007, 2008). There are more than a few hundred pharmacological studies on medicinal Rhodiola species (mainly on R. rosea) that show a wide range of activities reflecting their diverse traditional use. They possess adaptogenic and stress-protective (neuro-cardio and hepato protective) and antioxidant effects, as well as stimulating effects on the central nervous system, including on cognitive functions such as attention, memory and learning; anti-fatigue effects; antidepressive and anxiolytic effects; endocrine activity normalizing; and life-span increasing effects (Aslanyan et al., 2010; Sarris et al., 2011; Panossian et al., 2013). The main active compounds are reputedly phenylpropanoids (rosavin, rosarin, rosin) and phenylethanoids (salidroside and tyrosol). Quality issues of medicinal Rhodiola spp. Rhodiola roots and rhizomes are highly valuable products traded at an international level. Since the majority of R. rosea and R. crenulata raw material supplied still comes from wild-collection, their intensive collection leads to scarcity (Galambosi, 2006; Lu and Lan, 2013). Herbal preparations of Rhodiola species (mainly R. rosea) are extensively utilized around the globe. There is an increasing number of commercial products available on the American, Asian and European markets, either as food supplements or herbal medicines. R. rosea herbal monographs have been included in many Pharmacopeias worldwide. On the other hand, R. crenulata is the only species used medicinally in TCM (Table 3). Table 3 Generation of Rhodiola spp. recorded in selected pharmacopeias and publications. Pharmacopeia/publication Recorded Rhodiola species Medicinal use part Herbal product Department of Health and Ageing, Australian Government Rhodiola rosea Root (Rhizome) Dry extract Committee on Herbal Medicinal Products, 2012 Rhodiola rosea Rhizoma et radix Extract United States Pharmacopeia (32th Edition) Rhodiola rosea Rhizoma et radix Dry extract, tincture Chinese Pharmacopoeia, 2010 Rhodiola crenulata Rhizoma et radix Extract Russian Pharmacopoeia (12th Edition) Rhodiola rosea Rhizoma et radix Extract Due to this rapid increase of Rhodiola raw material demand, other Rhodiola species such as R. fastigiata, R. sachalinensis, R. quadrifida, Rhodiola sacra (Prain ex Hamet) S. H. Fu and Rhodiola serrata H. Ohba have been sold on the market (Xin et al., 2015). Since there is not any consistent worldwide quality control programme, inadequate quality assessment of Rhodiola spp. is a common issue. This raises concerns about possible adulteration and misidentification issues. The lack of genuine drug material, confusion over the Chinese Pin Yin name of the drug when sourcing from China and accidental or deliberate adulteration during the manufacturing stage may contribute to low quality of final products. The analytical techniques currently available focus on identifying R. rosea or R. crenulata through chromatographic methods. Other species of Rhodiola have generally not been considered. R. sachalinensis presents a particular problem as it may contain similar marker compounds to R. rosea (and some sources suggest that it is the same species—see http://www.kew.org/mpns-portal). Integrated analytical platform approach NMR-based metabolomics NMR-based metabolic fingerprinting has been used in the analysis of numerous food and medicinal species focusing on their quality assurance as well as their pharmacology. Such comparative studies include Danggui [Angelica sinensis (Oliv.) Diels] and Engelwurz/European Angelica (Angelica archangelica L.; Li et al., 2014). Metabolomic differences between different Tussilago farfara L. accessions (Zhi et al., 2012) and different Salvia miltiorrhiza Bunge production sites (Jiang et al., 2014) were also studied by NMR fingerprinting coupled with multivariate analysis. Compared to GC-MS and LC-MS, NMR has some advantages such as non-selectiveness, high reproducibility, and good stability (Simmler et al., 2014). At the same time, structural information on metabolites can be obtained from NMR directly. Therefore, NMR can be regarded as an ideal choice for chemical comparison and identification of the phytochemical differences of medicinal plants. HPTLC Since the NMR-metabolomic approach is not a validated pharmacopoeial method, there is a need to be compared to a standard method like high performance thin layer chromatography (HPTLC). This method is widely used for the authentication and quality control of herbal substances (Reich et al., 2008). Compared to NMR-based metabolic fingerprinting, HPTLC could be highly effective with relatively lower price (Booker et al., 2014). HPTLC can also be helpful for the identification of specific compounds. Therefore, we chose these two complementary approaches in this study. A third analysis strategy using DNA bar coding was used to help verify some of the samples (details are given in the Supplement S2). Materials and methods Sampling and preparation of plant material Forty-two batches of Rhodiola market samples (i.e., not authenticated) were collected between October 2014 and January 2015 from different suppliers including retail outlets, The internet, pharmaceutical companies in seven different locations (Beijing, Guangdong, Qinghai, Anhui, Hebei, Jilin, and Hong Kong SAR) and in China, Germany and Russia. These raw-material samples were mainly labeled as R. rosea, R. crenulata, R. sachalinensis, and R. quadrifida. 18 batches of authenticated plant material were provided by Agroscope Institute (Switzerland). The samples were rhizomes of R. rosea plants propagated from different wild Swiss populations (Mattmark, Carrasino, and Nomnon) or botanical gardens (Switzerland and Germany). In addition, authenticated R. rosea samples which were grown from seeds or provided to the institute by Dr. Bertalan Galambosi were also included. Lastly, in June 2015, samples of R. crenulata and R. fastigiata roots and rhizomes were collected from Garze, Sichuan, China (altitude 4500 m). These samples were authenticated by Professor Shuyuan Li, (Guangdong Pharmaceutical University, Guangzhou, China). Botanical reference materials (BRMs) for R. rosea, R. crenulata, and R. sachalinensis were obtained from the National Institute of Food and Drug Control (NIFDC, China), Dr. William Schwabe (Germany) and Agroscope (Switzerland). BRMs for R. quadrifida and R. fastigiata were provided by Professor Alexander Shikov (Saint-Petersburg Institute of Pharmacy, Russia) and Dr. Anthony Booker (UCL School of Pharmacy). R. fastigiata was authenticated by Professor Shuyuan Li (Guangdong Pharmaceutical University, Guangzhou, China). All the collected samples were deposited in the herbarium of the UCL School of Pharmacy (London, UK). A detailed description of the investigated samples including their origins and representative symbols are provided in Supplement (S1). Crude root samples were ground to powder using a household grinder (EK1665ROFOB, Salter, UK) and sieved (0.70 mm mesh). All the powder samples were kept in 1.5 ml tubes (Eppendorf AG.) at 4°C until use. Solvents, reagents, and reference compounds Deuterium oxide (D2O), methanol-d4 (99.8% D, MeOD), dimethyl sulfoxide-d6 (DMSO-d6), and tetramethylsilane (TMS) were obtained from Cambridge Isotope Laboratories Inc. (Andover, MA). Salidroside, gallic acid, rosarin, and rosavin were purchased from Sigma-Aldrich Chemicals (St Luis, USA). Tyrosol was purchased from Acros organics (New Jersey, US). Water used in this study was purified by using ULTRAPURE water system (Millipore, Germany). All other chemicals were of analytical grade. 1H-NMR spectroscopy Sample preparation Nine-hundred microliter of MeOD-d4 was added for extraction. The samples were vortexed (Rodamixer, UK) for 30 s and sonicated at an ultrasound bath (Fisher, XB22, UK) for 10 min. The solutions were centrifuged for 10 min at 14,000 rpm (EBA21, Hettich, Faust Laborbedarf AG, Germany). Six-hundred microliter of supernatant was transferred to a 5 mm diameter NMR spectroscopy tube and the samples were submitted for NMR spectroscopic analysis. The one and two dimensional 1H-NMR spectra were recorded on Brucker Avance 500 MHz spectrometer (Bruker Analytic, Germany), which was equipped with a QNP (31P, 13C, 15N, and 1H) 5 mm cryoprobe. The acquisition parameters were: size of the spectra 64 k data points, line broadening factor = 0.16 Hz, pulse width (PW) = 30 degrees, and the relaxation delay d1 = 1 s. The acquisition temperature was 298 K. In order to assess the coherence of the results obtained, two samples from the same batch were subjected to NMR analysis on the different days of examination. To minimize the error caused by root selection during sample grinding, any samples weighing more than 500 g were analyzed twice. Data analysis The resulting spectra were manually phased and auto-baseline corrected by Topspin 3.2 (Bruker, Germany) for organic fractions. Signals between δ 5.20–4.40 ppm and δ 3.35–3.22 ppm were removed prior to statistical analysis due to the presence of methanol-d4. The total area of peaks (δ 10.00–0.00 ppm) was integrated into small (0.04 ppm) buckets by bucketing (binning) function using AMIX or ACD-Labs in order to generate a number of integrated regions of the data set. The buckets obtained were then imported to Microsoft EXCEL (2013) where the samples were re-labeled and their species information was added. Principal component analysis (PCA) was performed with SIMCA-P 13.0 (Umetrics, Umeå, Sweden) for metabolomic analysis of the generated dataset. Scaling mode of Pareto (Par) and Unit Variance (UV) were tested to optimize the analysis model. HPTLC Sample preparation One milliliter of ethanol was added to 50 mg of weighed samples for extraction. The solutions were then mixed on a rotary mixer (Rodamixer, UK) for 30 s, sonicated in an ultrasound bath (Fisher, XB22, UK) for 10 min and centrifuged for 10 min at 14,000 rpm. The supernatant was used for HPTLC analysis. The reference standard solutions of salidroside, rosarin, rosavin, gallic acid, and tyrosol were prepared at a concentration 1 mg/ml in methanol. Both the reference material and the test samples were stored at 4°C. Data analysis Samples were applied to the plates as bands 8 mm wide by using Linomat 5 semi-automatic applicator with 100 μl syringe. The space between bands was 2.0 mm and the rate of application was 90 nl·s−1. The number of tracks per plate was 15, and 5 μl of standard and sample solutions were applied. The temperature and relative humidity were controlled to 21–24°C and 33%, respectively. Ten milliliter of solvent was poured into the right inlet for development and 25 ml of solvent was poured into the left inlet for saturation. Plates were previously air dried for 10 s and developed in a 20 × 10 cm twin-trough chamber (Analtech, USA) lined with Whatman filter paper (20 × 10 cm) and saturated with mobile phase (Ethylacetate, methanol, water, formic acid (77:13:10:2) vapor for 20 min. The development distance was 70.0 mm from the lower edge. The developed plates were derivatised by dipping in sulfuric acid reagent, using a CAMAG chromatogram immersion device and heated at 100°C on a plate heater for 5 min. Sulfuric acid reagent was prepared with a procedure as follow: 20 ml sulfuric acid was carefully added to 180 ml ice-cold methanol and mixed. The plates were visualized using CAMAG visualizer under white light, UV 254 nm and at UV 366 nm, photographed and uploaded to HPTLC computer software (VisionCats). Results and discussion 1H-NMR and multivariate statistical analysis By incorporating the whole region (0–10 ppm) and Pareto (Par) scaling, a significant clustering is observed in R. rosea samples (Figure 2). R. rosea can be differentiated distinctly from the rest of the species based on their principal component variability. Figure 2 Scores plot of five different species of Rhodiola (R. rosea, R. crenulata, R. quadrifida, R. sachalinensis, R. fastigiata), showing principle component 1 and principal component 2. According to the spectra of the species (Figure 3), the aromatic region (6–8 ppm) is dominated by the main marker compounds (rosavin and salidroside). Hence, this region was analyzed independently using Par scaling (Figure 4). Based on the scores plot produced, Rhodiola species were separated more clearly compared to the scores plot of the whole region. Figure 3 1H-NMR spectra of the reference compounds, salidroside and rosavin, together with the spectra of botanical reference material. 1: R. fastigiata, 2: R. quadrifida, 3: R. crenulata, 4: R. sachalinensis, 5: R. rosea, 6: rosavin, and 7: salidroside. (From bottom to top) (A) Whole region (0–10 ppm); (B) aromatic region (6–8 ppm). Figure 4 Scores plot of Rhodiola samples using the aromatic 1H-NMR region and Pareto scaling. R. crenulata and R. quadrifida were also separated from the rest of the species. However, in this model they were clustered together. This suggests that there is no crucial metabolomic difference between them in the aromatic region. At this point it was considered important to visually inspect the spectra of the BRM's and detect any differences that might be lost with the integration of the data. R. crenulata BRM has an additional quartet at 6 ppm not detected in the rest of the species. This quartet can also be found in all the other R. crenulata samples investigated (figures not shown). Therefore, an effective separation between R. crenulata and R. quadrifida samples can be accomplished by combining the PCA results with the detection of the additional peaks on the 1H-NMR spectra only present in R. crenulata samples between 5 and 6 ppm. We also studied the group-pair comparisons in PCA model with Par scaling (Figure 5). The score plots showed that Rhodiola species separated well (A: R. crenulata with other Rhodiola species; B: R. rosea with other Rhodiola species; C: R. crenulata with R. rosea). Figure 5 Score plots of group comparison between Rhodiola species. (A) R. crenulata (red) with other Rhodiola spp. (blue); (B) R. rosea (green) with other Rhodiola spp. (blue); (C) R. crenulata (red) with R. rosea (green). The main differences were between δ 7.5–7.3 ppm (PC1) and δ 7.0–6.8 ppm (PC2). The chemical shift of the main variable metabolites were mainly rosavin, rosarin, and cinnamyl alcohol derivatives. The metabolites detected were elucidated by the analyses of the 1H-NMR spectra as well as the comparison with the reference compounds, together with the in-house NMR chemical shift database (Mudge et al., 2013; Luo et al., 2015). The summary of the assignment of 1H-NMR spectral peaks obtained from the R. rosea, R. crenulata, and R. sachalinensis BRM extracts are provided in Supplement (S3). HPTLC analysis The band position and visibility of the standards rosavin, rosarin, and salidroside (Figure 6) appear with characteristic colors and increasing retention factors (Rfs) 0.19, 0.26, and 0.31, respectively. Under UV light 254 nm, salidroside is not visible. Under 366 nm, after derivitisation with sulfuric acid, rosavin and rosarin appear as pale pink bands and salidroside as a green one. Figure 6 Left: HPTLC results of standard compounds under UV 254 nm (rosavin Rf = 0.19, rosarin Rf = 0.26, gallic acid Rf = 0.58); Right: HPTLC results of standard compounds under UV 366 nm, after derivatisation with sulfuric acid (rosavin Rf = 0.19, rosarin Rf = 0.26, salidroside Rf = 0.31, gallic acid Rf = 0.58, tyrosol Rf = 0.76). Gallic acid shows good visibility under UV 254 nm, while it is not easily detected under UV 366 nm at a dark blue back-round. Tyrosol is visible in 254 nm but less clear in 366 nm. The raw plant material obtained from the market was also studied by our HPTLC method (list of samples in Supplement S4). Under UV 254 nm (Figure 7), there were two obvious bands among these samples (Rf = 0.27 and 0.48). However, due to lack of reference standards, their identity remains unknown. Further studies need to be conducted using NMR and LC-MS. The majority of the samples investigated contained concentrations of tyrosol similar to the standard raw material used (R24, R30, and R31). Samples R1–R6 contained lower levels of this compound possibly due to their longer storage time. Therefore, tyrosol could be considered as a marker to study duration of Rhodiola storage. It was also found that only five samples (R9, R25, R58, R59, and R24) contained high levels of rosavin, which turned out to be the ones from R. rosea. Moreover, this result can also be verified by the NMR results (Figure 5). However, it is not evident whether there is adulteration of R. sachalinensis in R. rosea since their metabolites are similar. Figure 7 HPTLC results for all Rhodiola market samples, mobile phase [Ethylacetate, methanol, water, formic acid (77:13:10:2)]. Under UV 366 nm after derivatisation eight samples (R1, R5, R6, R15, R27, R32, R61, and R64) had a low concentration of salidroside (Rf = 0.31). These samples could have been kept for a long time after collection and the salidroside content could have decreased due to lack of a good storage environment. Combining the results of HPTLC and 1H-NMR multivariate statistical analysis, we also analyzed the adulteration rate among all the market samples (Supplement, S4). Thirty percent of the Rhodiola samples collected from the market were not, as declared on the label, i.e., either R. rosea or R. crenulata. Some R. rosea samples were also being sold as R. crenulata. 47.7% of raw material samples were not labeled properly and their species information were not clearly illustrated to customers. This highlights a clear lack of proper local government policies and good quality control strategies. According to our study, different Rhodiola species (including R. rosea and R. crenulata) can be found in the Chinese market. However, they are neither sold separately nor well-identified. Therefore, there is a high potential of adulteration and substitution among these species. Qualitative and quantitative analysis of mixtures Since in the value chain, mixing of batches and, therefore, potentially also of species, is of major concern, the possibility of qualitatively and quantitatively detecting plant mixtures was also investigated. The additional species chosen for this study was R. crenulata which is considered to be the most common adulterant of R. rosea. The selected BRMs were weighed individually in different proportions and then added together in an Eppendorf reaction tube. The rest of the sample preparation was identical to the methodology for the 1H-NMR spectroscopy. The samples were renamed as seen in Table 4. After the acquisition of the spectra, they were baseline and phase corrected and zeroed to the TMS peak in Topspin 3.2. Table 4 1H-NMR-based detection of plant mixtures by. Sample name Mg of R. rosea BRM Mg of R. crenulata BRM RR100 100 00 RR80RC20 80 20 RR60RC40 60 40 RR40RC60 40 60 RR20RC80 20 80 RC100 00 100 In all samples, the salidroside peak intensity remains almost the same since this constituent is present in both species. The peaks of rosavin are gradually decreasing with the addition of R. crenulata, whereas the characteristic quartet at 6 ppm due to the presence of an unknown compound is increasing with the addition of R. crenulata and it is not detected in R. rosea at all (Figure 8). Figure 8 1H-NMR spectra of the whole region (left) and the aromatic region (right) of the R. rosea and R. crenulata mixtures. The acquired spectra were bucketed using Amix and only focused on this region (6 ppm). When the whole quartet was integrated into a single bucket, the observed increase of its intensity was not adequately represented. Therefore, the bucket size used changed to 0.002 ppm and only incorporated the first peak of the quartet (6.0028–6.0048 ppm). The buckets obtained from Amix were transferred into Excel, where the relationship between the bucket value and the percentage of R. crenulata in the mixture was expressed graphically as a calibration curve. The bucket value of the respective peak is increasing in a linear mode (Figure 9). Figure 9 Calibration curve showing the bucket value of the peak vs. the percentage of R. crenulata within a mixture of R. crenulata and R. rosea. Similar results can also be obtained with HPTLC analysis. The HPTLC fingerprints produced consist of the over-spotted BRM extracts in different volumes as seen in Table 5. The final volume applied was 5 μl. Table 5 Sample preparation for the detection of plant mixtures by HPTLC. R. rosea 100% RR100 R. rosea BRM 5 μL R. rosea 80% and R. crenulata 20% RR80 R. rosea BRM 4 μL R. crenulata BRM 1 μL R. rosea 60% and R. crenulata 40% RR60 R. rosea BRM 3 μL R. crenulata BRM 2 μL R. rosea 40% and R. crenulata 60% RR40 R. rosea BRM 2 μL R. crenulata BRM 3 μL R. rosea 20% and R. crenulata 80% RR20 R. rosea BRM 1 μL R. crenulata BRM 4 μL R. crenulata 100% RC100 R. crenulata BRM 5 μL As seen in Figure 10, when the loading volume of the R. rosea decreases, the representative markers of this species (rosavin and rosarin) decrease as well. However, the band for salidroside, (since it occurs in both species) remains almost the same. Figure 10 HPTLC fingerprints of all R. rosea and R. crenulata mixtures under UV 254 nm (tracks 1–6), white light and SAR (tracks 7–12), and UV 366 nm and SAR (tracks 13–18). By gradually increasing the R. crenulata proportion, several bands gradually appear above salidroside that could potentially be used as markers for the qualitative and semi-quantitative HPTLC analysis of mixtures of these two Rhodiola species. Further work needs to be carried out to determine the identity and species-specificity of these compounds. Conclusions This study provided a method for distinguishing five different species of Rhodiola and suggests possible methods for quantifying different species within mixtures. The metabolomic and phytochemical differences between these different species has been demonstrated through NMR spectroscopy and HPTLC analysis. Species represented with only a small number of samples will need further investigation in order to accurately define their chemical characteristics. There is a need to study the links between producers and consumers especially when in trans-national trade and re-enforce the hypothesis that poor quality and adulterated products can be products of poorly governed value chains, particularly at the early stages of supply. Moreover, through the establishment of well-controlled and well-managed value chains it is possible to better prevent accidental or deliberate contamination and adulteration from occurring. Author contributions AB, main author and collector of samples in China, assisted with multivariate analysis of NMR data, responsible for contribution toward discussions and conclusions. CG, Responsible for HPTLC analysis and writing the methods, results, and part of the discussion for HPTLC. LZ, Responsible for NMR analysis and writing of the methods, results and part of the discussion relating to NMR. SL, Responsible for authentication of Chinese Rhodiola specimens and DNA analysis shown in supplementary data. MH, Principle investigator and overall director of the project, played a major role in the writing of the introduction and conclusions. All authors proof read manuscript and made contributions to the final version. Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Important parts of this work have been kindly funded through a charitable donation by Dr. Willmar Schwabe GmbH & Co. KG, Germany. The authors proclaim no conflict of interest. LZ stay at the School of Pharmacy, UCL, UK was funded through an exchange agreement with Guangdong Pharmaceutical University, Guangzhou, PRC. We thank Lina Du, Yu Liao for collecting samples from Qinghai, Eric Brand, and Professor Zhongzhen Zhao (Hong Kong Baptist University) for supplying the samples from Hong Kong, Dr. José Vouillamoz (Agroscope Federal Research Institute, Switzerland) and Professor Alexander Shikov (Saint-Petersburg Institute of Pharmacy, Russia) for providing part of the plant material used in this research. We thank Amy Tso, Herbprime Co., Ltd, Mr. Chen, Sun Ten Co., Ltd, sourcing company, Taiwan and the Yi minority for their help in sourcing plant material on the Tibetan plateau. Supplementary material The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fphar.2016.00254 Click here for additional data file. Abbreviations TCMTraditional Chinese Medicine NMRNuclear magnetic resonance HPTLCHigh performance thin layer chromatography sppSpecies. ==== Refs References Alm T. (2004 ). Ethnobotany of Rhodiola rosea (Crassulaceae) in Norway . 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==== Front Front Plant SciFront Plant SciFront. Plant Sci.Frontiers in Plant Science1664-462XFrontiers Media S.A. 10.3389/fpls.2016.01269Plant ScienceMini ReviewConserved Roles of CrRLK1L Receptor-Like Kinases in Cell Expansion and Reproduction from Algae to Angiosperms Galindo-Trigo Sergio 1Gray Julie E. 2Smith Lisa M. 1*1Department of Animal and Plant Sciences, University of SheffieldSheffield, UK2Department of Molecular Biology and Biotechnology, University of SheffieldSheffield, UKEdited by: Elena M. Kramer, Harvard University, USA Reviewed by: Verónica S. Di Stilio, University of Washington, USA; Ive De Smet, Vlaams Instituut voor Biotechnologie and UGent, Belgium; Martin Parniske, Ludwig Maximilian University of Munich, Germany *Correspondence: Lisa M. Smith, Lisa.M.Smith@sheffield.ac.ukThis article was submitted to Plant Evolution and Development, a section of the journal Frontiers in Plant Science 29 8 2016 2016 7 126913 6 2016 10 8 2016 Copyright © 2016 Galindo-Trigo, Gray and Smith.2016Galindo-Trigo, Gray and SmithThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Receptor-like kinases (RLKs) are regulators of plant development through allowing cells to sense their extracellular environment. They facilitate detection of local endogenous signals, in addition to external biotic and abiotic stimuli. The Catharanthus roseus RLK1-like (CrRLK1L) protein kinase subfamily, which contains FERONIA, plays a central role in regulating fertilization and in cell expansion mechanisms such as cell elongation and tip growth, as well as having indirect links to plant–pathogen interactions. Several components of CrRLK1L signaling pathways have been identified, including an extracellular ligand, coreceptors, and downstream signaling elements. The presence and abundance of the CrRLK1L proteins in the plant kingdom suggest an origin within the Streptophyta lineage, with a notable increase in prevalence in the seeded land plants. Given the function of the sole CrRLK1L protein in a charophycean alga, the possibility of a conserved role in detection and/or regulation of cell wall integrity throughout the Strephtophytes is discussed. Orthologs of signaling pathway components are also present in extant representatives of non-vascular land plants and early vascular land plants including the liverwort Marchantia polymorpha, the moss Physcomitrella patens and the lycophyte Selaginella moellendorffii. Deciphering the roles in development of the CrRLK1L protein kinases in early diverging land plants will provide insights into their ancestral function, furthering our understanding of this diversified subfamily of receptors in higher plants. CrRLK1Lsignaling pathwaycell expansionfunctional conservationkinaseStreptophytaBiotechnology and Biological Sciences Research Council10.13039/501100000268BB/N004167/1 ==== Body Receptor-Like Kinases (RLKs) in Plants Plant cells sense their extracellular environment and moderate their developmental programs accordingly. Extracellular signals range from tissue-specific cues to indicators of abiotic and biotic environmental conditions such as drought or disease. A critical component of environment-sensing is plasma membrane-localized receptors. Receptor-like kinases (RLKs) are among the most expanded protein families in plants, comprising ∼600 members in Arabidopsis thaliana (Shiu and Bleecker, 2001). A small proportion of plant RLKs have been characterized, with comprehensive functional descriptions for relatively few members, including BRASSINOSTEROID-INSENSITIVE 1, CLAVATA1 and FLAG-ELLIN-SENSITIVE 2 (Wang et al., 2001; Chinchilla et al., 2006; DeYoung et al., 2006). Understanding RLKs is critical as they regulate many aspects of plant function from development to stress responses. Receptor-like kinases have a modular organization consisting of an amino-terminal extracellular domain (ECD), a transmembrane (TM) domain, and an intracellular kinase domain (Walker, 1994). Classification by their variable ECDs defines 15 RLK subfamilies including the leucine-rich repeat (LRR) and Catharanthus roseus RLK 1-like (CrRLK1L) subfamilies (Shiu and Bleecker, 2003). The RLKs share a common mechanism for signal perception and transmission. Firstly, the ECD recognizes a specific ligand, the biochemical nature of which depends on the ECD, with ligand binding inducing receptor-coreceptor dimerization. This intermolecular interaction promotes signal transduction through conformational change, leading to kinase domain activation via auto- or trans-phosphorylation (Afzal et al., 2008). Finally, phosphorylation of downstream pathway components results in signal transmission and activation of adaptive responses to the extracellular stimulus. The Catharanthus roseus RLK1-Like (CrRLK1L) Subfamily Named after Catharanthus roseus, the species in which its first member (CrRLK1) was identified (Schulze-Muth et al., 1996), the CrRLK1L subfamily has received increasing attention over the past decade. Members of the CrRLK1L subfamily have two ECD regions with similarity to the putative carbohydrate-binding malectin domain (MD). Malectin, first described in Schallus et al. (2008) in Xenopus laevis, acts in the endoplasmic reticulum N-glycosylation surveillance system. The two tandem MDs are annotated as a malectin-like domain (MLD) (Figure 1A), a conformation specific to algae and plants. Within the RLK family, the MLD comprises the main ECD in the CrRLK1L, LRK10L-2, LRR-1c, and LRR-1a subfamilies. The MLD is classified as a rapidly evolving domain in the CrRLK1L subfamily (Escobar-Restrepo et al., 2007). Although, MLDs are highly divergent in primary sequence, suggesting functional divergence, conservation of predicted secondary structures allows inference of a role in carbohydrate binding (Boisson-Dernier et al., 2011). However, while the structure of malectin is available (Schallus et al., 2008, 2010), no three-dimensional structure of the MLD has been resolved, preventing a more definitive comparison. FIGURE 1 (A) Domain organization of the CrRLK1L proteins. Domains are to scale and based on FER as given in the NCBI Conserved Domain Database. SP, signal peptide; MD, malectin domain; TM, transmembrane domain. (B) Overview of CrRLK1L protein kinase signaling pathway components as described in the main text. RALF, rapid alkalinization factor; LRE/LLG, LORELEI/LRE-like GPI-AP; ROPGEF, RAC/ROP guanine exchange factor; ROP/RAC, Rho of plants/RAC GTPase; SAM1, 2, S-adenosylmethionine synthetase 1, 2; NADPHox, NADPH oxidase; ROS, reactive oxygen species. Speculative steps are represented by dashed lines. Cellular Functions of CrRLK1L Receptor-Like Kinases Since Escobar-Restrepo et al. (2007) discovered a role for FERONIA (FER, AT3G51550) in pollen tube (PT) reception at the female gametophyte, the CrRLK1L subfamily has been linked to an increasing number of biological processes. Generally regarded as cell wall stability sensors, CrRLK1L proteins have been implicated in cell elongation and cell shape, polarized growth, and plant–pathogen interactions (reviewed in Boisson-Dernier et al., 2011; Cheung and Wu, 2011; Lindner et al., 2012; with a summarizing table of CrRLK1L functions in Nissen et al., 2016). Cell Wall Integrity Genetics provided the first evidence of CrRLK1L surveillance of cell wall stability (Hematy et al., 2007), with mutations in THESEUS1 (THE1, AT5G54380) partially rescuing the hypocotyl growth defect of cellulose deficient cesA6 (cellulose synthase A, catalytic subunit 6) mutants without influencing the cellulose deficiency (Hematy et al., 2007). THE1 activation may be cell wall perturbation-dependent, leading to reinforcement by ectopic lignification, and preventing excessive loosening and consequent damage to the cell wall. Cell Elongation Cell elongation during vegetative growth involves several CrRLK1L subfamily members. HERCULES RECEPTOR KINASES 1 and 2 (HERK1 and HERK2, AT3G46290 and AT1G30570), THE1, FER, CURVY1 (CVY1, AT2G39360) and uncharacterized CrRLK1L AT5G24010 are among the most up-regulated genes upon brassinosteroid (BR) treatment (Guo et al., 2009a,b). BR functions in cell elongation and Guo et al. (2009a,b) described reduced hypocotyl and petiole cell elongation in herk1the1 and fer mutants, and a stronger phenotype in the herk1herk2the1 triple mutant. BR treatment further reduces hypocotyl length in these lines, indicating redundancy between cell elongation pathways (Guo et al., 2009a). Balancing of cell wall rigidification/loosening has been proposed as the underlying regulatory mechanism for CrRLK1L-mediated cell elongation (Hematy et al., 2007). Cell wall-loosening enzymes, such as expansin and pectin lyase-like genes, are down-regulated in the herk1the1 double mutant, which could promote cell wall stiffening and reduced cell elongation (Guo et al., 2009a; Boisson-Dernier et al., 2011). In contrast, expansin and pectin lyase-like gene expression is up-regulated in the1 mutants in a cesA6 background, suggesting context-dependent function. Tip Growth in Root Hairs, Pollen Tubes, and Trichomes Tip growth represents an extreme form of polarized cell expansion that occurs during development of root hairs, PTs, and trichomes (Yang, 1998). For tip growth to occur, various mechanisms must be spatiotemporally coordinated, including polarized exocytosis of vesicles, cytoskeletal reorganization, and generation of second messenger gradients (Lee and Yang, 2008). Many key genetic and exogenous factors have been described (Li et al., 1999; Baluska et al., 2000; Rigas et al., 2001; Foreman et al., 2003; Bloch et al., 2011). Genetic factors include five CrRLK1L proteins: FER, ANXUR1 (ANX1, AT3G04690), ANX2 (AT5G28680), CVY1 and [Ca2+]cyt-associated protein kinase 1/ERULUS (CAP1/ERU, AT5G61350). Although, described in different structures (FER and CVY1 in root hairs; ANX1 and ANX2 in PTs), the former four proteins are localized to the plasma membrane of the growing tip where they increase reactive oxygen species (ROS) production by a common mechanism (Duan et al., 2010; Gachomo et al., 2014). Both fer and cvy1 mutants have defects in other tip growth-requiring structures such as the leaf trichomes (Duan et al., 2010; Gachomo et al., 2014). In contrast, CAP1 is localized to the tonoplast in root hairs where it activates nitrogen permease in response to ammonium/nitrogen levels and facilitates accumulation of Ca2+ (Bai et al., 2014), demonstrating the potential for CrRLK1L proteins to function in a wider range of cellular processes. Pollen Tube-Female Gametophyte Interactions During fertilization in angiosperms, the PT grows through the pistil, transporting two sperm cells. At the female gametophyte synergid cells (SCs), the PT bursts to release the sperm cells (Dresselhaus and Franklin-Tong, 2013). Three CrRLK1L subfamily members regulate this process in Arabidopsis: FER, ANX1, and ANX2. Maternal fer mutants fail to induce PT rupture and growth arrest (Escobar-Restrepo et al., 2007). FER is asymmetrically localized to the filiform apparatus in the SCs, and regulates a local increase in ROS (Escobar-Restrepo et al., 2007). Exogenous quenching of ROS in the SC results in a fer-like PT overgrowth phenotype, implying FER-triggered, ROS-mediated, PT growth arrest (Duan et al., 2014). Domain swaps between FER and closely related CrRLK1Ls indicate that, while the FER ECD is needed to complement reproductive PT reception defects in fer mutants, the kinase domain can be replaced with that of related CrRLK1Ls (Kessler et al., 2015). Furthermore, FER kinase activity is not required, suggesting that co-receptors may compensate or provide signal transduction capacity (Kessler et al., 2015). The two closest homologs to FER, ANX1 and ANX2, act redundantly as its male equivalents. While FER is expressed in all tissues except for mature pollen, ANX1 and ANX2 localization is restricted to the PT tip (Boisson-Dernier et al., 2009). Both ANX proteins are proposed to sense cell wall integrity and maintain its stability at the PT tip (Boisson-Dernier et al., 2009, 2011; Miyazaki et al., 2009). ROS production and a Ca2+ gradient are required downstream of ANX1 and ANX2 in polarized PT growth (Boisson-Dernier et al., 2013). Biotic and Abiotic Responses The CrRLK1L subfamily is linked to plant–pathogen interactions, with multiple CrRLK1L genes up-regulated upon elicitor treatments including bacterial flagellin (epitope flg22) and fungal chitin (Lindner et al., 2012). FER becomes phosphorylated upon treatment with flg22 and is speculated to act as a coreceptor with FLS2 (Keinath et al., 2010). FER triggers an intracellular ROS burst in Arabidopsis suspension cells in response to flg22 (Keinath et al., 2010). Kessler et al. (2010) identified FER as a pathogen resistance determinant in powdery mildew infections. They described the similarity between PT reception at the SCs and the first steps in infection, identifying shared molecular components (Kessler et al., 2010). In addition, a member of the mildew resistance locus O (MLO) family, NORTIA (NTA, AT2G17430), re-locates to the filiform apparatus upon PT reception at the SCs in a FER-dependent fashion and is necessary for correct PT response (Kessler et al., 2010). Together these results shed new light on plant–pathogen interactions. Additionally, CrRLK1L genes respond to a variety of stress and hormone treatments. From genome-wide expression data, Lindner et al. (2012) summarized the general down-regulation of CrRLK1L subfamily expression in response to abiotic stresses such as heat, drought, high osmolarity, cold and hypoxia. Since CrRLK1Ls are linked to ROS production, down-regulation could indicate a strategy to minimize the deleterious oxidative burst that is common to abiotic stress responses. Hormone treatments such as BRs also increase transcript levels of a subset of CrRLK1Ls (Guo et al., 2009a,b). CrRLK1L Signaling Pathways A number of proteins have been identified as either direct interaction partners of CrRLK1L kinases, or as phosphorylated/activated upon CrRLK1L signaling. Although our understanding is incomplete, conserved signaling mechanisms are emerging. CrRLK1L signaling generally involves activation of downstream elements through phosphorylation of guanine exchange factors, activation of plasma membrane NADPH oxidase-dependent ROS production and calcium ion fluctuations (Figure 1B). Upstream Effectors Perhaps the most striking recent discovery is identification of a ligand for FER. A phosphoproteomic screen of seedlings exposed to the peptide rapid alkalinization factor 1 (RALF1, AT1G02900) identified FER as its receptor (Haruta et al., 2014). Additionally, putative downstream elements of RALF1-FER intracellular signaling were identified, including a plasma membrane H+-ATPase that increases apoplastic pH and cell wall rigidity upon activation (Figure 1B). Surprisingly, as MDs were anticipated to bind carbohydrate ligands, RALF1 is a small secreted peptide that lacks N-glycosylation (Haruta et al., 2014). The RALF family comprises 34 members in Arabidopsis. The ubiquitous, pollen-specific or SC-specific expression patterns of some RALF-encoding genes correlate with those of CrRLK1L genes, leading to the hypothesis that additional RALF peptides may be ligands for CrRLK1Ls in different processes or developmental stages (Murphy and De Smet, 2014; Wolf and Hofte, 2014). For example, two RALF peptides, SlRALF in tomato and AtRALF4 in Arabidopsis, regulate PT elongation (Covey et al., 2010; Morato do Canto et al., 2014). Further evidence supporting a link between the CrRLK1L and RALF families was provided when Srivastava et al. (2009) described down-regulation of AtRALF23 upon BR treatment. AtRALF23 appears to counteract BR effects on cell growth and elongation (Srivastava et al., 2009), a process in which HERK1, HERK2, FER and THE1 participate (Guo et al., 2009a,b). Additionally, RALF1 suppresses BR effects on root cell elongation by inducing several BR-down-regulated genes involved in BR biosynthesis (Bergonci et al., 2014). Although unexpected, the discovery of a peptide ligand for FER does not exclude carbohydrate binding by CrRLK1L proteins. As suggested by Wolf and Hofte (2014), the RALF1-CrRLK1L interaction may not involve the MLD, and each CrRLK1L kinase may have multiple biochemically diverse ligands, a property described for animal receptors such as the epidermal growth factor receptor and beta-adrenergic receptor kinase (Touhara, 1997; Moghal and Sternberg, 1999). Coreceptors and Chaperones Other RLKs such as FLS2 and CrRLK1L subfamily member HERK1 may act as coreceptors with FER (Guo et al., 2009a; Keinath et al., 2010), however, only two glycosylphosphatidylinositol-anchored proteins (GPI-APs) have been biochemically confirmed to interact with FER and mediate downstream signaling (Li et al., 2015; Liu et al., 2016). LORELEI (LRE) and LRE-like GPI-AP1 (LLG1) bind the extracellular juxtamembrane domain of FER and, in different developmental contexts, are essential for deposition and stability of FER as well as effective RALF1-FER signaling (Li et al., 2015). Considering that there are two further LRE homologs in Arabidopsis (LLG2 and LLG3) with different expression patterns (Capron et al., 2008; Tsukamoto et al., 2010), we speculate that different LLGs could act as chaperones and co-receptors for different CrRLK1L proteins in diverse developmental contexts. Downstream Signaling Pathways Downstream components of CrRLK1L signaling have been identified, including RAC/ROP guanine exchange factors (ROPGEFs), a plant-specific subfamily of RHO-GTPases that convert GDP into GTP and activate RAC/ROPs (Carol et al., 2005). Two studies have identified five ROPGEFs that interact with the kinase domain of FER, and two RAC/ROPs as potential downstream components (Duan et al., 2010; Yu et al., 2012). One RAC/ROP (ROP11; AT5G62880) phosphorylates and inactivates PP2C phosphatase ABI2, an integral element in abscisic acid (ABA) signaling (Nishimura et al., 2010; Yu et al., 2012). Adding further complexity, the stunted phenotype of fer-4 mutants has been linked to the ethylene biosynthetic pathway through characterization of S-adenosylmethionine synthetase 1 and 2 (SAM1 and SAM2) as direct interactors of the FER kinase domain (Mao et al., 2015). Mao et al. (2015) propose a scenario in which FER inhibits SAM1 and SAM2 through phosphorylation, suppressing S-adenosylmethionine production and down-regulating ethylene biosynthesis. Together, these findings depict the first steps in a branched CrRLK1L pathway, where a single receptor can interact with multiple partners to activate different downstream elements (Figure 1B). Crosstalk of CrRLK1L signaling with the hormones ABA, ethylene, and BR is also likely. As previously discussed, increased ROS production occurs in various CrRLK1L-mediated processes. NADPH oxidases are required for the CrRLK1L-mediated ROS burst during tip growth in root hairs or PTs, and PT reception at the SCs (Lee and Yang, 2008; Boisson-Dernier et al., 2013). Interestingly, studies in rice identified a NADPH oxidase as a direct interactor of a RAC/ROP protein (Wong et al., 2007). This connection has recently been confirmed in Arabidopsis by characterizing ROP11 as an interaction partner and activator of the NADPH oxidase Rboh F in root hairs (Yan et al., 2016). Finally, NTA and the receptor-like cytoplasmic kinase MARIS also act downstream in this signaling cascade (Kessler et al., 2010; Boisson-Dernier et al., 2015), although the exact mechanisms are yet to be elucidated. The question of whether CrRLK1L pathways indirectly modify gene expression by post-translational modification of transcription factors remains to be addressed. An Evolutionary Perspective of the CrRLK1L Subfamily Despite recent advances, only four studies focus on CrRLK1L protein function in species other than Arabidopsis. Beyond the initial identification of CrRLK1 (Schulze-Muth et al., 1996), phylogenomics identified 16 CrRLK1L homologs with Gigantea-mediated circadian regulation of their expression in Oryza sativa (Nguyen et al., 2014). Secondly, Niu et al. (2016) described ∼40 CrRLK1L family members in diploid cotton species, six of which are linked to fiber development. Finally, a closely related protein, CpRLK1, was functionally characterized in a charophycean unicellular alga (Hirano et al., 2015). CpRLK1 is expressed during mating in this heterothallic alga and regulates gamete formation. Hirano’s study is of particular evolutionary interest since comparison of CrRLK1L function between algae and angiosperms demonstrates potentially analogous involvement in reproduction, cell growth, and cell wall stability sensing. CpRLK1 functions in the release of +-type gametes that lack a cell wall, with +-type cells of CpRLK1 knockdown lines generating an abnormally enlarged conjugation papilla but not releasing the gametal protoplast (Hirano et al., 2015). CpRLK1 is suggested to detect cell wall integrity during conjugation to regulate the release of +-type cells, evoking similarities with regulation of PT function by CrRLK1L proteins in angiosperms (Wolf and Hofte, 2014). Similarly, partial protoplast protrusion in Closterium CpRLK1 knockdown lines resembles cellular material discharge in impaired root hair development phenotypes of fer-4 mutants, supporting a possible ancestral role in cell wall integrity surveillance (Li et al., 2015). Plant RLKs may have originated in the Streptophyta lineage, which is ancestral to both land plants and charophycean algae (Sasaki et al., 2007). Although, limited by a paucity of algal genomes or transcriptomes, CpRLK1 identification in a charophycean alga substantiates divergence of RLK subfamilies and emergence of the CrRLK1L subfamily early in the evolution of Streptophytes (Shiu and Bleecker, 2001; Sasaki et al., 2007). Conservation of cell wall synthesizing enzymes (rosette cellulose, arabinogalactan, and hemicellulose synthases) across the Streptophyta lineage supports the hypothesis of evolutionary conservation of CrRLK1L cell wall surveillance functions (Sorensen et al., 2010; Popper et al., 2011; Fangel et al., 2012). There has been an expansion of the CrRLK1L subfamily from early diverging to recent Streptophyta lineages, with the greatest increase in prevalence evident between seedless vascular plants and seed plants (Figure 2A). Expansion of the subfamily may have allowed acquisition of new roles in reproductive processes such as PT growth and its recognition at the female gametophyte (see Pollen Tube-Female Gametophyte Interactions), developmental innovations characteristic of the seed plants (Doyle, 2006; Linkies et al., 2010). An extant member of the earliest divergent angiosperm lineage, Amborella trichopoda (Albert et al., 2013) has orthologs of most characterized A. thaliana CrRLK1L proteins (Figure 2B). The conifer Picea abies (Nystedt et al., 2013) has orthologs of the reproductive clade (FER, ANX1, and ANX2) and some non-reproductive members (THE1 and HERK2; Figure 2B). FER and ANX orthologs in A. trichopoda and P. abies suggest a potentially conserved role of these CrRLK1Ls in mediating PT growth and fertilization (see Pollen Tube-Female Gametophyte Interactions). Fertilization in ferns and some gymnosperms (e.g., cycads and ginkgo) depends on multiflagellated sperm rather than on siphonogamous PT transport of non-motile sperm cells, which arose independently in angiosperms and gymnosperms (Doyle, 2006). Given available data, it is impossible to speculate whether the reproductive clade formed independently when siphonogamy established in conifers and angiosperms or was already present before divergence of these lineages of land plants. FIGURE 2 (A) Occurrence of CrRLK1L signaling elements across plant lineages. Data is shown for Chlorophyta species Ostreococcus lucimarinus, Chlamydomonas reinhardtii, Volvox carteri, Coccomyxa subellipsoidea, Micromonas pusilla, and Micromonas sp. (Merchant et al., 2007; Palenik et al., 2007; Worden et al., 2009; Prochnik et al., 2010; Blanc et al., 2012); Charophyta Closterium peracerosum-strigosum-littorale complex (Hirano et al., 2015); the liverwort Marchantia polymorpha (Sasaki et al., 2007; These sequence data were produced by the US Department of Energy Joint Genome Institute http://www.jgi.doe.gov/ in collaboration with the user community); the model bryophyte Physcomitrella patens (Rensing et al., 2008; Lehti-Shiu and Shiu, 2012); lycophyte Selaginella moellendorffii (Banks et al., 2011; Lehti-Shiu and Shiu, 2012); conifer Picea abies (Nystedt et al., 2013); single living sister species to all other angiosperms Amborella trichopoda (Albert et al., 2013) and Arabidopsis thaliana (Hematy and Hofte, 2008). According to the GenBank, Phytozome and ConGenIE databases, the CrRLK1L subfamily is only present in Streptophyta, increasing in number with developmental complexity. Other signaling elements present similar trends in numbers: RALF peptides, ROPs and membrane bound NADPH oxidases are absent in Chlorophyta and present in land plants (Berken et al., 2005; Torres et al., 2006; Eklund et al., 2010; Cao and Shi, 2012). ROPGEFs are present in land plants and Chlorophyta, although their presence in the latter is restricted to Ostreococcus lucimarinus (∗). Full genomic or transcriptomic records are not available for charophyte and fern lineages included in this figure (ND). (B) Comparative phylogenetic analysis of the CrRLK1L proteins in plants. Sequences were aligned using ClustalX (Larkin et al., 2007). The evolutionary history was inferred using a Neighbor-Joining phylogenetic tree generated with the software MEGA5.2 (Saitou and Nei, 1987; Tamura et al., 2011). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to each branch. Putative CrRLK1L members in A. thaliana (AT4G39110, AT2G21480, AT5G61350|CAP1|ERU, AT5G54380|THE1, AT2G23200, AT5G24010, AT2G39360|CVY1, AT3g46290|HERK1, AT5g59700, AT1g30570|HERK2, AT3G51550|FER, AT3G04690|ANX1, AT5G28680|ANX2, AT5G39000, AT5G38990, AT5G39020, AT5G39030), A. trichopoda (AMBOCrRLK1L1-9; Phyotzome v11 IDs: evm_27.TU.AmTr_v1.0_scaffold – 00077.136, 00016.362, 00003.355, 00092.136, 00045.60, 00080.56, 00024.19, 00001.334, 00001.335), P. abies (PICEACrRLK1L1-7; ConGenIE IDs: MA_71280g0010, MA_21765g0010, MA_10432359g0010, MA_5246g0010, MA_44655g0010, MA_45223g0010, MA_10432359g0020), S. moellendorffii (Smo40963, Smo76106), P. patens (Phpat.020G063800, Phpat.020G062600, Phpat.009G042500, Phpat.009G042200, Phpat.009G042300), M. polymorpha (BAF79940|MpRLK7), and Closterium peracerosum-strigosum-littorale Complex (AB920609|CpRLK1) are shown. Lotus japonicus MLD-LRR-containing RLK SYMRK (LOC553849|LjaSYMRK) and its closest MLD-LRR-RLK homolog in A. thaliana (AT1G67720) were used as an outgroup together with ERECTA from A. thaliana (AT2G26330|ERECTA). Although Selaginella moellendorffii is a vascular plant, the composition of its CrRLK1L family closely resembles that of non-vascular plants: CrRLK1L subfamily members from the liverwort Marchantia polymorpha, the moss Physcomitrella patens, and the lycophyte S. moellendorffii form a sister clade to non-reproductive CrRLK1L proteins (THE1, CVY1, HERK1, and CAP1; see Cell Wall Integrity, Cell Elongation, Tip Growth in Root Hairs, Pollen Tubes, and Trichomes). Although further research is required to confirm protein function, the phylogeny suggests a potentially conserved function in cell wall stability and regulation of cell elongation across land plants that was recruited in seed plant specific processes as the family expanded. Algal CpRLK1 does not cluster with the early divergent Streptophyta sequences, but is positioned between the CrRLK1L subfamily and the closely related MLD-LRR-RLK subfamily, of which Lotus japonicus SYMRK is best characterized (Antolin-Llovera et al., 2014; Hirano et al., 2015). This may be due to additional extracellular sequences in both CpRLK1 and MLD-LRR-RLKs, which are not present in other CrRLK1L proteins. Ongoing genomic and transcriptomic projects will facilitate more comprehensive phylogenetic studies of RLKs in early diverging Streptophyte lineages and ferns. Phylogenies along with functional studies from diverse species will help us decipher (i) the importance of divergence between CpRLK1 and other CrRLK1Ls, (ii) the evolutionary history of the expansion of this subfamily of RLKs, and (iii) whether the reproductive clade of CrRLK1Ls is specific to seed plants or appeared earlier in the evolution of plants, i.e. in ferns. Interestingly, CrRLK1L signaling may be conserved from bryophytes to angiosperms (Figure 2A). Downstream signaling components for polarized cell expansion and tip growth mechanisms that are CrRLK1L-mediated are present in liverworts, bryophytes, lycophytes, gymnosperms and angiosperms, including ROPGEFs, RAC/ROPs and NADPH oxidases. Homologs of putative ligands in the form of RALF peptides are also present in these species (Cao and Shi, 2012; Albert et al., 2013; Nystedt et al., 2013). Furthermore, ROPGEFs and RAC/ROPs have conserved functions in cell polarity and tip growth regulation in the filamentous gametophytic (protonemal) stage of P. patens (Ito et al., 2014). In this light, it is plausible that CrRLK1L proteins mediate tip growth from bryophytes to angiosperms via conserved CrRLK1L-ROPGEF-RAC/ROP signaling pathways. A more detailed analysis of when signaling pathway components arose relative to CrRLK1L kinases is precluded by a lack of genomic data from early diverging Streptophyta lineages (excepting the identification of ROPGEF proteins in some Chlorophyta species), however, all signaling components were apparently present in early non-vascular land plants (Figure 2A). As a final example of how CrRLK1L function and signaling may be conserved across plant evolution, let us consider CAP1 function in root development. Rhizoids are tip-growing root-like structures providing anchorage and water and nutrient uptake for some charophytes and land plants with a free-living gametophytic generation (liverworts, mosses, hornworts, lycophytes, and ferns; Jones and Dolan, 2012). Rhizoids and root hair development share a regulatory transcription network in which transcription factor ROOTHAIR DEFECTIVE 6 (RHD6) family has a central function. RHD6 transcription factors drive expression of root hair-specific genes, including genes required for cell expansion, and are necessary and sufficient for root hair and rhizoid development in angiosperms, liverworts and mosses (Masucci and Schiefelbein, 1994; Menand et al., 2007; Yi et al., 2010). Interestingly, expression of CAP1 (see Tip Growth in Root Hairs, Pollen Tubes, and Trichomes) is positively regulated by RHD6 and RHD6-LIKE 4 (RSL4) in Arabidopsis, suggesting that CAP1 acts downstream of RHD6 and RSL4 in root hair growth (Menand et al., 2007; Bruex et al., 2012). Given functional conservation of RHD6 across distant land plant lineages, we can hypothesize that a similar regulatory network may occur in early diverging land plants, in which RHD6/RSL4 orthologs transcriptionally regulate CrRLK1Ls during tip growth of rhizoids. Future Directions CrRLK1L kinases are receiving increasing attention for their roles in regulating developmental and stress responses. A number of questions remain to be addressed: (i) cross-talk with hormonal pathways, (ii) putative carbohydrate-binding capacity via the MLD, and (iii) the ligand-receptor relationships with different RALF peptides. Functional conservation across plant lineages remains speculative, with further research in early diverging lineages key to inferring primordial function, and understanding conserved mechanisms shared by functionally diverse CrRLK1L proteins in higher plants. Author Contributions SG-T, JG, and LS conceived and designed the review. SG-T wrote the manuscript draft and all authors edited, read and approved the final manuscript. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. LS and SG-T are supported by the Department of Animal and Plant Sciences, University of Sheffield. Research in JG’s lab is supported by RCUK grant BB/N004167/1. Thanks are due to Andrew Fleming for discussion of the manuscript. We apologize to colleagues whose work we did not cite or discuss in depth due to the format length restriction. We also thank John Bowman (Monash) for granting us permission to use unpublished data from the Marchantia genome. ABBREVIATIONS ABAabscisic acid ANX1ANXUR1 ANX2ANXUR2 BRbrassinosteroid CAP1/ERU[Ca2+]cyt-associated protein kinase 1/ERULUS CpRLK1Closterium peracerosum-strigosum-littorale Complex RLK 1 CrRLK1LCatharanthus roseus RLK 1-like CVY1CURVY1 ECDextracellular domain FERFERONIA flg22flagellin 22 (epitope of FLAGELLIN) GPI-APglycosylphosphatidylinositol-anchored protein HERK1HERCULES Receptor Kinase 1 HERK2HERCULES Receptor Kinase 2 LLGLRE-like GPI-AP LRELORELEI LRRleucine-rich repeat MDmalectin domain MLDmalectin-like domain MLOmildew resistance locus O NTANORTIA PAMPpathogen-associated molecular pattern PMpowdery mildew PTpollen tube RALFrapid alkalinization factor RLKreceptor-like kinase ROPGEFRAC/ROP guanine exchange factor ROSreactive oxygen species SAM1S-adenosylmethionine synthetase 1 SAM2S-adenosylmethionine synthetase 2 SCsynergid cells THE1THESEUS1 TMtransmembrane domain. ==== Refs References Afzal A. J. Wood A. J. Lightfoot D. A. (2008 ). 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PMC005xxxxxx/PMC5002435.txt

==== Front Front MicrobiolFront MicrobiolFront. Microbiol.Frontiers in Microbiology1664-302XFrontiers Media S.A. 10.3389/fmicb.2016.01350MicrobiologyOriginal ResearchSlr1670 from Synechocystis sp. PCC 6803 Is Required for the Re-assimilation of the Osmolyte Glucosylglycerol Savakis Philipp 1†Tan Xiaoming 2Qiao Cuncun 2Song Kuo 2Lu Xuefeng 2Hellingwerf Klaas J. 1Branco dos Santos Filipe 1*1Molecular Microbial Physiology Group, Swammerdam Institute for Life Sciences, University of AmsterdamAmsterdam, Netherlands2Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology – Chinese Academy of SciencesQingdao, ChinaEdited by: Wendy Schluchter, University of New Orleans, USA Reviewed by: Qingfang He, University of Arkansas at Little Rock, USA; Michael Summers, California State University, Northridge, USA; Peter Lindblad, Uppsala University, Sweden *Correspondence: Filipe Branco dos Santos, f.brancodossantos@uva.nl†Present address: Philipp Savakis, Systems Bioinformatics, Vrije Universiteit Amsterdam, Amsterdam, Netherlands This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology 29 8 2016 2016 7 135013 7 2016 16 8 2016 Copyright © 2016 Savakis, Tan, Qiao, Song, Lu, Hellingwerf and Branco dos Santos.2016Savakis, Tan, Qiao, Song, Lu, Hellingwerf and Branco dos SantosThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.When subjected to mild salt stress, the cyanobacterium Synechocystis sp. PCC 6803 produces small amounts of glycerol through an as of yet unidentified pathway. Here, we show that this glycerol is a degradation product of the main osmolyte of this organism, glucosylglycerol (GG). Inactivation of ggpS, encoding the first step of GG-synthesis, abolished de novo synthesis of glycerol, while the ability to hydrolyze exogenously supplied glucoslylglycerol was unimpaired. Inactivation of glpK, encoding glycerol kinase, had no effect on glycerol synthesis. Inactivation of slr1670, encoding a GHL5-type putative glycoside hydrolase, abolished de novo synthesis of glycerol, as well as hydrolysis of GG, and led to increased intracellular concentrations of this osmolyte. Slr1670 therefore presumably displays GG hydrolase activity. A gene homologous to the one encoded by slr1670 occurs in a wide range of cyanobacteria, proteobacteria, and archaea. In cyanobacteria, it co-occurs with genes involved in GG-synthesis. Slr1670cyanobacteriaSynechocystisosmolyteglucosylglycerolsalt stressNederlandse Organisatie voor Wetenschappelijk Onderzoek10.13039/501100003246VENI grant 863.11.019 ==== Body Introduction Upon increases in extracellular osmolarity, many bacteria synthesize small organic molecules that raise the intracellular osmotic pressure. In cyanobacteria, the nature of the osmolyte correlates with the host’s osmotolerance: strains with a low salt tolerance produce sucrose; moderately halotolerant strains utilize glucosylglycerol (GG); and highly tolerant strains use glycine betaine (Hagemann, 2011). The moderately halotolerant cyanobacterium Synechocystis sp. PCC 6803 (hereafter: Synechocystis) uses GG as its primary osmolyte (Richardson et al., 1983), but is also capable of salt-induced sucrose synthesis. GG is synthesized via a two-step pathway from central metabolites (Figure 1): in the first step, glucosylglycerol phosphate is synthesized from ADP-glucose and glycerol-3-phosphate, in a condensation reaction catalyzed by glucosylglycerol phosphate synthase (GgpS). Cleavage of the phosphate moiety is subsequently accomplished by glucosylglycerol phosphate phosphatase (GgpP). Synechocystis harbors a transporter that is used for reuptake of GG that is lost due to leakage of this osmolyte from the cytoplasm (Hagemann et al., 1997). FIGURE 1 (A) Proposed overview of Glycerol and glucosylglycerol (GG) metabolism. (B) Genes involved in GG synthesis and mutant strains used in this study. Arrows represent the direction of transcription of the respective open reading frame. Another osmolyte that is frequently used by bacteria such as Escherichia coli is the disaccharide trehalose (Welsh et al., 1991). Although incapable of its synthesis, Synechocystis can take up exogenously supplied trehalose and use it as an osmoprotectant (Mikkat et al., 1997). In agreement with this it is observed that in cells to which trehalose has been added, the total concentration of GG decreases over time, suggesting that this latter molecule can be catabolized (Mikkat et al., 1997). Some marine cyanobacteria have been demonstrated to be able to ferment a part of their osmolytes. Thus, Microcoleus chthonoplastes ferments some of its GG in the dark, re-assimilating the glucose part, while the glycerol part is excreted, or lost through leakage through the cytoplasmic membrane (Stal and Moezelaar, 1997). Yet, a metabolic pathway for GG re-assimilation has so far remained elusive in cyanobacteria (Pade and Hagemann, 2014), although recently, a dedicated GG phosphorylase was discovered in Bacillus selenitireducens (Nihira et al., 2014). We reported elsewhere that under mild salt stress, wild-type Synechocystis cells synthesize small quantities of glycerol (Savakis et al., 2015). Here, we provide evidence supporting the notion that GG is the source of this glycerol under salt stress. In addition, we demonstrate that a previously unassigned protein, Slr1670, is directly involved in (and required for) GG re-assimilation. Materials and Methods Chemicals were purchased from Sigma-Aldrich, unless stated otherwise. Glucosylglycerol (GG, 51% aqueous solution) was purchased from Bitop (Germany). Culturing Conditions Escherichia coli was grown in LB medium at 37°C and shaking at 200 rpm. Selection of transformants was carried out at 37°C on LB medium solidified with 1.5% (w/v) agar. Where appropriate, ampicillin, kanamycin and chloramphenicol were added at 100, 50, and 35 μg/mL, respectively. For batch experiments, Synechocystis cells were grown in a shaking incubator (Innova 43, New Brunswick Scientific, 120 rpm, 30°C) under fluorescent white light (15 W cool fluorescent white light, F15T8-PL/AQ, General electric, incident light intensity 30–40 μE/m2/s) in BG11 medium, supplemented with 10 mM TES/KOH and adjusted to an initial pH of 8.0. Cells were inoculated to an OD730 of 0.1 from a pre-culture. Where indicated, NaCl was added to a concentration of 200 mM to non-adapted cells. For salt tolerance experiments, cells were grown in transparent 96-well plates [Greiner bio-one cellstar, F-bottom with breathe easy seals (Diversified Biotech)] under white light (GE PL/AQ F15T8) at 30°C and shaking at 700 rpm in BG11 medium, buffered to an initial pH = 8.0 with 10 mM TES/KOH and supplemented with NaHCO3 to a concentration of 50 mM. Synechocystis mutants were selected under white light (10 μE/m2/s) at 30°C on BG11 medium solidified with 1.5% (w/v) agar and supplemented with 10 mM TES/KOH pH = 8.0, plus 0.3% (w/v) Na2S2O3 and selection markers where appropriate. Extraction of Genomic DNA Cells were grown until an OD730 of around 1 and 1 mL was harvested by centrifugation [12,000 rpm, 1 min, room temperature (RT)]. The supernatant was discarded and the cells were resuspended in 200 μL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Next, 200 mg glass beads (0.1 mm diameter) were added and the sample was vortexed for 5 min. The sample was cleared by centrifugation (5 min, 12,000 rpm, RT), and the supernatant was transferred to a fresh tube. Then, Phenol/chloroform/isoamylalcohol (25/24/1, v/v/v; 200 μL) was added, the sample was mixed and centrifuged (5 min, 12,000 rpm, RT). The aqueous phase was transferred to a fresh tube and washed with 200 μL of chloroform/isoamylalcohol (24/1, v/v). After centrifugation (5 min, 12,000 rpm, RT), 40 μL 5 M NaCl and 400 μL ethanol were added. After thorough mixing, the sample was placed at -20°C for 30 min. Then, the DNA was pelleted (10 min, 12,000 rpm, RT) and washed with 500 μL 70% pre-cooled ethanol. After centrifugation (5 min, 12,000 rpm, RT), the supernatant was removed and dried on a bench-top incubator at 30°C for 15 min. The pellet was dissolved in 30 μL nuclease-free water and used immediately, or stored at -20°C. Plasmid Construction For an overview of the plasmids used and created in this study, see Table 1. For the primers, see Table 2. For the construction of pMD18Tslr1670, slr1670 was amplified from genomic DNA of Synechocystis with primers slr1670-BamHI and slr1670-XhoI and introduced into pMD18-T. For the construction of pMD18Tslr1670KmR, pMD18Tslr1670 was digested with NcoI, blunted with T4 DNA polymerase and ligated with the Km resistance cassette (obtained from pRL446 Elhai and Wolk, 1988 by digestion with PvuII). The glpK gene was amplified from the genomic DNA of Synechocystis by PCR with primers glpK-Fwd and glpK-Rev, and cloned into the pMD 18-T vector (Takara, Japan). The resulting plasmid was digested by NheI, blunted by T4 DNA Polymerase (Fermentas), and ligated to the blunted chloramphenicol resistance gene cassette, resulting in the plasmid pXT323. Table 1 Plasmids used in this study. Plasmid Description Source pRL446 Elhai and Wolk, 1988 pMD18T TA cloning vector TaKaRa clonetech pMD18Tslr1670 This study pMD18Tslr1670KmR Construction Δslr1670 This study pXT323 Construction ΔglpK This study Table 2 Primers used in this study. Primer Sequence Slr1670-BamHI AGGATCCATGAAAACATTGAATCGTATCCATCTG Slr1670-XhoI TCTCGAGGCATTCCTTCTTCGAGCGA Slr1670-NheI fw GATAACGCTAGCATGAAAACATTGAATC Slr1670-BamHI rv ATGGATCCCTAGCATTCCTTCTTCGAGC glpK-Fwd CGCCATATGACAGCAAAACATAATCAG glpK-Rev TCTCGAGGATGGAAGCAATGTCAC Construction of Mutant Synechocystis Strains For an overview of the strains in this study, see Table 3. Mutant strains of Synechocystis were constructed essentially as described previously (Vermaas, 1996; Angermayr et al., 2012). Briefly, 10 mL cells were grown to an OD730 of 0.2–0.6, centrifuged and concentrated 20- to 50-fold. Then, plasmid DNA (1–3 μg) was added to 100–300 μL of the cell suspension. The cells were incubated in tubes at 30°C for 5 h. Cells were grown for 16 h on a membrane filter, on plates without selective pressure. Next, cells were transferred onto plates containing the relevant selection marker. Colonies appeared after 1–2 weeks. Identity of transformants was confirmed by colony PCR, using appropriate primers. Segregation of mutant strains was achieved in liquid culture. For construction of the slr1670 strain, WT Synechocystis was transformed with pMD18Tslr1670KmR. For construction of the double insertion mutant Δslr1670Δslr1672, Synechocystis Δslr1672 was transformed with pMD18Tslr1670KmR, and segregation was monitored by PCR (with the primers NheI-slr1670 fw and BamHI-slr1670 rv). Table 3 Strains used in this study. Strain Genotype Source Synechocystis sp. PCC 6803 X. Xu, Institute of Hydrobiology – Chinese Academy of Sciences Synechocystis ΔggpS ΔggpS::KmR Du et al., 2013 Synechocystis ΔglpK ΔglpK::CmR This study Synechocystis Δslr1670 Δslr1670::KmR This study Synechocystis ΔglpKΔslr1670 ΔglpK::CmRΔslr1670::KmR This study Analysis of Intra- and Extracellular Metabolites Using HPLC For analysis of extracellular metabolites, samples from a culture were cleared by centrifugation (14,500 rpm, 10 min, 21°C) and remaining particles were removed with a syringe, fitted with a filter (Sartorius Stedin Biotech, minisart SRP 4, 0.45 μm pore size). Samples were then analyzed by HPLC [column: Rezex ROA-Organic Acid H+ (8%) (Phenomenex); column temperature: 85°C; detector (Jasco, RI-1530); eluent: 7.2 mM H2SO4; flow: 0.5 mL/min]. Identification and quantification was done using external standards (detection limit: ∼0.02 mmol/L). For extraction of GG from cyanobacteria, 1 mL of a culture (OD730 = 6) was harvested by centrifugation (15,000 rpm, 10 min, 4°C). Pellets were resuspended in 1 mL 80% (v/v) ethanol and incubated at 65°C for 4.5 h. After re-centrifugation (15,000 rpm, 10 min, 21°C), the supernatant was transferred to a fresh tube and the liquid was evaporated using a stream of nitrogen gas. The residue was dissolved in 1 mL of water, filtered and analyzed by HPLC as described above. Genome Scale Metabolic Modeling We studied the newly identified GG degradation pathway in the context of a genome-scale metabolic network model previously reported for Synechocystis (Nogales et al., 2012). Although GG was already present in this earlier reconstruction, along with the reactions needed for its synthesis and exchange over the cytoplasmic membrane, some missing key steps made it impossible for the degradation pathway to carry any flux in silico. In order to fix this, and additionally include the re-utilization of GG, reactions for transport, metabolism and storage were added to the model (Table 4; Figure 2). Flux balance analyses of this new version of the Synechocystis stoichiometric model were carried out in the on-line modeling platform FAME (Boele et al., 2012), using newly developed visualization tools specific for this organism (Maarleveld et al., 2014). Constraining the relevant reactions correctly simulated the phenotype of the different derivative strains constructed in this study. The stoichiometric impact of the different GG breakdown pathways that are postulated here on the genome-scale metabolic network of Synechocystis was assessed by including the two alternative routes in the model. Comparisons between model versions mimicking different strains and conditions were established using biomass maximization as the objective function, while constraining the exchange flux of GG according to experimental measurements, as detailed elsewhere (Santos et al., 2011). Increased fitness was deduced from calculations of the maximum of the objective function (BOFmax) for the unconstrained utilization reaction (i.e., GG phosphorolysis, hydrolysis, and/or glycerol phosphorylation constrained between 0 and ∞), divided by BOFmax for the respective flux constrained to zero. Table 4 Reactions added to the genome-scale model of Synechocystis. Reaction name Reaction ID Equation Gene association GG transport via diffusion (cytosol to periplasm) R_glcglyctpp M_glcglyc_c⇒M_glcglyc_p – Glucosylglycerol Hydrolase R_GLCGLYCHyd M_h2o_c + M_glcglyc_c⇒M_glc_DASH_D_c + M_glyc_c slr1670 Glucosylglycerol Phosphorylase R_GLCGLYCPhosphorylase M_pi_c + M_glcglyc_c⇒M_g1p_c + M_glyc_c slr1670 Glucosylglycerol storage (cytosol to sink) R_GLCGLYCstorage M_glcglyc_c⇒ – M_glcglyc, glycosylglycerol; M_h2o_c, water; M_glc_DASH_D, D-glucose; M_glyc, glycerol; M_pi, inorganic phosphate; M_g1p, glucose-1-phosphate.FIGURE 2 Schematic representation of the modifications of the genome-scale model of Synechocystis sp. PCC 6803. The reactions R EX GLCGLYC LPAREN e RPAREN, R GLCGLYCtex, R GLCGLYCtpp, R GLCGLYCABCpp syn, R GLCGLYCstorage, R GLCGLYCHyd, and R GLCGLYCphosophorylase were added. Phylogenetic Analyses PSI-BLAST of the Slr1670 sequence against non-redundant protein sequences was carried out on the 24th of September, 2015, using the following parameters: expect threshold: 10, word size: 3, matrix: BLOSUM62, Gap existence cost: 11, Gap extension cost: 1, Conditional compositional score matrix adjustment. For the second iteration, sequences with a coverage > 90% (91/96 hits) were selected. Alignments were constructed using MEGA6 (ClustalW algorithm; pairwise alignment: gap opening penalty: 6, gap extension penalty: 0.1; Multiple alignment: gap opening penalty: 6, gap extension penalty: 0.2; Protein weight matrix: BLOSUM62; Residue-specific penalties: on; Hydrophilic penalties: on; Gap separation distance: 4; end gap separation: off; use negative matrix: off; delay divergent cutoff: 30%). The best model for the phylogenetic analysis was found using the following parameters: tree to use: neighbor-joining tree; statistical method: maximum likelihood; Gaps/missing data treatment: partial deletion; site coverage cut-off: 95%; branch swap filter: very strong. The best model for the cyanobacterial subset (Figure 3) was LG+G (Le and Gascuel, 2008). The best model for the entire set of proteins was LG+G+F (Le and Gascuel, 2008). The outgroup (hexokinase 1 of Saccharomyces cerevisiae) was used to root the trees and was not included in the figures. FIGURE 3 Slr1670 homologs are present in a variety of cyanobacterial-, bacterial-, and archaeal species. A phylogenetic tree based on the similarity of these homologs is shown for cyanobacterial species for which a complete genome sequence was available (see supplement for the full tree). Additionally, yellow-green boxes visualize presence of genes homologous to ggpS, ggpP, and glpK, in the respective genome. Gray boxes indicate that no significant hit was obtained. Yellow triangles mean low coverage/similarity while green triangles indicate high coverage/similarity. Results When grown in presence of 200 mM NaCl, wild type Synechocystis accumulates small amounts of glycerol in the extracellular medium (Savakis et al., 2015 and Figure 4A) The transcript of glpK, encoding glycerol kinase, was reported to be upregulated under salt stress conditions (Billis et al., 2014). We expected that in the absence of other assimilation reactions, a strain deficient in glpK would produce an increased amount of glycerol when facing salt stress. Interestingly, instead, glycerol production remained unaltered in a strain in which glpK was disrupted with a chloramphenicol resistance cassette (Figures 4A,C and 5A,C). This finding suggests that glpK might not be involved in the assimilation of glycerol under the conditions tested. FIGURE 4 Inactivation of slr1670 or sll1566 (ggpS) abolishes glycerol production under mild salt stress. Squares represent wild type (A), diamonds the ggpS inactivation mutant (B), circles the glpK inactivation mutant (C), and triangles the slr1670 inactivation mutant (D). Filled symbols represent optical density values and correspond to the left y-axes; empty symbols represent extracellular glycerol concentrations c, measured in mmol/L and correspond to the right y-axes. Cells were grown in BG11 medium buffered to an initial pH of 8.0 with 10 mM TES/KOH and supplemented with 200 mM NaCl. Error bars represent the standard deviation of at least two biological replicates. Error bars that are not visible are smaller than the respective data point symbol. FIGURE 5 Glucosylglycerol degradation is abolished in the slr1670 inactivation mutant. The ggpS disruption strain cannot synthesize GG, but does produce glycerol in the presence of extracellular GG. Squares represent the wild type strain (A), diamonds the ggpS inactivation mutant (B), circles the glpK inactivation mutant (C), triangles the slr1670 inactivation mutant (D), and hexagons the slr1670/glpK double inactivation mutant (E). Filled symbols represent optical density values and correspond to the left y-axes; half-filled or empty symbols represent glycerol concentrations c, measured in mmol/L and correspond to the right y-axes. Cells were grown in BG11 medium buffered to an initial pH of 8.0 with 10 mM TES/KOH, and supplemented with 200 mM NaCl in the presence and absence of 10 mM GG. Error bars represent the standard deviation of at least two biological replicates. Error bars that are not visible are smaller than the respective data point symbol. To test whether instead the appearance of glycerol in the extracellular medium is dependent on the presence of intracellular GG, we analyzed a mutant strain in which ggpS is disrupted via insertion of a kanamycin resistance cassette (Du et al., 2013). This strain is sensitive to moderately high salt concentrations (∼400 mM NaCl, Supplementary Figure S1), as observed previously (20). Growth at lower concentrations of NaCl (i.e., 200 mM), however, was unaffected (Supplementary Figure S1F). In the supernatant of liquid cultures of this strain, no glycerol could be detected upon the addition of salt (Figure 4B). This indicated that GG is a precursor of glycerol under these conditions. Significantly, within the genomic context of ggpS and glpK there is also a gene, whose transcript was shown to be upregulated under salt stress conditions (Dickson et al., 2012; Qiao et al., 2013; Billis et al., 2014). The open reading frame upstream of glpK, slr1670, has a translated length of 885 amino acids. The Slr1670 protein belongs to the GHL5 family of hypothetical glucoside hydrolases (Naumov and Stepushchenko, 2011). In order to investigate the role of Slr1670, a mutant strain was constructed in which this ORF was disrupted by a kanamycin resistance cassette (Figure 1B). Growth of the Slr1670 disruption strain was unaffected by salt (Supplementary Figures S1A,D,G,J). However, no glycerol was detectable in the extracellular medium when this strain was grown in BG11 medium supplemented with 200 mM NaCl (Figure 4D). We then added GG to cells of the Δslr1670 strain, and analyzed the extracellular glycerol concentration in relation to the amount of glycerol formed in related deletion mutants (Figure 5). In the wild type strain, the concentration of extracellular glycerol was increased as compared to the control condition (no additional GG, Figure 5A). The ggpS disruption strain accumulated extracellular glycerol only when GG was added (Figure 5B). The amount of glycerol was lower than in the wild type strain. When GG was supplied to the ΔglpK strain, the extracellular accumulation of glycerol was increased and comparable to that of the wild type (Figure 5C). In all strains tested, the addition of extracellular GG led to an increase in growth rate in the exponential phase (Figures 5A–D). When GG was added exogenously to the slr1670 disruption mutant, no glycerol was formed (Figure 5D). These findings are indicative of Slr1670 playing a role in glycerol production from GG. To make sure that the absence of glycerol formation could not be attributed to polar effects of the slr1670 disruption [e.g., (over-) expression of glpK from the promoter of the kanamycin resistance cassette], a mutant deficient in both slr1670 and glpK was constructed. Under salt stress, this strain failed to accumulate glycerol in the extracellular medium (Figure 5E). The slr1670 deletion strain showed increased intracellular concentrations of GG (Figure 6), corroborating the hypothesis that Slr1670 is involved in GG degradation. Taken together, these findings suggest that Slr1670 is required for the degradation of GG to glycerol. FIGURE 6 Inactivation of slr1670 leads to increased accumulation of intracellular glucosylglycerol. Cells were grown in BG11, supplemented with 200 mM NaCl and 10 mM TES buffer, at an initial pH of 8.0 to OD730 values of 6–7. For each strain, three biological replicates were analyzed. For the extraction, three technical replicates were taken from every biological replicate. Intracellular concentrations were calculated assuming a culture with an OD730 of 1 contains 0.2 g of dry weight per liter and assuming that 1 mg dry weight corresponds to 1 μL intracellular volume. Error bars show standard deviations calculated over all data points for a given strain (9). The asterisk denotes statistical significance (P < 0.001). We used genome-scale modeling, to analyze whether a possible growth advantage could be conferred by the ability to degrade GG. We started with the model published by Nogales et al. (2012), and extended it with previously published data and with the new information acquired here. Since inactivation of the ABC transporter involved in GG uptake leads to extracellular accumulation of GG (Hagemann et al., 1997; Mikkat and Hagemann, 2000), a leakage reaction from the cytoplasm to the periplasm was introduced. In this state, the model would not predict synthesis of GG when maximizing growth rate. Therefore, a sink reaction for cytoplasmic GG was introduced (Table 4; Figure 2). Since it is at this point unknown whether cleavage of GG occurs via hydrolysis or phosphorolysis (as described by Nihira et al., 2014 for the B. selenitireducens enzyme), both reactions were included. Phosphorolysis of GG, similar to the reaction catalyzed by the enzyme of B. selenitireducens, yields phosphorylated glucose (Nihira et al., 2014). We therefore expected a clear preference for this reaction over the hydrolysis reaction. Accordingly, we simulated growth for phosphorolytic cleavage for a range of GG uptake fluxes, qGG, and divided the obtained rates by those obtained for hydrolytic cleavage (Supplementary Figure S2A). At low uptake rates (low qGG values), no difference in growth rate was predicted for either phosphorolysis or hydrolysis. Only at very high uptake rates was the phosphorolysis reaction beneficial, but even there, the predicted increase in growth rate was minor (<2%). Interestingly, the genome-scale model makes similar predictions for the benefit of glycerol utilization: for low values of qGG, no benefit for the assimilation of glycerol is predicted (Supplementary Figure S2B). Even at very high values, the increase in ratio is very small (<2%). To estimate a physiological range for qGG, we used the glycerol production data from the strains supplemented with extracellular GG (Figure 5). The ΔggpS mutant is unable to synthesize GG, and extracellular glycerol consequently must stem from the degradation of exogenous GG exclusively (Figure 5B). We therefore used the glycerol production values from this strain to constrain qGG in the genome-scale model. Next, we simulated photo(hetero)trophic growth (μ) at 30 μE/m2/s with the resulting qGG values for wild type, ΔggpS, ΔglpK, and Δslr1670. To estimate if the degradation of GG results in increased fitness for these qGG values, we divided the growth rate of the respective strains by the growth rate of the Δslr1670 mutant and compared these values to the experimentally determined data (Table 5). In the exponential phase (days 0–4) there is excellent agreement between simulation and experiment. Between day 4 and day 6 and between day 8 and day 10, the predicted increase is lower than in the experiment. As the culture increases in density, the absolute value of photons that a single cell perceives decreases. This results in slower (linear) growth (due to light limitation). Under the assumption that the contribution of GG utilization to growth remains constant, the difference in growth (expressed as the ratio) between a strain that utilizes GG, and one that cannot, will therefore be amplified under low light conditions. Since we used a constant photon uptake rate for the simulations, it is expected that at later time points, the effect of GG utilization is underestimated. In the strains that do have a functional slr1670, on day 8, there is a drop in optical density (Figures 5A–C), which results in negative values for the experimentally determined growth rates. Since the model can only predict growth, a comparison between the experimental and simulated ratios is not meaningful for this and the adjacent intervals. The cause of this transient drop in OD is at present unknown. A possible explanation is that the degradation of GG, and utilization of the glucose part thereof, leads to a drop in intracellular osmotic pressure. Transient efflux of water could then reduce the cell volume and hence, the optical density. Table 5 Growth rates of Synechocystis wild type, ΔggpS and ΔglpK divided by the growth rate of Δslr1670. μ/μ [%] Interval [days] qGG [mmol ⋅gDW-1 h-1] WTΔslr1670 ΔggpSΔslr1670 ΔglpKΔslr1670 sim.i exp.ii sim.i exp.ii sim.i exp.ii 0–2 0.008 103.9 100.9 ± 2.4 103.9 105.7 ± 2.3 103.9 102.8 ± 3.5 2–4 0.015 107.4 109.4 ± 11.0 107.4 106.1 ± 12.2 107.4 104.6 ± 10.3 4–6 0.014 106.9 122.8 ± 5.6 106.9 123.3 ± 5.4 106.9 128.7 ± 11.2 6–8 0.009 104.6 -73.7 ± 36.8 104.6 -42.8 ± 24.6 104.6 -106.9 ± 21.7 8–10 0.003 101.5 135.7 ± 41.8 101.5 93.0 ± 31.6 101.5 120.9 ± 59.8 qGG values were calculated using glycerol production data of the ΔggpS mutant (Figure 5).i sim.: simulated; at physiological uptake fluxes of GG, the model does not predict a difference for the mode of cleavage (phosphorolytic versus hydrolytic, Supplementary Figure S2A). Similarly, utilization of the glycerol part does not lead to increased growth rate (Supplementary Figure S2B). ii exp.: experimental; error margins represent standard deviations of three replicates.Until now, Slr1670 is annotated as a hypothetical protein, so we decided to study its phylogenetic distribution. A PSI BLAST of the translated sequence of Slr1670 yielded around 90 homologs, which are distributed among the cyanobacteria, α-proteobacteria and Archaea (see Figure 3 for a condensed tree, including only the cyanobacterial species and Supplementary Figure S3 for the full phylogenetic tree). In many of the cyanobacterial strains, also homologs of ggpS, ggpP, and glpK could be identified (Figure 3). Notably, a homolog was also found in M. chthonoplastes, a strain that has been shown to ferment GG under dark conditions, thereby utilizing the glucose moiety, but excreting glycerol (Moezelaar et al., 1996). Discussion Production of one GG molecule requires fixation of 9 CO2 molecules. From a cellular/physiological point of view, GG is an energetically costly compound with often transient use only. It is therefore conceivable that systems have evolved to reduce energy losses related to the synthesis of this osmolyte. The GgtABCD system takes up GG that is lost from the cells due to leakage from the cellular cytoplasm; inactivation of this system leads to pronounced extracellular accumulation of GG (Hagemann et al., 1997; Mikkat and Hagemann, 2000). Upon transition from a high-salt to a low-salt environment, osmoprotective compounds are no longer needed to maintain turgor pressure, so cells able to salvage the nine carbon atoms of GG are at an advantage. In line with this, the adjusted genome scale model of Synechocystis predicted an increase in growth rate when extracellular GG was made available (Table 5). Marine cyanobacteria may ferment a portion of their osmoprotectants (Stal and Moezelaar, 1997). For example, M. chthonoplastes ferments GG, using only the glucose moiety and excreting the glycerol (Moezelaar et al., 1996). In M. chthonoplastes, GG was used as a substrate for fermentation during the night. Interestingly, a gene homologous to slr1670 was also found in the genome of M. chthonoplastes (Figure 3), suggesting that the corresponding protein is involved in the degradation of GG in this strain. At this moment, it is not known if the degradation of GG, observed under continuous illumination in Synechocystis, a freshwater organism, serves a regulatory purpose or is the result of a constitutive reaction that becomes more meaningful in the dark. During growth with constant incident illumination, the average amount of light per cell decreases. The individual cells, however, perceive something different. At a given moment, the cells located close to the light source receive light at a high intensity, while a cell in the center of the culture receives fewer photons or none at all. Increases in culture density therefore may lead to increased periods of darkness from the perspective of an individual cell. This is corroborated by the observation that more glycerol is excreted per cell in the late versus the early phase of the experiment (Figures 4 and 5). This notion might explain why glucose recycling becomes active during the day. Cellular release of glycerol by both Synechocystis and M. chthonoplastes is in agreement with the prediction from the genome scale model, i.e., that glycerol utilization does not lead to increased growth at physiological levels of the GG assimilation reaction (Supplementary Figure S2B). The mutant strains deficient in degradation of GG are valuable tools to study the kinetics of GG synthesis. Under industrial production settings, Synechocystis may be exposed to variations in osmotic stress. It is important to be able to simulate the behavior of this organism under such condition in order to facilitate its usage in the direct conversion of CO2 to products of interest in a sustainable fashion (dos Santos et al., 2014). The addition in the model of the ability to synthesize, transport and degrade GG is a necessary step toward the accurate simulation of the growth of Synechocystis in such conditions. Additionally, disruption of slr1670 allows the construction of strains with increased levels of GG, an interesting commodity chemical (Tan et al., 2015). Conclusion We have demonstrated a function for a previously non-annotated protein that has turned out to be required for the re-assimilation of GG. The protein shows homology to glucoside hydrolases and is found in a wide range of cyanobacteria, where its presence correlates with the presence of genes required for GG synthesis. Furthermore, homologs of this protein are also found in the α-proteobacteria and in the domain of the Archaea (Supplementary Figure S3). Strikingly, halotolerant organisms are overrepresented in these two classes of organisms, particularly among the Archaea. Often, homologs obtained had previously been annotated as alpha-amylases. These annotations, however, are purely based on homology and are not supported by experimental evidence. It is also of interest to note that the genomes of some cyanobacterial species encode multiple reading frames that exhibit sequence similarity with Slr1670. Genome-scale modeling revealed that the increase in growth rate caused by the utilization of the glucose part of GG outweighs the use of the glycerol part by far. This is in line with observations in M. chthonoplastes, in which only the glucose part of GG is utilized by the cells. Studies with the purified enzyme will help to elucidate the kinetic properties of Slr1670 and shed light on the mechanism of cleavage of GG. Author Contributions PS and XT conceived the study, designed and carried out the experimental plan, and wrote the paper. CQ and KS implemented the experimental plan. XL, KH, and FBS conceived and supervised the study and wrote the paper. Conflict of Interest Statement KH is advisor to Photanol B.V. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding. This article was written within the research program of BioSolar Cells, co-financed by the Dutch Ministry of Economic Affairs. FBS is supported by the Netherlands Organization for Scientific Research (NWO) through VENI grant 863.11.019. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. 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==== Front J NeurophysiolJ. NeurophysioljnjnJNJournal of Neurophysiology0022-30771522-1598American Physiological Society Bethesda, MD 27306671JN-00103-201610.1152/jn.00103.2016Call for PapersBiology of Neuroengineering InterfacesValidating silicon polytrodes with paired juxtacellular recordings: method and dataset Neto Joana P. 125Lopes Gonçalo 15Frazão João 1Nogueira Joana 15Lacerda Pedro 1Baião Pedro 1Aarts Arno 4Andrei Alexandru 3Musa Silke 3Fortunato Elvira 2Barquinha Pedro 2Kampff Adam R. 151Champalimaud Neuroscience Programme, Champalimaud Centre for the Unknown, Lisbon, Portugal; 2Departamento de Ciência dos Materiais, CENIMAT/I3N and CEMOP/Uninova, Caparica, Portugal; 3IMEC, Leuven, Belgium; 4ATLAS Neuroengineering; and 5Sainsbury Wellcome Centre, University College London, London, United KingdomAddress for reprint requests and other correspondence: J. P. Neto, Champalimaud Centre for the Unknown, Av. Brasília, Doca de Pedrouços, 1400-038 Lisbon, Portugal (e-mail: joana.neto@neuro.fchampalimaud.org).15 6 2016 1 8 2016 15 6 2016 116 2 892 903 3 2 2016 19 5 2016 Copyright © 2016 the American Physiological SocietyAmerican Physiological SocietyLicensed under Creative Commons Attribution CC-BY 3.0: the American Physiological Society.Recording in vivo from the same neuron with two different methods is difficult. It requires blindly moving each probe to within ∼100 μm of one another and for this reason such “dual-recordings” are rare. However, comparing the signals measured by different techniques is necessary to understand what they measure. We developed a method to precisely align the axes of two manipulators and used it to gather a “ground truth” dataset for dense extracellular polytrodes. Cross-validating new methods for recording neural activity is necessary to accurately interpret and compare the signals they measure. Here we describe a procedure for precisely aligning two probes for in vivo “paired-recordings” such that the spiking activity of a single neuron is monitored with both a dense extracellular silicon polytrode and a juxtacellular micropipette. Our new method allows for efficient, reliable, and automated guidance of both probes to the same neural structure with micrometer resolution. We also describe a new dataset of paired-recordings, which is available online. We propose that our novel targeting system, and ever expanding cross-validation dataset, will be vital to the development of new algorithms for automatically detecting/sorting single-units, characterizing new electrode materials/designs, and resolving nagging questions regarding the origin and nature of extracellular neural signals. extracellular action potentialspike sortingground truthjuxtacellular recordingpolytrodesFCT-MCTESSFRH/BD/76004/2011European Union's Seventeen programme (FP7/2017-2013)600925http://doi.org/10.13039/501100005032 Fundação Bial (Bial Foundation)190/12 ==== Body NEW & NOTEWORTHY Recording in vivo from the same neuron with two different methods is difficult. It requires blindly moving each probe to within ∼100 μm of one another and for this reason such “dual-recordings” are rare. However, comparing the signals measured by different techniques is necessary to understand what they measure. We developed a method to precisely align the axes of two manipulators and used it to gather a “ground truth” dataset for dense extracellular polytrodes. understanding how the brain works will require tools capable of measuring neural activity at a network scale, i.e., recording from thousands of individual neurons (Buzsáki 2004). Technical advances have driven progress in large-scale neural recordings, and the development of microfabricated silicon polytrodes has led to an exponential increase in the number of neurons that can be simultaneously monitored (Stevenson and Kording 2011; Berényi et al. 2014; Michon et al. 2014). However, each improvement in recording technology inevitably raises new questions about the nature of the signal and demands new analysis methods to interpret these growing datasets. Extracellular recording is unique in its ability to record populations of neurons deep in the brain with submillisecond resolution; it also poses particularly daunting challenges for analysis. Each electrode is sensitive to the spiking activity of hundreds of neurons in its vicinity, and sorting this cacophony into individual sources is a challenge (Marblestone et al. 2013). Furthermore, fundamental questions regarding how each neuron participates in the bulk extracellular signal remain unresolved: How many neurons contribute to the signal detected by an electrode? How does a neuron's contribution decay with distance from the probe? Do different types of neurons have different extracellular signatures? Are extracellular recordings biased for particular types of neurons? How does the presence of the probe interfere with the activity of the surrounding neural tissue? Answers to these questions will require experiments to validate existing and future extracellular electrode technology as well as new analysis methods to interpret their data. Employing modern methods for integrated circuit design and fabrication, probes with thousands, or even millions, of discrete sites are now being developed (Dombovári et al. 2014; Ruther and Paul 2015; Shobe et al. 2015). These devices will densely sample the extracellular electric field, such that one nearby neuron will be detected by many individual electrodes, and will thus provide a detailed description of the spatiotemporal profile of a neuron's extracellular action potential (EAP). It is expected that this additional detail will significantly aid analysis methods for the detection and isolation, and possibly type identification, of individual neurons in the vicinity of the probe, yet methods capable of utilizing such a dense sampling are just now being developed (Rossant et al. 2016). “Ground truth” data, for which one knows exactly when a neuron in the vicinity of an extracellular probe generates an action potential, are necessary to validate the performance of these new recording devices and analysis procedures. However, the validation datasets currently available for extracellular recordings only exist for tetrodes and single-wire electrodes (Wehr et al.1999; Henze et al. 2000; Chorev and Brecht 2012; Henze and Buzsáki 2007) or are from in vitro preparations (e.g., slices; Anastassiou et al. 2015) in which the majority of background neural activity has been surgically removed. Evaluating the existing silicon polytrodes, as well as forthcoming ultra-high density CMOS probes, in vivo will require new datasets, and, ideally, new methods for efficiently gathering this vital cross-validation data. Targeting a single neuron close to an extracellular probe with another electrode requires accurately positioning both devices deep in neural tissue. When performed blindly, the efficiency of achieving paired-recordings in which one neuron is detected by both probes is rather low, making such validation experiments much more difficult than just haphazardly recording extracellular neural signals. For this reason, such datasets are very rare; however, the ones that do exist (for tetrodes in the hippocampus) have been invaluable (Harris et al. 2000; Gold et al. 2007). We anticipate that a large amount of such validation data will be required to characterize the large-scale neural recording devices currently being developed, and we thus set out to make paired-recordings easier. In the following we report a new method for efficiently and reliably targeting, blindly, two different recording devices to the same region in the brain. This method was then used to acquire a “ground truth” dataset from rat cortex with 32- and 128-channel silicon polytrodes, which can now be used to validate methods for interpreting dense extracellular recordings and help resolve persistent debates about the nature and origin of the extracellular signal. This dataset, which will grow as new devices are fabricated, is available online (http://www.kampff-lab.org/validating-electrodes). MATERIALS AND METHODS Set-Up Design and Calibration The dual-recording setup requires two aligned, multi-axis micromanipulators (Scientifica) and a long working distance optical microscope (Fig. 1A). A “PatchStar” (PS) and an “In-Vivo Manipulator” (IVM) are mounted on opposite sides of a rodent stereotaxic frame. The stereotaxic frame also defined the common X-, Y-, and Z-axes to which the manipulators were aligned: X-axis is parallel to the medio-lateral axis; Y-axis is parallel to the anterior-posterior axis, and Z-axis is parallel to the dorso-ventral axis. Fig. 1. In vivo paired-recording setup: design and method. A: schematic of the dual-probe recording station. The “PatchStar” (PS) micromanipulator drives the juxtacellular pipette and the “In-Vivo Manipulator” (IVM) manipulator drives the extracellular polytrode. The setup includes a long working distance microscope assembled from optomechanical components mounted on a 3-axis motorized stage. The alignment image provides a high-resolution view from above the stereotactic frame, top left; however, a side-view can also be obtained for calibration purposes, top right (scale bar = 100 μm). B: schematic of a coronal view [reproduced from Paxinos G, Watson C. The Rat Brain In Stereotaxic Coordinates (6th ed.), Elsevier, 2007, with permission] of the craniotomy and durotomies with both probes positioned at the calibration point. The distance between durotomies, such that the probe tips meet at deep layers in cortex, was ∼2 mm. The black arrows represent the motion path for both electrodes entering the brain (scale bar = 1 mm). C: diagram of simultaneous extracellular and juxtacellular paired-recording of the same neuron at a distance of 90 μm between the micropipette tip and the closest electrode on the extracellular polytrode (scale bar = 100 μm). The probes were held at an angle: the PS allows the combination of two motion axes (XZ-axis in approach mode) and this approach angle was set at 61° from the horizontal, whereas the IVM, a rigid three-axis linear actuator, was tilted −48.2° from the horizontal around the Y-axis. The use of two different models of manipulator was a practical constraint in this study, as the IVM permitted a greater range of travel for initial prototyping. However, future dual-probe setups will utilize two PS systems and the calibration and operation procedures will remain identical. Alignment microscope. A custom microscope was assembled from optomechanic components (Thorlabs), a long-working distance objective (Infinity-Corrected Long Working Distance Objective, ×10; Mitutoyo), and a high-resolution CMOS camera (PointGrey). The numerical aperture of the objective (0.28) had a theoretical resolution limit of ∼1 μm in X- and Y-directions and ∼10 μm in Z-direction, which was oversampled by the camera sensor. Oblique illumination was necessary to acquire an image of both the extracellular probe and juxtacellular pipette, directly above the craniotomy, with sufficient contrast to accurately “zero” the position of each probe (Fig. 1A, top left). The repeatability of visually aligning each probe to the center of the image (“zeroing”) was evaluated by manually moving the tip of the pipette several times (n = 11) from outside the field-of-view to the focal plane and image center and recording the manipulator coordinates. The optical alignment procedure had 0.5 ± 0.5 μm repeatability in XY and 2.6 ± 1.7 μm in Z. Mechanical alignment. Ensuring that the axes from both manipulators are parallel began with a mechanical alignment procedure. The PS was “squared” with the stereotactic frame using a digital machinist's dial (Fine Reading Indicator; RS Pro) mounted in the electrode holder, using exactly the same procedure that a machinist uses to align a milling machine XY-table and -column. The dial was placed in contact with a planar surface of the stereotactic frame and moved along this surface (see Supplemental Material Movie S1; Supplemental Material for this article is available online at the Journal website). Any change in the micrometer-sensitive dial's readings during movement indicated a misalignment, and the manipulator was repeatedly “realigned” (i.e., tapped with a soft surface hammer) until this differential was minimized. The IVM manipulator was then aligned using the same procedure in the Y-axis using both the vertical and horizontal planes of the stereotactic frame. Estimating misalignment. Each probe tip was positioned, sequentially, at the center of the microscope image (indicated by an overlay crosshair) and the respective motorized manipulator coordinates were set to zero (X = 0, Y = 0, Z = 0; Fig. 1A). The pipette was then moved to a different position in space, the microscope was moved and refocused to recenter the tip of the pipette in the crosshair. Next, without moving the microscope, we moved the extracellular probe to the same coordinates as the pipette. If there were no misalignment between the two manipulators, then the probe should arrive at the center of the image crosshair. If the probe is not centered, then the amount of re-positioning required (in X, Y, and Z) to venter the probe provides an accurate measure of residual axis misalignment. These “errors” were recorded for each position as the probes were sequentially moved to several different locations (n = 15) that spanned a large volume (5,000 × 5,000 × 5,000 μm, in 1,000-μm steps). Following mechanical alignment, the average distance error recorded in this volume was 75.6 ± 36.2 μm (n = 15). Note that our manipulators were mounted with different approach angles (θIVM = 48.2°) and converting the coordinates of the IVM into the PS frame requires the following transformation and assume perfect Y-axis alignment. XPATCH=cos(θIVM)⋅ZIVM+sin(θIVM)⋅XIVM YPATCH=YIVM ZPATCH=−sin(θIVM)⋅ZIVM+cos(θIVM)⋅XIVM Software correction. By using one manipulator as the reference, we can use the position errors measured at many different locations to estimate the coordinate transformation that best compensates for the misalignment of the second manipulator. We adopted the PS coordinate system as the reference frame and transformed the recorded IVM coordinates into the PS frame, in an affine manner, as follows for the X-axis: XPATCH = A·XIVM, where A represents a transformation matrix that best matches these pairs of coordinates. The distance error estimated after the software alignment was reduced to 10.5 ± 5.2 μm (n = 15). The protocol for the acquisition and transformation of axis position was implemented in Bonsai, an open-source visual programming framework, which can be freely downloaded at https://bitbucket.org/horizongir/bonsai (Lopes et al. 2015). Surgery Rats (400–700 g, both sexes) of the Long-Evans strain were anesthetized with a mixture of ketamine (60 mg/kg ip) and medetomidine (0.5 mg/kg ip) and placed in a stereotaxic frame that was atop a vibration isolation table (Newport). Equipment for monitoring body temperature as well as a live video system for performing craniotomies and durotomies were integrated into the setup. At the initial stage of each surgery, atropine was given to suppress mucus secretion (atropine methyl nitrate; Sigma-Aldrich). Anesthetized rodents then underwent a surgical procedure to remove the skin and expose the skull above the targeted brain region. Small craniotomies (4 mm medial-lateral and 2 mm anterior-posterior) were performed above dorsal cortex. The craniotomy centers were 2.5 mm lateral to the midline and ranged from +4 to −4 anterior-posterior, thus exposing either motor, sensory, or parietal cortex. Two reference electrodes Ag-AgCl wires (Science Products E-255) were inserted at the posterior part of the skin incision on opposite sides of the skull. Dense Silicon Polytrodes All experiments were performed with two different high-density silicon polytrodes. A commercially available 32-channel probe (A1x32-Poly3-5mm-25s-177-CM32; NeuroNexus), with 177-μm2 area electrodes (iridium) and an intersite pitch of 22–25 μm, was used in the first experiments. The impedance magnitude for these sites at 1 kHz was ∼1 MΩ, but for some experiments, a PEDOT coating was applied to lower this impedance to ∼50–100 kΩ. In later experiments, we used a 128-channel probe produced by the collaborative NeuroSeeker project (http://www.neuroseeker.eu/) and developed by IMEC using CMOS-compatible process technology. The probe's titanium nitride (TiN) electrodes were 400 μm2 (20 × 20 μm2) in size, were arranged at a pitch of 22.5 μm, and had an impedance magnitude of 50 kΩ at 1 kHz. Before each surgery, the impedance magnitude of each electrode site was measured for diagnostic purposes using a protocol implemented by the amplifier/DAC chip (InTan Technologies). Following each surgery, cleaning was performed by immersing the probe in a trypsin solution [trypsin-EDTA (0.25%), phenol red; TermoFisher Scientific] for 30–120 min and rinsing with distilled water. Probe Insertion and Simultaneous Juxtacellular-Extracellular Recordings After both the extracellular probe and juxtacellular pipette positions were sequentially “zeroed” to the center of the microscope image, the microscope was replaced by a macro-zoom lens (Edmund Optics) for visually guided insertion. The extracellular probe was inserted first, at a constant velocity of 1 μm/s, automatically controlled by the manipulator software. When the extracellular probe was in place the juxtacellular pipette filled with 1× PBS was then lowered through a second durotomy under visual guidance using the overhead surgery camera. We used capillary borosilicate glass tubing with flame polished ends, an outer diameter of 1.50 mm, inner diameter of 0.86 mm, and a length of 10 cm (Warner Instruments). The tubing was pulled into micropipettes using a laser-based micropipette puller (P-2000; Sutter Instruments). The resulting juxtacellular pipettes had resistances between 3 and 7 MΩ and tip diameter of about 1–4 μm. As the pipette approached the extracellular electrodes, we followed a protocol for performing loose-patch recordings from neurons as previously describe (Herfst et al. 2012). Positive pressure (25–30 mmHg) was reduced on the pipette to 1–10 mmHg (DPM1B Pneumatic Transducer Tester; Fluke Biomedical), and the amplifier for juxtacellular recordings (ELC-01X; NPI) was set to voltage-clamp mode (25-mV steps at 20 Hz). As the electrode was advanced towards a cell membrane, we observed an increase in the pipette resistance. If spikes were observed, the pressure was then released (0 mmHg) and a slight suction applied to obtain a stable attachment to the cell membrane. A data acquisition board (National Instruments) was used to control amplifier voltage commands. However, after a stable recording was achieved, simultaneous recording of both extracellular and juxtacellular electrodes used exclusively the Open Ephys (http://www.open-ephys.org) acquisition board ADCs (for the juxtacellular signal) along with the RHD2000 series digital electrophysiology interface chip that amplifies and digitally multiplexes the extracellular electrodes (Intan Technologies). Extracellular signals in a frequency band of 0.1–7,500 Hz and juxtacellular signals in a frequency band of 300-8,000 Hz were sampled at 30 kHz with 16-bit resolution and were saved in a raw binary format for subsequent offline analysis using a Bonsai interface. For the analyses described in the following, a third order Butterworth filter with a band-pass of 100-14,250 Hz (95% of the Nyquist frequency) was used in the forward-backward mode. For some recordings we noticed a high-frequency noise contribution and we thus used a band-pass of 100-5,000 Hz. All experiments were approved by the Champalimaud Foundation Bioethics Committee and the Portuguese National Authority for Animal Health, Direcção-Geral de Alimentação e Veterinária. RESULTS Setup Design The “dual-probe” positioning and recording setup presented in Fig. 1A was designed to reliably target neural cell bodies located within ∼100 μm of the polytrode electrode sites without optical guidance. In this setup, the motorized manipulators, video capture, online visualization/control parameters, and extra- and juxtacellular voltage recording were integrated and coordinated by custom open-source software developed within the Bonsai framework (Lopes et al. 2015). Following a mechanical alignment and software calibration of both manipulators' axes, each paired-recording experiment began with the optical “zeroing” of both probes. Each probe was positioned, sequentially, at the center of the microscope image (indicated by a crosshair) and the motorized manipulator coordinates set to zero (Fig. 1A). As shown in Fig. 1B, this alignment is performed directly above the desired rendez-vous point inside the brain, as close as possible above dura, usually between 1 and 4 mm but far enough to reduce background light reflected from the brain surface into the microscope image. During optical calibration it is possible to select any point on the extracellular electrode to be the origin (X = 0, Y = 0, Z = 0) by aligning that point of the probe in the reticle. However, the distance reported in the subsequent data is always the Euclidean distance between the tip of the pipette and the closest extracellular electrode. With practice, multiple cells in the vicinity (<200 μm) of the polytrode could be recorded through multiple insertions of the juxtacellular pipette (Fig. 1C is a schematized example of one paired-recording). Before the surgeries, we validated the alignment of the motors by moving both probes independently from the calibration point (0, 0, 0) towards a different point in space and recording the position difference between them after travel. During our experiments, the movement of the probes primarily occurred in the XZ-plane. We found that when we moved both probes to a new Z-position 3 mm below the calibration point (0, 0, −3), similar to an actual recording experiment (Fig. 1B), the distance error observed was 10.5 μm after the software calibration (and 31.6 μm before software calibration), which was acceptable for targeting the same region in cortex. During a recording, advancing the pipette very close to the extracellular probe surface (<30 μm) allowed direct detection of the 25-mV test pulse delivered by the juxtacellular amplifier (Supplemental Material Movie S2) on the extracellular array. The peak of this test pulse was largest on the targeted, and thus nearest, electrode site, providing further validation of our setup's positioning accuracy. Paired Juxtacellular-Extracellular Recording Twenty-three neurons were recorded with a distance <200 μm between the juxtacellular pipette tip and the closest extracellular electrode within the cortex of anesthetized rats. The precision aligned dual-probe setup could efficiently target neurons nearby the extracellular probe, and for each insertion of the pipette at least one paired-recording was obtained. Eleven animals were used to record all the pairs in this study (the full dataset is summarized in Supplemental Material Table 1). However, with practice, it was possible to insert the juxtacellular pipette several times at different locations (max 4) and to record many neurons (max 6) along a single track. The juxtacellular pipette had a long thin taper to minimize tissue displacement during penetration and promote longer stable recordings (Herfst et al. 2012) (Fig. 2A). As the juxtacellular electrode was advanced through the brain, several neurons were encountered at different locations along the motion path and, consequently, at different distances from the extracellular polytrodes. Figure 2B illustrates the large juxtacellular (peak-to-peak ∼4 mV) signal recorded from a neuron encountered at a distance of 51.0 ± 10.5 μm between the micropipette tip and the closest extracellular electrode. The positive-before-negative biphasic waveform shape (Fig. 2C) is indicative of a capacitively coupled cell-attached recording from a somatic/perisomatic located recording pipette (Herfst et al. 2012). However, for two paired-recordings in the dataset, the pipette recording exhibited the waveform of well isolated extracellular spike (negative-before-positive), likely due to incomplete contact between the membrane and pipette presenting lower peak-to-peak amplitudes (2015_09_04_Pair 5.0 and 2015_09_03_Pair 9.0). Fig. 2. Paired extracellular and juxtacellular recordings from the same neuron. A: recording pipette with a long thin taper used for juxtacellular recordings with typical tip diameter of 1–4 μm and resistance of 3–7 MΩ. B: representative juxtacellular recording from a cell ∼1,256 μm in depth, 51 μm from the extracellular probe (2014_10_17_Pair1.0), with a firing rate of ∼ 1 Hz. C: juxtacellular action potentials are overlaid, time-locked to the maximum positive peak, with the average spike waveform superimposed (n = 442 spikes). D: extracellular dense polytrode array with a span of 275 μm along the shank axis; the electrode channel number is represented at each site. E: representative extracellular recording that corresponds to the same time window as the above juxtacellular recording. Traces are ordered from upper to lower electrodes and channel numbers are indicated. F: extracellular waveforms, aligned on the juxtacellular spike peak, for a single channel (channel 18) and the juxtacellular triggered average (JTA) obtained by including an increasing number of juxtacellular events (n as indicated). G: spatial distribution of the amplitude for each channel's extracellular JTA waveform. The peak-to-peak amplitude within a time window (±1 ms) surrounding the juxtacellular event was measured and the indicated color code was used to display and interpolate these amplitudes throughout the probe shaft. H: waveform averages for all the extracellular electrodes are spatially arranged. The channel with the highest peak-to-peak JTA amplitude (channel 18) is marked with a black asterisk and the closest channel (channel 9) is marked with a red asterisk. I: extracellular JTA time courses for each channel are overlaid and colored according to the scheme in H. A simultaneous extracellular recording was made with the 32-channel probe illustrated in Fig. 2D, allowing us to specifically characterize the extracellular signature of an action potential generated by the juxtacellular recorded neuron. The band-pass filtered extracellular traces, ordered according to the electrode's geometry, are presented in Fig. 2E and correspond to the same time window as the juxtacellular recording (Fig. 2B). A short time window (4 ms) extracted from the extracellular trace around each detected juxtacellular event (occurrence of the action potential positive peak) for one extracellular channel is shown in Fig. 2F. Despite the low amplitude, a clear extracellular signature of the juxtacellular recorded neuron's spike can be recovered by averaging windows across multiple events. This juxtacellular triggered average (JTA) can be computed for all channels, allowing a high signal-to-noise estimate of the spatiotemporal distribution of the EAP. The JTA peak-to-peak amplitude for each channel interpolated within the electrode site geometry, sometimes called “the cell footprint” (Delgado Ruz and Schultz 2014), is shown in Fig. 2G. The JTA waveforms for each channel are shown, arranged using the relative probe spacing in Fig. 2H and overlaid in Fig. 2I. The example presented in Fig. 2 is from one paired juxtacellular and extracellular recording. Several recordings were made in a similar manner and we next examined the variety of extracellular signatures obtained for different neurons at different positions relative to 32 and 128-channel dense polytrodes. Distance Dependence of Extracellular Signal Amplitude Following a stable juxtacellular recording we were sometimes able to move the extracellular probe and obtain another recording configuration/distance for the same neuron. The relationship between extracellular signal amplitude and distance from the probe for 35 such recording configurations, obtained from 23 neurons, is shown in Fig. 3 (we also included in the dataset 3 paired-recordings with distances >200 μm). Across all of our paired-recordings, the distance between a neuron and the extracellular electrodes was the major factor determining the peak-to-peak extracellular signal amplitude. Fig. 3. Distance dependence of extracellular signal amplitude. The maximum peak-to-peak amplitude of the JTAs (±1 ms of the alignment time) across all extracellular channels for each paired-recording vs. the distance between the closest extracellular electrode and the juxtacellular pipette tip. Horizontal error bars report uncertainty in position estimate (±10.5 μm). The gray shaded region indicates a 5-μV threshold for excluding possible cross-talk electrical artifacts between the extra- and juxtacellular recording electronics. Large peak-to-peak amplitudes were only observed for neurons <50 μm from the nearest electrode, which is in accordance with previous measurements in hippocampus (Henze et al. 2000) and some theoretical models (Somogyvári et al. 2012). For neurons encountered within 50 to 150 μm of the probe surface, the magnitude of the neuron's extracellular signal ranged from 38 to 5 μV. All neurons encountered with a distance ≥150 μm did not show a canonical spike waveform, even after averaging, and we occasionally detected a small artifact (<5 μV at 0 ms) that was similar for all channels and likely due to cross talk between the extracellular and juxtacellular recording electronics. Nevertheless, we include these distant cells in the dataset since they could potentially be used to better understand the spike-local field potential relationship (Lewis et al. 2015; Berényi et al. 2014). Detection of the Juxtacellular Spikes on the Extracellular Probe The first step in the analysis of extracellular data is the identification of discrete spike events (Hazan et al. 2006). Therefore, to use paired recordings to evaluate algorithms for assigning these extracellular events to clusters belonging to distinct neurons (i.e., spike sorting), one must be able to detect the juxtacellular spike on the extracellular electrodes. We used a popular spike detection algorithm, SpikeDetekt, which extracts action potentials as spatiotemporally localized events (Rossant et al. 2016), to identify all spikes visible to our extracellular probe. SpikeDetekt uses a high threshold to detect spikes on a single channel and then a lower threshold to associate neighboring channels (using a flood-fill algorithm) that sense the same spike. We used the same detection parameters for our entire dataset: third-order Butterworth, forward-backward mode, band-pass filter (500-14,250 Hz) and strong and weak threshold levels of 4.5 and 2 times the standard deviation, respectively. The juxtacellular spike times were determined as the peaks of well-isolated threshold crossings (see Supplemental Material Table S1 for the threshold values used for each individual paired-recording). This spike detection process is illustrated in Fig. 4, A–C, for a data segment containing the contribution of a neuron that was simultaneously recorded with the juxtacellular pipette (2014_11_25_Pair3.0). To compare the extracellularly detected event times with the spike times observed in the juxtacellular recording, we generated a peri-event time histogram (PETH) using all spike events found by SpikeDetekt on the extracellular channels aligned relative to each juxtacellular spike (Fig. 4, D and E). In some paired-recordings, these PETHs reveal a high probability of spike co-occurrence at 0 ms, indicating that the juxtacellular neuron's spike is being found by SpikeDetekt. The count value in the PETH 0-ms bin for the recorded pair in Fig. 4D (2014_11_25_Pair3.0, channel 18) suggests that all of the juxtacellular spikes were found but that this bin also includes detections of coincident spiking events occurring in the background neural activity (386 detected, 348 actual). These false positives could potentially be distinguished through sorting. In contrast, the PETH 0-ms bin for the pair in Fig. 4E (2014_03_26_Pair2.0, channel 9) indicates that only 23% of the total number of juxtacellular spikes were detected (35 detected, 150 actual), some of which are likely to also reflect coincident background events. We note that a larger number of putative juxta spikes could be recovered in this recording by reducing the high threshold level used by SpikeDetekt but this would also include more background events and likely complicate subsequent sorting. The two examples in Fig. 4 thus highlight both an “easy” and “hard” case for spike detection algorithms, which must be further developed in coordination with sorting algorithms, to better utilize the rich information provided by dense polytrodes. Fig. 4. Extracellular detection of the juxtacellular neuron's action potentials. Representative juxtacellular recording (A) and wide-band (B; 0.1–7,500 kHz) signal recorded simultaneously with a 32-channel silicon polytrode. C: on the highpass filtered extracellular data is visible the occurrence of temporally overlapping spikes on separated electrodes. The highlighted traces are expanded in the right panel and included black arrows to indicate all spikes identified by SpikeDetekt using standard thresholds and green arrows to indicate the time of juxtacellular spikes. D: peri-event time histograms of the extracellular spike events found by SpikeDetekt, relative to the juxtacellular spike times in 1-ms bins centered at 0 ms, are shown for each electrode channel at their relative position on the extracellular probe. The channel with the largest peak in the bin at 0 (±0.5 ms from the juxtacellular event) is indicated by asterisk and expanded at bottom. E: same presentation as in D but for a neuron with a smaller extracellular action potential. Spatiotemporal Structure of Extracellular Signatures Neurons near the polytrode surface exhibited a rich diversity of action potential waveforms (amplitude and dynamics) spread across multiple electrode sites (Fig. 6 and Supplemental Material Movies S3 and S4). This spatiotemporal structure will not only provide additional information for improving spike detection and sorting procedures, but may also reveal specific contributions from different parts of the neural membrane to this extracellular signature. For example, in Fig. 5C, the first negative peak (blue trace) in the extracellular potential is hypothesized to arise from currents in the distal axon initial segment (Shu et al. 2007; Hu et al. 2009) and the later peaks (purple and red traces) might then be due to the backpropagation to soma and dendrites (Buzsáki et al. 1998). The propagation velocity, estimated from the distance of the recording sites and the delay between the negative peaks (blue and red traces in Fig. 5C) was ∼0.55 m/s, which is in agreement with the value found in the literature for backpropagation of action potentials in cortical pyramidal cells 0.67 ± 0.11 m/s (Buzsáki et al. 1998). Another example of complex structure in the extracellular signature is seen in Fig. 5E, in this case the primary signal is localized to a small region of electrodes and varies greatly between neighboring sites, which are separated by only 2.5 μm. These examples, and others in our dataset, clearly suggest that the amount of useful spatiotemporal information captured by dense large-scale neural recording devices is promising (Buzsáki 2004), not only for improving algorithms that detect and sort events but also to identify cell types based on the morphology suggested by their extracellular signature. Fig. 5. Spatiotemporal structure of extracellular signatures. A: geometry and dimensions of the 32-channel electrode array. B: JTA waveforms (2014_11_25_Pair1.1) for all the extracellular electrodes are spatially arranged according to the probe geometry. C: expanded comparison of the JTA waveforms for the indicated electrodes with a line denoting the peak time of the juxtacellular spike. D–F: similar presentation as (A–C) for one 128-channel polytrode pair example (2015_09_04_Pair5.0). DISCUSSION In the present study, our dual-recording setup allowed precise targeting of both an extracellular probe and a juxtacellular pipette to the same position in cortex. The setup is low cost and easily implemented by any electrophysiology laboratory with two motorized (servo/stepper) micromanipulators. We hope that our description will instigate the collection of such critical cross-validation data from the forthcoming deluge of novel neural recording devices. Dataset for Cross-Validating Polytrodes and Spike Detection/Sorting Algorithms A summary of the current cross-validation dataset is presented in Fig. 6. It includes twenty juxta-extracellular pairs recorded with both 32- and 128-channel polytrodes, at a range of interprobe distances and depths in cortex (800 to 1,800 μm from the pial surface). Future experiments, which use cell-attached labeling to anatomically reconstruct the juxtacellular neuron following a paired-recording, are now being pursued to extend this validation dataset. However, the existing dataset already includes a number useful cross-validation examples: nearby cells with large EAPs, which will provide “ground truth” data for evaluating current spike detection and sorting algorithms, as well as more challenging intermediate cells, for which new algorithms, specifically optimized to use the additional information available to dense silicon polytrodes, may be able to recover. The full dataset, as well as probe maps and analysis code, is available online (http://www.kampff-lab.org/validating-electrodes/) and summarized in Supplemental Material Table S1. Fig. 6. Dataset for validating spike detection and sorting algorithms for dense polytrodes. A: spatial distribution of the peak-to-peak amplitude within a time window (±1 ms) surrounding the juxtacellular event and the indicated color code was used to display and interpolate these amplitudes throughout the 32-channel probe shaft. In addition, the extracellular JTA waveforms for all the extracellular electrodes are spatially arranged. B: same presentation as in A for paired-recordings with the 128-channel probe. Are We Missing Neurons with Extracellular Recording? An apparent discrepancy in the number of neurons that are reported to be active with optical (∼50%) vs. electrical (<10%) recording techniques has prompted a number of researches to ask whether extracellular recording is “missing something” (Buzsáki 2004; Shoham et al. 2006). Many explanations have been proposed (extracellular recording and sorting methods are biased to highly active neurons, some neuron types have weak or more localized extracellular signatures, etc.), yet estimating how many neurons an electrode should detect depends critically on knowing the volume of neural tissue to which an electrode is sensitive (i.e., from how far away can a neuron's spike be detected?). The literature is rather inconsistent, but there a number of reports of recording neurons extracellularly (>50 μV) from >100 μm away (Henze and Buzsáki 2007; Du et al. 2011), while others suggest, based on modeling (Gray et al. 1995; Shoham et al. 2006; Somogyvári et al. 2012; Delgado Ruz and Schultz 2014) and “ground truth” measurements (Henze et al. 2000; Anastassiou et al. 2015), that the limit is in fact closer to ∼50 μm. Our data are consistent with the latter estimates (the maximum distance at which we observed a large peak-to-peak amplitude spike was only 48 μm) but also suggest a possible explanation for these discrepant views. We propose that when an extracellular probe insertion is aligned with the axis of a pyramidal neuron's apical dendrite (or any neuron with an elongated morphology), then its EAP will be visible over a large distance, roughly matching the extent that the cell's dendritic arbor passes nearby the probe (Buzsáki et al. 1998). However, as the probe is positioned further away laterally from the neuron soma (perpendicular to the major axis of the neuron) then the EAP amplitude falls off steeply. All of our recordings used an extracellular array that was inserted parallel to the apical dendrites of cortical pyramidal neurons (Fig. 1B) and perpendicular to the cortical laminae. Therefore, if we were juxtacellularly recording from a pyramidal neuron whose soma was nearby the electrode surface and whose apical dendrite was aligned with the probe surface, then we would expect to detect a strong EAP across a large portion of the electrode surface, albeit with varying temporal waveforms (Figs. 5B and 6). However, if we juxtacellularly record from neurons whose soma are further from the probe surface, the size of the EAP on the probe surface will decrease rapidly (Fig. 3). This interpretation can explain why one neuron might occasionally be detected over hundreds of micrometers (i.e., that neuron's morphology happened to be aligned with the probe/insertion track), but still supports the conclusion that an extracellular electrode is primarily sensitive to neurons (and their processes) within a 50 μm radius. Given this limited sensitivity range, are we still missing neurons? Based on cellular density estimates for cortex (40,000 to 60,000 neurons/mm3; DeFelipe et al. 2003), and the half-spherical volume in front of a polytrode electrode, then we would expect each site to be sensitive to ∼10–15 neurons. These estimates are consistent with reported results for dense silicon polytrodes in cortex (Blanche et al. 2005). Our data thus suggest that there may not be a “dark neuron” problem but rather that extracellular electrodes are sensitive to a much smaller volume than is sometimes proposed. However, much more data, from different brain regions containing diverse cell types, will be required to resolve this critical issue. We propose that our new dual-recording method will make gathering such important validation data much, much easier. GRANTS This work was supported by funding from the European Union's Seventh Framework Programme (FP7/2007–2013) Grant Agreement 600925 and the FCT-MCTES Doctoral Grant SFRH/BD/76004/2011 (to J. P. Neto) and Bial Foundation Grant 190/12. Institutional support and funding was provided by the Champalimaud Foundation and Sainsbury Wellcome Centre (funded by the Gatsby Charitable Foundation and the Wellcome Trust). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). ENDNOTE At the request of the authors, readers are herein alerted to the fact that additional materials related to this manuscript may be found at the institutional website of one of the authors, which at the time of publication they indicate is: http://www.kampff-lab.org/validating-electrodes. These materials are not a part of this manuscript, and have not undergone peer review by the American Physiological Society (APS). APS and the journal editors take no responsibility for these materials, for the website address, or for any links to or from it. AUTHOR CONTRIBUTIONS J.P.N., G.L., J.F., and A.R.K. conception and design of research; J.P.N., J.F., J.N., and P. Baião performed experiments; J.P.N., J.F., P.L., and A.R.K. analyzed data; J.P.N., J.F., P.L., and A.R.K. interpreted results of experiments; J.P.N., J.F., and A.R.K. prepared figures; J.P.N. and A.R.K. drafted manuscript; J.P.N., G.L., J.F., P.L., A. Aarts, A. Andrei, S.M., E.F., P. Barquinha, and A.R.K. edited and revised manuscript; J.P.N. and A.R.K. approved final version of manuscript. Supplementary Material Movie 1 Movie 2 Movie 3 Movie 4 Table 1 ACKNOWLEDGMENTS We are very grateful to George Dimitriadis, Kinga Kocsis, and all the members of the Intelligent Systems Laboratory for contributions. We also thank the laboratory of Kenneth Harris and the members of the Bernstein Center in Freiburg for helpful discussions. ==== Refs REFERENCES Anastassiou CA , Perin R , Buzsáki G , Markram H , Koch C Cell type- and activity-dependent extracellular correlates of intracellular spiking . J Neurophysiol 114 : 608 –623 , 2015 .25995352 Berényi A , Somogyvári Z , Nagy AJ , Roux L , Long JD , Fujisawa S , Stark E , Leonardo A , Harris TD , Buzsáki G Large-scale, high-density (up to 512 channels) recording of local circuits in behaving animals . 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==== Front Front MicrobiolFront MicrobiolFront. Microbiol.Frontiers in Microbiology1664-302XFrontiers Media S.A. 10.3389/fmicb.2016.01349MicrobiologyOriginal ResearchCharacterization and Strain Improvement of a Hypercellulytic Variant, Trichoderma reesei SN1, by Genetic Engineering for Optimized Cellulase Production in Biomass Conversion Improvement Qian Yuanchao 1Zhong Lixia 2Hou Yunhua 3Qu Yinbo 1Zhong Yaohua 1*1State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong UniversityJinan, China2Shandong Institute for Food and Drug ControlJinan, China3Bioengineering Institute, Qilu University of TechnologyJinan, ChinaEdited by: Xueyang Feng, Virginia Tech, USA Reviewed by: Guang Zhao, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, China; Leqian Liu, University of California, San Francisco, USA *Correspondence: Yaohua Zhong, zhongyaohua@sdu.edu.cnThis article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology 29 8 2016 2016 7 134914 6 2016 16 8 2016 Copyright © 2016 Qian, Zhong, Hou, Qu and Zhong.2016Qian, Zhong, Hou, Qu and ZhongThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.The filamentous fungus Trichoderma reesei is a widely used strain for cellulolytic enzyme production. A hypercellulolytic T. reesei variant SN1 was identified in this study and found to be different from the well-known cellulase producers QM9414 and RUT-C30. The cellulose-degrading enzymes of T. reesei SN1 show higher endoglucanase (EG) activity but lower β-glucosidase (BGL) activity than those of the others. A uracil auxotroph strain, SP4, was constructed by pyr4 deletion in SN1 to improve transformation efficiency. The BGL1-encoding gene bgl1 under the control of a modified cbh1 promoter was overexpressed in SP4. A transformant, SPB2, with four additional copies of bgl1 exhibited a 17.1-fold increase in BGL activity and a 30.0% increase in filter paper activity. Saccharification of corncob residues with crude enzyme showed that the glucose yield of SPB2 is 65.0% higher than that of SP4. These results reveal the feasibility of strain improvement through the development of an efficient genetic transformation platform to construct a balanced cellulase system for biomass conversion. Trichoderma reeseiuracil auxotrophyβ-glucosidasebiomass conversionpretreated corncob residuesNational Natural Science Foundation of China10.13039/501100001809No. 31370135 ==== Body Introduction Lignocellulosic biomass, which includes agricultural crop and forest residues, is the most abundant renewable resource on earth (Singhania et al., 2013). It is considered a promising alternative to fossil fuel because it can be converted to second-generation biofuels, such as bioethanol (Gusakov, 2013). However, the relatively high cost of cellulase preparation for cellulose deconstruction is a major obstacle that limits the large-scale commercial production of bioethanol with lignocellulosic biomass as the substrate (Wu et al., 2011). Most of the current cellulase preparations involve the use of filamentous fungi, such as Trichoderma reesei, but the ability to produce cellulase through the use of a wild strain is not always fit for industrial requirements (Weiss et al., 2013). Therefore, different well-known hypercellulolytic mutants, such as T. reesei QM9414 and RUT-C30, have been developed through classical mutation (Montenecourt and Eveleigh, 1977, 1979). Compared with random mutation, genetic modification provides a rational strategy for strain improvement. However, the main limitation of this strategy is the selection of a proper marker for genetic transformation. Diverse selection mechanisms, including auxotroph complementation (Penttilä et al., 1987), antibiotic resistance (Bailey et al., 2002), and nitrogen utilization (Te’o et al., 2002), have been applied to filamentous fungi. Auxotrophic markers, especially the mutation of the orotidine 5′-monophosphate decarboxylase gene (pyr4/G or ura3) that leads to uracil auxotrophy, are widely applied in fungal transformation because of their high efficiency and ease in obtaining transformants (Smith et al., 1991). Moreover, a pyr4 blaster cassette containing a direct repeat sequence can be recycled by screening with 5′-FOA as the counter selection for multiple gene operations (Hartl and Seiboth, 2005). Evidently, construction of a uracil auxotroph strain derived from a cellulase hyperproducing mutant could facilitate genetic modification and strain improvement. Trichoderma reesei is one of the most popular industrial filamentous fungi utilized for cellulase production. Its cellulase system cannot be present in optimal ratios for the deconstruction of lignocellulosic substrates, because it was originally isolated from decayed canvas (Mandels and Reese, 1957; Gomes et al., 1992). The well-known T. reesei mutants, including QM9414 and RUT-C30, were generated through random mutation with only cellulose as the screening substrate. Hence, their cellulase preparations are unfeasible for large-scale production and conversion of lignocellulosic materials into bioethanol. Given their capacity to secrete high-level cellulases, T. reesei strains still occupy the dominant position as a suitable starting point for the construction of engineered strains used to produce highly active and economically feasible cellulases for the bioethanol industry (Rosgaard et al., 2006). The T. reesei cellulase system is composed of endoglucanase (EG) and cellobiohydrolase (CBH), which jointly hydrolyze cellulose to produce cellooligosaccharide and cellobiose (Teugjas and Väljamäe, 2013). These products are further degraded into glucose by β-glucosidase (BGL) to complete the rate-limiting step of cellulose hydrolysis (Sørensen et al., 2013). However, the low amount of secreted BGL (i.e., BGL1) usually leads to incomplete cellobiose hydrolysis, which in turn inhibits the product of the T. reesei enzyme system (Rosgaard et al., 2006). Hence, various strategies, such as exogenous addition of BGLs (Duenas et al., 1995), co-cultivation of high-BGL activity strains with T. reesei (Gutierrez-Correa et al., 1999), and genetic transformation of T. reesei with homologous and heterologous bgl genes, have been proposed to supplement BGL activity (Zhang et al., 2010; Ma et al., 2011; Treebupachatsakul et al., 2015). In our previous work, a hypercellulolytic variant SN1 was isolated on Carboxy Methyl Cellulose (CMC) agar plates and also used for cellulase production. Here, the strain SN1 was identified as T. reesei and characterized with high endoglucanase activity. For strain improvement, a uracil auxotroph strain was constructed by deleting the pyr4 gene via homologous recombination in SN1. The β-glucosidase gene was further overexpressed by using the strong engineered cbh1 promoter to optimize the cellulase system for the saccharification of different pretreated corncob residues. Materials and Methods Strains, Plasmids, and Culture Conditions Trichoderma reesei QM9414 (ATCC 26921), which is a general cellulase producer, and RUT-C30 (ATCC 56765), which is another cellulase high-producing strain that is less sensitive to glucose repression, were utilized as the control strains for the comparison of cellulase production. The strain SN1 is a hypercellulolytic variant isolated from our laboratory (Song et al., 2016) and used as the initial host for strain improvement. Escherichia coli DH5a (TransGen, Beijing, China) was used for vector construction and propagation. The pMD18-T cloning vector (Takara, Otsu, Japan) was purchased for TA cloning. The pTHB vector was used for bgl1 overexpression; this vector contained a T. reesei bgl1 expression cassette under the control of a modified four-copy cbh1 promoter, as described by Zhang et al. (2010). The plasmid pAB4-1 contained the Aspergillus niger pyrG gene as a selection marker to encode orotidine-5′-phosphate decarboxylase (Hartl and Seiboth, 2005). All strains were grown and maintained on a potato dextrose agar plate (PDA) containing 20.0 g/L D-glucose and 20.0 g/L agar for 5–7 days at 30°C. The conidia were harvested, and 106 conidia were inoculated in a 500 mL flask containing 150 mL minimal medium (MM; Penttilä et al., 1987). MM with 300 μg/mL hygromycin B and 1.5 mg/mL 5′-FOA (Sigma, USA) was applied as the selective medium to screen the uracil auxotroph transformants (Singh et al., 2015). An esculin plate containing 3 g/L of esculin, 10 g/L of sodium carboxymethy cellulose (CMC–Na), 0.5 g/L of ferric citrate, and 20 g/L agar was utilized to screen the strains showing β-glucosidase (BGL) activity. A CMC plate containing 10 g/L of CMC–Na, 1 g of yeast extract, and 20 g/L agar was utilized to screen the strains showing cellulase (EG) activity. Morphology and Molecular Identification The fungal strains were grown on PDA plates or solid MM with 2% glucose, 2% glycerol, or 2% lactose as the carbon sources. Photographs of the colonies were obtained with a SONY DSC-HX400 camera for morphology comparison. Microscopic images were captured with a Nikon Eclipse80i light microscope (Nikon, Japan). The fungal genomic DNA was isolated according to the method of Penttilä et al. (1987). For phylogenetic identification of the variant strain SN1, 18S and internally transcribed spacer (ITS) rDNA gene fragments were amplified through polymerase chain reaction (PCR) with the universal primer pairs 18S-F1/18S-R1 and ITS-F1/ITS-R1 (Table 1; White et al., 1990), respectively. The SN1 genomic DNA was utilized as the PCR template. The amplified products were purified, sequenced, and compared with the sequences of fungal rDNA 18S and ITS from the NCBI database through the use of the BLAST algorithm. Multiple sequence alignment was conducted with ClustalX. Maximum composite likelihood method analysis was conducted with the MEGA 5 software. Table 1 Primers used in this study. Primers Sequences (5′–3′) Employment 18S-F1 TTCCAGCTCCAATAGCGTAT Strain identification 18S-R1 CAGACAAATCACTCCACCAAC Strain identification ITS-F1 TCCGTAGGTGAACCTGCGG Strain identification ITS-R1 TCCTCCGCTTATTGATATGC Strain identification pyr4-UF1 AGTGTTTGATGCTCACGCTC Mutant construction pyr4-UR1 GGAGATGTTGCTGAAGTCGA GGCGAGGGAGTTGCTTTA Mutant construction pyr4-DF1 ATGAGTCGTTTACCCAGAATAAG AAAGGCATTTAGCAAGA Mutant construction pyr4-DR1 TGAACAGTAAGGTGTCAGCA Mutant construction pyr4-UF2 TGATGCTCACGCTCGGAT Mutant construction pyr4-DR2 TCGTCTCGTTCAGCTCGTAATC Mutant construction Ypyr4-UF1 ACACAACCTACTGAGCAGAACC Mutant construction Yhph-F2 GTCTGGACCGATGGCTGTG Mutant construction Hph-F1 CGACTTCAGCAACATCTCC Mutant construction Hph-R1 ATTCTGGGTAAACGACTCAT Mutant construction Yhph-F1 GCAAAGTGCCGATAAACA Mutant construction Yhph-R1 GCGAAGGAGAATGTGAAG Mutant construction pyrG-S CTTCCTAATACCGCCTAGTCAT Mutant construction pyrG-A AGCCGCTGGTCAATGTTATC Mutant construction YpyrG-F1 ATCAACACCATGTCCTCCAA Mutant construction YpyrG-R1 ACACGAATCCCATAACGAAG Mutant construction Y1 GCCAGGGATGCTTGAGTGTA Mutant construction Y2 CCCAGCCACAGGACCAAGTATG Mutant construction pyr4-probe-F GCCATCCTCCTTCTTCCTCT Probe pyr4-probe-R TGATACACACAAGTCTGCCAGAT Probe bgl-probe-F TTGAGCCCAATCAGAAATGCGT Probe bgl-probe-R CCCAGCCACAGGACCAAGTATG Probe Construction of the pyr4 Deletion Strain The Δpyr4::hph cassette for pyr4 gene disruption was constructed with the double-joint PCR method (Yu et al., 2004). The cassette carried the E. coli hygromycin B phosphotransferase (hph) gene as the selection marker between the 5′- and 3′-flanking regions of pyr4. Specifically, the pyr4 5′- and 3′-flanking sequences were amplified with two primer pairs, pyr4-1500-UF1/pyr4-72-UR1 and pyr4-60-DF1/pyr4-1540-DR1 (Table 1), respectively, with the SN1 genomic DNA as the template. The hygromycin B phosphotransferase gene (hph) was amplified with hph-F1/hph-R1 primers (Table 1). The amplicons were then mixed in 1:3:1 molar ratio of 5′-flanking region:hph:3′-flanking region and jointed together during another round of PCR without primers. The resulting PCR product was used directly as a template for the third round of PCR to construct the pyr4 disruption cassette with the nest primers pyr4-1400-UF2/pyr4-1488-UR2 (Table 1). The pyr4 disruption cassette was transformed into SN1 protoplasts afterward through the method described by Penttilä et al. (1987). The target transformants were screened on MM containing 300 μg/mL hygromycin B and 1.5 mg/mL 5′-FOA. The purified candidate transformants were identified through PCR and Southern blot with the genomic DNA as the template to verify if the pyr4 locus was replaced by the deletion cassette. Overexpression of the β-Glucosidase bgl1 Gene in T. reesei SP4 The vectors pTHB and pAB4-1 were co-transformed into protoplasts of T. reesei SN1 through the above mentioned method. The transformants were directly screened on MM. The candidates were then verified through PCR by using primers Y1 and Y2 (Table 1), as described by Zhang et al. (2010). Esculin agar plates were prepared and utilized to confirm the bgl1 overexpression strain. Southern Blot Analysis The probe of pyr4 was a fragment amplified through PCR by using primers pyr4-probe-F and pyr4-probe-R to detect the pyr4 gene. The XbaI/HindIII-digested genomic DNA was hybridized by the pyr4 probe. The probe of bgl1 was a fragment amplified through PCR by using primer bgl1-probe-F/bgl1-probe-R to detect the bgl1 gene. The EcoRI-digested genomic DNA was hybridized by the bgl1 probe. The probe-hybridized DNA fragment was detected with the DIG High Prime DNA Labeling and Detection Starter Kit I (Roche Diagnostics, Mannheim, Germany). Shake Flask Cultivation for Cellulase Production The seed cultures for cellulase production were prepared in MM after incubation at 200 rpm and 30°C for 36 h in a 300 mL flask. Then 15 mL of the activated cells was transferred to 150 mL of the cellulase production medium (CPM) in a 500 mL flask. The CPM composition was as follows (g/L): 20 Microcrystalline cellulose, 2.5 (NH4)2SO4, 5 KH2PO4, 0.6 MgSO4.7H2O, 1 CaCl2.2H2O, and 20 corn steep liquor. Cellulase Activity Assay and Zymogram Analysis of β-Glucosidase Activity The filter paper (FP), EG, CBH, and BGL activities of T. reesei were measured as described before (Ghose, 1987) by using Whatman No. 1 FP (Whatman, UK), CMC–Na (Sigma, USA), p-nitrophenyl-β-D-cellobioside (pNPC; Sigma, USA), and p-nitrophenyl-β-D-glucopyranoside (PNPG; Sigma, USA) as the substrates, respectively. One unit of enzyme activity was defined as the amount of enzymes releasing 1 mole of reducing sugar (or p-nitrophenol in the CBH and BGL assays) per minute under the assay conditions. Renaturing SDS–PAGE electrophoresis was performed in 12% polyacrylamide separating gel containing 0.1% SDS with 0.3% CMC–Na as the substrate; this procedure was performed in a Mini-PROTEAN tetra electrophoresis cell (Bio-Rad Laboratories, Milan, Italy). Saccharification of the Pretreated Corncob Residues Acid-pretreated and delignined corncob residues were used as substrates in the saccharification process; their composition has been described by Zhao et al. (2008) (Supplementary Table S1). The cellulase crude complexes for the saccharification of the pretreated corncob residues were placed in 100 mL flasks containing 5% (w/v) substrate. The pH value and temperature were adjusted to 4.8 (with 50 mM citric acid buffer) and 50°C, respectively. Enzyme loading was adjusted to the same FP activity (10 FPU/g substrates). Glucose production was detected with an SBA-40C biological sensor analyzer (BISAS, Shandong, China) after incubation for 24 or 48 h. Cellulose conversion was calculated as follows: Cellulose⁢ conversion=Glu⁢cose⁢ yields(mg)Substrate⁢ weight(mg)××0.9×100% Cellulose⁢ content⁢ (%) Results Molecular and Morphological Identification of Cellulolytic Strain SN1 The cellulase production variant SN1 was identified with partial 18S rDNA and ITS sequences to characterize its phylogenetic relationships. Both the 18S rDNA gene and ITS sequences of SN1 showed 100% identity to those of T. reesei (Supplementary Figure S1). Hence, the strain SN1 was defined as a variant of T. reesei. Trichoderma reesei is a cellulolytic filamentous fungus originally isolated in the Solomon Islands during World War II. T. reesei QM9414 and RUT-C30 are among the most well-known high cellulase-producing mutants after 30 years of strain improvements (Peterson and Nevalainen, 2012). They are utilized as control strains in the current study for comparison with SN1 to analyze growth in different carbon sources. All strains were cultured on plates containing 2% glucose, 2% glycerol, and 2% lactose and on PDA plates for 4 days. As shown in Figure 1A, SN1 exhibited a colony morphology similar to that of QM9414 and RUT-C30 on glucose, glycerol, and lactose but showed increased radial growth and reduced pigment production on the PDA plates. The growth rate of SN1was comparable to that of QM9414 but higher than that of RUT-C30 on all the growth media tested in this study (Figure 1B). Microscopic observations revealed that SN1 had a spore morphology similar to that of the others but possessed a less-branched mycelium (Supplementary Figure S2). In summary, the strain SN1 is a novel T. reesei variant with high EG activity. FIGURE 1 Growth of Trichoderma reesei SN1, QM9414 and RUT-C30 on different carbon sources. (A) Strains were grown on plates containing minimal medium supplemented with 2% glucose, 2% glycerol, 2% lactose, or PDA at 30°C for 3 days. (B) The growth was represented by the relative growth rate which was measured as the colony diameter increase per centimeter per day. Cellulase Production and Its Cellulolytic Potential in Biomass Saccharification by T. reesei SN1 To investigate the capacity to secrete cellulase, the fungal strains were cultured on media containing CMC as the substrate, which is generally used to detect EG activity. The media plate was dyed with 1% congo red and decolored with 1 M NaCl after 3 days of cultivation. The cellulolytic halo around the SN1 colony was much larger than those of RUT-C30 and QM9414 (Figure 2A). This result indicates that T. reesei SN1 probably secreted high-level EG. Another medium containing CMC and esculin as the substrate was applied to test the ability to secrete β-glucosidase (Figure 2B). The size of the black halo around the SN1 colony was smaller than that of QM9414, suggesting that T. reesei SN1 secreted a relatively low level of BGL. T. reesei SN1, QM9414 and RUT-C30 were fermented in CPM to further verify cellulase production. The culture supernatants were also used for the cellulase activity assay. As shown in Figure 2C, T. reesei SN1 produced a total cellulase activity (FPA) comparable to that of QM9414 but exhibited a notably higher (nearly two times) EG activity than QM9414 and RUT-C30. Comparison of CBH and BGL activities showed that SN1 also produced a CBH activity similar to that of QM9414 but had a much reduced (almost half) BGL activity (Figure 2D). These results confirm that T. reesei SN1 is a novel hypercellulytic strain that produces prominent EG activity but minimal BGL activity. These results prompted us to examine the saccharification potential of the SN1 enzyme to convert cellulosic materials. The experimental design involved the enzymatic hydrolysis of two types of pretreated corncob residues. This hydrolysis was performed at 50°C and pH of 4.8 for 48 h. In the saccharification of the acid-pretreated corncob residues, the amount of glucose released using the SN1enzyme at 24 h was only 5.3 mg/mL (i.e., 15.2% cellulose conversion), which was lower than the values for QM9414 (6.3 mg/mL corresponding to 17.9% cellulose conversion) and RUT-C30 (6.4 mg/mL corresponding to 17.9% cellulose conversion). The glucose releases in SN1 (12.0 mg/mL corresponding to 34.4% cellulose conversion) and QM9414 (12.1 mg/mL corresponding to 34.8% cellulose conversion) were similar after an additional 24 h of enzymatic hydrolysis (i.e., after a total enzymatic reaction of 48 h). However, they were still lower than that in RUT-C30 (13.3 mg/mL corresponding to 38.4% cellulose conversion; Figure 3A). When the delignined corncob residues were used as the substrate, the final glucose release in SN1 (13.4 mg/mL corresponding to 36.7% cellulose conversion) after a 48 h reaction was still lower than those in QM9414 (15.3 mg/mL corresponding to 42.0% cellulose conversion) and RUT-C30 (16.2 mg/mL corresponding to 44.4% cellulose conversion; Figure 3B). Therefore, T. reesei SN1 exhibited outstanding EG activity. However, its saccharification efficiency for cellulosic materials was obviously lower than those of QM9414 and RUT-C30 probably because of the insufficiency of β-glucosidase in the SN1enzyme system. FIGURE 2 A comparison of cellulase production in T. reesei SN1, QM9414 and RUT-C30. (A) The endoglucanase production of T. reesei SN1, QM9414 and RUT-C30 on the condition of 1% CMC-Na with 0.5% triton at 30°C for 2 days. (B) Detection of β-glucosidase activity on the CMC-esculin plate in T. reesei SN1, QM9414 and RUT-C30 at 30°C for 24 h. (C) The FPA and EG activities of the T. reesei SN1, QM9414 and RUT-C30, were measured after being induced in cellulase fermentation medium for 5 days. (D) The CBH and BGL activities of the T. reesei SN1, QM9414 and RUT-C30, were measured as the above mentioned. All results are represented as the mean of three independent experiments; error bars express the standard deviation. FIGURE 3 Saccharification of different pretreated concob residues by T. reesei SN1, QM9414 and RUT-C30. (A) Saccharification of acid pretreated corncob residues with equal FPA activity. (B) Saccharification of delignined corncob residues. Data are represented as the mean of three independent experiments; error bars express the standard deviations. Generation of a pyr4 Disruption Strain from T. reesei SN1 The uracil auxotroph strain maximizes high-efficiency genetic transformation and can be used for multiple gene manipulations depending on the pyr4 marker gene that can be bidirectionally selected (Seidl and Seiboth, 2010). Thus, disruption of the pyr4 gene in T. reesei SN1 to construct the uracil auxotroph strain will contribute to strain improvement through genetic modification. In this study, a Δpyr4::hph cassette was constructed and utilized to disrupt the pyr4 gene in T. reesei SN1 (Figure 4A). The generated uracil auxotroph strain SP4 was confirmed through plate screening. As shown in Figure 4B, SP4 could normally grow on MM containing 0.1% uracil but could not grow on MM without uracil. The XbaI/HindIII-digested genomic DNA was hybridized with the pyr4 probe for further Southern blot assay. It yielded a 6.0 kb fragment for the strain SP4 and a 4.7 kb fragment for the parent strain SN1 (Figure 4C). These results suggest that the pyr4 gene was successfully knocked out in SP4. Furthermore, comparison of the cellulase production of SP4 and SN1 showed that the cellulase activities of the SP4 crude enzyme preparations were almost similar to those of SN1 (data not shown). FIGURE 4 Deletion of pyr4 gene in T. reesei SN1. (A) Schematic representation of the pyr4 locus in the Δpyr4 and parent strains. Relative positions of the XbaI/HindIII enzyme restriction sites are noted. Probe used for Southern analysis is shown as red box. (B) Phenotypic assay on MM supplemented with uracil (left) and MM without uracil (right) at 30°C for 2 days. (C) Southern blot of the genome digested with XbaI/HindIII. A fragment of 4.7 kb is present in the parent strain, the 6.0 kb band is shown in Δpyr4 strains. Overexpression of bgl1 under the Control of the Artificial Four-Copy cbh1 Promoter in T. reesei SP4 To overcome the lack of BGL in the SP4 enzyme system, the pTHB plasmid containing the bgl1 gene under the control of a strong modified cbh1 promoter, as described by Zhang et al. (2010) (Figure 5A), was co-transformed with the A. niger pyrG-containing plasmid pAB4-1 into T. reesei SP4. The 126 positive transformants were all further screened on esculin plates to test the BGL secretion (data not shown). This esculin method has been widely used to screen strains showing BGL activity (Lee et al., 2014). Two of the transformants, namely, SPB1 and SPB2, showed much larger black zones around the colony than the parental strain SP4 (Figures 6A,B). They were then selected and verified through PCR amplification of the bgl1 gene using the primers Y1 and Y2 (Figure 5B). For further analysis by Southern blot, the EcoRI-digested genomic DNA was hybridized by the bgl1 probe and yielded a 1.8 kb fragment for the parent strain SP4 (Figure 5C). An additional 5.0 kb band was observed for SPB1, and four additional bands (2.3, 4.5, 5.0, and 5.5 kb) were found for SPB2. These results suggest that one and four bgl1 copies were successfully integrated into the genomes of SPB1 and SPB2, respectively. FIGURE 5 Overexpression of bgl1 gene in the Δpyr4 mutant. (A) An expression cassette containing the bgl1 gene is controlled by the T. reesei modified cbh1 promoter. Relative positions of the EcoRI enzyme restriction sites are noted. Probe used for Southern analysis is shown as red box. (B) PCR assay of the BGL overexpression transformants. Both the transformants SPB1 and SPB2 show a 1.0 kb DNA fragments panning using the primers Y1 and Y2, M: DNA marker. (C) Southern blot of the genome digested with EcoR1. The 1.8 kb (red line) wild-type band is present in all the strains. The additional bands indicate the bgl1-overexpression cassette was successfully integrated into the chromosome. FIGURE 6 Phenotypic and cellulase production assay of bgl1 overexpression transformants. (A) The black halo was observed on the transformants SPB1 and SPB2 at the 12 h. (B) The black halos were observed on the transformants SPB1 and SPB2 at the 24 h. (C) Detection of β-glucosidase activity from the culture-free supernatant of T. reesei SN1, SPB1and SPB2 performed on 12% SDS–PAGE containing 0.5% esculin as substrate. (D) BGL, FPA, EG, CBH activities were measured after being induced in cellulase fermentation medium for 5 days. Data are represented as the mean of three independent experiments; error bars express the standard deviations. Characterization of Cellulase Production by the bgl1-Overexpression Strains The bgl1-overexpression strains were cultivated in a CPM for 5 days. The culture supernatants were collected for the cellulase activity assay. First, renaturing SDS-PAGE analysis on 12% polyacrylamide gel containing 0.3% esculin as a substrate was applied to detect the BGL secreted by the fungal strains. Figure 6C shows a clear band in the secreted proteins of transformant SPB1 or SPB2 but not in SP4. This result indicates that the two bgl1-overexpression strains are very likely to secrete a much higher level of BGL than the parental strain. In accordance with this result, the BGL activities of SPB1 and SPB2 measured with pNPG as a substrate were 4.7 and 17.1 times higher than that of SP4, respectively (Figure 6D). FP activity (FPA) is generally utilized to evaluate the total cellulase activities. The FPA of SPB1 and SPB2 showed 39.7 and 22.6% increase compared with that of the parent strain SP4 (Figure 6D). However, the EG and CBH activities of the two transformants were not significantly changed, besides a relatively lower CBH activity (10.0% decrease) in SPB2 (Figure 6D). Interestingly, the highest β-glucosidase activity (8.3 U/mL) was found in SPB2; the value was 3.2-fold higher than that of SPB2 (2.6 U/mL). However, the FPA in SPB2 (2.6 U/mL) was lower than that in SPB1 (3.0 U/mL). This difference in FPA in the two transformants may have been caused by the negative effect of the additional four copies of cbh1 promoters in the SPB2. It was reported that multiple copies of promoters might result in the titration effect of transcriptional regulators for cellulase gene expression (Rahman et al., 2009; Zhang et al., 2010). Saccharification of the Corncob Substrates by Cellulase Preparations from T. reesei SPB1 and SPB2 The ability of the cellulase preparations produced by T. reesei SPB1 and SPB2 to hydrolyze the pretreated corncob residues was investigated. In the saccharification of the acid-pretreated corncob residues, the glucose release (12.6 mg/mL corresponding to 36.2% cellulose conversion) using the SPB1 enzyme was slightly higher than that for SP4 (12.1 mg/mL corresponding to 34.4% cellulose conversion) but lower than that for RUT-C30 (13.3 mg/mL corresponding to 38.4% cellulose conversion) after a total enzymatic reaction of 48 h (Figure 7A). The SPB2 enzyme exhibited the highest glucose release (15.5 mg/mL corresponding to 44.6% cellulose conversion). This result indicates that the high BGL activity in the SPB2 enzyme contributed to the efficient hydrolysis of the acid-pretreated corncob residues. The final glucose yields of SPB1 (17.6 mg/mL corresponding to 48.2% cellulose conversion) and SPB2 (22.2 mg/mL corresponding to 61.0% cellulose conversion) after 48 h reaction were higher than those of SP4 (13.4 mg/mL corresponding to 36.7% cellulose conversion) and RUT-C30 (16.2 mg/mL corresponding to 44.4% cellulose conversion) when the delignined corncob residues were used as a substrate (Figure 7B).Therefore, both bgl1-overexpression strains (SPB1and SPB2) exhibited better performance than the parental strain SP4 regardless of whether acid-pretreated or delignined corncob residues were used as the substrate. These results suggest that BGL activity has a notable positive correlation with cellulose conversion and indicates that β-glucosidase plays a key role in the enzymatic hydrolysis of corncob residues. FIGURE 7 Saccharification of different pretreated concob residues by the BGL-overexpression strains. (A) Saccharification of acid pretreated corncob residues by T. reesei SP4 and the BGL-overexpresion strains SPB1 and SPB2 with equal FPA activity. (B) Saccharification of delignined corncob residues T. reesei SP4 and the BGL-overexpresion strains SPB1 and SPB2 with equal FPA activity. Data are represented as the mean of three independent experiments; error bars express the standard deviations. Discussion The application of cellulolytic microorganisms to cellulosic biomass bioconversion is considered a potential sustainable approach to develop novel bioproducts, such as biofuels (Premalatha et al., 2015). Diverse cellulolytic microorganisms, including fungi and bacteria, have been isolated from various environments (Jyotsna et al., 2010; Raddadi et al., 2013; Dantur et al., 2015; Premalatha et al., 2015). However, the ability to produce cellulose-degrading enzymes by using wild strains is not always fit for industrial requirements (Weiss et al., 2013). Hence, much effort has been exerted to mutate or engineer potential strains for application in the cellulase industry. In this study, a hypercellulolytic fungus, SN1, was identified as a T. reesei variant according to morphological characteristics and ribosomal DNA sequences. Trichoderma reesei was initially isolated from decayed canvas during World War II and is currently one of the most well-known industrial fungi used for cellulase production (Seidl et al., 2008). Several mutants with high cellulase activity, such as NG-14, QM9414, and RUT-C30, have been obtained after more than half a century of strain improvement. Among these mutants, QM9414 and RUT-C30 are the most widely used in cellulase production and as model strains for basic research on cellulase expression (Stricker et al., 2006; Gyalai-Korpos et al., 2011). Therefore, the SN1 strain was compared with these two mutants for hyphal growth and cellulase production in this study. The growth rate of SN1 was comparable to that of QM9414 but was faster than that of RUT-C30. SN1 produced remarkably reduced pigment and sporulated earlier (Figure 1). RUT-C30 can produce approximately 1.4 times the total cellulase activity of QM9414 but only 50% of the β-glucosidase activity of QM9414 (Peterson and Nevalainen, 2012). In this study, SN1 showed a total cellulase activity comparable to that of QM9414 and a notably higher (nearly two times) EG activity than both QM9414 and RUT-C30 (Figure 2). Considering that our lab studied T. reesei for years, and this strain is quite different from QM9414 (carbon catabolite-repressed, containing the full-length cre1 gene) and RUT-C30 (carbon catabolite-derepressed, containing the truncated cre1 gene) whether on morphological phenotype or cellulase production, so we further tested whether the SN1 strain was carbon catabolite-derepressed and found that it was carbon catabolite-repressed and contained a complete carbon catabolite repressor gene cre1 (data not shown). Therefore, we speculated that SN1 was a novel T. reesei variant, which was most probably spontaneously mutated from QM9414 due to the cellulose substrates used as the continued screening pressure. Many studies have shown that EGs can be widely used in industrial activities, such as pulp beatability (Kamaya, 1996), deinking (Gübitz et al., 1998), and bio-polishing (Li et al., 2011). The high-level endoglucanase production showed that SN1 is a potential strain for industrial cellulase production. However, despite the feature of SN1 cellulases, the respective activities in the cellulase products appeared to be present in suboptimal ratios for lignocellulose degradation. Hence, improvement of the cellulolytic capacity of T. reesei SN1 is required. This enhancement is often accomplished through genetic engineering. Auxotrophy selection markers, such as uracil auxotrophy, have been proven highly efficient for T. reesei genetic transformation (Hartl and Seiboth, 2005; Steiger, 2013). T. reesei TU6 is an auxotrophic strain widely used for its high transformation efficiency (Gruber et al., 1990). However, TU6 was obtained through random γ-radiation mutagenesis from QM9414, resulting in slow growth, decreased conidiation, and reduced cellulase production. Therefore, a new uracil auxotrophic strain, SP4, was constructed in this study through targeted gene deletion from a hypercellulolytic strain (SN1). No discernible difference in growth, conidiation, or cellular morphology was observed between SP4 and its parental strain SN1 (data not shown). Furthermore, SP4 produced a high cellulase level and could be genetically transformed with high efficiency because of the uracil auxotroph. Therefore, T. reesei SP4 provides a platform strain suitable for genetic manipulation to improve cellulolytic efficiency in biomass conversion. The major challenges in the bioconversion of lignocellulosic biomass include obtaining the optimum amount of BGL to complete the final step of cellulose hydrolysis (Singhania et al., 2013; Pryor and Nahar, 2015). Low β-glucosidase activity results in incomplete cellobiose hydrolysis, which further leads to product inhibition in the cellulolytic enzymes. Construction of engineering strains with high BGL activity is the preferred strategy to address this problem. Indeed, studies have shown that overexpression of BGL-encoding genes in T. reesei could result in improved BGL activity; this overexpression was mostly accomplished through genetic transformation with hygromycin B resistance (Liu et al., 2013). Wang and Xia (2011) constructed a T. reesei strain with a BGL activity of 5.3 U/mL by using the heterologous expression of the A. nigerβ-glucosidase (cellobiase) gene. Ma et al. (2011) reported that heterologous expression of the Penicillium decumbentsβ-glucosidase gene via Agrobacterium-mediated transformation in T. reesei leads to an eightfold increase in BGL activity. In another study, Zhang et al. (2010) utilized a modified cbh1 promoter to overexpress the native bgl1 gene in T. reesei and obtained a pyrithiamine-resistant strain with 5.7-fold higher BGL activity. In this study, the same bgl1 expression cassette was transformed into SP4 with uracil auxotrophy as a selection marker. This uracil auxotroph transformation system showed high efficiency. One transformant, SPB2, exhibiting a 17.1-fold higher BGL activity with additional four copies of bgl1 gene was selected from 126 positive transformants. The ratio of BGL activity to FPA in SPB2 reached 2.8, which was much higher than that in the parental strain (0.2). Chen et al. (2008) reported that cellobiose would be efficiently degraded into glucose if the ratio of BGL activity to FPA is more than 0.3. Thus, the enzyme system produced by the bgl1-overexpression strains constructed in this study can be directly applied to the saccharification of lignocellulosic materials without adding extra BGL. The SPB2 enzyme containing the highest BGL activity displayed excellent performance in the saccharification of both corncob substrates (Figure 7). The SPB1 enzyme was more effective than SP4 in the saccharification of the delignined corncob residues, but both had a similar effect on the hydrolysis of the acid-pretreated corncob residues. A possible reason for the difference maybe that a small amount of β-glucosidase in the enzyme complex was adsorbed on to the lignin component in the acid-pretreated corncob residues, which is in accordance with the postulation of Haven and Jørgensen (2013). The enhancement effect of excessive BGL on the saccharification of cellulosic materials (shown by the SPB2 enzyme) has been previously reported (Peterson and Nevalainen, 2012). Such results can be explained by the possibility that a large amount of β-glucosidase would reach the saturated adsorption of lignin and further degrade cellobiose, thus helping alleviate product inhibition and facilitate cellulose conversion. Conclusion A novel hypercellulolytic T. reesei variant, SN1, was characterized. A uracil auxotrophic strain, SP4, was constructed through gene deletion to achieve high-efficiency genetic transformation. Further overexpression of the bgl1 gene in SP4 significantly enhanced the BGL activity in the cellulolytic enzyme complex and was proven to be highly effective in the saccharification of the pretreated corncob residues. These results reveal the feasibility of strain improvement from a mutant industrial strain via ab initio genetic manipulation. This strategy appears to be practicable as an essential step in the construction of genetically engineered strains for the production of commercially viable enzyme preparations for biomass conversion. Author Contributions YuQ, YH, YiQ, and YZ designed and coordinated the study and wrote the manuscript. YuQ and LZ carried out the experiments and analyzed the results. All authors read and approved the final manuscript. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This work was supported by grants from the National Natural Science Foundation of China (No. 31370135), Fundamental Research Funds of Shandong University (No. 2015CJ005), and Agricultural Science and Technology Achievement Transformation Fund of Shandong Province (2014 No. 45). Supplementary Material The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb.2016.01349 Click here for additional data file. FIGURE S1 | Phylogenetic tree of strain SN1 based on the 18S rDNA gene sequence and internally transcribed spacer (ITS) region. (A) 18S rDNA sequences. The 18s rDNA gene sequences from Trichoderma reesei (XM_006965206.1), T. sp (AB923813.1), T. harzianum (KP133166.1), T. viride (AF218788), Penicillium oxalicun (JQ349066.1), Aspergillus niger (AM270052.1), Cladonia arbuscula (KT148631.1). (B) ITS region sequences, the ITS region sequences from T. reesei (KT336514.1), T. harzianum (KR232487.1), T. viride (DQ442274.1), T. saturnisporum (KF294853.1), T. asperelloides (KT426897.1), T. pseudokoningii (AF140046), P. oxalicun (KT189516.1), A. niger (KM613139.1). Click here for additional data file. Click here for additional data file. FIGURE S2 | Mycelium morphology of SN1, QM9414, and RUT-C30. All strains were cultivated 2–5 days on PDA plates (glucose) to analyze the microscopic morphology of mycelium and spore. The images were captured using DIC. Scale bars are listed 20 μm in the figure. Click here for additional data file. Click here for additional data file. ==== Refs References Bailey M. Askolin S. Hörhammer N. Tenkanen M. Linder M. 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==== Front Can J Infect Dis Med MicrobiolCan J Infect Dis Med MicrobiolCJIDMMThe Canadian Journal of Infectious Diseases & Medical Microbiology = Journal Canadien des Maladies Infectieuses et de la Microbiologie Médicale1712-95321918-1493Hindawi Publishing Corporation 10.1155/2016/8254343Research ArticleTransfusion-Transmissible Infections among Voluntary Blood Donors at Wolaita Sodo University Teaching Referral Hospital, South Ethiopia http://orcid.org/0000-0002-1717-9030Bisetegen Fithamlak Solomon 1 * Bekele Fanuel Belayneh 2 Ageru Temesgen Anjulo 3 Wada Fiseha Wadilo 4 1School of Medicine, College of Health Sciences, Wolaita Sodo University, P.O. Box 138, Wolaita Sodo, Ethiopia2School of Public Health and Environmental Science, College of Health Sciences and Medicine, Hawassa University, Hawassa, Ethiopia3Wolaita Sodo University Teaching Referral Hospital Laboratory, P.O. Box 378, Wolaita Sodo, Ethiopia4School of Medicine, College of Health Sciences and Medicine, Wolaita Sodo University, P.O. Box 138, Wolaita Sodo, Ethiopia*Fithamlak Solomon Bisetegen: fitha2007@yahoo.comAcademic Editor: Elisabetta Caselli 2016 15 8 2016 2016 825434324 2 2016 5 7 2016 14 7 2016 Copyright © 2016 Fithamlak Solomon Bisetegen et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Transfusion-transmissible infections, human immunodeficiency virus, hepatitis B virus, hepatitis C virus, and syphilis are among the greatest threats to blood safety and pose a serious public health problem. Objective. To determine the magnitude of blood borne infections among blood donors at Wolaita Sodo University Teaching Referral Hospital. Methods and Materials. A cross-sectional study was conducted from 10/11/2015 up to 10/12/2015. 390 donors were consecutively included and data on donor's age, sex, blood type, and serum screening results were obtained by structured questionnaire and laboratory investigation. The collected data were entered into Epi Data version 1.4 and then exported to SPSS version 20.0 for analysis. Result. The seroprevalence of blood borne pathogens is 29.5% of which HCV, HBV, HIV, and syphilis account for 8.5%, 9.5%, 6.4%, and 7.5%, respectively. Multiple infections were observed among 2.8% of the infected individuals. In addition, age ≥ 30 has a significant association with HCV. Conclusion. Significantly higher prevalence of transfusion-transmissible infections was identified from blood donors and they remain to be the greatest threat to blood safety, so comprehensive screening of donors' blood for HIV, HBV, HCV, and syphilis using standard methods is highly recommended to ensure the safety of blood recipient. ==== Body 1. Introduction Although blood transfusion is one of the known therapeutic interventions that cut across a number of clinical disciplines, the practice is not without risks [1]. The highest risk groups are children suffering from malaria and anemia; women with pregnancy related hemorrhage; and victims of major trauma [2]. Transfusion-transmissible infectious agents such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), and syphilis are among the greatest threats to blood safety for the recipient and pose a serious public health problem [3]. Among HIV transmission ways blood transfusion accounts for 5–10% in Sub-Saharan Africa [4]. Similarly, 12.5% of patients who received blood transfusion are at risk of posttransfusion hepatitis [5]. The high prevalence of HIV, HBV, HCV, and syphilis has heightened the problems of blood safety in Ethiopia. Thus, continuous monitoring of the magnitude of transfusion-transmissible infections in blood donors is important for estimating the risk of transfusion and optimizing donor recruitment strategies to minimize infectious diseases transmission [3]. However, there is scarce published information about the burden of major transfusion-transmissible infections in the study area. Morbidity and mortality resulting from the transfusion of infected blood have far-reaching consequences, not only for the recipients themselves, but also for their families, their communities, and the wider society [6, 7]. Only continuous improvement and implementation of donor selection, sensitive screening tests, and effective inactivation procedures can ensure the elimination, or at least reduction, of the risk of acquiring TTIs [8]. Evaluation of data on the prevalence of transfusion-transmissible infections, namely, HIV, HBV, HCV, and syphilis, among blood and plasma donors permits an assessment of the occurrence of infections in the blood donor population and consequently the safety of the collected donations. It also gives an idea of the prevalence of the transfusion-transmissible infections (TTIs) among blood donors which allows for assessment of epidemiology of these infections in the community. 2. Methods and Materials 2.1. Study Area and Setting Institution based cross-sectional study was conducted at Wolaita Sodo University Teaching Referral Hospital from 10/11/2015 up to 10/12/2015. The university hospital provides clinical services for 85,700 populations. In 2015, approximately 1,500 units of blood were transfused in the hospital. 2.2. Study Population Three hundred ninety blood donors were prospectively recruited in the study from October 10, 2015, up to November 10, 2015, and convenient sampling techniques were used to recruit blood donors who were eligible to donation, consented, interviewed, and gave blood for serum screening of transfusion-transmissible infections. All blood donors who fulfilled the national and regional blood bank criteria were included. Blood donors who did not meet the inclusion criteria (<18 years, >65 years, history of long-term medication use, and unwillingness to give oral informed consent) were excluded from the study. 2.3. Data Collection Data on blood donor's age, sex, blood group, serological results of HCV, HBs Ag, HIV, and syphilis were collected at the time of blood collection, by using a structured questionnaire. The laboratory uses immune chromatographic techniques to screen blood donors. In addition, the blood samples were retested with ELISA technique to confirm the results. 2.4. Laboratory Testing Five milliliters of venous blood was collected using sterile test tube from each blood donor. Serum was separated by centrifugation at a speed of 3500 revolutions per minute (rpm) for 5 minutes and 2 mL of serum was collected from each sample using sterile plastic vials. Blood group for each blood donor was determined at each study area using blood group antisera: anti-A, anti-B, and anti-D for Rh factor. Each donor was tested for HBs Ag and anti-HCV by ACON one-step insert rapid test strips in the laboratory. To confirm the results, samples were retested by using 4th-generation ELISA technique. Blood samples tested by using serological assays for HIV infection were screened by 4th-generation ELISA, Vironostika HIV Uni-Form II AG/Ab. Hepatitis B virus was screened by using an immunoassay ELISA Hepanostika HBs Ag Uni-Form II, hepatitis C virus by using the human anti-HCV 3rd-generation ELISA, and syphilis by using syphilis antibody ELISA (FTA). 2.5. Data Management The collected data were entered into Epi Data version 1.4 and then exported to SPSS version 20 for analysis. Summary statistics such as frequencies and percentages were computed. The results were presented using tables, charts, and graphs. Chi-square was used to see a difference of blood borne pathogens between age, sex, and blood groups. A p value of <0.05 was considered as a significant difference. 2.6. Quality Assurance All positive samples and 10% negative samples were retested in South Nations and Nationalities People Region/SNNPR/regional laboratory to ensure the quality. Standard operational procedures were strictly followed and QC materials were used for all serological tests. In addition, laboratory quality was assured by well-trained professionals, training, and supervision during sample collection. 2.7. Ethical Consideration The study was carried out after getting approval from the ethical clearance committee of Wolaita Sodo University. Consent was signed before sample collection and the participants' information is used only for study purpose. Respondents were not identified by name and the participant had the right to discontinue the participation any time. 3. Result 3.1. Donor's Characteristics A total of 390 blood donors were included during the study period; among these, 291 (74.6%) were male and 99 (25.4%) were female with female to male ratio of 1 : 3. The median age of the donors was 28 years with the range 18 and 60 years. Regarding donors ABO blood group distribution, 200 (51.3%) of the donors were “O” blood type. The remaining 104 (26.7%), 76 (19.7%), and 9 (2.4%) of the participants were “A,” “B,” and “AB” blood types, respectively. 3.2. Prevalence of Blood Borne Pathogens Blood borne pathogens were detected among 115 (29.5%) of the donors and 275 (70.5%) were free from the four infections. Donors with positive results for HCV and HBs Ag were 33 (8.5%) and 37 (9.5%), respectively. Twenty-five of them were positive for HIV whereas 31 (7.9%) were positive for syphilis test (Figure 1). Among those who have the infection, 104 (26.7%) were positive for only one of the pathogens and the other 11 (2.8%) were coinfected with two of the four blood borne infections. Coinfection for more than two pathogens was not detected. Five of the coinfected donors were positive for HBs Ag and syphilis antibody tests. In addition, two donors with HIV positive screening result were also positive for HCV antibody test and other two were positive for syphilis antibody test (Figure 2). Almost one-third of all male and 29 (29.3%) of all female donors in this study were positive at least for one of the four blood borne pathogens. Prevalence of HCV infection was 8.9% among males and 7.1% among female donors. Donors who are positive for HBs Ag test were 11.0% in males and 5.1% in females. About seven percent of the male donors and five percent of the female donors have been positive for HIV. Antibody evidence for Treponema pallidum was detected among 6.2% of male and 13.1% of female donors. Regarding infection distribution with age categories, 65 (23.6%) of donors less than or equal to 30 years and 50 (43.9%) of donors greater than 30 years have evidence of at least one blood borne pathogen. The predominant positive result was for HBs Ag, which accounts for 24 (8.7%) and 13 (11.4%) among donors less than or equal to 30 years of age and greater than 30 years, respectively. Sex has no significant association with any of transfusion-transmissible infections but age ≥ 30 years has a significant association with HCV prevalence (p = 0.04) (Table 1). Regarding the distribution of the infections with blood group, 8 (10.4%) of donors with A blood group are positive for HCV, whereas 10 (13%) of HBs Ag reactive donors were B blood group. Among A blood group donors, 17 (8.5%) were positive for HIV, and 10 (9.6%) were reactive for syphilis. RH+ donors showed that positive reaction for HCV accounts for 8.2% and 9.6% positive for HBs Ag. RH− donors showed relatively high prevalence of HCV, 4 (11.8%), and syphilis, 5 (14.7%) (Table 2). 4. Discussion In the current study, the seroprevalence of transfusion-transmissible infections was 115 (29.5%) which is similar to 29.85% reported in Burkina Faso [9], 21.6% in Cameroon [10], and 19.3% in Nigeria [11]. But the result is lower than 43.2% reported in Bahir Dar, Ethiopia [2], which could be due to the fact that most of the study participants in the previous study were commercial blood donors whereas only voluntary donors were included in this study. The current finding is much higher than previous studies conducted elsewhere, 14% in Islamabad [12], 9.6% in Sudan [13], 2.6% in Northeast Ethiopia, Gondar [3], 2.22% in Karnataka, India [14], 1.7% in Nepal [15], and 1.1%, 1%, and 0.93% in India [16–18]. Hence, even when compared with other resource constrained settings, the burden of blood borne pathogens is quite high in this study. Widest difference in blood borne pathogen in different studies in our country and others could be due to the use of different generation of ELISA test kits having different sensitivities and specificities and geographical factors. Donors with positive results for HCV, 33 (8.5%) in this study, are comparable with 13.3% reported in Bahir Dar, North Ethiopia [2], 8.7% in Burkina Faso [9], 8.34% in Islamabad [12], and 8% in Ghana [19]. But the result is considerably higher than studies conducted elsewhere, 4.8% [10], 3.4% [13], 1.7% [20], 1.1% [21], 0.51% [22], 0.48% [23], 0.21% [24], 0.2% [17], 0.16% [25], and 0.03% [14]. The higher prevalence of this study as compared with the above findings could be supported by the reason that most of the donations before six years were commercial donors and they were donating blood without being screened for HCV and these strains may be circulating in the community without noticing. Prevalence of HBs Ag among blood donors in this study, 9.5%, is lower than a prevalence of 25% in North Ethiopia [2], 14.9% in Burkina Faso [9], and 14.3% in Jos, Nigeria [26]. But it is in harmony with a prevalence of 10.1%, reported the Cameroon study [10]. On the contrary, the current finding is in discordance with studies conducted in Ethiopia and elsewhere, 6.2% in Northwest Ethiopia [20], 5.3% in Nigeria [21], 5% in Sudan [13], 4.7% in Gondar [20], 3.91% in Islamabad [12], 1.16% in China [22], 0.9% [18], 0.63% [17], 0.49% [24], and 0.3% [25] in India, and 0.09% [23] in Canada. These variations could also be due to actual changes in population risks or effectiveness of donor screening measures. HIV prevalence of 6.4% observed among blood donors at Wolaita Sodo University Teaching Referral Hospital is lower than 11.7% for Bahir Dar, Ethiopia [2], but considerably comparable with 4.1% reported in Cameroon [10] and 3.8% reported in Ghana [19] but higher than previous studies conducted in the country and elsewhere [12, 13, 17, 18, 21–23, 25, 27, 28] in different countries. HIV prevalence in the current study shows high discrepancy with the nationwide adult HIV prevalence estimate of 1.1% [29], but lower than 10.2% reported in SNNPR [30]. The high prevalence of HIV seropositivity in comparison with the national figure and other local regions could be due to lower voluntary counseling test coverage in this region than the other [30]. Treponemal positivity prevalence of 7.9% in our study is lower than the prevalence of 13.5% reported in Ghana [19], but in harmony with 7.5% prevalence reported in similar study conducted in the same country, Ghana [31], and 5.7% in Cameroon [10]. Our finding is higher than 3.96% prevalence in Burkina Faso [9], 1.2% in Bahir Dar [2], 0.89% in Islamabad [12], 0.3% in China [22], 0.23% in India [28], 0.22% in Gujrat, India, and Uttarakhand, India [17, 18]. This could possibly be explained in association with the higher prevalence of HIV in this study where one could facilitate the transmission of the others. In the current study, there was no association between sex, ABO blood group, RH factor, and any of blood borne pathogens. Age group ≥ 30 have a significant association with HCV which is unclear but could be explained by the fact that liver fibrosis increases with old age and in age groups 40–64 and could also be due to the highest seropositivity of HCV for individuals aged >30 [32, 33]. 5. Conclusion and Recommendation In general, the prevalence of transfusion-transmissible infection is high in the study area. The blood donors in this study were voluntary subjects, who are apparently healthy, but this study found that these diseases are prevalent among volunteer donors. Thus, strict selections of blood donors with standard methods are highly recommended to ensure the safety of blood for the recipient. Acknowledgments The authors acknowledge all the staff members of blood bank unit, Wolaita Sodo University, Ethical Review Board for the ethical clearance, and blood donors. Competing Interests All authors declare that they have no competing interests. Authors' Contributions Fithamlak Solomon Bisetegen conceived the study, Fithamlak Solomon Bisetegen, Fanuel Belayneh Bekele, Temesgen Anjulo Ageru, and Fiseha Wadilo Wada participated in the design of the study and performed the statistical analysis, and Fithamlak Solomon Bisetegen and Fanuel Belayneh Bekele interpreted the data. Fithamlak Solomon Bisetegen obtained ethical clearance and permission for study. Temesgen Anjulo Ageru supervised data collectors; Fithamlak Solomon Bisetegen, Fanuel Belayneh Bekele, Temesgen Anjulo Ageru, and Fiseha Wadilo Wada drafted the paper or revisited it critically for important intellectual content. All authors read and approved the final paper. Figure 1 Distribution of blood borne pathogens infection among blood donors at Wolaita Sodo Teaching Referral Hospital 2015. Figure 2 Infection status of blood donors by blood borne pathogens at Wolaita Sodo Teaching Referral Hospital 2015. Table 1 Distribution of blood borne pathogens by sex and age among blood donors at Wolaita Sodo Teaching Referral Hospital, 2015. Variable HCV HBs Ag HIV Syphilis R NR R NR R NR R NR No (%) No (%) No (%) No (%) No (%) No (%) No (%) No (%) Sex                  Male 26 (8.9) 265 (91.1) 32 (11.0) 259 (89.0) 20 (6.9) 271 (93.1) 18 (6.2) 273 (93.8)  Female 7 (7.1) 92 (92.9) 5 (5.1) 94 (94.9) 5 (5.1) 94 (94.9) 13 (13.1) 86 (86.9) Age category                  ≤30 22 (8.0) 254 (92.0) 24 (8.7) 252 (91.3) 11 (4.0) 265 (96.0) 13 (4.7) 263 (95.3)  >30 11 (9.6) 103 (90.4) 13 (11.4) 101 (88.6) 14 (12.3) 100 (87.7) 18 (15.8) 96 (84.2) Table 2 Distribution of blood borne pathogens by donor's blood type among blood donors at Wolaita Sodo Teaching Referral Hospital, 2015. Blood type HCV HBs Ag HIV Syphilis R NR R NR R NR R NR No (%) No (%) No (%) No (%) No (%) No (%) No (%) No (%) ABO blood group                  A+/− 5 (4.8) 99 (95.2) 7 (6.7) 97 (93.3) 5 (4.8) 99 (95.2) 10 (9.6) 94 (90.4)  B+/− 8 (10.4) 69 (89.6) 10 (13.0) 67 (87.0) 2 (2.6) 75 (97.4) 6 (7.8) 71 (92.2)  AB+/− 1 (1.1) 8 (98.9) 2 (2.2) 7 (97.8) 1 (1.1) 8 (98.9) 1 (1.1) 8 (98.9)  O+/− 19 (9.5) 181 (90.5) 18 (9.0) 182 (91.0) 17 (8.5) 183 (91.5) 14 (7.0) 186 (93.0) RH blood group                  Positive 29 (8.2) 326 (91.8) 34 (9.6) 321 (90.4) 24 (6.8) 332 (93.2) 26 (7.3) 329 (92.7)  Negative 4 (11.8) 30 (88.2) 3 (8.8) 31 (91.2) 1 (2.9) 33 (97.1) 5 (14.7) 29 (85.3) ==== Refs 1 Fleming A. F. HIV and blood transfusion in sub-Saharan Africa Transfusion Science 1997 18 2 167 179 10.1016/S0955-3886(97)00006-4 2-s2.0-0030709370 10174681 2 Azene D. Bayeh A. Fisseha W. 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PMC005xxxxxx/PMC5002454.txt

==== Front Biomed Res IntBiomed Res IntBMRIBioMed Research International2314-61332314-6141Hindawi Publishing Corporation 10.1155/2016/1486824Research ArticleDetection of Peste des Petits Ruminants Viral RNA in Fecal Samples of Goats after an Outbreak in Punjab Province of Pakistan: A Longitudinal Study http://orcid.org/0000-0001-5016-8969Wasee Ullah Riasat 1 2 * Bin Zahur Aamer 3 Latif Asma 3 Iqbal Dasti Javid 2 Irshad Hamid 3 Afzal Muhammad 4 Rasheed Tahir 3 Rashid Malik Adnan 5 Qureshi Zafar-ul-Ahsan 1 1Veterinary Research Institute, Zarar Shaheed Road, Lahore Cantt 54810, Pakistan2Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan3Animal Health Research Laboratories, Animal Sciences Institute, National Agricultural Research Centre, Islamabad 44000, Pakistan4Progressive Control of PPR in Pakistan, FAO-UN, Islamabad 44000, Pakistan5Department of Animal Husbandry, Azad Government of the State of Jammu and Kashmir, Muzzafarabad 13100, Pakistan*Riasat Wasee Ullah: riasatwasee@gmail.comAcademic Editor: Hasan T. Atmaca 2016 15 8 2016 2016 148682410 4 2016 16 7 2016 21 7 2016 Copyright © 2016 Riasat Wasee Ullah et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Peste des petits ruminants (PPR) is a highly contagious viral disease of domestic and wild small ruminants and thus has serious socioeconomic implications. In Pakistan, during the year 2012-2013, estimated losses due to PPR were worth Rs. 31.51 billions. Close contact between infected and susceptible animals is an important route of transmission of PPR. Therefore, carrier animals play an important role in unnoticed transmission of PPR. The objective of the study was to investigate the detection of PPR virus in goats recovered from PPR. A suspected PPR outbreak was investigated and confirmed as PPR after analysing appropriate samples collected from infected animals using rRT-PCR. A longitudinal study was conducted over the period of 16 weeks to ascertain the detection of PPR virus (PPRV) in faecal samples of recovered goats. Ninety-six (96) faecal samples from each sampling were collected at 4, 8, 12, and 16 weeks after the outbreak. Faecal samples were analysed using rRT-PCR. Of 96 from each sampling a total of 46, 37, 29, and 25 samples were positive for PPR viral genome at 4, 8, 12, and 16 weeks, respectively, after recovery. Attempts were made for the isolation of PPR virus on Vero cells, but results were negative. These results indicated the detection of PPR viral RNA up to 16 weeks after infection. Therefore, these results may help in the future epidemiology of PPR virus shedding and possible role as source of silent infection for healthy animals especially when there is no history of any outbreak in nearby flock or area. Food and Agriculture Organization of the United NationsGCP/PAK/127/USA ==== Body 1. Introduction Peste des petits ruminants (PPR) is a highly contagious viral disease of domestic and wild small ruminants caused by PPR virus (PPRV) of family Paramyxoviridae [1]. The disease is associated with high morbidity (100%) and mortality (up to 90%) [2]. PPR is also classified as transboundary animal disease (TAD) [3]. The typical form of PPR is associated with anorexia, pyrexia, ulceration, necrosis of mucous membranes, sores in mouth, mucopurulent nasal and ocular discharges, pneumonia, inflammation of the gastrointestinal tract (GIT), and diarrhea [4–6]. Animals of all ages are susceptible to the disease. However, the disease is more fatal in kids and lambs. A close contact with infected animals is considered an important mean of transmission of disease [7]. Optimizations of advanced methods for the detection of PPRV are important for large scale surveillance studies especially to investigate carrier state of the animals. So far, very few reports have been documented in this regard. However, following the acute phase of PPR in small ruminants some animals may experience a long asymptomatic persistent stage. PPR viral RNA detection has been reported for 11 weeks after recovery and in some animals even up to 12 weeks after recovery using haemagglutination (HA) test [8]. Furthermore, it has also been reported that animals infected with PPR virus are incubatory carriers and virus shedding was detected in their secretions and excretions 2-3 days prior to the onset of the clinical disease [9]. Under given conditions these carriers may contribute to the unnoticed transmission of PPR virus at high risk areas like weekly livestock markets and during communal grazing at pastures. Such asymptomatic carriers are threat to livestock farmers and may contribute to transboundary episode of the disease. This suggests the need to carry out studies to understand the persistence and carrier state of small ruminants and further transmission of the disease. There are no systematic studies reported about persistence and carrier state of PPR. We report a longitudinal study that has the objective to determine the duration of detection of PPR viral RNA in the faecal samples of animals recovered from PPR. 2. Materials and Methods 2.1. Outbreak Investigation and Collection of Samples A suspected outbreak of PPR was investigated in an organised goat farm in suburban area of Lahore district, Pakistan, on 20 November 2012. The affected animals were examined for the presence of clinical signs specific to PPR virus infection. The temperature and clinical signs were recorded on prescribed proforma. The outbreak control measures were implemented and symptomatic therapeutic interventions were advised to the farmer. Structured epidemiological investigations were conducted to determine the most likely source of PPR virus transmission. Information regarding previous history of the disease at the farm, vaccination status of the flock, flock size, number of affected animals, number of dead animals, and history of a PPR outbreak in the nearby farm/area was recorded on a prescribed proforma. Necropsy of recently dead animals was performed. The carcasses were examined for the evidence of discharges (ocular and nasal), diarrhea, and pneumonia. Gross pathological lesions in GIT and respiratory tract were recorded. Ocular, nasal, and oral swabs and faecal samples were collected from live animals. Ocular swabs were collected by inserting a sterile swab (BD sterile swab) beneath the conjunctiva and swirling it so that the ocular secretions may adhere to the swab. The oral and nasal swabs were collected by inserting a sterile swab (BD sterile swab) deep into the oral and nasal cavity. Faecal samples were collected directly from rectum of clinically affected goats in polyethylene zipper bags using sterile gloves. The gloves were changed after collecting each sample. The tissue samples including lungs, liver, spleen, lymph nodes (mesenteric and bronchial), kidneys, and intestine were collected from dead animals. Each sample from donor animal was given a unique identification number with date. All the samples were transferred to laboratory in cold conditions. The swabs, tissues, and fecal and sera samples were stored at −70°C till further analysis. 2.2. Analysis of Samples Oral, ocular, and nasal swabs were processed for analysis. The cotton area of the swab was separated out gently from swab stick with the help of sterile forceps and scissor. Swab was put into a sterile Eppendorf tube containing 1.5 mL of sterile phosphate buffer saline (PBS; 0.01 M pH 7.4). The swab was completely squeezed in 1.5 mL Eppendorf tube and centrifuged at 10,000 RPM for 3–5 minutes at 4°C. Supernatant was collected and stored at −70°C till further analysis. The tissue samples were processed by making approximately 10% homogenate of infected tissues in sterile PBS (0.01 M pH 7.4). The homogenate was centrifuged at 10,000 RPM for 3–5 minutes at 4°C in a 1.5 mL Eppendorf tube. The supernatant was collected and stored at −70°C till further analysis. RNA was extracted from all the samples using RNeasy kit (Qiagen GmbH, Hilden, Germany). A negative control was also included for detection of possible contamination during extraction. The extraction of RNA was performed according to manufacturer instructions. Briefly, 560 μL of lysis buffer and 700 μL of 70% ethanol have been added to 140 μL of tested sample and allowed to spin in spin filtered column for one minute. For washing, wash buffer and buffer RPE are added and allowed to spin. To elute the extracted RNA, 40 μL of RNA's free water was used. The extracted RNA was placed at −20°C until further use. The quantity and purity of extracted RNA were determined using NanoDrop (NanoDrop 1000, Thermo Scientific, Wilmington, DE, USA) [10]. Each sample was analysed for the presence of PPR virus specific genome using reverse transcriptase real time polymerase chain reaction (rRT-PCR) [11]. rRT-PCR was done using core reagent kit (TaqMan EZ-RT-PCR Core Reagent), sequence specific primers, and probes described by [11]. Briefly, 5.0 μL of TaqMan EZ buffer, 2.5 μL of 25 mM Mn (OAc)2, 03 μL of dNTPs, 01 μL of forward primer, 01 μL of reverse primer and 01 μL of probe, 01 μL of rTh DNA polymerase, and 10.5 μL of nuclease free water were added to make the volume 25 μL. Master mix (22.5 μL) and 2.5 μL of RNA template were added to the wells of optical 96-well reaction plate (MicroAmp™ N801-0560). The plate was covered with adhesive film (MicroAmp) and spun in refrigerated centrifuge at 2500 rpm for 1 minute. The rRT-PCR was performed using ABI7500 real time PCR system (Applied Biosystems) and ABI prism SDS software, an initial reverse transcription temperature at 45°C for 30 min, followed by reverse transcriptase inactivation and DNA polymerase activation at 95°C for 5 min, and then 50 cycles of 15 s at 94°C and 30 s at 60°C. The reporter dye (FAM) signal was measured against the internal reference dye (ROX) signal to normalize the signals for non-PCR-related fluorescence fluctuations that occur from well to well. The data were collected at the annealing step of each cycle and the threshold cycle (Ct) for each sample was calculated by determining the point at which the fluorescence exceeded the threshold limit. 2.3. Study Design The animals positive for PPR were followed for the period of four months from 20 November 2012 to 20 March 2013. Sera and fecal samples were collected from recovered animals at 4, 8, 12, and 16 weeks after outbreak. The sera samples were analysed for the presence of PPRV specific antibodies using anti- nucleocapsid (N) monoclonal antibody (MAb) based competitive ELISA (c-ELISA) [12]. The faecal samples were analysed using rRT-PCR [11]. 3. Results 3.1. Epidemiological Observation The animals were raised on a semiextensive system where the animals were taken out for grazing in the morning and supplemented with a concentrate stall feeding in the evening for fattening. The flock consisted of 140 goats with age ranging between 10 and 18 months. The flock had history of introduction of five new animals from a nearby livestock market. None of the animals had a history of vaccination against PPR. There was no outbreak of PPR in the nearby area/village. The morbidity rate was 100%. However, 44 out of 140 animals died during the outbreak with a mortality rate of 31.42%. 3.2. Clinical Picture The clinical examination of the affected animals revealed high fever ranging between 39°C and 42°C, conjunctivitis, mucopurulent nasal and discharges along with depression, anorexia, swollen lips, cough, and diarrhoea. The affected animals showed signs of severe dehydration and their hind quarters were soiled with diarrhoea material. The mouth lesions were found in all the affected animals with red raw areas on inner side of the lips, lower gums, and necrosis on the dorsal surface of the tongue. 3.3. Postmortem Findings The postmortem examination of n = 3 recently dead animals was conducted. On external appearance the carcasses were found to have evidence of dehydration with sunken eyes, rough/dry skin, and hind quarter soiled with diarrhoea material. The internal examination revealed pneumonic lungs with haemorrhages on mucosal surfaces of rumen, abomasum, and large intestine (caecum and colon). Haemorrhages were also observed on liver and kidneys of one animal. The body lymph nodes were inflamed and swollen particularly the mesenteric lymph nodes. For laboratory confirmation of PPR a total of 29 swabs and 33 faecal and 12 tissue (lungs = 3, liver = 2, lymph nodes = 4, spleen = 2, and kidney = 1) samples were collected. Sixteen (55.17%) swabs and 28 (84%) faecal and 10 (83%) tissue samples were found positive for PPRV genome using rRT-PCR. These results confirmed the outbreak of PPR in the flock. 3.4. Fecal Samples The samples were collected according to the method as described earlier. At each sampling n = 96 faecal samples were collected. RNA extraction and rRT-PCR were performed as mentioned earlier. At each sampling n = 96 faecal samples were collected from PPR recovered goats, out of which 46, 37, 29, and 25 samples were found positive for PPR viral genome at 4, 8, 12, and 16 weeks, respectively. These results indicated that small ruminants shed PPR viral genome up to four months (16 weeks) after clinical recovery from the disease (Figures 1 and 2). 4. Discussion A PPR outbreak causing high morbidity and low mortality in a goat herd in Punjab was confirmed on the basis of clinical signs and various lab diagnostic tests. The animals recovered from the outbreak were monitored and their faecal samples were collected for a period of 16 weeks to determine the persistence of PPRV in faecal samples of recovered animals. The results of the study indicated that introduction of new animals into the flock from weekly livestock markets resulted in the initiation of an outbreak in herd of goats under investigation. Similar mode of disease transmission has been reported previously by Asmar et al. [13] in Saudi Arabia where outbreaks in sheep were attributed to the introduction of new animals from livestock market. Under such conditions the newly introduced animals remained unaffected while rest of the flock experienced PPR virus infection. This may suggest that newly introduced animals may have experienced infection in the past and become carrier of the virus. PPR outbreaks have been reported in healthy herds after the introduction of newly purchased potentially incubating animals to the flocks [14]. During investigation typical clinical signs and symptoms of PPR virus infection were observed in infected animals. These include high fever up to 42°C, lesions in mouth, oral and nasal congestion, respiratory signs, and diarrhea leading to death of the animals. Similar findings were made in previous reports [9, 15, 16]. In this study it was observed that the morbidity rate was 100% while mortality rate was 31.42%. These findings are in complete concurrence with previous study which reported morbidity and mortality rates due to PPR ranging from 0 to 90% depending on the local husbandry practices, breed, age, and other factors [17]. The presence of PPR virus in current study was confirmed by clinical signs, postmortem examination, and rRT-PCR, while competitive ELISA (c-ELISA) was used for antibody detection. The results of the study indicated detection of PPRV in goats recovered from PPR. However, the number of animals giving positive results decreased from 46 on 4th week after vaccination to 25 on 16th week after vaccination. Previous studies also reported the persistence of PPRV antigen in faecal samples of PPR recovered goats [8, 18]. However, the duration of detection of PPRV antigen in various studies was different. For example, Ezeibe et al. [8] reported detection of PPRV antigen using HA in faecal samples of recovered goats up to 12 weeks after recovery. Another study reported persistence of PPRV in faecal samples of recovered goats up to 30 days in vaccinated and 60 days in unvaccinated goats after recovery [18]. Various samples have been analysed using different diagnostic tests to understand the persistence of PPRV in animals recovered from PPR [6]. For example, a recent study reported the persistence of PPRV after challenge for 40 days in nasal, ocular, and oral swabs [19]. In contrast, another study reported the persistence of PPRV in faecal samples of recovered goats for 12 weeks. Secondly the aim of the study was to determine the duration of detection of PPR viral RNA in the faecal samples of animals recovered from PPR to understand the role of faecal samples through this route. Therefore, faecal samples appeared to be the sample of choice for persistence studies. We used rRT-PCR for detection of PPRV in faecal samples of goats whereas previous studies used HA for detection of PPRV in faecal samples [8, 18]. rRT-PCR is considered a highly specific test for detection of PPRV [11]. However, a previous study evaluating HA for detection of PPRV in faecal samples of sheep and goats reported low specificity of HA for detection of PPRV compared to reverse transcriptase PCR [20]. Therefore, HA may not be considered a suitable test for detection of PPRV antigen in faecal samples of PPR recovered goats. In conclusion, this study reported the detection of PPRV in faecal samples of PPR recovered goats using rRT-PCR up to 16th weeks after recovery indicating the possible role of PPR recovered goats in transmission of disease to in-contact healthy animals. However, a study should be carried out to isolate the virus from faecal samples positive for PPRV RNA to further elucidate the role of recovered animals in transmission of disease to healthy animals. Therefore, these results may help in studying the future epidemiology of PPR virus shedding and transmission of PPR virus by fecal material and possible role as source of silent infection for healthy animals especially when there is no history of any outbreak in nearby flock or area. Acknowledgments Authors want to acknowledge Agricultural Linkages Program (ALP) of Pakistan Agricultural Research Council (PARC), Pakistan, for technical support and are highly thankful to FAO-UN Project on “Progressive Control of PPR in Pakistan (GCP/PAK/127/USA)” for financial support. Competing Interests Authors have no any conflict of interests. Figure 1 Monthwise detection of PPRV genome from fecal samples. Figure 2 Decreasing trend of PPR viral RNA shedding up to 16 weeks after outbreak in recovered animals. ==== Refs 1 Kwiatek O. Minet C. Grillet C. Peste des petits ruminants (PPR) outbreak in Tajikistan Journal of Comparative Pathology 2007 136 2-3 111 119 10.1016/j.jcpa.2006.12.002 2-s2.0-34047271778 17321539 2 Abu Elzein E. M. Hassanien M. M. Al-Afaleq A. I. Abd Elhadi M. A. Housawi F. M. Isolation of peste des petits ruminants from goats in Saudi Arabia Veterinary Record 1990 127 12 309 310 2-s2.0-0025712839 2238415 3 Zahur A. B. Ullah A. Hussain M. Sero-epidemiology of peste des petits ruminants (PPR) in Pakistan Preventive Veterinary Medicine 2011 102 1 87 92 10.1016/j.prevetmed.2011.06.011 2-s2.0-84860389033 21788090 4 Abubakar M. Ashiq S. Zahoor A. B. Arshed M. J. Banyard A. C. Diagnosis and control strategies for peste des petits ruminants virus: global and Pakistan perspectives Pakistan Veterinary Journal 2011 31 4 267 274 2-s2.0-83755196046 5 Roeder P. Obi T. Recognizing Peste des Petits Ruminants: A Field Manual 1999 Rome, Italy Food and Agriculture Organization of the United Nations 6 Ullah R. W. Zahur A. B. Latif A. Dasti J. I. Zahra R. Khan S.-H. Mild form of Peste des Petits Ruminants Virus (PPRV) in Pakistan Pakistan Journal of Zoology 2015 47 1 276 279 7 Lefevre P. C. Diallo A. Peste des petits ruminants Revue Scientifique et Technique: International Office of Epizootics 1990 9 4 951 965 8 Ezeibe M. C. O. Okoroafor O. N. Ngene A. A. Eze J. I. Eze I. C. Ugonabo J. A. C. Persistent detection of peste de petits ruminants antigen in the faeces of recovered goats Tropical Animal Health and Production 2008 40 7 517 519 10.1007/s11250-008-9128-3 2-s2.0-50249135449 18716908 9 Couacy-Hymann E. Bodjo S. C. Danho T. Koffi M. Y. Libeau G. Diallo A. Early detection of viral excretion from experimentally infected goats with peste-des-petits ruminants virus Preventive Veterinary Medicine 2007 78 1 85 88 10.1016/j.prevetmed.2006.09.003 2-s2.0-33846028191 17064800 10 Balamurugan V. Sen A. Venkatesan G. A rapid and sensitive one step-SYBR green based semi quantitative real time RT-PCR for the detection of peste des petits ruminants virus in the clinical samples Virologica Sinica 2012 27 1 1 9 10.1007/s12250-012-3219-z 2-s2.0-84860796396 22270801 11 Bao J. Li L. Wang Z. Development of one-step real-time RT-PCR assay for detection and quantitation of peste des petits ruminants virus Journal of Virological Methods 2008 148 1-2 232 236 10.1016/j.jviromet.2007.12.003 2-s2.0-39149138114 18243345 12 Choi K.-S. Nah J.-J. Ko Y.-J. Kang S.-Y. Jo N.-I. Rapid competitive enzyme-linked immunosorbent assay for detection of antibodies to peste des petits ruminants virus Clinical and Diagnostic Laboratory Immunology 2005 12 4 542 547 15817764 13 Asmar J. A. Radwan A. I. Abi A. H. Rasheid A. A. A PPR like disease in central Saudi Arabia: evidence of its immunologic relation to rinderpest; prospects for a control method Proceedings of the 4th Conference on the Biological Aspects of Saudi Arabia March 1980 Riyadh, Saudi Arabia 325 337 14 Taylor W. Barrett T. Rinderpest and Peste des Petits Ruminants 2008 4th Diseases of Sheep 15 Nanda Y. P. Chatterjee A. Purohit A. K. The isolation of peste des petits ruminants virus from Northern India Veterinary Microbiology 1996 51 3-4 207 216 10.1016/0378-1135(96)00025-9 2-s2.0-0030220320 8870184 16 Hamdy F. M. Dardiri A. H. Response of white-tailed deer to infection with peste des petits ruminants virus Journal of Wildlife Diseases 1976 12 4 516 522 10.7589/0090-3558-12.4.516 2-s2.0-0038943719 16502689 17 Diallo A. Control of peste des petits ruminants and poverty alleviation? Journal of Veterinary Medicine Series B: Infectious Diseases and Veterinary Public Health 2006 53 1 11 13 10.1111/j.1439-0450.2006.01012.x 2-s2.0-33750997862 18 Abubakar M. Arshed M. J. Zahur A. B. Ali Q. Banyard A. C. Natural infection with peste des petits ruminants virus: a pre and post vaccinal assessment following an outbreak scenario Virus Research 2012 167 1 43 47 10.1016/j.virusres.2012.03.018 2-s2.0-84861480851 22504337 19 Liu W. Wu X. Wang Z. Virus excretion and antibody dynamics in goats inoculated with a field isolate of peste des petits ruminants virus Transboundary and Emerging Diseases 2013 60 2 63 68 10.1111/tbed.12136 2-s2.0-84887377787 24589103 20 Latif A. Akhtar Z. Ullah R. W. Evaluation of haemagglutination assay (HA) for the detection of peste des petits ruminants virus (PPRV) in faecal samples of recovered goats Research Journal for Veterinary Practitioners 2014 2 1 11 13 10.14737/journal.rjvp/2014/2.1s.11.13

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==== Front Case Rep MedCase Rep MedCRIMCase Reports in Medicine1687-96271687-9635Hindawi Publishing Corporation 10.1155/2016/8708251Case ReportMounier-Kuhn Syndrome in an Elderly Female with Pulmonary Fibrosis http://orcid.org/0000-0001-9684-6577Boglou Panagiotis 1 http://orcid.org/0000-0002-7320-785XPapanas Nikolaos 2 http://orcid.org/0000-0001-6996-237XOikonomou Anastasia 3 Bakali Stamatia 4 http://orcid.org/0000-0001-7121-6253Steiropoulos Paschalis 1 * 1Department of Pneumonology, Medical School, Democritus University of Thrace, 68100 Alexandroupolis, Greece2Second Department of Internal Medicine, Medical School, Democritus University of Thrace, 68100 Alexandroupolis, Greece3Department of Radiology, Medical School, Democritus University of Thrace, 68100 Alexandroupolis, Greece4Department of Microbiology, Medical School, Democritus University of Thrace, 68100 Alexandroupolis, Greece*Paschalis Steiropoulos: pstirop@med.duth.grAcademic Editor: Stephen P. Peters 2016 15 8 2016 2016 87082519 5 2016 20 7 2016 Copyright © 2016 Panagiotis Boglou et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Mounier-Kuhn syndrome (MKS), or tracheobronchomegaly, is a rare clinical and radiologic condition characterized by pronounced tracheobronchial dilation and recurrent lower respiratory tract infections. Tracheobronchomegaly presents when the defect extends to the central bronchi. MKS can be diagnosed in adult women when the transverse and sagittal diameters of the trachea, right mainstem bronchus, and left mainstem bronchus exceed 21, 23, 19.8, and 17.4 mm, respectively. Its diagnosis is based on chest radiograph and chest computed tomography (CT). Patients, usually middle-aged men, may be asymptomatic or present with clinical manifestations ranging from minimal symptoms with preserved lung function to severe respiratory failure. Pulmonary function tests (PFTs) typically reveal a restrictive pattern. This report presents an elderly woman with previously diagnosed pulmonary fibrosis with symptoms of increased sputum production and haemoptysis. High-resolution chest CT showed tracheal and main stem bronchi dilatation along with bronchial diverticulosis. PFTs indicated a restrictive pattern characteristic of the underlying pulmonary fibrosis. The patient is the oldest, referred to the female gender, at presentation of MKS hitherto reported. This case highlights the need to include MKS in the differential diagnosis of recurrent lower respiratory tract infections, even in older subjects. ==== Body 1. Introduction Mounier-Kuhn syndrome (MKS) is an infrequent congenital syndrome, whose hallmark is airway enlargement. On histological examination, absence or atrophy of the elastic fibres within the tracheal wall is typically found [1–7]. This condition results in airway dilatation in the trachea and bronchi. Tracheal diverticula may also occur, mainly in the posterior trachea [8]. The typical features of this entity were first described by Mounier-Kuhn in 1932 [9]. Patients usually complain of recurrent respiratory tract infections along with various functional perturbations: from minimal involvement with preservation of lung function, through severe disease with bronchiectasis to overt respiratory failure [1–7, 10]. Patients are typically middle-aged males [2–4]. Its diagnosis is based on chest radiograph and chest computed tomography (CT). MKS is frequently overlooked, but should be considered in the investigation of recurrent lower respiratory tract infections [1–9]. This report presents a female patient, the oldest hitherto described, with MKS who had been previously diagnosed with pulmonary fibrosis. 2. Case Presentation An 83-year-old woman was referred to the pneumonology department of our hospital due to increasing productive cough and haemoptysis. During the last 30 years, she had experienced dyspnoea on exertion and increased expectoration of mucoid sputum that became purulent during infectious exacerbations, often with bloody streaked sputum. She denied fever, wheezes, chest pain, and weight loss, or any other symptom indicative of gastroesophageal reflux disease (GERD). Her past medical history included pulmonary fibrosis diagnosed 6 years before in another country; left ovarian cancer treated with hysterosalpingo-oophorectomy 20 years before; arterial hypertension; diabetes mellitus and osteoporosis. There was no family history of any respiratory disease and she had never smoked. No history of GERD was noted in her medical records. On physical examination, her vital signs were normal. Finger clubbing was not present. Mild inspiratory crackles at the lower third of both lung fields were revealed. Laboratory investigations were as follows: erythrocyte sedimentation rate: 15 mm/h; C-reactive protein: 1.03 mg/L with oxygen saturation at 96% on room air and arterial blood gases analysis with PaO2: 77.3 mmHg, PCO2: 41.5 mmHg, and PH: 7.45 (FiO2: 21%). Immunologic tests (rheumatoid factor, anti-CCP (anticyclic citrullinated peptide), C3, C4, p-ANCA, and c-ANCA) were within normal range while antinuclear antibody (ANA) levels were mildly raised (1/160) but there was no characteristic immunofluorescence pattern. Pulmonary function tests (PFTs), conducted 1 month before her admission, revealed a forced expiratory volume in 1 sec (FEV1) of 1.83 L (58% predicted), a forced vital capacity (FVC) of 1.95 L (55% predicted), and FEV1/FVC of 105%. We were not able to perform static lung volumes measurement and diffusion test, due to the patient's inability to cooperate. Sputum results were negative for mycobacteria. Bronchoscopy was not performed because the patient did not consent. An old chest radiograph, conducted 25 years ago, was available (Figure 1). This revealed widening of the trachea and main stem bronchi. These findings were confirmed by a new chest radiograph on the first day of the hospitalisation without any deterioration in terms of tracheobronchial dilatation (Figure 2). A high-resolution chest CT showed enlarged trachea and main stem bronchi (trachea with both sagittal and axial dimensions of 2.1 cm, right and left main bronchi measuring in axial dimensions 2.1 cm and 1.8 cm, resp.), extensive bronchiectasis, interstitial fibrosis of the lower lobes, and chronic pleural thickening (Figures 3 –6). The patient refused to undergo any further examination. 3. Discussion This is the oldest female patient at presentation of MKS hitherto described. Although this condition is typically encountered in middle-aged men [3–5, 11–13] our patient was an elderly female, which emphasises that MKS needs to be considered in older subjects as well. Another important issue relating to the patient presented was the previous diagnosis of pulmonary fibrosis. This diagnosis rendered the recognition of MKS more difficult. The hallmark of MKS, also known under alternative names (tracheal diverticulosis, tracheobronchiectasis, tracheocele, tracheomalacia, and tracheobronchopathia malacia), [9, 14–17] is dilatation of trachea and central bronchi with normal diameter of peripheral airways. In women, transverse and sagittal tracheal diameter must, by definition, be greater than 21 and 23 mm, respectively, [10]. There are 3 subtypes: type 1, with minimal symmetrical dilation in the trachea and main bronchi (as in the patient reported); type 2, with pronounced tracheal dilatation and diverticula; and type 3, whereby marked tracheal and bronchial dilatation extends further until the distal bronchi bilaterally [18]. Aetiology of MKS is unclear. There may be a primary defect or atrophy of elastic and smooth muscle tissue [19, 20]. MKS may also present in association with miscellaneous conditions, for example, Ehlers-Danlos syndrome, Marfan syndrome, connective tissue diseases, ataxia telangiectasia, and ankylosing spondylitis [21–24]. Diseases resulting in severe upper lobe fibrosis, such as sarcoidosis, cystic fibrosis, or diffuse pulmonary fibrosis, and airway inflammatory conditions, notably allergic bronchopulmonary aspergillosis, are also implicated in its pathogenesis [21–24]. Pulmonary fibrosis of the patient was considered idiopathic, due to the following reasons. First, serological findings were negative. Additionally any occupational exposure was excluded. There are no pathognomonic symptoms present in MKS. Patients are usually asymptomatic while excessive sputum production may occur secondary to bronchiectasis and lower respiratory tract infection [18]. Occasional haemoptysis and dyspnoea may be seen as well [22]. In our case, the patient presented with productive cough and haemoptysis. Its diagnosis rests on imaging studies. In the rare case of gross tracheal enlargement, chest X-rays may be diagnostic. More frequently, however, chest CT is required to reliably ascertain tracheal dimensions and to investigate any development of complications, for example, bronchiectasis [25]. At the same time, PFTs reveal a restrictive pattern. The effect of enlarged airways on spirometry derives from the weakness of the tracheobronchial walls and hypotonia in the myoelastic elements, resulting in dynamic airway compression (expiratory collapse during forced exhalation) and dynamic restriction. The restrictive pattern in our patient is magnified from the underlying fibrosis and possibly by the associated retention of secretions. Nevertheless, these findings are not always met in MKS, because cases with normal spirometric values have been also reported [5]. Diffusion test could have been helpful; however this was not performed, due to the inability of the patient to cooperate. Asymptomatic patients require no treatment. Therapy includes respiratory physiotherapy and antibiotics during infectious exacerbations, [26–32], while tracheal stenting is very rarely employed [2, 33]. In conclusion, this case highlights that a chest CT scan should be performed in patients reporting chronic recurrent lower respiratory tract infections to investigate underlying conditions, including MKS. Indeed, this condition, despite long-term follow-up for repeated lower respiratory infections and chronic cough, had long been left undiagnosed in our patient, until a high-resolution chest CT was performed. Our case is the oldest female patient described in literature with MKS, indicating that appropriate diagnostic workup may be required in elderly subjects as well. Competing Interests The authors declare that there are no competing interests. Authors' Contributions Paschalis Steiropoulos conceived and wrote the paper and acquired data; Panagiotis Boglou provided material support and reviewed literature; Anastasia Oikonomou acquired data; Stamatia Bakali acquired data and provided material support; Nikolaos Papanas made critical revisions of the paper. Figure 1 Chest X-ray conducted 24 years ago, displaying a trachea enlargement without any signs of fibrosis. Figure 2 Current X-ray displaying trachea enlargement accompanied by fibrotic elements in the lungs. Figure 3 High-resolution computed tomography (HRCT) of the chest at the level of the upper lobes. An enlargement of the trachea at the thoracic inlet is observed measuring in both sagittal and axial dimensions 2.1 cm. A subpleural reticular pattern associated with focal thickening of the pleura and of the major fissures bilaterally. Figure 4 HRCT of the chest at the level of the carina. An enlargement of the right and left main bronchi is observed measuring in axial dimensions 2.1 cm and 1.8 cm, respectively. A subpleural reticular pattern associated with focal thickening of the pleura and traction bronchiectasis and bronchiolectasis is also noted. Figure 5 HRCT of the chest at level below carina shows dilated right and left main bronchi and undulating wall of left main bronchus, indicating bronchial diverticulosis. A subpleural reticular pattern associated with focal thickening of the pleura and traction bronchiectasis and bronchiolectasis is also noted. Figure 6 HRCT of the chest at the level of the lower lobes. An extended and coarse subpleural reticular pattern is noted associated with traction bronchiectasis and bronchiolectasis as well as with honeycombing. ==== Refs 1 Adani G. L. Baccarani J. Lorenzin D. Renal transplantation in a patient affected by Mounier-Kuhn syndrome Transplantation Proceedings 2005 37 10 4215 4217 10.1016/j.transproceed.2005.11.024 2-s2.0-29544438015 16387081 2 Randak C. O. Weinberger M. A child with progressive multiple tracheal diverticulae: a variation of the Mounier-Kuhn syndrome Pediatric Pulmonology 2013 48 8 841 843 10.1002/ppul.22663 2-s2.0-84881381413 22949127 3 Odell D. D. Shah A. 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==== Front Case Rep Infect DisCase Rep Infect DisCRIIDCase Reports in Infectious Diseases2090-66252090-6633Hindawi Publishing Corporation 10.1155/2016/2547645Case ReportRefractory Toxic Shock-Like Syndrome from Streptococcus dysgalactiae ssp. equisimilis and Intravenous Immunoglobulin as Salvage Therapy: A Case Series http://orcid.org/0000-0002-8978-8978Islam Marjan 1 * Karter Dennis 1 Altshuler Jerry 2 Altshuler Diana 3 Schwartz David 4 5 Torregrossa Gianluca 6 1Department of Medicine, Mount Sinai Beth Israel, New York, NY 10003, USA2Department of Pharmacy, Mount Sinai Beth Israel, New York, NY 10003, USA3Department of Pharmacy, NYU Langone Medical Center, New York, NY 10016, USA4Department of Medicine, NYU Langone Medical Center, New York, NY 10016, USA5NYU School of Medicine, New York, NY 10016, USA6Department of Cardiac Surgery, Mount Sinai Beth Israel, New York, NY 10003, USA*Marjan Islam: mislam@chpnet.orgAcademic Editor: Antonella Marangoni 2016 15 8 2016 2016 254764522 5 2016 6 7 2016 21 7 2016 Copyright © 2016 Marjan Islam et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Infections from Streptococcus dysgalactiae ssp. equisimilis (SDSE) can cause a wide variety of infections, ranging from mild cellulitis to invasive disease, such as endocarditis and streptococcal toxic shock-like syndrome (TSLS). Despite prompt and appropriate antibiotics, mortality rates associated with shock have remained exceedingly high, prompting the need for adjunctive therapy. IVIG has been proposed as a possible adjunct, given its ability to neutralize a wide variety of superantigens and modulate a dysregulated inflammatory response. We present the first reported cases of successful IVIG therapy for reversing shock in the treatment of SDSE TSLS. ==== Body 1. Introduction Streptococcus dysgalactiae ssp. equisimilis (SDSE) are gram-positive β-hemolytic group C and G streptococci that commonly colonize the human respiratory, gastrointestinal, and female genital tracts. Epidemiologically and pathologically, this species is very similar to Group A streptococcus, capable of causing infections ranging from mild cellulitis or pharyngitis, to life-threatening invasive disease such as necrotizing fasciitis, meningitis, endocarditis, and septic shock. It has rarely been reported to cause streptococcal toxic shock-like syndrome (TSLS), with some strains found to produce streptococcal exotoxins [1]. We present what we believe to be the first 2 reported cases of successful intravenous immunoglobulin (IVIG) therapy for adult refractory SDSE TSLS. 2. Case Report 1 An 82-year-old male with history of heart failure, atrial fibrillation, diabetes, and multiple prior admissions for cellulitis presented to the ED with right lower-extremity erythema with associated altered mental status, fever of 101.8°F, hypotension (72/40 mmHg), and tachycardia (146 bpm). He was admitted to the MICU for septic shock from presumed cellulitis, with initial labs significant for a lactate of 4.5 mmol/L, acute kidney injury (SCr 1.9 mg/dL), leukocytosis (white blood cell count 17 k/μL, 88% neutrophils), and a procalcitonin of 38.64 ng/mL. He was requiring 26 mcg/min of norepinephrine for refractory hypotension, and vancomycin with piperacillin/tazobactam for cellulitis was initiated. On day 2, his blood cultures grew SDSE, and vasopressin (0.04 U/min) and dobutamine were added to maintain cardiac output due to a presumed sepsis-induced myocardial depression. Despite hemodynamic support, the patient remained in shock, raising concern for development of a refractory TSLS. Surgery was consulted for potential necrotizing fasciitis and IVIG (Gamunex®-C) (1 g/kg day 1, 0.5 g/kg days 2-3) and clindamycin were initiated. The following evening, the patient's vasopressor requirements had lessened, eventually titrated off completely over the following 24 hours. His lactate had cleared, and he did not require any surgical intervention. He was narrowed to penicillin on day 3 and transferred out of the MICU the following day. Workup for a source was inconclusive, with an abdominal CT scan showing no fluid collections or abscess. He was deemed stable for discharge on day 8. 3. Case Report 2 A 37-year-old male with history of coarctation of the aorta presented to the ED with fevers and diaphoresis for 2 weeks. He was initially treated for flu-like symptoms but developed rigors, abdominal pain, and jaundice, prompting him to return to our institution. On admission, he was febrile to 101°F, thrombocytopenic (platelet count 15 k/μL), with leukocytosis (white blood cell count 17.5 k/μL, 96% neutrophils), and with acute renal failure (SCr 1.79 mg/dL). He was started on broad spectrum antibiotics, which were subsequently narrowed to ceftriaxone (a rash developed with penicillin) and clindamycin after sensitivities revealed penicillin-susceptible SDSE (penicillin MIC < 0.06). Gentamicin was later added in the setting of persistent fevers and tachycardia. A transesophageal echocardiogram (TEE) revealed a 1.6 cm mobile tricuspid valve vegetation and a possible abscess at the aortic root after initial transthoracic echocardiogram (TTE) was inconclusive. His admission EKG showed a new 1st degree AV block. By day 9, his heart block progressed to type II second-degree AV block, with periods of complete heart block. With concern for infectious spread to the conduction system and a perivalvular abscess, he was taken emergently to the OR for radical debridement of the aortic roots, aortomitral curtain, and interatrial septum. He underwent an extensive bovine patch repair of the aortomitral curtain, interatrial septum, and pericardium. He also underwent enlargement of the aortic annulus and roots and placement of a mechanical aortic valve and permanent pacemaker. Pathology from the aortic valve revealed acute inflammatory cells with gram-positive cocci. Postoperatively, the patient remained febrile, and antimicrobials were transiently broadened. On day 14, a TTE revealed a new mitral valve vegetation, with a perforated anterior leaflet and severe mitral regurgitation. On day 26, repeat TEE revealed progression of mitral valve endocarditis, with a flail anterior leaflet with a second perforation, mandating surgical intervention. On day 28, the patient underwent repeat sternotomy with debridement of the mitral valve endocarditis and extensive reconstruction of the cardiac skeleton, requiring a repeat aortic and mitral valve replacement. Postoperatively, he became hypotensive and progressed into a refractory vasoplegic shock. He was given 2 doses of methylene blue and started on epinephrine (5 mcg/min), norepinephrine (19 mcg/min), and vasopressin (0.1 U/min). Despite adequate vasopressors, he remained vasoplegic, and IVIG was attempted as salvage therapy. He underwent 2 days of 100 g (1 g/kg adjust body weight) IVIG (Gamunex-C), which allowed for titration off vasopressor support over the following 2 days. An extensive infectious workup followed to identify other potential causes for a possible culture-negative endocarditis, including serology for B. henselae, B. quintana, Brucella, Coccidioides, Q Fever, and Rocky Mountain Spotted Fever, and PCR for Tropheryma whipplei, all of which returned negative. On day 46, the patient underwent a final TTE, which showed no valvular vegetation. He was deemed stable for discharge, with follow-up in the cardiac surgery clinic. 4. Discussion SDSE belong to a group of pyogenic streptococci, often referred to as β-hemolytic streptococci. While they were considered nonpathogenic for years, recent population-based studies have revealed invasive SDSE to have a similar disease profile as invasive Streptococcus pyogenes [1]. SDSE primarily present as skin and soft tissue infections, though invasive forms may present as osteomyelitis, pulmonary or intra-abdominal abscesses, meningitis, endocarditis, or necrotizing fasciitis. Septic shock and multisystem organ failure may result from TSLS. While injection drug users and immunosuppression pose increased risk, previously healthy individuals can develop severe infections as well [2]. Molecular studies have shown SDSE to display nearly identical virulence factors as S. pyogenes, though the etiology behind the emergence of more human-invasive strains remains undetermined. The principal mechanism appears to be translocation of mobile DNA elements into bacterial genomes by bacteriophages [1]. Indeed certain superantigen genes such as speA, speC, and speM have been found in SDSE strains nearly identical to those in S. pyogenes. Further, SDSE have consistently displayed the M protein common to S. pyogenes, conferring resistance to phagocytosis. In addition, S. pyogenes and SDSE share adhesion virulence factors such as fibronectin and plasminogen binding proteins, allowing for colonization of epithelium and invasion into the bloodstream [1]. Streptococcal TSLS is the most severe manifestation of invasive disease from streptococci, with case-mortality rates reported as high as 81% [3]. The pathogenesis behind SDSE-mediated shock likely involves the release of superantigens known as streptococcal pyrogenic exotoxins (SPEs). SPEs activate T-cell receptor molecules that directly interact with the MHC class II on antigen-presenting cells, leading to massive T-cell proliferation and a cytokine storm [2]. The effects of such a large influx of cytokines can precipitate severe vasoplegia and hemodynamic collapse, conferring the mortality seen in streptococcal TSLS [4]. SDSE remains nearly universally susceptible to penicillin and other β-lactam agents [1]. Clinical investigations have identified the need for an adjunctive therapy however, as high mortality rates persist despite prompt antimicrobial therapy. Since many streptococcal superantigens contribute to the pathogenesis of invasive streptococcal infections, IVIG has been suggested as the plausible adjunct, given its ability to modulate the inflammatory response elicited by virulence factors and counteract a wide variety of superantigens simultaneously [5]. It is postulated that only individuals who lack neutralizing antibodies to the putative virulence factors, such as the SPEs or M-protein, develop invasive streptococcal infections and TSLS [6]. Indeed studies have demonstrated patients with bacteremia and TSLS lacked antibodies directed against speB, suggesting passive immunization of patients lacking neutralizing antibodies may modify the course of this toxin-mediated disease [7]. Prior reports demonstrated IVIG's capability to block in vitro T-cell activation of staphylococcal and streptococcal superantigens. Emerging evidence has also suggested IVIG may contain superantigen-neutralizing antibodies [8]. Multiple case reports have demonstrated improved clinical outcomes in patients with TSLS who have received IVIG [9–11], while larger clinical series have supported its use. Kaul et al. demonstrated a higher 30-day survival in patients with TSLS who received IVIG compared to controls (67% versus 34%, p = 0.02), demonstrating an odds ratio for survival of 8.1 (CI 1.6–45, p = <0.01) [8]. Further, they demonstrated reduction in bacterial mitogenicity and T-cell production of IL-6 and TNF-α in patients who received IVIG [8]. Darenberg et al. compared IVIG therapy to placebo in patients with streptococcal TSLS and demonstrated a 3.6-fold lower mortality rate in the IVIG group, though the study was underpowered (p = 0.3) [12]. The IVIG group also had significantly lower sepsis-related organ failure assessment scores on days 2 (p = 0.02) and 3 (p = 0.04) compared to placebo, while also demonstrating increased plasma neutralizing activity against superantigens expressed by autologous isolates (p = 0.03) [12]. Upon revisiting the cases presented, it is plausible that our patients lacked the appropriate antibodies necessary to eradicate a putative virulence factor, leading to an invasive SDSE infection and ultimately succumbing into a refractory TSLS. Administration of IVIG likely helped modulate their profoundly dysregulated inflammatory responses, neutralizing the virulent super-antigens expressed by SDSE and allowing for hemodynamics to normalize. The successful use of IVIG for refractory TSLS demonstrates the utility of this novel adjunctive therapy, while highlighting the changing epidemiology and pathogenicity of SDSE. Additional Points The information in this paper was not presented in any meetings at the time of submission. Competing Interests None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this paper. ==== Refs 1 Brandt C. M. Spellerberg B. Human infections due to Streptococcus dysgalactiae Subspedes equisimilis Clinical Infectious Diseases 2009 49 5 766 772 10.1086/605085 2-s2.0-70149091079 19635028 2 Korman T. M. Boers A. Gooding T. M. Curtis N. Visvanathan K. Fatal case of toxic shock-like syndrome due to group C streptococcus associated with superantigen exotoxin Journal of Clinical Microbiology 2004 42 6 2866 2869 10.1128/JCM.42.6.2866-2869.2004 2-s2.0-2942623965 15184494 3 Natoli S. Fimiani C. Faglieri N. Toxic shock syndrome due to group C Streptococci. A case report Intensive Care Medicine 1996 22 9 985 989 10.1007/s001340050200 2-s2.0-0029840939 8905439 4 Ojukwu I. C. Newton D. W. Luque A. E. Kotb M. Y. S. Menegus M. Invasive Group C Streptococcus infection associated with rhabdomyolysis and disseminated intravascular coagulation in a previously healthy adult Scandinavian Journal of Infectious Diseases 2001 33 3 227 229 10.1080/00365540151060969 2-s2.0-0035088672 11303816 5 Norrby-Teglund A. Basma H. Andersson J. McGeer A. Low D. E. Kotb M. Varying titers of neutralizing antibodies to streptococcal superantigens in different preparations of normal polyspecific immunoglobulin G: implications for therapeutic efficacy Clinical Infectious Diseases 1998 26 3 631 638 10.1086/514588 2-s2.0-0031968581 9524835 6 Holm S. E. Norrby A. Bergholm A.-M. Norgren M. Aspects of pathogenesis of serious group A streptococcal infections in Sweden, 1988-1989 Journal of Infectious Diseases 1992 166 1 31 37 10.1093/infdis/166.1.31 2-s2.0-0026771289 1607705 7 Barry W. Hudgins L. Donta S. T. Pesanti E. L. Intravenous immunoglobulin therapy for toxic shock syndrome The Journal of the American Medical Association 1992 267 24 3315 3316 10.1001/jama.1992.03480240077038 2-s2.0-0026647438 1597914 8 Kaul R. McGeer A. Norrby‐Teglund A. Intravenous immunoglobulin therapy for streptococcal toxic shock syndrome—a comparative observational study Clinical Infectious Diseases 1999 28 4 800 807 10.1086/515199 10825042 9 Nadal D. Lauener R. P. Braegger C. P. T cell activation and cytokine release in streptococcal toxic shock-like syndrome The Journal of Pediatrics 1993 122 5 727 729 10.1016/s0022-3476(06)80014-4 2-s2.0-0027315581 8496751 10 Yong J. M. Holdsworth R. J. Parratt D. Necrotising fasciitis The Lancet 1994 343 8910 1427 1428 10.1016/S0140-6736(94)92548-8 2-s2.0-27344449778 11 Lamothe F. D'Amico P. Ghosn P. Tremblay C. Braidy J. Patenaude J.-V. Clinical usefulness of intravenous human immunoglobulins in invasive group A streptococcal infections: case report and review Clinical Infectious Diseases 1995 21 6 1469 1470 10.1093/clinids/21.6.1469 2-s2.0-0029561245 8749635 12 Darenberg J. Ihendyane N. Sjölin J. Intravenous immunoglobulin G therapy in streptococcal toxic shock syndrome: a European randomized, double-blind, placebo-controlled trial Clinical Infectious Diseases 2003 37 3 333 340 10.1086/376630 2-s2.0-0041402681 12884156

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==== Front Case Rep PsychiatryCase Rep PsychiatryCRIPSCase Reports in Psychiatry2090-682X2090-6838Hindawi Publishing Corporation 10.1155/2016/9406813Case ReportTwo Sudden and Unexpected Deaths of Patients with Schizophrenia Associated with Intramuscular Injections of Antipsychotics and Practice Guidelines to Limit the Use of High Doses of Intramuscular Antipsychotics http://orcid.org/0000-0002-7167-2993Wahidi Nasratullah 1 http://orcid.org/0000-0002-3664-9392Johnson Katie M. 2 Brenzel Allen 1 2 http://orcid.org/0000-0002-7756-2314de Leon Jose 1 3 4 5 * 1Department of Psychiatry, College of Medicine, University of Kentucky, Lexington, KY 40509, USA2Department of Behavioral Health, Developmental and Intellectual Disabilities, Frankfort, KY 40621, USA3Eastern State Hospital, University of Kentucky Mental Health Research Center, Lexington, KY 40511, USA4Psychiatry and Neurosciences Research Group (CTS-549), Institute of Neurosciences, University of Granada, 18971 Granada, Spain5Biomedical Research Centre in Mental Health Net (CIBERSAM), Santiago Apóstol Hospital, University of the Basque Country, 01004 Vitoria, Spain*Jose de Leon: jdeleon@uky.eduAcademic Editor: Erik Jönsson 2016 15 8 2016 2016 94068139 5 2016 3 7 2016 Copyright © 2016 Nasratullah Wahidi et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Intravenous haloperidol has been associated with torsades de pointes (TdP). These two sudden deaths were probable adverse drug reactions (ADRs) following intramuscular (IM) antipsychotics. The autopsies described lack of heart pathology and were highly compatible with the possibility of TdP in the absence of risk factors other than the accumulation of antipsychotics with a high serum peak after the last injection, leading to death within hours. The first case was a 27-year-old African-American male with schizophrenia but no medical issues. His death was probably caused by repeated IM haloperidol injections of 10 mg (totaling 35 mg in 2 days). The second case involves a 42-year-old African-American female with metabolic syndrome. Her probable cause of death was the last ziprasidone IM injection of 20 mg in addition to (1) three extra haloperidol doses (2 hours before the ziprasidone injection, 5 mg oral haloperidol; approximately 21 hours earlier, 5 mg oral haloperidol; and 2 days prior, one 10 mg IM haloperidol injection), (2) 10 mg/day of scheduled oral haloperidol for 6 days before death, and (3) a long-acting paliperidone injection of 156 mg 18 days before death. The study of haloperidol glucuronidation and its impairment in some African-Americans is urgently recommended. ==== Body 1. Introduction Thioridazine was introduced in the US market in 1959 [1]. In 1964 [2], Kelly et al. reported that thioridazine was associated with quinidine-like electrocardiogram (EKG) abnormalities in 28 patients and with two cases of ventricular tachycardia leading to death in patients taking 1500 mg/day and 3600 mg/day, respectively. The awareness of these deaths before publication led the pharmaceutical company to talk to Ban and St. Jean [3]. Ban proposed completing a prospective EKG study using four doses of thioridazine and four doses of chlorpromazine and trifluoperazine as controls. EKG abnormalities resembling those caused by quinidine and hypokalemia were present in all 6 patients taking thioridazine (versus 3/6 for chlorpromazine and 1/6 for trifluoperazine) [3]. Before Ban and St. Jean published their study, Desautels et al. identified a patient who survived ventricular tachycardia while receiving 1500 mg/day of thioridazine for 6 weeks [4]. Some other studies were published regarding the matter [1], including one by Simpson as senior author [5] describing thioridazine-induced EKG changes as very likely in the elderly and, therefore, recommending against thioridazine as a drug for the elderly. According to Shorter, Ban tried to convince the pharmaceutical company of the clinical relevance of thioridazine-induced arrhythmias but was not successful [1]. As a matter of fact, the company did not change the prescribing information to warn physicians of the risk of thioridazine causing torsades de pointes (TdP) until 2000 [1]. Finally, in 2006, more than 40 years after the first deaths associated with thioridazine, the drug was withdrawn from the US market [6]. After slowly increasing awareness, the relationship between drugs, including antipsychotics, and TdP, a potentially lethal adverse drug reaction (ADR), became irrefutable in the 1990s and the US Food and Drug Administration (FDA) had no choice but to intervene. The turning point came in 1996 [7]. Terfenadine was a second-generation antihistamine that had been approved in the USA in 1985 [6] after no systematic studies on drug-drug interactions (DDIs) and on drug metabolism. The randomized controlled trials (RCTs) in healthy subjects with limited comedication had demonstrated that it was a very safe drug. Once approved, terfenadine was widely used in the general population including in patients who were taking erythromycin, ketoconazole, and itraconazole. These 3 drugs are powerful cytochrome P450 (CYP) 3A4 (CYP3A4) inhibitors and terfenadine was metabolized by CYP3A4. In 1996, the FDA became aware [7] that there were at least 125 deaths in the US caused by terfenadine. Patients taking any of these major CYP3A4 inhibitors had major accumulations of terfenadine with very high serum concentrations leading to TdP. The FDA required warning labels in the prescribing information for terfenadine and the CYP3A4 inhibitors; terfenadine was finally withdrawn from the market in 1998 [6]. The terfenadine deaths explained by DDIs led to the FDA's awareness of the need to study CYP metabolism and DDIs for new drug submissions. The requirements were progressively increased in the late 1990s, after several other drugs metabolized by CYP3A4 and with potential to cause TdP were withdrawn from the market [6]. Sertindole, a second-generation antipsychotic, was introduced in Europe in 1996 [8]. It rapidly became associated with sudden cardiac deaths and an in vitro study demonstrated that sertindole has high affinity as an antagonist of the cardiac potassium channel [9]. In humans, the delayed rectifier potassium current is mediated by the ion channel KCNH2 encoded by the human ether-a-go-go–related gene (HERG) [9]. After the publication of this article describing that sertindole was an antagonist of the channel encoded by HERG [9], it became evident that first-generation antipsychotics, such as thioridazine, must have the same antagonist properties at the same cardiac channel; this also explained why hypokalemia is a risk factor for antipsychotic-induced TdP [10]. Since TdP is a very rare event, in the late 1990s the FDA proposed using prolongation of the QT interval as a TdP risk marker. This was a complex decision since there is general agreement that the QT interval needs to be corrected by heart frequency, which is called corrected QT (QTc), but there are several formulas for doing it and no agreement among experts concerning which one is best. Another issue is how good QTc prolongation proves to be as a sign of the blockade of the cardiac potassium channel, which is determined by drug affinity and the serum concentrations in a specific patient. Thioridazine appears to have the highest affinity among antipsychotics and the use of very high doses >1,000 mg/day, such as the doses used in the 1960s, led to very high serum concentrations and extremely high potential for TdP in many, if not all, patients. In the late 1990s, the company developing ziprasidone was trying to introduce it into the US market, but ziprasidone has affinity for the human cardiac potassium channels and caused prolongations of the QTc interval. To allay the FDA's concerns, ziprasidone's marketer decided to complete a milestone prospective randomized study [11] comparing 6 oral antipsychotics (haloperidol, quetiapine, olanzapine, risperidone, thioridazine, and ziprasidone) by themselves and in the presence of CYP inhibitors (fluvoxamine for CYP1A2, ketoconazole for CYP3A4, and paroxetine for CYP2D6). With the specific doses used in that study, thioridazine was associated with greater QTc prolongations with a mean increase of 30.1 milliseconds (ms), ziprasidone was second with 15.9 ms, and haloperidol was third with 7.1 ms. Following this study, ziprasidone was permitted in the US market in 2001 with some warnings about QTc prolongation in the prescribing information. Then, some of the first-generation antipsychotics started to be withdrawn from some markets due to TdP risk. This led to major discussions in the US and international journals [12–14] and major disagreement on the advertising by the company marketing antipsychotics about the clinical relevance of antipsychotic-induced QTc prolongations and the virtues or weaknesses of various second-generation antipsychotics regarding their effects on the QTc interval. The problems [14] are that (1) sudden death on oral antipsychotics has a very low incidence: around 1 in every 10,000 patients. This requires 10,000 patients taking medications for an extended period to identify a few cases, making it very difficult to study; and (2) these large pharmacoepidemiological naturalistic studies include sudden death cases with multiple confounding factors and no guarantee that they are explained by TdP versus other causes of sudden death or by TdP explained by the combination of multiple factors besides an antipsychotic. Review articles started recommending a QTc limit (e.g., >500 ms) as a risk marker for TdP [12]. This became controversial since other authors recommended other values, such as >450 ms [13]. Moreover, the literature clearly indicated that women have a greater mean QTc; therefore, a sex-corrected QTc limit for predicting risk for TdP may be needed (e.g., >450 ms in men and >470 ms in women) [13]. These controversies concerning QTc prolongation are not easy to resolve since there is limited reliable data on TdP cases caused by antipsychotics. Summarizing our current knowledge [14], we can describe most cases of drug-induced TdP as occurring in the context of substantial prolongation of the QTc interval, typically to values >500 ms, but QTc alone is a relatively poor predictor of arrhythmic risk in any individual patient. Some drugs that substantially prolong the QTc interval produce very low rates of TdP while others have much smaller QTc effects but are considerably more prone to cause TdP [15]. The psychiatric literature describing clinical cases of TdP is very complex [14], since the cases are frequently associated with polypharmacy and DDIs, with a pharmacodynamic component involving multiple drugs with HERG channel inhibitory properties and sometimes a pharmacokinetic component, an inhibitor increasing the plasma concentrations of one or several of the drugs. Other risk factors for TdP such as female gender, bradycardia, hypokalemia, and hypomagnesemia may be important, too [16]. Since 2001, oral ziprasidone has occasionally been associated with TdP, but most of the cases include patients with other TdP risk factors [17]. A large 1-year mortality study randomly assigning its 18,000 patients to ziprasidone or olanzapine concluded that these drugs have similar nonsuicide mortality, but the study acknowledged that it did not have enough power to detect rare events such as TdP [18]. In a comprehensive review of the literature, sponsored by ziprasidone's marketer and focused on the company's RCT and the postmarketing surveillance data, Camm et al. [19] concluded that ziprasidone is safe if used as indicated. On the other hand, a more recent pharmacoepidemiology review by independent investigators suggested that there were 3 antipsychotics definitively associated with TdP: amisulpride, haloperidol, and ziprasidone [20]. As population clinical data [19] and laboratory pharmacological studies [21] indicate that drug-induced QTc prolongations are without doubt driven by higher serum concentrations, the parenteral administration of any of these antipsychotics, which is associated with much higher peak concentrations than oral administration, should substantially increase the risk of antipsychotic-induced TdP in patients receiving intramuscular (IM) formulations. IM ziprasidone was approved in the US for agitation in schizophrenia patients in 2002. After a healthy young Chinese male patient with schizophrenia demonstrated a QTc interval prolongation of 83 ms after receiving a single IM injection with 20 mg of ziprasidone, Li et al. [22] completed a systematic review on the effects of IM ziprasidone on the QTc interval prolongation. They identified 19 trials in English or Chinese with a total of 1428 patients, many using haloperidol IM as a control. Their review identified two cases of patients who experienced symptoms probably related to QTc prolongation after IM ziprasidone. If one assumes that published IM ziprasidone cases are representative, one can estimate you need only 1,000 patients on IM ziprasidone to identify symptoms of TdP versus 10,000s required to identify sudden deaths associated with oral antipsychotics. In the systematic review, mean QTc change from baseline to end of each trial ranged from −3.7 to 12.8 ms after IM ziprasidone (compared with −3.5 to 14.7 ms on haloperidol IM). Four RCTs were used to calculate a meta-analysis of QTc interval prolongation; it showed no significant differences between IM ziprasidone and IM haloperidol groups. In summary, IM ziprasidone appeared to have some risk for TdP that did not look different from IM haloperidol. The history of haloperidol-induced TdP is also as protracted as that of thioridazine-induced TdP. Haloperidol is a first-generation antipsychotic marketed in the 1960s but with the peculiarity of having formulations for IM and intravenous (IV) administration that are more risky for TdP since they provide much higher peak serum concentrations than oral formulations. IV haloperidol is mainly used for sedation by internists rather than by psychiatrists and internists use it in continuous administration controlled by nurses; there is potential for huge doses since haloperidol is not a potent drug for sedating patients. Psychiatrists tend to use haloperidol IM and combine it with other more sedating drugs (e.g., lorazepam and/or diphenhydramine). During the 1990s and 2000s, there was a progressive increase in cases demonstrating the association between extremely high accumulated doses of haloperidol IV and TdP [23–26], to the point that the FDA required a warning in the US haloperidol prescribing information in 2007 [27]. Based on the case reports of potentially fatal cardiac events, the FDA warned that haloperidol is not approved for IV administration, but if IV administration is used, EKG monitoring should be performed. Meyer-Masseti et al. [27] considered the FDA's recommendations in 2007 confusing. The FDA warned that QTc prolongation and risk of TdP were increased with IV administration of haloperidol or administration of haloperidol at greater-than-recommended doses in any formulation. However, Meyer-Masseti et al. [27] stressed that neither the “typical” dosing range nor the minimum dose associated with these cardiac ADRs was specified in the haloperidol prescribing information. In their review of the literature [27], Meyer-Masseti et al. identified a total of 70 cases of IV haloperidol associated with QTc prolongation and/or TdP. When postevent QTc data were reported, QTc was prolonged >450 ms in 96% of the cases. Most (97%) of the patients had additional risk factors for TdP, mainly the coprescription of other proarrhythmic agents. Patients experiencing haloperidol-associated TdP received a wide range of cumulative doses from 5 mg to 645 mg. In their review, Meyer-Masseti et al. do not address the crucial issue: what do we know about TdP only caused by IV haloperidol in the absence of other major risk factors? It is obvious that IV haloperidol should not be used in patients with TdP risk factors, but we do not know what doses of IV haloperidol may be risky in patients with no known TdP risk factors. The association between oral and IM haloperidol with TdP has received much less attention in the literature than the association with IV haloperidol. Jackson et al. [28] published a TdP case associated with 4 mg oral haloperidol (2 doses of 2 mg separated by 8 hours) in a 66-year-old woman. Harvey et al. [29] studied 12 volunteers with schizophrenia who were given a single IM injection of 7.5 mg haloperidol or 4 mg lorazepam in a blinded, randomized, placebo-controlled crossover design. Mean changes in the QTc interval in those receiving the haloperidol IM dose ranged from 3.6 to 5.1 ms, depending on the formula used to correct for heart frequency. Harvey et al. [29] concluded that, on average, this dose of IM haloperidol led to minimal prolongation of the QT interval and this effect was of theoretical concern in individuals with risk factors for TdP but seemed unlikely to be a problem in the vast majority of patients. In our literature searches in PubMed, we have not been able to identify clinical studies of QTc prolongation using IM haloperidol formulations with repeated administration, such as those used by psychiatrists to control agitation in the clinical environment or case reports of TdP associated with IM haloperidol. There are IM ziprasidone RCTs using haloperidol IM as a control, but most of the published articles focused on the mean increases in QT prolongation rather than the extreme cases and frequently considered haloperidol-induced QTc changes as not clinically relevant (reviewed in Table 1 of [22]). In this paper, we present two cases of sudden death after IM injections of antipsychotics (haloperidol in the first case and a haloperidol-ziprasidone combination in the second case) with autopsies indicating lack of heart pathology and high compatibility with the possibility of TdP. After using two different ADR scales in each patient [30, 31], the authors consider these two sudden deaths to be probable ADRs. 2. The Patient Sample That Led to Two Cases The state of Kentucky is located in the center of the United States and has a population of approximately 4 million people. Most Kentuckians are Caucasians; less than 10% are African-Americans and small numbers of people are from other racial/ethnic backgrounds. The majority of adult patients with severe mental illnesses (SMIs) get admitted to 4 state psychiatric hospitals and 1 forensic facility. According to our centralized database with admissions data, in the last 15 years the annual number of different patients admitted to these four psychiatric state hospitals has ranged between 5,556 and 7,106. Our experience with large published studies [32, 33] in these admitted populations indicates that there are three main groups of diagnoses justifying admission, each approximating one-third of the cases: (1) severe mood disorders, (2) schizophrenia and related psychoses, and (3) complications of substance use disorders [32, 33]. Since 2002, a state mortality review process has provided outside review of the deaths at any state facility, in addition to the internal regulatory process at each facility. The facilities include not only these state psychiatric hospitals but also 3 nursing homes and 4 long-term care facilities for adults with intellectual disabilities. Since 2008, some community deaths have been reviewed as well. Focusing only on deaths occurring during admission at state psychiatric hospitals over 14 years (from 2002 to January 2016), we identified 95 deaths. These 95 patients were 63% male and 87% Caucasian (11% African-American), which roughly corresponds to the admission demographics in our state psychiatric hospitals, based on our published studies including thousands of patients [32, 33]. The mean age of the 95 deceased patients was 58.0 years (standard deviation 16.7 years), apparently higher than the average age of admitted patients every year, which tends to be in the late 30s [32, 33]. Obviously, older patients are more prone to die. Of these 95 deaths, two (approximately 2%) appeared highly compatible with TdP associated with antipsychotic treatment. Both patients were prescribed high doses of IM antipsychotic injections and had autopsies showing no heart abnormalities. According to the committee's evaluation, besides these 2 deaths compatible with TdP, there were 32 other unexpected sudden deaths that may need to be considered in differential diagnosis. Only 7 of these 32 deaths had autopsies but the committee concluded that (1) 17 appear compatible with myocardial infarcts, (2) 9 appear compatible with pulmonary thromboembolism, (3) two appear compatible with sudden death in the context of epilepsy, (4) one appears compatible with a rupture of aortic aneurysm, and (5) three were of unknown origin (including one in which the request for autopsy was denied by the coroner). An antipsychotic-induced arrhythmia was not suspected in any of these 32 deaths since they were not temporally associated with recent IM injections or extremely high doses of oral antipsychotics, but there is no way of ruling out TdP with 100% certainty. An autopsy providing another cause of death (e.g., myocardial infarct) cannot rule out the possibility that the patient had an antipsychotic-induced TdP on top of a myocardial infarct. Nonetheless, the two cases described in detail in this paper were judged to be highly compatible with sudden death after the high doses of IM antipsychotics in the absence of other known risk factors for TdP. 3. Case Presentations 3.1. Case  1 3.1.1. Prior History This patient was a 27-year-old African-American male. His weight was 88 Kg (194 pounds). He had been diagnosed with chronic schizophrenia since age 22. He also had a history of polysubstance abuse and was a smoker. He had 9 prior state hospital admissions. During two of these admissions, he received one injection of 5 mg haloperidol IM to control psychosis and agitation, each without any obvious problem. During the 8th admission, he intermittently refused oral antipsychotic treatment medication that was court-ordered, which led to 2 injections of 10 mg haloperidol (and diphenhydramine 50 mg) without any problem, but the second injection was administered 3 days later, when the peak concentrations from the first injection were long gone. During the 9th admission, when he was 25 years old, an EKG showed “sinus bradycardia” (frequency of 58 beats per minute). The QT interval was read as 376 ms and the QTc as 373 ms. At that time he was taking, as oral medications, risperidone 4 mg/day, benztropine 2 mg/day, and divalproex sodium 1500 mg/day and 3 days before the EKG, the patient had also received an injection of long-acting risperidone of 37.5 mg but no other antipsychotic injections in the prior week. Three days before the EKG, electrolytes including potassium were normal. In summary, this EKG during reasonable doses of oral and long-acting risperidone demonstrated a completely normal QTc two years before the patient's death. 3.1.2. Last Admission The patient's 10th admission to state facilities lasted less than 3 days due to his death in the early morning of the third day. It was an involuntary admission with forced medication approved by the court. The patient was transferred from jail where he had had no access to tobacco smoking for weeks. Upon arrival, he was uncooperative and psychotic. His oral medications, risperidone 6 mg/day (3 mg twice a day) and benztropine 2 mg/day (1 mg twice a day), were reordered since it appeared that he had not been taking them in jail. A court-approved forced intramuscular (IM) order of haloperidol 10 mg and diphenhydramine 50 mg was to be administered if he refused any dose of oral risperidone. The admitting psychiatrist was questioned about this high haloperidol IM dose after the patient's death and provided the explanation that 3 years prior, at the same facility (his 8th admission to a state facility), the patient had received the same IM dosage of haloperidol 10 mg IM and diphenhydramine 50 mg without problems when refusing court-ordered oral medications. The psychiatrist was not aware that only 2 such injections were given during that prior admission and they were separated by 3 days. At admission the patient was uncooperative, refused a physical exam, and only cooperated with the measure of blood pressure (130/90 mm of Hg) and pulse (106 beats per minute). During the rest of the admission, he refused offers of completing the physical exam, vital signs and laboratory tests, but the available information indicated no obvious medical problems and he was essentially a medically healthy young man with no history of any serious medical problems during his 9 prior admissions to our facilities. During the first 2 days of this admission, the patient refused all oral medications and received forced IM medication twice a day with total accumulated dosages of IM haloperidol 35 mg and diphenhydramine 200 mg. The patient received 4 IM haloperidol injections but one of the nurses made a mistake, giving only 5 mg. Therefore, the patient received 3 injections of 10 mg and 1 of 5 mg, making a total of 35 mg/day in 2 days. On the second day, particularly, he received a set of injections with dosages of IM haloperidol 10 mg and diphenhydramine 50 mg in the early a.m. and another set at night. Eighteen minutes after the night injection, a staff member checking the room reported that the patient displayed leg movements and 15 minutes later (33 minutes after the haloperidol injection) he was found unresponsive. Cardiopulmonary resuscitation (CPR) was started but the automated external defibrillator (AED) advised no shock. The emergency medical service (EMS) arrived, pronouncing the patient dead 1 hour and 13 minutes after the last haloperidol IM injection of 10 mg. 3.1.3. Autopsy The autopsy showed no obvious anomalies in the heart or other organs that would explain the patient's sudden death. In macroscopic and microscopic examination, the coronary arteries and myocardium were essentially normal. 3.1.4. ADR Scales Using the Liverpool ADR Causality Assessment Tool [30], haloperidol-induced sudden death after 4 haloperidol IM injections with an accumulated dose of 35 mg in 2 days was deemed probable. The patient had not taken any other antipsychotic for weeks. Similarly, based on the Naranjo scale [31], a score of positive 6 (+6) was determined (scoring +1 on items 1, 5, 8, and 10 and +2 on item 2), consistent with a probable ADR. All authors agreed with the scores on both ADR scales. As the patient had no signs of myocardial ischemia or pulmonary thromboembolism, it appears to the authors that haloperidol had to cause the sudden unexpected death by TdP after the accumulation of high serum haloperidol concentrations, particularly with the fourth and last IM injection. The possible contribution of diphenhydramine to this case and to the second case is discussed in detail in the Discussion (see Section 4.5). As far as we can tell, the literature typically describes TdP as short-duration phenomena (usually seconds) with two outcomes: (1) death due to cardiac arrest or (2) disappearance but with substantial risk of recurrence [34, 35]. Therefore, it may not be not surprising that the AED did not recommend an electrical shock which may suggest that TdP was not present at the time CPR was initiated in this patient. We do not know how long the patient had been unconscious or in cardiac arrest; we only know that his unconsciousness was identified 33 minutes after the last haloperidol IM injection. 3.2. Case  2 3.2.1. Prior History This patient was a 42-year-old African-American female who had given birth to one son. She had been diagnosed with chronic schizophrenia since age 18 and had 8 prior admissions to Kentucky state psychiatric facilities. The court had previously declared her legally incompetent and a sister was appointed to make decisions for her. However, in Kentucky, guardians cannot involuntarily admit patients to psychiatric hospitals; a court needs to approve these admissions. 3.2.2. Last Admission The patient's 9th admission to a state psychiatric facility was precipitated by the worsening of her psychotic behavior. She was taken to the emergency room in a university hospital with short-term beds under an involuntary order allowing a 72-hour examination before deciding whether or not to ask a court for an involuntary psychiatric admission. After the filing of court documents for an involuntary admission, she was transferred to a psychiatric state hospital. The admission lasted a total of 7 days until her death. She stayed 4 days in the emergency room and 3 days at the psychiatric hospital. The psychiatric diagnosis was chronic paranoid schizophrenia. Her medical diagnoses included obesity, diabetes mellitus type 2, hypertension, hyperlipidemia, asthma, gastroesophageal reflux disease (GERD), seasonal allergy, and valproate-induced alopecia. Her weight was 131.5 kg (290 pounds) and her body mass index 45.5. At the emergency room, the sister reported that the patient was taking divalproex sodium 1000 mg/day (500 mg twice a day) and paliperidone palmitate injections. The sister explained that divalproex sodium had recently been changed to topiramate due to alopecia, but this change had not yet been implemented; moreover, the patient had been noncompliant with the medication at home. The community mental health center providing outpatient treatment reported that the patient had received paliperidone palmitate 156 mg every month for 2 years with the last injection 16 days before the admission. Her medical medications were, for diabetes, insulin glargine 10 units/day and metformin 2000 mg/day (1000 mg twice a day); for hypertension, lisinopril 5 mg/day; and for allergies, loratadine 10 mg/day. In the emergency room, she was paranoid, tried to hit the admitting psychiatrist, and showed bizarre behaviors, disrobing and defecating on the bathroom floor. A 5 mg haloperidol IM injection was given and oral psychiatric medication was started, including oral haloperidol, 10 mg/day (5 mg twice a day), and divalproex sodium, 1000 mg/day (500 mg twice a day). The admitting psychiatrist also wrote an “as-needed” oral order for agitation and/or aggression combining 5 mg haloperidol, 2 mg lorazepam, and 25 mg diphenhydramine. These three combined oral drugs (or a single oral drug) could be administered at the discretion of the nurses but with a frequency of not more than every 6 hours. For her medical problems, a diet for diabetic patients was started and aspirin 81 mg/day was added to her prior medical medications (insulin glargine, 10 units/day; metformin, 2000 mg/day; lisinopril, 5 mg/day; and loratadine, 10 mg/day). An EKG on admission (first day at the emergency room after a haloperidol 5 mg IM injection) revealed QT of 336 ms, QTc of 457 ms, and sinus tachycardia (frequency of 111 beats per minute). The routine blood laboratory analyses on the day of admission showed normal electrolytes but presented several abnormalities including glucose of 472 mg/dL (normal range 65–110), white blood cell count of 11,700 leukocytes/mm3 (normal range 4,100–10,800), neutrophil count of 7,900 neutrophil/mm3 (normal range 1,700–6,000), platelet count of 503,000 platelets/mm3 (normal range 140,000–370,000), and glycosylated hemoglobin at 10.4% (normal range 4.8–5.9). Her valproic acid level was undetectable (<3 μg/mL). Her urine showed high glucose, moderate ketones, a few epithelial cells and traces of bacteria. In summary, the laboratory abnormalities suggested that she had not been compliant with her valproate or her antidiabetic medication. Three days later, some of the abnormal levels were reduced: glucose was 330 mg/dL, white blood cell count of 11,740 leukocytes/mm3, neutrophil count of 4,500 neutrophil/mm3 (a normal value), and platelet count of 497,000 platelets/mm3. A lipid profile showed normal values for triglycerides, 133 mg/dL (range 35–150), for cholesterol, 175 mg/dL (range 105–200), and for high density lipoproteins, 37 mg/dL but an elevated low density lipoprotein of 112 mg/dL (range 0–99). At the psychiatric hospital, the patient was very agitated, psychotic, paranoid, and uncooperative. She would try to walk into other patients' rooms, including male rooms, and touch staff and other patients. These behaviors could not be controlled by having one staff member closely watching her, since she did not listen to redirection. In the state psychiatric hospital over the 3-day period, the antipsychotic dosage progressively accumulated in addition to the divalproex sodium, 1000 mg/day. On day 3 before her death, she received 10 mg of scheduled oral haloperidol and an extra IM injection of 10 mg of haloperidol (with 2 mg IM lorazepam). On day 2 before her death, she received 10 mg of scheduled oral haloperidol. In the early morning (5 AM), she received an extra oral combination of 5 mg of haloperidol, 2 mg of lorazepam, and 25 mg of diphenhydramine. At night, she was agitated and trying to get into the rooms of other patients and at 11 PM she again received extra oral doses of 5 mg of haloperidol, 2 mg of lorazepam, and 25 mg of diphenhydramine. Forty-five minutes later, as she still could not sleep, she was given 50 mg of oral hydroxyzine pamoate. Shortly thereafter, on the third day of admission, in the early AM, the psychiatrist on call was contacted because the patient continued exhibiting the same behaviors and could not receive another haloperidol dose until 6 hours had passed; the psychiatrist ordered the combination of 2 IM injections, 20 mg IM of ziprasidone and 25 mg diphenhydramine at 1:19 AM (2 hours and 19 minutes after the last extra dose of oral haloperidol). Finally, at 2:20 AM she was described as resting quietly in her room. At 7:15 AM, when staff asked if the patient wanted breakfast, she opened her eyes and responded with a gesture that she did not want it and went back to sleep. At 9:00 AM her breathing sounded abnormal to a staff member, who called for a nurse; the nurse found the patient unresponsive with no radial pulse and a faint carotid pulse. This was 7 hours and 41 minutes after the 20 mg ziprasidone IM injection and approximately 10 hours after the last extra dose of oral haloperidol. CPR was started but the AED advised no shock. The EMS arrived and found her in asystole, so at 9:40 AM the patient was declared dead. 3.2.3. Autopsy The autopsy revealed no obvious anomalies in the heart or other organs that would explain the patient's sudden death. It is important to stress that in spite of her metabolic syndrome, there were no signs of atherosclerosis in the coronary arteries nor any sign of myocardial ischemia in macroscopic or microscopic examinations. There was no evidence of pulmonary thromboembolism. There were some minor abnormalities including mild atherosclerosis at the thoracoabdominal aorta, liver changes compatible with a fatty liver, and a small thyroid mass that microscopic examination demonstrated to be a follicular neoplasm. Postmortem blood toxicology for diphenhydramine was 172 ng/mL (reference range: 30–300 ng/mL) and for hydroxyzine was 35.4 ng/mL (reference range: 22–80 ng/mL). 3.2.4. ADR Scales Using the Liverpool ADR Causality Assessment Tool [30], antipsychotic-induced sudden death was deemed probable. The last ziprasidone IM injection of 20 mg was considered crucial, because it was given in addition to (1) three extra haloperidol doses (approximately 2 hrs before the ziprasidone injection, 5 mg oral haloperidol; approximately 21 hours earlier, 5 mg oral haloperidol; and 2 days before, one 10 mg IM haloperidol), (2) 10 mg/day of scheduled oral haloperidol for 6 days prior to death, and (3) a long-acting paliperidone injection of 156 mg 18 days prior to death. Similarly, on the Naranjo scale [31], a score of +6 was determined (scoring +1 on items 1, 5, 8, and 10 and +2 on item 2), consistent with a probable ADR. All the authors agreed with the scores on both ADR scales. As the patient had no signs of myocardial ischemia or pulmonary thromboembolism, it appears that the combination of antipsychotics, including the addition of IM ziprasidone on top of IM haloperidol, oral haloperidol, and long-acting paliperidone caused the sudden, unexpected death by TdP. The ziprasidone peak from the IM injection probably combined with the slower peak of the extra oral haloperidol administered 2 hours earlier causing the TdP early the following morning. Oral absorption is much slower than IM absorption and the oral haloperidol absorption may have been further delayed by the combination of an antimuscarinic drug, diphenhydramine. The possible contribution of diphenhydramine to this case and to the first case is discussed in detail in the Discussion (see Section 4.5). 4. Discussion 4.1. Limitations of the Patient Sample To identify these 2 sudden deaths, possibly explained by TdP associated with antipsychotics, we started with 95 deaths reviewed retrospectively using the judgment of a committee composed of psychiatrists, nurses, and pharmacists who have expertise in the subject. During the committee's 13 years of reviewing deaths, judgments regarding the medical cause of death were made after discussion by 2 or 3 psychiatrists who were present at the meetings. The senior author was involved in all reviews of all patients and attended all meetings. We know that the weakest part of our reviews is the limited number of autopsies, 17% (16/95). For years, we have encouraged their use, but we have not been too successful in increasing their numbers, particularly due to cost. We were fortunate to have autopsies of these 2 cases that were highly compatible with TdP. Even with all the limitations of our retrospective reviews, we have accumulated substantial experience with >500 deaths in our state facilities and >350 deaths of psychiatric patients in the community. 4.2. Limitations of the Case Reports It is difficult to make causal connections in case reports. Both deaths were sudden and unexpected, with no signs of cardiac ischemia in the autopsies. They were, however, associated with the accumulation of high serum antipsychotic concentrations after IM injections, compatible with probable ADRs, according to the scales. There are other rare causes of sudden death (e.g., genetic long QT syndrome, Brugada syndrome, or catecholaminergic polymorphic ventricular tachycardia) that cannot be completely ruled out [36], but the proximity of the high doses of IM antipsychotics and the lack of known family histories of sudden deaths in both patients suggested that these two sudden deaths were most likely explained by antipsychotic-induced TdP. Electrolyte abnormalities may contribute to TdP. The first patient refused laboratory testing but was a healthy young male and the second patient did not have any electrolyte abnormality, although magnesium is not routinely measured in our hospitals. In summary, a reasonable interpretation is that these two sudden deaths were unfortunate cases of TdP secondary to the accumulation of high serum peaks of antipsychotics after the last IM injection. Sinus bradycardia is a risk factor for TdP [16]. The first patient had an EKG with sinus bradycardia 2 years before his death. The EKG described a frequency of 58 beats per minute which appears to be mild bradycardia to us. We do not know if he had sinus bradycardia at any time during the admission leading to his death since the patient refused EKG and vital signs except at the time of admission when he was tachycardic. It is possible that a sinus bradycardia may have contributed to the development of TdP in the first case but we have no way to prove or disprove it. 4.3. African-American Race Knowing that less than 10% of our patients are African-American, we were surprised that both of these deceased patients were African-American. If we assume a frequency of 10% among our patients at psychiatric hospitals in Kentucky, the chance probability that one deceased patient is African-American is 0.10. The chance probability that both are African-Americans is very low: 0.01 (obtained by multiplying 0.10 by 0.10). This is lower than the typical set value of p < 0.05 and suggests that the association of African-American race with 100% (2/2) of the antipsychotic-induced sudden deaths may not be explained by chance. We also formally tested the hypothesis of the association between African-American race and TdP by using as a control the prevalence of African-Americans in sudden deaths not compatible with TdP. A 2-sided Fisher exact test provided a significant difference (p = 0.016) when comparing 100% (2/2) of African-Americans in sudden deaths compatible with TdP versus 7.5% (2/26) of African-Americans in sudden deaths that were not considered to be explained by TdP and in which the race was known. In spite of the very small sample size of 2 deaths, both tests of significance were compatible with the possibility that African-American patients may have more risk of death from antipsychotic-induced TdP in our sample. Let us for the moment assume that, as a matter of fact, African-Americans may have a higher risk of dying in our psychiatric hospitals by antipsychotic-induced TdP. Is there any pharmacological mechanism that may explain this association? Yes, there is one; it is the possibility that African-Americans are prone to a higher prevalence of being poor metabolizers (PMs) of haloperidol. Haloperidol metabolism has never been systematically studied to the extent that new drugs are currently studied before they are introduced in the US market. This is important because some old antiepileptics were found to have completely unexpected pharmacokinetic properties when intensively studied prior to introduction in the US market in the 2000s [37]. Psychiatric textbooks and review articles [38] usually indicate that CYP2D6 PMs have decreased ability to eliminate haloperidol. A study of 31 subjects taking haloperidol as a baseline for a clozapine RCT provided an unexpected result [39]. The persistence of serum haloperidol concentrations after haloperidol discontinuation was used to calculate haloperidol elimination half-life in each patient as an approximated marker of haloperidol metabolism. After eliminating patients who had taken haloperidol decanoate, there were a total of 26 patients, of which 16 patients had a half-life <3 days, suggestive of relatively normal haloperidol metabolism, and 10 patients had a half-life ≥3 days, compatible with being haloperidol PMs. Not surprisingly, the only 2 CYP2D6 PMs were in the group with a half-life ≥3 days, suggesting that, in effect, not having CYP2D6 impairs one's ability to eliminate haloperidol. Unexpectedly, the only significant difference (with a p = 0.014) between the 16 haloperidol normal metabolizers and the 10 haloperidol PMs was that all 4 African-Americans were haloperidol PMs and had half-lives ≥3 days (as a matter of fact, all 4 patients had half-lives >7 days) [39]. As haloperidol is at least partly metabolized by glucuronidation and some of the glucuronidation enzymes have lower activity in African-Americans [40], it is possible to hypothesize that some African-Americans may have lower ability to eliminate haloperidol due to decreased activity in haloperidol glucuronidation. Recently, an in vitro study of haloperidol metabolism using human hepatocytes has suggested that the uridine diphosphate glucuronosyltransferase (UGT) 2B7 (UGT2B7) may be important in the metabolism of haloperidol [41]. Although there is substantial variation of UGT2B7 alleles worldwide [42], the clinical relevance of these genetic variations for people with African ancestry is not known [43]. In summary, there is very limited information on haloperidol metabolism, but it is possible that some African-Americans may have impaired ability to eliminate haloperidol. If our two patients had impaired ability to eliminate haloperidol, that impairment may have contributed to higher serum haloperidol concentrations, leading to greater risk for TdP. As the haloperidol serum concentration level increases, the blockade of cardiac potassium channels would likewise increase. 4.4. Accumulation of One or Several Antipsychotic Formulations We think that, in these two patients, the last antipsychotic IM injection served as “the straw that broke the camel's back” by providing an even higher peak in serum antipsychotic concentration which contributed to further blockade of cardiac potassium channels encoded by HERG reaching a very high level, leading to TdP. In the first patient, this was achieved after 4 haloperidol IM injections with an accumulated dosage of 25 mg in 2 days before the fourth and last injection of 10 mg, making a total of 35 mg. In the second patient, it was achieved after the last ziprasidone IM injection of 20 mg in addition to (1) three extra haloperidol doses (approximately 2 hours before the ziprasidone injection, 5 mg oral haloperidol; approximately 21 hours earlier, 5 mg oral haloperidol; and 2 days before, one 10 mg IM haloperidol), (2) 10 mg/day of scheduled oral haloperidol for 6 days prior to death, and (3) a long-acting paliperidone injection of 156 mg 18 days prior to death. The second patient received 5 mg oral haloperidol at 11 PM; therefore, the nurse could not administer another as-needed haloperidol dose until 5 AM the next morning and called the psychiatrist on call. In retrospect, we think that the on-call psychiatrist who responded to the nurse and prescribed 20 mg IM of ziprasidone at 1:19 AM on top of the prior extra dose of oral haloperidol made a huge mistake. Both ziprasidone and haloperidol appear to be potent blockers of cardiac potassium channels in clinical doses [11]. Therefore, in this second case, the effects of ziprasidone blocking the cardiac potassium channels encoded by HERG were added to the blocking effects of the prior haloperidol dose. In retrospect, it would have been better to control the agitation of this patient only with lorazepam, which has no relevant effects on the QTc interval [29]. 4.5. Comedications in Addition to Antipsychotics The only comedication received by the first patient was IM diphenhydramine. The second patient received, besides IM diphenhydramine, several medications (valproate, insulin, metformin, lisinopril, lorazepam, and hydroxyzine), which have not been associated with TdP or relevant QTc prolongation. Diphenhydramine has been associated with (1) TdP, occasionally during voluntary overdoses [44–46]; (2) a strong signal for TdP in the databases from drug regulatory agencies [47]; (3) QTc prolongations during voluntary overdoses [48–52], and (4) QTc prolongations in EKGs completed at the emergency room when prescribed by itself or with other drugs that prolong the QTc interval [53]. However, some of these diphenhydramine overdose studies with EKG data, including 126 cases of overdoses with no TdP case [48] and 1 case monitored for several days [50], suggested that diphenhydramine causes tachycardia and this has a major effect on protecting from TdP in situations of QTc prolongation. The accumulated diphenhydramine dosage was 200 mg IM during the last 3 days of Case  1 and 75 mg during the last 2 days of Case  2 (25 mg IM 2 days prior to death and 50 mg oral on the day before death). We doubt that these doses of diphenhydramine had any relevance in the sudden deaths of these two patients because (1) TdP induced by diphenhydramine overdoses appears to require huge quantities measured in grams [44, 45], (2) diphenhydramine-induced tachycardia may protect from TdP in situations of QTc prolongation [48, 50], and (3) some animal models of clinical relevance in humans for drugs that prolong QTc cannot detect any effect for diphenhydramine doses trying to replicate clinical dosing [54]. 4.6. Reflections on the History of Antipsychotic-Induced TdP The next section discusses a practice guideline limiting high doses of haloperidol and/or ziprasidone IM injections in the hope of preventing the repetition of similar TdP cases in our Kentucky facilities. Ideally, practice guidelines should be based on principles following the hierarchical thinking proposed by evidence-based medicine, which gives higher value to RCTs. In the real world, those researchers insisting on the evidence-based medicine (EBM) approach are not aware that evidence related to ADRs is seriously distorted by the effects of pharmaceutical companies to cover the ADRs resulting from their drugs and their lack of attention to ADRs during studies undertaken for market approval [55]. Moreover, RCTs focus on mean effects, but when a very rare ADR, such as TdP, is studied, mean values are irrelevant and outliers are important [56]. Therefore, some authors can dismiss the risk of haloperidol-induced TdP because it is mainly based on case reports and not RCT data [57]. How unwise is it to ask clinicians to wait for RCT data on haloperidol-induced TdP? It is very unwise; a randomized study of >18,000 patients followed for 1 year had no statistical power to detect ziprasidone-induced TdP [18]. Haloperidol is off-patent; therefore, nobody is going to finance a large RCT of 10,000s of patients for several years to detect statistical differences between haloperidol and other drugs in causing TdP. Imagine that the thioridazine marketer had paid attention to Ban [1] in the late 1960s, performed more studies, and withdrawn thioridazine from the market instead of waiting until 2006. The outcome of that decision, based on “limited” case reports would probably have led to saving hundreds or thousands of lives worldwide during the last 30 years. Psychiatrists would have prescribed other antipsychotics with less risk for TdP rather instead of prescribing thioridazine, which may be the oral antipsychotic that most frequently causes sudden death [58]. Similarly, it would be a mistake to ignore the risk of TdP associated with high doses of haloperidol IM, suggested by our 2 case reports. 4.7. Practice Guidelines Both deaths were considered possibly preventable if the prescribing psychiatrist had better understood the risk of a standing as-needed order for agitation using IM antipsychotics with potential to cause TdP. This type of order is written for all our patients but is rarely used by nurses. If they are used in a patient, IM antipsychotic injections are rarely repeated within a few hours. In the first case, the psychiatrist allowed a high dosage of 10 mg haloperidol IM every 12 hours, which is rarely prescribed in our hospitals (most psychiatrists prescribe 5 mg every 6 hours at most). The psychiatrist thought it was safe because the same order had been written for the same patient at the same hospital 3 years before, but 3 years before it was scarcely used. In the second case, the as-needed order of 5 mg every 6 hours was written as administered by the oral route, but when it was not enough to control the agitation of the patient, the nurses called the on-call psychiatrist who circumvented the limit of a haloperidol dose every 6 hours by adding 20 mg IM ziprasidone in close proximity to the last oral dose of haloperidol. The medical literature has addressed the risk of TdP during IV haloperidol treatment, but the psychiatric literature including practice guidelines [59] has paid little attention to the risk of TdP after IM haloperidol and/or IM ziprasidone. Therefore, a practice guideline was developed for IM antipsychotics to limit dosing for haloperidol and/or ziprasidone IM in order to prevent future TdP cases and sudden deaths in our public psychiatric hospitals in Kentucky. The practice guideline that is shown in the Appendix was based on (1) a comprehensive review of the literature on the use of IM antipsychotics to treat agitation, including review articles [60–74] and prior practice guidelines [59, 75–79]; (2) learning from the mistakes in these two cases; and (3) a review of the pharmacokinetic and pharmacodynamic mechanisms behind the DDIs contributing to antipsychotic-induced TdP [14, 80, 81]. 5. Conclusion The medical literature has addressed the risk of TdP during IV haloperidol treatment but the psychiatric literature has paid little attention to TdP risk after IM haloperidol and/or IM ziprasidone [59]. In this paper, we presented two cases of sudden death after IM injections of antipsychotics, which were considered to be probable ADRs. The autopsies described lack of heart pathology and were highly compatible with the possibility of TdP in the absence of risk factors other than the accumulation of antipsychotics with an even higher serum peak after a last injection led to death within a few hours. It is possible that these cases were explained by a combination of high serum concentrations of antipsychotics with particular individual vulnerability. Some studies suggest that rare genetic variations at potassium channels may contribute to predisposition to drug-induced TdP [82]. The first case describes a 27-year-old African-American male with schizophrenia but no medical issues. His death occurred in the early morning of the third day and was probably caused by repeated IM haloperidol injections of 10 mg (totaling 35 mg in 2 days). The second case involves a 42-year-old African-American female with a metabolic syndrome. Her death was probably caused by a last ziprasidone IM injection of 20 mg in addition to (1) three extra haloperidol doses (approximately 2 hours before the ziprasidone injection, 5 mg oral haloperidol; approximately 21 hours earlier, 5 mg oral haloperidol; and 2 days prior, one 10 mg IM haloperidol), (2) 10 mg/day of scheduled oral haloperidol for 6 days prior to death, and (3) a long-acting paliperidone injection of 156 mg 18 days prior to death. The association of TdP deaths with African-American race in spite of the small sample size appears to suggest a significant overrepresentation when compared with our patients at Kentucky public psychiatric hospitals. As a matter of fact, patients with poor capacity to metabolize haloperidol might be overrepresented among African-Americans. The study of haloperidol metabolism through glucuronidation in African-Americans is urgently recommended since the percentage of impaired individuals may be overrepresented in African-American individuals. If it is demonstrated that African-Americans have lower ability to metabolize haloperidol, this would indicate the need to consider race and possibly genetics, in the dosing of IM antipsychotics for agitation and for the need to include these differences in the practice guidelines for agitation. Acknowledgments This paper was completed without any external funding. Lorraine Maw, M.A., at the UK Mental Health Research Center, helped with editing. Appendix Proposed practice guidelines for dosing parenteral antipsychotics in order to avoid TdP during the control of agitation at psychiatric hospitals are as follows. IV Haloperidol. The use of IV haloperidol is not recommended and it cannot be used unless continuous EKG monitoring is available. IM Chlorpromazine. The use of IM chlorpromazine is not recommended because of the lack of studies and very limited number of RCTs [60] and the potential for oral chlorpromazine to cause TdP and orthostatic hypotension [61]. IM chlorpromazine cannot be used unless there is (1) an absence of any of the risk factors for TdP (see below) and (2) a baseline EKG that shows QTc < 450 ms on the same medication that the patient is currently taking. Combining IM Haloperidol and IM Ziprasidone. Orders for IM haloperidol and IM ziprasidone cannot be combined in the same patient. If a patient has received IM haloperidol, no IM ziprasidone can be administered for 24 hours and if a patient has received IM ziprasidone, no IM haloperidol can be administered for 24 hours. IM Haloperidol  (1) IM Haloperidol cannot be used in any patient with risk factors for TdP (see below).  (2) Standard agitation as-needed orders for IM haloperidol doses ≥10 mg cannot be written unless there is a baseline EKG that shows QTc < 450 ms on the same medication that the patient is currently taking. See below for an exception for patients on potent inducers.  (3) Standard agitation as-needed orders for IM haloperidol doses of 5 mg should not recommend administration with a frequency <12 hours unless there is a baseline EKG that shows QTc < 450 ms on the same medication that the patient is currently taking. The combination of IM haloperidol with IM benzodiazepines and/or IM antihistaminic/anticholinergics has not been systematically studied but is frequently used by our clinicians. To decrease the total number of haloperidol doses and reduce the potential for sedation, clinicians are encouraged to combine haloperidol IM injections of 5 mg with simultaneous administration of a benzodiazepine IM injection (such as IM lorazepam) and/or IM anticholinergic/antihistamine injection (such as IM diphenhydramine). As these latter medications do not appear to be associated with risk for QTc prolongation, they can be administered at frequencies of less than 12 hours.  (4) If there is no baseline EKG while on current medications, it is OK to write standard orders for agitation for haloperidol 5 mg every 12 hours while using other IM drugs with less potential for QTc prolongation more frequently (e.g., for 1 mg of lorazepam every 6 hours and/or for 25 mg of diphenhydramine every 6 hours).  (5) Fluoxetine is at least a mild inhibitor of haloperidol metabolism [81]. In patients taking fluoxetine or who have taken it in the last month (norfluoxetine stays in the body for weeks after discontinuation), clinicians need to be aware that the doses of haloperidol IM that they write may produce higher effects. The limited available published information [81] suggests that adding fluoxetine is equivalent to increasing serum haloperidol concentrations by 33% after 12 weeks on fluoxetine. This is equivalent to multiplying the effects of the haloperidol IM dose by a factor of 1.33 and to compensate, this will require multiplying IM haloperidol doses by 0.75 (calculated by dividing 1/1.33 = 0.75). There are no haloperidol studies of the effects of fluoxetine over several months and at steady state for inhibition of haloperidol metabolism; therefore, it is possible that patients taking fluoxetine may require even lower IM haloperidol doses. It may be safer to avoid IM haloperidol in patients taking fluoxetine since fluoxetine also prolongs QTc. IM Ziprasidone IM ziprasidone cannot be used in any patient with risk factors for TdP (see below). The prescribing information recommends maximum total daily doses of ≤40 mg/day and intervals for doses of ≥2 hours for 10 mg and ≥4 hours for 20 mg injections. Standard agitation as-needed orders for IM ziprasidone of 20 mg should not recommend administration with a frequency of <12 hours unless there is a baseline EKG that shows QTc < 450 ms on the same medication that the patient is currently taking. IM Olanzapine IM olanzapine appears to have less risk for QTc prolongation than IM haloperidol and/or IM ziprasidone. If there is a need for IM antipsychotics in a patient with risk factors for TdP, olanzapine IM is a better choice than haloperidol or ziprasidone IM [65]. The olanzapine prescribing information recommends maximum total daily doses of ≤30 mg/day and an interval for 10 mg doses of ≥2 hours after the initial injection and ≥4 hours after the second IM injection. Although oral olanzapine is not usually linked to orthostatic hypotension, IM olanzapine has been associated with syncope. The prescribing information recommends doses ≤5 mg in geriatric patients (≥65 years) and doses of 2.5 mg in patients with risk for orthostatic hypotension. IM olanzapine is a fairly sedating drug and there is no need to combine it with benzodiazepines. As a matter of fact, it should not be combined with IM benzodiazepines since this combination has occasionally been associated with hypotension [71] and even rarely with death [67]. The decision to add an IM olanzapine injection within 24 hours of a benzodiazepine IM injection would require chart documentation for the reason and a written order to closely monitor the patient for orthostatic hypotension and/or respiratory depression to avoid any risk, since we have very limited data about the safety of this combination, which is discouraged by olanzapine's marketer [63]. Obviously, if the patient is lying in bed, there is no need to monitor for orthostatic changes unless the patient needs to get up. If the patient is taking fluvoxamine, IM olanzapine should be used in lower doses due to the inhibition of olanzapine metabolism. The clinician should consider using half the dosage he/she typically uses [14]. IM Aripiprazole IM aripiprazole appears to have less risk for QTc prolongation than IM haloperidol and/or IM ziprasidone. The aripiprazole prescribing information recommends (a) maximum total daily doses of ≤30 mg/day, (b) a dose of 9.75 mg (range 5.25–15 mg), and (c) and an interval between injections ≥2 hours. If the patient is taking powerful CY3PA4 inhibitors or CYP2D6 inhibitors (including paroxetine and fluoxetine), halving the doses is recommended. Duloxetine and bupropion are moderate CYP2D6 inhibitors; therefore, using half the dosage used for typical patients appears to be a good idea [14]. There is limited information comparing IM aripiprazole with other IM second-generation antipsychotics, but the available information suggests that IM aripiprazole may be less effective for controlling agitation than IM olanzapine or IM ziprasidone [61, 62]. Patients with Risk for TdP. Having any of the following is considered a risk factor for TdP: (1) history of sudden death in the family, (2) personal history of syncope, (3) active or residual serious heart disease, (4) geriatric age (≥65 years), (5) bradycardia (<60 beats per minute), (6) hypokalemia, (7) hypomagnesemia, or (7) the use of nonpsychiatric drugs with risk for QTc prolongation including antiarrhythmics (amiodarone dofetilide, quinidine, procainamide, and sotalol), some antibiotics (e.g., gatifloxacin and moxifloxacin), or others (e.g., arsenic trioxide, mefloquine, methadone, pentamidine, and tacrolimus). Some of these factors can be easily reversed; therefore, correcting electrolyte abnormalities or discontinuing drugs associated with QTc prolongation should be considered before adding orders for IM antipsychotics. IM benzodiazepines do not appear to prolong QTc interval; therefore, it appears safe to use them in patients with risk for TdP [67]. Patients on Scheduled Antipsychotics or Antidepressants with Risk for TdP. Some oral antipsychotics (including amisulpride, haloperidol, iloperidone, phenothiazines, pimozide, or ziprasidone), haloperidol decanoate, fluphenazine decanoate, and antidepressants (including SSRIs and TCAs) have been definitively associated with clinically relevant potential to prolong QTc; therefore, it is better to avoid adding IM haloperidol and IM ziprasidone for agitation in a patient taking any of these drugs. IM benzodiazepines or IM olanzapine should be considered for as-needed orders for agitation in these patients. If the clinician considers the riskier path of adding IM haloperidol or IM ziprasidone to these psychiatric medications, he/she needs to consider using lower doses and provide written justification. Patients on Inducers. If the patient is on a potent inducer (e.g., rifampin or some antiepileptic drugs: carbamazepine, phenytoin, or phenobarbital), he/she may metabolize some antipsychotics such as aripiprazole, haloperidol, or olanzapine very fast [80] and may need higher doses of these IM antipsychotics beyond what is typically recommended. If the clinician needs to write higher doses of any of these IM antipsychotics, he/she should consult the pharmacy first before writing an order for unusually high doses of any IM antipsychotics. Potent inducers appear to have small effects on ziprasidone metabolism [80]; therefore, although this has not been studied well, patients taking potent inducers may respond to normal doses of IM ziprasidone. Normal doses of ziprasidone IM may be a rational choice for patients taking potent inducers. Patients with Alcohol Intake/Intoxication. IM antipsychotics should be judiciously used to manage agitation in patients with recent alcohol intake or alcohol intoxication. IM olanzapine [71, 72] and IM ziprasidone with or without benzodiazepines [70] have been associated with decreases in oxygen saturation. Individualized Treatment. This set of guidelines for IM antipsychotics was developed to discourage the use of high doses of IM haloperidol, IM ziprasidone, or the combination of IM haloperidol with IM ziprasidone for standard agitation orders for all patients. It does not appear safe to continue to use nurses' judgment to decide the safe frequency of this type of order when this appears to have led to two sudden deaths in our psychiatric hospitals. In emergency situations, a clinician can overrule the dosing limits in this guideline by using a one-time order prescribing high doses as long as he/she understands the risk and has appropriate safeguards in place (see the IM olanzapine and IM benzodiazepine combinations) and documents them in the chart. Once the emergency situation is resolved, the clinician should be ready to have his/her actions reviewed by the hospital pharmacy that may encourage or discourage similar actions in the future based on the peculiarities of the individual case. Abbreviations EKG:Electrocardiogram IM:Intramuscular IV:Intravenous ms:Milliseconds SSRI:Selective serotonin reuptake inhibitor TCA:Tricyclic antidepressant TdP:Torsades de pointes. Disclosure No commercial organizations had any role in the completion or publication of this paper. Competing Interests Drs. Wahid, Johnson, Brenzel, and de Leon declare no competing interests during the last 36 months. ==== Refs 1 Shorter E. 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==== Front Int J NephrolInt J NephrolIJNInternational Journal of Nephrology2090-214X2090-2158Hindawi Publishing Corporation 10.1155/2016/2132387Research ArticleImpact of Pediatric Chronic Dialysis on Long-Term Patient Outcome: Single Center Study http://orcid.org/0000-0003-1869-3282Levy Erez Daniella 1 2 * Krause Irit 1 2 Dagan Amit 1 2 Cleper Roxana 1 2 Falush Yafa 1 Davidovits Miriam 1 2 1Institute of Nephrology, Schneider Children's Medical Center of Israel, 49202 Petah Tikva, Israel2Sackler Faculty of Medicine, Tel Aviv University, 6997801 Tel Aviv, Israel*Daniella Levy Erez: levy.erez.daniella@gmail.comAcademic Editor: David B. Kershaw 2016 15 8 2016 2016 213238724 1 2016 14 5 2016 21 6 2016 Copyright © 2016 Daniella Levy Erez et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objective. Owing to a shortage of kidney donors in Israel, children with end-stage renal disease (ESRD) may stay on maintenance dialysis for a considerable time, placing them at a significant risk. The aim of this study was to understand the causes of mortality. Study Design. Clinical data were collected retrospectively from the files of children on chronic dialysis (>3 months) during the years 1995–2013 at a single pediatric medical center. Results. 110 patients were enrolled in the study. Mean age was 10.7 ± 5.27 yrs. (range: 1 month–24 yrs). Forty-five children (42%) had dysplastic kidneys and 19 (17.5%) had focal segmental glomerulosclerosis. Twenty-five (22.7%) received peritoneal dialysis, 59 (53.6%) hemodialysis, and 6 (23.6%) both modalities sequentially. Median dialysis duration was 1.46 years (range: 0.25–17.54 years). Mean follow-up was 13.5 ± 5.84 yrs. Seventy-nine patients (71.8%) underwent successful transplantation, 10 (11.2%) had graft failure, and 8 (7.3%) continued dialysis without transplantation. Twelve patients (10.9%) died: 8 of dialysis-associated complications and 4 of their primary illness. The 5-year survival rate was 84%: 90% for patients older than 5 years and 61% for younger patients. Conclusions. Chronic dialysis is a suitable temporary option for children awaiting renal transplantation. Although overall long-term survival rate is high, very young children are at high risk for life-threatening dialysis-associated complications. ==== Body 1. Introduction End-stage renal disease (ESRD) is a major cause of morbidity and mortality in children. In the pediatric population, ESRD is mainly due to congenital anomalies of the kidney and urinary tract (CAKUT) and glomerular diseases [1–3]. The prevalence of ESRD in children in the United States is 8.3 per 100,000 [4] and 1.5/100,000 in Israel [5]. Kidney transplantation provides the best long-term results and optimum quality of life [6–10]. Because of the shortage of kidney sources in Israel, more than 75% of children waiting for a renal transplant are on dialysis for more than 2 years due to the shortage of available kidneys [5] placing them at high risk of dialysis-associated complication. Data on the long-term outcome of this patient group are scarce. In children, peritoneal dialysis (PD) is preferred over hemodialysis (HD) in terms of quality of life, growth, and preservation of residual renal function [7]. Hemodialysis poses greater risks of access failure, vascular thrombosis, and obliteration of the great veins, which can be compromised for life [2, 6, 11]. In addition, multisystem involvement in ESRD in this population can lead to growth retardation [12, 13], cardiovascular complications [14, 15], and hematological complications [16–18]. The life span of children with ESRD is significantly lower than that of the age- and gender-matched general population. In studies of children on dialysis from Australia and New Zealand, survival rates were 85.7% at 3 years, 79% at 10 years, and 66% at 20 years [19]. Lower survival rates were found in children less than 12 months; 3-year survival for this age group was 68% in the North American Pediatric Renal Transplant Cooperative Study (NAPRTCS) annual report [20]. Similar results were reported in other studies from Netherlands [21, 22], with little change in more recent studies [3] despite significant progress made in renal replacement therapy. The most important risk factors for poor treatment outcome were younger age at onset of dialysis and type of nonrenal comorbidities [1, 3, 19, 20]. The leading causes of death in children on dialysis are cardiovascular disease and infections [6, 19, 21]. The aim of the present study was to evaluate the long-term outcome of a large cohort of pediatric patients on chronic dialysis in a single tertiary medical center. Attention was focused on mortality rate and causes of death. 2. Materials and Methods The study cohort included all patients with ESRD maintained on dialysis for at least 3 months in the Dialysis Unit of Schneider Children's Medical Center of Israel from January 1995 through December 2013. The following data were retrospectively recorded from the medical files of each patient: clinical parameters such as cause of ESRD, age at diagnosis, associated diseases, age at initiation of dialysis, type of dialysis (PD, HD, and both), duration of dialysis, dialysis complications; laboratory parameters such as complete blood cell count and blood chemistry (iron status was evaluated by recommended parameters); parameters related to treatment of chronic kidney disease such as weight and height standard deviation scores (SDS) at dialysis onset and study end, use of recombinant growth hormone (rGH) therapy, blood pressure percentiles and SDS by gender, age and height percentile, presence of left ventricular hypertrophy (LVH) measured by echocardiography (mass index > 51 gr/m2.7), use and number of antihypertensive drugs, administration of recombinant erythropoietin (rEPO), and need for blood transfusions throughout treatment. 2.1. Definitions 2.1.1. Exit Site/Tunnel Infection Exit site/tunnel infection was diagnosed by the presence of purulent drainage with negative peritoneal fluid culture [23]. Peritonitis was diagnosed by the presence of at least 2 of the following criteria: cloudy effluent and/or abdominal pain or fever; effluent leukocyte count of >100 cells/mm3 with >50% neutrophils; bacterial growth in the peritoneal culture [24]. 2.1.2. Anemia Anemia was diagnosed when hemoglobin level was less than 11 g/dL [18] or when there was a need for blood transfusions. 2.1.3. Hypertension Hypertension was diagnosed by either blood pressure values over the 95th percentile for age and heights at 3 visits or a need for antihypertensive therapy. Data were presented by SDS scores. 2.2. Outcome Measures 2.2.1. Primary Outcome Primary outcome was as follows: death or survival at the end of the study period, survival with successful kidney transplant, or continuing dialysis at the end of the study period. 2.2.2. Secondary Outcome Secondary outcome was as follows: dialysis complications (number of infections, number of access failures, and need for exchange), changes in growth, recorded in z scores, cardiovascular disease defined as z scores of hypertension, and LVH and anemia (percent of the population and hemoglobin level.) Outcome was analyzed overall and by dialysis modality (PD versus HD), age at onset of dialysis (>5 years versus <5 years), and year of initiation of dialysis (1995–2003 versus 2004–2013). 2.3. Statistical Analysis Data were analyzed using BMDP statistical software [25]. Analysis of variance (ANOVA) was used to compare continuous variables (biological parameters) between groups, with Bonferroni's corrections for multiple comparisons and Pearson's chi-square test for discrete variables (such as complications). Mann-Whitney nonparametric U test was used to analyze parameters such as number of blood pressure drugs. Comparing changes over time, we used ANOVA with repeated measures or Wilcoxon test, as appropriate. Survival curves were formulated according to the Kaplan-Meier method. Patient survival was calculated per 100 patient dialysis years. A p value of ≤0.05 was considered significant. The study was approved by the Ethics Committee of Rabin Medical Center. 3. Results 3.1. Background Characteristics One hundred and ten children met the study criteria, 63 boys (57.3%) and 47 girls (42.7%) of mean age 10.67 ± 5.27 years (range: 1 month–24 yrs.) at onset of dialysis. Twenty-three patients were <5 years and 87 > 5 years; Seventy-four patients (67%) were adolescents (age: over 12 years). Eighty-two patients (74%) were of Jewish and 28 (26%) were of Arab origin. Maintenance dialysis duration was as follows: median 1.46 years (range: 0.25–17.54 years) and mean 2.48 ± 3.02 years. Mean duration of follow-up was 13.5 years ± 5.84. The major causes of ESRD are shown in Table 1. Table 2 shows clinical parameters of the patients analyzed according to dialysis modality. Significance was found only in patient mean age (p < 0.001). The later period (2004–2013) was characterized by a twofold increase in the proportion of patients treated with PD (15% versus 31% of all dialyzed patients). 3.2. Dialysis Access For PD, a two-cuffed peritoneal catheter was inserted by a specialized pediatric surgeon in the operating room. Prophylaxis with Cefamezine was administered in all patients during the later period of the study (2004–2013). All the caregivers were instructed for appropriate performance of the procedure emphasizing aseptic techniques and were managed by automated PD. For HD, an arteriovenous graft or fistula was used in 44 patients (all of whom weighed > 20 kg, 52%) [23] and a central venous perm-cuffed catheter (Permacath [26]) in 41 (48%). Noncuffed central catheters were only used when there was an acute indication for dialysis and used as a bridge until a permanent access could be secured. Overall, 5 vascular access exchanges per 1000 patient-years were needed; in 35 patients (59% of patients on HD exclusively) there was no need for a change of access throughout the treatment course. The main reason for catheter removal was infection. 3.3. Dialysis Complications 3.3.1. Exit Site Exit site infection rate was 5.9 episodes per 1000 patient-years. 3.3.2. Hemodialysis At least one episode of exit site infection occurred in 22 patients on HD both exclusively and sequentially (25%). Bacteremia was documented at least once in 19 patients on HD (22%). The average bacteremia rate was 4.4 episodes per 1000 patient-years. 3.3.3. Peritoneal Dialysis 16 patients developed exit site infection (31%). The average peritonitis rate was 20 episodes per 1000 patient-years. Tunnel infections were 2.2 episodes per 1000 patient-years. No bacteremia episodes were noted in patients on PD. The rate of total catheter-related infections was significantly higher for PD than for HD (p < 0.001). The earlier treatment period was characterized by a significantly higher rate of exit site infections (68.8% versus 28%, p < 0.001) and of bacteremia (19.1% versus 40.8%, p < 0.02). The peritonitis rate in the patients on PD was also higher in the earlier period, but the difference did not reach statistical significance (68% versus 81%, p = 0.35). 3.4. Anemia Anemia was documented in 101 patients (92%) before onset of dialysis. Mean hemoglobin level was 9.11 ± 1.69 gr/dL at onset of dialysis and 10.66 ± 1.52 gr/dL (p < 0.001) at study end. The rate of rEPO administration was 85.3% at onset of dialysis, increasing to 97.8% at study end (p = 0.01). Findings by dialysis modality are shown in Table 2. Blood transfusion was necessary in 21 patients (19.8%) during the first 3 months on dialysis and in 5 patients (4.5%) during the last 3 months on dialysis. 3.5. Growth and Nutrition Mean weight SDS was −0.56 ± 0.84 at dialysis onset and −0.84 ± 0.69 at study end (p = 0.01). Respective values for height were −1.22 ± 1.11 and −1.62 ± 1.26; this difference was not statistically significant (p = 0.13). Growth parameters analyzed by dialysis modality are shown in Table 2. Patients were placed on an age- and weight-adjusted diet under careful follow-up by a renal dietitian. A gastrostomy tube was inserted in 13 cases (14%). Forty-seven patients (43%) were treated with rGH. 3.6. Hypertension Seventy-seven patients (70%) were hypertensive (systolic, diastolic, or both) at onset of dialysis. Mean systolic blood pressure SDS decreased from 3.35 ± 2.19 at onset of dialysis to 2.14 ± 2.83 at study end (p < 0.001). Respective values for diastolic blood pressure SDS were 2.51 ± 1.72 and 1.36 ± 2.22 (p < 0.001). The number of medications needed to control hypertension ranged from 1 to 4 (median 1) at onset of dialysis and decreased to 0 to 5 (median 0) at study end. Seventy patients (63%) needed treatment at dialysis onset compared to 46 (42%) at study end (p < 0.001). LVH was found in 50 patients (46%) at onset of dialysis and 37 (34%) at study end (p = 0.01). During treatment, LVH developed in 11 patients (10.5%) who had not had LVH at dialysis onset. 3.7. Outcome Eighty-nine patients (90.3%) received a kidney graft during the study period, of whom 79 (88.8%) had a functioning graft at study end and 10 (11.2%) lost graft function and returned to dialysis. Causes for loss of graft included 5 patients with noncompliance; 1 with FSGS recurrence; 2 with acute rejection; 1 with graft renal artery thrombosis; 1 with chronic antibody mediated rejection. Eighty-six patients had one renal transplant and 2 patients had 2 transplants. Sixty-four grafts originated from deceased donors, 15 were from living-related donors, and 9 were from living nonrelated donors. Another 8 patients (7.3%) continued dialysis; this group age was on average 11 ± 6.4 years at onset of dialysis and were on maintenance dialysis for an average of 2.85 ± 3.9 years (range: 0.25–6.64). One child with CAKUT ceased dialysis with stable kidney function. 12 patients died during the study period, with a mortality rate of 10.9%. Causes of death are detailed in Table 3. Two patients who died suffered from Schimke immunoosseous dysplasia complicated by moyamoya phenomenon. Their death was attributed to cerebrovascular accidents. Three patients who died from hyperkalemia were on Kayexalate and a low potassium diet. On Kaplan-Meier analysis, the 5-year overall survival rate on dialysis was 84%: 90% in children > 5 years and 61% in children < 5 years at dialysis onset (Figure 1). Patients treated with PD exclusively had survival rates of 100% at 1 year and 78% at 5 years; the rate for HD exclusively was 93% at both time points and for combined modalities 95% and 68%. There was no statistically significant difference in overall survival among the groups (p = 0.56). Mortality rates analyzed by patient-years on dialysis were 4.3 deaths per 100 patient-years for the whole cohort, 2.39 deaths per 100 patient-years for patients > 5 years at onset of dialysis, and 11.6 deaths per 100 patient-years for patients < 5 years at onset of dialysis. The mortality rate for PD exclusively was 9 per 100 patient-years, for HD exclusively was 7.26 per 100 patient-years, and for combined treatment was 3.93 per 100 patient-years. By treatment period, 5-year survival rates were 73% in patients treated in 1995–2003 and 90% in patients treated in 2004–2013; the difference was not statistically significant (p = 0.81). 4. Discussion To the best of our knowledge, this is one of the largest single-center longitudinal outcome series of paediatric ESRD managed with long-term maintenance dialysis in the medical literature. Previous studies described smaller cohorts of 34 [27] and 98 children [8] with shorter follow-up times of 9 and 14 years, respectively. Males were slightly overrepresented in our study (57.3% versus 42.7% for females), in agreement with findings that ESRD is more common in males [6, 21, 22, 24, 25]. Also expected was the younger age of patients who started with PD compared to patients who started with HD (7.58 versus 12.41 years) given the considerable technical difficulties with dialysis in younger children [2, 11]. Maintenance dialysis duration in our study, median 1.46 years, range 0.25–17.54 years, and mean 2.48 ± 3.02 years, was similar to that in centers in Europe and USA [6, 8, 28] despite the limited graft sources in a small country such as Israel. The limited amount of potential donors in Israel can be explained by various religious beliefs causing individuals not to donate organs. Our dialysis unit can provide both HD and PD. PD is known to have several advantages over HD in children, including better preservation of native renal function, lack of long-term compromise of the main venous vascular tree, and freedom from frequent hospital visits with significantly less interference with everyday life activities and quality of life. Nevertheless, only 35% of our patients were started on PD compared to 80% in other series [8]; this can be explained by the reasonable travel distances to the pediatric dialysis unit from most parts of our country; therefore patients who cannot be managed with PD at home are easily switched to HD when it is in their best interest. Furthermore, 67% of our cohort were adolescents, many of whom may have declined PD because of its major impact on body image. Primary graft/arteriovenous fistulas are considered the best permanent vascular access for HD with lowest risks of secondary failure and complications [26]; they were used in 52% of our patients, a significantly higher rate than reported in the literature (36% and 21.3%) [22, 28]. The rate of infections was low in our study compared to NAPRTCS [20]: 2 episodes per 100 patient-years versus 8.1 per 100 patient-years. Low rates of secondary failure and infection rates have been associated with high surgical skills and expertise and use of standardized PD education programs, administration of prophylactic antibiotics prior to insertion of PD catheters, and aseptic techniques. Anemia may be caused by a multitude of factors and poses a challenging problem in patients on chronic dialysis similar to other studies. Ninety-two percent of our patients were anemic at onset of dialysis, with a mean hemoglobin level of 9.11 ± 1.69 gr%. By the end of the study, this value increased significantly to 10.66 ± 1.52 gr% (p < 0.001), concomitant with a decrease in the need for blood transfusions (from 19.8% of patients to 4.5%; p < 0.001). A similar positive trend was found in the annual NAPRTCS report with a hematocrit range of 29.9% to 32% at onset of dialysis, which improved after 6 months to 32.3%–33% as found in [20]. It is possible that the more intensive rEPO treatment given during dialysis compared to the predialysis period was responsible for this finding. In addition, we suspect that compliance was better for intravenous than subcutaneous rEPO administration. Cardiovascular disease is a major cause of death in adolescents and young adults [15]. In their nationwide Dutch study partly focused on cardiovascular disease, Groothoff et al. [15] reported LVH rates of 47% and 39% in male and female adolescents, respectively, and in an analysis of a large European registry, Fadrowski et al. [16] found that uncontrolled hypertension was present in 45.5% of patients on HD and 35.5% on PD; rates of antihypertensive drug use in these groups were 69.7% and 68.2% male/female, respectively, and in 51.9% of all patients. In our study, 70% of children presented with hypertension at onset of dialysis. By study end, there was a significant improvement in blood pressure SDS, with a decrease in the LVH rate from 46% to 34% (p = 0.01). Improvement in LVH over time on dialysis can be explained by good fluid volume control and close monitoring of cardiovascular parameters and better patient compliance and adherence to treatment. Growth retardation is a complication of chronic kidney disease and has a significant influence on both final adult height and quality of life [7, 13]. In the NAPRTCS reports, growth rate improved significantly, from −2.8 to −1.9 (p = 0.078), in patients treated with rGH (9.4% of patients on PD and 8.7% of patients on HD) but worsened in the remainder [8, 20]. We did not observe a significant improvement in growth in the present study, perhaps our patients were not as growth retarded initially as in the NAPRTCS data which may explain their relative lack of response to GH. Also some of our patients may have already reached their adult height. According to the NAPRTCS reports [20, 22] survival in paediatric patients on dialysis, calculated by deaths per 100 patient-years, varied from 13.6 at age 1 year, to 8.2 at age 1-2 years, 6.1 at age 2–5 years, and 2.8 at age > 6 years. Overall survival rates were 95% at 1 year and 85.7% at 3 years [20, 22]. In the long-term study from Australia and New Zealand by McDonald and Craig [19], 10-year and 20-year survival rates of children on dialysis were 79% and 66%, respectively. The United States Renal Data System (USRDS) study by Mitsnefes et al. [3] reported an overall mortality of 9.88 per 100 patient-years in patients younger than 5 years and 3.86 per 100 patient-years in patients older than 5 years. Shroff et al. [8] in a single-Center 14-year study reported 17 deaths in 98 patients, for an overall survival rate of 83%. In our present 18-year study, of 110 young patients on maintenance dialysis, 12 died. Eight deaths (7.2%) were related to dialysis complications. The 5-year overall survival rate was 84%: 61% in patients younger than 5 years and 90% in patients older than 5 years at onset of dialysis. Calculating survival in patient-years of dialysis yielded a better outcome than in the study of Mitsnefes et al. [3]: 4.3 deaths for the whole cohort, 11.6 in the younger group, and 2.39 in the older group. Only 2 patients who died were exclusively on PD and those 2 died due to complications of their primary illness. It was not found statistically significant, probably because of a small number of deaths. The limitation of the present study is its retrospective design, a small sample size which for a single center is relatively large but not compared with multicenter studies, and a specific population which may defer from other centers/groups. In conclusion, despite the relatively long period on maintenance dialysis, pediatric patients with ESRD in our center have a similar outcome in terms of survival and dialysis complications to that reported in other industrialized countries. We suggest that efforts be made to broaden the use of arteriovenous fistulas and minimize the use of central cuffed catheters in order to avoid infections and thrombosis. Implementation of standardized PD education programs, prophylactic antibiotics prior to insertion of PD catheters, and strict adherence to aseptic techniques may reduce the rate of PD -associated infections. The findings may have important implications for decreasing the risks of dialysis-associated complications especially for very young ESRD patients, thereby lowering hospitalization and improving survival. Competing Interests All authors have no conflict of interests to disclose. Figure 1 Kaplan Meier survival curve of study population and Kaplan Meier survival curves of children beginning dialysis at age > 5 years or <5 years. Table 1 Causes of ESRD leading to need for dialysis. Major causes of ESRD Number (%) CAKUT 45 (40.9%) Nephronophthisis 6 (5.45%) FSGS 19 (17.27%) Congenital nephrotic syndrome 4 (3.63%) Metabolic diseases 5 (4.54%) Alport 2 (1.81%) Denys-Drash syndrome 3 (2.72%) Glomerulonephritis 12 (10.9%) Hypoxic injury 1 (0.9%) Familial HUS 2 (1.81%) Secondary HUS 2 (1.81%) PCKD 2 (1.81%) Other/unknown 7 (6.36%) Table 2 Study parameters related to dialysis modality. Parameters HD exclusively (n = 59, 53.6%) PD exclusively (n = 25, 22.7%) Both modalities ∗ (n = 26, 23.6%) p value Age at dialysis onset (yr.), mean ± SD 12.41 ± 4.58† 7.58 ± 5.36† 9.68 ± 5.21 <0.001 Duration of dialysis (yr.), mean ± SD 1.41 ± 0.79 1.32 ± 0.47 1.41 ± 0.76 0.87 Number of patients by period of treatment, n (%)          1995–2003 33 (55.9%) 9 (15.3%) 17 (28.8%) 0.09‡  2004–2013 26 (51.0%) 16 (31.4%) 9 (17.6%) Hb level, g/dL, mean ± SD          At dialysis onset 9.18 ± 1.62 9.22 ± 1.61 8.8 ± 1.96 0.6  At study end 10.88 ± 1.41 10.55 ± 1.75 10.32 ± 1.46 0.3 Growth SDS, mean ± SD          At dialysis onset           Height SDS −1.25 ± 0.94 −1.01 ± 1.50 −1.4 ± 0.83 0.52   Weight SDS −0.64 ± 0.70 −0.31 ± 1.20 −0.55 ± 0.64 0.25  At study end           Height SDS −1.47 ± 1.22 −1.46 ± 1.44 −2.3 ± 0.8† 0.009   Weight SDS −0.81 ± 0.62 −0.37 ± 0.79 −1.22 ± 0.46 <0.001 ∗PD and HD sequentially. † p < 0.001. ‡The number of patients treated by PD increased significantly in the later period. Table 3 Characteristics of patients on maintenance dialysis who died during the study period (12/110). Pt. number Age at dialysis initiation (years) Sex Cause of ESRD Duration of dialysis (years) Modality of dialysis Access Age at death (years) Cause of death 1 1.58 M Familial HUS 0.86 HD P 2.42 Cardiac arrest due to hyperkalemia 2 3.69 M CAKUT 2.35 Combined P + T 6.04 Sepsis 3 1.98 M HUS s/p BMT 0.42 Combined P + T 2.41 Sepsis 4 2 F Bilateral Nephrectomy due to Wilms tumor 0.59 Combined P + T 2.59 Cardiac arrest due to hyperkalemia 5 3.54 F Familial HUS 9.57 HD AVF 13.11 Access failure 6 14.51 F Systemic Lupus Erythematosus 6.23 HD AVF 20.74 Mesenteric event 7 7.96 M FSGS 1.52 Combined P + T 9.48 Access failure 8 7.8 M FSGS-Schimke syndrome 2.86 PD T 10.66 CVA 9 9.08 M FSGS-Schimke syndrome 1.73 PD T 10.81 CVA 10 3.2 M Congenital nephrotic syndrome 0.81 HD P 4.01 Cardiac arrest due to hyperkalemia 11 9.55 F Nephrotoxic kidney injury 5.91 HD AV 15.46 Metastatic Neuroblastoma 12 4.62 M CAKUT 1.43 Combined P + T 6.05 Sepsis AVF: arterial venous fistula; BMT: bone marrow transplantation; CAKUT: congenital anomalies of the kidney and urinary tract; CVA: cerebrovascular accident due to moyamoya phenomenon; ESRD: end-stage renal disease, FSGS: focal segmental glomerulosclerosis; HD: hemodialysis, HUS: hemolytic uremic syndrome; P: permacath; PD: peritoneal dialysis; s/p: status post; T: tenckhoff peritoneal catheter. ==== Refs 1 Miklovicova D. 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==== Front Anat Res IntAnat Res IntARIAnatomy Research International2090-27432090-2751Hindawi Publishing Corporation 10.1155/2016/4106981Research ArticleA Cadaveric Investigation of the Dorsal Scapular Nerve Nguyen Vuvi H. 1 Liu Hao (Howe) 2 Rosales Armando 1 http://orcid.org/0000-0003-3006-0576Reeves Rustin 1 * 1Center for Anatomical Sciences, University of North Texas Health Science Center, Fort Worth, TX 76107, USA2Department of Physical Therapy, University of North Texas Health Science Center, Fort Worth, TX 76107, USA*Rustin Reeves: rustin.reeves@unthsc.eduAcademic Editor: Udo Schumacher 2016 15 8 2016 2016 410698124 6 2016 19 7 2016 Copyright © 2016 Vuvi H. Nguyen et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Compression of the dorsal scapular nerve (DSN) is associated with pain in the upper extremity and back. Even though entrapment of the DSN within the middle scalene muscle is typically the primary cause of pain, it is still easily missed during diagnosis. The purpose of this study was to document the DSN's anatomy and measure the oblique course it takes with regard to the middle scalene muscle. From 20 embalmed adult cadavers, 23 DSNs were documented regarding the nerve's spinal root origin, anatomical route, and muscular innervations. A transverse plane through the laryngeal prominence was established to measure the distance of the DSN from this plane as it enters, crosses, and exits the middle scalene muscle. Approximately 70% of the DSNs originated from C5, with 74% piercing the middle scalene muscle. About 48% of the DSNs supplied the levator scapulae muscle only and 52% innervated both the levator scapulae and rhomboid muscles. The average distances from a transverse plane at the laryngeal prominence where the DSN entered, crossed, and exited the middle scalene muscle were 1.50 cm, 1.79 cm, and 2.08 cm, respectively. Our goal is to help improve clinicians' ability to locate the site of DSN entrapment so that appropriate management can be implemented. ==== Body 1. Introduction In standard anatomical textbooks and atlases, the dorsal scapular nerve (DSN) is documented as a motor nerve originating from the ventral ramus of spinal nerve root C5, from the superior trunk of the brachial plexus [1–4]. In addition to C5, various texts have also documented the DSN to occasionally receive contributions from C4 [5–9]. This nerve typically pierces the middle scalene muscle and travels posteroinferiorly to innervate the levator scapulae, rhomboid minor, and rhomboid major muscles [6–11]. Collectively, these muscles function to elevate and retract the scapula [4–6]. In contrast, several anatomical studies in the primary literature indicated that the spinal root origins and muscle innervations of the DSN may vary. One study found that the DSN not only receives contribution from C5 but also may receive variable contributions anywhere from C4-T1 [12]. Ballesteros' and Ramirez's study found that nearly 48% of the DSNs originated from C5 whereas approximately 30% shared a trunk with the long thoracic nerve [13]. Lee et al. (1992) reported that nearly 25% of the DSNs in their study originated from other spinal nerve roots aside from C5 [14] whereas Tubbs et al. (2005) reported that 95% of the DSNs originated from C5 and 5% branched from C5 and C6 [10]. A recent study by Shilal et al. (2015) also documented that the DSN arose from C5 and C6 and receives communications with the long thoracic nerve [15]. In addition, there are varying reports regarding the muscles that DSN innervates. For example, one case study from Japan reported that the DSN innervated the serratus posterior superior muscle [16]. In a study by Frank et al. (1997), they reported that the DSN innervated the levator scapulae muscle in only 11 out of 35 neck specimens [17]. The entrapment of the DSN is often located at the middle scalene muscle, because the nerve often pierces this muscle [18]. This nerve impingement or entrapment often leads to pain in the upper extremity and back. Patients typically experience sharp or aching pain along the medial border of their scapula that can radiate to the lateral aspect of their arm and forearm [19]. In addition, patients also report pain in their neck and back, as well as dysfunction of their shoulders [12]. Occupations which involve raising the arms over long periods of time, such as painters and electricians, make these particular individuals more likely to develop DSN entrapment [20]. There are also reports of DSN injury among athletes [21]. For example, Jerosch et al. (1990) reported that, along with injury to the long thoracic nerve, the DSN was also injured as a result of an anterior shoulder dislocation during judo [22]. Another report described an isolated DSN entrapment in a body builder using anabolic steroids. It was thought that the middle scalene muscle was injured due to repetitive stretching during exercises of neck flexion and forceful repetitive shoulder shrugging [23]. Lastly, concurrent with injury to the suprascapular nerve, the DSN was also injured in two sibling volleyball players. According to Ravindran, the brother and sister were active volleyball players for over 6 years and interestingly had almost identical symptoms in that both developed right shoulder and scapular pain with particular wasting of the right infraspinatus muscles. Both siblings also had mild winging of the right scapula with weakness of the rhomboid muscles [24]. In addition to these DSN injuries in sports, there are also case reports in which a lesion to or neuropathy of the DSN caused scapular winging [20, 25]. Because the DSN branches from the brachial plexus, clinicians often describe the impingement of this nerve as contributing to thoracic outlet syndrome (TOS). Specifically, the impingement of the DSN affects the interscalene space in TOS [12, 26, 27]. Meaningful epidemiological figures of this syndrome are difficult to obtain due to debate among clinicians with regard to the exact definition, diagnosis, and treatment of TOS [28]. As a result, some experts believe that TOS may be underdiagnosed or misdiagnosed [29–31]. The incidence of TOS has been broadly estimated to range from 0.3% to 8% in the US population [28, 32] and the most commonly affected age range is between 20 and 50 years [30]. Current treatments used by clinicians in relieving patients from DSN entrapment may involve either conservative and/or surgical treatments. Conservative treatments beyond physical rehabilitation may involve administering a local nerve block injection, which is commonly guided via ultrasound, in order to relieve patients of their symptoms [27, 33, 34]. It is very important for health care providers to have good working knowledge of the area around the scalene muscles in the neck, especially if they are going to apply nerve block injections in this area. They must be aware of other important neurological structures such as the roots and trunks of the brachial plexus and the phrenic nerve. Surgical treatment for DSN entrapment typically involves lesion of the muscle that is impinging the DSN, most often the middle scalene muscle [12]. In either case, it is important for clinicians to be mindful of the location and route of this nerve as it passes anterior, through, or posterior to the middle scalene muscle. The purpose of this study is to undertake a more extensive investigation and description of the anatomy of the DSN in order to gain a better understanding of the spinal root origins, anatomical route, and muscular innervations of this nerve. In addition, we created a model of the DSN's path in relation to the middle scalene muscle by using measurements established at the transverse plane of the laryngeal prominence. The measurements for the DSNs in this study will assist clinicians with efficiency in pinpointing the surface location of this nerve in their patients for the purpose of diagnosis and treatment of possible nerve entrapment. 2. Materials and Methods The dorsal scapular nerve was dissected and examined in 20 embalmed adult cadavers (12 females and 8 males) obtained through the Willed Body Program, Center for Anatomical Sciences, at the University of North Texas Health Science Center (UNTHSC) in Fort Worth, Texas. The age of the donors span from 52 to 93 years with a mean age of 75 years. The self-reported ethnicities of the donors are (95%) Caucasian and (5%) African American. The cadavers are individually wrapped in cotton shrouds with Maryland State Wetting agent (Hydrol Chemical Company, Yeadon, PA) and are stored in metal tanks located in the UNTHSC Gross Anatomy Laboratory. The cadavers used in this project were initially dissected by first-year medical students enrolled in the school's gross anatomy course. Once the medical students were finished with their dissections, the final preparation of the cadavers for this study began. The sternocleidomastoid muscles were detached from their origin and reflected laterally. The superior trunk of the brachial plexus (C5 and C6) was identified between the anterior and middle scalene muscles and any fascia overlying these muscles was removed. The DSN was first identified in relation to the scalene muscles, and then the route to the muscles that it innervates was traced. If the DSN or the scalene muscles on the cadaver were damaged (left or right side), then the DSN data on that side was excluded from the study. The majority of the DSN dissection in this study remained intact on the left side of the neck region compared to the right one. On the right side of the neck, an incision was made to access vasculature for the embalming of our cadavers. Therefore, important structures such as the scalene muscles and the DSN were often damaged on that side. A transverse plane through the laryngeal prominence was established using a 90° angle ruler to create a reference site for three points of measurement to document the oblique route the DSN takes in relation to the middle scalene muscle. The points were derived measuring the distance of the DSN from this transverse plane as the nerve enters, crosses, and exits the middle scalene muscle. The point at which the DSN “enters” the middle scalene muscle is defined as where the nerve initially contacts the medial border of the middle scalene muscle. The point at which the DSN “exits” the middle scalene muscle is defined as where the nerve contacts the lateral border of the middle scalene muscle. Finally, the point where the DSN “crosses” the middle scalene muscle is defined as the midpoint where the nerve contacts the medial (enters) and lateral (exits) border of the muscle. Yellow pins were placed to delineate the transverse plane (white dashed lines) of the laryngeal prominence (Figure 1(a)). The distances of the DSN from this plane as it enters, crosses, and exits the middle scalene muscle were measured using an electronic sliding caliper (Mitutoyo Corp.); three repeated measurements were made for each observation from this plane (Figure 1(b)). Average values and standard deviations were calculated from these measurements. In order to test the reliability (consistency) of these measurements, Cronbach's alpha test was conducted through the Statistical Package for Social Sciences (SPSS) software (IBM Corp. 2015. IBM SPSS Statistics for Windows, Version 23.0, Armonk, NY: IBM Corp.). Dissection images were taken with a digital camera (Nikon Coolpix AW110). 3. Results The DSN was dissected from 20 embalmed adult cadavers that were previously dissected by first-year medical students for classroom study. From these 20 cadavers, a total of 23 DSNs were examined. As indicated in Table 1, 70% of the DSNs originated from the spinal nerve roots of C5, whereas 22% arose from C4 and 8% from C6. With regard to the route of the DSN in relation to the middle scalene muscle, 74% pierced the muscle, whereas 13% of the DSNs traveled anterior to the middle scalene muscle, and 13% traveled posterior to the muscle. In addition, we observed that 52% of the DSNs provided innervation to the levator scapulae, rhomboid minor, and rhomboid major muscles combined. Furthermore, we observed that, in 48% of the cadavers in our study, the DSN supplied only the levator scapulae muscle. Figures 2(a) and 2(b) are examples of a dissection of the DSN followed from its spinal root origins, anatomical route in relation to the middle scalene muscle, and muscular innervations in a 90-year-old female cadaver in the supine and prone position, respectively. Measurements were taken of the DSN as it courses obliquely from the medial to lateral border of the middle scalene muscles. From the transverse plane of the laryngeal prominence, the mean distance at which the DSN enters (medial border) the middle scalene muscle was 1.50 cm with a standard deviation of 0.88 cm; the DSN crosses (midpoint) the middle scalene muscle at 1.79 cm (±0.89 cm) and exits (lateral border) this muscle at a mean distance of 2.08 cm (±0.96 cm). These mean values and standard deviations were calculated from the 23 DSNs dissected and documented in this study (N = 23). Cronbach's alpha value was 0.999 which indicates very high consistency of the triplicate measurements conducted in this study. 4. Discussion In this study, we report the percentage of cases in which the spinal root of the DSN arose from C5 (70%) to be very similar to that reported by Lee et al. (1992) where the DSN arose from C5 approximately 75.8%. Similar to our data, almost 25% of the DSNs in that study originated from spinal root origins other than C5, such as from the superior trunk of the brachial plexus (C5 and C6), C4 and C5, and C6 alone [14]. In terms of the muscular innervations, almost half of the DSNs in our study supplied the levator scapulae muscles only. Interestingly, Frank et al. (1997) reported that although the DSN consistently pierced the middle scalene muscle, the muscular innervations of this nerve were highly variable. Their study documented that the DSN innervated the levator scapulae in only 11 out of 35 neck specimens [17]. These reports and our current data suggest that the anatomy of the DSN is variable and may be a possible reason in which clinicians often overlook the impingement of this nerve during differential back diagnosis [18]. 5. Conclusion Our research will assist clinicians in becoming aware of potential variations in the overall anatomy of the DSN in terms of its spinal root origins, anatomical route, and muscular innervations. No prior study has measured the oblique route of the DSN as it crosses the middle scalene muscle relative to the transverse plane of the laryngeal prominence. For future studies, these measurements will allow us to evaluate the surface projection of the DSN relative to its typical site of impingement (the middle scalene muscle) while using the transverse plane of the laryngeal prominence as a reference point. The long-term goal of this study is to provide data to assist clinicians and therapists to accurately and efficiently pinpoint the location of this nerve in patients with possible DSN impingement. Acknowledgments The authors thank the selfless gifts made by body donors to the Willed Body Program, Center for Anatomical Sciences at the University of North Texas Health Science Center in Fort Worth. This research would not be possible without their generosity. Competing Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Figure 1 (a) A 90° angle ruler was placed directly on top of the laryngeal prominence creating a transverse plane as denoted by the yellow pins. The DSN branches from C5 and pierces the middle scalene muscle. (b) An electronic sliding caliper was used to measure the distances (cm) from the transverse plane (white dashed line) of the laryngeal prominence to the DSN as it enters the middle scalene muscle (green pin), crosses this muscle (blue pin), and exits the middle scalene muscle (orange pin). SCM is sternocleidomastoid muscle. Figure 2 (a) An anterolateral view of the right neck region of a 90-year-old female cadaver in the supine position. The DSN branches from C4 and pierces the middle scalene muscle. (b) In the prone position, the DSN travels posteroinferiorly after piercing the middle scalene muscle to supply the levator scapulae, rhomboid minor, and rhomboid major muscles. The rhomboid muscles are reflected laterally from their origin to show the route of the DSN. Table 1 Variation in the spinal roots and innervations of the DSN.   N and percentage Origin   C4 5 (22%) C5 16 (70%) C6 2 (8%) Route   Anterior to middle scalene m. 3 (13%) Piercing middle scalene m. 17 (74%) Posterior to middle scalene m. 3 (13%) Muscles innervated   Levator scapulae m. only 11 (48%) Levator scapulae m. & rhomboid mm. 12 (52%) Total N 23 Cadaver number (N) and percentage for specific spinal root origins, route, and muscles innervated for the DSN. ==== Refs 1 Snell R. S. 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==== Front Biomed Res IntBiomed Res IntBMRIBioMed Research International2314-61332314-6141Hindawi Publishing Corporation 10.1155/2016/9418163Review ArticleGenetic Association of CHAT rs3810950 and rs2177369 Polymorphisms with the Risk of Alzheimer's Disease: A Meta-Analysis Liu Yong 1 2 3 Chen Qicong 4 Liu Xu 1 5 Dou Mengmeng 1 6 Li Silu 7 Zhou Jiahui 1 6 Liu Hong 2 http://orcid.org/0000-0002-1245-251XWu Yongfu 1 8 * http://orcid.org/0000-0002-5821-703XHuang Zunnan 1 2 6 * 1Dongguan Scientific Research Center, Guangdong Medical University, Dongguan, Guangdong 523808, China2School of Pharmacy, Guangdong Medical University, Dongguan, Guangdong 523808, China3Key Laboratory for Research and Development of Natural Drugs of Guangdong Province, Zhanjiang, Guangdong 524023, China4Department of Biochemistry and Molecular Biology, Guangxi Medical University, Nanning, Guangxi 530021, China5The Second School of Clinical Medicine, Guangdong Medical University, Dongguan 523808, China6Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Dongguan, Guangdong 523808, China7School of Basic Medicine, Guangdong Medical University, Dongguan, Guangdong 523808, China8Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong 524023, China*Yongfu Wu: wyf163126@126.com and *Zunnan Huang: zn_huang@yahoo.comAcademic Editor: Lei Yao 2016 15 8 2016 2016 941816324 2 2016 3 6 2016 3 7 2016 Copyright © 2016 Yong Liu et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Choline acetyltransferase (CHAT) rs3810950 and rs2177369 polymorphisms have been implicated in susceptibility to Alzheimer's disease (AD). Due to the inconsistent results from previous studies, a meta-analysis was performed to estimate the association between these polymorphisms and AD risk more precisely. Pooled results of our meta-analysis indicated CHAT rs2177369 polymorphism was correlated with decreasing AD risk in one of five genetic models (dominant: OR = 0.77, 95% CI: 0.62–0.96), while rs3810950 mutant was associated with AD development in three models (allelic: OR = 1.18, 95% CI: 1.01–1.37, homozygous: OR = 1.63, 95% CI: 1.09–2.42, and recessive: OR = 1.65, 95% CI: 1.20–2.26). In subgroup analysis by ethnicity, the association between CHAT rs3810950 polymorphism and AD risk was just found in the recessive model (OR = 1.47, 95% CI: 1.05–2.07) among Caucasians, while four genetic models (allelic: OR = 1.23, 95% CI: 1.01–1.48; homozygous: OR = 2.24, 95% CI: 1.48–3.39; dominant: OR = 1.21, 95% CI: 1.06–1.40; and recessive: OR = 2.18, 95% CI: 1.45–3.29) assumed this association in Asians. In conclusion, our meta-analysis indicated CHAT rs2177369 polymorphism might play a protective role in AD, while rs3810950 variant was a risk factor for AD but its single heterozygous mutations might not influence susceptibility to AD. National Natural Science Foundation of China31170676Traditional Chinese Medicine Bureau Foundation of Guangdong Province20151264Sail Plan ==== Body 1. Introduction Alzheimer's disease (AD) is a neurodegenerative disorder characterized by severe damage of cognition. It is the most common form of age-related dementia [1]. Today, 35 million patients fight against dementia and most of them suffer from AD [1, 2]. This brings huge losses to the social economy and seriously affects the long-term health-care system. Neuropathology of AD is characterized by the accumulation of extracellular β-amyloids (Aβ) in plaques and intracellular hyperphosphorylated tau protein [3]. Although the causes for the development of AD are still unclear, many studies showed that AD was triggered by multiple genetic and environmental factors [2, 4–6]. Genetic studies revealed that genetic factors played significant roles in the development of Alzheimer's disease [5]. Recently, several studies, including the large-scale genome-wide association studies (GWAS) of AD, have reported some susceptibility genes such as Apolipoprotein E (ApoE), Bridging Integrator 1 (BIN1), Clusterin (CLU), complement component receptor 1 (CR1), and choline O-acetyltransferase (CHAT) [7–10]. Among them, CHAT is the key enzyme responsible for the synthesis of a neurotransmitter acetylcholine and the target for many effective pharmacological therapies of AD [11]. CHAT gene has several genetic polymorphic loci such as rs1880676, rs2177369, rs868750, and rs3810950. In this paper, we focus only on the influence of the rs3810950 (G>A) and rs2177369 (A>G) polymorphisms on AD risk. We do not include other CHAT polymorphisms for this meta-analysis because all the other available polymorphisms do not meet the performance standard of conducting a mate-analysis due to their limited case-control studies. The mutation of A/G in both rs3810950 and rs2177369 is fascinating due to its functionality. These polymorphisms in the CHAT gene may affect the synthesis of the enzyme, thereby amplifying the cholinergic neurotransmission deficits in AD [12]. The rs3810950 polymorphism has been proven to be associated with Alzheimer's disease in nine studies [10, 12–19]. In these studies, the gene variation was found to associate with Alzheimer's disease among people in Asia, America, and five European countries. However, two other studies identified no association between CHAT rs3810950 and AD in British people [20, 21]. Similarly, three previous articles, which investigated the relationship between rs2177369 and AD risk in the British and Italian population, respectively, also provided inconsistent results [21–23]. These controversial results from the earlier reports of different geographic areas might be caused by the relatively small size of each individual study and its low power to detect the true effect. We thus conducted a meta-analysis to give a more precise estimation of the association between these two CHAT polymorphisms and AD susceptibility. 2. Methods and Materials 2.1. Literature Search and Inclusion Criteria Two investigators searched the PubMed, Embase, CNKI, Wanfang, and AlzGene databases to find all relevant records using the following keywords: “Choline acetyltransferase OR CHAT”, “Alzheimer's disease OR AD” and “polymorphism OR polymorphisms OR variant OR mutation”. The searches were last updated on May 15, 2016. Two authors took responsibility for literature searches to ensure the integrity of the data collection. The inclusion criteria to select eligible articles in this meta-analysis were as follows: (1) the association of CHAT rs3810950 or rs2177369 polymorphism with Alzheimer's disease; (2) a case-control design; (3) complete genotype data including the number of homozygous mutant, heterozygous and wild genotypes to calculate ORs. On the other hand, the literatures were excluded if they met any of the following criteria: (1) abstracts, editorials, review articles, and unrelated meta-analyses; (2) studies without polymorphisms; (3) studies with other diseases or other polymorphisms; (4) studies with incomplete genotype data. Only one could be accepted if the publications were duplicated. Any disagreement regarding the inclusion of articles was resolved by discussion among the authors. 2.2. Data Extraction One author extracted the following information from each study: (1) the first author's name; (2) the year of publication; (3) the country and ethnicity of the participants (patients and controls); (4) the number of Alzheimer's disease cases and controls; (5) the frequency of genotypes in AD cases and controls; (6) genotyping method; (7) diagnosis criteria of AD. Then, another author checked the data carefully to ensure they are complete and correct. In case-control studies, Hardy Weinberg Equilibrium (HWE) was used for quality assessment of genotype data. A high-quality study was considered that its control group conformed to HWE. A study without HWE in controls was defined as a low quality one. Low quality studies were excluded in the sensitivity analysis. Newcastle-Ottawa Scale (NOS) criteria [24] were used to evaluate the quality of the case-control studies included in the meta-analysis. The evaluation of content in the NOS was classified into three independent aspects: object selection, comparability, and exposure assessment. In this retrospective analysis, an included study should get at least five points [25] in the NOS quality assessment. 2.3. Statistical Analysis In this meta-analysis, the pooled odds ratios (ORs) with 95% confidence intervals (CIs) were used to estimate the association of CHAT rs3810950 or rs2177369 polymorphisms with the risk of AD. We did not use environmental factors to adjust the poor ORs due to the very limited information provided in each of the included studies. We performed this meta-analysis using five genetic models: the allelic, homozygous, heterozygous, dominant, and recessive model. Chi-square test and I 2 test were used to calculate the heterogeneity of these genetic models. A P value <0.05 and/or I 2 > 50% indicated substantial heterogeneity, and then a random effect model was used; otherwise, a fixed effect model was used to calculate the ORs and 95% CIs of any genetic model with unobserved heterogeneity [26, 27]. Additionally, meta-regression based on the covariates of sample size, ethnicity, and genotyping method was adopted to explore the source of heterogeneity. Subgroup analysis was also conducted according to ethnicity and genotyping method. At the same time, we did a stratified analysis by ApoE-ε4 status. Statistical power analysis was executed to estimate the suitability of the sample size employed to the power of the study. We assumed an unmatched case-control design and considered a two-sided P value of 0.05. “Venice criteria” [28] (Table S1, see Supplementary Material available online at http://dx.doi.org/10.1155/2016/9418163) were also applied to assess the credibility of the cumulative evidence of each meta-analyzed association under the genetic models we investigated. The evidence level is graded as strong, moderate, or weak. Sensitivity analysis was applied to investigate the influence of the individual studies to the pooled results by omitting one study at a time. Both Begg's and Egger's tests were used to assess publication bias and a P value of less than 0.01 was considered statistically significant. The trim and fill method was also employed to identify and correct funnel plot asymmetry arising from publication bias. Data analysis was performed by professional software Review Manager 5.3 (Cochrane Informatics and Knowledge Management Department), STATA 14.0 (Stata Corporation College Station, Texas, USA), and Quanto software package (Version 1.2.4, http://biostats.usc.edu/software). 3. Results 3.1. Characteristics of the Studies As shown in Figure 1, 655 related articles were first discovered from database searching up to May 15, 2016. Then, 611 articles were obtained after the duplicated publications were weeded out. Among them, 66 were abstracts, editorials, review articles, or unrelated meta-analyses, 493 lacked polymorphisms, and 39 related to other diseases and polymorphisms or had incomplete genotype data. Thus, we further discarded these studies (598) according to the exclusion criterions. Finally, we got thirteen eligible articles for our meta-analysis, which included 11 studies related to CHAT rs3810950 polymorphism and four studies linked to CHAT rs2177369 polymorphism. The baseline characteristics of all of these studies are listed in Table 1. The included studies conformed to HWE except for two: one reported by Mubumbila et al. [13] on CHAT rs3810950 polymorphism and the other one as Cook (1) study by Cook et al. [21] on CHAT rs2177369 polymorphism. Statistical power based on the given sample size of each study ranged from 5.00% to 90.29% under the dominant model and from 5.73% to 98.68% under the recessive model. The NOS results showed that the quality score of all the included studies satisfied the standard to reach five points or more (Table 2). 3.2. A Meta-Analysis of CHAT rs3810950 Polymorphism with AD Risk In this meta-analysis, a total of 11 studies [10, 12–21] involving 3951 patients and 5963 controls were included to investigate the associations between CHAT rs3810950 and the risk of Alzheimer's disease (as shown in Table 1). Combined data showed that CHAT rs3810950 polymorphism was associated with an increased risk of AD in three of five genetic models (allelic A versus G: OR = 1.18, 95% CI: 1.01–1.37, P = 0.03; homozygous AA versus GG: OR = 1.63, 95% CI: 1.09–2.42, P = 0.02; and recessive AA versus AG + GG: OR = 1.65, 95% CI: 1.20–2.26, P < 0.01) (Figure 2) but no association was observed in the remaining two models (heterozygous AG versus GG: OR = 0.99, 95% CI: 0.90–1.10, P = 0.87; dominant AA + AG versus GG, OR = 1.08, 95% CI: 0.92–1.28, P = 0.34) (Table 3). Power analysis on the pooled sample size showed that the statistical power was 46.23% and 99.99%, respectively, in the dominant and recessive models. In general, our meta-analysis demonstrated that CHAT rs3810950 polymorphism increased the risk of Alzheimer's disease but only a heterozygous mutation of rs3810950 might not influence the susceptibility to AD. 3.3. Subgroup Analysis The results from a subgroup analysis by ethnicity in Caucasians and Asians for all five genetic models are shown in Table 3. Only the recessive model (AA versus GG + GA: OR = 1.47, 95% CI: 1.05–2.07, P = 0.03) established the association of CHAT rs3810950 polymorphism with AD risk among Caucasians, while four genetic models (allelic A versus G: OR = 1.23, 95% CI: 1.01–1.48, P = 0.04; homozygous AA versus GG: OR = 2.24, 95% CI: 1.48–3.39, P < 0.01; dominant AA + AG versus GG, OR = 1.21, 95% CI: 1.06–1.40, P < 0.01; and recessive AA versus GG + GA: OR = 2.18, 95% CI: 1.45–3.29, P < 0.01) assumed this association among Asians. The statistical power of an Asian subgroup was 77.90% and 99.45% and that of a Caucasian subgroup was 6.34% and 99.99% calculated from power analysis, respectively, under the dominant and recessive models. Thus, only the homozygous mutant genotype AA might be related with the susceptibility to AD in a Caucasian population but only the heterozygous genotype AG might not be linked to increasing AD risk in an Asian population. In addition, a further analysis of studies involving British only indicated that no association between CHAT rs3810950 polymorphism and Alzheimer's disease was found in British people for all five genetic models (Table S2 in Supporting Information). The statistical power calculated for the British group was 5.41% and 26.56% under the dominant and recessive models, respectively. In the subgroup analysis according to the genotyping method [29], conflict results were obtained based on two different subgroups known as quantitative PCR and nonquantitative PCR (Table 3). The pooled ORs calculated for the quantitative PCR group showed that CHAT rs3810950 polymorphism contributed to increasing AD risk in three of five genetic models (allelic A versus G: OR = 1.32, 95% CI: 1.05–1.65, P = 0.02; homozygous AA versus GG: OR = 1.89, 95% CI: 1.00–3.55, P = 0.05; and recessive AA versus GG + GA: OR = 1.94, 95% CI: 1.18–3.19, P = 0.01) while no association was found in all the five genetic models for the nonquantitative PCR group (Table 3). The statistical power of a quantitative PCR subgroup was 77.24% and 99.99% and that of a nonquantitative PCR subgroup was 5.11% and 75.03%, respectively, under the dominant and recessive models. 3.4. Stratified Analysis Because four included studies in this meta-analysis provided the genotype data of CHAT rs3810950 polymorphism and ApoE-ε4 allele [10, 12, 14, 15], we further operated a risk-stratification analysis to calculate the association of rs3810950 polymorphism with AD based on the absence and presence of ApoE-ε4. The combined influence of CHAT rs3810950 polymorphism and ApoE-ε4 allele on Alzheimer's disease is shown in Table 4. For all the comparisons, the GG + GA genotype within non-ApoE-ε4 carriers served as a reference. Among ApoE-ε4 carriers, individuals with the GG + GA genotype showed a significantly increased risk of Alzheimer's disease (OR = 3.46, 95% CI: 1.78–6.71, P < 0.001). A substantial interaction was also found between ApoE-ε4 carriers and the AA genotype (OR = 4.87, 95% CI: 1.67–14.22, P = 0.004). All of these evidences manifested that ApoE-ε4 allele could be a vital factor in the Alzheimer's disease caused by CHAT rs3810950 polymorphism. Under the existence of ApoE-ε4, the risk of Alzheimer's disease increased notably when the genotype of rs3810950 was GG + GA or AA. 3.5. Strength of the Evidence When Venice criteria were applied to assess credibility, results under all five genetic models were graded as “A” for “amount of evidence” of all meta-analyses except “B” for British subgroup analysis, “A,” “B,” or “C” for “replication consistency,” and “A” for “protection from bias” of the overall analysis (Table S3). These results suggested that there was moderate or weak evidence of the association between rs3810950 polymorphism and AD susceptibility. 3.6. A Meta-Analysis between CHAT rs2177369 Polymorphism and AD Risk A meta-analysis of the association between CHAT rs2177369 polymorphism and AD risk included four independent studies [21–23] with a total of 981 cases and 806 controls (as shown in Table 1). Combined data revealed that CHAT rs2177369 polymorphism was correlated with a decreased risk of AD in the dominant model (GG + GA versus AA: OR = 0.77, 95% CI: 0.62–0.96, P = 0.02, statistical power = 69.05%) but no connection was detected in the rest four genetic models (allelic G versus A: OR = 0.85, 95% CI: 0.61–1.18, P = 0.34; homozygous GG versus AA: OR = 0.73, 95% CI: 0.40–1.32, P = 0.30; heterozygous GA versus AA: OR = 0.80, 95% CI: 0.63–1.01, P = 0.06; recessive GG versus GA + AA: OR = 0.85, 95% CI: 0.49–1.47, P = 0.55, statistical power = 30.29%) (Table 5, Figure 3). Thus, the overall analysis indicated that CHAT rs2177369 polymorphism could reduce the risk of AD but the association might be only slightly correlated. We also performed two subanalyses which excluded Cook (1) and Scacchi studies, respectively, because these two studies provided entirely opposite effects of CHAT rs2177369 polymorphism on AD risk [21, 23]. The meta-analysis from the exclusion of Cook (1) study showed that CHAT rs2177369 mutant was not associated with AD risk in all five genetic models (allelic G versus A: OR = 0.96, 95% CI: 0.70–1.31, P = 0.78; homozygous GG versus AA: OR = 0.91, 95% CI: 0.52–1.59, P = 0.73; heterozygous GA versus AA: OR = 0.83, 95% CI: 0.62–1.10, P = 0.19; dominant GG + GA versus AA: OR = 0.87, 95% CI: 0.67–1.14, P = 0.32, statistical power = 19.62%; recessive GG versus GA + AA: OR = 1.04, 95% CI: 0.63–1.71, P = 0.88, statistical power = 5.97%) (Table 5, Figure S1). On the other hand, the meta-analysis from the exclusion of Scacchi study showed that CHAT rs2177369 variant was statistically significant associated with decreasing AD risk in all five genetic models (allelic G versus A: OR = 0.73, 95% CI: 0.61–0.86, P < 0.01; homozygous GG versus AA: OR = 0.54, 95% CI: 0.39–0.76, P < 0.01; heterozygous GA versus AA: OR = 0.76, 95% CI: 0.58–0.99, P = 0.05; dominant GG + GA versus AA: OR = 0.68, 95% CI: 0.53–0.88, P < 0.01, statistical power = 86.83%; recessive GG versus GA + AA: OR = 0.65, 95% CI: 0.48–0.87, P < 0.01, statistical power = 73.79%) (Table 5, Figure S2). Thus, the sub-analysis of the exclusion of Cook (1) study showed that CHAT rs2177369 polymorphism did not affect AD risk while that from the exclusion of Scacchi study indicated that this polymorphism played a protective role in the Alzheimer's disease. 3.7. Heterogeneity, Meta-Regression, Sensitivity Analysis, and Publication Bias Our meta-analysis showed evidence of genetic heterogeneity in all the genetic models of the two polymorphisms except for CHAT rs2177369 polymorphism in the heterozygous model (I 2 = 0%, as shown in Tables 3 and 5). Through the calculation of between-study heterogeneity in clinical AD samples, we found significant heterogeneity among studies on CHAT rs3810950 polymorphism in the allelic model (I 2 = 74%), homozygous model (I 2 = 72%), dominant model (I 2 = 62%), and recessive model (I 2 = 66%) and on CHAT rs2177369 polymorphism in the allelic model (I 2 = 82%), homozygous model (I 2 = 77%), and recessive model (I 2 = 82%). Meanwhile, moderate heterogeneity was observed in the remaining two models as the heterozygous model (I 2 = 37%) on CHAT rs3810950 polymorphism and the dominant model (I 2 = 32%) on CHAT rs2177369 polymorphism. On CHAT rs3810950 polymorphism, the results of the meta-regression showed that the sample size, ethnicity, and genotyping method did not contribute to the heterogeneity of genetic models (data not shown here). Sensitivity analysis was then performed to assess the impact of the independent studies which caused obvious heterogeneity in those four models on CHAT rs3810950 polymorphism. We explored the influence of these studies on the pooled OR by removing each one at a time and found no significant change of our meta-analysis results. We did not perform both meta-regression and sensitivity analysis on CHAT rs2177369 polymorphism due to only four case-control studies involved. We further performed Begg's and Egger's tests to assess publication bias in the study of the genetic association between CHAT rs3810950 polymorphism and AD risk. As shown in Table S4, no obvious publication bias was detected according to the obtained P values for these genetic models. In addition, we did not observe any obvious asymmetry from the shape of Begg's funnel plot (Figure S3). In general, the effect of publication bias could be negligible in the included studies on CHAT rs3810950 polymorphism. However, we estimated the risk of publication bias neither in the subgroup analyses nor in the meta-analysis on CHAT rs2177369 polymorphism because the number of case-control studies in these studies was less than ten. 4. Discussion The association of CHAT rs3810950 polymorphism with Alzheimer's disease was previously reported in one published meta-analysis [30]. However, the earlier work only included three case-control studies with 1183 incident AD patients and 1705 controls. In order to give a more precise estimation of the association between CHAT rs3810950 polymorphism and AD, we collected 11 eligible studies with a total of 3951 cases and 5963 controls to perform this meta-analysis. Based on the enriched data we got, five genetic models were carried out to further clarify the impact of rs3810950 variant on AD. As a result, our meta-analysis showed that CHAT rs3810950 polymorphism was associated with the risk of Alzheimer's disease but such an association might not exist for only single-allele mutant in the general population (Figure 2 and Table 3). In other words, people with the homozygous mutant genotype (AA) should have a much higher risk of developing Alzheimer's disease than those with the heterozygous (AG) and wide-type genotype (GG). The results could be quite reliable providing that the pooled OR showed statistical significance and the statistical power was nearly 100% under the recessive model. These more refined findings in our study could be useful for future genetic studies on AD. In order to get more detailed information about this association, we further carried out subgroup analyses by ethnicity and by genotyping method, respectively. On one hand, subgroup analysis by ethnicity indicated that Caucasians might have a lower risk of Alzheimer's disease than Asians (Table 3). In addition, among Caucasians, the British might have much less possibility of suffering from Alzheimer's disease even if their CHAT rs3810950 genotype was AA when compared to the people from other geographic regions (Table 3 and Table S2). Nevertheless, there were only two British studies [20, 21]. This might result in false negative findings and thus people should be more cautious about this result. Low statistical power calculated for the British group clearly supported this point (Table S2). On the other hand, subgroup analysis by genotyping method implied that the genotyping error or bias might exist in our meta-analysis due to the inconsistent results obtained for the quantitative PCR and nonquantitative PCR subgroups, respectively. Besides, the high and low statistical power calculated, respectively, for the quantitative PCR and nonquantitative PCR subgroups could indicate that no association was observed between CHAT rs3810950 polymorphism and AD risk for the nonquantitative PCR subgroup probably just due to its small sample size effect (Table 3). A further stratified analysis of the association between CHAT rs3810950 allele and Alzheimer's disease according to the ApoE-ε4 status showed that, with the same rs3810950 polymorphism, ApoE-ε4 carriers exhibited a significantly higher incidence of AD than the non-ApoE-ε4 carriers (Table 4). Thus, interaction between CHAT rs3810950 polymorphism and ApoE-ε4 allele could be a huge risk factor for Alzheimer's disease. However, only four studies were included in this risk-stratification analysis and we should also treat this result with caution. Up to date, four independent studies have investigated the link between CHAT rs2177369 polymorphism and the risk of AD. Among them, two studies [21, 22] indicated no relationship between this polymorphism and the susceptibility to the disease. The other two studies [21, 23] found the association between CHAT rs2177369 variant and AD risk but provided the opposite results. Cook et al. [21] denoted that carrying the minor alleles (GG + GA) was significantly protective with respect to carrying the homozygous wide-type allele (AA), while Scacchi et al. [23] showed that the homozygous mutant (GG) was a risk factor compared with the GA + AA genotypes. Our meta-analysis results indicated that the rs2177369 polymorphism played a protective role in the disease, which agreed well with Cook (1) study. Though the results from the overall analysis based on the four independent studies only supported a weak association, the subanalysis of the exclusion of Scacchi study clearly suggested that this polymorphism was a protective factor for AD (Table 5). On the other hand, we considered that the results of no association between CHAT rs2177369 polymorphism and AD risk derived from the subanalysis excluding the Cook (1) study were unreliable since they did not reach the statistical significance and the statistical power was quite low (19.62% for dominant model and 5.97% for recessive model, Table 5). In addition, the substantial heterogeneity existed among the included studies under the allelic, homozygous, and recessive models (Table 5). However, additional studies with larger sample sizes need to be further performed for verifying the potential protective association between CHAT rs2177369 polymorphism and the risk of Alzheimer's disease. Presence of heterogeneity was detected in this retrospective study. It was known that age, gender, ethnicity, lifestyle habits (smoking and alcohol), education, vascular risk, ApoE-ε4 status, and other genetic or environmental factors influence AD onset [31–34]. Therefore, we have reasons to believe that these potential causes may account for the heterogeneity and the different results among the included studies. We tried to extract the data of these AD risk factors for further analysis. However, we could not conduct the subgroup analysis by age, gender, vascular risk, education or habits, and so forth due to their insufficient data. For CHAT rs3810950 polymorphism with AD risk, we could not clarify the sources of significant between-study heterogeneity neither from the ethnicity or genotyping method based subgroup analyses and ApoE-ε4-based stratified analysis nor from the meta-regression according to the variables of sample size, ethnicity, and genotyping method. Therefore, other factors such as age, habits, and education may cause the high heterogeneity among studies on rs3810950 polymorphism. For CHAT rs2177369 polymorphism with AD risk, however, we found that substantial heterogeneity only existed between the study investigated by Scacchi et al. [23] and others performed by Cook et al. [21] and Piccardi et al. [22], respectively. When excluding the Scacchi study, only unimportant between-study heterogeneity was observed in the meta-analysis (I 2 ≤ 26% for all five genetic models, Table 5 and Figure S2). Thus, we also considered that the potential factor such as age, education, habits, or even treatment with anti-inflammatory drugs in the Scacci study, and so forth may account for the high heterogeneity between this study and others on rs2177369 polymorphism. Our meta-analysis had several advantages. Firstly, this study is the first meta-analysis to investigate the association of CHAT rs2177369 polymorphism with the development of Alzheimer's disease. In addition, though a previous meta-analysis [30] has explored the relationship of CHAT rs3810950 variant with the susceptibility to AD, our study was performed based on a much larger sample size. Secondly, five genetic models were used in this meta-analysis. As a result, our study not only demonstrated the association between these two polymorphisms with AD, but also clarified that the homozygous and heterozygous mutant genotypes might play the potentially different roles in AD susceptibility. Thirdly, subgroup analyses, subanalyses, and meta-regression were conducted to explore the source of heterogeneity under the genetic models in this retrospective analysis. Ethnicity-based subgroup analysis also helped us to investigate the possibly different impact of CHAT rs3810950 polymorphism on AD risk in different ethnic groups. Fourthly, a risk-stratification analysis of the association by ApoE-ε4 status was also carried out in our study. This analysis assisted us to detect the promising effect of gene-gene interaction on the development of disease. Fifthly, we not only used Begg's and Egger's tests to assess the risk of publication bias but also employed the trim and fill method to identify and correct funnel plot asymmetry arising from publication bias. Sixthly, the NOS criteria were performed to evaluate the quality of the included studies and Venice criteria were applied to assess the cumulative evidence of the associations in our meta-analysis. Finally, statistical power analysis was also executed to estimate the effect of the sample size on the power of the study and this increased the credibility of our result. Some limitations should be also recognized in this meta-analysis. Firstly, a subgroup analysis based on age, gender, or lifestyle habits may also contribute to the association of CHAT rs3810950 and rs2177369 polymorphisms with Alzheimer's disease [35, 36], but we did not perform such an extensive analysis because of the limited data. Secondly, the geographic regions of the participants were restricted. We could not find a study on CHAT rs3810950 loci to investigate the population in Africa, Australia, or South America, while studies on CHAT rs2177369 loci are only involved in the British and Italian populations. This limitation might lead our results into less accuracy. Thirdly, we could not use environmental factors to adjust the pooled ORs of the association between the genetic polymorphism and the disease because of the unavailability of environmental information in the included studies. Finally, an obvious heterogeneity was observed in this meta-analysis. The study designs, populations (age and gender), habits, and geographical location may contribute to the heterogeneity. Alzheimer's disease is the most prevalent neurodegenerative disease in the elderly and it has caused serious damage to our health [1, 2, 6]. Substantial progress has been made towards characterization of Alzheimer's disease, but presently there are still no efficient therapies for Alzheimer's disease, and the pathogenic mechanism of Alzheimer's disease still remains unclear [4, 37, 38]. Thus, it is urgent to enrich our understanding of AD pathogenesis or we will almost surely fail to develop effective treatments for Alzheimer's disease. The results from this meta-analysis would help us to reach this goal. Supplementary Material Considerations for epidemiologic credibility in the assessment of cumulative evidence on genetic associations. Acknowledgments This work was supported by National Natural Science Foundation of China (31170676), Traditional Chinese Medicine Bureau Foundation of Guangdong Province, China (20151264), and the funds from Sail Plan “the Introduction of the Shortage of Top-Notch Talent” Project (YueRenCaiBan [2014] 1) of Guangdong Province, China. Competing Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Authors' Contributions Yong Liu and Qicong Chen are equal contributors. Figure 1 Flow diagram of the process used to select eligible studies. Figure 2 Forest plots of CHAT rs3810950 polymorphism and AD risk in three genetic models. (a) The allelic model (A versus G); (b) the homozygous model (AA versus GG); (c) the recessive model (AA versus AG + GG). Figure 3 Forest plots of CHAT rs2177369 polymorphism and AD risk in five genetic models. (a) The allelic model (G versus A); (b) the homozygous model (GG versus AA); (c) the heterozygous model (GA versus AA); (d) the dominant model (GG + GA versus AA); (e) the recessive model (GG versus GA + AA). Table 1 The baseline characteristics of all the studies included in this meta-analysis.   First author Year Area Ethnicity Number of cases Number of controls Cases Controls HWE Genotyping Diagnosis criteria Statistical power G/G G/A A/A G/G G/A A/A P method Dominant/recessive rs3810950 Mubumbila [13] 2002 France & Germany Caucasian 122 112 48 32 42 64 34 14 0.016 SSCP-PCR1 None 71.33%/98.68% Harold [20] 2003 UK Caucasian 131 118 69 51 11 65 47 6 0.627 PCR-RFLP2 NINCDS-ADRDA 6.62%/22.59% Kim [12] 2004 Korea Asian 246 561 171 61 14 419 133 9 0.856 PCR/sequencing2 NINCDS-ADRDA & DSM-IV 33.45%/94.29% Cook [21] 2005 UK Caucasian 210 315 112 76 22 161 128 26 1 PCR-RFLP2 NINCDS-ADRDA 8.26%/13.68% Ahn Jo [14] 2006 Korea Asian 316 264 211 99 6 192 70 2 0.129 PCR/sequencing2 NINCDS-ADRDA 35.87%/58.33% Ozturk [15] 2006 USA Caucasian 999 708 562 377 60 363 296 49 0.304 PCR/sequencing2 NINCDS-ADRDA 51.57%/11.65% Tang [16] 2008 China Asian 273 271 190 75 8 179 83 9 1 PCR-RFLP2 NINCDS-ADRDA 14.06%/5.73% Grünblatt [17] 2009 Austria Caucasian 120 456 63 45 12 268 164 24 1 TaqMan1 NINCDS-ADRDA 23.56%/47.82% Grünblatt [18] 2011 Austria & Italy Caucasian 137 464 79 46 12 267 173 24 0.61 TaqMan1 NINCDS-ADRDA 5.00%/35.07% Mengel-From [10] 2011 Denmark Caucasian 661 1308 38 203 420 55 455 798 0.369 TaqMan1 NINCDS-ADRDA 90.29%/7.57% Lee [19] 2012 Korea Asian 736 1386 505 205 26 1023 342 21 0.26 RT-PCR1 None 73.11%/94.39% rs2177369 Cook (1) [21] 2005 UK Caucasian 202 295 29 85 88 76 124 95 0.0073 PCR-RFLP2 NINCDS-ADRDA 77.81%/71.82% Cook (2) [21] 2005 UK (Cardiff) Caucasian 179 175 26 79 74 29 83 63 0.8754 PCR-RFLP2 NINCDS-ADRDA 17.71%/7.82% Piccardi [22] 2007 Italy Caucasian 158 118 44 75 39 40 57 21 1 PCR-RFLP2 NINCDS-ADRDA & DSM-IV 36.06%/15.30% Scacchi [23] 2009 Italy Caucasian 442 218 167 200 75 61 117 40 0.2737 PCR-RFLP2 NINCDS-ADRDA 8.45%/56.24% Note: 1quantitative PCR and 2nonquantitative PCR. Table 2 Quality assessment scheme for the included literatures (Newcastle-Ottawa Scale). First author Year Selection Comparability Exposure Total I II III IV V VI VII VIII Mubumbila [13] 2002 ∗ ∗   ∗ ∗   ∗   ∗∗∗∗∗ Harold [20] 2003 ∗ ∗   ∗ ∗   ∗   ∗∗∗∗∗ Kim [12] 2004 ∗ ∗ ∗ ∗ ∗∗   ∗   ∗∗∗∗∗∗∗ Cook [21] 2005 ∗ ∗   ∗ ∗   ∗   ∗∗∗∗∗ Ahn Jo [14] 2006 ∗ ∗ ∗ ∗ ∗∗   ∗   ∗∗∗∗∗∗∗ Ozturk [15] 2006 ∗ ∗   ∗ ∗∗   ∗   ∗∗∗∗∗∗ Tang [16] 2008 ∗ ∗   ∗ ∗∗   ∗   ∗∗∗∗∗∗ Grünblatt [17] 2009 ∗ ∗   ∗ ∗   ∗   ∗∗∗∗∗ Grünblatt [18] 2011 ∗ ∗   ∗ ∗   ∗   ∗∗∗∗∗ Mengel-From [10] 2011 ∗ ∗   ∗ ∗∗   ∗   ∗∗∗∗∗∗ Lee [19] 2012 ∗ ∗ ∗ ∗ ∗∗   ∗ ∗ ∗∗∗∗∗∗∗∗∗ Piccardi [22] 2007 ∗ ∗   ∗ ∗   ∗   ∗∗∗∗∗ Scacchi [23] 2009 ∗ ∗   ∗ ∗   ∗   ∗∗∗∗∗ Note: I: is the case definition adequate? II: representativeness of the cases. III: selection of controls. IV: definition of controls. V: comparability of cases and controls on the basis of the design or analysis. VI: ascertainment of exposure. VII: same method of ascertainment for cases and controls. VIII: nonresponse rate. Table 3 Subgroup analyses of the association between CHAT rs3810950 polymorphism and Alzheimer's disease risk.   Genetic comparison I 2 (%) Effect model OR [95% CI] P OR Statistical power Overall A versus G 74 Random 1.18 [1.01, 1.37] 0.03 NA AA versus GG 72 Random 1.63 [1.09, 2.42] 0.02 NA AG versus GG 37 Fixed 0.99 [0.90, 1.10] 0.87 NA AA + GA versus GG 62 Random 1.08 [0.92, 1.28] 0.34 46.23% AA versus GG + GA 66 Random 1.65 [1.20, 2.26] <0.01 99.99% Ethnicity-based              Caucasian (7) A versus G 77 Random 1.16 [0.94, 1.42] 0.16 NA AA versus GG 74 Random 1.42 [0.90, 2.25] 0.13 NA AG versus GG 0 Fixed 0.88 [0.77, 1.00] 0.06 NA AA + GA versus GG 60 Random 1.02 [0.82, 1.28] 0.85 6.34% AA versus GG + GA 66 Random 1.47 [1.05, 2.07] 0.03 99.99% Ethnicity-based              Asian (4) A versus G 52 Random 1.23 [1.01, 1.48] 0.04 NA AA versus GG 46 Fixed 2.24 [1.48, 3.39] <0.01 NA AG versus GG 5 Fixed 1.15 [0.99, 1.32] 0.07 NA AA + GA versus GG 33 Fixed 1.21 [1.06, 1.40] <0.01 77.90% AA versus GG + GA 40 Fixed 2.18 [1.45, 3.29] <0.01 99.45% Genotyping-based              Quantitative PCR (5) A versus G 76.5 Random 1.32 [1.05, 1.65] 0.02 NA AA versus GG 80.6 Random 1.89 [1.00, 3.55] 0.05 NA AG versus GG 46.9 Fixed 1.08 [0.93, 1.26] 0.32 NA AA + GA versus GG 64.3 Random 1.18 [0.90, 1.55] 0.24 77.24% AA versus GG + GA 77.3 Random 1.94 [1.18, 3.19] 0.01 99.99% Genotyping-based              Nonquantitative PCR (6) A versus G 62.8 Random 1.06 [0.88, 1.28] 0.52 NA AA versus GG 61.6 Random 1.40 [0.82, 2.37] 0.22 NA AG versus GG 18.8 Fixed 0.93 [0.82, 1.06] 0.29 NA AA + GA versus GG 48.4 Fixed 1.01 [0.83, 1.21] 0.95 5.11% AA versus GG + GA 55.7 Random 1.42 [0.88, 2.29] 0.16 75.03% Note: NA: not applicable. Table 4 Risk of Alzheimer's disease associated with CHAT rs3810950 polymorphism by ApoE-ε4 status. Genetic comparison Non-ApoE-ε4 carriers ApoE-ε4 carriers Cases Controls OR (95% CI) P Cases Controls OR (95% CI) P GG + GA 851 1605 1 (reference) NA 862 377 3.46 (1.78–6.71) <0.001 AA 292 673 1.03 (0.62–1.71) 0.08 203 185 4.87 (1.67–14.22) 0.004 Note: NA: not applicable. Table 5 Meta-analysis of the association between CHAT rs2177369 polymorphism and Alzheimer's disease risk. Meta-analysis Genetic comparison I 2 (%) Effect model OR [95% CI] P OR Statistical power Overall G versus A 82 Random 0.85 [0.61, 1.18] 0.34 NA GG versus AA 77 Random 0.73 [0.40, 1.32] 0.3 NA GA versus AA 0 Fixed 0.80 [0.63, 1.01] 0.06 NA GG + GA versus AA 32 Fixed 0.77 [0.62, 0.96] 0.02 69.05% GG versus AA + GA 82 Random 0.85 [0.49, 1.47] 0.55 30.29% Analysis without Cook (1) study G versus A 72 Random 0.96 [0.70, 1.31] 0.78 NA GG versus AA 62 Random 0.91 [0.52, 1.59] 0.73 NA GA versus AA 0 Fixed 0.83 [0.62, 1.10] 0.19 NA GG + GA versus AA 7 Fixed 0.87 [0.67, 1.14] 0.32 19.62% GG versus AA + GA 69 Random 1.04 [0.63, 1.71] 0.88 5.97% Analysis without Scacchi study G versus A 22 Fixed 0.73 [0.61, 0.86] <0.01 NA GG versus AA 13 Fixed 0.54 [0.39, 0.76] <0.01 NA GA versus AA 0 Fixed 0.76 [0.58, 0.99] 0.05 NA GG + GA versus AA 0 Fixed 0.68 [0.53, 0.88] <0.01 86.83% GG versus AA + GA 26 Fixed 0.65 [0.48, 0.87] <0.01 73.79% Note: NA: not applicable. ==== Refs 1 Ballard C. Gauthier S. 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Epidemiology of alzheimer's disease and other forms of dementia in China, 1990–2010: a systematic review and analysis The Lancet 2013 381 9882 2016 2023 10.1016/s0140-6736(13)60221-4 2-s2.0-84878660799 7 Beecham G. W. Hamilton K. Naj A. C. Genome-wide association meta-analysis of neuropathologic features of Alzheimer's disease and related dementias PLOS Genetics 2014 10 9 e1004606 10.1371/journal.pgen.1004606 8 Lambert J.-C. Ibrahim-Verbaas C. A. Harold D. Meta-analysis of 74,046 individuals identifies 11 new susceptibility loci for Alzheimer's disease Nature Genetics 2013 45 12 1452 1458 10.1038/ng.2802 24162737 9 Seshadri S. Fitzpatrick A. L. Ikram M. A. Genome-wide analysis of genetic loci associated with Alzheimer disease Journal of the American Medical Association 2010 303 18 1832 1840 10.1001/jama.2010.574 20460622 10 Mengel-From J. Christensen K. Thinggaard M. Mcgue M. Christiansen L. Genetic variants in the choline acetyltransferase (ChAT) gene are modestly associated with normal cognitive function in the elderly Genes, Brain and Behavior 2011 10 8 876 882 10.1111/j.1601-183X.2011.00728.x 2-s2.0-80755139668 11 Davies P. Maloney A. J. Selective loss of central cholinergic neurons in Alzheimer's disease The Lancet 1976 2 8000 p. 1403 12 Kim K.-W. Suh Y.-J. Park W.-Y. Choline acetyltransferase G +4 A polymorphism confers a risk for Alzheimer's disease in concert with Apolipoprotein E ε 4 Neuroscience Letters 2004 366 2 182 186 10.1016/j.neulet.2004.05.041 2-s2.0-3242789442 15276243 13 Mubumbila V. Sutter A. Ptok U. Heun R. Quirin-Stricker C. Identification of a single nucleotide polymorphism in the choline acetyltransferase gene associated with Alzheimer's disease Neuroscience Letters 2002 333 1 9 12 10.1016/s0304-3940(02)00955-2 2-s2.0-0037110992 12401548 14 Ahn Jo S. Ahn K. Kim J.-H. 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==== Front Case Rep SurgCase Rep SurgCRISCase Reports in Surgery2090-69002090-6919Hindawi Publishing Corporation 10.1155/2016/4182741Case ReportLeft Upper Lobectomy for Congenital Lobar Emphysema in a Low Weight Infant http://orcid.org/0000-0002-9708-0523Kanakis Meletios 1 * Petsios Konstantinos 1 Bobos Dimitrios 1 Sarafidis Kosmas 2 Nikopoulos Stefanos 2 Kyriakoulis Konstantinos 1 Lioulias Achilleas 3 Giannopoulos Nicholas 1 1Department of Pediatric and Congenital Heart Surgery, Onassis Cardiac Surgery Center, Athens, 17674 Kallithea, Greece2Department of Neonatology and Neonatal Intensive Care Unit, Aristotle University of Thessaloniki, Hippokration Hospital, 54642 Thessaloniki, Greece3Department of Thoracic Surgery, Sismanoglio General Hospital of Athens, 15126 Marousi, Greece*Meletios Kanakis: meletis_kanakis@yahoo.grAcademic Editor: Christophoros Foroulis 2016 15 8 2016 2016 418274129 5 2016 26 6 2016 21 7 2016 Copyright © 2016 Meletios Kanakis et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Congenital lobar emphysema (CLE) is a rare lung congenital malformation. Differential diagnosis of the disease remains challenging in an infant with acute respiratory distress. We report a case of a 3-week-old female infant with a weight of 2.1 kg who presented respiratory distress related to CLE. Left upper lobectomy was performed and she had an uneventful recovery. ==== Body 1. Introduction Congenital lobar emphysema (CLE) is a rare anomaly of lung development and often appears in the neonatal period, with hyperinflation of one or more pulmonary lobes, in the absence of bronchial obstruction [1]. Actually, true emphysematous changes are lacking and some investigators classify this disease as congenital hyperinflation. CLE occurs in 1 case per 20–30 thousand births [2]. The etiology of congenital lobar emphysema is idiopathic in half of cases, whereas the other 50% have several mechanisms proposed to explain the air-trapping, which can be divided into intrinsic and extrinsic subtypes [3]. Early diagnosis is crucial and in many cases it is difficult to differentiate between CLE and hyperinflation resulting from extrinsic bronchial obstruction (lymph nodes, vessels, masses, or cysts) that compresses the bronchus and produces valve obstruction. However, it is stated that even more than half of CLE cases are not related to airway abnormalities. Surgical removal is the most common treatment choice with operative mortality rate about 3 to 7% [2]. 2. Case Presentation A 3-week-old female infant with a weight of 2.1 kg was referred to our Department for the surgical management of CLE. The infant was in respiratory distress. Oxygen saturation was 82% in room air. Past medical history was unremarkable. In the last week, mother had noticed occasional episodes of tachypnea. Rapid development of dyspnea episodes brought the infant to the Emergency Department in respiratory distress. Respiratory rate was 70/min and oxygen saturation was of 85% on room air. Chest examination showed asymmetry with bulging of left hemithorax with decreased air entry on the left and crepitations on the right. Chest X-ray showed overdistention of the left lung field with asymmetry in parenchymal transparency with a hyperlucent zone. A prominent mediastinal shift with compression and atelectasis of the right lung was evident. Initial diagnosis was consistent with respiratory tract infection complicated with pneumothorax. Further diagnostic workup with computed tomography scan confirmed the presence of an emphysematous left upper lobe and atelectasis of the left lower lobe with subsequent contralateral mediastinal shift to the right causing atelectasis of the right lung (Figures 1(a) and 1(b)). Diagnosis was consistent with CLE. Echocardiographic workup showed the presence of small patent ductus arteriosus (PDA), which was hemodynamically insignificant. Decision was favorable for surgical treatment due to the presence of respiratory distress in the setting of mediastinal shift and subsequent compression of the unaffected lung lobes. She underwent a left posterolateral thoracotomy and a left upper lobectomy was also performed (Figures 2(a) and 2(b)). Patent ductus arteriosus was also ligated. She had an uneventful postoperative course and she was discharged from the hospital on postoperative day 12. Pathology examination of the resected lobe revealed lung parenchyma with atelectatic changes and emphysematous dilatation of alveolar spaces. 3. Discussion Nowadays, CLE is usually being diagnosed during prenatal evaluation by ultrasonography and may be associated with polyhydramnios and fetal hydrops. Early diagnosis of CLE is crucial and in many cases is complicated due to the variety of its clinical presentation that varies from mild tachypnea to severe respiratory distress [4]. The most common clinical presentation is neonatal acute respiratory distress, which is caused by localized air trapping that compresses the ipsilateral and contralateral normal lungs. Symptoms worsen as the emphysematous lobe gradually enlarges. Cyanosis is the second most common finding. However, similar symptoms with CLE may occur in bronchopneumonia, cyanotic congenital heart diseases, and several congenital abnormalities of the lung. Congenital lobar emphysema may be confused with tension pneumothorax. Chest tube insertion may further increase respiratory distress and lead to injury of the lung parenchyma. In CLE the pulmonary vessels extend to the periphery of the hyperinflated lobe and there is no visualization of a pleural line unlike in pneumothorax [5]. Moreover, differential diagnosis includes congenital cystic adenomatoid malformation, sequestration, bronchogenic cyst, unilateral hyperlucent lung syndrome, and pulmonary interstitial emphysema. The diagnosis, although not definitive, is made by clinical examination and chest X-ray and can be confirmed by CT, as we performed in our case. Chest radiograph usually shows many abnormalities that raise the suspicion of CLE. In addition, CT provides anatomic details that serve as a guide for safe resection of the affected lobe. CT can show the abnormally narrowed bronchus and affected lobe, as well as the collapsed lobe. It is also useful in differential diagnosis of mediastinal mass or an enlarged heart and may rule out the presence of associated anomalous vascular anomalies [6]. In literature the use of bronchoscopy as an important tool in the differential diagnosis is also discussed, but it is not recommended for primary screening test and it is mainly indicated for children whose symptoms appear in later neonatal period [2]. Furthermore, improper use of this procedure may aggravate the respiratory distress in CLE patients. On the contrary, Tey et al. in a recent article concluded that flexible bronchoscopy should be used to study suspected CLE cases to determine their causes and decide whether to treat the patient conservatively, by lobectomy, or by other strategies [7]. In our case the clinical presentation of CLE occurred during the third week of life with extreme respiratory distress. As with this child, respiratory distress is the commonest mode of clinical presentation. Dyspnoea, wheezing, grunting respiration, tachypnea, and sometimes progressive cyanosis are the most common symptoms. In almost 95% of CLE cases the clinical signs are evident in the early neonatal period (from few days after birth to six months) but there are a number of cases for which diagnosis may be delayed up to 5-6 months [5, 8]. The low weight of the child was a challenge for both anesthesia and surgical procedure. The various factors that increase morbidity and mortality in these patients are usually consequent to the immaturity of the various systems and the associated congenital defects. Infants with low body weight undergoing thoracic surgery are a major challenge for both surgeons and the postsurgery care unit. It has been observed that postsurgery outcome for low weight infants is worse compared to the outcome of normal body weight infants. Therefore, the need for close monitoring perioperatively and postoperatively is greater whereas procedure complications may occur. CLE perioperative mortality ranges from 3 to 7% [9]. Due to the low weight of the child we focused not only on the appropriate respiratory weaning but also on the prompt nutrition intake. The anesthetic considerations in neonatal surgical emergencies are based on the physiological immaturity of various body systems, poor tolerance of the anesthetic drugs, associated congenital disorders, and concerns regarding the use of high concentration of oxygen. Regarding the induction of anesthesia when positive pressure ventilation is applied before opening of the chest, it may cause rapid inflation of emphysematous lobar cyst with sudden mediastinal shift and cardiac arrest. Therefore, induction of anesthesia should provide adequate spontaneous ventilation with minimal airway pressure. Occasional gentle assistance is necessary. Once the chest is opened and the affected lobe is delivered, the patient can be paralyzed and the lungs can be ventilated by controlled ventilation [10]. In our case, manual-assisted ventilation with low inflating pressure (7–18 cm H2O) was used until the chest was opened. Congenital heart disease has an association with congenital lobar emphysema. In literature 12–20% concomitant CHD or vascular slings have been reported. Therefore, many studies recommend the performance of echocardiography during the differential examination [1, 3, 4]. In our case echo revealed only the presence of a PDA. Although it was not hemodynamically significant, PDA ligation was performed after the completion of lobectomy via the left posterolateral thoracotomy. The lobar emphysema was located in the left upper lobe and this is the most frequently affected site. According to previous studies, left upper lobe is most often involved, followed by the right middle lobe [1, 4, 11]. The involvement of the lower lobes is very rare. The cause of distribution of the affected lobes is not well known. It is possible that location is related to embryonic stage [7]. Moreover, in many cases a shift of the mediastinum occurs as was the case here. Surgery is the treatment of choice, especially in the setting of mediastinal shift with subsequent compression of the unaffected lung lobes. In earlier case series surgical excision of the affected lobe was recommended as soon as possible in all infants younger than 2 months and in infants older than 2 months who present severe respiratory symptoms. The clinical presentation of the infant was used as a guide to the appropriate time for surgery. In some cases with older infants, with mild or moderate respiratory distress, conservative management can be performed along with a close follow-up of the patient [2, 6]. In our case, although lobectomy in an infant with a low weight was more demanding, postoperative course was uneventful with an excellent outcome. The diagnosis of CLE in infants may be a challenge. The choice of treatment should be based on the severity of onset. This case suggests that lobectomy could be safely performed in a low weight infant with severe respiratory distress. Competing Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Figure 1 Computed tomography of the chest showing the presence of an emphysematous left upper lobe and atelectasis of the left lower lobe with subsequent contralateral mediastinal shift to the right causing atelectasis of the right lung. Figure 2 Intraoperative photos showing the macroscopic appearance of the hyperexpanded left upper lobe. ==== Refs 1 Cataneo D. C. Rodrigues O. R. Hasimoto E. N. Schmidt A. F. Jr. Cataneo A. J. M. Congenital lobar emphysema: 30-year case series in two university hospitals Jornal Brasileiro de Pneumologia 2013 39 4 418 426 10.1590/s1806-37132013000400004 2-s2.0-84884790980 24068262 2 Andrade C. F. da Costa Ferreira H. P. Fischer G. B. Congenital lung malformations Jornal Brasileiro de Pneumologia 2011 37 2 259 271 10.1590/S1806-37132011000200017 2-s2.0-79955727315 21537663 3 Durell J. Lakhoo K. Congenital cystic lesions of the lung Early Human Development 2014 90 12 935 939 10.1016/j.earlhumdev.2014.09.014 2-s2.0-84918556819 25448785 4 Latif I. Shamim S. Ali S. Congenital lobar emphysema Journal of the Pakistan Medical Association 2016 66 2 210 212 2-s2.0-84954505772 26819171 5 Idro R. I. Kisembo H. Mugisa D. Bulamu A. Congenital lobar emphysema: a diagnostic challenge and cause of progressive respiratory distress in a 2 month-old infant African Health Sciences 2002 2 3 121 123 2-s2.0-55249126253 12789097 6 Ulku R. Onat S. Ozçelik C. Congenital lobar emphysema: differential diagnosis and therapeutic approach Pediatrics International 2008 50 5 658 661 10.1111/j.1442-200x.2008.02630.x 2-s2.0-52449130328 19261115 7 Tey S. L. Wu C. Y. Su Y. T. Tsai C. C. Lin Y. C. Congenital lobar emphysema in a toddler: the role of flexible bronchoscopy in diagnosis and treatment E-Da Medical Journal 2015 2 15 20 8 Abushahin A. M. Tuffaha A. S. Khalil N. K. Ismeal A. M. Bilateral congenital lobar emphysema: a rare cause for respiratory distress in infancy Annals of Thoracic Medicine 2012 7 4 250 252 10.4103/1817-1737.102187 2-s2.0-84870224039 23189104 9 Alsoufi B. Manlhiot C. Mahle W. T. Low-weight infants are at increased mortality risk after palliative or corrective cardiac surgery The Journal of Thoracic and Cardiovascular Surgery 2014 148 6 2508 2514.e1 10.1016/j.jtcvs.2014.07.047 2-s2.0-84920053663 25238883 10 Chandran-Mahaldar D. Kumar S. Balamurugan K. Raghuram A. R. Krishnan R. Kannan Congenital lobar emphysema Indian Journal of Anaesthesia 2009 53 4 482 485 20640213 11 Moideen I. Nair S. G. Cherian A. Rao S. G. Congenital lobar emphysema associated with congenital heart disease Journal of Cardiothoracic and Vascular Anesthesia 2006 20 2 239 241 10.1053/j.jvca.2006.01.019 2-s2.0-33646479228 16616669

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==== Front Case Rep EndocrinolCase Rep EndocrinolCRIECase Reports in Endocrinology2090-65012090-651XHindawi Publishing Corporation 10.1155/2016/5218985Case ReportCerebrovascular Accident due to Thyroid Storm: Should We Anticoagulate? http://orcid.org/0000-0002-2840-7190Gonzalez-Bossolo Alex * Gonzalez-Rivera Alexis Coste-Sibilia Santiago Internal Medicine Training Program, Department of Medicine, University District Hospital, University of Puerto Rico School of Medicine, P.O. Box 365067, San Juan, PR 00936-5067, USA*Alex Gonzalez-Bossolo: alex.gonzalez7@upr.eduAcademic Editor: Osamu Isozaki 2016 15 8 2016 2016 52189851 6 2016 19 7 2016 Copyright © 2016 Alex Gonzalez-Bossolo et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Thyroid storm is a life-threatening condition that occurs secondary to an uncontrolled hyperthyroid state. Atrial fibrillation is a cardiovascular complication occurring in up to 15% of patients experiencing thyroid storm, and if left untreated this condition could have up to a 25% mortality rate. Thyroid storm with stroke is a rare presentation. This case report details a left middle cerebral artery (MCA) stroke with global aphasia and thyroid storm in a 53-year-old Hispanic male patient. Although uncommon, this combination has been reported in multiple case series. Although it is well documented that dysfunctional thyroid levels promote a hypercoagulable state, available guidelines from multiple entities are unclear on whether anticoagulation therapy is appropriate in this situation. ==== Body 1. Introduction Thyroid storm is a life-threatening condition that is associated with an uncontrolled hyperthyroid state caused by a precipitating event, such as recent surgery, trauma, infection, iodine load, or poor adherence to antithyroid medications [1]. Although tachycardia is the most common cardiovascular manifestation of thyroid storm, atrial fibrillation occurs in up to 15% of patients [2, 3]. Thyroid hormone dysregulation affects the coagulation pathway and promotes settings in which a stroke may occur [4]. There is, however, no clear recommendation for anticoagulation therapy in patients experiencing thyroid storm. This case report describes a 53-year-old Hispanic man who experienced a left middle cerebral artery (MCA) stroke and global aphasia with coexisting thyroid storm. 2. Case Presentation A 53-year-old man with a history of untreated hyperthyroidism (toxic nodule), chronic smoking, and arterial hypertension arrived to the emergency room of our institution complaining of disorientation, restlessness, palpitations, vomiting, abdominal pain, and fever (not quantified) that had begun over 48 hours prior. Worsening symptoms, including right-sided weakness and difficulty understanding verbal language and communication with his spouse in the previous 24 hours, prompted the patient's assistance to our institution. Upon evaluation by the emergency room physician, the patient had a decreased neurological status with poor response to pain and verbal stimuli, for which he was endotracheally intubated to secure his airway. A head computed tomography (CT) scan without intravenous contrast revealed a gross acute left middle cerebral artery (MCA) territory infarction with no associated hemorrhage and minimal compression of the left lateral ventricle (Figures 1 and 2). The patient had a National Institutes of Health stroke scale value of 9/42, so the emergency department consulted the internal medicine department for further management. Thrombolytic therapy was not indicated due to the timing of symptom appearance. Upon evaluation in our department, we determined that the patient was acutely ill with a Glasgow Coma Scale score 11/15 when assessed while off sedation and on mechanical ventilation support with adequate oxygenation. His vital signs showed that he had a remarkable high fever (39.8°C), tachycardia (heart rate: 107 bpm), and elevated blood pressure 180/90 mmHg. A physical examination revealed right hemiplegia with bibasilar crackles and an irregular rhythm. He also exhibited remarkable pedal edema. An electrocardiogram revealed atrial fibrillation with adequate ventricular response. Due to a patient history of untreated hyperthyroidism secondary to problems with medical insurance as well as clinical signs and symptoms, the patient was diagnosed with thyroid storm. The patient had a Burch and Wartofsky score of 70, which is indicative of thyroid storm [5]. Treatment was initiated immediately, using a beta blocker (60 mg of propranolol) to control adrenergic symptoms, a thionamide (200 mg of propylthiouracil) to block hormone synthesis, and a glucocorticoid (100 mg of hydrocortisone) to reduce T4-T3 conversion. Lugol's iodine solution (10 drops of 8 mg iodine per 0.05 mL drop) used to stop thyroid hormone synthesis was initiated 1 hour after the antithyroid drugs. Additionally, along with statin therapy (atorvastatin 80 mg), clopidogrel 75 mg and enoxaparin 40 mg were administered as secondary stroke prevention. Intravenous diuretics were given to treat fluid overload symptoms. Laboratory results revealed no leukocytosis or leukopenia, adequate hemoglobin and hematocrit levels, no electrolytes disturbances, and a negative toxicology screen. Thyroid function tests were abnormal (thyroid stimulating hormone: <0.225 µIU/mL, total thyroxine: 16.50 mcg/dL, and free thyroxine: 5.49 ng/dL) (Table 1). A chest radiograph revealed opacity projecting at the right lower lung field with silhouetting of the diaphragm and increased interstitial lung markings, which were consistent with pulmonary edema. A two-dimensional echocardiogram revealed atrial fibrillation at baseline, mild left ventricular systolic dysfunction, no valvulopathies, and mild left atrium and right atrium dilation. Based on the Japanese Thyroid Association criteria for thyroid storm, this case met the criteria for definitive diagnosis of TS1-grade thyroid storm [5]. Our patient was treated aggressively for thyroid storm upon arrival to our institution. Successful extubation was achieve on day 6 following admission. When the patient's mental status recovered, he confirmed that his fever palpitations and nausea preceded the paralysis, confirming that the events of thyrotoxicosis leaded to the stroke. In addition, his sinus rhythm returned to normal after completed hyperthyroidism treatment and his pedal edema improved. The patient was transferred to a rehabilitation center for inpatient physical therapy on the 8th day of admission. Following physical therapy sessions, he was discharged and given thionamide therapy with methimazole for his hyperthyroidism and was followed up by primary endocrinologist for further management. Based on the clinical progression of the patient's illness, we feel that if early anticoagulation had been started, the cerebrovascular accident with associated global aphasia might have been prevented. 3. Discussion Thyroid storm is a life-threatening condition that occurs due to an accentuated hyperthyroid state. Recent studies report a mortality range from 8 to 25% [6]. Thyroid storm usually presents in patients with a current history of poorly treated thyrotoxicosis. It can also be precipitated by an infection, recent surgery, or recent trauma, among other things. The clinical presentation resembles an accentuated catabolic state. Fever is a very common sign and is often accompanied by sweating. Other manifestations include agitation, diarrhea, nausea, vomiting, abdominal pain, jaundice, seizures, and coma [5, 7]. A link between the cardiovascular system and elevated thyroid function has also been characterized. Sinus tachycardia is the most common electrocardiographic finding, but atrial fibrillation can also occur and is the most common cardiovascular complication, arising in 5–15% of cases [2, 3]. Another important link that was recently discovered is between thyroid hormone and coagulation pathways. Thyrotoxicosis promotes a hypercoagulable state due to a shortened activated partial thromboplastin time, increased fibrinogen levels, and increased factor VIII and factor X activity [4]. These abnormalities predispose a patient to stroke regardless of heart rhythm. Although these links are known and the mechanisms have been documented, there are no specific or clear-cut recommendations regarding anticoagulation therapy in these patients. Guidelines from multiple entities also do not include hyperthyroidism as a risk factor for stroke. The most recent American Thyroid Association (ATA) guidelines for thyrotoxicosis management do not clearly state that anticoagulation therapy should be included only suggesting its use in the event of heart failure [8]. In 2014, the American Heart Association (AHA), American College Cardiology (ACC), and the Heart Rhythm Society (HRS) published guidelines for atrial fibrillation (AF) management. They stated that the evidence associating thyrotoxicosis with AF was not sufficient enough to recommend anticoagulation therapy administration. Anticoagulation should be guided by the CHA2DS2-VASc risk factors, which do not include hyperthyroidism [9]. Although no large randomized controlled trials have assessed this association, many case reports/series have documented it. Yuen et al. reported a case series of 21 subjects with thyrotoxicosis and atrial fibrillation, in which 23% of the patients had developed systemic emboli [10]. In another study, Bar-Sela and associates describe 30 out of 142 thyrotoxic patients with concomitant atrial fibrillation, 12 (40%) of whom experienced an embolic event [11]. In a recent prospective cohort study from Taiwan, 3176 patients with hyperthyroidism were followed for 5 years and compared with subjects without hyperthyroidism. The risk for developing a stroke was 1.4-fold greater in the hyperthyroidism cohort compared with the control group after adjusting for several confounders [12]. In conclusion, although some clinical evidence exists concerning cardioembolic stroke in thyrotoxic patients, no standard of care exists in this situation, likely because the clinical protocol is based on case reports and methodologically flawed small-scale studies. However, the decision for anticoagulation in thyrotoxicosis should be based on the patient's risk factors, as shown in previous studies, and not solely on the presence of thyroid storm. The case presented here highlights an important unresolved issue regarding the administration of anticoagulation therapy in patients with thyrotoxicosis with atrial fibrillation. Acknowledgments The authors wish to thank Editage® for English language editing. The authors are also grateful to Yolianne Lozada Capriles, MD, for invaluable advice and discussion about the paper and Raymond Rivera Vergara for literature review. Competing Interests The authors declare that there are no competing interests regarding the publication of this paper. Figure 1 Head computed tomography image without contrast, showing left middle cerebral artery territory infarction. Figure 2 High density within left middle cerebral artery territory, corresponding to the site of arterial occlusion. Table 1 Thyroid function tests and serum chemistries.   Upon hospital arrival Upon discharge Thyroid stimulating hormone (µIU/mL) <0.225 N/Aa Total T4 (µg/dL) 16.50 12.2 Free T4 (ng/dL) 5.49 2.3 Sodium (mEq/L) 147 143 Potassium (mEq/L) 3.8 3.5 Blood urea nitrogen (mg/dL) 32 29.5 Creatinine (mg/dL) 0.82 0.50 Calcium (mg/dL) 8.5 8.4 Phosphorus (mg/dL) 3.70 3.5 Magnesium (mg/dL) 2.22 2.0 Albumin (g/dL) 2.7 2.5 Carbon dioxide (mEq/L) 25.2 26.7 Prothrombin time (s) 13 N/Aa Partial thromboplastin time (s) 30 N/Aa INR (s) 1.0 N/Aa White blood cell (×103/μL] 8.8 7.5 Hemoglobin (g/dL) 14.7 14.0 Hematocrit (%) 44 42 Platelet count (×109/L) 300 350 aValue not available. ==== Refs 1 Sarlis N. J. Gourgiotis L. Thyroid emergencies Reviews in Endocrine and Metabolic Disorders 2003 4 2 129 136 10.1023/a:1022933918182 2-s2.0-0038374793 12766540 2 Petersen P. Hansen J. M. Stroke in thyrotoxicosis with atrial fibrillation Stroke 1988 19 1 15 18 10.1161/01.str.19.1.15 2-s2.0-0023857463 3336898 3 Petersen A. W. Puig-Carrión G. D. López-Candales A. Should we revisit anticoagulation guidelines during thyroid storm? Boletín de la Asociación Médica de Puerto Rico 2015 107 1 62 66 2-s2.0-84931077789 26035989 4 Franchini M. Montagnana M. Manzato F. Vescovi P. P. Thyroid dysfunction and hemostasis: an issue still unresolved Seminars in Thrombosis and Hemostasis 2009 35 3 288 294 10.1055/s-0029-1222607 2-s2.0-67649229621 19452404 5 Akamizu T. Satoh T. Isozaki O. Diagnostic criteria, clinical features, and incidence of thyroid storm based on nationwide surveys Thyroid 2012 22 7 661 679 10.1089/thy.2011.0334 2-s2.0-84863428154 22690898 6 Angell T. E. Lechner M. G. Nguyen C. T. Salvato V. L. Nicoloff J. T. LoPresti J. S. Clinical features and hospital outcomes in thyroid storm: a retrospective cohort study The Journal of Clinical Endocrinology & Metabolism 2015 100 2 451 459 10.1210/jc.2014-2850 2-s2.0-84922531604 25343237 7 Nordyke R. A. Gilbert F. I. Jr. Harada A. S. M. Graves' disease. Influence of age on clinical findings Archives of Internal Medicine 1988 148 3 626 631 10.1001/archinte.1988.00380030132023 2-s2.0-0023857370 3341864 8 Bahn R. S. Burch H. B. Cooper D. S. Hyperthyroidism and other causes of thyrotoxicosis: management guidelines of the American Thyroid Association and American Association of Clinical Endocrinologists Thyroid 2011 21 6 593 646 10.1089/thy.2010.0417 21510801 9 January C. T. Wann L. S. Alpert J. S. 2014 AHA/ACC/HRS guideline for the management of patients with atrial fibrillation: a report of the American college of Cardiology/American heart association task force on practice guidelines and the heart rhythm society Journal of the American College of Cardiology 2014 64 21 e1 e76 10.1016/j.jacc.2014.03.022 2-s2.0-84908220640 24685669 10 Yuen R. W. Gutteridge D. H. Thompson P. L. Robinson J. S. Embolism in thyrotoxic atrial fibrillation The Medical Journal of Australia 1979 1 13 630 631 2-s2.0-0018805096 492021 11 Bar-Sela S. Ehrenfeld M. Eliakim M. Arterial embolism in thyrotoxicosis with atrial fibrillation Archives of Internal Medicine 1981 141 9 1191 1192 10.1001/archinte.141.9.1191 2-s2.0-0019443501 7259379 12 Sheu J.-J. Kang J.-H. Lin H.-C. Lin H.-C. Hyperthyroidism and risk of ischemic stroke in young adults: a 5-year follow-up study Stroke 2010 41 5 961 966 10.1161/strokeaha.109.577742 2-s2.0-77951765125 20360542

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==== Front Case Rep SurgCase Rep SurgCRISCase Reports in Surgery2090-69002090-6919Hindawi Publishing Corporation 10.1155/2016/3176842Case ReportAdenocarcinoma of the Right Colon in a Patient with Bloom Syndrome http://orcid.org/0000-0001-8088-427XMartinez Carlos Augusto Real 1 2 * http://orcid.org/0000-0002-9845-3117Pinheiro Lilian Vital 1 Rossi Debora Helena 1 http://orcid.org/0000-0001-6820-8164Camargo Michel Gardere 1 Ayrizono Maria de Lourdes Setsuko 1 Leal Raquel Franco 1 Coy Cláudio Saddy Rodrigues 1 1Division of Colorectal Surgery, University of Campinas, Campinas, SP, Brazil2Postgraduate Program in Health Sciences, São Francisco University, Avenida São Francisco de Assis, 218 Jardim São José, Bragança Paulista, 12916-350 São Paulo, SP, Brazil*Carlos Augusto Real Martinez: caomartinez@uol.com.brAcademic Editor: Baran Tokar 2016 15 8 2016 2016 31768426 4 2016 18 7 2016 20 7 2016 Copyright © 2016 Carlos Augusto Real Martinez et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction. Bloom syndrome (BS) is an inherited disorder due to mutation in BLM gene. The diagnosis of BS should be considered in patients with growth retardation of prenatal onset, a photosensitive rash in a butterfly distribution over the cheeks, and an increased risk of cancer at an early age. Clinical manifestations also include short stature, dolichocephaly, prominent ears, micrognathia, malar hypoplasia and a high-pitched voice, immunodeficiency, type II diabetes, and hypogonadism associated with male infertility and female subfertility. The aim of this report is to describe case of patient with BS who developed adenocarcinoma of the cecum, successfully treated by right colectomy. Case Report. A 40-year-old man underwent colonoscopy to investigate the cause of his diarrhea, weight loss, and anemia. The patient knew that he was a carrier of BS diagnosed at young age. The colonoscopy showed an expansive and vegetating mass with 5.5 cm in diameter, located within the ascending colon. Histopathological analysis of tissue fragments collected during colonoscopy confirmed the presence of tubular adenocarcinoma, and he was referred for an oncological right colectomy. The procedure was performed without complications, and the patient was discharged on the fifth postoperative day. Histopathological examination of the surgical specimen confirmed the presence of a grade II tubular adenocarcinoma (stage IIA). The patient is currently well five years after surgery, without clinical or endoscopic signs of relapse in a multidisciplinary approach for the monitoring of comorbidities related to BS. Conclusion. Despite the development of colorectal cancer to be, a possibility rarely described the present case shows the need for early screening for colorectal cancer in all patients affected by BS. ==== Body 1. Introduction Bloom syndrome (BS) is an extremely rare autosomal recessive genetic disorder caused by a mutation in the BLM gene encoding a DNA repair enzyme homology to the RecQ helicases that receives the same name of the gene [1, 2]. The absence of BLM protein activity leads to a DNA repair defect, which causes increased risk of mutations [2, 3]. The main clinical features in the BS include short stature, dolichocephaly (long, narrow head), sun-sensitive malar telangiectatic erythema, and immune deficiency, low birth weight, prenatal and postnatal retardation of the growth development, prominent ears, micrognathia, congenital telangiectatic erythema in face, type II diabetes, male infertility and female subfertility, immunodeficiency, upper respiratory recurrent infections, and an increased risk of cancer at an early age [4, 5]. One of the reasons for the interest in this rare syndrome is the great risk to development of malignancies at various sites, most commonly breast, gastrointestinal tract, and skin [6]. Patients with BS are estimated to develop malignancy at a rate 150–300 times higher than the general population and about 25% of them develop malignancy, at a mean age of 20.7 years [6, 7]. This increased risk of cancer leads to a shortened life expectancy and patients with BS rarely survived after their fifties [2]. Approximately 12% of BS patients can develop colon cancer, and the mean age at diagnosis is 35.4 years [2, 8]. The aim of this report is to present a patient with a BS who developed colon cancer at 40 years of age. 2. Case Report A 34-year-old-man, with a previous history of recurrent chest infections, presented with a six-month history of diarrhea, abdominal pain specially located in the right lower quadrant, asthenia, lack of appetite, and weight loss (5 kg). He was the second child of four children and referred that one of his brother died at the age of 40 years by liver failure devoid hepatic metastasis of colorectal cancer. He reports type II diabetes making use of metformin. During the adolescence he had been hospitalized on four occasions for the treatment of pulmonary infection. The patient reports that he had recently been referred for surgical resection of a skin tumor with 1.5 cm in diameter located on the forehead (Figure 1). The lesion was removed a week after its diagnosis and the histopathological study confirmed the presence of a basal cell carcinoma. He was born in Brazil and their ancestors do not belong to Ashkenazi ethnicity. At admission, physical examination showed a short stature (142 cm), weight of 48 kg, growth retardation, dolichocephaly, triangular face, beaked nose, prominent ears (Figure 2(a)), and hypogonadism. He had a high-pitched voice, small mandible, dystrophic nails, clinodactyly (Figure 2(b)), and palmar transverse crease (Figure 2(c)). He had a telangiectatic erythema in malar region of the face and forehead that worsened after solar radiation and a linear scar in brow related to previous surgical resection of a basal cell carcinoma (Figure 2(d)). He also presents with several café au lait macules in the abdominal skin and gluteal region (Figure 3). Abdominal examination revealed palpable mobile mass in the right lower quadrant. Blood tests revealed hypoalbuminemia at 1.8 g/L, glycemia at 280 mg/dL, and hypochromic microcytic anemia at 8.4 g/dL. Renal and hepatic biochemical tests were normal. The suspect of BS was confirmed by chromosomic analysis showing chromosomal breakages and sister chromatid exchanges. Upper gastrointestinal endoscopy was normal and colonoscopy revealed a polypoid-ulcerated mass located in ascending colon (Figure 4). Biopsies specimens of the mass confirmed a well-differentiated adenocarcinoma. Computed tomography scan of the abdomen and pelvis only showed the cecal mass without hepatic metastasis or regional lymph node enlargement. With a diagnosis of ascending colon adenocarcinoma he was submitted to an open oncological right colectomy. He had uneventful recovery and was discharged on the 5th day. The histopathological examination confirmed the diagnosis of tubular adenocarcinoma without neoplastic involvement in 23 lymph nodes resected (stage IIA). The revision of histopathology slide of the tumor located on forehead confirmed the previous diagnosis of basal cell carcinoma. The patient was instructed to daily use sunscreen on all skin surfaces exposed to solar radiation. He was referred to surveillance program of the digestive tract neoplasms, performing colonoscopy and upper endoscopy every year. At this time the patient is well, with no signs of recurrence of the colon cancer or polyps five years after colectomy. 3. Discussion BS also known as congenital telangiectatic erythema is a rare, autosomal recessive genetic disorder found in humans and experimental models [6, 9, 10]. The disease was first described in New York City, in 1954, by David Bloom, a Polish-born dermatologist, as a congenital telangiectatic erythema resembling lupus erythematosus in dwarfs [9, 11]. BS is caused by mutations in BLM gene located in chromosome 15 (15q26.1) that comprises 4,437 base pairs, which encodes a protein BLM with 1,417 amino acids homology to the RecQ helicases [12]. Thisprotein restores breaks in double-stranded DNA and is the only known RecQL helicase that can unwind asingle-stranded DNA sequence of four consecutive guanines [13]. The absence of BLM activity leads to a DNA defect repair, which causes genomic instability with increased rates of chromosomal breakage, rearrangements, gene mutation, and increase of the risk of cancer development [3]. The most characteristic cytogenetic features in BS is the 15-fold increased rate of sister chromatid exchanges and this increased level is a pathognomic feature of BS [7, 14]. The transmission of BS is autosomal recessive, and a homozygous state is fundamental for clinical manifestation 1. Heterozygous carriers usually are asymptomatic; however a link between heterozygous carrier status and increased risk of colorectal and breast cancer has been established [1, 15, 16]. The syndrome was initially reported in the Ashkenazi Jewish population. The BLM gene mutation is known to be highly prevalent among Ashkenazi population, where the carrier state is estimated to be more than 1 : 110 to 1 : 231 [1, 17–19]. The BS population has been monitored since 1960 via Bloom's Syndrome Registry (BSR) and the data is periodically updated [20]. Actually data collected in 2009 from the BSR showed 265 persons with BS are from 222 families (sibships) [11]. This data also showed that of the 265 cases collected of the BS only 21 (7.9%) patients live in South America and 16 (6.03%) in Brazil [11]. The patient of this report did not belong to the Judaic ethnicity. The hallmark of the clinical presentation of BS is characteristic with marked retardation of the growth development and an early onset photosensitive facial rash that worsened with solar exposition [1]. Data from BSR evaluating the height in 95 patients showed that the means in men was 149 cm (128–164) while in women was 138 cm (115–160) [11]. The patient described in this report had height of 142 cm, staying within the described mean of the BSR data. The malar telangiectatic erythema of patients with BS usually more intensely affects skin exposed to solar irradiation. Similarly to that found in patients with lupus, the erythema of the patients with BS compromises more frequently the face, especially malar region. The erythema may also compromise the skin of the forehead, as occurs in the patient of this report, ears, lips, and neck [7, 9, 16]. These injuries worsened after sun exposure that can form painful superficial skin ulcers. To prevent the worsening of the telangiectatic erythema and to avoid the increased risk of basal cell carcinoma, as occurs in the patient of this report BS patients need to daily use sunscreen. German reviewing the first 100 cases of BS found eight cases of skin cases and only three cases associated with colorectal cancer [6]. Therefore, the simultaneity of colorectal cancer and skin cancer is a not common association. Other skin lesions are described in patients with BS. The presence of café au lait macules mainly located in the trunk is described as found in the patient of this report [7]. Old scars can also be identified at the sites where prevailing telangiectatic erythema resulted from healing of sun exposure ulcers. Patients with BS generally present with a small but proportionate body, associated dolichocephaly, kneel-shaped face, sharp nose, and low-set ears giving those affected characteristic facies a high-pitched voice, features also on the patient in this case [1, 6]. Type II diabetes mellitus and recurrent respiratory infections are described generally at young age [6, 17]. The patient of this report was referred treatment for type II diabetes since age of 23 years and four previous hospital admissions to treat pneumonia. Repetitive lung infections are related to immunodeficiency often found in patients with BS. Men with BS present with genital hypoplasia and oligospermia. There are reports of adult female patients with BS with a history of normal childbirth, but, generally, the subfertility is a rule [21]. The most significant implication of a diagnosis of BS from the patient's point of view is the high risk of developing cancer [1, 6]. Patients with BS have an increased risk of developing a variety of malignancies, notably leukemia, lymphomas, and carcinomas of various sites, that is high from the twenties onwards, which is the case of our patient. The course of life in these patients is made remarkable by early onset single or multiple neoplasias involving any organ system, which is the main cause of mortality. Many patients will have more than one cancer and most will die by the third decade of life [6, 22]. Newer data indicate that BLM mutations contribute to breast cancer susceptibility, and heterozygous carriers of a BLM mutation have a higher probability of developing colorectal cancer [15, 16, 23]. Among the most serious complications that patients with BS develop lifelong, malignant neoplasms of different locations are the most frequent, accounting for 46%, followed by type II diabetes (15.8%) and lung infections (2.6%) [11]. BSR data showed that of 265 patients followed 122 (46.3%) developed cancer [11]. Of those patients 69 (56.5%) were men and 53 (43.4%) women. Epithelial tumors are the most frequent type (52.5%), followed by lymphoid (24.9%) and hematopoietic neoplasm (11.3%) [11]. Different types of neoplasms are described in about 12% of patients. Acute leukemia and lymphomas seem to predominate in the first two decades of life and carcinomas, mainly located on gastrointestinal tract, more common after the second decade [24]. Tumors that are rare in the general population, such osteosarcoma, medulloblastoma, and Wilms' tumor are more common in patients with BS [9]. About 12% of BS patients develop colorectal cancer, usually from the second decade of life and the median age at diagnosis is 35.4 years, similar to that occurring in patient of this report [8, 24]. German in 1997 analyzing the first 100 cases of cancer on the BSR found 13 cases of colorectal cancer, seven in proximal colon (cecum, ascending, hepatic flexure, and transverse) and six in distal colon (descending, sigmoid, and rectum) [6]. Since then, we can only find three other published cases, two with fatal course [2, 8, 9]. Although the occurrence of skin cancer in patients with BS is well documented in the worldwide literature, the finding of primary skin carcinoma occurring concomitant with colorectal cancer, to the best of our knowledge, only two cases, has been reported [2, 6]. The best surgical approach of colorectal cancer in carriers BS is still controversial. As these patients have an increased risk for developing a second malignance at young age, some authors recommend conducting a proctocolectomy [2]. If the patient refuses the total colectomy partial colectomy and annual follow-up colonoscopy for colon and rectum remaining can be offered. In patient of this report, we discuss the advantages and disadvantages of the two surgical options, and the patient opted for the realization of the right colectomy with annual follow-up. So far, this strategy seemed to us valid because the annual colonoscopic and upper endoscopy monitoring conducted in the last five years did not find any suspicious lesion. This is the first BS case associated with colon and basal cell skin cancer that has been reported in Brazilian population. It is important to recognize this rare condition in order to diagnose the disease at an early stage and tailor the treatment regimen to try to avoid the development of malignances. A screening program should therefore be offered, particularly for upper gastrointestinal, breast, and colon cancer. Competing Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Figure 1 Basal cell carcinoma in forehead. Figure 2 (a) Dolichocephaly, beaked nose, and prominent ears. (b) Clinodactyly and dystrophic nails. (c) Palmar transverse crease. (d) Telangiectatic erythema and linear scar in forehead due to previous surgical excision of basal cell carcinoma. Figure 3 Café au lait macules in the anterior abdominal skin. Figure 4 Polypoid-ulcerated mass in ascending colon. ==== Refs 1 German J. Bloom's syndrome Dermatologic Clinics 1995 13 1 7 18 7712653 2 Thomas E. R. A. Shanley S. Walker L. Eeles R. Surveillance and treatment of malignancy in bloom syndrome Clinical Oncology 2008 20 5 375 379 10.1016/j.clon.2008.01.007 2-s2.0-43049178222 18359209 3 Hickson I. D. RecQ helicases: caretakers of the genome Nature Reviews Cancer 2003 3 3 169 178 10.1038/nrc1012 2-s2.0-0037364415 12612652 4 Morimoto W. Kaneko H. Isogai K. Kasahara K. Kondo N. Expression of BLM (the causative gene for Bloom syndrome) and screening of Bloom syndrome International Journal of Molecular Medicine 2002 10 1 95 99 2-s2.0-0036651403 12060858 5 Tikoo S. Sengupta S. Time to bloom Genome Integrity 2010 1 1, article 14 10.1186/2041-9414-1-14 2-s2.0-79953794452 6 German J. Bloom's syndrome. XX. The first 100 cancers Cancer Genetics and Cytogenetics 1997 93 1 100 106 10.1016/S0165-4608(96)00336-6 2-s2.0-0031052108 9062585 7 Arora H. Chacon A. H. Choudhary S. Bloom syndrome International Journal of Dermatology 2014 53 7 798 802 10.1111/ijd.12408 2-s2.0-84902795737 24602044 8 Balc S. Aktas D. Mucinous carcinoma of the colon in a 16-year-old Turkish boy with Bloom syndrome: cytogenetic, histopathologic, TP53 gene and protein expression studies Cancer Genetics and Cytogenetics 1999 111 1 45 48 10.1016/s0165-4608(98)00175-7 2-s2.0-0032946216 10326590 9 Draznin M. Robles D. T. Nguyen V. Berg D. An unusual case of bloom syndrome presenting with basal cell carcinoma Dermatologic Surgery 2009 35 1 131 134 10.1111/j.1524-4725.2008.34393.x 2-s2.0-58149291611 19076197 10 Warren M. Chung Y.-J. Howat W. J. Irradiated Blm-deficient mice are a highly tumor prone model for analysis of a broad spectrum of hematologic malignancies Leukemia Research 2010 34 2 210 220 10.1016/j.leukres.2009.06.007 2-s2.0-75049086036 19709744 11 Weill Cornell Medical College Bloom's syndrome registry, 2009, http://weill.cornell.edu/bsr/clinical_description/ 12 Ellis N. A. Groden J. Ye T.-Z. The Bloom's syndrome gene product is homologous to RecQ helicases Cell 1995 83 4 655 666 10.1016/0092-8674(95)90105-1 2-s2.0-0028785586 7585968 13 Karow J. K. Wu L. Hickson I. D. RecQ family helicases: roles in cancer and aging Current Opinion in Genetics and Development 2000 10 1 32 38 10.1016/s0959-437x(99)00039-8 2-s2.0-0034001767 10679384 14 Diaz A. Vogiatzi M. G. Sanz M. M. German J. Evaluation of short stature, carbohydrate metabolism and other endocrinopathies in Bloom's syndrome Hormone Research 2006 66 3 111 117 10.1007/s11207-006-0137-y 2-s2.0-33747034158 16763388 15 Gruber S. B. Ellis N. A. Rennert G. BLM heterozygosity and the risk of colorectal cancer Science 2002 297 5589 p. 2013 10.1126/science.1074399 2-s2.0-18544386262 12242432 16 Prokofyeva D. Bogdanova N. Dubrowinskaja N. Nonsense mutation p.Q548X in BLM, the gene mutated in Bloom's syndrome, is associated with breast cancer in Slavic populations Breast Cancer Research and Treatment 2013 137 2 533 539 10.1007/s10549-012-2357-1 2-s2.0-84880630260 23225144 17 Inamadar A. C. Palit A. Bloom syndrome in an Indian child Pediatric Dermatology 2005 22 2 147 150 10.1111/j.1525-1470.2005.22212.x 2-s2.0-16344369357 15804305 18 Peleg L. Pesso R. Goldman B. Bloom syndrome and Fanconi's anemia: rate and Ethnic origin of mutation carriers in Israel Israel Medical Association Journal 2002 4 2 95 97 2-s2.0-0036188591 11876000 19 Oddoux C. Clayton C. M. Nelson H. R. Ostrer H. Prevalence of Bloom syndrome heterozygotes among Ashkenazi Jews American Journal of Human Genetics 1999 64 4 1241 1243 10.1086/302312 2-s2.0-0033361768 10090915 20 German J. Passarge E. Bloom's syndrome. XII. Report from the registry for 1987 Clinical Genetics 1989 35 1 57 69 2-s2.0-0024591985 2647324 21 Mulcahy M. T. French M. Pregnancy in Bloom's syndrome Clinical Genetics 1981 19 3 156 158 2-s2.0-0019450446 7273457 22 Bachrati C. Z. Hickson I. D. RecQ helicases: suppressors of tumorigenesis and premature aging Biochemical Journal 2003 374, part 3 577 606 10.1042/bj20030491 2-s2.0-0141567744 12803543 23 Li L. Eng C. Desnick R. J. German J. Ellis N. A. Carrier frequency of the Bloom syndrome blm(Ash) mutation in the Ashkenazi Jewish population Molecular Genetics and Metabolism 1998 64 4 286 290 10.1006/mgme.1998.2733 2-s2.0-0031678636 9758720 24 Benjazia E. Turki H. Atig A. Bloom syndrome complicated by colonic cancer in a young Tunisian woman Clinics and Research in Hepatology and Gastroenterology 2011 35 10 682 684 10.1016/j.clinre.2011.06.001 2-s2.0-80054860138 21778134

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==== Front Biomed Res IntBiomed Res IntBMRIBioMed Research International2314-61332314-6141Hindawi Publishing Corporation 10.1155/2016/1495710Research ArticleExperimental Forelimb Allotransplantation in Canine Model http://orcid.org/0000-0001-5542-8555Hong Sa-Hyeok * http://orcid.org/0000-0003-4372-6955Eun Seok-Chan * Department of Plastic and Reconstructive Surgery, Seoul National University College of Medicine, Seoul National University Bundang Hospital, Seoul 463-706, Republic of Korea*Sa-Hyeok Hong: shuing@naver.com and *Seok-Chan Eun: seokchan.eun@gmail.comAcademic Editor: Gerald Brandacher 2016 15 8 2016 2016 149571012 5 2016 3 7 2016 22 7 2016 Copyright © 2016 S.-H. Hong and S.-C. Eun.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.As reconstructive transplantation is gaining popularity as a viable alternative for upper limb amputees, it is becoming increasingly important for plastic surgeons to renew surgical skills and knowledge of this area. Forelimb allotransplantation research has been performed previously in rodent and swine models. However, preclinical canine forelimb allotransplantation studies are lacking in the literature. The purpose of this paper is to provide an overview of the surgical skills necessary to successfully perform forelimb transplantation in canines as a means to prepare for clinical application. A total of 18 transplantation operations on canines were performed. The recipient limb was shortened at the one-third proximal forearm level. The operation was performed in the following order: bones (two reconstructive plates), muscles and tendons (separately sutured), nerves (median, ulnar, and radial nerve), arteries (two), and veins (two). The total mean time of transplantation was 5 hours ± 30 minutes. All of the animals that received transplantation were treated with FK-506 (tacrolimus, 2 mg/kg) for 7 days after surgery. Most allografts survived with perfect viability without vascular problems during the early postoperative period. The canine forelimb allotransplantation model is well qualified to be a suitable training model for standard transplantation and future research work. Ministry of Science, ICT and Future Planning2012R1A1A2021731 ==== Body 1. Introduction Vascularized composite allotransplantation (VCA) is becoming a widely accepted alternative to upper limb amputation. Upper limb transplantation is also a prime example of VCA, comprising the subcutaneous, neurovascular, and mesenchymal tissues, such as the bone, cartilage, muscle, fascia, and skin [1]. In practice, they are faced with various medical, administrative, social, ethical, and regulatory challenges. Although many issues still require resolution, reconstructive surgeons should be prepared and consider VCA as the final option in reconstructive surgery. The outcomes of proximal forearm transplantation are hopeful [2, 3]. Before clinical application, however, the transplantation procedure requires thorough validation in animal models; thus, it is necessary to find a suitable animal model to further study correlative problems [4–6]. An ideal experimental model for upper limb transplantation should consider the following: feasible animal size, anatomic variance, vessel pedicle consistency, reasonable cost, operation time, ease of animal care, and so forth. Limb allotransplantation has been performed formerly in rodent [7, 8] and swine [9–11] models. There has previously been one study on limb allotransplantation using canines hind limbs [12]; however, canine forelimb allotransplantation has never been discussed in the literature. Developing an upper limb allograft model in canines is more appealing than other large animals, simply because the anatomy of canine forelimb—including upper extremity nerve and vascular anatomy—most closely resembles that of humans (aside from nonhuman primates). As such, using canines to develop an animal model for upper limb allotransplantation seemed most logical. The purpose of this paper is to provide an overview of surgical techniques involved in canine forelimb transplantation to be prepared for clinical application. 2. Materials and Methods 2.1. Experimental Animals Two-year-old Beagles, weighing 12–15 kg, were used in this study. We closely followed the Guide for the Care and Use of Laboratory Animals, National Research Council. To ensure that transplants were exchanged between unrelated animals, their pedigrees were checked for at least two generations. Use of all animals in these experiments followed the guidelines for humane treatments of animals. A total of 18 transplantation operations were performed. All surgical procedures were performed under sterile conditions. Anesthesia was induced with ketamine and maintained with enflurane inhalation via tracheal intubation. During the procedure, animals were kept warm with a light source and a heating pad. The skin was cleansed with povidone-iodine (10%) solution and an antibiotic (potassium penicillin, 100,000 IU/kg, administered intramuscularly) was prophylactically administered before surgery. Lactated Ringer's solution was administered as a fluid supplement during and after the operation. 2.2. Preparation of the Donor and Recipient Two teams performed the surgery simultaneously on both the donor and the recipient, attempting to shorten the operation time. On the donor's arm, a circumferential incision was made. The flexor and extensor muscle groups were elevated off of the ulna and the radius. The radius and ulna were cut using an electrical saw at the midradius level (Figure 1). The radial and ulnar arteries were arranged at the midforearm level for reanastomosis. The median nerve, ulnar nerve, and radial nerve were all prepared with as much length as possible. Under tourniquet control, anterior and posterior skin flaps were elevated on the recipient's arms, and all neurovascular structures and muscles were identified and prepared. The medially and laterally originating muscles were dissected free from the ulna and radius. Then, the radius and ulna were cut using an electrical saw at the midradius level. The recipient's limb was shortened at proximal 1/3 level of the forearm. 2.3. Transplantation of the Vascularized Composite Allograft The sequence of transplantation then followed a standard replantation sequence with vascular repair that occurred immediately after bone fixation (osseous fixation, arterial repair, and venous repair, followed by nerve and muscular repair). Bone alignment ultimately dictates the pronation-supination range of motion in the proximal both-bone fractures, replantation and transplantation. Both plates were initially placed on the recipient's bones and then temporarily fixed to the donor. After bone fixation, the muscle groups were approximated using two reconstruction titanium plates (Figure 2). Attachment of the three nerves (median, ulnar, and radial nerves) and anastomosis of the two arteries and two veins were performed using nylon 9-0 under microscopy (Figure 3). The final skin suture marked the end of the transplantation surgery. 2.4. Treatments and Observation The recipients received intravenous lactated Ringer's solution to compensate for perioperative fluid loss. They were immobilized and meticulously monitored throughout the postoperative course, with particular attention to adequate oral and fluid supplementation to facilitate recovery. A postoperative forelimb X-ray was taken at the intensive care unit (ICU) (Figure 4). All of the animals that received transplantation were treated with FK-506 (tacrolimus, 2 mg/kg) for 7 days after surgery. The transplanted upper limb flaps were evaluated every three hours for any clinical signs of rejection. Erythema, edema, loss of hair, desquamation, ulceration, and progressive shrinkage of the flap were considered to be clinical signs of rejection. 3. Results The mean time required to accomplish the upper limb transplantation procedure was 5 h and 30 min, and the mean time of warm ischemia was 45 min. A successful outcome was achieved by using the distal part of the radial and ulnar arteries as the recipient's arteries and the radial and ulnar veins as the recipient's veins. The mean diameter of the artery and vein was 4.0 mm (range: 3.8–4.2) and 6.2 mm (range: 5.8–6.4), respectively. On posttransplantation day 1, the animals returned to their normal routine of eating and drinking, but with immobilization. Mild soft tissue edema and hematomas under the flaps were observed, but no cases required drainage. One recipient showed acute rejection signs on posttransplantation day 5 and was sacrificed by intravenous administration of 100 mg/kg sodium pentobarbital on day 7. The remaining 17 recipients showed no signs of rejection until postoperative week 2 (Figure 5). The histologic outcomes were well correlated with the macroscopic appearance. The skin component showed signs of inflammation earlier and with greater intensity than other components, and it revealed epidermal edema and necrosis with massive neutrophilic infiltration in the dermis (grade III rejection reaction); the muscle and cartilage showed grades 2 and 1 rejection responses, respectively (Figure 6). 4. Discussion Although there are only a limited number of reported forearm transplantation cases, they suggest favorable results [3]. Nonetheless, a highly scrutinized and well-established procedure from animal models is required before attempting upper limb allotransplantation on humans. For this purpose, we need to find the most suitable animal model to further our understanding of any potential correlative problems. In previous reports, rodent models have been attempted; however, they were too small and too different for establishing such models as the standard experiment for surgical skill training. In 1971, Lance et al. reported a canine hind limb model; however, nine dogs out of twenty lost the transplanted limb within the first postoperative week due to technical failures [12]. In our case, there was no transplanted limb loss due to technical failure. There are many advantages to a canine forelimb transplantation model [13]. First, since canines are relatively larger animals with larger vessel size, compared with rodents, the operation, harvesting and insetting of the flaps, as well as microanastomosis, can be performed with relative ease. Second, the anatomical structure of canine forearms is comparable to that of humans. Third, the preoperative settings and anesthesia procedures are simple. Fourth, compared with other larger animals, the associated cost is lower, thus more cost-effective. Finally, the operation does not require advanced skills in microsurgery. As a result, a canine model can be implemented in many laboratories with minimal VCA experience. Arterial and venous anastomosis is the most critical part of flap survival and overall success of the surgery. It is debatable whether to perform the flap inset or microanastomosis first [14]. Although the significance of early revascularization of the harvested flap cannot be underestimated, it is quite challenging to fix the flap safely without having the bone fixated using the titanium plates. Furthermore, the time required to fixate the bone was quite short in this model. Therefore, we performed microanastomosis after bone fixation. We have also performed nerve repair in this model, and, as aforementioned, due to the anatomical similarities between canines and humans, it is possible to practice surgical techniques before clinical application. The animals were treated with low dose tacrolimus (2 mg/kg) only for 7 days, and the observation time was 2 weeks. Although this may be insufficient for the evaluation of long-term postoperative outcome, it is sufficient for evaluating the outcome of technical aspects of the operation, with respect to surgical techniques. If the operation was not successful, then there should be early failure in the transplanted limb within 2 weeks. Only 1 out of 18 recipients was sacrificed, and therefore we can safely assume that the outcome of the surgical procedures was excellent in this study. The primary purpose of this canine model was to simulate upper limb transplantation in animals best suited to prepare for clinical applications in humans. It was mainly intended to provide an overview of surgical simulation and techniques, and, as such, it was not focused on long-term postoperative function monitoring and failed to incorporate long-term immunosuppressive strategies to increase survival of the flaps. The effect of immunosuppressants in composite tissue transplantation is well documented in the literature [15–17]. A limitation to using a canine model is the lack of immune markers; there are a limited number of tools suitable for immunologic research—such as tolerance study—in a canine model compared with rodent models, such as mice or rats [13, 18, 19]. It is expected that further experimental investigations and future innovations might address the limitations posed by this model. In sum, we have provided an overview of the canine forelimb allotransplantation technique and investigated its technical feasibility and applicability. The canine forelimb allotransplantation model is convenient, cost-effective, and reproducible. Moreover, it is suitable for training reconstructive surgeons who are not familiar with VCA procedures and lack advanced microsurgery skills. However, it is worthy to note that this model provides only a simplified version of flap harvesting and transferring procedures. The flap harvesting, transfer, and microscopic skills could be corrected in a better attainable method. Due to its feasible size and convenience for operation, canine forelimb allotransplantation model is a reasonable, reproducible, and representative upper limb transplantation study model. Acknowledgments This work was supported by the National Research Foundation, 2012R1A1A2021731, of the Ministry of Science, ICT and Future Planning, Korean Government. Disclosure This work followed the Guide for the Care and Use of Laboratory Animals, National Research Council. Competing Interests There are no competing interests to declare. Figure 1 The radius and ulna are cut using an electrical saw at the midradius level. Figure 2 Bone fixation using two reconstruction titanium plates. Figure 3 The attachment of three nerves (median, ulnar, and radial nerve) and the anastomosis of two arteries and two veins. Figure 4 Postoperative course performed with immobilization at an intensive care unit (ICU). Figure 5 Posttransplantation appearance of forelimb allograft recipient at posttransplantation day 14. Figure 6 Biopsy samples taken from skin showed necrotic epidermis and edema typical of grade III rejection reaction (a), whereas biopsy specimens from the muscle showed grade II rejection of multifocal infiltration with myocyte necrosis (b) and cartilage showed grade I rejection of only focal erosion (c). ==== Refs 1 Azari K. Brandacher G. Vascularized composite allotransplantation Current Opinion in Organ Transplantation 2013 18 6 631 632 10.1097/MOT.0000000000000028 2-s2.0-84888290270 24220046 2 Lanzetta M. Petruzzo P. Dubernard J. M. Second report (1998–2006) of the international registry of hand and composite tissue transplantation Transplant Immunology 2007 18 1 1 6 10.1016/j.trim.2007.03.002 2-s2.0-34250640725 17584595 3 Schneeberger S. Ninkovic M. Gabl M. First forearm transplantation: outcome at 3 years American Journal of Transplantation 2007 7 7 1753 1762 10.1111/j.1600-6143.2007.01837.x 2-s2.0-34250221084 17511764 4 Cendales L. C. Kirk A. D. Moresi J. M. Ruiz P. Kleiner D. E. Composite tissue allotransplantation: classification of clinical acute skin rejection Transplantation 2006 81 3 418 422 10.1097/01.tp.0000185304.49987.d8 2-s2.0-32844466214 16477229 5 Eun S.-C. Baek R.-M. Park C.-G. Prolongation of the rat composite tissue allograft survival by the combination of tolerogenic immature dendritic cells and short-term treatment with FK506 Transplantation Proceedings 2013 45 5 1792 1796 10.1016/j.transproceed.2013.01.021 2-s2.0-84879232819 23769045 6 Lee W. P. A. Butler P. E. M. Randolph M. A. Yaremchuk M. J. Donor modification leads to prolonged survival of limb allografts Plastic and Reconstructive Surgery 2001 108 5 1235 1241 10.1097/00006534-200110000-00021 2-s2.0-0034790071 11604625 7 Larsen M. Friedrich P. F. Bishop A. T. A modified vascularized whole knee joint allotransplantation model in the rat Microsurgery 2010 30 7 557 564 10.1002/micr.20800 2-s2.0-78349297867 20842706 8 Muramatsu K. Doi K. Kawai S. Limb allotransplantation in rats: combined immunosuppression by FK-506 and 15-deoxyspergualin Journal of Hand Surgery 1999 24 3 586 593 10.1053/jhsu.1999.0586 2-s2.0-0032956047 10357540 9 Siemionow M. Klimczak A. Advances in the development of experimental composite tissue transplantation models Transplant International 2010 23 1 2 13 10.1111/j.1432-2277.2009.00948.x 2-s2.0-70949085304 19719468 10 Solla F. Pan H. Watrelot D. Leveneur O. Dubernard J.-M. Gazarian A. Composite tissue allotransplantation in newborns: a swine model Journal of Surgical Research 2013 179 1 e235 e243 10.1016/j.jss.2012.01.030 2-s2.0-84870668108 22482769 11 Kuo Y.-R. Chen C.-C. Shih H.-S. Prolongation of composite tissue allotransplant survival by treatment with bone marrow mesenchymal stem cells is correlated with T-cell regulation in a swine hind-limb model Plastic and Reconstructive Surgery 2011 127 2 569 579 10.1097/PRS.0b013e318200a92c 2-s2.0-79951618610 21285761 12 Lance E. M. Inglis A. E. Figarola F. Veith F. J. Transplantation of the canine hind limb. Surgical technique and methods of immunosuppression for allotransplantation. A preliminary report The Journal of Bone & Joint Surgery—American Volume 1971 53 6 1137 1149 2-s2.0-0015120988 4937289 13 Mathes D. W. Noland M. Graves S. Schlenker R. Miwongtum T. Storb R. A preclinical canine model for composite tissue transplantation Journal of Reconstructive Microsurgery 2010 26 3 201 207 10.1055/s-0030-1247717 2-s2.0-77749315077 20108180 14 Lee K. M. Eun S.-C. Experimental canine facial transplantation Transplantation Proceedings 2014 46 4 1208 1211 10.1016/j.transproceed.2013.11.045 2-s2.0-84900302981 24815162 15 Demir Y. Ozmen S. Klimczak A. Mukherjee A. L. Siemionow M. Tolerance induction in composite facial allograft transplantation in the rat model Plastic and Reconstructive Surgery 2004 114 7 1790 1801 10.1097/01.PRS.0000142414.92308.AB 2-s2.0-14944339200 15577350 16 Hettiaratchy S. Butler P. E. M. Tolerance induction in composite facial allograft transplantation in the rat model Plastic and Reconstructive Surgery 2006 117 3 1043 1045 10.1097/01.prs.0000201204.57587.e8 2-s2.0-33645747993 16525312 17 Radu C. A. Kiefer J. Gebhard M. M. Local administration of Mitomycin-C-Treated peripheral blood mononuclear cells (PBMCs) prolongs allograft survival in vascularized composite allotransplantation Microsurgery 2016 36 5 417 425 10.1002/micr.30003 18 Kim S. K. Aziz S. Oyer P. Hentz V. R. Use of cyclosporin A in allotransplantation of rat limbs Annals of Plastic Surgery 1984 12 3 249 255 10.1097/00000637-198403000-00006 2-s2.0-0021368856 6609665 19 Inceoglu S. Siemionow M. Chick L. Craven C. M. Lister G. D. The effect of combined immunosuppression with systemic low-dose cyclosporin and topical fluocinolone acetonide on the survival of rat hind- limb allografts Annals of Plastic Surgery 1994 33 1 57 65 10.1097/00000637-199407000-00011 2-s2.0-0028243123 7944198

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==== Front Adv VirolAdv VirolAVAdvances in Virology1687-86391687-8647Hindawi Publishing Corporation 10.1155/2016/9058403Research ArticleIsolation and Metagenomic Identification of Avian Leukosis Virus Associated with Mortality in Broiler Chicken http://orcid.org/0000-0003-1526-4028Bande Faruku 1 2 http://orcid.org/0000-0001-8357-4701Arshad Siti Suri 1 * http://orcid.org/0000-0001-7379-0507Omar Abdul Rahman 1 3 * 1Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor Darul Ehsan, Malaysia2Department of Veterinary Services, Ministry of Animal Health and Fisheries Development, PMB 2109, Usman Faruk Secretariat, Sokoto 840221, Sokoto State, Nigeria3Laboratory of Vaccine and Immunotherapeutic, Institute of Bioscience, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor Darul Ehsan, Malaysia*Siti Suri Arshad: suri@upm.edu.my and *Abdul Rahman Omar: aro@upm.edu.myAcademic Editor: Finn S. Pedersen 2016 15 8 2016 2016 905840319 4 2016 29 6 2016 Copyright © 2016 Faruku Bande et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Avian leukosis virus (ALV) belongs to the family Retroviridae and causes considerable economic losses to the poultry industry. Following an outbreak associated with high mortality in a broiler flock in northern part of Malaysia, kidney tissues from affected chickens were submitted for virus isolation and identification in chicken embryonated egg and MDCK cells. Evidence of virus growth was indicated by haemorrhage and embryo mortality in egg culture. While viral growth in cell culture was evidenced by the development of cytopathic effects. The isolated virus was purified by sucrose gradient and identified using negative staining transmission electron microscopy. Further confirmation was achieved through next-generation sequencing and nucleotide sequence homology search. Analysis of the viral sequences using the NCBI BLAST tool revealed 99-100% sequence homology with exogenous ALV viral envelope protein. Phylogenetic analysis based on partial envelope sequences showed the Malaysian isolate clustered with Taiwanese and Japanese ALV strains, which were closer to ALV subgroup J, ALV subgroup E, and recombinant A/E isolates. Based on these findings, ALV was concluded to be associated with the present outbreak. It was recommended that further studies should be conducted on the molecular epidemiology and pathogenicity of the identified virus isolate. Universiti Putra Malaysia ==== Body 1. Introduction Avian leukosis virus (ALV) is an economically important retrovirus affecting meat and egg-type chicken. The virus belongs to an Alpharetrovirus genus in the family Retroviridae. Based on the envelope glycoprotein (gp85) it was possible to classify exogenous ALV into different subgroups, namely, A, B, C, D, E, and J. Particularly, the viral envelope glycoprotein is responsible for attachment and receptor specificity as well as the production of neutralizing antibodies [1, 2]. Of the viral subgroups so far identified, subgroups A, B, and J are considered most prevalent and more economically important [3]. Subgroup J was first isolated in meat-type chicken in the United Kingdom in 1989 but currently is causing devastation to poultry industry worldwide [4]. Apart from its immunosuppressive effect, ALV is commonly associated with lymphoid leukosis, myelocytic myeloid leukosis, and renal as well as other forms of tumours [5]. This study reports some virological and molecular sequencing approaches used to identify the viral cause of mortality in a broiler flock in Malaysia. 2. Materials and Methods Following a suspected outbreak in a broiler chicken farm with capacity of about 10,000 birds, tissue samples, including trachea, kidney, and proventriculus, were submitted to the virology laboratory, Universiti Putra Malaysia, for virus isolation and identification. The mortality rate was reported to reach about 10% in the 27-day-old flock (n = 6000) and more than 20% in the 30-day-old flock (n = 4000). The chickens flock health programme consisted of vaccination against NDV, IBD, and IBV. Postmortem findings revealed mild petechial haemorrhage on the proventriculus and markedly swollen kidney (figures not shown). Samples were processed and inoculated in a 9-day-old embryonated chicken egg as well as MDCK cells and then monitored for the evidences of virus growth. Identification of the virus was carried out by electron microscopy using the negative staining methods while confirmation was done by next-generation sequencing using the MiSeq illumina sequencing platform. A ScriptSeq v2 RNA-Seq library preparation kit was employed and used according to manufacturer's guidelines (epicenter, USA). 3. Results and Discussion Avian leukosis, specifically of subgroup J origins, has been reported previously in Malaysia [6]. This study investigated a viral cause of mortality observed in a broiler farm in northern Malaysia in 2013. Due to the involvement of kidney, initial tentative diagnosis focused on avian infectious bronchitis [7]; however inoculation of kidney suspension in chicken's embryonated egg revealed evidence of virus growth characterized by severe haemorrhage and embryo mortality (Figure 1). Embryo mortality was observed to increase as the passaging of virus increases to passage 3. Specifically, at passage 2, there was about 70%–100% mortality of embryos starting from day 2 to day 3 postinoculation (pi). Avian leukosis virus has been reported to cause severe haemorrhage and death of the embryo within 4 to 5 days following infection of egg embryo [5]. Similarly, inoculation of MDCK cells with kidney derived suspension showed evidence of virus growth characterized by the presence of cytopathic effects including cell ballooning, granulation, rounding, giant cells formations, and cell detachment starting from day 3 pi (Figure 2). Although most ALVs produce no visible morphological changes in cell culture, infection of chicken embryo fibroblast cells with cytopathic ALVs strains such as the RAV-2 was reported to cause CPE and detachment of cells 3 days after infection [8]. Other studies confirmed the tropism and growth of ALVs in chicken embryo fibroblast cells based on the presence of CPE [9, 10]. It is also generally known that MDCK cells express receptors for some avian viruses such as avian influenza [11]. Virus identification carried out using negative staining electron microscopy revealed a spherically rounded, virus-like particle with characteristic projections resembling spike structures (Figure 3). Subsequent analysis by comparison revealed that the virus morphology observed in this study was similar to that reported by Tsang et al. [12]. Despite evidence of virus growth in egg embryo and cell culture, attempts to detect common viral diseases using specific primers against NDV, AI, and IBV were unsuccessful [7]; hence, a next-generation RNA sequencing approach was employed. Following sequencing, analysis of the generated sequences (Genbank accession number: KX061539) using NCBI BLAST revealed high sequence homology with exogenous ALV viral envelope protein region. Furthermore, phylogenetic characterization using the entire envelope sequences (1230 bp) revealed close identity with exogenous ALV-TW-3593 from Taiwan (100%), ALV-Hkd-026 strains from Japan (99%), and a putative endogeneous ALV sequence designated ALVE-B11 (99%). In terms of virus subgroup, the identified local ALV sequences demonstrated closeness to ALV J, ALV E, and recombinant ALV A/E as well as an endogenous ALV of chicken (Figure 4). In terms of protein sequences, which comprise the receptor binding SU domain (gp85) and transmembrane protein TM domain (gp37), Malaysian ALV isolate also phylogenetically clustered with Taiwanese isolate (TW3593) and Chinese isolate (WB11008e) as well as Japanese isolate (Hkd026) (figure not shown). In view of the nature of retroviruses, it is usual that mutation and recombination in ALV may influence virus diversity and tissue tropism as well as the severity of infection they cause [13]. These factors might probably account for the reported high mortality rate observed in the farm as well as during passaging of virus in embryo. 4. Conclusion Based on the findings observed in this report, it was concluded that the present outbreak was associated with ALV. The study further demonstrates that ALV continues to circulate among poultry in Malaysia though it might be underdiagnosed. There is the need for flock monitoring against ALV and the possibility of including ALV infection as one of the differentials in related outbreaks. It is recommended to further study the molecular epidemiology and pathogenesis of the present local ALV strain in Malaysia. Acknowledgments The authors would like to acknowledge support from the Laboratory of Vaccine and Immunotherapeutics, Institute of Bio Sciences, UPM. Similar appreciation goes to Dr. Dilan Satharasinghe for technical assistance in sequencing. Competing Interests The authors declare no competing interests. Authors' Contributions All authors have contributed equally. Figure 1 Inoculation of viral suspension in 9-day old embryonated chicken egg showing evidence of severe haemorrhage in infected embryo (a) as compared with negative control (b) after 24 hours pi. Figure 2 Infection of MDCK cells with virus suspension revealed evidences of virus growth as demonstrated by CPE which is characterized by cell ballooning, granulation, formation of giant cells (arrow), rounding, and cell detachment as infection progresses 3 dpi (a); 5 dpi (b) and 7 dpi (c); (d) control uninfected cells. Mag ×20 and 100 μm. Figure 3 Negative staining electron microscopy showing the morphological appearance of the isolated virus. Note: presence of spike projections surrounding the viral particle (arrow) whose diameter ranges from 80 to 120 nm. Mag ×100 μm. Figure 4 Neighbor-joining phylogenetic analysis using partial nucleotide sequences of the ALV envelope gp85 glycoprotein gene. Malaysian ALV isolate is presented in bold red ink. The confidence level of the inferred tree was determined using 1000 bootstrap. Evolutionary analysis was conducted in MEGA6 software. ==== Refs 1 Coffin J. M. Levy J. A. Structure and classification of retroviruses The Retroviridae 1992 1 New York, NY, USA Plenum Press 19 49 2 Payne L. N. Howes K. Gillespie A. M. Smith L. M. Host range of Rous sarcoma virus pseudotype RSV(HPRS-103) in 12 avian species: support for a new avian retrovirus envelope subgroup, designated Journal of General Virology 1992 73 11 2995 2997 10.1099/0022-1317-73-11-2995 2-s2.0-0026485603 1331300 3 Dai M. Feng M. Liu D. Cao W. Liao M. Development and application of SYBR Green i real-time PCR assay for the separate detection of subgroup J Avian leukosis virus and multiplex detection of avian leukosis virus subgroups A and B Virology Journal 2015 12 1, article 52 10.1186/s12985-015-0291-7 2-s2.0-84928011202 4 Payne L. N. Brown S. R. Bumstead N. Howes K. Frazier J. A. Thouless M. E. A novel subgroup of exogenous avian leukosis virus in chickens Journal of General Virology 1991 72 4 801 807 10.1099/0022-1317-72-4-801 2-s2.0-0025892322 1849967 5 Payne L. N. Fadly A. M. Leukosis/Sarcoma Group 1997 Ames, Iowa, USA Iowa State University Press 6 Thapa B. R. Omar A. R. Arshad S. S. Hair-Bejo M. Detection of avian leukosis virus subgroup J in chicken flocks from Malaysia and their molecular characterization Avian Pathology 2004 33 3 359 363 10.1080/0307945042000220435 2-s2.0-3242732333 15223566 7 Bande F. Arshad S. S. Omar A. R. Bejo M. H. Abubakar M. S. Abba Y. Pathogenesis and diagnostic approaches of avian infectious bronchitis Advances in Virology 2016 2016 11 4621659 10.1155/2016/4621659 8 Weller S. K. Temin H. M. Cell killing by avian leukosis viruses Journal of Virology 1981 39 3 713 721 2-s2.0-0019481404 6270346 9 Fadly A. M. Isolation and identification of avian leukosis viruses: A review Avian Pathology 2000 29 6 529 535 10.1080/03079450020016760 2-s2.0-0034489429 19184848 10 Lupiani B. Hunt H. Silva R. Fadly A. Identification and characterization of recombinant subgroup J avian leukosis viruses (ALV) expressing subgroup A ALV envelope Virology 2000 276 1 37 43 10.1006/viro.2000.0539 2-s2.0-0034633802 11021992 11 Lee C.-W. Jung K. Jadhao S. J. Suarez D. L. Evaluation of chicken-origin (DF-1) and quail-origin (QT-6) fibroblast cell lines for replication of avian influenza viruses Journal of Virological Methods 2008 153 1 22 28 10.1016/j.jviromet.2008.06.019 2-s2.0-49349085137 18638503 12 Tsang S. X. Switzer W. M. Shanmugam V. Evidence of avian leukosis virus subgroup E and endogenous avian virus in measles and mumps vaccines derived from chicken cells: investigation of transmission to vaccine recipients Journal of Virology 1999 73 7 5843 5851 2-s2.0-0032994354 10364336 13 Svarovskaia E. S. Cheslock S. R. Zhang W.-H. Hu W.-S. Pathak V. K. Retroviral mutation rates and reverse transcriptase fidelity Frontiers in Bioscience 2003 8 d117 d134 10.2741/957 2-s2.0-0347117654 12456349

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==== Front J Immunol ResJ Immunol ResJIRJournal of Immunology Research2314-88612314-7156Hindawi Publishing Corporation 10.1155/2016/5457010Research Article In Vitro Effects of Some Botanicals with Anti-Inflammatory and Antitoxic Activity Guidetti Gianandrea 1 http://orcid.org/0000-0001-8122-9170Di Cerbo Alessandro 2 * Giovazzino Angela 3 Rubino Valentina 3 Palatucci Anna Teresa 4 Centenaro Sara 1 Fraccaroli Elena 1 Cortese Laura 5 Bonomo Maria Grazia 6 Ruggiero Giuseppina 3 Canello Sergio 1 Terrazzano Giuseppe 3 6 1Division of Research and Development, SANYpet SpA, 35023 Bagnoli di Sopra, Italy2School of Specialization in Clinical Biochemistry, “G. d'Annunzio” University, 66100 Chieti, Italy3Department of Translational Medical Sciences, University of Naples Federico II, 80131 Naples, Italy4Ph.D. School of Science, University of Basilicata, 85100 Potenza, Italy5Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, 80100 Naples, Italy6Department of Science, University of Basilicata, 85100 Potenza, Italy*Alessandro Di Cerbo: alessandro811@hotmail.itAcademic Editor: Giuseppe A. Sautto 2016 15 8 2016 2016 545701031 5 2016 19 7 2016 21 7 2016 Copyright © 2016 Gianandrea Guidetti et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Several extrinsic factors, like drugs and chemicals, can foster autoimmunity. Tetracyclines, in particular oxytetracycline (OTC), appear to correlate with the emergence of immune-mediated diseases. Accumulation of OTC, the elective drug for gastrointestinal and respiratory infectious disease treatment in broiler chickens, was reported in chicken edible tissues and could represent a potential risk for pets and humans that could assume this antibiotic as residue in meat or in meat-derived byproducts. We investigated the in vitro anti-inflammatory properties of a pool of thirteen botanicals as a part of a nutraceutical diet, with proven immunomodulatory activity. In addition, we evaluated the effect of such botanicals in contrasting the in vitro proinflammatory toxicity of OTC. Our results showed a significant reduction in interferon- (INF-) γ production by human and canine lymphocytes in presence of botanicals (⁎ p < 0.05). Increased INF-γ production, dependent on 24-hour OTC-incubation of T lymphocytes, was significantly reduced by the coincubation with Haematococcus pluvialis, with Glycine max, and with the mix of all botanicals (⁎ p < 0.05). In conclusion, the use of these botanicals was shown to be able to contrast OTC-toxicity and could represent a new approach for the development of functional foods useful to enhance the standard pharmacological treatment in infections as well as in preventing or reducing the emergence of inflammatory diseases. ==== Body 1. Introduction The immune system has the fundamental role of not only protecting and defending the organism against infections but also controlling homeostasis and health maintenance against infections, autoimmune diseases, and tumor onset [1]. Depending on the pathogen or on antigen, two different immune responses can occur: the humoral and the cellular responses [2]. Moreover, the immune system can be classified into two fundamental phases: the innate and acquired (or adaptive) responses [3]. Innate immunity is present in vertebrates and in nonvertebrates, represents the first-line defence in the species and is based on cells (i.e., macrophages, polymorphonuclear cells, and natural killer lymphocytes) and on some mechanisms, mediated by soluble substances (i.e., complement proteins, antibodies, natural compounds, etc.) that defend the plants and animals from infections [4]. Conversely, adaptive immunity is present only in vertebrates and is a host defence related to several specific cellular mechanisms that specifically recognize the antigens and are fundamentally expressed by B and T lymphocytes, plasma cells, and antibodies [5]. The CD4+ T helper (TH) lymphocytes represent key cells in the polarization of inflammatory/noninflammatory immune response: TH1 and TH2 are the most common [6]. The TH1 response is characterized by the a secretion of INF-γ, which optimizes the bactericidal macrophages capability, induces the production of opsonizing and complement-fixing antibodies, and fosters the establishment of an optimal CTL response. The TH2 response is characterized by interleukin- (IL-) 4, IL-5, IL-10, and IL-13 release, which results in the activation of B cells to make neutralizing noncytolytic antibodies, leading to humoral immunity [6]. Exacerbation and endurance of TH1 response have been associated with the emergence of inflammatory diseases [6] and autoimmune disorders [7]. In particular, INF-γ appears to play a pivotal role in inducing autoimmune responses [8–16]. Several extrinsic factors, like drugs and chemicals, can foster the development of autoimmunity [17–22]. In this regard, the use of tetracyclines appears to correlate with the emergence of autoimmune diseases [23–29]. Concerning this, OTC represents the main drug used to control gastrointestinal and respiratory diseases in broiler chickens. Its accumulation was demonstrated in chicken edible tissues [30] and could represent a potential risk also for pets and humans that could assume this antibiotic as a residue in meat or in meat-derived byproducts. Recently, we published two papers evidencing the in vitro toxicity of bone meal-derived OTC from intensive poultry farming, in terms of apoptosis induction [31], as well as the proinflammatory cytokines, that is, INF-γ, release from peripheral blood mononuclear cells (PBMCs) cultures [32]. Moreover, we evidenced that the presence of significant concentrations of OTC in gym trained human subjects was linked to the presence of food intolerances [33]. Therefore, we hypothesized a possible modulatory activity exerted by a pool of botanicals derived from medical plants, which are successfully used in several commercially available nutraceutical diets. Intriguingly, many botanicals could have the capability to modulate the immune system [34]. In this regard, it is well known that the immunomodulatory activity of acemannan, a mucopolysaccharide extracted from Aloe vera, related to modulation of nitric oxide release that modulate classes I and II MHC cell surface antigens involved in antigen presentation [35, 36]. The same immunomodulating activity was observed for fermented Carica papaya able to increase Treg cells, reduce INF-γ +CD4+ T cells, and possibly alter the growth of several cancer cell lines [37–39]. As to Maitake mushroom (Grifola frondosa), many reports have shown its ability to downregulate cytokine secretion, such as Tumor Necrosis Factor- (TNF-) α and INF-γ, as well as to inhibit adhesion molecule expression and cell-mediated immunity enhancement [40–44]. Downregulation of overexpressed cytokines in different inflammatory and immune-related inflammatory conditions was also reported for curcumin extracted from turmeric (Curcuma longa) [45–47]. Antiproliferative and chemopreventive effects are known to be also exerted by other curcuminoids, for example, demethoxycurcumin, bisdemethoxycurcumin, and alpha-turmerone [48, 49]. Cytokine downregulation is also performed by Glycine max (soybean) isoflavones that interfere with leukocyte endothelial adhesion ability [50–54]. In more detail, isoflavones, that is, genistein, can suppress dendritic cell function and cell-mediated immunity. It is noteworthy that some botanical principles, which have been investigated in this study such as astaxanthin (from Haematococcus pluvialis), resveratrol (from Polygonum cuspidatum), and Cucumis melo, are characterized by antioxidant and anti-inflammatory properties as well as modulation properties towards CD8+ T-cell proliferation [55, 56]. Anti-inflammatory but also oxidative stress preventing activity has been also ascribed to Cucumis melo extract due to its high activity on superoxide dismutase [57, 58]. Recently, we published a paper evidencing the role for a nutraceutical diet in regulating the immune response in canine Leishmaniosis along with standard pharmacological treatment [59]. In particular, the presence of Ascophyllum nodosum, Cucumis melo, Carica papaya, Aloe vera, Haematococcus pluvialis, Curcuma longa, Camellia sinensis, Punica granatum, Piper nigrum, Polygonum cuspidatum, Echinacea purpurea, Grifola frondosa, and Glycine max in the diet correlated with a significant decrease in TH1 response, in terms of INF-γ production. Such evidence highlighted the anti-inflammatory effects of these specific botanicals. In addition, we suggested the anti-inflammatory effects of several botanicals added to specific diets in relieving inflammatory conditions in chronic pathologies affecting dogs [59–63]. Based on these premises, the aim of our study was to investigate the potential anti-inflammatory properties of those 13 botanicals having immune-modulatory effect as supplemented diet regulating the immune response in Leishmaniosis [59]. In particular, we tested the Ascophyllum nodosum, Cucumis melo, Carica papaya, Aloe vera, Haematococcus pluvialis, Curcuma longa, Camellia sinensis, Punica granatum, Piper nigrum, Polygonum cuspidatum, Echinacea purpurea, Grifola frondosa, and Glycine max and their ability to counteract the proinflammatory toxicity of OTC in vitro. 2. Materials and Methods 2.1. Culture Medium and Botanicals To evaluate the cellular production of cytokines, human and canine PBMCs were incubated overnight with an ad hoc culture medium. Briefly, the first step was the solubilization of 1 gr of powder of each plant-derived substance in an appropriate chemical vehicle depending on solubility degree. In particular, Ascophyllum nodosum (pure powder of Ascophyllum nodosum seaweed, laminarin content min. 2.3%, and fucoidans content min. 11.4% [64]), Aloe vera (Aloe vera gel 200 : 1 powder, aloin content min. 1% [65]), Cucumis melo (lyophilized extract of melon, superoxide dismutase min. 1 UI/mg [57]), Polygonum cuspidatum (powder obtained from dried Polygonum cuspidatum roots, resveratrol content min. 8% [66]), Camellia sinensis (standardized decaffeinated green tea leaves powder, catechins content min. 75% [67]), Carica papaya (Papaya fermented granular, rich in papain [68]), Glycine max (Soy powder, 40% isoflavones [69]), and Grifola frondosa (maitake carpophore dry extract, polysaccharides content min. 20.0% [70]) were solubilized in 10 mL of PBS 1x, with the exception of Glycine max that was added to 30 mL of PBS 1x to gain the full solubilization. Haematococcus pluvialis (standardized beadlets of Haematococcus pluvialis extract, astaxanthin content min. 2.5% [71]) was solubilized in 5 mL of dimethyl sulfoxide and 5 mL of PBS 1x. Echinacea purpurea (Echinacea purpurea dried extract, polyphenols content min 4% [72]), Piper nigrum (black pepper powder, piperine content min. 95% [73]), Curcuma longa (turmeric dried extract, curcuminoids content min. 95% [74]), and Punica granatum (standardized powdered extract from pomegranate, ellagic acid content min. 20% [75]) were solubilized in 4 mL of ethanol and 6 mL of water. The solubilized botanicals were added to RPMI 1640 culture medium (Sigma-Aldrich, Milan, Italy) to obtain the ad hoc medium in the proportion of 1 : 10 (vehicle-solubilized substance : RPMI 1640) to preserve the good quality of cellular condition in the culture. The cytokine cell production was evaluated in presence of the ad hoc medium containing the solubilized individual substance or a mixture containing all the solubilized botanicals. The vehicles employed for the solubilization were used as specific controls in the same proportion of ad hoc medium (1 : 10, vehicle : RPMI 1640). The mixture was composed by all ad hoc medium from the botanicals in a variable percentage according to that contained in the commercial canine food, previously used as immunomodulating diet able to reduce INF-γ production [59]. Briefly, the mixture contained 66.3% of Ascophyllum nodosum, 3.1% of Aloe vera, 6.1% of Cucumis melo, 1.5% of Polygonum cuspidatum, 1.5% of Camellia sinensis, 3.1% of Carica papaya, 4.6% of Glycine max, 6.3% of Grifola frondosa, 1.1% of Haematococcus pluvialis, 3.1% of Echinacea purpurea, 0.6% of Piper nigrum, 2.3% of Curcuma longa, and 1.5% of Punica granatum. The obtained mixture was added to RPMI 1640 culture medium to obtain the ad hoc medium in the proportion of 1 : 10 (vehicle/mixture : RPMI 1640) to preserve the good quality of cellular condition in the culture. Ascophyllum nodosum, Aloe vera, Cucumis melo, Polygonum cuspidatum, Camellia sinensis, and Haematococcus pluvialis were purchased from Italfeed S.r.l, Milano (Italy). Carica papaya, Glycine max, Echinacea purpurea, Punica granatum, Piper nigrum, and Curcuma longa were purchased from Nutraceutica S.r.l, Monterenzio, Bologna (Italy) while Grifola frondosa was purchased from A.C.E.F. S.p.a., Fiorenzuola D'Arda, Piacenza (Italy). All the botanicals are in form of powder and are free from genetically modified organisms (Reg. 1829/2003-1830/2003 EC), gluten, bovine transmissible spongiform encephalopathy, and food allergens (DIR 2003/89/EC and 2006/142/EC). 2.2. Human and Canine Donors and Cell Preparation The human blood collection from 10 healthy donor volunteers (5 males and 5 females, 20–30 years old) was performed at the Haemotrasfusional Center of University of Naples “Federico II,” according to standard procedures and used within the 3 hours from the collection. Peripheral blood was collected from ten healthy dogs (5 males and 5 females, 5–9 years old and ranging between 15 and 35 kg in weight). All dogs were enrolled with the owner consent in the Department of Veterinary Medicine and Animal Productions, University of Naples “Federico II.” Human or canine PBMCs were isolated by centrifugation on Lymphoprep (Nycomed Pharma) gradients, as previously described [59, 76]. Obtained PBMCs were considered as mixed population of T and non-T lymphocytes. 2.3. Monoclonal Antibodies, Detection of Intracellular Cytokine Production, and Flow Cytometry For the immune-fluorescent staining a panel of fluorescent-labelled monoclonal antibodies (mAbs) was used to evaluate the human CD3, CD8, INF-γ, and IL-4, as well as a panel of isotype-matched mAb controls (Becton Dickinson Pharmingen, San Jose, California). In addition, we used several fluorescent-labelled mAbs against canine CD3, CD4, CD8, CD45, INF-γ, and IL-4 molecules and isotype-matched controls (Serotec Ltd., London, UK). To analyze the production of INF-γ and IL-4 cytokines, 2 × 106/mL purified PBMCs were incubated overnight (10–12 hours) in the ad hoc medium of each botanical or of mixture (see Section 2.1). In particular, to obtain the cytokine production, PBMCs were always cultured in presence of 500 ng/mL of phorbol-12-myristate-13-acetate (PMA) and 1 μg/mL of Ionomycin (Sigma-Aldrich), as described in [77]. To avoid extracellular cytokine export, the cultures were performed in the presence of 5 μg/mL of Brefeldin-A (Sigma-Aldrich), as described in [77]. To test the ability of botanicals in contrasting the toxic role of OTC, we used the commercial preparation of the drug (Oxytetracycline 20%®, TreI, Reggio Emilia, Italy). 1 μg of OTC [31] was added to cell culture and incubated for overnight (10–12 hours) as previously described [32]. In addition, Haematococcus pluvialis or Glycine max or the mixture ad hoc medium was used in the coincubation of cells with OTC and all along the overnight (10–12 hours) culture. At the end of overnight (10–12 hours) incubation, the above incubated cells were fixed and permeabilized by using a commercial cytokine staining kit following the manufacturer's instructions (Caltag Laboratories, Burlingame, CA, USA). Briefly, the cell fixing and permeabilization procedure were of 20 min at 4°C each. At the end of procedure, PBMCs were washed twice by centrifugation (800 ×g) in RMPI 1640 culture medium. PBMCs were stimulated overnight with PMA and Ionomycin, cultured in a medium containing the botanicals solubilization buffer (vehicle), and used as control points. The proportion of vehicle and RPMI 1640 was the same of ad hoc medium (1 : 10 ratio). The intracellular cytokine production was evaluated by using the triple staining technique and analyzed by flow cytometry (FACSCalibur platform) and CellQuest Software (Becton Dickinson Pharmingen, San Jose, California). The analyzed cells were always gated (R1 in dot plot of Figures 1(a) and 2(a)) on forward scatter (FSC) and side scatter (SSC) FACS parameters (cell size and cell complexity, resp.) to reasonably select the region of viable lymphocytes in order to avoid any interference due to the possible presence of death cells. 2.4. Statistical Analysis Data are presented as the means ± standard error of the mean (SEM) and were firstly checked for normality using the D'Agostino-Pearson normality test. The Kruskal-Wallis followed by Dunn's multiple comparisons analysis was performed. A ∗ p < 0.05 was considered significant. Statistics was performed by GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA). 3. Results and Discussion 3.1. The Anti-Inflammatory Effect of Botanicals as Significant Reduction of INF-γ Production in Human T and Non-T Lymphocytes We focused on INF-γ production, as the main proinflammatory cytokine able to foster the TH1 and non-T cell immune responses involved in several etiopathogenic mechanisms at the basis of inflammatory-mediated disease [6]. As shown in Figure 1, the overnight incubation (10–12 hours) with each botanical as well as with a mix of all botanicals induced a significant decrease in INF-γ production in the TH lymphocytes (CD3+ CD8− cells gated as R1 in the dot plot graphs of Figures 1(b) and 1(c) and reported as mean ± SEM of 10 experiments in Figure 1(d)) and in non-T cells, mainly represented by NK lymphocytes (CD3− CD8− cells gated as R2 in the dot plot graphs of Figure 1(c)). In particular, the individual incubation with Ascophyllum nodosum, Cucumis melo, Carica papaya, Haematococcus pluvialis, Curcuma longa, Camellia sinensis, Punica granatum, Piper nigrum, Polygonum cuspidatum, Echinacea purpurea, Grifola frondosa, and Glycine max was able to reduce INF-γ production. Intriguingly, despite the obtained slightly decrease in cytokine production, the Aloe vera incubation did not induce a significant reduction from the statistical point of view (Figure 1(d)). In this regard, we cannot rule out that a larger number of experiments (more than the 10 performed in this study and summarized in Figure 1(d)) or a higher concentration of substance could confirm the reduction in lymphocyte INF-γ production by the incubation with this botanical. The basal IL-4 production was undetectable or only slightly detectable in T and non-T lymphocytes, as expected in PBMCs from healthy human donors after exposure to PMA and Ionomycin [77] and was not modulated after the overnight incubation with the botanicals (Figure 1(c)). Each specific vehicle, used to solubilize the botanicals, was used as control and the obtained value was substracted from each experimental point to obtain the correction following the formula “the value obtained from cell culture in presence of botanicals – the value obtained from cell culture in presence of the vehicle alone = corrected experimental point value.” It is of note that even if the used vehicles appeared to not induce significant cell death in the culture, the flow cytometry analysis was always performed by gating on viable cells to avoid any possible interference dependent on death cells (see Figure 1(a) and Section 2.3). Moreover, the ad hoc medium from botanicals did not exert effect in absence of PMA and Ionomycin stimulation (data not shown). 3.2. The Anti-Inflammatory Effect of Botanicals as Significant Reduction of INF-γ Production in Canine CD4+ T Lymphocytes The individual incubation with Ascophyllum nodosum, Cucumis melo, Aloe vera, Haematococcus pluvialis, Curcuma longa, Camellia sinensis, Punica granatum, Polygonum cuspidatum, Echinacea purpurea, Grifola frondosa, and Glycine max was able to significantly decrease the INF-γ production in the CD4+ lymphocytes (dot plot graphs in Figure 2(c), summarized in Figure 2(d)). In contrast, the incubation with Carica papaya or with Piper nigrum seemed not to induce a statistically significant reduction (Figure 2(c)). Also, in this case, as referred to in human experiments, we cannot rule out that a larger number of experiments (more than the 10 performed in this study, summarized in Figure 2(d)) or a higher concentration of the substances could confirm the reduction in lymphocyte INF-γ production by the incubation with these two botanicals. IL-4 production was undetectable in T lymphocytes, as expected in PBMCs from healthy dogs after exposure to PMA and Ionomycin [59], and was not modulated after the overnight incubation with the botanicals (data not shown). The specific vehicles, employed to solubilize the substances, were used as controls and the resulting values were substracted from experimental points, as described (see Section 3.1). Flow cytometry analysis was always performed by gating on viable cells to avoid any possible interference dependent on death cells (see Figure 2(a) and Section 2.3). 3.3. The Anti-Inflammatory Effect of Botanicals as Significantly Contrasting Effect on INF-γ Production Dependent on OTC Exposure of Human T Lymphocytes Notably, the individual incubation with Haematococcus pluvialis or with Glycine max was able to contrast the previously demonstrated proinflammatory effect of OTC in human T lymphocytes [32]. Indeed, the increased INF-γ production, dependent on 24-hour OTC-incubation of T lymphocytes, was strongly reduced by the coincubation with Haematococcus pluvialis or Glycine max (Figures 3(a) and 2(b), resp.). Note that the individual incubation with the botanicals, other than Haematococcus pluvialis and Glycine max, was unable to contrast OTC-toxicity (data not shown), while the mixture of all substances exerted a significant effect. Nevertheless, as referred to in previous sections, we cannot rule out that a larger number of experiments or a higher concentration of each substance could confirm the anti-OTC effect also for the other tested botanicals. The specific vehicles, used to solubilize the substances, were considered as controls and the resulting values were substracted from experimental points, as described (see Section 3.1). 4. Conclusions This study was inspired by two recently published in vivo observations in which we suggested a potential anti-inflammatory effect of some nutraceutical diets, containing the studied botanicals, in infectious and inflammatory diseases [59–61]. In particular, we observed that a diet enriched by Ascophyllum nodosum, Cucumis melo, Carica papaya, Aloe vera, Haematococcus pluvialis, Curcuma longa, Camellia sinensis, Punica granatum, Piper nigrum, Polygonum cuspidatum, Echinacea purpurea, Grifola frondosa, and Glycine max was able to reduce proinflammatory T cell responses in canine Leishmaniosis [59] and the clinical feature of ear inflammation in chronic otitis in dogs [60]. Here, we observed the in vitro effect of Ascophyllum nodosum, Cucumis melo, Haematococcus pluvialis, Curcuma longa, Camellia sinensis, Punica granatum, Polygonum cuspidatum, Echinacea purpurea, Grifola frondosa, and Glycine max in reducing in vitro proinflammatory cytokine production by human and canine PBMCs. These botanicals appeared to exert a potential anti-inflammatory effect that was evident in the reduction of INF-γ production in human T and non-T cells and in canine T lymphocytes. Conversely, Aloe vera, Carica papaya, and Piper nigrum appeared to be ineffective in reducing this cytokine production. These results seem to be contradictory with the data observed in dogs [59], where the diet containing all these botanicals, including Aloe vera, Carica papaya, and Piper nigrum, exerted a therapeutic effect by reducing the inflammatory aspects of Leishmaniosis. Such apparent contradiction may be explained by the different sensitivity between in vitro and in vivo, as well as by the fact that in vivo botanicals are probably synergized in the combined administration as in the diet. In this regard, this latter consideration fits with in vitro effect obtained by the mixed incubation with all substances that induced the INF-γ decrease. Moreover, as stated in Section 3, we cannot rule out that a larger number of experiments or a higher concentration of substances could confirm the reduction in lymphocyte INF-γ production also by Aloe vera, Carica papaya, and Piper nigrum. Taken together, our observation highlighted the relevance for the use of botanicals to modulate the inflammatory responses in both dogs and humans. Indeed, exacerbation and the persistence of TH1 response frequently result in the emergence of inflammatory diseases [6] and autoimmunity disorders [7] and the increase of INF-γ production is associated with autoimmunity in humans [8–16]. In addition, some of the botanicals used in this study were previously suggested as antioxidants and immune-modulating substances to reach the physiological status in several models of disease in human [55, 78] and animals [59, 60, 79–83]. Moreover, this study was also inspired by our recent paper, which evidenced the in vitro toxicity of OTC in terms of inflammatory response increase by human lymphocytes [32]. In this regard, here we evaluated the potential ability of Ascophyllum nodosum, Cucumis melo, Carica papaya, Aloe vera, Haematococcus pluvialis, Curcuma longa, Camellia sinensis, Punica granatum, Piper nigrum, Polygonum cuspidatum, Echinacea purpurea, Grifola frondosa, and Glycine max to contrast the OTC-toxicity exerted in vitro in human lymphocytes. Our data suggested that the incubation with the mixture of these botanicals clearly reduced the OTC-induced INF-γ production in T cells. It is of relevance that the individual incubation with Haematococcus pluvialis or with Glycine max significantly reduced this cytokine production. Such evidence may shed new light on the misunderstood scenario resulting from the increasing emergence of inflammatory diseases in humans, dogs, and cats [84–89]. Moreover, it has been suggested that tetracycline, in particular OTC, could take part in this scenario and could represent harmful compounds for human health and animals fed meat derived from intensive livestock [25–30, 33, 90]. In conclusion, this study could open an interesting approach regarding the use of anti-inflammatory and antioxidant botanicals in immune-mediated pathologies and in infectious diseases as well as to counteract the effect of several putative toxic substances present in food, such as the OTC, which can cause inflammatory disorders and diseases. Ethical Approval This study has been reviewed by Ethical Animal Care and Use Committee of the University of Naples Federico II and received formal Institutional approval (Protocol no. 2015/0071388) in accordance with local and national law, regulations, and guidelines (Circular no. 14 of September 25, 1996, and Italian civil code article 1175). Consent Informed consent from human donors was obtained in accordance with the Declaration of Helsinki, as approved within the study protocol by the Institutional Review Board at the Federico II University of Naples. Competing Interests The authors declare that there is no conflict of interests regarding the publication of this paper. This research was performed in collaboration with some scientists from the Division of Research and Development of SANYpet S.p.A. (as indicated in the authors' affiliations) according to scientific and ethical principles of the scientific community. Authors' Contributions Gianandrea Guidetti, Alessandro Di Cerbo, Angela Giovazzino, and Valentina Rubino contributed equally to this work. Figure 1 The effects of botanicals on cytokine production by human PBMCs. (a) shows the gating on viable lymphocytes (R1 in dot plot graph) based on FSC and SSC parameters (see Section 2); (b) represents the gating on TH lymphocytes (CD3+ CD8− as R2 in the dot plot graph) and on non-T cells (CD3− CD8− cells as R3 in the dot plot graph); and (c) shows the INF-γ and IL-4 production in human TH lymphocytes and non-T cells incubated with ad hoc medium derived from botanicals or from mixture (see Section 2). Cytokine production was evaluated as percentage of INF-γ (y-axis) and IL-4 (x-axis) producing cells. The percentage of INF-γ (upper left quadrant inside the dot plots) and IL-4 (low right quadrant inside the dot plots) producing CD4 T (R2) and non-T (R3) cells are reported. The different cell incubations with ad hoc medium derived from botanicals or from mixture (see Section 2) are indicated on the top of each graph. (d) reports the statistic representation of 10 experiments on human CD4+ T Lymphocytes evaluated as percentage of INF-γ producing cells, ∗ p < 0.05. The different cell-incubations with ad hoc medium derived from botanicals or from mixture (see Section 2) are indicated on the top of each column. The abbreviation “ctr” in (c) and (d) indicates the basal cytokine production by PMBCs stimulated by PMA and Ionomycin and in presence of the ad hoc medium based on the same solubilizing-vehicle but free from the botanicals (see Section 2); specifically, ctr 1 (Ascophyllum n., Carica p., Aloe v., Cucumis m., Glycine m., and Grifola f.), ctr 2 (Echinacea p., Piper n.), ctr 3 (Haematococcus p.), and ctr 4 (the mixture of all the botanicals). Figure 2 The effects of botanicals on INF-γ production by canine PBMCs. (a) shows the gating on viable lymphocytes (R1 in dot plot graph) based on FSC and SSC parameters (see Section 2). (b) represents the gating on CD4+ T lymphocytes (CD3+ CD8− as R2 in the dot plot graph). (c) reports the results from one representative experiment showing the percentage (the number in upper quadrant) of INF-γ producing canine CD4+ T lymphocytes gated on R2 (y-axis); x-axis indicates the SSC parameter (see Section 2). The different coincubations of cells with ad hoc medium or mixture (see Section 2) are indicated on the top. (d) shows the statistic representation the INF-γ production by canine CD4+ T Lymphocytes evaluated as percentage of INF-γ producing cells in 10 representative experiments, ∗ p < 0.05. The abbreviation “ctr” in (c) and (d) indicates the basal INF-γ production by PMBCs stimulated by PMA and Ionomycin and in presence of the ad hoc medium based on the same solubilizing-vehicle but free from the botanicals (see Section 2): specifically, ctr 1 (Ascophyllum n., Carica p., Aloe v., Cucumis v., Glycine m., and Grifola f.), ctr 2 (Echinacea p., Piper n.), and ctr 3 (Haematococcus p.). Figure 3 Statistic representation of the INF-γ production in human CD4+ T Lymphocytes after the OTC exposure and the contrasting effects after botanicals challenge in 10 representative experiments. (a) Haematococcus p.; (b) Glycine m.; and (c) the mixture of all the botanicals. Cytokine production was evaluated as percentage of INF-γ producing T CD4+ cells. All the incubations (basal, OTC alone, and OTC + botanical) were performed in the ad hoc medium based on the vehicle used to solubilize the botanical, so that the abbreviations “ctr” indicate the basal INF-γ production by PMBCs stimulated by PMA and Ionomycin and in presence of the ad hoc medium based on the same solubilizing-vehicle but free from the botanicals (see Section 2). ∗ p < 0.05. ==== Refs 1 Delves P. J. Roitt I. M. The immune system. First of two parts The New England Journal of Medicine 2000 343 1 37 49 10.1056/nejm200007063430107 2-s2.0-0342656166 10882768 2 Medzhitov R. Recognition of microorganisms and activation of the immune response Nature 2007 449 7164 819 826 10.1038/nature06246 2-s2.0-35349016235 17943118 3 Hoebe K. Janssen E. Beutler B. The interface between innate and adaptive immunity Nature Immunology 2004 5 10 971 974 10.1038/ni1004-971 2-s2.0-5444234216 15454919 4 Janeway C. A. Jr. Medzhitov R. 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==== Front Adv MedAdv MedAMEDAdvances in Medicine2356-67522314-758XHindawi Publishing Corporation 10.1155/2016/3415046Research ArticleThe Protective Effects of Alpha-Lipoic Acid and Coenzyme Q10 Combination on Ovarian Ischemia-Reperfusion Injury: An Experimental Study http://orcid.org/0000-0002-2750-7317Tuncer Ahmet Ali 1 * Bozkurt Mehmet Fatih 2 Koken Tulay 3 Dogan Nurhan 4 Pektaş Mine Kanat 5 Baskin Embleton Didem 1 1Department of Pediatric Surgery, Afyon Kocatepe University Hospital, 03000 Afyonkarahisar, Turkey2Department of Pathology, Faculty of Veterinary Medicine, Afyon Kocatepe University, 03000 Afyonkarahisar, Turkey3Department of Biochemistry, Afyon Kocatepe University Medical Faculty, 03000 Afyonkarahisar, Turkey4Department of Biostatistics, Afyon Kocatepe University Medical Faculty, 03000 Afyonkarahisar, Turkey5Department of Obstetrics & Gynecology, Afyon Kocatepe University Hospital, 03000 Afyonkarahisar, Turkey*Ahmet Ali Tuncer: drtaali@yahoo.comAcademic Editor: João Quevedo 2016 15 8 2016 2016 341504620 4 2016 25 6 2016 20 7 2016 Copyright © 2016 Ahmet Ali Tuncer et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objective. This study aims to evaluate whether alpha-lipoic acid and/or coenzyme Q10 can protect the prepubertal ovarian tissue from ischemia-reperfusion injury in an experimental rat model of ovarian torsion. Materials and Methods. Forty-two female preadolescent Wistar-Albino rats were divided into 6 equal groups randomly. The sham group had laparotomy without torsion; the other groups had torsion/detorsion procedure. After undergoing torsion, group 2 received saline, group 3 received olive oil, group 4 received alpha-lipoic acid, group 5 received coenzyme Q10, and group 6 received both alpha-lipoic acid and coenzyme Q10 orally. The oxidant-antioxidant statuses of these groups were compared using biochemical measurement of oxidized/reduced glutathione, glutathione peroxidase and malondialdehyde, pathological evaluation of damage and apoptosis within the ovarian tissue, and immunohistochemical assessment of nitric oxide synthase. Results. The left ovaries of the alpha-lipoic acid + coenzyme Q10 group had significantly lower apoptosis scores and significantly higher nitric oxide synthase content than the left ovaries of the control groups. The alpha-lipoic acid + coenzyme Q10 group had significantly higher glutathione peroxidase levels and serum malondialdehyde concentrations than the sham group. Conclusions. The combination of alpha-lipoic acid and coenzyme Q10 has beneficial effects on oxidative stress induced by ischemia-reperfusion injury related to ovarian torsion. Afyon Kocatepe University12.TUS.03 ==== Body 1. Introduction Ovarian torsion is defined as the total or partial rotation of the ovary, the fallopian tube, or both, around its vascular axis. This clinical situation is an emergency of pediatric surgery and gynecology as it causes abdominal pain which requires surgery. Therefore, detorsion of the ovary is surgically performed to maintain its proper circulation [1, 2]. Ovarian torsion and detorsion (T/D) induce ischemia-reperfusion injury which leads to the occurrence of morphological, histological, and biochemical alterations within the ovarian tissue. This is due to a pathological process, which is named ischemia-reperfusion injury. As the ischemic tissue receives an excessive supply of molecular oxygen during reperfusion, acute ischemic injury is worsened by the products of oxidative stress including free radicals and oxygen reactive species. These products cause lipid peroxidation which impairs the permeability of cell membranes and disrupts the integrity of cells [3–6]. Alpha-lipoic acid (ALA) is found naturally within the mitochondria. It is an essential cofactor for pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. The coupling of ALA and its reduced form (dihydrolipoic acid) is described as the “ideal,” “unique,” and “universal antioxidant” [7]. It has been demonstrated that ALA protects various membrane systems from oxidative injury including neuronal membranes, erythrocyte membranes, and mitochondrial membranes [8–10]. The efficiency of ALA has been shown in diabetes mellitus, atherosclerosis, ischemia-reperfusion diseases, multiple sclerosis, cognitive losses, and senile dementia [11]. Coenzyme Q10 (CoQ10) is a strong antioxidant which provides stabilization of cell membranes by participating in the mitochondrial electron transport chain. This molecule also acts as a cofactor in the synthesis of adenosine triphosphate (ATP) by oxidative phosphorylation. It has been shown that CoQ10 inhibits lipid peroxidation, scavenges free oxygen radicals, and increases the utilization of oxygen in energy production [12–14]. Free radicals which occur as a result of ischemia-reperfusion and oxidative stress affect the phospholipids on the cell membrane and they create phosphoryl choline and ceramide on the scale of neutral sphingomyelinase (n-SMase). Ceramide leads to caspase activation and activates apoptosis. CoQ10 decreases apoptosis by affecting both the creation of free radicals and n-SMase step. Likewise, alpha-lipoic acid prevents apoptosis both by sweeping free oxygen radicals as an antioxidant and by affecting the caspase system [15, 16]. The present study aims to evaluate whether ALA, CoQ10, or the combination of both can be used to protect prepubertal ovarian tissue from ischemia-reperfusion injury in an experimental rat model of ovarian torsion. 2. Materials and Methods This study was approved by the Ethical Committee of Afyon Kocatepe University for Animal Experiments (number: 59269667/281, date: 15.02.2013). The experiments in this study were performed in accordance with the guidelines for animal research. 2.1. Study Design The rats that were included in the experiment had intact circadian rhythm and ad libitum feeding within their natural environment. Forty-two female preadolescent Wistar-Albino rats (mean age: 4 weeks, weight range: 40–45 g) were divided into 6 equal groups randomly. All surgical operations were performed under intramuscular 50 mg/kg ketamine (Ketasol 10%, Richter Pharma AG, Wels, Austria) and 10 mg/kg xylazine (Alfazyne 2%, Alfasan International BV, Woerden, Netherlands) anesthesia. Rats were placed in the dorsal recumbent position. A 1.5 cm midline laparotomy incision was made under sterile conditions so that the abdomen was opened and the large intestines were separated gently. In order to visualize the left ovary, large intestines are placed on the left side of the rat's abdomen. After the left ovary was found, it was made to undergo torsion of 720 degrees clockwise and then fixated to the anterior abdominal wall with a single 5/0 prolene suture. Afterwards, the abdominal incision was repaired with continuous sutures. Rats were covered with gauze to be protected from hypothermia and they recovered from the effects of anesthesia in approximately 30 minutes. Medication was given orally with gavage after an hour postoperatively; detorsion was performed at the 3rd hour and bilateral oophorectomy and blood samples were taken at the 9th hour postoperatively. The reason for choosing postoperative 9th hour for biochemical, histopathological, and immunohistochemical evaluation is based on pharmacokinetic properties of the molecules. When ALA is taken orally, it is rapidly absorbed and reaches plasma peak levels in 30 minutes to 1 hour [17]. In contrast, coenzyme Q10 reaches peak plasma levels in about 4–6 hours after its oral administration [18]. Group 1 (sham group) included 7 rats that had laparotomy without torsion. Group 2 (saline group) consisted of 7 rats that underwent T/D and received saline orally (Polifleks 0.9% NaCl, Eczacıbaşı-Baxter, Istanbul, Turkey). Group 3 (olive oil group) included 7 rats that had T/D and received olive oil orally (Taris, Izmir, Turkey). Group 4 (ALA group) consisted of 7 rats that underwent T/D and received ALA orally (100 μM/kg/day, ≥99% titration in olive oil, Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Group 5 (CoQ10 group) included 7 rats that had T/D and received CoQ10 orally (10 mg/kg/day, ≥98% titration in olive oil, Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Group 6 (ALA + CoQ10) included 7 rats that underwent T/D and received both ALA and CoQ10 orally. As both ALA and CoQ10 were administered in olive oil solutions in the treatment groups, an olive oil group was included to rule out whether olive oil itself would exert antioxidant effects. 2.2. Biochemical Analysis for Oxidant-Antioxidant Status Blood samples were collected into heparinized tubes and were examined in the biochemistry laboratory of the study center. Serum levels of oxidized/reduced glutathione (GSH) and malondialdehyde (MDA) concentrations were determined by high-performance liquid chromatograph (HPLC) in the isocratic phase in the Agilent 1100 series instrument with fluorescent detection (Ex:515, Em:553 nm for MDA; Ex:385, Em:515 for GSH) using a kit from Chromsystems Chemicals GmbH (glutathione kits, Chromsystems Chemicals, Munich, Germany; malondialdehyde kits, Chromsystems Chemicals, Munich, Germany). The results were evaluated as µmol/g hemoglobin for GSH and µmol/L hemoglobin for MDA. Serum levels of glutathione peroxidase were determined using glutathione peroxidase kits (glutathione peroxidase assay kits, Cayman Chemicals, Ann Arbor, USA). ELISA microanalyzer was used for absorbance assays (ChemWell 2910, Awareness Technology Inc., Palm City, USA). The results were evaluated as U/g hemoglobin for glutathione peroxidase. 2.3. Histopathological Evaluation Since the study was carried out on ovarian tissues of preadolescent rats, the pathologist was unable to evaluate the ovarian reserve based on the counts of primordial, preantral, and antral follicles. In order to specify the ovarian damage histopathologically, at least five microscopic sections were examined and semiquantitative scores were obtained. The presence of follicular cell degeneration, hyperemia, hemorrhage, and inflammation were used as the criteria for ovarian injury. Each specimen was scored using a scale ranging from 0 to 3 (0: none; 1: mild; 2: moderate; 3: severe) [19]. The ovarian sections were analyzed by the same pathologist who was blinded to experiment groups. 2.4. TUNEL Assay for Apoptosis Paraffin blocks were prepared for histopathological examination and 5 µm sections from these blocks were transferred to positive charged slides for TUNEL assay. Nuclease-free proteinase K (0.6 units/mL, pH 8.0) incubation was applied (in 37°C oven, for 10 minutes) for antigen retrieval. After washing with PBS, 10-minute 3% hydrogen peroxide application was made at room temperature for blocking of endogenous peroxidase activities. Sections were transferred into humidity chamber and a commercial apoptosis kit (In Situ Cell Death Detection Kit, POD (cat. number 1 684 817), Roche Diagnostics GmbH, Mannheim, Germany) was applied according to the manufacturer's procedure: slides are incubated in TUNEL mixture for 1 hour at 37°C and then they are incubated for 30 minutes in converter pod at 37°C. All slides were stained with AEC chromogen. For counterstain, Mayer's hematoxylin staining was performed. After each step, sections were washed with PBS. The immunoreactivity for apoptosis was graded from 0 to 4 with a light microscope in terms of density and distribution of staining: (0) no positivity on stroma and follicles, (1) mild positivity only in follicles, (2) moderate positivity in follicles and slight positivity in stroma, (3) moderate positivity in follicles and stroma, and (4) severe positivity in follicles and stroma. 2.5. Immunohistochemical Assay for Nitric Oxide Synthase (NOS) Streptavidin-biotin-peroxidase complex (VECTASTAIN Elite ABC Kit PK-6101, Vector Laboratories, Peterborough, UK) method was used. Briefly, 5 μm tissue sections were mounted on positive charged slides from paraffin blocks. Deparaffinized and rehydrated sections were transferred into 3% hydrogen peroxide for blocking endogenous peroxides and then antigen retrieving processes were carried out. Nonspecific immunoglobulins were blocked with nonimmune sera. Antibodies against NOS (C-terminal-polyclonal 1/40 dilution (ab15203), Abcam, Cambridge, UK) were applied to the sections. Then, biotinylated secondary antibody and streptavidin-peroxidase were dropped on the tissue sections. After this process, sections were visualized with 3-amino-9-ethylcarbazole chromogen (AEC substrate kit (code number 00-2007), Invitrogen, Basel, Switzerland). The immunoreactivity for NOS was graded from 0 to 4 with a light microscope in terms of density and distribution of staining: (0) no positivity in stroma and follicles, (1) mild positivity in follicles, (2) moderate positivity in follicles and slight positivity in stroma, (3) moderate positivity in follicles and stroma, and (4) severe positivity in follicles and stroma. 2.6. Statistical Analysis Collected data were analyzed by MedCalc (version 12.7.5.0, Ghent, Belgium). Continuous variables were expressed as mean ± standard deviation (range: minimum–maximum) whereas categorical variables were denoted as mean rank. Kruskal-Wallis test and Mann-Whitney U test were used for the comparisons. Conover-Inman post hoc test was used to determine the groups between which statistical significance exists. Two-tailed P values less than 0.05 were accepted to be statistically significant. 3. Results Table 1 compares the pathological scoring of the sham, control, and study groups (Table 1). When compared to the right ovaries, the left ovaries had significantly higher apoptosis scores in the saline, olive oil, ALA, CoQ10, and ALA + CoQ10 groups (P = 0.003, P = 0.002, P = 0.001, P = 0.002, and P = 0.008, resp.). The right ovaries of the sham, control, and study groups were statistically similar in aspect of apoptosis scores (P = 0.07). The left ovaries of the ALA + CoQ10 group had significantly lower apoptosis scores than the left ovaries of the saline and olive oil groups (P = 0.001 for both) (Figures 1 and 2). The ALA, CoQ10, and ALA + CoQ10 groups had statistically similar apoptosis scores (P 4-5 = 0.430, P 4-6 = 0.556, and P 5-6 = 0.612). The right and left ovaries had statistically similar NOS level in the sham, control, and study groups (P = 0.530, P = 0.644, P = 0.367, P = 0.709, P = 0.947, and P = 0.578, resp.). The right ovaries of the ALA + CoQ10 group had significantly higher NOS content than the right ovaries of the saline and olive oil groups (P = 0.001 for both). The left ovaries of the ALA + CoQ10 group had significantly higher NOS content than the left ovaries of the saline and olive oil groups (P = 0.001 for both) (Figures 3 and 4). The ALA, CoQ10, and ALA + CoQ10 groups had statistically similar NOS content but NOS content of the ALA + CoQ10 group tended to be higher than those of the ALA and CoQ10 groups (P 4-5 = 0.126, P 4-6 = 0.248, and P 5-6 = 0.378). When compared with the left ovaries of the saline and olive oil groups, the left ovaries of the ALA, CoQ10, and ALA + CoQ10 groups had significantly lower ovarian damage scores (P = 0.03). However, there were no statistically significant differences among the study groups (P 4-5 = 0.1, P 4-6 = 1.00, and P 5-6 = 0.1) (Figure 5). The right and left ovaries of the sham group were statistically similar whereas the left ovaries of the remaining groups had significantly higher tissue damage scores than those of the contralateral ovaries (P values are enlisted in Table 1). The biochemical assessments of the oxidant-antioxidant statuses of the sham, control, and study groups were compared (Figure 6). All six groups were statistically similar with respect to serum glutathione concentrations (P = 0.069). When compared with the sham group, the ALA + CoQ10 group had significantly higher glutathione peroxidase levels and serum malondialdehyde concentrations (P = 0.007 and P = 0.027, resp.). There were no statistically significant differences between the glutathione peroxidase levels of the control groups (groups 2 and 3) and the ALA, CoQ10, and ALA + CoQ10 groups (P 2-4 = 0.687, P 2-5 = 0.360, P 2-6 = 0.117, P 3-4 = 0.402, P 3-5 = 0.576, and P 3-6 = 0.802; P 4-5 = 0.609, P 4-6 = 0.276, and P 5-6 = 0.564). There were no statistically significant differences between serum malondialdehyde concentrations of the control groups (groups 2 and 3) and the ALA, CoQ10, and ALA + CoQ10 groups but malondialdehyde levels of the ALA + CoQ10 group tended to be lower than those of the ALA and CoQ10 groups (P 2-4 = 0.446, P 2-5 = 0.420, P 2-6 = 0.067, P 3-4 = 0.372, P 3-5 = 0.349, and P 3-6 = 0.050; P 4-5 = 0.965, P 4-6 = 0.286, and P 5-6 = 0.306). When compared with the control group, the ALA + CoQ10 group had lower serum malondialdehyde concentrations (P = 0.05). 4. Discussion Ischemia refers to the decrease in blood supply of an organ which results in the breakdown of ATP and lipid peroxides so that the generation of lactic acid and hypoxanthine is enhanced. As blood supply normalizes during reperfusion, xanthine oxidase converts hypoxanthine to uric and superoxide radicals. These radicals consist of hydrogen peroxide, hydroxyl radicals, and superoxide anions. Superoxide radicals cause lipid peroxidation, which impairs the permeability of cell membranes, disrupt cellular integrity, and, thus, lead to cell damage. The production of nitric oxide and peroxynitrite is also accelerated in case of reperfusion followed by ischemia [20–23]. In this study, the apoptosis scores of the left ovaries were significantly lower than those of the right ovaries in the saline, olive oil, ALA, CoQ10, and ALA + CoQ10 groups. This finding indicates that a model of ischemia-reperfusion has been established successfully in all of the control and study groups. ALA has been addressed as an efficient glutathione substitute which can increase cellular glutathione content and improve the antioxidant status of the myocardium [24]. It has been also reported that ALA protects against hepatic ischemia-reperfusion injury in rats [25]. The administration of ALA before the torsion of spermatic cord exerted significant protective effects against ischemia-reperfusion injury [26]. These protective effects may be attributed to the reduction of lipid peroxidation and the reinforcement of antioxidant defense mechanisms including glutathione and glutathione peroxidase [24–26]. A number of studies have focused on the utilization of CoQ10 for the prevention and treatment of ischemia-reperfusion injury in many organs. Kalayci et al. made up a model of brain ischemia-reperfusion injury and found that malondialdehyde levels were significantly reduced in rats that received a single dose of 10 mg/kg CoQ10 intraperitoneally [14]. Erol et al. concluded that CoQ10 treatment was able to decrease malondialdehyde levels in an experimental model of testicular ischemia-reperfusion injury [13]. Similarly, Ozler et al. reported that an intraperitoneal injection of CoQ10 could decrease oxidative stress markers significantly in a rat model of ovarian ischemia-reperfusion injury [19]. As for the present study, the administration of ALA or CoQ10 via oral route resulted in a decrease in apoptosis scores and an increase in NOS content of the ovaries that underwent torsion and subsequent detorsion. The NOS make up a group of enzymes that catalyze the production of nitric oxide from L-arginine. Nitric oxide is an important cellular signaling molecule that modulates the protection against oxidative damage. The increase in NOS content of the ovaries treated with ALA + CoQ10 indicates the antioxidant activity of the aforementioned molecules [27, 28]. In addition, ALA treatment caused an increase in serum glutathione peroxidase levels and a decrease in serum malondialdehyde concentrations. However, these alterations were not statistically significant. Such discrepancy may be the result of relatively smaller cohort size, oral route of administration for antioxidant molecules, relatively shorter follow-up period after surgery, and relatively insufficient dose of ALA administration. Oral route was preferred for the administration of antioxidant molecules because the availability and utility of these agents in clinical settings were a concern of this study. The other power-limiting factors were the inability to measure the tissue concentrations of oxidant and antioxidant molecules and the failure in specifying ovarian contents of endothelial and inducible NOS separately. These failures might be attributed to the relatively small size of prepubertal ovarian tissues. The findings of the present study indicate a cumulative increase in NOS and a cumulative decrease in malondialdehyde levels for the combination of ALA and CoQ10. Yet, these alterations have been regarded as statistically insignificant. These findings imply that the combination of ALA and CoQ10 could improve ATP synthesis and reverse some of the cell damage caused by the reduction of energy availability. Moreover, this combination would extinguish the hazardous effects of oxidative stress which are primarily induced by the impairment of respiratory chain. An underlying factor for this synergetic effect might be that the combination treatment could diminish one or more of the final common pathways of mitochondrial dysfunction so that mitochondrial ATP production is improved [29, 30]. Another explanation may be the fact that the combination of two antioxidant agents would be more effective than an individual antioxidant agent because different antioxidants complement each other. Thus, there would be an increase in the total antioxidant capacity that is required to respond to the outburst of free radicals and reactive oxygen species [31]. Although the left ovaries have undergone torsion and subsequent detorsion in this study, the NOS content of the right ovary in the ALA + CoQ10 group has been found to be significantly higher. This finding can be explained by the reduction in blood supply of the right ovary. This reduction is caused by the vasoconstriction which is induced by the increase in the sympathetic activity after the torsion/detorsion procedure. On the other hand, no significant differences could be detected among the study groups in aspect of apoptosis. This failure could be due to the relatively small cohort size and the differences in the pathological examination techniques [32]. 5. Conclusion The findings of this study imply that the combination of ALA and CoQ10 has beneficial effects on oxidative stress induced by ischemia-reperfusion injury in an experimental model of ovarian torsion. These beneficial effects appear to be more pronounced within the ovarian tissue but less prominent in peripheral circulation. This finding may be due to the relatively shorter follow-up period after the administration of ALA or CoQ10. Further research is warranted to clarify the effects of ALA and CoQ10 on the prepubertal ovarian tissues which have been exposed to ischemia-reperfusion injury. Acknowledgments The authors declare that this study has received financial support from the Scientific Research Project Coordination Unit of Afyon Kocatepe University (12.TUS.03). Competing Interests The authors report no competing interests. Figure 1 Mean rank of apoptosis. Figure 2 Slight TUNEL positivity in follicular cells (arrow) in group 1 (a). Moderate TUNEL positivity in follicular cells (arrows) and slight positivity in stroma (yellow arrows) in group 6 (b). Severe TUNEL positivity in follicular cells (arrows) and stroma (yellow arrows) in group 2 (c). ×50. TUNEL method. AEC chromogen. Gill's (I) Hematoxylin. Figure 3 Mean rank of NOS immunoreactivity. Figure 4 Slight NOS positivity in follicular cells (lines) (a). Moderate NOS positivity in follicular cells in group 3 (lines) (b). Severe NOS positivity in follicular cells (lines) and stroma (yellow lines) in group 6 (c). ×50. ABC peroxidase. AEC chromogen. Gill's (I) Hematoxylin. Figure 5 When compared with the left ovaries of the saline and olive oil groups, the left ovaries of the alpha-lipoic acid, coenzyme Q10, and alpha-lipoic acid + coenzyme Q10 groups had significantly lower ovarian damage scores (P = 0.03). The same letters indicated that the difference was statistically insignificant but different letters showed that there was statistical significance between the groups. Figure 6 When compared with the sham group, the alpha-lipoic acid + coenzyme Q10 group had significantly higher glutathione peroxidase levels and serum malondialdehyde concentrations (P = 0.007 and P = 0.027, resp.). When compared with the control group, the alpha-lipoic acid + coenzyme Q10 group had lower serum malondialdehyde concentrations (P = 0.05). The same letters indicated that the difference was statistically insignificant but different letters showed that there was statistical significance between groups (“a” for glutathione; “1, 2” for malondialdehyde; and “x, y” for glutathione peroxidase). Table 1 Pathological scoring of all groups.     Apoptosis score Mean ± std. deviation P Nitric oxide synthase Mean ± std. deviation P Histopathological evaluation Mean ± std. deviation P Group 1 (sham) (n = 7) Left ovary 1.0 ± 0.0 0.259 0.2 ± 0.1 0.530 0.0 ± 0.0 1.00 Right ovary 0.7 ± 0.2 0.3 ± 0.1 0.0 ± 0.0 Group 2 Control (saline) (n = 7) Left ovary 3.8 ± 0.9 0.003∗ 1.3 ± 0.6 0.644 2.7 ± 0.48 0.001∗ Right ovary 3.6 ± 0.7 1.7 ± 0.8 0.71 ± 0.48 Group 3 Control (olive oil) (n = 7) Left ovary 3.7 ± 0.5 0.002∗ 1.7 ± 0.5 0.367 2.8 ± 0.37 0.001∗ Right ovary 3.6 ± 0.7 2.0 ± 0.6 0.85 ± 0.69 Group 4 (ALA) (n = 7) Left ovary 2.7 ± 1.1 0.001∗ 2.3 ± 1.1 0.709 1.7 ± 0.48 0.005∗ Right ovary 0.6 ± 0.1 2.4 ± 1.3 0.57 ± 0.53 Group 5 (CoQ10) (n = 7) Left ovary 2.6 ± 1.4 0.002∗ 2.4 ± 1.2 0.947 1.7 ± 0.75 0.015∗ Right ovary 1.1 ± 0.3 2.1 ± 0.9 0.7 ± 0.48 Group 6 (ALA + CoQ10) (n = 7) Left ovary 2.5 ± 1.3 0.008∗ 2.6 ± 1.4 0.578 1.7 ± 0.48 0.014∗ Right ovary 0.9 ± 0.1 2.6 ± 1.5 0.57 ± 0.78   ∗ P < 0.05 was accepted to be statistically significant. ==== Refs 1 Laganà A. 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==== Front J Aging ResJ Aging ResJARJournal of Aging Research2090-22042090-2212Hindawi Publishing Corporation 10.1155/2016/5081021Review ArticleThe Emerging Roles of the Calcineurin-Nuclear Factor of Activated T-Lymphocytes Pathway in Nervous System Functions and Diseases http://orcid.org/0000-0003-3262-1795Kipanyula Maulilio John 1 * Kimaro Wahabu Hamisi 1 Etet Paul F. Seke 2 1Department of Veterinary Anatomy, Faculty of Veterinary Medicine, Sokoine University of Agriculture, P.O. Box 3016, Chuo Kikuu, Morogoro, Tanzania2Department of Basic Health Sciences, Qassim University, Buraydah, Al-Qassim 51452, Saudi Arabia*Maulilio John Kipanyula: kipanyula@suanet.ac.tzAcademic Editor: Elke Bromberg 2016 15 8 2016 2016 508102113 5 2016 21 7 2016 Copyright © 2016 Maulilio John Kipanyula et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.The ongoing epidemics of metabolic diseases and increase in the older population have increased the incidences of neurodegenerative diseases. Evidence from murine and cell line models has implicated calcineurin-nuclear factor of activated T-lymphocytes (NFAT) signaling pathway, a Ca2+/calmodulin-dependent major proinflammatory pathway, in the pathogenesis of these diseases. Neurotoxins such as amyloid-β, tau protein, and α-synuclein trigger abnormal calcineurin/NFAT signaling activities. Additionally increased activities of endogenous regulators of calcineurin like plasma membrane Ca2+-ATPase (PMCA) and regulator of calcineurin 1 (RCAN1) also cause neuronal and glial loss and related functional alterations, in neurodegenerative diseases, psychotic disorders, epilepsy, and traumatic brain and spinal cord injuries. Treatment with calcineurin/NFAT inhibitors induces some degree of neuroprotection and decreased reactive gliosis in the central and peripheral nervous system. In this paper, we summarize and discuss the current understanding of the roles of calcineurin/NFAT signaling in physiology and pathologies of the adult and developing nervous system, with an emphasis on recent reports and cutting-edge findings. Calcineurin/NFAT signaling is known for its critical roles in the developing and adult nervous system. Its role in physiological and pathological processes is still controversial. However, available data suggest that its beneficial and detrimental effects are context-dependent. In view of recent reports calcineurin/NFAT signaling is likely to serve as a potential therapeutic target for neurodegenerative diseases and conditions. This review further highlights the need to characterize better all factors determining the outcome of calcineurin/NFAT signaling in diseases and the downstream targets mediating the beneficial and detrimental effects. ==== Body 1. Introduction Cellular responses to calcium (Ca2+) mobilization are highly versatile due to the ability of intracellular Ca2+ signaling to activate an extensive repertoire of downstream signaling targets [1, 2]. Among such molecules, the calmodulin- (CaM-) dependent phosphatase calcineurin and its transcription factors termed nuclear factors of activated T cells (NFATs) are reported as pivotal in a wide range of physiological processes, including homeostasis, angiogenesis, myogenesis, adipogenesis, osteogenesis, chondrocyte differentiation, cardiovascular system development, pancreatic β-cell proliferation, hair follicle cell differentiation and remodeling, and activities of cells of the immune and nervous systems [3–12]. Overexpressed and decreased activities of calcineurin/NFAT pathway were also reported in pathologies affecting these functions [3, 6, 13–18]. A growing body of evidence suggests that calcineurin/NFAT pathway plays critical roles in normal and pathological nervous system. Over the last decade, this signaling pathway was reported as major player in corticogenesis, synaptogenesis, and neuritogenesis during mammalian nervous system development [19–23], as well as myelination, synaptic plasticity, neurotransmission, and central and peripheral nervous system cell proliferation, migration, and differentiation in the mature nervous system [24–26]. Altered calcineurin/NFAT activation is increasingly linked to pathological features of neurodegenerative diseases such as amyotrophic lateral sclerosis, Huntington's, Parkinson's, and Alzheimer's diseases, characterized by massive synaptic dysfunction, glial activation, and neuronal death in some regions of the brain [26–29]. Calcineurin/NFAT involvement has also been reported in psychiatric disorders, epilepsy, and traumatic brain and spinal cord injuries [30–35]. Moreover, recent data also suggest that NFAT isoforms are selectively activated in neurons and glial cells in nervous system diseases [36, 37] and animal models [38–41]. Herein, we discuss the current understanding of the role of calcineurin/NFAT signaling pathway in both physiology and pathologies of the adult and developing nervous system, with an emphasis on recent reports and cutting-edge findings. 2. Calcineurin/NFAT Pathway 2.1. Calcineurin Calcineurin, also termed as protein phosphatase B (PP2B), is a Ca2+/CaM-dependent serine/threonine phosphatase. It was described for the first time in the bovine brain about 40 years ago [42, 43]. Calcineurin is made up of a 61 kD CaM-binding catalytic subunit termed calcineurin A (CnA) and a 19 kD Ca2+-binding regulatory subunit named calcineurin B (CnB) stably associated (noncovalently) [44, 45]. To date three isozymes of the catalytic subunit (CnAα, CnAβ, and CnAγ) have been reported. There are two isoforms of the regulatory subunit, namely, CnB1 and CnB2. In vertebrates, each subunit is encoded by a separate gene PPP3CA, PPP3CB, and PPP3CC for CnB1 and PPP3R1, PPP3R2 for CnB2 [46, 47]. The two calcineurin isoforms are widely distributed in mammalian tissues. Immunohistochemical studies showed that under normal conditions calcineurin isoforms are highly expressed in neuroinflammation-sensible neurons like corticohypothalamic pyramidal cells and cerebellar Purkinje cells, as well as in peripheral nervous system (PNS) glia like Schwann cells, but not in central nervous system (CNS) glia [37, 48, 49]. The subcellular distribution of calcineurin is an important control point in regulating its activity. Various studies addressed the subcellular localization of calcineurin in various mammalian cells [2, 50, 51]. In neurons, calcineurin was found in the cytoplasm, endoplasmic reticulum, Golgi apparatus, nucleus, synaptic vesicles, microsomes, mitochondrion outer membrane, and plasma membrane [2, 48, 49, 51, 52]. 2.2. NFAT Transcription Factors NFAT proteins are a family of transcription factors normally found in the cytoplasm in a hyperphosphorylated (inactive) state [53–55]. This family comprises five distinct gene products termed (i) NFATc, NFAT2, or NFATc1; (ii) NFATp, NFAT1, or NFATc2; (iii) NFATx, NFAT4, or NFATc3; (iii) NFAT3 or NFATc4; and (iv) osmotic response element-binding protein (OREBP), tonicity-responsive binding-protein (TonEBP), or NFAT5 [56, 57]. In the context of this review, nomenclature of NFATc1-c4 and NFAT5 will be used. NFATc1–c4 occur as monomers with unique amino and carboxyl termini containing transcription activation domains (TADs) and two conserved domains: (i) a regulatory domain termed NFAT homology region (NHR), which shows a lesser degree of pair-wise sequence identity but has several strongly conserved sequence motifs characteristic of the NFAT family; and (ii) the Rel homology region (RHR) in the C-terminus, where the DNA binding domain (DBD) is located [14, 56]. The NHR encompasses calcineurin docking sites, a nuclear localization signal (NLS) responsible for calcineurin-mediated nuclear translocation, and an extended serine-rich region [44, 58–61]. On the other hand, the DBD binds DNA and interacts with partner proteins to transactivate gene transcription. Examples of partner transcription factors include AP-1 cell life and death regulators [dimeric transcription factors composed of activating transcription factor (ATF), Fos, or Jun subunits] and important oncogenic regulators like myocyte enhancer factor-2 (MEF2) and GATA binding protein 4 (GATA4) [60, 62]. Regions out of the regulatory and DNA binding domains like TADs demonstrate relatively little sequence conservation [57, 59]. NFAT5 is a homodimer with a distinct domain structure. This isoform retains only the RHR region of homology to the Ca2+-regulated isoforms, whilst the remaining 600 amino acids are completely different. The DBD of NFATs is distantly related to the DBD of nuclear factor kappa B (NF-κB)/Rel family, allowing them to be classified sometimes as members of this extended family [63, 64]. NFATs regulate the transcriptional induction of genes encoding for immune modulators/activators such as granulocyte-macrophage colony-stimulating factor (GM-CSF), forkhead box P3 (FOXP3), immunoglobulin kappa (Igκ), gamma interferon (IFNγ), CD5, CD25, CD28, CD40, interleukin- (IL-) 2, IL-3, IL-4, IL-5, IL-13, IL-8, Th2-type cytokine IL-31, Fas ligand, macrophage inflammatory protein 1 alpha (MIP-1α), protein tyrosine kinase Syk, cyclooxygenase 2 (COX-2), and tumor necrosis factor alpha (TNF-α) and its family member BlyS. NFATs may also control genes encoding signaling molecules as variate as Ca2+ regulators [inositol 1,4,5-trisphosphate (IP3) receptor (IP3R), regulator of calcineurin 1 (RCAN1)], growth factors (VEGF, neurotrophins), myelination genes (P0 and Krox-20), glucose regulation genes (insulin, HNF1, PDX, and GLUT2), cell cycle and death regulator/activators [p21Waf1, c-Myc, cyclin-dependent kinase 4 (CDK4), B-cell lymphoma 2 (Bcl-2), and cyclins A2, D1, and D2], oncogenes (Wnt, β-catenin), microRNAs (miR-21, miR-23, miR-24, miR-27, miR-125, miR-195, miR-199, and miR-224), and surfactants (sftpa, sftpb, sftpc, and abca3) [9, 65–74]. NFAT isoforms are ubiquitously expressed and are generally regulated by Ca2+ signaling, with exception of NFAT5 [59, 75, 76]. NFAT5 regulates hypertonic stress-induced gene transcription, whereas the other NFATs act as integrators of Ca2+ driven signaling pathways in gene expression and cell differentiation programs [57, 77, 78]. All Ca2+-regulated NFATs (NFATc1–c4) are expressed in neurons [79]. The previously assumed functional redundancy of NFAT functions was proved wrong by their wide-ranging expression profile among cell types under both physiological and pathological conditions. For example, loss of specific NFAT subtypes resulted in cardiovascular, skeletal muscle, cartilage, neuronal, and/or immune system defects [54, 59, 80, 81]. In addition, Vihma and colleagues reported that NFAT subcellular localizations and transcriptional activities are isoform- and cell type-specific [37]. In that context, the strongest transcriptional activators were NFATc3 and NFATc4 in primary hippocampal neurons and NFATc1 and NFATc3 in human embryonic kidney-derived HEK293 cells. 2.3. Signaling Activation The activation of calcineurin/NFAT signaling pathway involves three key steps: (i) NFAT protein dephosphorylation by calcineurin; (ii) nuclear translocation of NFATs; and (iii) increased affinity for DNA. Ligand-receptor interaction activates phospholipase C (PLC) resulting in the release of IP3, which in turn leads to the release of Ca2+ from intracellular stores through IP3Rs [59, 82]. Notably, Ca2+ release from the intracellular stores requires a stimulus capable of generation of second messengers that trigger Ca2+ release from intracellular stores in the endoplasmic/sarcoplasmic reticulum, via channels of IP3R1–3 and ryanodine receptors (RyR1–3) [59, 83–87]. Like for other Ca2+/CaM-dependent enzymes, calcineurin activation requires increase in cytosolic Ca2+ levels. Such activation may result from the binding of Ca2+ to calcineurin B subunit or from the binding of Ca2+-activated CaM to calcineurin [50]. In turn, calcineurin activates NFATs by dephosphorylating multiple N-terminal phosphoserine residues in the regulatory domain. Such dephosphorylation increases NFAT affinity for DNA [59]. The dephosphorylation results in a conformational change in the NFAT molecule that exposes the nuclear localization signal, allowing NFAT nuclear translocation to take place, presumably through nuclear pores [88]. In the nucleus NFATs become transcriptionally active by forming complexes with other factors and coactivators, providing a direct link between intracellular Ca2+ signaling and gene expression. Calcineurin also promotes nuclear retention of NFAT by masking nuclear export signals (NES) and preventing NES-dependent nuclear export [88, 89]. Moreover, calcineurin mediated NFAT activation is mainly required at resting state (resting membrane potential), where the amount of Ca2+ released from intracellular stores is not sufficient for direct NFAT activation [75, 90]. 2.4. Regulation and Pharmacology 2.4.1. Endogenous Regulation Regulators of calcineurin (RCANs) or modulatory calcineurin-interacting proteins (MCIPs; MCIP1–3) belonging to the calcipressin family of proteins play a pivotal role in regulating calcineurin activity. The RCANs are evolutionarily conserved proteins that can directly bind and inhibit calcineurin. The RCAN1 genes are found on chromosome 21. The RCAN gene encodes different isoforms of protein, namely, RCAN1, RCAN2, RCAN3, and RCAN4 where RCAN1 and RCAN4 are the main isoforms. Furthermore, it appears that RCAN1 has two RCAN1 isoforms: 1S with 197 amino acids and RCAN.1L with 252 amino acids. Previous studies have shown that an aberration in RCANs activities decreases calcineurin-NFAT signaling activities. Recent studies have shown that RCANs can show both inhibitory and facilitatory roles in activation of the calcineurin-NFAT signaling pathway. A mechanistic explanation on how RCAN proteins precisely modulate calcineurin function is still debatable. Overall RCANs have been implicated to function primarily as chaperones for calcineurin biosynthesis or recycling, requiring binding, phosphorylation, ubiquitylation, and proteasomal degradation for their stimulatory effect [50, 91, 92]. Other regulators including scaffolding proteins, CAIN/CABIN-1, and A-kinase anchoring protein 79 (AKAP79) have also been identified to interact and inhibit calcineurin function in a phosphorylation-dependent manner in mammalian cells (details given below). 2.4.2. Endogenous Inhibition The regulation of calcineurin/NFAT activity is achieved: (i) in the nucleus by activities of serine/threonine kinases promoting the export of nuclear NFAT; (ii) in the cytoplasm, through phosphorylation of NFAT serine (SP) repeats and N-terminal domain, which are critical for NFAT activation and nuclear import [88, 93, 94]. Various kinases were implicated in NFAT nuclear export. Examples include glycogen synthase kinase (GSK), protein kinase A (PKA), casein kinase, and also mitogen-activated protein kinases (MAPKs) like c-Jun NH2- terminal kinases (JNKs) and cellular stress-associated p38 kinase [14, 54]. Although different kinases have been implicated in regulating NFAT activity, the distinction between drivers and passengers in the cytoplasm and in the nucleus is still puzzling. Notably, it is not clearly understood whether the kinases which mediate rephosphorylation of nuclear or activated NFATs are similar to those that phosphorylate NFATs under basal state conditions [14, 54]. Other early reports from targeted genetic and pharmacological manipulations suggest that members of the JNK family regulate both the import and export of NFATs while p38 MAPKs mediate NFAT rephosphorylation, thus NFAT nuclear export [13, 89, 95]. Calcineurin/NFAT signaling blocking agents mainly act at calcineurin and NFAT levels. They include cellular (protein inhibitors) and pharmacological inhibitors. Although numerous endogenous proteins may have potential to inhibit calcineurin or NFAT activities, only four are well-characterized: (i) A-kinase anchoring protein 79 (AKAP79), a scaffold protein that prevents calcineurin-substrate interactions [96, 97]; (ii) calcineurin inhibitor (CAIN) or calcineurin-binding (CABIN) proteins, which block calcineurin activity [98, 99]; (iii) calcineurin homologous protein (CHP); and (vi) modulatory calcineurin-interacting proteins (MCIP1–3), which prevent NFAT nuclear import by preventing its phosphorylation [94, 100–103]. 2.4.3. Pharmacological Inhibition Two pharmacological inhibitors of NFAT translocation, namely, cyclosporine A, FK506 (tacrolimus), and its ethyl analog ascomycin, are commonly used as immunosuppressants, particularly in organ transplantation [104–107]. These chemically distinct microbial products inhibit calcineurin activity by binding with subnanomolar affinity to cytosolic proteins called immunophilins. The resultant drug-protein composite binds tightly to calcineurin and blocks its phosphatase activity by preventing substrate access [14, 59]. Cyclosporine A binds to cyclophilin and FK506 binds to FK506-binding protein (FKBP) [88, 108]. These inhibitors indiscriminately block all downstream calcineurin signaling, including various signaling pathways collaborating with calcineurin/NFAT signaling pathway [39, 93]. Such complete blockade of calcineurin activities may explain at least partly the serious side effects of cyclosporine A and FK506 [104, 105], particularly in pediatric patients [106, 107]. Recent studies in rat brain slices and cultured astrocytes revealed that cyclosporine A increases reactive oxygen species (ROS) formation and alters glucose and energy metabolism partly by Krebs cycle inhibition and anaerobic glycolysis activation [109]. These detrimental effects probably participate in cyclosporine A neurotoxicity. Additionally, inhibition of NFAT activity suppressor GSK-3 mediated by wild-type mice chronic treatment with lithium or cyclosporine A resulted in increases in nuclear translocation of NFATc3 and Fas-dependent apoptosis in brain neurons, accompanied by pronounced motor deficits [110]. In the same study, neither neuronal loss nor motor deficits were observed in Fas deficient Tet/DN-GSK-3 mice, suggesting that GSK-3 contributes to the neurological toxicity induced by cyclosporine A. Therefore, GSK-3 inhibitors may improve calcineurin inhibitor neurotoxicity. Cyclosporine A may also mediate neuronal affection by decreasing biometal availability [111–113]. Moreover, experimental evidence has shown increases in spinal NMDA receptor activity as a result of calcineurin inhibitor induced pain syndrome (CIPS) [114–116]. A whole-cell patch-clamp study in spinal cord slices revealed that the effect may be mediated by the potentiation of pre- and postsynaptic NMDA receptor activity in the spinal cord [114]. In the same study, it was shown that FK506 treatment increased drastically the amplitude of excitatory postsynaptic currents mediated by NMDA receptor in dorsal horn neurons. Inhibitors of the serine/threonine protein kinase casein kinase II (CK2) involved in the upregulation of synaptic NMDAR activity in neurons abrogated pain hypersensitivity caused by FK506 [117, 118]. Nonetheless, cyclosporine A also mediated neuropathic pain independently of NMDA receptor [108, 118], suggesting that the processes mediating CIPS are complex and warrant further studies. Interestingly, inhibitors that do not block calcineurin enzymatic activity per se, but rather interfere with enzyme targeting of one or more of its substrates, have recently been developed and used. For instance, the inhibitors of NFAT-calcineurin association (INCA) compounds may interfere selectively with calcineurin/NFAT interaction without preventing dephosphorylation of other calcineurin substrates [119, 120]. Substrate-selective enzyme inhibition represents an important progress over cyclosporine A or FK506-mediated complete blockade of calcineurin/NFAT signaling. This development is expected to lead to the development of safer classes of calcineurin/NFAT inhibitors. Certainly, the beneficial actions of cyclosporin A and FK506 are counterbalanced by serious toxicities attributed partly to their interference with calcineurin signaling in other cells and tissues. Although INCA have nonspecific cytotoxic effects, they are generally considered to be less toxic than treatment with cyclosporine A or FK506. 3. Nervous System Development Calcineurin/NFAT signaling pathway is multifunctional. In the PNS, calcineurin/NFAT signaling was reported to have critical roles in the survival, proliferation, and differentiation of both neural and glial precursor cells [22], highlighting its potential role in tissue regeneration. Similar lines of evidence suggest that calcineurin/NFAT plays critical roles in the regulation of the CNS development, including corticogenesis and synaptogenesis. 3.1. Corticogenesis Evidence supporting the involvement of the calcineurin/NFAT4 signaling pathway in corticogenesis includes a study in developing mouse cerebellar granule neurons that reported a pivotal role in controlling the temporal regulation of nuclear factor 1 occupancy. This serves as a key link between membrane potential and dendritic maturation, by a voltage-sensitive developmental switch [121]. In addition, the multifunctional HMG-box transcription factor Tox, a novel regulator of mammalian corticogenesis, is regulated by calcineurin/NFAT signaling [19]. Genetic and biochemical analyses in the developing embryo revealed that fibroblast growth factor- (FGF-) mediated calcineurin signaling may trigger neural induction by increasing Smad1/5 transcription via silencing of bone morphogenetic protein (BMP) signaling [122]. Furthermore, store-operated Ca2+ entry (SOCE) activation regulates gene transcription in the developing nervous system and mediates neural progenitor cell proliferation through calcineurin/NFAT signaling [123–125]. The role of the calcineurin/NFAT signaling in corticogenesis is probably mediated by NFATc3, the predominant NFAT isoform in neural progenitor cell cultures, which is also a potent inducer of neural progenitor cell differentiation into neurons and astrocytes [22, 126]. Interestingly, activity-dependent NFATc3 accumulation in the nucleus was reported in pericytes from cortical parenchymal microvessels [127], and differential expressions of NFATc3 and NFATc4 were reported in developing rat brain and traumatic brain injury models, where NFATc4 was primarily expressed by neurons and NFATc3 by astrocytes [125, 128]. These observations suggest that different NFATs are recruited at the same time in resident cells of damaged and growing nervous tissue, indicating that characterizing the effects of such NFAT changes in specific cell types may provide new therapeutic targets for neurodevelopmental disorders. 3.2. Synaptogenesis The complex interactions between inhibitory gamma-aminobutyric acid (GABA) and excitatory NMDA receptor activities are required during synaptogenesis. Furthermore, this interaction plays an important role in the induction of immediate early genes necessary for effective changes in synaptic plasticity and long-term memory formation through calcineurin-dependent transcription of the key brain-derived neurotrophic factor (BDNF) [20, 23, 129, 130]. Of particular interest for neurodevelopmental treatments is a recently reported novel synthetic neurotrophic (BDNF and neurotrophin-like) compound that is able to induce neurite growth and confer neuroprotection [21]. In addition, the neurotrophin nerve growth factor (NGF) is known to upregulate the key regulator of plasminogen activation system and synaptogenic protein plasminogen activator inhibitor 1 (PAI-1) in primary mouse hippocampal neurons via calcineurin/NFAT signaling [131]. Protein kinase C (PKC)/calcineurin signaling-mediated dephosphorylation of axon growth regulatory molecule growth-associated protein 43 (GAP43) at developing GABAergic synapses resulted in pathological processing mimicking neonatal hypoxia, including misfolding of gephyrin, a protein critical for the organization of GABA receptors [132]. Calcineurin signaling also mediates the GABAergic synaptic modulation induced by transient receptor potential vanilloid type 1 (TrpV1), a ligand-gated channel abundantly expressed in developing primary sensory neurons [133, 134]. Certainly, these observations suggest that calcineurin signaling is involved in the development of GABAergic synaptic functions in the CNS. Moreover, GABA promoted the shrinkage and elimination of synapses by suppressing local dendritic Ca2+ signaling in rat hippocampal CA1 pyramidal neurons via a mechanism depending on calcineurin and on actin-binding protein cofilin [135]. Similarly, more recent studies also suggested that calcineurin is a major signaling molecule in the selection of synapses, a critical mechanism in the reorganization of the developing and adult CNS mediated by the major inhibitory neurotransmitter GABA [132, 135, 136]. 3.3. Endosome Trafficking It is now widely accepted that calcineurin signaling is a major player in the control of the trafficking and signaling of endosomes performing the retrograde signaling, an event critical for the development, but also for the nervous system function [2, 137, 138]. Calabrese and colleagues reported calcineurin signaling modulation as a key event in the differential regulation of dynamins, major players of synaptic vesicle recycling in nerve terminals of developing neurons [137]. Notably, these authors observed that tetrodotoxin- (TTX-) mediated chronic suppression of neuronal activity results in the suppression of dynamin 1 clustering at nerve terminals and in an increase of clustering of dynamin 3, partly mediated by calcineurin signaling silencing. In addition, NGF-mediated calcineurin/NFAT signaling is critical for the control of endosome trafficking in neurons [77, 131, 138]. Such NGF/calcineurin/NFAT control of the trafficking of endosomes is under the control of the effector protein coronin-1 and regulatory events such as the phosphorylation of cAMP responsive element binding protein (CREB), which are also mediated by NGF receptor tropomyosin receptor kinase type 1 (TrkA) [138]. Notably, as other tyrosine kinase receptors, the receptors of neurotrophins may also activate NFATs via inhibition of the promoter of NFAT nuclear export GSK-3β, independent of Ca2+ and calcineurin signaling. For instance, a study assessing the physiological roles of estrogen-related receptor gamma (ERRγ), an orphan nuclear receptor highly expressed in the nervous system during embryogenesis and over lifespan, revealed its involvement in the regulation of dopaminergic neuronal phenotype. Such effect was mediated by GSK-3β-NFAT interactions, independently of Ca2+/calcineurin signaling [139]. Similar, NGF facilitated NFAT-mediated gene expression induced by mild depolarization in dorsal root ganglion sensory neurons without changes in PLC activity-dependent events, including Ca2+/calcineurin signaling. Instead, NGF effects were induced by phosphoinositide 3-kinase (PI3K)/Akt signaling triggered by TrkA receptor activation, which abrogated GSK-3β activity [77]. Furthermore, calcineurin is universally involved in vesicle endocytosis [12], and alterations in its endocytic activity may participate in the pathogenic processes of various psychiatric diseases. For instance, alterations in presynaptic functions of the γ isoform of the calcineurin catalytic subunit, such as synaptic vesicle cycling, have been suggested to contribute to schizophrenia, where variations in calcineurin Aγ gene PPP3CC are common in neurons [140]. Deregulation of intracellular Ca2+ also associates with the disruption of fast axonal transport (FAT) in the pathogenesis of Alzheimer's disease (AD) [141–144]. 4. Nervous System Function Calcineurin/NFAT has critical roles in neuronal and glial cell activity and survival, as well as resulting events, fundamental for nervous system function like neurotransmission and synaptic plasticity, and myelination as summarized in Figure 1. 4.1. Synaptic Plasticity and Neurotransmission 4.1.1. Synaptic Connectivity and Plasticity Calcineurin/NFAT signaling and other pathways activated by T-type Ca2+ channel activation play critical roles in the shaping of synaptic connectivity of thalamocortical and nucleus reticularis thalami GABAergic neurons mediated by slow wave sleep [145], a process pivotal for the consolidation of recently acquired memories and for the restoration of synaptic homeostasis. This mechanism may be evolutionary conserved [54, 146, 147]. PLC/calcineurin signaling has been reported to regulate the trafficking of GABA A receptor in layer 3 pyramidal cells of murine barrel cortex [136]. Furthermore, the inhibition of group I metabotropic glutamate receptors (mGluRs), IP3Rs, or calcineurin in CA1 neurons resulted in the blockade of the heterosynaptic shrinkage [85, 87] that drives circuit remodeling during activity-dependent refinement of the developing nervous system and during experience-dependent plasticity in the hippocampus. This inhibition has been reported to negatively affect long-term potentiation (LTP) [87]. Experimental evidence also suggests that calcineurin is a regulator of synaptic plasticity. For instance, axon initial segment rapid shortening was partly mediated by calcineurin-dependent mechanisms, including phosphorylation of voltage-gated Na+ channels, in dentate granule cells [148]. Rapid modulation of the axon initial segment, the site of action potential initiation, is the major plasticity mechanisms used by neurons to control their excitability time from seconds to days. In another study, decreases in resting Ca2+ levels associated with prolonged blockade of synaptic activity resulted in the synthesis of retinoic acid, which triggered the related homeostatic synaptic plasticity, via a calcineurin-dependent mechanism in neurons [149]. Moreover, spinogenesis enhancement in hippocampal neurons by the steroid hormones dihydrotestosterone and testosterone was blocked by individual antagonism of PKC, PKA, calcineurin, LIM kinase (LIMK), or the MAPKs Erk and p38 [150]. Similar, estradiol-mediated rapid modulation of synaptic plasticity, an essential process for synaptic regulation, was abrogated by individual targeting of PI3K, PKC, PKA, calcineurin, CaM kinase II (CaMKII), LIMK, Erk, or p38, in hippocampal neurons [151]. 4.1.2. Learning and Neurotransmission Evidence of calcineurin/NFAT involvement in learning and memory processes was provided by experimental models of neurodegenerative disorders. A study in PS1-M146V knock-in FAD mice showed that decreased calcineurin activity is a common phenomenon in aging-related memory decline and may account for memory defects in AD, together with mutations in the gene encoding for GSK-3β substrate presenilin 1 (PS1) [152], which are critical for amyloid-β (Aβ) generation. In a study using a spinocerebellar ataxia type 3 (SCA3) transgenic mouse model, typical impairments of motor learning and cerebellar motor coordination resulted from altered long-term depression (LTD) of glutamatergic transmission in parallel fiber-Purkinje neurons [153]. Such alteration resulted mainly from transcriptional downregulation of PLC β4, IP3R1, and calcineurin B, suggesting that PLC/IP3-Rs/calcineurin signaling is required for cerebellar LTD induction, thus motor learning and coordination. Calcineurin/NFAT signaling is also involved in neuronal excitability and neurotransmission. Under physiological conditions, AKAP79/150-mediated calcineurin/NFAT signaling may prevent neuronal hyperexcitability in hippocampal neurons by increasing the transcriptional expression of key regulators of neuronal excitability like M-type K+ channels [154]. PKC/calcineurin/NFAT signaling contributes to the maintenance of cyclic nucleotide-gated (HCN) channels in the hyperpolarized status critical for their mediation of neuronal excitability decrease in the distal dendrites of hippocampal CA1 pyramidal neurons [155], and membrane-derived bioactive phospholipid lysophosphatidic acid type 1 (LPA1) triggers RhoA/Rho kinase (ROCK)/calcineurin signaling to induce the internalization of the GABAAγ2 subunit at inhibitory synapses [156]. 4.2. Calcineurin-PKA Interactions The interplay between calcineurin and PKA signaling plays a critical role in the negative-feedback mechanism driving homeostatic synaptic plasticity [157], that is, accounting for the compensation of excessive inhibition or excitation of neuronal activity. Although calcineurin/NFAT signaling mainly regulates axon terminal remodeling, while PKA/CREB signaling controls synaptic vesicle accumulation [158], many lines of evidence suggest mutual inhibitory interactions between the activities of these signaling pathways. Examples include the activity of these signaling molecules when anchored to AKAP79/150. PKA kinase activity triggered by anchoring to AKAP79/150 resulted in the enhancement of Ca2+-dependent inactivation of L-type Ca2+ channels, while the activation of the phosphatase activity of AKAP79/150-anchored calcineurin reversed such PKA action, reducing Ca2+-dependent inactivation [96, 159]. The basal activity of AKAP79/150-anchored PKA maintained L-type Ca2+ channel-calcineurin/NFAT signaling functional coupling by preserving the phosphorylation of these channels, contrary to anchored calcineurin [159, 160]. Interestingly, such AKAP79/150 activity mediated the modulation of roundabout axonal guidance receptors Robo2/3 and ligands Slit2/3 in brain regions involved in reward, learning, and memory processes like islands of Calleja and the hippocampus [97]. Furthermore, PKA can induce NFAT nuclear export [14, 54]. PKA activation by forskolin-stimulated cAMP increased the stability and half-life of RCAN1 protein, enhancing its inhibitory effects on calcineurin [161]. In a recent study in pilocarpine-induced status epilepticus, a murine model of temporal lobe epilepsy (TLE), the nuclear translocation of CREB-regulated transcription coactivator 1 (CRTC1), which is a key regulator of CREB activity, was regulated by calcineurin activity in hippocampal neurons [24]. Opposite effects of calcineurin and PKA were also reported as key events in (i) the dynamic fission-fusion events that determine the shape and function of mitochondria [162, 163], thus cell survival; (ii) neuronal output stabilization induced by tonic dopamine via type 1 dopaminergic receptors [164]; and (iii) the phosphorylation/dephosphorylation of serine 897 in the NR1 subunit of the NMDA receptor (pNR1), whose increases in phosphorylation were reported in acute morphine withdrawal [30]. 4.3. Myelination Available data suggest that calcineurin/NFAT pathway participates in signaling cascades pivotal for Schwann cell myelination. Early studies reported that (i) murine Schwann cells express all Ca2+-dependent NFAT isoforms; (ii) the promoter and upstream enhancer elements of the myelinating factor Krox-20 contain NFAT binding sites; and (iii) NFATs recruited by Ca2+-dependent signaling can make transcriptionally active complexes with Krox-20 [9, 93, 165]. Reporter assays showed that Krox-20 is NFAT target gene and that calcineurin is upregulated in Schwann cells expressing Krox-20 [8, 166]. Moreover, we reported using rat Schwann cell cultures and an in vitro model of myelination that promyelinating actions of calcineurin/NFAT signaling, including increases in the expression of the myelinating genes Krox-20, Periaxin, and P0, require cAMP elevation [9]. Notably, in the absence of cAMP elevation, increase in cytosolic Ca2+ failed to induce Krox-20 expression. Furthermore, cyclosporine A and FK506 abrogated Krox-20 expression. Comparable observations were reported more recently by other authors [165, 167]. Experimental evidence also suggests that the activation of calcineurin/NFAT required for Schwann cell myelination occurs partly through neuregulin 1 (NRG1) stimulation [8, 168–170]. A study in mice lacking calcineurin B in cells of the neural crest lineage showed that calcineurin/NFAT signaling is required for NRG1-mediated Schwann cell myelination [170]. In this model NFAT activation failed, Krox-20 levels in Schwann cell were decreased, and radial sorting and myelination were markedly delayed [8, 170]. NRG1 addition to neuron-Schwann cell cocultures promoted the activation of NFAT isoforms and cooperative transcriptional activities of NFATc4 and SOX10 required for Krox-20 upregulation [168, 169]. In addition, NRG1/calcineurin/NFAT signaling upregulates myelinating genes in Schwann cells [8, 168, 171]. Unexpectedly, FK506 stimulated Schwann cell proliferation and promoted the survival of oligodendrocyte in murine models of traumatic spinal cord injury [172–174], suggesting that this signaling pathway is involved in the maintenance, thus activity, of myelinating cells in both peripheral and central nervous systems. Moreover, NFAT1 hyperactivation decreased experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein (MOG), a key regulator of CNS myelination [175, 176]. 5. Controversial Roles in Nervous System Diseases 5.1. Neurodegenerative Diseases 5.1.1. Pathogenic Roles Increased calcineurin activity was reported in both aging and AD models [27, 177, 178]. For instance, calcineurin/NFAT signaling may mediate the aberrant activity of deregulated plasma membrane Ca2+ pumps (PMCAs), a suggested link between brain aging and the onset of neurodegenerative diseases [27, 179, 180]. The strongest AD genetic risk factor, the apoE4 allele, encodes for apolipoprotein E4 that has poor inhibitory abilities on calcineurin activity, unlike the neuroprotective apolipoproteins E2 and E3 [181–183]. Apolipoprotein E4 also drives CNS functional alterations associated with normal aging such as disturbed sleep [28, 29]. Overactivated calcineurin/NFAT signaling may contribute to synaptic plasticity affection in pathological conditions. Notably, in a postmortem study in human hippocampi, high nuclear levels of NFATs observed at the early stage of AD increased with cognitive decline severity [177]. Additionally, short exposure to Aβ oligomers resulted in calcineurin activation with transient changes in postsynaptic proteins and morphological in spines, while longer exposure resulted in NFAT activation and marked spine loss in primary cortical neurons of wild-type mice [184]. Aβ-treatment of murine hippocampal neurons also resulted in Ca2+ signaling-dependent defects in BDNF transport first in dendrites and then in axons [40]. Studies in mouse and rat models of severe childhood epilepsy revealed that calcineurin/NFAT signaling mediates seizure-induced dendrite growth suppression in pyramidal neurons and thus the resulting learning and memory impairment associated with this intractable condition [185]. NFAT signaling alterations in neurodegenerative diseases are selective. A report by Abdul and colleagues strongly suggested that such selective alteration may play a key role in Aβ-induced neurodegeneration [186]. These authors observed increases in calcineurin A activity and more marked shifts of NFATc2 and NFATc4 to nuclear compartments in human hippocampus with increased dementia severity, while even in rapid-autopsy postmortem human brain tissue NFATc1 was unchanged. NFATc2 was more active in AD patients with mild cognitive impairment, contrary to NFATc4 whose expression was mostly associated with severe dementia. Still in the same study, changes in calcineurin/NFAT4 were directly correlated to soluble Aβ levels in postmortem hippocampus, while oligomeric Aβ strongly stimulated NFAT activation in primary rat astrocyte cultures. In another study, NFATc4 levels were significantly increased in brains of APP/PS1 transgenic mice (AD model) and NFATc4 overexpression increased Aβ production in human myeloid leukemia SAS-1 cells [36], suggesting a role for NFATc4 in amyloidogenesis. Mechanisms proposed for NFAT-mediated amyloidogenesis in human and murine astrocytes include increases in the expression of the gene encoding for TMP21, a p24 cargo protein involved in Aβ and Aβ precursor protein (APP) trafficking [187–190]. 5.1.2. Beneficial Effects The role of calcineurin in aberrant α-synuclein-mediated midbrain dopaminergic neuron toxicity, a hallmark of Parkinson's disease (PD), is controversial. In a study addressing the underlying intracellular mechanisms driving α-synuclein-mediated neurodegeneration, transgenic expression of PD α-synuclein A53T missense mutation promoted calcineurin/NFAT signaling, suggesting that this signaling pathway may contribute to the neurotoxic effects of aberrant α-synuclein [191]. Surprisingly, in a study using cells from various models (ranging from yeast to neurons), although aberrant α-synuclein also seemed to induce cellular toxicity via overactivation of Ca2+-dependent signaling pathways, calcineurin inhibition with FK506 also resulted in toxicity [192], suggesting that calcineurin may mediate both beneficial and toxic effects under stimulation by aberrant α-synuclein. Characterizing the precise downstream targets mediating calcineurin beneficial or toxic effects may provide more insights of the novel therapeutic targets for synucleinopathies. Calcineurin also mediates some beneficial and toxic effects of AMPA and NMDA receptors, which are postsynaptic site-located glutamate-gated ion channels critical for synaptic plasticity. Calcineurin translocation to synapses and increases in its activity mediated by NMDA receptor trafficking were reported as critical components of mechanisms driving rapid compression-induced dendritic spine plasticity in cortical pyramidal neurons, that is, the rapid trimming of dendritic spines occurring about 12 hours after mechanical compression [38]. A study using soluble Aβ treated in cultured rat hippocampal neurons and cultured hippocampal neurons from APPSwe AD-transgenic mice suggested that calcineurin signaling mediates AD-like synaptic dysfunction induced by tau protein partly via AMPA receptor downregulation [193]. In that study, soluble Aβ oligomer-induced deficits in AMPA receptor trafficking were mediated by tau phosphorylation and mislocalization to dendritic spines. FK506 abrogated all these alterations. Concomitant tau hyperphosphorylation and calcineurin overactivation were also reported in mouse models of Huntington's disease [41]. On the other hand, IL-6/Janus kinase (JAK) signaling induced neuroprotective anti-NMDA activities in cultured cerebellar granule neurons via calcineurin-dependent inhibition of activities of NMDA receptor subunits NR2B and NR2C and concomitant inhibitions of NMDA-induced L-type voltage-gated Ca2+ channel activity and intracellular Ca2+ store release [194]. Moreover, in AD pathogenesis, insulin-like growth factor 1 (IGF-1) that acts as a regulator of tau phosphorylation is silenced in activated astrocytes by Aβ/calcineurin-induced release of IGF-1-binding protein 3 (IGFBP-3); but intriguingly, Aβ directly induces increases in tau phosphorylation, and resulting neuronal death, via a mechanism involving the silencing of NFAT export kinase GSK-3β [195], suggesting opposite roles for calcineurin in AD pathogenesis. IGF-1 also protected motor neurons in SOD1 transgenic mice, a widely used model of amyotrophic lateral sclerosis, via a calcineurin-dependent mechanism [196]. Although it is now widely accepted that therapeutic benefits of IGF-1 treatment in neurodegenerative conditions may emerge partly from calcineurin-dependent inhibition of glial inflammatory reaction mediated by preventing TNF-α-induced nuclear translocation of NF-κB [197–199], it is also clear that the expression of TNF-α, resulting in detrimental neuroinflammation and functional alterations in neurons, is mediated by astrocytic and microglial calcineurin/NFAT signaling [200–202]. Studies in transgenic mice and in vitro models of neuroinflammatory diseases provided mechanistic insights into the context of these opposite roles. These studies showed that TNF-α activate calcineurin/NFAT/NF-κB canonical inflammatory pathway in quiescent astrocytes, while in activated astrocytes, IGF-1 released locally recruited calcineurin signaling to inhibit NF-κB-NFAT transcriptional activity through activation of the purinergic receptor P2Y6 [203, 204], suggesting that the activation status of the cell is an important determinant of calcineurin/NFAT activity, that is, downstream targets. 5.1.3. Pharmacological Inhibition and Endogenous Regulation The pharmacological inhibition of calcineurin/NFAT signaling improved animal condition in a number of studies in neurodegenerative diseases and models. Examples include AD mouse models where pharmacological inhibition of this signaling pathway decreased Aβ plaques, reduced glial activation, alleviated both Aβ synaptotoxicity and neurotoxicity, and improved synaptic function [25, 205–207], suggesting a therapeutic potential for calcineurin inhibitors in AD. Reports by Kim and colleagues from studies performed in presenilin 1-mutant model of AD provided some mechanistic insights into the cognitive decline improvement resulting from reducing calcineurin activation in affected brains [25, 208]. These authors observed that the inhibition of abnormally increased calcineurin activity characteristic of the disease resulted in the stabilization of the phosphorylation of GluA1, a subunit of Ca2+-permeable AMPA receptors, and promoted synaptic trafficking of Ca2+-permeable AMPA receptors, as well as the resulting improvement in animal cognition [25]. Such improvement resulted at least partly from the restoration of Ca2+-permeable AMPA receptor-mediated hippocampal LTP [208]. Decrease in calcineurin complexes with transmembrane AMPA receptor regulatory proteins (TARPs) like γ-8, which can stop the trafficking of both AMPA and to a lesser extent NMDA receptors [209–211], may also participate in this process. Furthermore, biometal mediated neurite elongation and neuritogenesis in neuron cultures via calcineurin silencing [111] and calcineurin/NFAT signaling induced a reduction in NGF expression and neurite outgrowth in rat neonatal ventricular cardiomyocytes and cultured sympathetic neurons [212, 213]. Not surprisingly considering the aforementioned observations in studies assessing the roles of calcineurin in neurodegenerative disorders (Section 5.1.2), endogenous regulators like plasma membrane calcium ATPase (PMCA) and RCAN1 mediate opposite effects in inflammatory processes. RCAN1 overexpression was reported to be pivotal in the prevention of sepsis and LPS-induced lethality [214] and in the protection against brain ischemia/reperfusion injury in murine models [215]. However, interactions of PMCA and vascular endothelial growth factor (VEGF), which dampened calcineurin/NFAT signaling, also induced the overexpression of both the counter-inflammatory factor RCAN1.4 and the proinflammatory factor COX-2 in activated murine endothelia [214, 216, 217]. Additionally, RCAN1 overexpression increased the susceptibility to oxidative stress in primary neurons [218] and exacerbated Ca2+ overloading-induced neuronal apoptosis [219], suggesting that the overexpression of calcineurin regulator RCAN1 may link Ca2+ overloading and oxidative stress in neurodegenerative disorders (Figure 1). Such detrimental effects of RCAN1 were mediated by RCAN1.4, and not by the other isoforms detected in human brain RCAN1.1 [91, 219], probably via PI3K/Akt/mTOR signaling [220, 221]. Such mechanistic insight was provided by a system study based on combinations of single-cell experimentation and in silico simulations where RCAN1 effect on inflammation mediated by calcineurin/NFAT appeared to change according to cellular levels, from inhibitory activity at low levels to facilitative activity at high levels [221]. Notably, in that study RCAN1 facilitative activity was switched on by nuclear export of GSK-3β, indicating that targeting the factors involved in this inhibitory mechanism of GSK-3β-mediated NFAT nuclear export may have a therapeutic potential in neurodegenerative diseases. Considering that RCAN1 overexpression is a hallmark of Down syndrome [219, 222], it can be hypothesized that this event also contributes to the pathogenesis of AD-like neuropathology typically observed in Down syndrome patients after their middle age [223, 224]. In addition, the activity of pituitary adenylate cyclase-activating peptide (PACAP), a neurotrophic peptide involved in nervous system development, learning, and memory, was significantly disturbed by changes in RCAN1 expression [225]. RCAN1 overexpression impaired neurotrophic support of sympathetic neurons by inhibiting TrkA endocytosis, resulting in NGF signaling silencing and associated neurodevelopmental deficits [92]. Furthermore, overexpression of RCAN1 or dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A), another Down syndrome-associated protein, negatively regulated NFAT-dependent transcriptional activity and decreased NGF-mediated upregulation of PAI-1 levels [131], a key synaptogenic mechanism. Intriguingly, deficiency of RCAN1.1, but not RCAN1.4, affected radial migration of rat cortical neurons and caused periventricular heterotopia [226], suggesting that this RCAN1.1 isoform may mediate positive RCAN1 effects in developing cortex. Future studies using selective activations and inhibitions of RCAN1 isoforms may reveal the mechanisms accounting for isoform-specific effects in the developing cortex. 5.2. Neurodegenerative Conditions and Other Nervous System Diseases 5.2.1. Neurodegenerative Conditions Alterations in the autophagy of mitochondria, the process that normally triggers damaged organelle elimination, are common in neurodegenerative diseases and conditions [227–230]. In a study using axotomized precerebellar neurons, a model of focal cerebellar lesion-induced remote degeneration, rapamycin-mediated autophagy, resulted in an aberrant mitochondrial fission partly caused by increased calcineurin activity [227]. The activity of a calcineurin docking motif present in the mitochondrial fission mechanoenzyme dynamin-related protein 1 (Drp1) contributed to mitochondrial fragmentation and ischemic neuronal injury in neuronal and nonneuronal cells [163]. Additionally, calcineurin inhibitors mitigated mitochondrial fragmentation in ferric ammonium citrate-exposed HT-22 hippocampal neurons, a model of iron overload and neurodegeneration [231]. Further evidence for calcineurin involvement in ischemic injury includes reports suggesting that abnormal increases in the activity of this phosphatase, mediated by disturbances in axonal Ca2+ homeostasis, may play a key role in secondary damage of neurons and capillary vessels observed during acute phase of diffuse axonal injury [173, 232]. Calcineurin signaling also mediated the activation of the cytoskeletal actin severing protein cofilin and the resulting neuronal death in oxygen-glucose deprivation/reperfusion and chemical induced oxidative stress, to in vitro models of ischemia [34]. Moreover, cyclosporine A prevented the apoptosis of astrocytes exposed to simulated ischemia in vitro via a calcineurin and Erk1/2-dependent mechanism [233] and through the inhibition of cytosolic phospholipase A2- (PLA2-) mediated release of arachidonic acid [234]. Selective calcineurin signaling in neurons and astrocytes is a key player in neurodegenerative conditions. An early study addressing neuronal apoptosis induced by the abused psychostimulant methamphetamine revealed pivotal roles for calcineurin activation and resulting Fas ligand upregulation mediated by nuclear translocations of NFATc3 and NFATc4 in rats [235]. The potent hepatotoxin microcystin-LR (MCLR) mediated an upregulation of calcineurin and NFATc3 levels in rat hippocampal neurons that resulted in marked increases in apoptotic and necrotic cell death [32]. MCLR effect was prevented by FK506 treatment. In addition, NFATc4 mediated light-induced retinal ganglion cell apoptosis by upregulating Fas ligand (FasL) expression on retinal neurons [236], and the overactivation of calcineurin/NFATc3 signaling induced the typical neuronal toxicity and functional alterations observed in murine developing hippocampal neurons following the inhalation of anesthetic isoflurane, including cognitive impairment [237]. A recent study in this model of postoperative cognitive dysfunction revealed that abnormal calcineurin/NFAT signaling associated with isoflurane exposure may mediate its detrimental effects by promoting the degradation of the survival molecule signal transducer and activator of transcription 3 (STAT3) [238]. Moreover, calcineurin/NFATc3 signaling in activated astrocytes played a key role in the induction of alterations in synaptic remodeling and homeostasis observed in the hippocampus in controlled cortical impact injury in rats [39, 128, 239]. As expected, calcineurin inhibition restored synaptic function and plasticity in the latter murine model of traumatic brain injury and in murine models of traumatic spinal cord injury [172–174] partly by abrogating astrocyte activation and reactive gliosis, which are pivotal events in neuroinflammation-mediated neuronal loss. Notably, calcineurin/NFAT signaling is critical for astrocyte activation [205, 240]. In another study in ischemic striatum and cortex and in cultured astrocytes where FK506 also induced neuroprotective effects, the calcineurin inhibitor prevented astrocyte apoptosis mediated by glutamate signaling [241]. Altogether, these observations suggest a role for calcineurin/NFAT signaling in astrocyte and neuronal losses observed in nervous system injury. Unexpectedly, sublethal ischemia increased neuronal resistance to excitotoxicity via calcineurin-dependent mechanisms including cyclin E1 protein increased expression and declustering of the delayed rectifying K+ channel Kv2.1 at highly phosphorylated somatodendritic clusters [242]. In addition, preconditioning of neurons with biometal ions (Cu2+, Zn2+) protected these cells against NMDA receptor-induced excitotoxicity, through metal chaperone PBT2-induced calpain cleavage of calcineurin [111, 112]. Thus, calcineurin/NFAT pathway participates in the interplay between proinflammatory and counter-inflammatory signals in the nervous system, further suggesting that unraveling the downstream targets accounting for beneficial and neurotoxic effects of this signaling pathway may have a therapeutic potential in neurodegenerative conditions. 5.2.2. Psychotic Disorders Early genetic studies showed that polymorphisms of the genes coding for either the catalytic or regulatory subunit of calcineurin isoenzymes are strongly associated with the risk for developing schizophrenia and other psychotic disorders whose pathological features include disturbances in Ca2+ signaling [46, 243–246]. A more recent genome-wide weighted coexpression network analysis on neural progenitors and neurons from individuals with Timothy syndrome, an autism spectrum disorder resulting from mutations in the gene encoding L-type CaV1.2 Ca2+ channels, suggested that the disease may be caused by disturbances in transcriptional activities of Ca2+-dependent signaling molecules like FOX proteins, MEF2, CREB, and NFATs [247]. Furthermore, GABA A receptor activation promoted a decrease in anxiety indicators and hippocampal neurogenesis via the calcineurin/NFAT4 signaling in mice, suggesting that pharmacological targeting of this signaling pathway may improve emotional disorders [248]. Similar evidence from pharmacological and postmortem studies suggests that treatment with antipsychotics aimed at ameliorating some of the symptoms of the CNS disorders leads to alterations of the calcineurin expression pattern in the human brain [249–253]. Decreased calcineurin levels in the nucleus accumbens were reported in opioid withdrawal, a dysphoric state associated with complications in patient pain and increased risk of drug abuse and addiction [30], suggesting a role for calcineurin in long-lasting behaviors associated with reward. Furthermore, in a patch-clamp electrophysiology and fast-scan cyclic voltammetry study in mouse brain slices, the endogenous modulatory peptide neurotensin induced a long-term prevention of pathogenic increases in presynaptic dopamine release, characteristic of schizophrenia and other severe mesencephalic pathologies, by increasing inhibitory D2 dopamine autoreceptor function via a calcineurin-dependent mechanism [35]. 5.2.3. Epilepsy Many lines of evidence also support calcineurin involvement in nervous system diseases. Notably, various reports have suggested that calcineurin is likely to mediate physiological and pathological activities of GABA receptors. For instance, complex interactions between PKC and calcineurin may play a key role in GABA B autoreceptor-mediated functional regulation of nicotinic acetylcholine receptors (nAChRs), whose activation triggers the release of neurotransmitters from presynaptic nerve terminals, in mouse striatal GABAergic nerve terminals [254]. Somatic modulation of GABA A receptor-mediated fast inhibitory signaling in epileptiform activity was induced by calcineurin signaling in low-magnesium model of seizure in rat hippocampal neurons [31], suggesting a role of calcineurin in benzodiazepine resistance and the potential of its pharmacological targeting in status epilepticus. Certainly, calcineurin/NFAT signaling involvement was also shown in pathogenic processes of other models of status epilepticus, including intracerebral injection of kainic acid [255], bicuculline [256], and pilocarpine [33]. Notably, calcineurin inhibitor ascomycin mediated anticonvulsant and neuroprotective effects, in different epilepsy models, including picrotoxin and latrunculin A models [257–259]. 5.3. Emerging Challenge: Better Models? A growing number of studies are raising concerns about mechanistic reports of Ca2+-dependent and other signaling pathways from currently used models of neurodegenerative diseases, in particular transgenic animals and cell lines. A study addressing the suitability of rat striatal primordia-derived ST14A cell line for the study of voltage-gated Ca2+ channel of striatal medium spiny neurons called for serious caution on the assumption of the presence of complete signaling cascades of G-protein coupled receptors in cell lines [260]. Notably, ST14A cells were reported to lack PLC-β1, a major effector of G-proteins for Ca2+ release from intracellular stores [59, 82], whose roles include (i) the regulation of forward locomotion in wild-type mice, among other dopamine receptor functions [261]; (ii) the mediation of Ca2+ flux required for mammalian sperm acrosome reaction [262]; and (iii) the mediation of the positive regulation of osteoblast differentiation [263]. In addition, most observations from studies in preclinical models of neurodegenerative diseases based on exogenous neurotoxins are not confirmed by clinical studies [264–266]. Similar observations emerge from studies in transgenic animals, where complex functional adaptations from gene knock-in or knock-out may limit the translational importance of findings [267–269]. In the case of neurodegenerative conditions, at least some of the controversy regarding changes in calcineurin/NFAT activity and/or expression appears to be due to the methodologies employed to measure activities and expression levels. Calcineurin, in particular, is highly sensitive to proteolysis during injury and neurodegeneration. However, most commercial antibodies to calcineurin only detect full-length calcineurin and miss the detection of high activity proteolytic fragments. Also, many studies have tended to measure calcineurin activity in whole brain tissue homogenates using commercially available phosphatase assays. While these kinds of assays are very good for kinetic analyses, they are very poor at assessing endogenous calcineurin activity toward endogenous substrates. Certainly, methodological challenges may have affected the quality of data generated by different groups. Although it appears that better models are needed, we also propose that better characterization of intracellular signaling in currently available and future experimental models may improve the translational importance of the findings. 6. Concluding Remarks Calcineurin/NFAT pathway is pivotal during nervous system development and in various functions of mature central and peripheral nervous system. Notably, this signaling pathway is involved in myelination, corticogenesis, synaptogenesis, neuritogenesis, endosome trafficking, homeostatic synaptic plasticity, learning, and memory. Experimental evidence also shows that alterations in the activity of calcineurin/NFAT pathway and in activities of its endogenous regulators in the nervous system microvascular endothelial cells, astrocytes, microglia, Schwann cells, oligodendrocytes, and neurons participate in the pathogenesis of neurodegenerative diseases and conditions, but also psychotic disorders. Studies in transgenic animals and in cell lines also suggested that neurodegeneration-associated detrimental changes in calcineurin/NFAT signaling are NFAT isoform-selective as changes in NFATc3 and NFATc4, but not NFATc1 or NFATc2, are usually common. In addition pharmacological inhibition mitigated neuronal and astrocyte loss and improved cognitive functions in many models. However, other studies reported beneficial roles of calcineurin/NFAT in neurodegenerative diseases and conditions, in particular those reporting neurotoxic effects of pharmacological inhibition and increased endogenous regulation. These studies suggested that calcineurin can mediate both neuroprotective and neurodegenerative signals according to poorly understood determinant factors, which included the activation status of astrocytes in the central nervous system. Future studies should be devised to characterize better the factors determining the outcome of calcineurin/NFAT signaling in neurodegenerative diseases and conditions, as well as the downstream targets mediating the beneficial and detrimental effects of this signaling pathway, considering the implications for therapy. Competing Interests Authors have no conflict of interests. Figure 1 Calcineurin/NFAT signaling pathway. Calcineurin/nuclear factor of activated T-lymphocytes (NFAT) signaling activation in the nervous system is mainly induced by neurotrophins via their tyrosine kinase receptors, glutamate receptors, and nonligand-dependent receptors, such as voltage-gated Ca2+ channels in hippocampal neurons. The genes transcribed and the effects of these signaling pathways are cell-type dependent. Abbreviations: AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; BDNF, brain-derived neurotrophic factor; GSK3, glycogen synthase kinase 3; IGF1R, insulin-like growth factor 1 receptor; IP3R, inositol 1,4,5-trisphosphate receptor; NF-κB, nuclear factor kappa B; NFATc, Ca2+-regulated NFATs; NGF, neurotrophin nerve growth factor; NMDA, N-methyl-D-aspartic acid; RCAN1, regulator of calcineurin 1; RyR; ryanodine receptor; TrkA, tropomyosin receptor kinase type 1; TrkB, tropomyosin receptor kinase type 2. ==== Refs 1 Berridge M. J. Bootman M. D. Roderick H. L. Calcium signalling: dynamics, homeostasis and remodelling Nature Reviews Molecular Cell Biology 2003 4 7 517 529 10.1038/nrm1155 2-s2.0-0038125598 12838335 2 Cárdenas A. M. Marengo F. D. 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==== Front Case Rep Oncol MedCase Rep Oncol MedCRIONMCase Reports in Oncological Medicine2090-67062090-6714Hindawi Publishing Corporation 10.1155/2016/6968534Case ReportSpontaneous Rupture of Hepatic Metastasis from Pancreatic Adenocarcinoma http://orcid.org/0000-0002-1951-447XRahul Anil * Robin Fernandes Adarsh Hiremath Internal Medicine, MD Anderson Cancer Center, 1400 Pressler Street, Houston, TX 77030, USA*Anil Rahul: drrahulanil@gmail.comAcademic Editor: Jorg Kleeff 2016 15 8 2016 2016 696853419 5 2016 12 7 2016 14 7 2016 Copyright © 2016 Anil Rahul et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.A 58-year-old man with advanced-stage pancreatic adenocarcinoma presented with fatigue and dyspnea. Examination revealed tachycardia (102 b/min) with mild tenderness in right upper quadrant. His hemoglobin (Hb) was 7.9 g/dL (10 days prior to presentation 12.2 g/dL), International normalized ratio (INR), platelet count was normal, and the stool guaiac test was negative. On admission, abdominal computed tomography (CT) scan showed hepatic metastatic lesion with a rupture and hemoperitoneum communicating to the subdiaphragmatic space. This rapid progression of anemia along with presenting symptoms and CT imaging were attributed to diagnosis of spontaneous rupture of liver metastasis from pancreatic adenocarcinoma. Patient received blood transfusion and hemoglobin was monitored in successive intervals. His general condition and anemia improved with conservative management and he was discharged in 3 days. Repeated CT after 4 months showed resolving hemoperitoneum and stable hemoglobin levels. The patient deceased 9 months after being diagnosed. A literature search revealed limited data regarding the incidence and management of spontaneous rupture of metastatic lesion secondary to pancreatic adenocarcinoma which has been managed conservatively and thus we are reporting our experience. ==== Body 1. Introduction Spontaneous ruptures secondary to malignant liver metastasis are uncommon occurrences that clinically present with similar symptoms/signs as the more common spontaneous rupture of liver secondary to hepatocellular carcinoma (HCC). HCC is consistently reported to have a poor prognosis and, due to its prevalence, continues to be the most lethal and life-threatening hallmark of advanced disease, attributing to 25–75% mortality in the acute phase HCC [1]. Hepatic metastatic rupture, on the other hand, has a comparably poorer prognosis owing to confounding clinical presentation and delayed diagnosis. Several invasive interventional methods have been developed to prevent fatal sequelae of intraperitoneal hemorrhage. Thus, these invasive interventions have become the most prevalent methods of treatment, far surpassing conservative therapies. However, the interventional strategies do not significantly improve median survival and result in recurrent complications and interruptions to chemotherapy. Herein, we present a rare case of successful conservative management of spontaneous rupture of liver metastasis secondary to advanced pancreatic adenocarcinoma with no delay in resuming immunotherapy for cancer. 2. Case Report A 58-year-old man presented with a 2-week history of fatigue and dyspnea. Two years prior to the presentation, he was diagnosed with pancreatic adenocarcinoma in the body and tail which subsequently metastasized to the liver, left lung, 6th cervical vertebra (c-6), splenic artery, ascitic fluid, and diaphragm which was confirmed by biopsies at aforementioned sites. In the interim, he underwent several lines of chemotherapy, radiation, and molecularly targeted therapy. Surgical procedures included wedge resection of the left lung lesions, cervical corpectomy and fusion, abdominal paracentesis, and gastrostomy tube placement. Past medical history includes type 2 diabetes mellitus and tubulovillous adenoma for which he underwent polypectomy. On admission, the patient was in minimal distress with tachycardia (102 beats/min), blood pressure of 99/62 mmHg, and mild tenderness in the right upper quadrant of abdomen. Laboratory data revealed hemoglobin (Hb): 7.9 g/dL; hematocrit (Hct): 24.9%; platelet count: 210,000/mm3; prothrombin time (PT): 14 s; INR: 1.08; activated partial thromboplastin time: 27.7 s; stool guaiac test that was negative; LFT's and RFT's that were within normal limits. 10 days prior to this presentation, his Hb and Hct levels were 12.2 g/dL and 37.7%, respectively, suggesting rapid progression of anemia. Abdominal contrast enhanced CT was immediately performed and revealed an enlarging hepatic metastatic lesion (78.9 × 68.8 mm) with a rupture (Figure 1) and hemoperitoneum (126.1 × 51.4 mm) with communication to the subdiaphragmatic space (Figure 3). Previous CT imaging showed a hepatic metastatic lesion measuring (56.0 × 64.4 mm) (Figure 2). Follow-up imaging revealed resolving hemoperitoneum (Figure 4). Considering the guarded condition and vitals being the lower limits of normalcy, the medical team consisting of a hospitalist, interventional radiologist, and oncologist decided upon managing the patient's treatment conservatively. The patient was given a blood transfusion; Hb and Hct were monitored serially in successive intervals during hospitalization. Subsequently, his general condition and anemia improved (Hb to 9.9 g/dL and Hct to 30.9%). Throughout his hospitalization, the patient's condition was stable and improved solely with conservative management. Since this approach was undertaken, there was no undue delay in resuming the patient's immunotherapy for pancreatic cancer. Repeated CT after 4 months showed resolving hemoperitoneum and stable Hb. The patient deceased 9 months after being diagnosed with hemoperitoneum secondary to metastatic hepatic rupture. 3. Discussion Pancreatic cancer is one of the deadliest cancers, evidenced by the fact that its mortality equals its incidence [2]. Early diagnosis and treatment are limited for this condition because of the tumor's aggressively invasive nature and early metastatic properties. Pancreatic cancer metastasizes early in its course and liver is the most common site for distant metastasis, followed by the peritoneal cavity [3]. Massive hemorrhage related to ruptured liver metastases is quite exceptional and less than 50 cases are reported in the literature. Spontaneous rupture of a metastatic liver tumor is rare and uncommon when compared to hepatic rupture of primary lesion (HCC) leading to hemoperitoneum, which is a devastating complication of both primary and metastatic hepatic tumors [4, 5]. Pivotal clinical features of hemoperitoneum indicating hepatic metastatic rupture include history of malignancy, abdominal pain, hypotension, severe anemia, elevated liver enzymes, and, in extreme cases, surgical abdomen. Choi et al. reported that peripheral location of the tumor, protruding, contour, discontinuity of hepatic surface, and surrounding hemoperitoneum are helpful diagnostic indicators of ruptured HCC [6]. The aforementioned clinical and imaging (CT) findings helped us to yield to a diagnosis of metastatic hepatic rupture in our patient. Treatment of hemoperitoneum secondary to spontaneous rupture of metastatic liver tumor depends on several factors, including tumor size, location, and severity of exsanguination. The major objective of treatment is to control the hemorrhage quickly and effectively; this can be accomplished by hepatic wedge resection/lobectomy or suture ligation of the bleeding source/hepatic artery. Recent studies suggested that a two-staged therapeutic approach in managing ruptured hepatic lesion consists of initial management with conservative approach, hemostasis achieved via transarterial embolization or surgery, followed by staged hepatic resection [7]. Transcatheter hepatic arterial embolization (TAE) may seem to be ideal for these patients, due to its various merits. Greatest benefit of TAE would be being done under local anesthesia and less invasive nature of the procedure [8]. That said, it also has some demerits, like recurrent bleeding and liver failure [8, 9], peritoneal abscess [10], implanted metastases, and patients who need to have preserved liver functions (not beyond Child-Pugh B) [11]. In addition, long-term results are poor if it is used as a solitary treatment approach without being followed by surgery [12]. On the other hand, even though surgery showed better mortality benefit [13], it has numerous risk like infections, and secondary bleeding, high morbidity [14], and last but not the least it puts a far greater toll on inveitable interruptions in chemo/immunotherapies, increase in hospital stay and cost. However, conservative treatment focuses on achieving hemostatsis by correcting coagulopathy, close monitoring, and follow-up medical imaging to confirm hemostasis after initial resuscitation [9]. A study by Hsueh et al. showed patients who received hepatectomy, either immediate or staged after posttransarterial embolization, and reported higher survival rates of 85.2% at 30 days and 62.2% at 1 year. By comparison, similar populations treated conservatively exhibited a reduction in liver function, prolonged INR, and increased 30-day mortality [15]. Contrary to the above study Leung et al. in their retrospective study on 112 patients with ruptured HCC, comparing the in-hospital mortality and median survival in patients treated with conservative and surgical approach, concluded that the conservative approach gave similar results to that of surgical approach [(62% versus 51%) and (7 days' versus 12 days) resp.] [16]. Having arrived at the diagnosis, with consideration of the risk factors in this patient, we agreed on managing the patient's treatment conservatively despite the odds of poor prognosis, and he responded very well and was discharged home in stable condition to resume immunotherapy for pancreatic cancer without any interruptions. 4. Conclusion In conclusion, we convey that spontaneous metastatic liver rupture secondary to advanced pancreatic adenocarcinoma is rare and has a poor prognosis. Clinical findings and CT are of great help in making a quick diagnosis of hemoperitoneum due to metastatic liver rupture. Although the literature supports invasive intervention (extrapolating from data for HCC) in advanced-stage pancreatic cancer, conservative treatment alone (if medically stable) can be employed to combat the sequelae of intraperitoneal hemorrhage and improve survival with fewer complications and minimal interruption or delays in necessary chemotherapy or cancer-directed treatments. Competing Interests All the authors declare no competing interests. Figure 1 Axial contrast enhanced CT image showing metastasis in liver dome (large yellow arrow) had grown since earlier study (Figure 2). Figure 2 Previous CT (contrast enhanced) showing liver metastases (yellow arrow) smaller at that time and no perihepatic fluid. Figure 3 Axial contrast enhanced CT image (same study as in Figure 1) showing (yellow arrow) subcapsular liver collection. Figure 4 Axial contrast enhanced CT obtained 4 months after study in Figures 1 and 3 showing decreasing size of subcapsular liver collection. ==== Refs 1 Aoki T. Kokudo N. Matsuyama Y. Prognostic impact of spontaneous tumor rupture in patients with hepatocellular carcinoma: an analysis of 1160 cases from a nationwide survey Annals of Surgery 2014 259 3 532 542 10.1097/sla.0b013e31828846de 2-s2.0-84894076208 23478524 2 Siegel R. Ma J. Zou Z. Jemal A. Cancer statistics, 2014 CA Cancer Journal for Clinicians 2014 64 1 9 29 10.3322/caac.21208 2-s2.0-84892805731 3 Yachida S. Jones S. Bozic I. Distant metastasis occurs late during the genetic evolution of pancreatic cancer Nature 2010 467 7319 1114 1117 10.1038/nature09515 2-s2.0-78049398107 20981102 4 Tung C.-F. Chang C.-S. Chow W.-K. Peng Y.-C. Hwang J.-I. Wen M.-C. Hemoperitoneum secondary to spontaneous rupture of metastatic epidermoid carcinoma of liver: case report and review of the literature Hepato-Gastroenterology 2002 49 47 1415 1417 2-s2.0-0036376944 12239954 5 Chen Z.-Y. Qi Q.-H. Dong Z.-L. Etiology and management of hemmorrhage in spontaneous liver rupture: a report of 70 cases World Journal of Gastroenterology 2002 8 6 1063 1066 10.3748/wjg.v8.i6.1063 2-s2.0-0036899293 12439926 6 Choi B. G. Park S. H. Byun J. Y. Jung S. E. Choi K. H. Han J.-Y. The findings of ruptured hepatocellular carcinoma on helical CT The British Journal of Radiology 2001 74 878 142 146 10.1259/bjr.74.878.740142 2-s2.0-0035088205 11718385 7 Veltchev L. M. Spontaneous rupture of hepatocellular carcinoma and hemoperitoneum-management and long term survival Journal of IMAB 2009 1 2009 3 102 8 Leung C. S. Tang C. N. Fung K. H. Li M. K. W. A retrospective review of transcatheter hepatic arterial embolisation for ruptured hepatocellular carcinoma Journal of the Royal College of Surgeons of Edinburgh 2002 47 5 685 688 2-s2.0-0036813938 12463708 9 Tanaka A. Takeda R. Mukaihara S. Treatment of ruptured hepatocellular carcinoma International Journal of Clinical Oncology 2001 6 6 291 295 10.1007/s10147-001-8030-z 2-s2.0-18244389258 11828948 10 Yokoi Y. Suzuki S. Sakaguchi T. Subphrenic abscess formation following superselective transcatheter chemoembolization for hepatocellular carcinoma Radiation Medicine 2002 20 1 45 49 2-s2.0-0036235517 12002604 11 Yeh C.-N. Chen H.-M. Chen M.-F. Chao T.-C. Peritoneal implanted hepatocellular carcinoma with rupture after TACE presented as acute appendicitis Hepato-Gastroenterology 2002 49 46 938 940 2-s2.0-0036021314 12143247 12 Rossetto A. Adani G. L. Risaliti A. Combined approach for spontaneous rupture of hepatocellular carcinoma World Journal of Hepatology 2010 2 1 49 51 10.4254/wjh.v2.i1.49 2-s2.0-84864068656 21160956 13 Jin Y.-J. Lee J.-W. Park S.-W. Survival outcome of patients with spontaneously ruptured hepatocellular carcinoma treated surgically or by transarterial embolization World Journal of Gastroenterology 2013 19 28 4537 4544 10.3748/wjg.v19.i28.4537 2-s2.0-84880789742 23901230 14 Chen W.-K. Chang Y.-T. Chung Y.-T. Yang H.-R. Outcomes of emergency treatment in ruptured hepatocellular carcinoma in the ED The American Journal of Emergency Medicine 2005 23 6 730 736 10.1016/j.ajem.2005.02.052 2-s2.0-25144477551 16182979 15 Hsueh K.-C. Fan H.-L. Chen T.-W. Management of spontaneously ruptured hepatocellular carcinoma and hemoperitoneum manifested as acute abdomen in the emergency room World Journal of Surgery 2012 36 11 2670 2676 10.1007/s00268-012-1734-6 2-s2.0-84867863303 22864567 16 Leung K. L. Lau W. Y. Lai P. B. S. Yiu R. Y. C. Meng W. C. S. Leow C. K. Spontaneous rupture of hepatocellular carcinoma: conservative management and selective intervention Archives of Surgery 1999 134 10 1103 1107 10.1001/archsurg.134.10.1103 2-s2.0-0032851790 10522855

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==== Front Biomed Res IntBiomed Res IntBMRIBioMed Research International2314-61332314-6141Hindawi Publishing Corporation 10.1155/2016/6861702Research ArticleInteraction Effects between Organochlorine Pesticides and Isoflavones In Vitro and In Vivo http://orcid.org/0000-0001-6792-1451Zhang Yunbo http://orcid.org/0000-0001-6445-6427Guo Jipeng http://orcid.org/0000-0002-2492-6938Zhang Xiao http://orcid.org/0000-0002-3688-7825Guo Jingjing http://orcid.org/0000-0002-5941-9485Zhang Ming http://orcid.org/0000-0002-1946-816XYang Yang http://orcid.org/0000-0003-3891-7490Na Xiaolin * Department of Environmental Hygiene, Public Health College, Harbin Medical University, Harbin, Heilongjiang 150081, China*Xiaolin Na: naxiaolin1495@sohu.comAcademic Editor: Kaiyu He 2016 15 8 2016 2016 686170230 3 2016 25 5 2016 16 6 2016 Copyright © 2016 Yunbo Zhang et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Organochlorine pesticides (OCPs) have caused increasing global concern due to their high toxicity, persistence, bioaccumulation, and significant adverse effects on human health. This study was to explore the interaction effects between OCPs and isoflavones. Six kinds of OCPs and 2 kinds of isoflavones-genistein and daidzein were included to study their effect on MCF-7 cells in vitro. Eighty-one female Sprague-Dawley rats were randomized to 9 groups according to factorial design to study the interaction effect between isoflavones and γ-HCH. Compared to organochlorine pesticides alone group, proliferation rate of MCF-7 cells was lower in 100 μmol/L genistein + organochlorine pesticides and 100 μmol/L daidzein + organochlorine pesticides group (p < 0.05). In vivo study showed that there are interaction effects on kidney weight and liver weight when treated with isoflavones and γ-HCH. The changes in uterine morphology and positive expression of ERα showed inhibition effects between isoflavones and γ-HCH. In conclusion, the data suggests that there are interactions between isoflavones and OCPs in vitro and in vivo. National Natural Science Foundation of China812021903097243981273079Harbin Medical University ==== Body 1. Introduction The research on endocrine-disrupting chemicals (EDCs) has attracted more and more interest of scientists. Organochlorine pesticides (OCPs), which are one of the EDCs, can be lipophilic, stable, difficult to be degraded, volatile, and accumulative. They are easy to be accumulated in environment, and humans can absorb them via food chain enrichment through the digestive tract, respiratory tract, and skin [1, 2]. The OCPs are usually divided into three main groups including DDT and its derivatives (DDTs and DDEs), HCHs, and chlorinated cyclodiene such as chlordane. DDEs are the main bioaccumulative metabolite of the organochlorine insecticide DDT. Exposure of men or animals to OCPs can cause some side effects including reproductive toxicity and even cancer [3, 4]. Soybean isoflavones are a kind of plant estrogen, extracted in soybean, mainly including genistein and daidzein. Genistein is the most frequently studied isoflavone due to its biological activities of improving cardiovascular, bone, and postmenopausal health. Daidzein, a major component of soy with structural similarities to estrogen, can exert effects of anti-inflammatory, lowering lipid levels and increasing mitochondrial biogenesis. Many studies have indicated that the isoflavones or single components (genistein or daidzein) have many important biological activities; for example, they can improve the women's menopause syndrome and prevent osteoporosis. Messina et al. found that higher isoflavones intake was associated with 25% reduction in recurrence in 9,514 breast cancer survivors over 7.4-year follow-up period [5]. Moreover, although the antiproliferation effect in different breast tumor cell types treated with different concentration of genistein and daidzein varied, their effects were related to ERα and c-erbB-2 expression [6]. Breast cancer is the most common and leading type of cancer in women, and the survival rates are very poor in developing countries. Human breast cancer cell line MCF-7 was used as the hormone-responsive breast cancer cell line in many studies. Estrogen can make the pathological progress of mammary gland cells accelerate, which is a major cause of estrogen dependent breast cancer. A large number of studies showed that OCPs could combine with estrogen receptor to cause some side effects including reproductive toxicity [7, 8]. According to Shekhar, p,p′-DDT could evoke responses and further enhance the responses together with estradiol or o,p′-DDT in estrogen receptor-positive breast cancer cells [9]. However, studies showed that both genistein and DDT can be combined with estrogen receptor. High concentrations of genistein can inhibit the proliferation of cells while DDTs promote the proliferation of cells, although the effects of isoflavones on breast cancer remain controversial and human clinical investigations are needed [10]. We can still predict that more phytoestrogen dietary intake may drop the weakness of exogenous estrogen. OCPs affect human health mainly by continuous exposures at low dose [11]. And human can be exposed to isoflavones by daily food. It has practical significance to carry out research on interactions between OCPs and isoflavones. 2. Material and Methods 2.1. In Vitro Experiment 2.1.1. Chemicals β-HCH, γ-HCH, o,p′-DDT, p,p′-DDT, p,p′-DDE, and chlordane were purchased from Dr. Ehrenstorfer GmbH Ltd. Co. (Germany). Genistin (Gen, purity ≥ 99%), daidzein (Dai, purity ≥ 99%), estradiol (E2), and methyl thiazolyl tetrazolium (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO). 2.1.2. Cell Culture and Treatment MCF-7 cells were cultured in a 37°C, 5% CO2 saturated humidity incubator with DMEM (containing 100 IU/mL penicillin/streptomycin and 10% FBS). The cells had digestive transfer culture after being grown to 80% cell density. Discard DMEM and rinse it two times by PBS (pH 7.4) solution. 0.05% trypsin (1 mL) was added to cover cell layer for 2-3 minutes. After discarding dispersed liquid, 5 mL DMEM was added in culture bottle. Cells were made into single cell suspension and placed into culture flask at 37°C. 2.1.3. MTT Experiment MCF-7 cells were cultured in phenol red-free DMEM (Sigma-Aldrich) with 100 IU/mL penicillin/streptomycin and 10% charcoal stripped estrogen-free FBS (Invitrogen). Cells were treated with the following solutions: (i) DMEM medium alone (the control group); (ii) 100 μmol/L genistein group; (iii) 100 μmol/L daidzein group; (iv) 1 μmol/L o,p′-DDT, 1 μmol/L p,p′-DDT, 1 μmol/L γ-HCH, 1 μmol/L chlordane, 10 μmol/L p,p′-DDE, and 20 μmol/L β-HCH; (v) 100 μmol/L genistein + 1 μmol/L o,p′-DDT, 100 μmol/L genistein + 1 μmol/L p,p′-DDT, 100 μmol/L genistein + 1 μmol/L γ-HCH, 100 μmol/L genistein + 1 μmol/L chlordane, 100 μmol/L genistein + 10 μmol/L p,p′-DDE, and 100 μmol/L genistein + 20 μmol/L β-HCH; (vi) 100 μmol/L daidzein + 1 μmol/L o,p′-DDT, 100 μmol/L daidzein + 1 μmol/L p,p′-DDT, 100 μmol/L daidzein + 1 μmol/L γ-HCH, 100 μmol/L daidzein + 1 μmol/L chlordane, 100 μmol/L daidzein + 10 μmol/L p,p′-DDE, and 100 μmol/L daidzein + 20 μmol/L β-HCH. Cell growth and inhibition rate was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded in 96-well plates (cell density of 3 × 104/mL) for 24 h and exposed to various concentrations of genistein, daidzein, or OCPs for 48 h. Then the cells were incubated with 20 μL 5 mg/mL MTT in the dark for 4 h at 37°C. Absorbance values (optical density, OD) were obtained using a microplate reader (model 680: Bio-Rad Laboratories, Inc., Hercules, CA, USA) at a wavelength of 490 nm. Wells containing cells in DMEM were served as the normal control. To convert OD values to proliferation rate, the following equation was used: proliferation rate (PR) = OD of test concentration/OD of control ∗ 100%. Each concentration was measured in 3–5 repeated wells, and every assay was tested ≥3 times. 2.2. In Vivo Experiment 2.2.1. Chemical Isoflavones were purchased from Rongsheng Ltd. Co. (Xi'an, China) with purity ≥98%. γ-HCH was purchased from Qinchengletter Ltd. Co. (Beijing, China) with purity of 99.4%. 2.2.2. Animals Prior to study initiation, the experimental protocol was reviewed and approved by the Committee on Animal Research and Ethics of Harbin Medical University (Harbin, China). Eighty-one female Sprague-Dawley (SD) rats, weighing 60–80 g, were purchased from Weitonglihua Experimental Animal Ltd. Co. (Beijing, China, Batch number SCXK (jing) 2012-0001). The animals were caged in a room maintained at 23 ± 2°C. After adaption for 1 week, rats were randomly divided into 9 groups in single cage. Each group was divided by 3∗3 factorial design with different dose of isoflavones and γ-HCH (Table 1). Isoflavones were added into diet. γ-HCH was dissolved with corn oil and administrated by intragastric administration. SD rats were fed in the standard animal laboratory for 4 weeks. At the end of the experiment, rats were sacrificed after being fasted for 12 h. Body organs such as brain, liver, kidney, spleen, and the parametrial and perirenal fat pads of rats were dissected and weighed. Blood was collected from the sacrificed animals and serum was separated from the blood (3,000 rpm, 15 min) for the determination of estradiol and testosterone using radioimmunoassay and serum glucose using automatic biochemical analyzer (Hitachi 7100, Japan). The reagent kits were bought from Northern Institution of Biotechnology (Beijing, China). Histopathological examination was performed on 5 μm paraffin sections with standard hematoxylin-eosin (HE) staining. The examination was performed blindly. Immunohistochemistry was performed to investigate the expression of ERα in uterus with ERα antibody from Zhongshan Technology Ltd. Co. (Beijing, China). The representative pictures were digitized by inverted microscope (Nikon Ti-S, Japan). The immunohistochemistry staining was quantified by using Image-Pro Plus software (Media Cybernetics, USA). 2.3. Statistics The statistical analyses were performed with the SPSS program. Data was expressed as means ± SD. Data in this work were normally distributed determined with the Kolmogorov-Smirnov test. The data of cell culture study was analyzed with one-way ANOVA. Animal study was analyzed using a two-way ANOVA with isoflavones and OCPs treatments as factors, followed by a post hoc test with Fisher's least significant difference (LSD). p value < 0.05 was considered significant for all statistical analyses. 3. Results 3.1. In Vitro The interaction of genistein and daidzein with OCPs in MCF-7 cells was shown in Figure 1. Compared to control group, OCPs could increase proliferation rate of MCF-7 cell line while genistein and daidzein could inhibit the growth of MCF-7 cell observably (p < 0.05). Compared with OCPs alone group (o,p′-DDT, p,p′-DDT, β-HCH, and γ-HCH), proliferation rate of MCF-7 cells was lower in both 100 μmol/L genistein + OCPs group and daidzein + OCPs group (p < 0.05). Furthermore, proliferation rate of MCF-7 cells in genistein treatment group was lower than daidzein treatment group in o,p′-DDT, p,p′-DDT, and β-HCH group, higher in γ-HCH group. 3.2. In Vivo 3.2.1. Effect of Isoflavones and γ-HCH on Body Weight Effect of isoflavones and γ-HCH on body weight was shown in Figure 2. Rats grew normally and the body weight increased step by step from beginning of experiment to the end. Two-way ANOVA indicated that no significant differences were found in rats' body weight treated with different dosage of isoflavones and γ-HCH (p > 0.05). 3.2.2. Interaction of Isoflavones and γ-HCH The main effects of isoflavones and γ-HCH and their interaction were shown in Table 2. According to Table 2, the effect of isoflavones treatment on fasting blood glucose was significant (p = 0.022); that is, serum glucose levels in 600 mg/kg diet isoflavones treatment groups were increased markedly compared to 0 mg/kg diet isoflavones treatment groups (6.1 ± 0.2 mmol/L in 900 mg/kg diet treatment groups, 6.8 ± 0.2 mmol/L in 600 mg/kg diet treatment groups, and 6.2 ± 0.2 mmol/L in 0 mg/kg diet treatment groups). No main effect of γ-HCH and interaction effects were found in serum fasting blood glucose. In addition, the main effect of γ-HCH in serum estradiol and testosterone levels was prominent; that is, γ-HCH could reduce serum level of estradiol and testosterone notably (estradiol: 33.64 ± 2.39 pg/mL in 8 mg/kg bw, 36.45 ± 2.39 pg/mL in 4 mg/kg bw, and 45.37 ± 2.51 pg/mL in 0 mg/kg bw; testosterone: 1.45 ± 0.22 ng/mL in 8 mg/kg bw, 1.66 ± 0.22 ng/mL in 4 mg/kg bw, and 2.30 ± 0.22 ng/mL in 0 mg/kg bw). No main effect of isoflavones and interaction effects were found. Furthermore, both isoflavones and γ-HCH could alter spleen weight significantly. Isoflavones had a tendency of increase, while γ-HCH could reduce spleen weight (data not shown). Overall, there are no interactions between isoflavones and γ-HCH except kidney (p = 0.047) and liver weight (p = 0.033). The changes of liver weight in isoflavones 900 mg/kg diet group were different from isoflavones 600 mg/kg diet along with the increase of γ-HCH dose. In isoflavones 600 mg/kg diet group, liver weight was increased significantly with the increase of γ-HCH dose (γ-HCH 8 mg/kg bw group: 7.36 ± 0.69 g; γ-HCH 4 mg/kg bw group: 6.97 ± 1.19 g; γ-HCH 0 mg/kg bw group: 6.55 ± 0.84 g). However, in isoflavones 900 mg/kg diet, liver weight was highest in γ-HCH 8 mg/kg bw group (7.70 ± 0.52 g) and lowest in γ-HCH 4 mg/kg bw group (6.80 ± 0.32 g). In addition, the changes of kidney weight in γ-HCH 8 mg/kg bw were different from γ-HCH 4 mg/kg bw along with the increase of isoflavones dose. In γ-HCH 8 mg/kg bw group, kidney weight was highest in isoflavones 600 mg/kg diet group (900 mg/kg diet: 1.55 ± 0.14 g; 600 mg/kg diet: 1.74 ± 0.13 g; 0 mg/kg diet: 1.54 ± 0.13 g). But in γ-HCH 4 mg/kg bw group, kidney weight was highest in isoflavones 0 mg/kg diet group (900 mg/kg diet: 1.63 ± 0.14 g; 600 mg/kg diet: 1.63 ± 0.27 g; 0 mg/kg diet: 1.69 ± 0.18 g). 3.2.3. Interaction of Isoflavones and γ-HCH on Uterine Morphology To make the comparison simpler and easier, only uterine morphology in groups 1, 3, 7, and 9 was analyzed. The interaction of isoflavones and γ-HCH on uterine morphology was shown in Figure 3. Compared with control group, tissue morphology was changed significantly in isoflavones treatment group and γ-HCH treatment group. High columnar endometrial glandular epithelial cells were found in isoflavones treatment group, while low columnar endometrial glandular epithelial cells were shown in γ-HCH treatment group. Furthermore, although high columnar endometrial glandular epithelial cells were found in group 1 with mixed treatment of 900 mg/kg diet isoflavones and 8 mg/kg bw γ-HCH, the degree of hyperplasia was lower than isoflavones treatment group and higher than γ-HCH treatment group (Figure 3(a)). In addition, the changes of expression of ERα in uterus were similar to the changes of uterine morphology (Figure 3(b)). Compared with control group, the positive expression of ERα in isoflavones treatment group was significantly higher (p < 0.001) and lower in γ-HCH treatment group (p = 0.025). The positive expression rate of ERα in group 1 with 900 mg/kg diet isoflavones and 8 mg/kg bw γ-HCH was lower than isoflavones treatment group and higher than γ-HCH treatment group (Figure 3(c)). 4. Discussion EDCs are chemicals that may interfere with the body's endocrine system and produce adverse developmental, reproductive, neurological, and immune effects in both humans and wildlife. EDCs are derived from a wealth of sources, including OCPs, xenoestrogens, long-chain alkylphenols, and phytoestrogen. MCF-7 cells derived from human breast cancer cells, which are positive to ER and sensitive to estrogen, have been widely used to evaluate environmental estrogen and explore the development mechanism of estrogen on breast cancer occurrence [12, 13]. Genistein is a phytoestrogen with a plant-derived phenolic compound that structurally mimics the hormone 17 β-estradiol. Previous studies revealed that genistein could significantly inhibit the proliferation of MCF-7 cells in a dose-dependent manner. High dose of genistein could inhibit proliferation rate of MCF-7 cells [6, 12, 14]. Besides, daidzein and genistein exhibited biphasic effects (stimulatory or inhibitory) on proliferation and ERα expression in MCF-7 cells. That is, 1 μmol/L daidzein significantly stimulates cell growth and 200 μmol/L daidzein could inhibit cell proliferation [6]. In this study, we found that both 100 μmol/L genistein and 100 μmol/L daidzein could inhibit proliferation of MCF-7 cells. The dichlorodiphenyltrichloroethane (DDT), a known endocrine disruptor, links to animal and human disorders [15]. Payne et al. found that o,p′-DDT, p,p′-DDE, β-HCH, and p,p′-DDT produce proliferative effects in MCF-7 cells. Furthermore, combined effects were demonstrated by regression analyses even when each mixture component was present at levels or below its individual no-observed-effect concentration [16]. However, the research on interaction between soy isoflavones (genistein and daidzein) and OCPs was little. According to Charles et al., both in vitro and in vivo, low concentrations of the six synthetic chemicals (SCs, including o,p-DDT) failed to increase estrogenic responses which were induced by plant-derived phytoestrogens (PEs) alone. In vitro, interactions between high dosages of SCs and PEs were greater than mixtures of SCs in the absence of PEs [17]. In our study, soy isoflavones (genistein and daidzein) could repair the damage of OCPs; that is, isoflavones could inhibit breast cell proliferation triggered by OCPs except p,p′-DDE and chlordane. In vivo test was conducted according to the results of in vitro experiment. We used soy isoflavones as a mixture of genistein and daidzein. Overall, isoflavones could influence blood glucose and spleen weight in female rats; γ-HCH could affect estradiol and testosterone levels in serum and spleen weight in high dose treatment. Furthermore, there is interaction effect on kidney weight and liver weight when treated with isoflavones and γ-HCH. No other effects were found in body weight, brain weight, fat weight, and so forth. Previous study showed that soy isoflavones could significantly decrease body weight [18], while HCB, p,p′-DDE, and β-HCH showed quadratic associations with BMI [19]. In this study, no significant changes were found in isoflavones and OCPs treatment. Ye et al. found that both daidzein and genistein did not have a significant effect on glycemic control and insulin sensitivity over a 6-month supplementation period in Chinese women [20]. Another research showed that isoflavones mixture could lower blood glucose level in serum in high-fat diet fed C57BL/6J mice for 92 days [21]. But in this study, low dosage of isoflavones (600 mg/kg diet) could increase blood glucose level in female rats. The possible causes of differences between previous study and the current study are as follows: (1) SD rats were used in the present study instead of C57/BL6 mice, which have different absorption and ingestion for different species; (2) natural synthesis isoflavones were used as the intervention medicine, and they were different from the mixture of daidzin and glycitin used in their study. Otherwise, the interaction effect of isoflavones and γ-HCH on kidney weight and liver weight indicated that they could resist their side effects to produce healthy condition. A wide number of pesticides, including OCPs, such as γ-HCH, may induce reproductive and developmental alterations by altering steroid hormone metabolism or binding to the estrogen/androgen receptors. Exposure to γ-HCH with dose of 25 mg/kg bw in vivo could increase absolute and relative uterus weight in F1 pups on postnatal day 22 and change vaginal patency and reduce diameters of primary oocytes at fully sexual maturity [22]. According to findings of Huang et al., both chlordane and γ-HCH treatment could increase estrogen, reduce testosterone, and alter morphology of the masculine appendage in shrimp [23]. In our study, estradiol and testosterone levels in serum were reduced in female rats treated with γ-HCH. Furthermore, the changes of uterine morphology and expression of ERα showed that there was interaction effect between isoflavones and γ-HCH. 5. Conclusions In summary, high dosage of genistein and daidzein could inhibit proliferation of MCF-7 cells treated with OCPs. Besides, there are interaction effects between isoflavones and γ-HCH in uterine morphology and expression of ERα. The mechanisms of interactions between OCPs and isoflavones are not clear. Competitive inhibition effect may possibly be involved in the mechanism. Isoflavones show stronger affinity to ER, which may inhibit the combination of γ-HCH to ER. Acknowledgments This study was supported by the National Natural Science Foundation of China (81202190, 30972439, and 81273079) and the Innovation Fund of Harbin Medical University. Competing Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Figure 1 Interaction of genistein and daidzein with OCPs on proliferation rate of MCF-7 cells. ∗ p value < 0.05 was considered significant compared with control group; # p value < 0.05 was considered significant compared with organochlorine pesticides alone group. Figure 2 Effect of isoflavones and γ-HCH on body weight in female SD rats. Group 1: 900 mg/kg diet isoflavones and 8 mg/kg bw γ-HCH; group 2: 900 mg/kg diet isoflavones and 4 mg/kg bw γ-HCH; group 3: 900 mg/kg diet isoflavones and 0 mg/kg bw γ-HCH; group 4: 600 mg/kg diet isoflavones and 8 mg/kg bw γ-HCH; group 5: 600 mg/kg diet isoflavones and 4 mg/kg bw γ-HCH; group 6: 600 mg/kg diet isoflavones and 0 mg/kg bw γ-HCH; group 7: 0 mg/kg diet isoflavones and 8 mg/kg bw γ-HCH; group 8: 0 mg/kg diet isoflavones and 4 mg/kg bw γ-HCH; group 9: 0 mg/kg diet isoflavones and 0 mg/kg bw γ-HCH. Figure 3 Interaction of isoflavones and γ-HCH on uterine morphology and expression of ERα. (a) showed interaction of isoflavones and γ-HCH on uterine morphology. Compared with control group (group 9), high columnar endometrial glandular epithelial cells were found in group 3, while low columnar endometrial glandular epithelial cells were shown in group 7. Furthermore, although high columnar endometrial glandular epithelial cells were found in group 1, the degree of hyperplasia was lower than isoflavones treatment group and higher than γ-HCH treatment group. (b) showed interaction of isoflavones and γ-HCH on expression of ERα. Compared to group 9, ERα expression was higher in group 3 (p ≤ 0.05), while being lower in group 7 (p ≤ 0.05). Moreover, ERα expression in group 1 was lower than group 3 and higher than group 7. (c) showed positive expression of ERα in immunohistochemistry. ∗ p value < 0.05 was considered significant compared with control group (group 9). Group 1 was treated with 900 mg/kg diet isoflavones and 8 mg/kg bw γ-HCH; group 3 was treated with 900 mg/kg diet isoflavones alone; group 7 was treated with 8 mg/kg bw γ-HCH alone; group 9 was treated with 0 mg/kg diet isoflavones and 0 mg/kg bw γ-HCH. Table 1 Groups by 3∗3 factorial design with different dose of isoflavones and γ-HCH (n = 9/group). γ-HCH (mg/kg bw)  Isoflavones (mg/kg diet) 900 600 0 8 1 4 7 4 2 5 8 0 3 6 9 Note: factorial design with 2 factors of 3 levels. Three levels of γ-HCH: 8 mg/kg bw, 4 mg/kg bw, and 0 mg/kg bw. Three levels of isoflavones: 900 mg/kg diet, 600 mg/kg diet, and 0 mg/kg diet. Table 2 Two-way ANOVA analysis of effect of isoflavones and γ-HCH treatment on rats (p value).   Main effect Interaction effect Isoflavones γ-HCH Blood glucose 0.022∗ 0.423 0.419 Estradiol 0.159 0.003∗ 0.652 Testosterone 0.749 0.020∗ 0.110 Spleen weight 0.015∗ 0.012∗ 0.795 Kidney weight 0.529 0.566 0.047∗ Liver weight 0.283 0.050 0.033∗   ∗ p value < 0.05 was considered significant. ==== Refs 1 Mahmoud A. F. A. Ikenaka Y. Yohannes Y. B. Distribution and health risk assessment of organochlorine pesticides (OCPs) residue in edible cattle tissues from northeastern part of Egypt: High accumulation level of OCPs in tongue Chemosphere 2016 144 1365 1371 10.1016/j.chemosphere.2015.10.016 2-s2.0-84953807326 26492422 2 Tang F.-L. Zhang M. Xu J.-F. Pollution characteristics and health risk assessment of organochlorine pesticides (OCPs) in the water of Lake Qiandao and its major input rivers Huan Jing Ke Xue 2014 35 5 1735 1741 10.13227/j.hjkx.2014.05.014 2-s2.0-84902344847 25055660 3 Yang J.-Z. 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==== Front J Immunol ResJ Immunol ResJIRJournal of Immunology Research2314-88612314-7156Hindawi Publishing Corporation 10.1155/2016/3530752Research ArticleDelta Procalcitonin Is a Better Indicator of Infection Than Absolute Procalcitonin Values in Critically Ill Patients: A Prospective Observational Study http://orcid.org/0000-0002-6128-0718Trásy Domonkos 1 * http://orcid.org/0000-0002-6863-075XTánczos Krisztián 1 http://orcid.org/0000-0003-4534-9006Németh Márton 1 Hankovszky Péter 1 http://orcid.org/0000-0002-8320-1515Lovas András 1 http://orcid.org/0000-0002-7716-2353Mikor András 1 Hajdú Edit 2 http://orcid.org/0000-0003-3535-5103Osztroluczki Angelika 1 http://orcid.org/0000-0001-6523-800XFazakas János 3 http://orcid.org/0000-0002-1468-4058Molnár Zsolt 1 1Faculty of Medicine, Department of Anaesthesiology and Intensive Therapy, University of Szeged, 6 Semmelweis Street, Szeged 6725, Hungary2Faculty of Medicine, Division of Infectious Diseases, First Department of Internal Medicine, University of Szeged, Szeged, Hungary3Faculty of Medicine, Department of Transplantation and Surgery, Semmelweis University, Budapest, Hungary*Domonkos Trásy: trasydom@gmail.comAcademic Editor: Roberta A. Diotti 2016 15 8 2016 2016 353075215 5 2016 17 7 2016 Copyright © 2016 Domonkos Trásy et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Purpose. To investigate whether absolute value of procalcitonin (PCT) or the change (delta-PCT) is better indicator of infection in intensive care patients. Materials and Methods. Post hoc analysis of a prospective observational study. Patients with suspected new-onset infection were included in whom PCT, C-reactive protein (CRP), temperature, and leukocyte (WBC) values were measured on inclusion (t 0) and data were also available from the previous day (t −1). Based on clinical and microbiological data, patients were grouped post hoc into infection- (I-) and noninfection- (NI-) groups. Results. Of the 114 patients, 85 (75%) had proven infection. PCT levels were similar at t −1: I-group (median [interquartile range]): 1.04 [0.40–3.57] versus NI-group: 0.53 [0.16–1.68], p = 0.444. By t 0 PCT levels were significantly higher in the I-group: 4.62 [1.91–12.62] versus 1.12 [0.30–1.66], p = 0.018. The area under the curve to predict infection for absolute values of PCT was 0.64 [95% CI = 0.52–0.76], p = 0.022; for percentage change: 0.77 [0.66–0.87], p < 0.001; and for delta-PCT: 0.85 [0.78–0.92], p < 0.001. The optimal cut-off value for delta-PCT to indicate infection was 0.76 ng/mL (sensitivity 80 [70–88]%, specificity 86 [68-96]%). Neither absolute values nor changes in CRP, temperature, or WBC could predict infection. Conclusions. Our results suggest that delta-PCT values are superior to absolute values in indicating infection in intensive care patients. This trial is registered with ClinicalTrials.gov identifier: NCT02311816. ==== Body 1. Introduction Treatment of severe sepsis and septic shock remains a major challenge in the critically ill, and it is still one of the leading causes of death worldwide [1]. Despite increased awareness of the importance of early resuscitation, mortality in North America and Europe ranges between 28 and 41% [2]. Based on a consensus agreement sepsis is defined as infection in the presence of systemic inflammatory response syndrome (SIRS) [3]. However, the signs of SIRS are nonspecific and can often be seen in several (none septic) critically ill conditions. Fever, tachycardia, or leukocytosis on their own has low sensitivity and specificity [4, 5]. Detailed microbiological results are often only available after 24 hours or later, and negative results do not necessarily rule out infection. Nevertheless, early diagnosis of infection in critically ill patients is of utmost importance, and delay in starting appropriate antibiotic therapy may lead to lethal events [6]. However, giving antibiotics unnecessarily to every acutely ill patient is an unacceptable practice for several reasons [7]. Therefore, fast reacting biomarkers of infection have been used for almost 50 years to help the clinician, of which C-reactive protein (CRP) and procalcitonin (PCT) are the most often used and studied [8]. Procalcitonin is a fast reacting biomarker with a half-life of around 24 hours [9]. Its sensitivity and specificity for bacterial infection seem to be superior compared to CRP [10, 11]. However, it must be considered that the same absolute values of PCT cannot be used in all circumstances. It has been reported that PCT levels are higher in surgical compared to medical patients [12], and elevated PCT can also be present without infection, in conditions such as trauma [13] and surgery [14] or after cardiac arrest [15]. There is some evidence that evaluating PCT kinetics may be superior to absolute values [12, 16]. In this study, our aim was to investigate whether the absolute value of PCT measured in critically ill patients on the day when infection was suspected, or the change in PCT (delta-PCT) from the day before to the day when infection was suspected, was a better indicator of infection. 2. Methods 2.1. Patient Selection This prospective observational study was part of the Early Procalcitonin Kinetics (EProK) study, which was undertaken between October 2012 and October 2013 and approved by the Regional and Institutional Human Medical Biological Research Ethics Committee, University of Szeged, Hungary (WHO-3005; 19.04.2012, Chairperson Professor T. Wittmann). A detailed description of the EProK study and the final results are published elsewhere [17]. The investigation was performed at the University of Szeged (Szeged, Hungary), Albert Szent-Györgyi Health Center in four tertiary intensive care units. The study was registered at ClinicalTrials.gov with the registration number: NCT02311816. Written informed consent was obtained from all subjects or from their relatives. 2.1.1. Inclusion Criteria In the EProK study all patients over 18 years with suspected infection on admission or during their stay on the intensive care unit were screened for eligibility. Patients were enrolled, when the attending intensive care specialist suspected infection, based on (1) suspected source which could be identified, (2) new onset organ dysfunction, and (3) body temperature, PCT, CRP, and the decision to start empirical antibiotic therapy. Once the original EProK study was completed, in a post hoc analysis those patients in whom PCT and CRP values were available from the previous day (t −1) were included in the current analysis. 2.1.2. Exclusion Criteria Exclusion criteria included patients younger than 18 years, who had received antibiotic therapy in the previous 48 hours, and those who received acute renal replacement therapy 24 hours before enrollment. Patients were also excluded following cardiopulmonary resuscitation and with end stage diseases with a “do not resuscitate” order. Immunocompromised patients (human immunodeficiency virus infection, bone marrow transplantation, malignant haematological disorders, and chemotherapy) were also excluded. 2.2. Subgroups and Definitions Diagnosis of infection was based on a post hoc analysis of mainly microbiological results but also clinical parameters and biochemical results which were evaluated by two experts (infectologist, EH, and an intensivist, FJ) blinded for the PCT data apart from the first PCT measurement (t 0, see below). The experts also took into consideration the recommendations of international guidelines [18, 19]. Based on these results, patients were grouped into “infection-” (I-) and “noninfection-” (NI-) groups. For subgroup analysis, patients were divided into “medical” and “surgical” groups. The medical-group represented patients who had had no surgical intervention before and during the study period and for source control did not require surgery. In the surgical-group infection either was related to an operation or required surgery for source control [12]. 2.3. Protocol and Data Collection Whenever infection was suspected by the attending physician, the signs of infection and the suspected source were recorded, which included high/low body temperature (<36°C; >38°C), high/low white blood cell count (<4,000; >12,000 million/mL), acute worsening of the clinical picture (hemodynamic instability, worsening PaO2/FiO2 ratio, and deterioration in mental status or any other clinical sign indicating infection). Microbiological specimens were collected from all suspected sources immediately before the administration of the first dose of antibiotics (t 0). 2.3.1. Data Collection After enrollment, demographic data, signs of infection, the suspected source of infection, and corresponding microbiological samples were registered. The length of intensive care unit and hospital stay, 28 days, and the overall mortality were also documented. 2.3.2. Procalcitonin Measurement It is common practice in our ICU to measure PCT daily in critically ill patients. Procalcitonin levels were documented from the previous day of enrollment (t −1) and immediately before the initiation of ABs (t 0). Core temperature, C-reactive protein (CRP), and white blood cell count (WBC) were also recorded with every PCT measure. The flow chart of the data collection is summarised in Figure 1. Serum PCT levels were measured with Cobas 6000 analyzer (Hitachi High-Technologies Corporation, Tokyo, Japan). Analyzer reagents (Elecsys® B·R·A·H·M·S PCT assay) were developed in collaboration with B·R·A·H·M·S corporation (Hennigsdorf, Germany) and Roche Diagnostics (Mannheim, Germany). Procalcitonin was determined by electrochemiluminescence immunoassay (ECLIA) serum on the automated Roche Elecsys and Cobas immunoassay analyzers. 2.3.3. Microbiological Staining and Antibiograms Microbiological tests were performed and sent at t 0, before the first antibiotic dose was administered and if needed they were repeated on the following days, to identify infection. 2.4. Statistical Analysis Data were analyzed using IBM SPSS Statistics Version 20 (Armonk, NY, USA) and Systat Software Inc. SigmaPlot 12.5 (London, UK) software. For continuous data, the Shapiro-Wilk tests were performed to assess normal distribution. Demographic data were analyzed between groups with Student's t-test or nonparametric data with the Mann-Whitney U test as appropriate. Categorical data were compared using χ 2 tests. Biomarkers were analyzed by using Two-Way Repeated Measures Analysis of Variances (All Pairwise Multiple Comparison Procedures: Holm-Sidak method). Logistic regression, receiver operating characteristic (ROC) curve, and the respective areas under the curves (AUC) were calculated for PCT, CRP, body temperature, and white blood cell count levels. The best cut-off values were determined using the Youden index (J = max⁡[Sens + Spec − 1]). The test parameters (sensitivity, specificity, positive, and negative predictive values) were compared by their 95% confidence intervals. Logistic regression analysis was used to determine the best combination of parameters and cut-offs for predicting infection. The level of p < 0.05 was defined as statistically significant. Data are given as mean ± standard deviation or median (interquartile range) as appropriate. The “delta” was considered as the changes in the absolute values (subtracting t −1 from t 0); the percentage values were calculated as [(t 0/t −1) × 100 − 100]. 3. Results Over the one-year study period all ICU patients were screened for eligibility and 209 patients were recruited into the EProK study. Out of the 209 patients in the current post hoc analysis we include 114 cases where PCT values were available from the previous day. Demography and outcomes characteristics for the entire cohort are summarised in Table 1. Out of the 114 patients, 85 (75%) patients were identified as having proven infection and in 29 (25%) patients the presence of infection was highly unlikely. Disease severity scores and outcomes were similar in the two groups, but the NI-group required less organ support. The clinical and laboratory signs of infection on which the clinicians suspected infection at the time of inclusion (t 0) are summarised in Table 2. Although all indices were higher in the I-group, but only the altered level of consciousness, hemodynamic instability, and the PCT was significantly different between the two groups. Regarding the suspected source of infection, generally there was nonsignificant difference between the groups, but significantly more patients were suspected of having abdominal related infection in the NI-group. Detailed data on the isolated pathogens and their sources are summarised in the Supplemental Digital Content Tables S5–7 (see Supplementary Material available online at http://dx.doi.org/10.1155/2016/3530752). 3.1. PCT, CRP, WBC, and Temperature Values at t −1 and t 0 3.1.1. Total Sample Measurement results at t −1 and t 0 in the I- and NI-groups are shown in Figure 2. PCT absolute values were similar at t −1, but by t 0 in the I-group levels were significantly higher compared to the NI-group and there was also a significant increase from t −1, while there was no such change in the NI-group. There was no significant difference in CRP and WBC count between the two groups nor could we find significant changes from t −1 to t 0. There was no difference between the groups for body temperature but there was a statistically significant increase in the NI-group by t 0. It is of note that body temperature remained <38°C in almost all patients. 3.1.2. Medical and Surgical Patients Measurement results in medical (n = 80) and surgical (n = 34) patients are summarised in Table 3. In the surgical subgroup PCT absolute values were significantly higher than in the medical cohort, but the pattern of change was similar. In the NI-group there was a slight, but statistically significant increase in medical patients from t −1 to t 0, while there was no significant change in surgical patients, where levels actually decreased slightly. However, in the I-group there was an almost 3-fold increase in the PCT levels. Regarding the CRP, body temperature, and WBC count, there was no significant changes over time and no differences between medical and surgical patients. 3.2. Predictive Value for Indicating Infection The predictive value for infection for the absolute values of PCT, CRP, temperature, and WBC count can be seen in Figure 3 and is summarised in Table 4. Only PCT had a significant predictive value, but with a poor AUC (Figure 3). However, regarding the percentage and delta changes CRP, temperature and WBC counts diagnostic value did not change, while PCT's AUC for both percentage and delta changes had a significantly better performance for predicting infection. Similar patterns were observed in the medical and surgical subgroups (Table 4). 3.3. Best Cut-Off Value The best cut-off values were defined for PCT only as there was no significant predictive value for the other parameters, as determined by the Youden index. For the PCT absolute value it was 0.84 ng/mL with a sensitivity of 61% (95% CI: 50–72) and specificity 72% (53–87) to indicate infection in the ICU. Regarding the percentage change a PCT increase of >88% from t −1 to t 0 had a sensitivity of 75% (65–84) and specificity of 79% (60–92) and a PCT delta change of >0.76 ng/mL had a sensitivity of 80% (70–88) and specificity of 86% (68–96) to indicate infection. Data were also analyzed using the logistic regression model for finding the best combination of these four parameters together to predict infection in the ICU. However, none of the combinations tested improved the performance for predicting infection (data not shown). 4. Discussion The main finding of this observational study was an increase in PCT levels from the day before (t −1) to the day when infection was suspected (t 0) predicted infection, while in patients with no proven infection PCT remained unchanged. Furthermore, regarding the conventional indicators of infection such as WBC, body temperature, and CRP, neither the absolute values nor their change from t −1 to t 0 could predict infection. Diagnosing infection in the critically ill is challenging. Appropriate decision making has paramount importance as any delay in adequate antibiotic treatment of sepsis and septic shock evokes worsening morbidity and mortality results [6, 20]. On the other hand unnecessary antibiotic administration in patients without infection has led to the emergence of multidrug-resistant bacteria [21, 22], complications related to the side effects of the antibiotics themselves and an increased burden of healthcare expenses [23]. Despite its importance, there is no gold standard for diagnosing/proving infection in the critical care setting. In our study 75% of patients had proven infection. This complex post hoc analysis of all results is fundamentally different from “labelling” patients as septic, based solely on the Surviving Sepsis Guideline criteria at the time of initial assessment as seen in several studies [24, 25]. Although our method also has some uncertainties, it provides a more robust approach utilising all data, clinical, biochemical, and microbiology alike, to aid in the diagnosis of patients with bacterial infection. However, it is also important to acknowledge that there is no gold standard to diagnose infection; therefore despite all our efforts, some patients in the NI-group may have had culture negative infection. In our investigation it was found that conventional indicators of infection such as body temperature and white cell count had less value in diagnosing infection. Levels of WBC count remained elevated on both days and there was no significant change over time. This phenomenon can be explained by the nonspecific activation of the immune cascade as often seen in ICU patients [26]. Although there was a statistically significant increase in body temperature in the NI-group, levels largely remained below 38°C in almost all patients. These results are in accordance with recent findings that increased temperature alone does not predict infection [27]. Although microbiology remains the gold standard for confirming pathogens, results only come back at least 24–48 hours after sampling. Furthermore, in several cases results remained negative, despite obvious signs of infection. In order to help the diagnostic process several novel biomarkers of infection have been developed [8]. However, all biomarkers share the same limitations that “one size will not fit all,” due to the complex pathomechanism and the heterogeneity of patients. The two most commonly used markers in infection/sepsis diagnostics are PCT and CRP [8]. Procalcitonin is detectable in the serum a few (2–4) hours after the onset of bacterial infection. It reaches its peak within 24 hours and then starts to decline with adequate treatment by around a 50% daily decrease according to its half-life [9]. In contrast, CRP has a delayed response. It reaches its maximum value usually after 48 hours of an insult and in general it lags behind the actual events of the inflammatory and clinical process. Furthermore, CRP levels are generally elevated in most ICU patients regardless of the aetiology. In our study neither the absolute values of CRP nor its delta changes were able to indicate new onset infection. Patients had elevated CRP values with a median of almost 200 mmol/L for the whole cohort, which makes interpretation very difficult. Furthermore the kinetics did not show any significant change over time. Therefore, our results question the place of CRP measurements for diagnosing infection on the ICU. The most important finding of the current study was to show the superiority of PCT kinetics over the absolute values to indicate new onset infection in the ICU. However, this requires at least daily measurements of PCT, which has been common practice in our ICU in critically ill patients in whom infection cannot be excluded. Our current findings are in accordance with those reported by Tsangaris et al. [16]. They also measured PCT daily and observed a twofold increase of PCT levels from the day before to the day when there was a sudden onset of fever in patients with proven infection, but no change in PCT was found in patients without infection. They concluded that, in patients treated chronically in the ICU, PCT values on the day of fever onset must be compared to values measured the previous day in order to define whether this rise in temperature was due to infection or not. An important difference between their and our study is that in our patients body temperature merely reached 38°C; in fact most of these patients were apyrexial, despite 75% having proven infection. Therefore, we recommend to evaluate PCT kinetics not only in the onset of fever, but whenever infection is suspected on the ICU. Based on the current results, the best cut-off values were also determined for change in PCT, which were >88% and >0.76 ng/mL delta change from t −1 to t 0. The reasons why a given absolute value of any biomarker, not just PCT, may be of limited value as compared to its changes can be explained by the pathomechanism of systemic inflammation. It was a very important discovery that after trauma, burns, ischemia-reperfusion, pancreatitis, major surgery, and so forth, the same or similar molecules are released predominantly from the mitochondria, as after an infectious insult. Based on aetiology these are called “damage-associated molecular patterns” (DAMP), or “pathogen associated molecular patterns” (PAMP). Once similar mediators/proteins are released they act on the same receptors of monocytes inflicting a similar inflammatory response, including PCT release and subsequent organ dysfunction [28, 29]. Indeed, PCT levels were found to be severalfold higher in surgical compared to medical patients in septic shock despite the similar clinical manifestation and severity of the clinical picture [12]. This explains why PCT levels were elevated in our surgical patient population without proven infection, with median values of around 3.5 (NI-group) and 3.8 (I-group) ng/mL at t −1. The corresponding PCT values in medical patients were substantially lower (0.26 and 0.89 ng/mL, resp.). Although levels were higher in the I-group at t −1, this difference did not reach statistical significance while there was a severalfold increase in the I-group in both medical and surgical patients with no change in kinetics in the NI-groups. In two large recent multicenter trials the authors could not show any benefit from a PCT-based approach in antibiotic management in the ICU [30, 31]. However, in both studies the threshold for intervention was a PCT of >1 ng/mL. As 40% of the patients in both trials were surgical, in whom this threshold for intervention may be too low, one cannot exclude that these patients may have had received antibiotics unnecessarily. This overuse of antibiotics may be one of the reasons for the worse outcome in the PCT-guided group in both studies. Our study provides further evidence that changes or kinetics of PCT may be superior to absolute values. The current study has several limitations. Firstly, one may argue that there was a selection bias; in other words, physicians suspected infection more often when they observed a PCT increase in a patient. Although this cannot be excluded completely, at the time when the study was performed, PCT collection was not the routine practice within the department, and delta-PCT was not included among the criteria of inclusion either. The whole idea of retrieving PCT data from the day before came after we analyzed the original EProK database. Secondly, despite all our efforts of allocating patients into the I- and NI-groups, this took place in a post hoc fashion. The available clinical results were analyzed in a blinded manner for delta-PCT (apart from PCT values at t 0) and thoroughly by our experts; however, one cannot exclude the possibility of inappropriate judgment during the decision making. The lack of gold standard for diagnosing infection is aggravated by this obscurity when configuring groups. Furthermore, the sample size was generally small, especially to be able to draw firm conclusions regarding the medical, surgical subgroups, although the trend in our results was certainly promising. Finally, it remains uncertain why PCT values were measured on the previous day before starting empiric antibiotic therapy in more than 50% of the 209 patients of the EProK study. Therefore, some selection bias cannot be excluded. The median day of inclusion into the study from ICU admission was 1 day, indicating that 50% of patients had PCT measurements on the ward/Accident and Emergency Unit, before admission. However, this may also reinforce the importance of measuring PCT values consecutively. 5. Conclusion The main finding of this observational study was that an increase in PCT levels from the day before (t −1) to the day when infection was suspected (t 0) predicted infection, while in patients with no proven infection PCT remained unchanged. Based on the data presented a single PCT measurement may not be adequate to differentiate between an infectious and noninfectious inflammatory response. This means that the kinetics of procalcitonin values based on daily measurements are superior to absolute values in diagnosing infection on the ICU and absolute values of procalcitonin may be of limited use. Both absolute values and kinetics of C-reactive protein are poor indicators of infection; furthermore, conventional indicators of infection such as white cell count and body temperature have limited use for predicting infection in the ICU. The clinical implication of these results is that daily PCT measurements in patients at high risk of infection allow the opportunity to evaluate PCT kinetics, which may improve diagnostic accuracy and rationalise antibiotic therapy on the ICU and improve outcome. Supplementary Material Supplemental Digital Content Tables S5-7: As a supplemental digital material a detailed summary of the results of the microbiological stainings can be seen. Table S5 shows the suspected sources of infection based on the clinical picture, laboratory tests and microbiological results. In the Table S6 the sources of positive and negative microbiological samplings sent for staining can be seen in the infection group. And in Table S7 the cultured pathogens and their source of sampling are collected. Acknowledgments The authors express their special thanks to János Sándor and Ferenc Rárosi, for their invaluable advice on statistics, and Mrs. Harriet Adamson for language editing and the help of the doctors and nurses on the intensive care unit, without which this study could have not been completed. The EProK study group is gratefully acknowledged. Competing Interests János Fazakas and Zsolt Molnár receive lecture fees from Thermo Fisher Scientific, BRAHMS Gmbh. The other authors declare no competing interests regarding the publication of this paper. Figure 1 Flow chart. Figure 2 PCT, CRP, body temperature, and WBC count absolute values in the total cohort. Boxplots present median (interquartile range) 10th and 90th percentile. ∗ indicates p < 0.05. Figure 3 The predictive value of the absolute values, percentage, and delta changes of PCT, CRP, temperature, and WBC count for infection in the total cohort. Table 1 Demographics, organ support, and outcome in the entire cohort.   Total NI-group I-group p value Age (years) 65 (22.5) 67 (25.5) 65 (22) 0.772 Gender (M/F) 69/45 15/14 54/31 0.261 Body height (cm) 170 (12) 167 (19) 170 (11) 0.766 Body weight (kg) 73 (25) 80 (25) 70 (20) 0.345 SAPS II points 62.2 ± 20.5 62.7 ± 25.5 66.1 ± 18.6 0.513 SAPS II PM (%) 77.2 (52.1) 64.0 (75.9) 78.5 (42.1) 0.437 ICU days before enrollment 1 (3) 1 (3) 1 (3) 0.669 Mechanical ventilation 80 (70.2%) 12 (41.4%) 68 (80.0%) <0.001 Vasopressor therapy 69 (60.5%) 13 (44.8%) 56 (65.9%) 0.045 ICU LOS (day) 9 (12) 8 (8) 9 (12) 0.089 ICU survival 84 (73.7%) 24 (82.8%) 60 (70.6%) 0.199 Hospital LOS (day) 17 (20) 14 (17) 19 (22) 0.050 Hospital survival 67 (58.8%) 20 (68.9%) 47 (55.3%) 0.197 28-day survival 64 (56.1%) 19 (65.5%) 45 (52.9%) 0.239 Data are given as mean ± standard deviation or median (interquartile range) as appropriate. M: male; F: female; SAPS: simplified acute physiology score; PM: predicted mortality; ICU: intensive care unit; LOS: length of stay; mechanical ventilation and vasopressor therapy represent data at the day of enrollment. Table 2 Clinical signs and suspected source of infection at enrollment (t 0).   Total n = 114 NI-group n = 29 I-group n = 85 p value Fever (<36°C; >38°C) 55 (48.2%) 13 (44.8%) 42 (49.4%) 0.670 WBC (>12 or <4 × 109/L) 82 (71.9%) 22 (75.9%) 60 (70.6%) 0.585 Impaired gas exchange 82 (71.9%) 18 (62.1%) 64 (75.3%) 0.171 Impaired consciousness 59 (51.8%) 9 (31.0%) 50 (58.8%) 0.010 Hemodynamic instability 74 (64.9%) 13 (44.8%) 61 (71.8%) 0.009 PCT (ng/mL) 3.37 (9.22) 1.12 (1.36) 4.62 (10.72) 0.018 CRP (mg/L) 182.75 (158.5) 147.60 (156.50) 208.80 (140.60) 0.301 Respiratory 72 (63.2%) 17 (58.6%) 55 (64.7%) 0.557 Soft tissue 13 (11.4%) 2 (6.9%) 11 (12.9%) 0.377 Abdominal 14 (12.3%) 7 (24.1%) 7 (8.2%) 0.024 Urinary tract 5 (4.4%) 0 5 (5.9%) 0.182 Bloodstream 6 (5.3%) 2 (6.9%) 4 (4.7%) 0.648 Central nervous system 4 (3.5%) 1 (3.4%) 3 (3.5%) 0.984 WBC: white blood cell count, PCT: procalcitonin, and CRP: C-reactive protein. The PCT and CRP values are presented as median (interquartile range). Table 3 PCT, CRP, body temperature, and white blood cell count in medical and surgical patients with and without infection.     NI-group I-group     t −1 t 0 t −1 t 0 Medical PCT (ng/mL) 0.26 (0.57) 0.54 (1.16)∗ 0.89 (1.52) 3.17 (5.9)∗# CRP (mg/L) 136.7 (159.1) 141 (125.9) 150 (184.3) 164.2 (145.3) BT (°C) 36 (1.02) 37 (0.82)∗ 36.9 (1.23) 37 (1.6) WBC (×109/L) 14.32 (8.9) 15.4 (8.64) 12.06 (6.36) 13.76 (10.16) Surgical PCT (ng/mL) 3.5 (9.91) 2.89 (9.33) 3.83 (22.55) 14.9 (58.06)∗# CRP (mg/L) 95 (342.5) 163 (327.4) 199.5 (130.1) 243.2 (112.7) BT (°C) 36.5 (2) 36.5 (2.4) 36 (1) 36.9 (1.1) WBC (×109/L) 8.99 (7.37) 14.56 (9.65) 11.9 (10.06) 10.91 (9.9) Data are presented as median (interquartile range). PCT: procalcitonin, CRP: C-reactive protein, BT: body temperature, and WBC: white blood cell count; ∗ p < 0.05 within groups and # p < 0.05 between groups. Table 4 The predictive value of the absolute values, percentage, and delta changes of PCT, CRP, temperature, and WBC count for infection in the total cohort.     Absolute value Percentage changes Absolute value changes     AUC 95% CI p value AUC 95% CI p value AUC 95% CI p value Total PCT 0.64 0.52–0.76 0.022 0.77 0.66–0.87 <0.001 0.85 0.78–0.92 <0.001 CRP 0.60 0.47–0.72 0.103 0.54 0.41–0.66 0.530 0.54 0.42–0.65 0.536 BT 0.52 0.39–0.63 0.804 0.56 0.44–0.68 0.300 0.56 0.44–0.68 0.322 WBC 0.60 0.48–0.70 0.125 0.51 0.40–0.61 0.852 0.51 0.39–0.61 0.924 Medical PCT 0.67 0.54–0.80 0.016 0.76 0.63–0.88 <0.001 0.83 0.73–0.92 <0.001 CRP 0.58 0.44–0.72 0.248 0.57 0.42–0.70 0.359 0.57 0.44–0.70 0.306 BT 0.51 0.37–0.64 0.858 0.64 0.50–0.77 0.055 0.64 0.49–0.77 0.060 WBC 0.57 0.44–0.70 0.329 0.56 0.43–0.68 0.441 0.57 0.43–0.69 0.365 Surgical PCT 0.78 0.58–0.97 0.025 0.80 0.59–1.00 0.014 0.94 0.85–1.00 <0.001 CRP 0.56 0.23–0.87 0.654 0.56 0.29–0.81 0.654 0.54 0.31–0.76 0.749 BT 0.52 0.22–0.80 0.898 0.63 0.39–0.85 0.306 0.63 0.39–0.86 0.296 WBC 0.63 0.44–0.82 0.277 0.67 0.47–0.86 0.166 0.71 0.49–0.90 0.108 AUC: area under the ROC curve, CI: confidence interval, PCT: procalcitonin, CRP: C-reactive protein, BT: body temperature, and WBC: white blood cell count. ==== Refs 1 Angus D. 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==== Front Gastroenterol Res PractGastroenterol Res PractGRPGastroenterology Research and Practice1687-61211687-630XHindawi Publishing Corporation 10.1155/2016/4561468Research ArticleRecognition Accuracy Using 3D Endoscopic Images for Superficial Gastrointestinal Cancer: A Crossover Study http://orcid.org/0000-0001-7891-9795Nomura Kosuke * Kaise Mitsuru Kikuchi Daisuke Iizuka Toshiro Fukuma Yumiko Kuribayashi Yasutaka Tanaka Masami Toba Takahito Furuhata Tsukasa Yamashita Satoshi Matsui Akira Mitani Toshifumi Hoteya Shu Department of Gastroenterology, Toranomon Hospital, Tokyo 105-8470, Japan*Kosuke Nomura: med20365nomura@yahoo.co.jpAcademic Editor: Werner A. Draaisma 2016 15 8 2016 2016 456146816 5 2016 19 7 2016 Copyright © 2016 Kosuke Nomura et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Aim. To determine whether 3D endoscopic images improved recognition accuracy for superficial gastrointestinal cancer compared with 2D images. Methods. We created an image catalog using 2D and 3D images of 20 specimens resected by endoscopic submucosal dissection. The twelve participants were allocated into two groups. Group 1 evaluated only 2D images at first, group 2 evaluated 3D images, and, after an interval of 2 weeks, group 1 next evaluated 3D and group 2 evaluated 2D images. The evaluation items were as follows: (1) diagnostic accuracy of the tumor extent and (2) confidence levels in assessing (a) tumor extent, (b) morphology, (c) microsurface structure, and (d) comprehensive recognition. Results. The use of 3D images resulted in an improvement in diagnostic accuracy in both group 1 (2D: 76.9%, 3D: 78.6%) and group 2 (2D: 79.9%, 3D: 83.6%), with no statistically significant difference. The confidence levels were higher for all items ((a) to (d)) when 3D images were used. With respect to experience, the degree of the improvement showed the following trend: novices > trainees > experts. Conclusions. By conversion into 3D images, there was a significant improvement in the diagnostic confidence level for superficial tumors, and the improvement was greater in individuals with lower endoscopic expertise. ==== Body 1. Introduction There have been major advances in the use of endoscopic devices for the diagnosis and treatment of gastrointestinal carcinoma as a result of new technology and the accumulation of cases that used these new technologies. However, information from conventional 2D images lacks information on depth perception, and, unlike real 3D information, 2D images provide merely probabilistic information. We believe that prediction with greater accuracy has become increasingly possible through experience, but we are not able to recognize tissue structures with 100% accuracy, and the potential for misrecognition of tissue structures still exists. Against this background, many institutions have begun to perform 3D endoscopic surgery in recent years. The utility of a 3D endoscopic system was reported in studies showing that the system improves the speed and accuracy of surgery through the enhancement of depth perception [1–3]. However, there have been few reports on endoscopic diagnosis to date [4]. In the field of diagnostic endoscopy, the incorporation of a 3D system is also expected to add an unprecedented amount of information on depth and improve the accuracy of diagnosis. During the present study, we used ESD resection specimens to perform a comparison between 2D and 3D images to determine whether the accuracy of lesion recognition can be improved through the use of 3D endoscopic images. 2. Materials and Methods 2.1. Study Design A total of 12 participants (4 novices, 4 trainees, and 4 experts) were recruited from a single institution, and 2 participants from each category were randomly allocated into two groups. An expert was defined as an endoscopist who had performed magnifying endoscopy for more than 500 cases, and a novice was defined as a physician who had never performed a single routine endoscopic examination. Test tissues were obtained from 20 ESD specimens: 15 superficial gastric cancers (14 cases of 0-IIc and 1 case of 0-IIb well-differentiated cancer), 3 superficial colon cancers (all IIa), and 2 superficial esophageal cancers (both 0-IIb). Each of the resected specimens was fixed on a black rubber plate with pins and immersed in a water box on a micromotion stage ((XCRS-80AR, MISUMI)). They were viewed under low magnification with Narrow Band Imaging system using a commercially available endoscopic unit (Evis Lucera Elite, Olympus Medical Systems, Co. Ltd., Tokyo Japan) and scopes fixed with a custom-made stand (Figures 1(a) and 1(b)). To create a 3D still image, parallax images were obtained by moving the fixed specimen in a horizontal direction with the micromotion stand. To acquire parallax images in the same lighting status, we used two scopes: one illumination scope simultaneously moved with the micromotion stage and another imaging scope did not move with it. A GIF-H260Z scope (Olympus Co., Japan) was used as the imaging scope and was attached to a scope cable, while a GIF-Q260 scope (Olympus Co., Japan) was used as the illuminating scope and was attached to a connector (Figure 2). Using an endoscopic image catalog containing two images, we extracted only odd rows from images for the left eye and even rows from images for the right eye and combined them to generate one line-by-line image. A 3D monitor and polarized 3D glasses enabled us to perceive a stereoscopic image through the observation of odd and even rows via the left and right eye, respectively. The two images on the retina were combined in the brain, which enabled perception of the virtual stereoscopic model (Figures 3(a) and 3(b)). Study 1: Figure 4 shows the study flow chart. Participants of group 1 evaluated only 2D images at first; then, after an interval of more than 2 weeks, they evaluated 3D images. Conversely, participants of group 2 first evaluated 3D images and after a set interval evaluated 2D images. The evaluation items were (1) diagnostic accuracy of tumor extent and (2) diagnostic confidence levels for the following items: (a) tumor extent, (b) morphology of the tumor surface, (c) microsurface structure (MSS) and/or microvascular structure (MVS), and (d) comprehensive recognition of the lesion. Study 2: the 2D and 3D images were examined sequentially, and improvement rates resulting from conversion into 3D images were evaluated in terms of confidence levels for items (a) to (d) above. In this study, we defined visual recognition of the demarcation line in the lesion as (a) tumor extent, recognition of irregular morphology in the lesion as (b) morphology of the tumor surface, recognition of MSS and/or MVS on the mucosal surface as (c) MSS and/or MVS, and recognition of (a)–(c) as a whole as (d) comprehensive recognition of the lesion. When determining tumor extent, the test participant described a demarcation line on a printed image of each lesion. Calculations to determine accuracy were then made based on histological mapping (Figure 3(c)). Specifically, the total number of accurate diagnoses of lesions and nonlesions (as a numerator) was divided by the sum of the total number of starting and ending points (Figure 3(c), red lines) in the mapping image and the number of lines made in the nonlesion area (Figure 3(c), white solid lines) (as a denominator). We evaluated confidence levels using a 5-point scale. In Study 1, the levels were as follows: 1 = extremely uncertain, 2 = uncertain, 3 = somewhat certain, 4 = almost certain, and 5 = absolutely certain. In Study 2, the levels were as follows: when compared to the 2D images, 1 = much more difficult to recognize clearly, 2 = somewhat more difficult to recognize clearly, 3 = the same as before (no change), 4 = somewhat easier to recognize clearly, and 5 = much easier to recognize clearly. In the statistical analysis, numerical data and categorical data (5-point scale) were analyzed using the t-test and Wilcoxon signed-rank test, respectively. A p value of <0.05 was considered to be statistically significant. 3. Results 3.1. Study 1 3.1.1. Effect of Examining 2D and 3D Images on Diagnostic Accuracy of Tumor Extent Results are shown in Table 1. The diagnosis of tumor extent was accurate in 78.4% of all participants who viewed 2D images and in 81.1% of those who viewed 3D images, with a slight but not significant improvement in the diagnostic accuracy by examining 3D images. We found no significant differences when we compared group 1 (2D: 76.9%, 3D: 78.6%) and group 2 (2D: 79.9%, 3D: 83.6%), and the results were approximately equivalent. On examining the accuracy by participant skill level, the accuracy was slightly higher in the 3D group regardless of their skill levels, with no significant difference between the two groups. 3.1.2. Effect of Examining 2D and 3D Images on Diagnostic Confidence Level Results are shown in Table 2. Diagnostic confidence levels for all items ((a) to (d)) were higher for the 3D images compared with the 2D images with a significant difference observed in all cases: (a) 3.26 versus 3.70 (p < 0.01), (b) 3.24 versus 3.93 (p < 0.01), (c) 3.17 versus 3.58 (p < 0.01), and (d) 3.08 versus 3.60 (p < 0.01). Examining the confidence level for each item by observer skill level, we found that all experts had high confidence levels when viewing 3D images, with a significant difference observed for items (a) and (b): (a) 3.63 versus 3.85 (p < 0.05), (b) 3.93 versus 4.25 (p < 0.01), (c) 3.83 versus 3.90 (p = 0.292), and (d) 3.68 versus 3.87 (p = 0.054). Trainees showed higher confidence levels for all items ((a) to (d)) with a significant difference; the overall degrees of certainty were (a) 3.45 versus 3.70 (p < 0.05), (b) 3.33 versus 3.88 (p < 0.01), (c) 3.33 versus 3.60 (p < 0.05), and (d) 3.28 versus 3.66 (p < 0.05). Diagnostic confidence levels were also higher for novices for all items ((a) to (d)), with a significant difference in every item: (a) 2.71 versus 3.55 (p < 0.01), (b) 2.46 versus 3.68 (p < 0.01), (c) 2.35 versus 3.23 (p < 0.01), and (d) 2.29 versus 3.28 (p < 0.01). When we examined the average improvement in items (a) to (d) for the observers, experts showed a slight improvement (0.20 ± 0.81), but the trainees (0.37 ± 0.91) and novices (0.98 ± 0.98) showed greater improvement on average, indicating that improvement was greater for participants with a lower level of skill and significant differences were also observed (Table 3). 3.2. Study 2 Results are shown in Table 4. There was improvement in diagnostic confidence levels for items (a) to (d) for all specimens and the 95% confidence interval did not exceed a value of 3, so significant differences were observed. We also noted significant differences for all items ((a) to (d)) for experts, trainees, and novices when we examined the improvement by participant skill level. 4. Discussion To date, 3D technology has been practically applied in various fields including movies, and applications in medical treatment have received significant attention in recent years, leading to an expansion in their use. In clinical settings, the application of 3D imaging has advanced from the perspectives of both three-dimensional image construction from investigation data and the implementation of stereoscopy. For the former, technology has allowed the creation of 3D images from data obtained from CT and MRI images, and this is reported to increase diagnostic ability and procedural precision [5–7]. In addition, the use of 3D printers has facilitated the practical application of materialized 3D data. Regarding implementation of stereoscopy in the latter, there have been several reports indicating utility in surgery, and use of 3D endoscopes facilitates stereoscopy [8–10], which suggests that techniques can be performed faster and more accurately. In the present report, we studied the utility of 3D images for the diagnosis of superficial GI tumor when using a flexible scope. This is the first report of its kind to date. In this study, we found that when compared with 2D images, 3D images conferred a higher confidence level when determining tumor extent, for morphological recognition of MSS and MVS, when performing overall diagnosis, and particularly when identifying irregular morphology. These data indicate that converting images into 3D makes the surface morphology of superficial GI tumors more distinct, resulting in a concomitant increase in the accuracy of lesion recognition as 3D endoscope images provide depth perception. In this study, the utility of conversion into 3D images was significantly greater for participants who had less experience with endoscopy. Experts could use their accumulated experience to visualize 3D structures from 2D images, whereas trainees and novices with little or no experience could not do so, resulting in a greater improvement after conversion into 3D imaging. These findings suggest that the training period for trainees and novices can be shortened by using 3D images. In addition, although significant differences were observed among experts during this crossover study, when compared to existing 2D images, slight improvement was observed in both the diagnostic accuracy and confidence level in lesion recognition. We believe that accuracy in recognition can be expected to improve by converting 2D images into 3D images, even for experts, because there was significant improvement during direct comparative studies. Although no significant intergroup difference was observed in the diagnostic accuracy of tumor extent, we plan to reinvestigate the accuracy by increasing the number of specimens in future studies. The findings of this study also suggest that 3D visualization enables us to observe the features and morphology of lesions in greater detail, thereby contributing to the early detection of superficial cancer in clinical practice. This study has some limitations. Subjective outcomes were used to investigate the utility of the 3D system. In principle, this study should have been blinded, but because of the obvious difference between 2D and 3D images, we conducted a crossover study instead. In addition, the sample size in this study was small as only 20 test lesions were examined, and thus the detection power for the differences between 2D and 3D images may be insufficient. In addition, 3D images and setting tested here are so artificial that the evidence obtained in this study is not directly applicable to clinical settings. We used ESD resection specimens and obtained only frontal still images. In the real world, lesions have blood supply and thus have a different color compared with resected specimens. Endoscopy views target lesions at various oblique angles. The present study used GI neoplastic lesions resected by ESD; thus we could not test the ability of 3D imaging in differentiating neoplasia from nonneoplasia. To overcome these limitations, we need to conduct clinical studies using 3D GI endoscopy. In the near future, we plan to perform studies using a prototype 3D endoscope for evaluating the impact of 3D imaging on diagnostic and therapeutic GI endoscopy. 5. Conclusions The conversion from 2D into 3D imaging may improve the diagnostic confidence level for superficial GI neoplasia, and the improvement by 3D imaging is greater in individuals with lower endoscopic expertise. The development of flexible 3D endoscopy may be worthwhile to improve endoscopic diagnosis. Competing Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Figure 1 (a) The method of endoscopic imaging. (b) The simplified schematic of (a). Figure 2 Connection method of the endoscopes. Figure 3 (a) A 2D sample of the resection specimen. (b) Example of parallax images (a 3D monitor and 3D glasses are required for three-dimensional visualization). (c) Adenocarcinoma components are detected in red line. Figure 4 Flow diagram of the study. Table 1 Average accuracy in determining the extent of disease.   2D (%) 3D (%) p All observers 78.4 ± 27.7 81.1 ± 25.5 0.177 Group 1 76.9 ± 27.6 78.6 ± 27.5 0.499 Group 2 79.9 ± 27.6 83.6 ± 23.1 0.079 Experts 80.8 ± 25.9 84.4 ± 23.5 0.061 Trainees 80.3 ± 26.3 81.4 ± 25.0 0.604 Novices 74.2 ± 30.0 77.4 ± 27.4 0.319 Table 2 The degree of certainty of Study 1 (average ± SD).   (a) Tumor extent (b) Morphology of tumor surface (c) MSS or/and MVS (d) Comprehensive recognition   2D 3D p 2D 3D p 2D 3D p 2D 3D p All observers 3.26 ± 1.20 3.70 ± 1.00 <0.01 3.24 ± 1.18 3.93 ± 1.03 <0.01 3.17 ± 1.14 3.58 ± 1.01 <0.01 3.08 ± 1.15 3.60 ± 0.96 <0.01 Experts 3.63 ± 1.18 3.85 ± 1.07 <0.05 3.93 ± 0.95 4.25 ± 0.87 <0.01 3.83 ± 1.00 3.90 ± 0.98 0.180 3.68 ± 1.06 3.87 ± 0.96 0.064 Trainees 3.45 ± 1.12 3.70 ± 0.98 <0.05 3.33 ± 1.03 3.88 ± 1.12 <0.01 3.33 ± 0.91 3.60 ± 0.82 <0.05 3.28 ± 1.04 3.66 ± 0.85 <0.01 Novices 2.71 ± 1.12 3.55 ± 0.92 <0.01 2.46 ± 1.05 3.68 ± 1.00 <0.01 2.35 ± 0.96 3.23 ± 1.10 <0.01 2.29 ± 0.87 3.28 ± 0.97 <0.01 Table 3 Average improvement of Study 1 (average ± SD).   (a) Tumor extent (b) Morphology of tumor surface (c) MSS or/and MVS (d) Comprehensive recognition Average Experts 0.23 ± 0.92 0.33 ± 0.80 0.08 ± 0.63 0.19 ± 0.85 0.20 ± 0.81∗ Trainees 0.25 ± 0.94 0.55 ± 0.97 0.28 ± 0.82 0.39 ± 0.84 0.37 ± 0.91∗,∗∗ Novices 0.84 ± 1.03 1.21 ± 1.21 0.88 ± 0.99 0.99 ± 0.96 0.98 ± 0.98∗∗ ∗ p < 0.05. ∗∗ p < 0.01. Table 4 The degree of certainty of Study 2.    (a) Tumor extent   (b) Morphology of tumor surface   (c) MSS or/and MVS   (d) Comprehensive recognition   Average 95% CI Average 95% CI Average 95% CI Average 95% CI All observers 3.48 3.40–3.56 4.07 3.99–4.15 3.32 3.24–3.40 3.53 3.41–3.63 Experts 3.31 3.18–3.44 3.95 3.84–4.06 3.15 3.05–3.25 3.48 3.35–3.61 Trainees 3.51 3.38–3.64 4.03 3.88–4.18 3.20 3.11–3.29 3.48 3.33–3.57 Novices 3.61 3.46–3.76 4.23 4.09–4.36 3.61 3.44–3.78 3.64 3.48–3.80 ==== Refs 1 Sørensen S. M. D. Savran M. M. Konge L. Bjerrum F. Three-dimensional versus two-dimensional vision in laparoscopy: a systematic review Surgical Endoscopy 2016 30 1 11 23 10.1007/s00464-015-4189-7 2-s2.0-84955192879 25840896 2 Feng C. Rozenblit J. W. Hamilton A. J. A computerized assessment to compare the impact of standard, stereoscopic, and high-definition laparoscopic monitor displays on surgical technique Surgical Endoscopy 2010 24 11 2743 2748 10.1007/s00464-010-1038-6 2-s2.0-78650185137 20361211 3 Tanagho Y. S. Andriole G. L. Paradis A. G. 2D versus 3D visualization: impact on laparoscopic proficiency using the fundamentals of laparoscopic surgery skill set Journal of Laparoendoscopic & Advanced Surgical Techniques 2012 22 9 865 870 10.1089/lap.2012.0220 2-s2.0-84869020391 23072406 4 Yao K. Matsui T. Furukawa H. Yao T. Sakurai T. Mitsuyasu T. A new stereoscopic endoscopy system: accurate 3-dimensional measurement in vitro and in vivo with distortion-correction function Gastrointestinal Endoscopy 2002 55 3 412 420 10.1067/mge.2002.121598 2-s2.0-0036518734 11868021 5 Oishi M. Fukuda M. Yajima N. Interactive presurgical simulation applying advanced 3D imaging and modeling techniques for skull base and deep tumors Journal of Neurosurgery 2013 119 1 94 105 10.3171/2013.3.jns121109 2-s2.0-84879936142 23581591 6 Yamashita K. Sakuramoto S. Mieno H. Preoperative dual-phase 3D CT angiography assessment of the right hepatic artery before gastrectomy Surgery Today 2014 44 10 1912 1919 10.1007/s00595-014-0858-8 2-s2.0-84927952294 24522892 7 Tian F. Wu J.-X. Rong W.-Q. Three-dimensional morphometric analysis for hepatectomy of centrally located hepatocellular carcinoma: a pilot study World Journal of Gastroenterology 2015 21 15 4607 4619 10.3748/wjg.v21.i15.4607 2-s2.0-84928198890 25914470 8 Wagner O. J. Hagen M. Kurmann A. Horgan S. Candinas D. Vorburger S. A. Three-dimensional vision enhances task performance independently of the surgical method Surgical Endoscopy 2012 26 10 2961 2968 10.1007/s00464-012-2295-3 2-s2.0-84871621061 22580874 9 Feng X. Morandi A. Boehne M. 3-dimensional (3D) laparoscopy improves operating time in small spaces without impact on hemodynamics and psychomental stress parameters of the surgeon Surgical Endoscopy 2015 29 5 1231 1239 10.1007/s00464-015-4083-3 25673344 10 Alaraimi B. El Bakbak W. Sarker S. A randomized prospective study comparing acquisition of laparoscopic skills in three-dimensional (3D) vs. two-dimensional (2D) laparoscopy World Journal of Surgery 2014 38 11 2746 2752 10.1007/s00268-014-2674-0 2-s2.0-84937965729 25002241

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==== Front Biomed Res IntBiomed Res IntBMRIBioMed Research International2314-61332314-6141Hindawi Publishing Corporation 10.1155/2016/5413849Research ArticleIdentification of Dietetically Absorbed Rapeseed (Brassica campestris L.) Bee Pollen MicroRNAs in Serum of Mice Chen Xuan http://orcid.org/0000-0002-8121-6457Dai Guan-hai http://orcid.org/0000-0002-1067-8574Ren Ze-ming http://orcid.org/0000-0001-8982-4409Tong Ye-ling http://orcid.org/0000-0001-6413-273XYang Feng http://orcid.org/0000-0003-0734-0502Zhu Yong-qiang * Institute of Basic Medicine, Zhejiang Academy of Traditional Chinese Medicine, Hangzhou 310007, China*Yong-qiang Zhu: zyq202@sina.comAcademic Editor: Peng Liao 2016 15 8 2016 2016 541384924 5 2016 5 7 2016 19 7 2016 Copyright © 2016 Xuan Chen et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.MicroRNAs (miRNAs) are a class of small noncoding RNA that, through mediating posttranscriptional gene regulation, play a critical role in nearly all biological processes. Over the last decade it has become apparent that plant miRNAs may serve as a novel functional component of food with therapeutic effects including anti-influenza and antitumor. Rapeseed bee pollen has good properties in enhancing immune function as well as preventing and treating disease. In this study, we identified the exogenous miRNAs from rapeseed bee pollen in mice blood using RNA-seq technology. We found that miR-166a was the most highly enriched exogenous plant miRNAs in the blood of mice fed with rapeseed bee pollen, followed by miR-159. Subsequently, RT-qPCR results confirmed that these two miRNAs also can be detected in rapeseed bee pollen. Our results suggested that food-derived exogenous miRNAs from rapeseed bee pollen could be absorbed in mice and the abundance of exogenous miRNAs in mouse blood is dependent on their original levels in the rapeseed bee pollen. Zhejiang Provincial Natural Science Foundation of ChinaLQ13C170002Zhejiang Provincial Science and Technology Department's Foundation2013F10001 ==== Body 1. Introduction MicroRNAs (miRNAs) are a class of small noncoding RNA that mediate posttranscriptional gene regulation by promoting cleavage or inhibiting translation of the target mRNA in plants or animals and play a critical role in nearly all biological processes, including metabolism and immune functions [1–3]. Recent studies suggest that plant miRNA may serve as a novel functional component of food which makes a critical contribution to maintaining and shaping animal body structure and function [4]. In 2012, a pilot study found that plant miRNAs from the diet will be absorbed by cells of the mammalian digestive tract and then packaged into microvesicles [4]. The microvesicles protect plant miRNAs from degradation and deliver them via the bloodstream to a variety of tissues (liver, kidney, heart, and brain), in which they will regulate cell gene expression [4]. Using the next-generation sequencing technology, Wang et al. demonstrated that human plasma contains a wide range of RNA from many exogenous species, including bacteria, fungi, and foods such as corn, rice, soybean, tomato, and grape [5]. Lukasik and Zielenkiewicz performed a bioinformatics analysis of publicly available raw data from studies on miRNAs composition in human and porcine breast milk exosomes to identify the fraction of food-derived miRNAs, and 35 and 17 miRNA species were identified, respectively [6]. miR-172 is the most highly enriched miRNA in Brassica oleracea, and after feeding mice with Brassica oleracea, miR-172 was found in the stomach, intestine, serum/blood, spleen, liver, kidney, and feces of mice [7]. Studies have shown that food-derived plant miRNAs have immunomodulating effects such as anti-influenza virus and antitumor [8, 9]. Zhou et al. found the first active miRNA in traditional Chinese medicine named miR-2911, a honeysuckle- (HS-) encoded atypical miRNA, that directly targets various influenza A viruses (IAVs) [8]. Subsequently, Yang et al. reported that miR-2911 levels fluctuated among various herbs. Feeding these different herb-based diets to the mice leads to different miR-2911 levels in the sera and urine which is associated with dietary intake levels [10]. Mlotshwa and others synthesized 3 tumor suppressor miRNAs (miR-34a, miR-143, and miR-145) with a characteristic of plant miRNA [11], and they reported that oral administration of the cocktail reduced tumor burden in well-established ApcMin/+ mouse model of colon cancer [12]. Furthermore, Western donor sera contained the plant miRNA miR159, whose abundance in the serum was inversely correlated with breast cancer incidence and progression in patients, and they demonstrated for the first time that a plant miRNA can inhibit cancer growth in mammals in vivo and in vitro [9]. Rapeseed (Brassica campestris L.) pollen is microgametophytes of rape. Bee-collected rapeseed pollen is widely used in food and healthy products [13]. In vivo and in vitro experiments demonstrated that the immune modulating effects by bee pollen might be attributed to its prevention and treatment for diseases [13–15]. Besides, RNA is rich in rapeseed bee pollen [16]. Nevertheless, whether miRNAs in rapeseed bee pollen could be absorbed by animals remains unclear. In this study, ICR mice were fed with rapeseed bee pollen, and then plant miRNAs including rapeseed miRNAs in mice blood were detected using next-generation sequencing technology. 2. Materials and Methods 2.1. Rapeseed Bee Pollen The rapeseed bee pollen was bought from Bee Research Institute of Anhui Agriculture University. The implementation of the standard is GB/T11758-89-bee pollen. Single pollen rates are over 95%, and the production date was November 10, 2015. 2.2. Animal Studies All animal experiments were performed using male ICR strain mice on a 12 h light/dark cycle in a pathogen-free animal feeding facility at Zhejiang Academy of Traditional Chinese Medicine. The animal study protocols were approved by the Animal Care and Use Committee of Zhejiang Academy of Traditional Chinese Medicine. At 6 weeks of age (weighted 26.37 ± 2.7 g), each mouse was fed rapeseed bee pollen (10 g/kg) by gavage. After a fixed time interval (3 h or 6 h on d1, d4, or d8), serum about 200 μL was collected from each mouse, and then total RNA was extracted using mirVana™ PARIS™ Kit (AM1556, Ambion™). 2.3. Illumina Hiseq2500 Sequencing The sequencing procedure was conducted according to standard steps provided by Illumina company, Inc. Briefly, a pair of adaptors were ligated to the 3′ and 5′ ends of total RNA. Reverse transcription followed by PCR is used to create cDNA constructs based on the small RNA ligated with 3′ and 5′ adapters. This process selectively enriches those fragments that have adapter molecules on both ends. Then the fragments of around 147–157 bp (22–30 nt length small RNA + adaptors) were purified by PAGE. The purified DNA was directly used for the cluster generation and sequencing using Illumina Hiseq2500 according to the manufacturer's instructions. The image files generated by the sequencer were then processed to produce digital data. The subsequent procedures included removing adapter dimers, junk, low complexity, common RNA families (rRNA, tRNA, snRNA, and snoRNA), and repeats. Subsequently, unique sequences with length in 18–26 nucleotides were mapped onto all plant miRNA precursors in miRBase 20.0 by BLAST search to identify known miRNAs and novel 3p- and 5p-derived miRNAs. Length variation at both 3′ and 5′ ends and one mismatch inside of the sequence were allowed in the alignment. The unique sequences mapping onto specific species mature miRNAs in hairpin arms were identified as known miRNAs. The unique sequences mapping onto the other arm of known specific species precursor hairpin opposite to the annotated mature miRNA-containing arm were considered to be novel 5p- or 3p-derived miRNA candidates. 2.4. Analysis of Level of miRNAs in Rapeseed Bee Pollen by RT-qPCR Total RNA was extracted from 80 mg rapeseed bee pollen using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Quantitative RT-PCR was performed using Taqman miRNA probes (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. To calculate the absolute expression levels of target miRNAs, a series of synthetic miRNA oligonucleotides at known concentrations were reverse transcribed and amplified. The absolute amount of each miRNA was then calculated with reference to the standard curve. Quantitative PCR was performed using an ABI-StepOnePlus machine (Applied Biosystems). 2.5. Statistical Analysis Differences are considered statistically significant at P < 0.05, using Student's t-test. 3. Results 3.1. Raw Data Filtering We sequenced a small RNA library from blood RNA of mouse fed with rapeseed bee pollen using the Illumina Hiseq2500 system. We acquired a total of 11,089,480 raw sequences. Overview of these reads from raw data to cleaned sequences is shown in Table 1. We illustrated small RNA reads with Rfam dataset; to remove rRNA, scRNA, snoRNA, snRNA, and tRNA, the pie charts were drawn for total reads and unique reads (Figure 1). 3.2. Plant miRNAs Spectrum in Serum of Mice Fed with Rapeseed Bee Pollen After removing the junk reads, the clean reads yield 34 plant miRNAs (Table 2). Plant miRNAs are 2′-O-methyl modified on their terminal nucleotide; in contrast, mammalian miRNAs with free 2′ and 3′ hydroxyls render plant miRNAs more difficult to be ligated to the cloning adapter compared with mammalian miRNAs. As a result, in the 11,089,480 raw reads, there were only 132 reads of plant miRNAs. However, some plant miRNAs can be detected even though the mammalian miRNAs caused a strong disturbance; in turn this proves that the content of plant miRNAs in mouse blood was not low. Among the plant miRNAs, miR-166a and miR-159 were with the highest levels in mouse blood; besides, these two miRNAs were both mapped onto rapeseed genome. 3.3. Comparison of Abundance Levels of miR-166a and miR-159 in Rapeseed Bee Pollen Based on the predominant two miRNAs (miR-166a and miR-159) in the blood, we assumed that miR-166a and miR-159 can be found in rapeseed bee pollen, and the content of miRNAs in the rapeseed pollen also will follow the trend in the serum. To confirm this, the levels of miR-166a and miR-159 in rapeseed bee pollen were assessed by stem-loop quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay. As a result, miR-166a and miR-159 can be detected in RNA of rapeseed bee pollen (Additional Figure 1 in Supplementary Material available online at http://dx.doi.org/10.1155/2016/5413849). Moreover, the CT values of miR-166a and miR-159 were 23.8 ± 0.23 and 31.22 ± 0.33, respectively, suggesting that the abundance level of miR-166a was higher than miR-159. These results suggested that food-derived exogenous miRNA from rapeseed bee pollen could be absorbed by mouse, and the abundance of specific miRNAs is dependent on their origins from the rapeseed bee pollen. 3.4. Comparison of Abundance Levels of miR-166a between Mice Fed with Rapeseed Bee Pollen and Control Given that miR-166a is the highest abundance rape-encoded miRNA in mice fed with rapeseed bee pollen, and it is rich in rapeseed bee pollen, we speculate that the miR-166a in mouse serum are mainly absorbed from rapeseed bee pollen. To test this speculation, we compared the abundance level of miR-166a in serum of mice fed with rapeseed bee pollen and control. As it is reported that the levels of plant-based miRNAs were elevated in serum of mice for 6 h [4], we compared the levels of miR-166a in serum of mice fed with rapeseed bee pollen after 6 h and control. As shown in Figure 2 and additional Figure 2, the levels of miR-166a were elevated in serum of mice fed with rapeseed bee pollen for 6 h compared with control by RT-qPCR. 4. Discussion An estimated 60% of all protein-coding genes are targeted by miRNAs in human [17]. In addition, many miRNAs are deregulated in immune system, inducing diseases like autoimmune diseases, inflammation, and tumors [3]. Food-derived miRNAs have the potential to restore the downregulated miRNAs in diseases. For example, immune-related miRNAs are abundant in breast milk, and they might play a critical role in the development of the infant immune system [18]. Furthermore, Western donor sera contained the plant miRNA miR159, whose abundance in the serum was inversely correlated with breast cancer incidence and progression in patients [9]. These studies raise the intriguing prospect of using edible plants miRNAs to prevent and treat mammal diseases. Bee pollen is rich in nutrition and medicinal composition, which ensued a wide use of bee pollen in food, health products, medicine, cosmetics, and other fields [13, 19, 20]. In the field of medicals, bee pollen is used for prevention and treatment of prostate diseases [14], cardiovascular and cerebrovascular diseases, immune diseases, and so forth [15]. Besides, bee pollen is rich in RNA with a range of 0.6%–1% (w/w) [16]. In this study, we confirmed that miRNAs from rapeseed bee pollen can be absorbed by mice, and the abundance of exogenous miRNAs in mouse blood is dependent on their original levels in pollen. Moreover, the detailed functions of these exogenous miRNAs in mammals should be investigated to help clarify the immune function or medical efficacy of bee pollen. Nevertheless, the present study provided first hand evidence for the potential usages of rapeseed bee pollen as a supplement of plant miRNAs. Supplementary Material The amplification curve of miR-159 (red) and miR-166a (green) by qPCR Acknowledgments This work was funded by the Zhejiang Provincial Natural Science Foundation of China (LQ13C170002) and Zhejiang Provincial Science and Technology Department's Foundation (2013F10001). Competing Interests The authors declare that they have no competing interests. Figure 1 Pie chart of sequence category. (a) Pie chart of sequence category of total reads. (b) Pie chart of sequence category of unique reads. Figure 2 The abundance levels of miR-166a in mouse serum after feeding with rapeseed bee pollen or chow diet for 6 h (n = 5). ∗ p < 0.05. Table 1 Overview of reads. Lib Type Total % of total Unique % of unique Raw reads Nuclear acid 11,089,480 100.000 209,873 100.000 3ADT & length filter   320,992 2.895 145,808 69.474 Junk reads   858 0.008 610 0.291 Rfam RNA 47,589 0.429 6,477 3.086 mRNA RNA 3,694 0.033 905 0.431 Repeats RNA 369 0.003 74 0.035 rRNA RNA 19,901 0.179 2,026 0.965 tRNA RNA 15,176 0.137 2,527 1.204 snoRNA RNA 5,507 0.050 750 0.357 snRNA RNA 527 0.005 243 0.116 Plant miRNA RNA 221 0.002 33 0.016 Another Rfam RNA RNA 6,478 0.058 931 0.444 Clean reads   10,716,785 96.639 56,136 26.748 Table 2 Plant miRNAs in mice fed with rapeseed bee pollen. miRNA ID miRNA sequence Length (nt) Frequency bna-miR-166a TCGGACCAGGCTTCATTCCCC 21 35 bna-miR-159 TTTGGATTGAAGGGAGCTCTA 21 22 gma-miR6300 GTCGTTGTAGTATAGTGGT 19 8 nta-miR6145e ATTGTTACATGTAGCACTGGCT 22 7 nta-miR6146b TTTGTCCAATGAAATACTTATC 22 6 nta-miR6020b AAATGTTCTTCGAGTATCTTC 21 5 nta-miR6149a TTGATACGCACCTGAATCGGC 21 5 ath-miR-166a TTCGGACCAGGCTTCATTCCCC 22 3 osa-miR530 TGCATTTGCACCTGCACCTCC 21 3 ahy-miR408 TGCACTGCCTCTTCCCTGGCT 21 3 mdm-miR408a TGCACTGCCTCTTCCCTGGCT 21 3 bna-miR397a ATTGAGTGCAGCGTTGATG 19 2 peu-MIR2916 CAACCATAAACGATGCCGACCAGG 24 2 nta-miR168a TCGCTTGGTGCAGGTCGGGAC 21 2 gma-miR482b TCTTCCCTACACCTCCCATACC 22 2 nta-miR482a TTTCCAATTCCACCCATTCCTA 22 2 nta-miR827 TTAGATGAACATCAACAAACA 21 2 ppt-miR894 TTCACGTCGGGTTCACCA 18 2 gma-miR3522 TGAGACCAAATGAGCAGCTGA 21 2 gma-miR4996 TAGAAGCTCCCCATGTTCTCA 21 2 bna-miR403 TTAGATTCACGCACAAACTCG 21 1 peu-MIR2916 ACCGTCCTAGTCTCAACCATA 21 1 aau-miR162 TCGATAAACCTCTGCATCCAG 21 1 bdi-miR398a TATGTTCTCAGGTCGCCCCTGT 22 1 gma-miR403a TTAGATTCACGCACAAACTT 20 1 gma-miR1507a TCTCATTCCATACATCGTCTGA 22 1 nta-miR6159 TAGCATAGAATTCTCGCACCTA 22 1 hbr-miR6173 GCTGTAAACGATGGATACT 19 1 ptc-miR6478 CCGACCTTAGCTCAGTTGGT 20 1 stu-miR7997c TTGCTCGGATTCTTCAAAAAT 21 1 bna-miR156b TTGACAGAAGATAGAGAGCAC 21 1 gma-miR166m GCGGACCAGGCTTCATTCCCC 21 1 stu-miR399a GGGCTACTCTCTATTGGCATG 21 1 bna-miR156a TGACAGAAGAGAGTGAGCAC 20 1 ==== Refs 1 Bartel D. 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==== Front Can Respir JCan. Respir. JCRJCanadian Respiratory Journal1198-22411916-7245Hindawi Publishing Corporation 10.1155/2016/8671742Clinical StudyAssessment of Postresuscitation Volume Status by Bioimpedance Analysis in Patients with Sepsis in the Intensive Care Unit: A Pilot Observational Study http://orcid.org/0000-0002-8293-7061Rochwerg Bram 1 2 http://orcid.org/0000-0002-2134-3745Cheung Jason H. 1 Ribic Christine M. 1 2 3 Lalji Faraz 3 Clarke France J. 2 Gantareddy Susheel 4 Ranganath Nischal 1 Walele Aziz 5 McDonald Ellen 1 Meade Maureen O. 1 2 Cook Deborah J. 1 2 Wilkieson Trevor T. 3 Clase Catherine M. 1 3 http://orcid.org/0000-0002-0335-4248Margetts Peter J. 1 3 http://orcid.org/0000-0003-0131-8718Gangji Azim S. 1 3 * 1Department of Medicine, McMaster University, Hamilton, ON, Canada2Department of Clinical Epidemiology and Biostatistics, McMaster University, Hamilton, ON, Canada3Division of Nephrology, St. Joseph's Healthcare Hamilton, Hamilton, ON, Canada4Department of Family Medicine, Northern Ontario School of Medicine, Sudbury, ON, Canada5Brampton Civic Hospital, Brampton, ON, Canada*Azim S. Gangji: gangji@mcmaster.caAcademic Editor: Jörg D. Leuppi 2016 15 8 2016 2016 86717429 12 2015 23 6 2016 12 7 2016 Copyright © 2016 Bram Rochwerg et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Bioimpedance analysis (BIA) is a novel method of assessing a patient's volume status. Objective. We sought to determine the feasibility of using vector length (VL), derived from bioimpedance analysis (BIA), in the assessment of postresuscitation volume status in intensive care unit (ICU) patients with sepsis. Method. This was a prospective observational single-center study. Our primary outcome was feasibility. Secondary clinical outcomes included ventilator status and acute kidney injury. Proof of concept was sought by correlating baseline VL measurements with other known measures of volume status. Results. BIA was feasible to perform in the ICU. We screened 655 patients, identified 78 eligible patients, and approached 64 for consent. We enrolled 60 patients (consent rate of 93.8%) over 12 months. For each 50-unit increase in VL, there was an associated 22% increase in the probability of not requiring invasive mechanical ventilation (IMV) (p = 0.13). Baseline VL correlated with other measures of volume expansion including serum pro-BNP levels, peripheral edema, and central venous pressure (CVP). Conclusion. It is feasible to use BIA to predict postresuscitation volume status and patient-important outcomes in septic ICU patients. Trial Registration. This trial is registered with clinicaltrials.gov NCT01379404 registered on June 7, 2011. Regional Medical Association Research GrantMcMaster University Department of MedicineMcMaster University Division of Nephrology ==== Body 1. Introduction Severe sepsis confers an extremely high mortality rate of approximately 40% in critically ill patients [1]. Early goal directed therapy (EGDT) with aggressive fluid resuscitation has been shown to reduce mortality by 16% in a single-center randomized trial [2]. Two subsequent multicenter trials found no clear benefit to EGDT; however, patients randomized to both protocolized and nonprotocolized care in both of these studies received early and aggressive fluid resuscitation [3, 4]. Beyond the initial resuscitative period, observational studies have identified that a persistent hypervolemic state has been independently associated with mortality, prolonged mechanical ventilation, and need for renal replacement therapy [5–7]. Although aggressive fluid resuscitation remains the cornerstone of EGDT, subsequent management of fluid status in patients with severe sepsis has not been well defined. An added challenge is that accurate volume assessment in patients in the intensive care unit (ICU) is difficult to ascertain. Subjective assessment of volume status by physical examination is limited by poor accuracy and reliability [8]. Other objective measures such as central venous pressure and pulmonary catheter pressure are invasive and not predictive of volume status [9–12]; therefore, tools to help guide clinicians in assessing patients' fluid status are required. Bioimpedance analysis (BIA) assesses the electrical properties of tissues by measuring the reactance and resistance of an alternating current passed through the body. BIA values can be plotted on reactance-resistance (R-X c) graphs to derive a vector length (VL). The VL is a reflection of an individual's overall volume status. A longer VL is indicative of euvolemia or volume depletion and a shorter VL is associated with volume expansion [13]. In patients with end-stage renal disease on dialysis, VL has been correlated inversely with volume status [14–18]. In patients with congestive heart failure, BIA has been shown to predict volume status; in these patients, VL was shown to increase with diuresis, suggesting that VL is a reflection of overall volume [19]. In addition, BIA devices are practical tools to use at the bedside, being compact, portable, inexpensive, and noninvasive. BIA has not been well studied in the ICU setting. In a single-center cross-sectional study comprising 34 critically ill medical and surgical patients [20], VL was found to correlate with central venous pressure (CVP) measures (r = −0.38, p = 0.025). However, the use of BIA to assess total volume status and prognosis in a prospective manner in critically ill patients with sepsis has not been reported. We performed a prospective observational pilot study to determine the feasibility of using BIA in the ICU. Our pathophysiologic hypothesis was that septic patients would have an initial period of volume expansion (reflected in low VL values), eventually resolving during the first few days of their ICU admission (as seen by increasing VL values) as fluid was mobilized and the patient moved towards no longer requiring invasive mechanical ventilation (IMV). Feasibility measures included recruitment efficiency [21], eligibility and enrolment, and consent rate. Our secondary objectives were to determine if VL was associated with the need for IMV and other clinical measures of volume, thereby assessing the construct validity of VL as a marker of volume. 2. Methods This prospective single-center study was conducted from January 17, 2011–Feb 1, 2012. Inclusion criteria were adult patients (age > 18 years) admitted to the ICU with systemic inflammatory response syndrome (SIRS) and a high clinical suspicion of infection, requiring invasive positive pressure ventilation, and with a central venous catheter (internal jugular or subclavian) allowing CVP measurement. Exclusion criteria were preexisting end-stage kidney disease on chronic dialysis, pregnancy, limb amputation(s), the presence of a temporary or permanent pacemaker, and inability to obtain informed consent. The study received approval from the Hamilton Integrated Research Ethics Board (HIREB) prior to patient enrolment. All patients who participated in this study provided explicit informed consent either directly or via their substitute decision maker. Baseline data collected at enrolment included age, sex, race, height, weight, CVP (cm H2O), APACHE II score, heart rate (HR), mean arterial pressure (MAP), urine output (mL/hr), multiple organ dysfunction score (MODS), and need for life support modalities. Enrolled patients were classified as medical or surgical (having received a surgical procedure within 72 hours of ICU admission). A seven-point Likert scale assessing peripheral edema was created and performed in duplicate for 45 of the measurements in order to evaluate reliability (see Supplementary Material available online at http://dx.doi.org/10.1155/2016/8671742 for edema scale). These duplicate measures were performed independently and the evaluators were blinded to one another's assessment. We used the intraclass correlation coefficient (ICC) to estimate agreement on the duplicate peripheral edema scores (r = +1 represents perfect agreement; r = 0 represents no agreement). Laboratory data collected at baseline included routine ICU blood work in addition to N-terminal brain natriuretic peptide (N-BNP) levels. BIA measurements were performed at enrolment (within 48–96 hours of ICU admission), and then at day 3 and day 7 after enrolment using the Bodystat Quadscan 4000 (Bodystat, Isle of Man, British Isles). Patients were assessed in the supine position on nonconductive surfaces. Tetrapolar placement of disposable foil-gum electrodes (wrist-hand and ankle-foot) was used. Measurements were performed at 5, 50, 100, and 200 Hz and done in triplicate with the average of these three measures used for analysis (see Supplementary Material for an example of an individual patient's bioimpedance readings with means and standard deviations). Bioimpedance standard operating procedures were developed for this study and distributed to all study sites to ensure consistency of technique (see Supplementary Material). Also, face-to-face training was provided to all research staff involved in performing measurements that included direct observation. Transformation of the data was completed according to the Piccoli method [22]. The first 28 patients enrolled had a total of three BIA measurements performed within the first week after enrolment. BIA VL data for 8 of these initial patients was lost due to human and/or device error. After analysis of the temporal changes in BIA in these first 20 patients, we concluded that more frequent BIA measurements conducted over an extended period of time would be required in order to demonstrate proof of concept. Consequently, a protocol change was instituted for the remaining patients such that BIA measurements were performed at baseline and on day 3 and day 7 and every four days until day 30 (or ICU discharge or ICU death). If patients were discharged from ICU prior to day 30, they were followed on the ward with a single subsequent BIA measurement. Physicians responsible for the clinical care of the recruited patients, nurses, study investigators, and the research coordinator were blinded to the BIA test results. Our primary outcome for this pilot was the feasibility of performing BIA vector length measures in the ICU setting. The initial study proposal focused on efficacy outcomes; however, given the protocol changes that were required after the first 20 patients were enrolled, we changed our primary focus to feasibility. Feasibility determination included recruitment efficiency, eligibility and enrolment, and consent rate. Secondarily, our proof of concept determination involved evaluating whether a correlation existed between initial BIA vector length and other measures of volume status, both intra- and extravascular including CVP, N-BNP serum levels, edema score, cumulative fluid balance, and total volume of fluid infused. Cumulative fluid balance and total volume of fluid infused were reported throughout the study period. Other patient-important outcomes included determining if an association existed between VL and ventilator status (i.e., having a status of requiring IMV versus not requiring IMV) and the proportion of patients who developed acute kidney injury as determined by the RIFLE criteria. Stage risk (RIFLE-R) and stage failure (RIFLE-F) were evaluated. RIFLE-R is defined as 1.5–2x increase in serum creatinine from baseline, a 25% decrease in estimated glomerular filtration rate (eGFR), or a urine output < 0.5 mL/kg/hr for any consecutive 6-hour period. RIFLE-F is defined as 3x increase in serum creatinine from baseline, a 75% decrease in eGFR, or a urine output <0.3 mL/kg/hr for any consecutive 24-hour period or anuria. Baseline BIA measurement data points for each patient were correlated with other baseline measures of volume status using the Pearson statistic. We used the ICC statistic to estimate agreement on the duplicate peripheral edema scores. A generalized estimating equation (GEE) was used to determine if a relationship between VL and ventilator status and VL and proportion of patients meeting RIFLE-R or RIFLE-F criteria was present. The GEE allowed us to estimate the parameters in our generalized linear model with adjustment for potential correlation between variables with multiple repeated measures. Potential confounders controlled for in the GEE included age, vector length, and study day. Statistical significance was set at p < 0.05. 3. Results A total of 655 patients were screened (Figure 1). Of these, 141 patients (21.5%) met the inclusion criteria, of which 63 patients subsequently met exclusion criteria. A further 18 eligible patients were not enrolled due to consent refusal (four patients), no substitution decision maker available (eleven patients), or language barrier/family distress (three patients). Therefore, 60 patients were enrolled (with a consent rate of 93.8%) over the 12-month recruitment period. Due to device malfunction (3 patients) and human error (5 patients), the BIA measurement values were not available for 8 patients. The average monthly recruitment rate was 6.15 patients enrolled per month. There were no reported patient complications from BIA measurement acquisition. Each measurement required less than 10 minutes to perform. Our study protocol was altered after we reviewed data from the first 20 patients. The initial protocol called for three BIA measures over the first week after enrolment. Initial measures suggested patients remained volume overloaded at day 7 with little change in their VL measurements (Figure 2). We therefore decided to expand the study period to better capture the change in volume status over time. After the amended protocol was approved by the HIREB, the BIA measurements were performed at baseline and on day 3 and day 7 and every four days until day 30 (or ICU discharge or ICU death). Thirty-two patients had up to 8 BIA assessments during their ICU stay. Even while performing BIA measurements every four days, variability among individual patient's data points was still significant (Figure 2). A total of 52 patients completed the study with a mean APACHE II score of 26.8 (SD 6.9). Baseline characteristics are presented in Table 1. Considering correlation with other known measures of volume status, shorter baseline VL measurements (suggesting volume overload) were associated with higher CVP readings (r = −0.21, p = 0.03), higher N-BNP levels (r = 0.302, p = 0.04), and higher scores on our edema scale (r = −0.673, p < 0.001) (Table 2). Agreement for our edema score was excellent (r = 0.73). Correlations with cumulative fluid balance, total volume of fluid infused, and serum albumin levels were not significantly associated with VL. For each individual 50-unit increase in VL (ohm/m), there was an associated 22% increase in the probability of not requiring IMV. This was not statistically significant (p = 0.13). Similarly, for each 50-unit increase in VL (ohm/m), there was a 32% decrease in meeting RIFLE-R criteria (p = 0.06) and a 65% decrease in meeting RIFLE-F criteria (p = 0.07). Figure 3 shows an example of contrasting clinical trajectories plotted on R-X c graphs for two individual patients enrolled in our study. The lower left quadrant of the graph represents volume overload and shorter vector lengths while the upper right quadrant represents hypovolemia and longer vector lengths. Patient 58 represents a patient who remained volume overloaded throughout their ICU stay and was never successfully extubated. Patient 36 had VL measurements that increased over time and the patient was extubated on study day twelve. 4. Discussion This was a prospective observational cohort study designed to examine BIA as a tool to measure overall postresuscitation volume status in ICU patients. Current measures of volume status are inaccurate and at times rely on invasive procedures. A novel method of determining postresuscitation volume status is needed and BIA represents an inexpensive, noninvasive, and reliable alternative to our current state of practice. Although our initial focus in this trial was to assess efficacy, the need for early protocol modifications led us to feasibility targets with a secondary objective to study the impact on patient-important outcomes and the correlation of BIA measurements with other known measures of volume status. Through this pilot, we have demonstrated the feasibility and that the BIA technique is an easily implementable tool in the ICU setting. Although measurements for 8 patients were lost, this was due to administrative error and battery failure rather than a problem with the BIA machine itself. Our enrolment rate (percentage enrolled compared to those screened) was 8% with fourteen patients missed due to inability to contact a substitute decision maker. If we had included these fourteen patients in the study, as would be possible using an alternate consent model such as deferred consent, and assuming consent had been obtained, our enrolment rate would have been at our target of 10%. Our consent rate for this feasibility study was excellent (93.75%), meeting our target of greater than 80%. This pilot study was not powered to evaluate clinically relevant patient outcomes. Despite this, we observed two trends (neither reached statistical significance): patients with longer VL (less hypervolemia) may be less likely to require IMV (p = 0.13) and may be less likely to develop acute kidney injury based on RIFLE criteria (p = 0.07). This latter finding is consistent with the observational data [5–7, 23, 24] and limited RCT data [25] that suggest a link between ongoing hypervolemia and renal dysfunction. VL was significantly and inversely associated with other markers of volume expansion, CVP, and edema score. This provides some criterion validity with respect to VL as a measure of total volume in critically ill patients. No association between VL and cumulative fluid balance or total fluid infused was evident; however, these measures are often unreliably and inconsistently recorded in the ICU [26]. Previous clinical applications of BIA have relied on regression-based equations which make assumptions based on healthy populations and lack validation in critically ill patients [27]. By using the Piccoli method [13], and R-X c graphs comparing resistance against reactance, we have been able to circumvent these issues. Only one other prospective observational study [20] has investigated the use of BIA in thirty-four ICU patients with a predominant admitting diagnosis of trauma. Those enrolled had serial BNP, CVP, and BIA measurements performed throughout their ICU stay. Baseline measurements showed a weak correlation between CVP and BIA VL (p = 0.025); however, this did not persist beyond the initial measurement. Also, there was no correlation between BIA VL and oxygenation index. Importantly, this study did not focus on postresuscitative patients with sepsis or examine the effect on patient-important outcomes such as ventilator requirements. Our original hypothesis suggested that septic patients would have an initial period of volume expansion that would resolve within the first few days after ICU admission. Based on initial results, we found that patients were significantly volume expanded at time of study enrolment (two days after ICU admission) and remained persistently volume overloaded even up to a week after admission. As previously mentioned, we therefore modified our protocol and began performing additional BIA measurements every four days for up to 30 days (or ICU discharge or ICU death). This observed delay in fluid mobilization in postresuscitation septic patients was significant and may warrant further investigation. The clinical importance of “deresuscitation” is increasingly being recognized and BIA could be used as a tool to help guide this process. Strengths of this feasibility study include the novel application of a relatively inexpensive and easy-to-use, bedside device to help address the question of postresuscitation volume status in critically ill patients. Our study design enabled real-time protocol enhancements and improved data collection. We focused on a specific patient population and excluded patients who were admitted to ICU and received volume resuscitation for etiologies other than sepsis. Correlating the BIA VL measurements with other known intra- and extravascular measures of volume status allowed for proof of concept assessment. Other strengths include assessing the reliability testing of the edema score. Limitations of this study include the relatively small number of patients who were all enrolled from a single ICU. Given the pilot nature of our study, it is difficult to draw any conclusions about clinically important outcomes based on these data. Despite the suggestion that BIA may be a predictor of ventilator status [28], there are many factors in addition to volume status that are associated with ventilator status in critically ill patients. Significant within-patient variability of BIA VL was noticed with the measurements being performed every four days and, therefore, in the future, consideration will be given to performing daily measurements to better capture the true variation in this measure. 5. Conclusions Clinicians faced with the challenge of managing hypervolemia beyond the acute resuscitative phase of critical illness lack a noninvasive and reliable method to guide volume management. Based on this pilot, BIA measures are significantly associated with traditional measures of resuscitative volume status such as CVP and BNP and postresuscitation hypervolemia such as edema score thereby adding to its validity as a measure of volume. Bioimpedance analysis may have a role in the management of sepsis beyond the initial resuscitative phase with regard to volume assessment and management, including aiding in reducing the associated untoward consequences. The results of this feasibility study have fueled the protocol of a large observational study that is currently underway to determine whether BIA vector length can predict patient-important outcomes such as ventilator dependence and AKI. Supplementary Material This supplementary material provides the 7-point Likert scale that was used to assess peripheral edema in patients enrolled in this study. Acknowledgments The authors would like to acknowledge the patients and families who agreed to participate in this pilot study as well as the Regional Medical Associates and the Division of Nephrology and the Department of Medicine at McMaster University for funding support for this pilot study. They would also like to acknowledge Mike Radford for his help with BIA measurements and data collection and Bronwyn Barlow-Cash for her help with data collection. Financial Support for the study was from the following: Regional Medical Association Research Grant, McMaster University Department of Medicine, and McMaster University Division of Nephrology. Abbreviations BIA:Bioimpedance analysis ICU:Intensive care unit VL:Vector length RIFLE:Risk, injury, failure, loss, end stage BNP:Brain natriuretic peptide CVP:Central venous pressure EGDT:Early goal directed therapy R-Xc graph:Resistance versus reactance graph IMV:Invasive mechanical ventilation SIRS:Systemic inflammatory response syndrome HIREB:Hamilton Integrated Research Ethics Board HR:Heart rate MAP:Mean arterial pressure APACHE:Acute physiology and chronic health evaluation MODS:Multiple organ dysfunction score ICC:Intraclass correlation coefficient Hz:Hertz eGFR:Estimated glomerular filtration rate GEE:Generalized estimating equation RCT:Randomized controlled trial AKI:Acute kidney injury. Disclosure None of those supplying funding support had any input in the study design, data collection or analysis, interpretation of the data, writing of the paper, or decision regarding publication. Competing Interests The authors declare they have no competing interests. Authors' Contributions All authors contributed to developing the study protocol and generating the analysis plan. Bram Rochwerg was on the steering committee, was the junior principle investigator, participated in data collection and analysis, and drafted the first version of the paper. Jason H. Cheung was on the steering committee and participated in data collection and analysis. Christine M. Ribic was on the steering committee, helped to oversee all aspects of the study, and participated in data analysis. Faraz Lalji was on the steering committee and participated in data analysis. France J. Clarke performed daily BIA measurements, collected data, and helped with data analysis. Susheel Gantareddy helped with data collection and database management. Nischal Ranganath created the database and helped with data collection and data entry. Aziz Walele was primarily involved in study protocol development and helped with some data collection. Ellen McDonald performed daily BIA measurements, collected data, and helped with data analysis. Maureen O. Meade contributed to data analysis. Deborah J. Cook contributed to data collection and analysis. Trevor T. Wilkieson ran the statistical analysis and contributed to analysis. Catherine M. Clase contributed to data analysis. Peter J. Margetts was on the steering committee, helped to oversee all aspects of the study, and participated in data analysis. Azim S. Gangji was the senior study principle investigator, sat on the steering committee, and oversaw all aspects of study procedures. The steering committee drafted the first edit of the paper and then all authors read, offered revisions, and approved the final version. Figure 1 Flowchart of patient enrolment. Figure 2 Change in vector length over time. Change in mean vector length over time is shown for 5 randomly selected patients. The green vertical line is shown at day 7 and demarcates the initial 7-day protocol (followed for the first 16 patients) from the subsequent 30-day protocol. Persistently small vector lengths (representing hypervolemia) are seen for the first 7 days which led us to increasing the number of measurements and length of follow-up for this feasibility study. There is significant variability within each patient's BIA measures, suggesting more frequent measures are required to more accurately assess volume status over time. Figure 3 R-X c (resistance versus reactance) graphs of two individual patients. An illustrative example of R-X c graphs demonstrating the volume trajectories of two enrolled patients. Patient 58 had 8 measurements done while in the ICU and was never extubated. It is evident that all of the measurements for patient 58 remain in the left lower quadrant of the R-X c graph suggesting a persistently small vector length (or a hypervolemic state). Patient 36 had 4 measurements done while in the ICU and was extubated after the 2nd measurement. Patient 36 started in the hypervolemia range; however, the patient moved to the upper right quadrant of the R-X c graph consistent with improved volume status and extubation (numbered dots indicate visit day). Table 1 Baseline patient characteristics at time of study enrolment. Characteristic N = 52 Age (years), mean (SD) 64 (13) Sex    Male, N (%) 27 (52%) Ethnicity    Caucasian, N (%) 47 (90%)  Black, N (%) 3 (6%)  Aboriginal, N (%) 1 (2%)  Other, N (%) 1 (2%) Weight (kg), mean (SD) 82.4 (27.6) Patient type    Medical, N (%) 46 (89%)  Surgical, N (%) 6 (11%) Chronic health index, median (min, max) 1 (0–2) APACHE II score, mean (SD) 26.8 (6.9) MODS score, mean (SD) 7.5 (2.9) Vasopressor/inotropic dependence on admission, N (%) 40 (77%) Serum creatinine on admission to ICU μmol/L, mean (SD) 143.4 (127.7) Table 2 Correlation of baseline vector length with other baseline measures of volume status. Clinical feature Pearson correlation coefficient (R) p value N-BNP serum level −0.30 0.04 Central venous pressure −0.21 0.03 Edema score −0.67 <0.001 Cumulative fluid balance −0.22 0.11 Volume of fluid infused since admittance (crystalloid & colloid) −0.19 0.18 Albumin serum level 0.271 0.079 ==== Refs 1 Dombrovskiy V. Y. Martin A. A. Sunderram J. Paz H. L. 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==== Front Int J Chronic DisInt J Chronic DisIJCDInternational Journal of Chronic Diseases2356-69812314-5749Hindawi Publishing Corporation 10.1155/2016/8096473Research ArticleRevealing Maximal Diameter of Upper Limb Superficial Vein with an Elevated Environmental Temperature http://orcid.org/0000-0002-5850-4207Irfan Hira 1 * Ooi Guo Shen 2 Kyin May M. 2 http://orcid.org/0000-0003-4560-2374Ho Pei 2 1Dow University of Health Sciences, Karachi 74200, Pakistan2Department of Cardiac, Thoracic and Vascular Surgery, National University Hospital, Singapore 119228*Hira Irfan: hirairfan89@hotmail.comAcademic Editor: Ivor J. Katz 2016 15 8 2016 2016 809647318 4 2016 3 7 2016 Copyright © 2016 Hira Irfan et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Ultrasonography is the primary tool for preoperative analysis of vein morphology for fistula creation in patients with end-stage renal disease. This study examines the effect of environmental temperature on the superficial vein size. Superficial veins of thirteen healthy volunteers were marked at three sites: cephalic vein in left lateral arm near cubital fossa, cephalic vein in left forearm at wrist, and basilic vein in left medial arm near cubital fossa. Mean diameters were recorded using ultrasound probe at 26°C and 43°C. Body temperature was increased using a Bair Hugger blanket. Mean values from the two temperatures were analyzed using paired sample t-test. All three superficial vein sites displayed statistically significant increase in diameter when the temperature was increased from 26°C to 43°C. Paired t-test showed p values of 0.001 for cephalic vein at wrist, 0.01 for cephalic vein near cubital fossa, and 0.01 for basilic vein near cubital fossa. This study proved that environmental temperature exerts a statistically significant effect on vein size measured by ultrasound during preoperative assessment for vascular access. Not to the extent of 43°C, the authors would recommend setting the room temperature higher during ultrasound vascular assessment to avoid underestimating the superficial vein size. ==== Body 1. Introduction According to a global overview by Grassmann et al. in 2005, more than 70% of end-stage renal disease (ESRD) patients were managed with hemodialysis [1]. Vascular access is an essential component of hemodialysis. National Kidney Foundation (NKF) guidelines recommend duplex US as the essential study for upper arm vascular assessment before fistula creation [2]. The diameter of superficial vein was identified as a key predictor of access patency. Clinicians often make the decision of using native vein or artificial graft based on assessment of vein size by ultrasound [3]. The size of arteries remains relatively stable unless in extreme physiological conditions such as hypovolemic shock [4]. Veins, on the contrary, are capacitance vessels and their size may vary significantly with different physiological and environmental conditions. Underestimation of vein size may result in wasting patient's native vein suitable for fistula creation, whereas overestimation may result in a low maturation rate of the arteriovenous fistula (AVF) being created. Numerous studies had explored thoroughly the potential of body positioning on vein size [5, 6]. However, not much work had investigated the effect of external environment. Our study aimed to assess variation in vein size in response to environmental temperature in a setting similar to regular vein size assessment performed for ESRD patient before fistula creation. 2. Materials and Methods National Healthcare Group domain specific review board (NHG DSRB) approval was obtained for this project. 13 healthy adults were randomly selected to participate in this experimental study. All subjects were of average build and denied chronic ailments including diabetes, hypertension, and peripheral arterial disease. Research protocol was explained to all subjects in detail, their informed consent was obtained, and measures were taken to keep identity of all the participants strictly confidential. Before initiating, participants were rested in a relatively stress-free environment for ten minutes. All measurements were obtained in a semirecumbent position on the left arm which was devoid of external pressures such as tight clothing or watches. Blood pressure measurements were taken in the right and the left arms and recorded. Three markings were made on the left upper limb: (i) lateral side of forearm 5 cm proximal to the styloid process of radius to mark cephalic vein in forearm (CV at wrist), (ii) lateral side of arm 5 cm proximal to the skin crease of cubital fossa to mark cephalic vein in arm (CV at cubital fossa), and (iii) medial side of arm 5 cm proximal to the skin crease of cubital fossa to mark basilic vein in arm (BV near cubital fossa). Internal diameter of veins was measured with the least pressure of ultrasound probe applied to these three sites. Readings were recorded to the accuracy of 0.01 cm using two-dimensional B mode ultrasound probe (SonoSite MicroMaxx) with an 11 MHz transducer. Average of the long and short axis of the ellipse was recorded at two separate occasions and the mean value was noted as the final diameter for each site. The two temperatures under focus in the study were 26°C, which was the room temperature of the area used to conduct the experiment, and 43°C, which was selected to bring about a measurable change to prove our hypothesis. Measurements were first taken at 26°C and the participants were allowed to resume their normal activities for 10 minutes before making them rest in a Bair Hugger (neck to toe) for 10 minutes. Warm gel was applied and the measurements for the temperature of 43°C were recorded. One clinician carried out all measurements at 26°C and a different clinician recorded all readings at 43°C to achieve a balance between interobserver variation and observer bias. SPSS STATISTICS 19 software for Windows was employed to analyze the data. Paired sample t-test was used to analyze if the differences in mean vein diameters at the two temperatures were obtained by chance alone. p value of <0.05 was considered significant, confidence interval was set at 95%, and quantile-quantile (Q-Q) plots were drawn to illustrate the normal distribution of data. 3. Results Q-Q plots confirmed that the data was normally distributed. Blood pressure readings for all participants were within normal range. Figures 1, 2, and 3 compare the diameters of veins at the three sites at 26°C and 43°C for all participants. Mean diameter of CV at wrist at 26°C was 0.28 cm (SD 0.10) which increased to 0.34 cm (SD 0.08) at 43°C. Mean diameter of CV near cubital fossa at 26°C was 0.31 cm (SD 0.09) which increased to 0.35 cm (SD 0.10) at 43°C. Mean diameter of BV near cubital fossa similarly increased from 0.51 cm (SD 0.13) at 26°C to 0.55 cm (SD 0.13) at 43°C. Paired t-test for mean diameters at the two temperatures showed p values of 0.001 for CV at wrist, 0.01 for CV near cubital fossa, and 0.01 for BV near cubital fossa. Hence, the increase in mean diameters of veins due to increase in temperature is not a result of chance alone and is statistically significant. 4. Discussion Ultrasonography is the usual investigation for assessing superficial vein size prior to fistula procedures. Clinical examination of patient's arm and forearm with tourniquet on is useful in evaluating suitability of native vein for AVF creation. However, it could be difficult and misleading in certain patients. In an educational article, Brown commented on the beneficial impact of preoperative ultrasound vein size assessment for decision of vascular access creation [2]. Robbin et al. found that ultrasound vein mapping leads to a 31% increase in use of native vein for fistula creation compared with surgeons' decision of vascular access based on clinical examination only [7]. The NKF-KDOQI guideline suggested that the preferable mean venous diameter should be ≥2.5 mm and arterial diameter ≥1.6 mm for successful AVF creation [8]. Different from artery, vein size may vary tremendously in response to body posture, stress, and external environment [5, 6]. One would therefore prefer that ultrasound vein size assessment captures the maximum potential of the vein to facilitate clinical decision and, hence, better outcome [9]. Underestimation of vein size may result in wasting of patient's native vein good for fistula creation. Thus many researchers conducted studies to find out the optimal protocol for superficial vein size assessment. Van Bemmelen et al. and Korten et al. had examined the effect of body posture, tourniquet, and regional warm reagent around the upper limb on size of superficial vein [5, 6]. Their results state that warm water immersion of the limb and increased hydrostatic pressure during sitting increase venous diameter. Little attention has been paid to the external environment where the study is performed. Our study aimed to assess the vein size variation in response to environmental temperature in a setting similar to regular ultrasound vein size assessment performed for ESRD patients before fistula creation. In our study, an environmental temperature change affecting the whole body of the subjects was applied instead of regional temperature increase solely over the upper limb as this better mimics the actual environment of subjects. The result of this study showed that the cephalic and basilic vein size of majority of subjects increased at a higher environmental temperature as compared to their sizes at air-conditioned room temperature of 26°C. Currently, all the ultrasound vein size assessments for renal disease patients are carried out at a room temperature between 22°C and 26°C in our hospital. Underestimation of the size of the superficial vein is hence likely to exist in certain proportion of the patients. Not to the extent of 43°C, the authors would recommend setting the room temperature higher in the venue of ultrasound vascular assessment to avoid underestimating the superficial vein size. Alternatively, covering the patients with warm blanket or Bair Hugger before the ultrasound study would also help to reveal the optimal size of the superficial vein. Healthy volunteers instead of renal disease patients were recruited for this study because the authors preferred to minimize any influence of medical comorbidities on the results. The study of Korten et al. showed that the change in vein size is comparable between healthy individual and dialysis patients towards various provocation methods [6]. Nonetheless, based on the result of the current study, a study with the same protocol should be performed on ESRD or renal impairment patients planned for vascular access surgery to confirm the response of their superficial vein towards environmental temperature. The degree of change in vein size in response to environmental temperature represents the reactivity of a particular vein to external stimulation. It is uncertain whether the specific vein will show similar reactivity towards other stimuli such as arterializations, needling, or small size hematoma after AVF creation. If so, the reactivity of vein towards temperature change might help clinicians to predict the reactivity of AVF towards other stimuli. To understand more about the association, we need to conduct a study on the reactivity of the native superficial vein to temperature change and follow up the early and long term outcome of the AVF. 5. Conclusion Environmental temperature of the room in which vascular assessment is being conducted seems to be important in creating optimum conditions to facilitate clinical decision of vascular access creation. A similar study needs to be conducted on ESRD patients to further validate the clinical effectiveness of our results and set clearer guidelines. Competing Interests The authors declare that there are no competing interests regarding the publication of this paper. Figure 1 Diameter of cephalic vein at forearm. Figure 2 Diameter of cephalic vein at arm. Figure 3 Diameter of basilic vein at arm. ==== Refs 1 Grassmann A. Gioberge S. Moeller S. Brown G. ESRD patients in 2004: global overview of patient numbers, treatment modalities and associated trends Nephrology Dialysis Transplantation 2005 20 12 2587 2593 10.1093/ndt/gfi159 2-s2.0-27944497842 2 Brown P. W. Preoperative radiological assessment for vascular access European Journal of Vascular and Endovascular Surgery 2006 32 6 p. 744 10.1016/j.ejvs.2006.07.019 2-s2.0-33750514174 3 Lauvao L. S. Ihnat D. M. Goshima K. R. Chavez L. Gruessner A. C. Mills J. L. Sr. Vein diameter is the major predictor of fistula maturation Journal of Vascular Surgery 2009 49 6 1499 1504 10.1016/j.jvs.2009.02.018 2-s2.0-66149096659 19497513 4 Jonker F. H. W. van Keulen J. W. Schlosser F. J. V. Thoracic aortic pulsatility decreases during hypovolemic shock: implications for stent-graft sizing Journal of Endovascular Therapy 2011 18 4 491 496 10.1583/10-3374.1 2-s2.0-80052188440 21861735 5 Van Bemmelen P. S. Kelly P. Blebea J. Improvement in the visualization of superficial arm veins being evaluated for access and bypass Journal of Vascular Surgery 2005 42 5 957 962 10.1016/j.jvs.2005.06.021 2-s2.0-27644434833 16275454 6 Korten E. Spronk S. Hoedt M. T. C. de Jong G. M. T. Tutein Nolthenius R. P. Distensibility of forearm veins in haemodialysis patients on duplex ultrasound testing using three provocation methods European Journal of Vascular and Endovascular Surgery 2009 38 3 375 380 10.1016/j.ejvs.2009.03.029 2-s2.0-68249093027 19464202 7 Robbin M. L. Gallichio M. H. Deierhoi M. H. Young C. J. Weber T. M. Allon M. US vascular mapping before hemodialysis access placement Radiology 2000 217 1 83 88 10.1148/radiology.217.1.r00oc2883 2-s2.0-0034537999 11012427 8 Vascular Access Work Group Clinical practice guidelines for vascular access American Journal of Kidney Diseases 2006 48 1, supplement 1 S188 S191 9 Mendes R. R. Farber M. A. Marston W. A. Dinwiddie L. C. Keagy B. A. Burnham S. J. Prediction of wrist arteriovenous fistula maturation with preoperative vein mapping with ultrasonography Journal of Vascular Surgery 2002 36 3 460 463 10.1067/mva.2002.126544 2-s2.0-0036733265 12218967

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==== Front Case Rep CardiolCase Rep CardiolCRICCase Reports in Cardiology2090-64042090-6412Hindawi Publishing Corporation 10.1155/2016/3210320Letter to the EditorComment on “Symptomatic Trifascicular Block in Steinert's Disease: Is It Too Soon for a Pacemaker?” http://orcid.org/0000-0003-2839-7305Finsterer Josef 1 * Stöllberger Claudia 2 1Krankenanstalt Rudolfstiftung, Finsterer J Postfach 20, 1180 Vienna, Austria22nd Medical Department with Cardiology and Intensive Care Medicine, Krankenanstalt Rudolfstiftung, Finsterer J Postfach 20, 1180 Vienna, Austria*Josef Finsterer: fipaps@yahoo.deAcademic Editor: Kjell Nikus 2016 15 8 2016 2016 32103207 4 2016 22 5 2016 Copyright © 2016 J. Finsterer and C. Stöllberger.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ==== Body With interest we read the article by Lasam et al. about a 62yo male with genetically confirmed myotonic dystrophy type I (MD1) who developed a trifascicular block (AV-block I, left anterior hemiblock, and right bundle branch block) which manifested clinically as lightheadedness [1]. After implantation of a dual chamber pacemaker, lightheadedness improved. We have the following comments and concerns. Since there is some correlation between the size of the CTG-repeat expansion in the DMPK gene on chromosome 19q13.3 and the phenotype, the authors should provide the size of the expansion in the presented patient. Additionally, it would be interesting to know the expansion size in all relatives which were tested for the mutation. In this respect, we also should be informed about the phenotype of the sister, nephew, and niece. Did any of them also develop cardiac disease and did they manifest in the same way as the index patient? Since patients with MD1 may develop cerebral disease (epilepsy, leukoencephalopathy, dementia, cerebral atrophy, brain tumours, and sleep disturbances) [2], we should be informed about the cerebral condition of the index patient. Were intellectual functions normal or impaired? Which were the findings on cerebral MRI, EEG, and carotid ultrasound? Lightheadedness is a nonspecific symptom and could result from affection of the cerebrum or the cerebral arteries as well. Since MD1 patients carry an increased risk of developing heart failure, ventricular arrhythmias, and even sudden cardiac death [3, 4], these patients should undergo long-term ECG recordings or electrophysiological stimulation to decide if they only require a pacemaker, an implantable cardioverter defibrillator (ICD), or even a cardiac resynchronisation therapy (CRT) system. Were ventricular arrhythmias ever recorded by the implanted pacemaker in the index case? Was the family history positive for sudden cardiac death, syncope, exertional dyspnoea, leg edema, or palpitations? Since MD1 is characterised by the phenomenon of anticipation [5], the CTG-repeat size may have expanded in subsequent generations and patients in preceding generations may be only mildly affected. Were all available family members investigated for such mild manifestations of the disease? Did the patient manifest with other typical clinical manifestation of MD1 such as frontal baldness, cataract, myopathic face, distal weakness, wasting, or myotonia [5]? Overall, the value of this interesting case could be increased by providing more clinical information, more genetic information, and an expanded family history and investigation. The more comprehensively MD1 patients and their families are investigated, the more we can learn about this still enigmatic disease. Competing Interests There are no conflicts of interest. ==== Refs 1 Lasam G. Roberti R. LaCapra G. Ramirez R. Symptomatic trifascicular block in Steinert’s disease: is it too soon for a pacemaker? Case Reports in Cardiology 2016 2016 4 6372181 10.1155/2016/6372181 2 Krogias C. Bellenberg B. Prehn C. Evaluation of CNS involvement in myotonic dystrophy type 1 and type 2 by transcranial sonography Journal of Neurology 2014 262 2 365 374 10.1007/s00415-014-7566-6 2-s2.0-84925499900 25385052 3 Mironov N. Y. Mironova N. A. Sokolov S. F. Ventricular tachycardia as a first manifestation of myotonic dystrophy Kardiologiya 2015 55 8 86 96 2-s2.0-84944128582 26761978 4 Stojanovic V. R. Peric S. Paunic T. Cardiologic predictors of sudden death in patients with myotonic dystrophy type 1 Journal of Clinical Neuroscience 2013 20 7 1002 1006 10.1016/j.jocn.2012.09.014 2-s2.0-84879022277 23683744 5 Finsterer J. Rudnik-Schöneborn S. Myotonic dystrophies: clinical presentation, pathogenesis, diagnostics and therapy Fortschritte der Neurologie Psychiatrie 2015 83 1 9 17 10.1055/s-0034-1385734 2-s2.0-84929378897 25602187

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==== Front Biomed Res IntBiomed Res IntBMRIBioMed Research International2314-61332314-6141Hindawi Publishing Corporation 10.1155/2016/1510264Research ArticleVaccination with Astragalus and Ginseng Polysaccharides Improves Immune Response of Chickens against H5N1 Avian Influenza Virus http://orcid.org/0000-0003-2627-1389Abdullahi Auwalu Yusuf 1 2 Kallon Sanpha 3 4 Yu Xingang 1 http://orcid.org/0000-0001-5157-6059Zhang Yongliang 3 * http://orcid.org/0000-0001-9393-1573Li Guoqing 1 * 1Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510542, China2Animal Science Department, Kano University of Science and Technology Wudil, PMB 3244, Kano 20027, Nigeria3Animal Science Department, Njala University, Freetown, Sierra Leone4College of Animal Science, South China Agricultural University, Guangzhou 510542, China*Yongliang Zhang: zhangyl@scau.edu.cn and *Guoqing Li: gqli@scau.edu.cnAcademic Editor: Abdelwahab Omri 2016 15 8 2016 2016 151026411 5 2016 7 7 2016 14 7 2016 Copyright © 2016 Auwalu Yusuf Abdullahi et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.To determine the effect of astragalus and ginseng polysaccharides (APS, GPS) on immune response and improvement of H5N1 vaccine, 360-day-old broilers were randomly divided into 8 groups of 45 chicks, comprising APS groups (1–3); GPS groups (4–6); vaccine group (7); and blank control (8) (without polysaccharide and vaccine). From day 12 after hatch groups 1–3 were given APS and groups 4–6 with GPS both at 100, 200, and 400 (mg/kg), respectively. At day 15 after hatch, groups 1–7 were vaccinated with 0.3 mL H5N1 vaccine subcutaneously; daily weight gain (DWG) and serum Ig antibody (by HI-test) were measured on 3, 7, 14, and 28 days after vaccination. Serum antibody titers and expression of cytokines (IL-2, IL-10, I FN-γ, and TNF) were determined by ELISA and RT-PCR. Results revealed that all the polysaccharide groups were numerically increased in antibody levels and the expression of cytokines was significant (P < 0.05) in the APS and GPS groups compared to corresponding vaccine group and blank control. DWG was higher (P < 0.05) in 400 mg/kg APS groups than control groups. Thus oral supplements of GPS and APS have shown their potential in the improvement of immune response and could be used as adjuvant in a formulation of H5N1 vaccine. South China Agricultural University work team project2011A020102009China Postdoctoral Research Fund2015M582391National Natural Science Foundation of China31272551 ==== Body 1. Introduction H5N1 is an avian (bird) flu virus that has caused outbreaks in domestic poultry in parts of Asia and the Middle East. As H5N1 is so deadly to poultry, it is considered “highly pathogenic.” Since 2006, nearly 600 human infections with highly pathogenic H5N1 viruses have been reported to the World Health Organization (WHO) from 15 countries in Asia, Europe, and the Middle Near East. About 60% of these people died from this illness [1]. The first avian influenza virus to infect humans occurred in Hong Kong in 1997. The epidemic was linked to chickens and classified as avian influenza A (H5N1). Infected birds can shed influenza virus in their saliva, nasal secretions, and feces. Susceptible birds become infected when they have contact with contaminated secretions or excretions or with surfaces that are contaminated with secretions or excretion from infected birds [2]. The availability and use of effective vaccines can be a valuable tool in controlling outbreaks of avian influenza. Adjuvant killed vaccines can provide a strong humoral response and have been proved to be effective in preventing disease from mildly pathogenic avian influenza (MPAI) and highly pathogenic avian influenza (HPAI) challenges [3]. The HPAI virus is highly lethal in poultry and can cause large outbreaks leading to substantial economic loss and can spread directly from poultry to human, constituting a possible “pandemic threat” to the human population [4]. Astragalus polysaccharide (APS) possesses main components such as mannose, D-glucose, D-galactose, xylose, and L-arabinose. This polysaccharide is used as an immunomodulating agent in mixed herbal decoctions to treat common cold, diarrhea, fatigue, and anorexia [5]. It can also stimulate cell proliferation, induce the expression of surface antigens on lymphocytes, and affect the expression of cytokines and promote the production of antibodies [6]. Panax ginseng polysaccharide (GPS), on the other hand, contains several components such as ginsenosides, essential oil, peptidoglycans, polysaccharides, nitrogen-containing compounds, fatty acid, and phenolic compounds [7, 8]. It is well-known traditional Chinese herbal medicine. Logical studies have shown that GPS had multifunctions such as promoting the production of cytotoxic cells against tumors and stimulated macrophages to produce helper types 1 and 2 (Th1 and Th2) cytokines [9, 10]. GPS was also shown to modulate the antioxidant defense system such as superoxide dismutase and glutathione peroxidase probably via inducing regulatory cytokines [11, 12]. As anti-inflammatory responses at an early phase it results in the enhancement of antimicrobial activities and protection of mice from staphylococcus aureus-induced sepsis [13, 14]. Our previous work with APS and GPS on H9N2 indicated that APS treatment reduced H9N2 AIV replication; GPS was enhanced by pretreatment of CEF, and both have promoted early humoral immune responses in young chickens [15, 16]. In this research, we tested vaccinated and nonvaccinated chickens with H5N1 vaccine to evaluate the immunoregulatory effect of APS and GPS on chickens and assess the immunization potential of APS and GPS against H5N1 avian influenza. 2. Materials and Methods 2.1. Ethics All animal experiments and husbandry involved in this study were treated in accordance with the guidelines of the South China Agricultural University Animal Care and Use Committee, which operates under the Animal Welfare Law and Regulations of the Department of Health and Human Services. The South China Agricultural University Animal Care and Use Committee has approved all protocols of this study. 2.2. APS and GPS APS was bought from MEDICASS Company in Beijing China and GPS was provided by the Animal Science College of South China Agricultural University. GPS was extracted and purified as described in our previous works [15, 16]. Trizol, isopropanol, chloroform [trichloromethane], DEPC water, and 75% ethanol were used in the extraction of RNA. DNase1 [5 u/μL], DNase1 buffer [10x], RNase inhibitor, agarose powder, and ethidium bromide were the other reagents used. 2.3. Purification of the Polysaccharides The polysaccharides were purified as follows: removal of protein and pigment by salvage method [17] and active carbon adsorption and then through D101 macroaperture resin column, ADS-7 polymer adsorbents column, A-25 DEAE cellulose, and G-75 Sephadex column [18]. The polysaccharide contents (%) of APS and GPS were measured by vitriol-anthrone [17] taking anhydrous glucose as standard control. The percentage content (%) of APS and GPS were 79.50 and 75, respectively. For details refer to our previous works [15, 16]. 2.4. Vaccine The inactive avian influenza vaccine (H5N1 subtype) was obtained from South China Agricultural University Poultry Farm and 0.3 mL was injected subcutaneously on the dorsal region of the neck of 15-day-old chickens. 2.5. Experimental Design Three hundred and sixty, 1-day-old Yue Huang avian broilers with an average weight of 60.5 g were randomly divided into 8 groups of 45 chicks each. They were housed in wire cages in air-conditioned room at 37°C and lighted for 24 h at the beginning of pretrial period. The temperature was gradually decreased to room temperature; lighting period was 12 hours per day. Chickens were fed with commercial starter diet, purchased from the university poultry farm. From day 12 after hatch groups 1–3 were supplemented orally with APS at 100 mg/kg, 200 mg/kg, and 400 mg/kg and groups 4–6 with GPS at 100 mg/kg, 200 mg/kg, and 400 mg/kg, respectively. Each dose of polysaccharide was dissolved in 1000 mL of water (till the end of the experiment). At 15 days after hatch, groups 1–7 were vaccinated with 0.3 mL H5N1 vaccine subcutaneously on the dorsal region of the neck, where group 7 only received vaccine without polysaccharide and group 8 was blank control without polysaccharide and vaccine. At 3, 7, 14, and 28 days after vaccination ten birds from each group (groups 1–8) were weighed before being euthanized by cervical dislocation; then blood samples were collected to separate serum and stored at −20°C until use. At the same time the spleen was weighed and immediately stored in liquid nitrogen. ELISA and RT-PCR tests were performed to determine the percentage of serum antibody titers and expression of cytokines (IL-2, IL-10, I FN-γ, and TNF). 2.6. Determination of Growth Performance Weight of each bird was measured on days 3, 7, 14, and 28 after vaccination and effect on weight gain was recorded. Average live body weight, total weight gain, and daily weight gain of different groups were compared. 2.7. Measurement of Antibody Titer Antibody titer was measured by hemagglutination inhibition (HI) test [19]. The HI test was a standard beta test, using 4 hemagglutinating units of antigen in 96-well plates, where the test serum had been diluted twofold. HI endpoint titers were determined as the reciprocal of the highest serum dilution that produced complete inhibition of hemagglutination. 2.8. RNA Extraction Samples of spleen from the various groups of chickens were frozen in liquid nitrogen. About 60 g to 80 g of the tissue were cut into smaller pieces with sterilized scissors and washed with PBS. One mL of Trizol (RNA-solv Reagent) was added and the pieces were grinded by a polytron grinder to homogenize the tissue. The solution was placed in dry ice for 2-3 min to prevent the degradation of the RNA. The mixture was harvested at 12000 rpm for 10 min at 4°С. This was done to precipitate the undegraded substances such as DNA, proteins, and lipids. A short break was taken at this point; thereafter the content was poured into a new tube and 200 μL of trichloromethane (chloroform) was added in order to separate RNA from DNA and protein. The content was shaken for 15 s and harvested at 12000 rpm for 15 min at 4°С. The aqueous upper phase (colorless liquid) was transferred in a new tube and 500 μL of isopropanol was added and shaken for few seconds. The isopropanol was used to precipitate the RNA and the content was incubated at −20°С for 30 min and centrifuged at 12000 rpm for 10 min. RNA was precipitated and the supernatant was discarded leaving RNA pellet at the bottom of the tube. The tube was knocked slightly to displace the RNA and then washed with 1 mL of 75% ethanol. The content was harvested at 7500 rpm for 5 min at 4°С and the supernatant was discarded and the tube was placed upside down to dry the RNA for 5–10 min. After drying, the RNA pellet was dissolved in 50 μL DEPC-treated H2O, vortexed and briefly centrifuged for 10 s. 2.9. Real-Time PCR The total RNA was extracted from tissues using Trizol reagent (Takara Biotechnology, Dalian, China) for the detection of cytokines and H5N1 expression was tested by ultraviolet spectrophotometer at an optical density range of 1.8–2.0. The isolated RNA was digested with DNase1 (Takara Biotechnology, Dalian, China) at 37°C for 30 min. One (1) μg of total RNA was used for reverse transcript with Rever Tra Ace QPCR RT kit (Toyobo Osaka, Japan) and amplifications were performed with 1 μL cDNA in a total volume of 20 μL. SYBR Green Real-Time PCR Master Mix (Roch Mortlake, Australia) was conducted with the Strata gene Mx3005P QPCR system (Strata gene) according to the manufacturer's instruction. All reactions were done in triplicate. Relative expression fold change was calculated by 2−ΔΔCt method and β-actin was used as the endogenous reference gene to normalize the expression level of target gene. The primers used in the RT-PCR were listed in Table 1. 2.10. Detection of Interleukins (IL-2 and IL-10) in Chicken's Serum by ELISA The ELISA Kits were purchased from Kemeidongya Biotechnology Company, Beijing, China, and were used to assay the levels of chicken interleukin 2 and interleukin 10 according to the manufacturer's instructions. The standard solutions were prepared from an original standard (640 ng/L) by serial dilution. Then the standard solutions (50 μL/well) were pipetted into the wells of the first row of the microelisa striplate leaving the first two wells blank and serum sample (40 μL/well) was pipetted into the remaining wells (two wells/group sample). Biotin-interleukin antibody (10 μL) was added followed by Str-HRP-Conjugate Reagent (50 μL/well). The microelisa striplate was sealed with closure plate membrane, shaken slightly, and incubated for 1 h at 37°С. Thereafter, the closure membrane was removed and liquid was drained and microelisa plate was washed five times by filling each well with wash buffer (350 μL) using a squirt bottle, multichannel pipette, manifold dispenser, or auto washer. After the last wash, the remaining wash buffer was removed by aspirating or decanting and the plate was invert and blotted against clean paper towels. Chromogen solution A (50 μL/well) was pipetted to each well followed by Chromogen B (50 μL) which was protected from light to avoid light sensitive and thereafter it was incubated for 10 min at 37°С. Stop solution (50 μL) was quickly added into each well to stop the reaction which was indicated by an instant change of blue to yellow color. Taking the blank well as zero, measurement of the optical density (OD) under 450 nm wavelength was carried out within 10 min after adding the stop solution. According to the standard concentration and the corresponding OD values, the standard curve linear regression equation was calculated and then the OD values of the sample were applied on the regression equation to calculate the corresponding sample's concentration. 2.11. Statistical Analysis All data were shown as mean ± SEM. Comparisons between two groups were analyzed using unpaired Student's t-tests and among multiple groups by ANOVA followed by a post hoc analysis using the Turkey's multiple comparison test using SPSS 17.0 software (SPSS, Chicago, IL, USA). A probability values (P < 0.05) was considered to be statistically significant. All experiments were performed at least three times. 3. Results 3.1. Differences in Body Weight Differences in weight gain during experimental period were shown in Table 2. The weight differences of the various groups were recorded on days 3, 7, 14, and 28. There was an increase in weight gain on days 7, 14, and 28 by all the groups and the weight gains of the experimental groups were significant (P < 0.05). On days 7 and 14 the APS groups of 200 mg/kg and 400 mg/kg were higher than the corresponding groups, while on day 28 the body weight of APS (400 mg/kg) group was higher than corresponding GPS groups including vaccine and blank control. On day 3 there was no significant difference in body weight gain between the APS and GPS groups (P > 0.05). Hence, APS group of 400 mg/kg was higher (P < 0.05) than all at 7, 14, and 28 days. 3.2. Changes in Serum Antibody Titer Changes in serum antibody titers were shown in Figures 1 and 2. On day 3 after immunization, the antibody titer of all levels of APS treated groups was not different (P > 0.05) from vaccine group and blank control. But on days 7, 14, and 28 after vaccination APS were significantly different (P < 0.05) from corresponding nonpolysaccharide treated groups. On day 28 the antibody titer level of 400 mg/kg APS treated group was higher (P < 0.01) than vaccine group and blank control. However, it was observed that on days 14 and 28 the antibody titer level of blank control was significantly lower (P < 0.01) than vaccine group (Figure 1). For GPS on day 3, after immunization the antibody titer level of 100 mg/kg GPS treated group was higher (P < 0.05) than vaccine group and blank control. Similarly on day 7, the antibody titer levels of the 200 mg/kg GPS treated group was higher (P < 0.05) than vaccine group and blank control. On day 28 the antibody titer level of 200 mg/kg GPS treated group was significantly higher (P < 0.01) than vaccine and blank control, while the antibody titer levels of the 100 mg/kg and 400 mg/kg GPS treated groups were higher (P < 0.05) than vaccine group and blank control. However, on days 14 and 28 the antibody titer level of the blank control was significantly very low (P > 0.01), while on day 7 it was significantly lower (P > 0.05) than vaccine group (Figure 2). 3.3. Expression of Cytokines Figure 3 revealed that on day 3 after vaccination the TNF-α gene expressions of the polysaccharide treated groups (all levels of APS and 100 mg/kg and 400 mg/kg GPS) were higher (P < 0.05) than corresponding nonpolysaccharide treated groups, while, on day 7, the gene expressions of polysaccharide treated groups (200 mg/kg and 400 mg/kg of both APS and GPS) were higher (P < 0.01) than nonpolysaccharide treated groups. Similarly on days 14 and 28, the gene expressions of the polysaccharide treated groups (200 mg/kg and 400 mg/kg APS and 400 mg/kg GPS) were higher (P < 0.01) than corresponding nonpolysaccharide treated groups. Figure 4 depicted that on day 3 the IFN-β gene expressions of the experimental groups (200 mg/kg and 400 mg/kg APS; and all levels of GPS) were higher (P < 0.01) than vaccine group and blank control. Equally, on day 7 the gene expression of the 400 mg/kg APS treated group was higher (P < 0.01), while both APS and GPS group at 200 mg/kg were significant (P < 0.05) compared with vaccine group and blank control. Furthermore on days 14 and 28 after vaccination, the gene expressions of the polysaccharide treated groups, all levels of both APS and GPS were higher (P < 0.01) than nonpolysaccharide treated groups. Figure 5 illustrated that, on day 3, the IL-2 gene expression of the 200 mg/kg and 400 mg/kg GPS treated groups were higher (P < 0.01), while the 200 mg/kg and 400 mg/kg APS and the 100 mg/kg GPS treated groups were higher (P < 0.05) than vaccine group and blank control. We observed that on days 3, 7, and 28 the gene expressions of all levels of GPS treated groups were higher (P < 0.05) than the corresponding APS groups. And on days 7, 14, and 28 IL-2 gene expressions of the 400 mg/kg APS and GPS treated groups were higher (P < 0.01); likewise on days 7 and 28, the IL-2 gene expressions of APS and GPS treated groups have shown similar trend. Figure 6 showed that, on days 3, 7, 14, and 28 after immunization, the IL-10 gene expressions of both 200 mg/kg and 400 mg/kg of APS and GPS treated groups were higher (P < 0.01) than vaccine group and blank control, while the IL-10 gene expressions of the 100 mg/kg APS and GPS were higher (P < 0.05) than vaccine group and blank control. On days 3, 7, 14, and 28 the IL-10 gene expressions of the 200 mg/kg and 400 mg/kg GPS treated groups were higher (P < 0.05) than corresponding 200 mg/kg and 400 mg/kg APS treated groups. 3.4. Interleukins (IL-2 and IL-10) in Chicken's Serum by ELISA Figures 7 and 8 revealed ELISA results for IL-2 and IL-10 and very high levels were produced after H5N1 vaccination. On days 3, 7, 14, and 28 both IL-2 and IL-10 of APS at 200 mg/kg and 400 mg/kg of GPS groups were higher (P < 0.01) than vaccine group and blank control. 4. Discussion The present study demonstrated that the combination of APS or GPS with H5N1 vaccine may have better protective effects against lethal H5N1 influenza virus infection in chickens. It was previously reported that APS and other Chinese herbs had adjuvant effect when coadministered with an influenza vaccine, and it increased both the innate and systemic humoral responses that provide complex protection against H5N1 in chickens [20]. In this study dynamic changes in body weight were observed on day 28 in which the experimental groups (400 mg/kg APS and GPS) were higher than vaccine group and blank control, probably due to better health situation of chickens (Table 2). The results further revealed that administration of GPS and APS was able to protect the chickens from viral damage after vaccination. This is in parallel with Hu [21] who reported that animals treated with Chinese herbal medicine or Chinese herbal ingredients before or after vaccination showed a reduced incidence of infectious diseases and an increased immune response. However, no significant difference in body weight gain was observed on day 3 in both GPS and APS treated groups (Table 2). Serum HI Ab titer is a valid indicator of the humoral immunity in chickens [22]. Previous studies have shown that the HI Ab is directly effective against NDV in chickens [23, 24]. Our result showed that the HI Ab titers in the treatment groups of GPS and APS were significantly higher than vaccine group after immunization (Figures 1 and 2), suggesting that GPS and APS could enhance the humoral immunity. It demonstrated that, on days 14 and 28 after vaccination, the anti-H5N1 HI Ab titers of the APS treated groups (200 mg/kg and 400 mg/kg) and 100 mg/kg, 200 mg/kg, and 400 mg/kg GPS treated groups were higher than vaccine group and blank control, indicating a more rapid response and potent effects in increasing the production of anti-H5N1 antibodies of the GPS and APS treatment groups. These findings provide evidence for the use of GPS and APS as effective herbal medicinal immune stimulators. More interestingly, vaccination of H5N1 caused decrease in body weight as shown in the results, but addition of both GPS and APS was able to recover the decreased body weights, probably due to body regulation to vaccination stress. The effects of APS and GPS on body weight gain were different, especially the doses from 200 mg/kg to 400 mg/kg; it is fascinating that body weight gain was always lower using GPS rather than APS, probably due to their chemical components, although both contain polysaccharides as their constituents but the latter contains more simple sugars than the former, which might contribute to the effects. Refer to Ríos and Waterman [25] for detailed review on astragalus. The reactivity of chicken spleen to antigens is higher in magnitude with increase in age [26]. The spleen constitutively expressed IL-4, IL-10, and IFN-γ cytokine genes as early as embryonic day 12 which is associated with shaping the spleen environment. The expression patterns of these cytokines coincide with the completion of colonization of the spleen by cellular migrants from the thymus [26]. In this study we recorded that on days 7, 14, and 28 the TNF-α gene expression in the spleen of the APS and GSP (200 mg/kg and 400 mg/kg) treated groups was higher than corresponding vaccine group and blank control. TNF involves in a network of cytokines and chemokines that stimulates the recruitment of immune cells in the infectious foci and can block the viral replication by interfering with the viral life cycle especially in viral entry [27]. Previous reports found APS and GPS to promote IL-2 bioactivity [28]. Accordingly, the increased bioactivity of IL-2 of peripheral blood lymphocytes may partly explain the increased lymphocytes proliferation. The Th2 cells secrete cytokines such as IL-4 and IL-10 which help B cell proliferation and are associated with humoral immunity [28]. In our finding it was revealed that gene expressions of IL-2 and IL-10 in APS and GPS groups were evidently higher than vaccine group and control, meaning that APS and GPS most probably stimulate cytokine production. Cytokines often have multiple effects, which originated from various effector cells. The variation of cytokines in our study coincided with finding of Shao et al. [29]. In comparison there was no significant difference in body weight; however, the cytokine gene expressions in the GPS groups were significant compared with corresponding APS groups. Hence APS which had been known over the centuries for its immune potency can be substituted by a more immune potent GPS as adjuvant in the formulation of a H5N1 vaccine. Our results indicated that oral supplement of both GPS and APS will be beneficial to health and vaccination efficiency in H5N1 vaccine in chickens. More interestingly, GPS exerted similar and even better improvement in this respect. 5. Conclusion Conclusively our results showed the following. (1) The addition of APS and GPS is beneficial to eliminate the chicken weight loss after vaccination. (2) APS and GPS have adjuvant properties to enhance the antibody level when used in combination with influenza vaccine (H5N1). (3) Expressions of IL-2, IL-10, and TNF-α and IFN-β in GPS and APS treated groups were higher than control group, suggesting GPS and APS stimulated cytokine production and macrophage activation. Intriguingly, expressions of IL-2 and IL-10 by ELISA have proved similar results. (4) The effects of GPS and APS on growth rate and immune response were related to their dosages used. Appropriate effective dosages such as 200 mg/kg and 400 mg/kg should be taken into consideration in the use of GPS and APS as adjuvant in the formulation of a new H5N1 vaccine. (5) The study further revealed that the combination of APS or GPS with H5N1 vaccine may have better protective effects against lethal H5N1 influenza virus infection in chickens. Generally, oral supplementation of GPS and APS dosages (100 mg/kg, 200 mg/kg, and 400 mg/kg) was beneficial to the health of the chickens to eliminate the chicken weight loss after vaccination. Similarly doses of 200 mg/kg and 400 mg/kg GPS and APS in vivo would be expected to serve as adjuvant for new vaccine formulation against H5N1 AIV. Acknowledgments This work was supported by South China Agricultural University work team project (no. 2011A020102009) and China Postdoctoral Research Fund (2015M582391) and National Natural Science Foundation of China (Grant no. 31272551). Competing Interests The authors declare that they have no competing interests. Authors' Contributions Auwalu Yusuf Abdullahi and Sanpha Kallon contributed equally to this paper. Figure 1 Antibody titers of APS treated groups and control groups. Superscripts with different letters (a–d) differ significantly (P < 0.05). Group 1 = 100 mg/kg APS + vaccine. Group 2 = 200 mg/kg APS + vaccine. Group 3 = 400 mg/kg APS + vaccine. Group 7 = vaccine only. Group 8 = no vaccine, no APS (blank control). Figure 2 Antibody titers of GPS treated groups and control groups. Superscripts with different letters (a–d) differ significantly (P < 0.05). Group 4 = 100 mg/kg GPS + vaccine. Group 5 = 200 mg/kg GPS + vaccine. Group 6 = 400 mg/kg GPS + vaccine. Group 7 = vaccine only. Group 8 = no vaccine, no GPS (blank control). Figure 3 Effect of APS and GPS on gene expression of TNF-α. Superscripts with different letters (a–e) differ significantly (P < 0.05). Group 1 = 100 mg/kg APS + vaccine. Group 2 = 200 mg/kg APS + vaccine. Group 3 = 400 mg/kg APS + vaccine. Group 4 = 100 mg/kg GPS + vaccine. Group 5 = 200 mg/kg GPS + vaccine. Group 6 = 400 mg/kg GPS + vaccine. Group 7 = vaccine only. Group 8 = no vaccine, no GPS (blank control). Figure 4 Effect of APS and GPS on gene expression of IFN-β. Superscripts with different letters (a–e) differ significantly (P < 0.05). Group 1 = 100 mg/kg APS + vaccine. Group 2 = 200 mg/kg APS + vaccine. Group 3 = 400 mg/kg APS + vaccine. Group 4 = 100 mg/kg GPS + vaccine. Group 5 = 200 mg/kg GPS + vaccine. Group 6 = 400 mg/kg GPS + vaccine. Group 7 = vaccine only. Group 8 = no vaccine, no GPS (blank control). Figure 5 Effect of APS and GPS on IL-2 gene expression. Superscripts with different letters (a–f) differ significantly (P < 0.05). Group 1 = 100 mg/kg APS + vaccine. Group 2 = 200 mg/kg APS + vaccine. Group 3 = 400 mg/kg APS + vaccine. Group 4 = 100 mg/kg GPS + vaccine. Group 5 = 200 mg/kg GPS + vaccine. Group 6 = 400 mg/kg GPS + vaccine. Group 7 = vaccine only. Group 8 = no vaccine, no GPS (blank control). Figure 6 Effect of APS and GPS on gene expression of IL-10. Superscripts with different letters (a–f) differ significantly (P < 0.05). Group 1 = 100 mg/kg APS + vaccine. Group 2 = 200 mg/kg APS + vaccine. Group 3 = 400 mg/kg APS + vaccine. Group 4 = 100 mg/kg GPS + vaccine. Group 5 = 200 mg/kg GPS + vaccine. Group 6 = 400 mg/kg GPS + vaccine. Group 7 = vaccine only. Group 8 = no vaccine, no GPS (blank control). Figure 7 Effect of APS and GPS on IL-2 by ELISA. Superscripts with different letters (a–d) differ significantly (P < 0.05). Group 1 = 100 mg/kg APS + vaccine. Group 2 = 200 mg/kg APS + vaccine. Group 3 = 400 mg/kg APS + vaccine. Group 4 = 100 mg/kg GPS + vaccine. Group 5 = 200 mg/kg GPS + vaccine. Group 6 = 400 mg/kg GPS + vaccine. Group 7 = vaccine only. Group 8 = no vaccine, no GPS (blank control). Figure 8 Effect of APS and GPS on IL-10 by ELISA. Superscripts with different letters (a–e) differ significantly (P < 0.05). Group 1 = 100 mg/kg APS + vaccine. Group 2 = 200 mg/kg APS + vaccine. Group 3 = 400 mg/kg APS + vaccine. Group 4 = 100 mg/kg GPS + vaccine. Group 5 = 200 mg/kg GPS + vaccine. Group 6 = 400 mg/kg GPS + vaccine. Group 7 = vaccine only. Group 8 = no vaccine, no GPS (blank control). Table 1 Sequence of the oligonucleotide primers used in Real-Time PCR. Gene name Primer (5′ → 3′) Product (bp) Accession number IL-2 F: CTTTGGCTGTATTTCGG 163 NM-204153.1 R: CTGGGTCTCAGTTGGTGT IL-10 F: GCTGAGGGTGAAGTTTGAG 192 NM-001004414.2 R: TGATGACTGGTGCTGGTCT TNF-α F: CTCAGGACAGCCTATGCCA 171 AY765397.1 R: CACGACAGCCAAGTCAACG IFN-β F: CATACTGAGCCAGATTGTTTCG 176 NM-205149.1 R: TCAAGTCGTTCATCGGGAG β-actin F: TGATATTGCTGCGCTCGTTG 202 JF436880.1 R: CTTTCTGGCCCATACCAACC Table 2 Effect of APS and GPS on body weight gain (g). Groups D3 D7 D14 D28 APS (100 mg/kg) 117.8 ± 8.49 148.3 ± 11.59b 214.9 ± 12.44c 396.1 ± 21.31b GPS (100 mg/kg) 107.2 ± 9.51 146.9 ± 13.03b 207.1 ± 20.6c 398.4 ± 23.82b APS (200 mg/kg) 113.3 ± 10.99 150.6 ± 12.5b 240.5 ± 16.43b 397.8 ± 22.53b GPS (200 mg/kg) 112.6 ± 8.9 145.7 ± 12.96b 213.4 ± 25.42c 371.5 ± 28.5b APS (400 mg/kg) 119.2 ± 10.78 166.4 ± 13.01a 261.4 ± 18.7a 432.0 ± 23.65a GPS (400 mg/kg) 117.6 ± 8.48 147.3 ± 14.15b 235.8 ± 22.73b 410.51 ± 27.34b Vaccine 109.7 ± 9.72 139.7 ± 14.73c 197.5 ± 21.91c 382.2 ± 23.9b Blank control 117.2 ± 9.07 146.8 ± 13.03b 235.8 ± 22.73b 382.8 ± 24.79b Superscripts with different letters (a, b, and c) differ significantly (P < 0.05). APS = astragalus polysaccharide; GPS = ginseng polysaccharide; D = day. ==== Refs 1 World Health Organization Global Influenza Program Surveillance Network Evolution of H5N1 avian influenza viruses in Asia Emerging Infectious Diseases 2005 11 10 1515 1521 10.3201/eid1110.050644 16318689 2 World Health Organization H5N1 avian influenza: timeline 2005, http://www.who.int/influenza/human_animal_interface/avian_influenza/H5N1_avian_influenza_update.pdf 3 Suarez D. L. Schultz-Cherry S. Immunology of avian influenza virus: a review Developmental and Comparative Immunology 2000 24 2-3 269 283 10.1016/s0145-305x(99)00078-6 2-s2.0-0034163283 10717293 4 Alexander D. J. Brown I. H. Recent zoonoses caused by influenza A viruses Revue Scientifique et Technique 2000 19 1 197 225 11189716 5 Chen Y. Wang D. Hu Y. 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==== Front Biomed Res IntBiomed Res IntBMRIBioMed Research International2314-61332314-6141Hindawi Publishing Corporation 10.1155/2016/9845927Research ArticleExpression of Codon-Optimized Plant Glycosyltransferase UGT72B14 in Escherichia coli Enhances Salidroside Production Xue Feiyan 1 2 Guo Huili 1 2 Hu Yingying 1 2 Liu Ran 1 2 Huang Lina 1 2 Lv Heshu 1 2 Liu Chunmei 1 2 Yang Mingfeng 1 2 http://orcid.org/0000-0002-5578-6958Ma Lanqing 1 2 3 * 1Key Laboratory of Urban Agriculture (North), Ministry of Agriculture, Beijing 102206, China2College of Biological Science and Engineering, Beijing University of Agriculture, Beijing 102206, China3Beijing Collaborative Innovation Center for Eco-Environmental Improvement with Forestry and Fruit Trees, Beijing 102206, China*Lanqing Ma: lqma@bua.edu.cnAcademic Editor: Yeo J. Yoon 2016 15 8 2016 2016 98459276 5 2016 18 7 2016 21 7 2016 Copyright © 2016 Feiyan Xue et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Salidroside, a plant secondary metabolite in Rhodiola, has been demonstrated to have several adaptogenic properties as a medicinal herb. Due to the limitation of plant source, microbial production of salidroside by expression of plant uridine diphosphate glycosyltransferase (UGT) is promising. However, glycoside production usually remains hampered by poor expression of plant UGTs in microorganisms. Herein, we achieved salidroside production by expression of Rhodiola UGT72B14 in Escherichia coli (E. coli) and codon optimization was accordingly applied. UGT72B14 expression was optimized by changing 278 nucleotides and decreasing the G+C content to 51.05% without altering the amino acid sequence. The effect of codon optimization on UGT72B14 catalysis for salidroside production was assessed both in vitro and in vivo. In vitro, salidroside production by codon-optimized UGT72B14 is enhanced because of a significantly improved protein yield (increased by 4.8-fold) and an equivalently high activity as demonstrated by similar kinetic parameters (K M and V max), compared to that by wild-type protein. In vivo, both batch and fed-batch cultivation using the codon-optimized gene resulted in a significant increase in salidroside production, which was up to 6.7 mg/L increasing 3.2-fold over the wild-type UGT72B14. Beijing Municipal Natural Science Foundation2164059Beijing Outstanding Talent Project2013D005021000003Beijing University of AgricultureGJB2013006GZL2015012Beijing Municipal Education CommissionCEFF-PXM2016_014207_000038National Natural Science Foundation of China3130062031370674Beijing Municipal Education CommissionKM20140020001 ==== Body 1. Introduction Glycosylation of small molecules has a profound impact on their biological properties including bioactivity, solubility, and stability. The majority of glycosylation reactions in nature are mediated by glycosyltransferases, which comprise a large and divergent polyphyletic multigene superfamily [1, 2]. In plants, glycosylation catalysed by uridine diphosphate (UDP) glycosyltransferases (UGTs) [2–4] is involved in the synthesis of many secondary metabolites. Studies on the recombinant expression of UGTs have resulted in the production of various plant secondary metabolites as glycosides [2, 5, 6]. However, many valuable plant glycosides remain difficult to produce in high yield and in a timely manner. Microbes, especially Escherichia coli and Saccharomyces cerevisiae, can be engineered to produce plant natural products by introducing key enzymes [7, 8]. E. coli is the most widely used, well-characterized heterologous host and proved to result in more products yield than S. cerevisiae [4, 9, 10]. Notably, poor expression of wild-type plant genes in E. coli has restricted the yield of products [11, 12]. One strategy for overcoming this limitation is codon optimization [12–14]. Salidroside (8-O-β-d-glucoside of tyrosol) is one of the main plant secondary metabolites in Rhodiola and has been demonstrated to have adaptogenic and ergogenic capacity [15]. Consequently, demand for salidroside compounds has grown, but Rhodiola has a long growth period and low salidroside content, and an alternative method of salidroside production is thus needed. Glycosylation typically occurs as one of the last steps during natural product synthesis [5], and this is the case for salidroside [16, 17]; the last step involves glycosylation of tyrosol, catalysed by UGT (Figure 1). In our previous work, three different UGT genes, UGT72B14, UGT73B6, and UGT74R1, were isolated from Rhodiola sachalinensis and characterized [16, 17]. Among the three UGTs, UGT72B14 demonstrated the highest activity for salidroside production, with a catalytic efficiency (V max/K M) of 12.3, which was 620% and 180% higher than UGT74R1 and UGT73B6, respectively [17]. Accordingly, we investigated the effect of codon optimization on the heterologous expression of UGT72B14 in E. coli and assessed the impact on salidroside production in the present study. 2. Methods 2.1. Strains and Plasmids The host E. coli (DE3) strain was from a frozen stock stored at the Key Laboratory of Urban Agriculture (North) of the Ministry of Agriculture of China, Beijing, China. This strain was used for salidroside production following transformation with the appropriate plasmid. The pET-28a (+) plasmid was purchased from Novagen (Darmstadt, Germany) and used for cloning and expression of UGT72B14 in the E. coli host strain. 2.2. Codon Optimization The wild-type UGT72B14 gene was isolated from Rhodiola sachalinensis (GenBank Accession Number EU567325) and optimized by replacing codons predicted to be less frequently used in E. coli with more favored codons, according to the Codon Usage Database and graphical codon usage analyzer. The optimized gene with introduction of restriction sites (Nco I and Xho I) was synthesized by Inovogen Tech. Co. (Beijing, China). 2.3. Protein Expression The synthetic gene was digested and ligated into the prokaryotic expression vector pET-28a (+) at the same restriction sites (Nco I and Xho I), under the control of T7 promoter. The resulting plasmid was transformed into the E. coli host. Following identification of a positive transformant, cells were grown at 37°C with shaking at 180 rpm in LB (Luria-Bertani) medium containing 50 μg/mL kanamycin. When the desired OD600 was reached, the temperature was reduced to 20°C, shaking was slowed to 120 rpm, and expression was induced with isopropyl β-d-1-thiogalactopyranoside (IPTG) at a final concentration of 0.8 mM. Cultures were harvested at various time points to measure OD600 and do sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Cell pellets, obtained by centrifugation at 12,857 ×g for 5 min, were resuspended in sterile H2O and subjected to SDS-PAGE analysis. 2.4. Protein Purification and Measurement of Enzyme Activity Recombinant E. coli cultures were incubated at 37°C with shaking at 180 rpm in LB medium containing 50 μg/mL kanamycin. When the OD600 reached 0.3 ± 0.1, E. coli harbouring the optimized UGT72B14 plasmid was induced at 20°C with shaking at 120 rpm as described above for a further 6–8 h. Cell pellets were harvested by centrifugation (8,000 ×g; 10 min; 4°C) and resuspended in lysis buffer (20 mM Tris, pH7.5, 300 mM NaCl, 20 mM imidazole, 1 mg/mL leupeptin, 2 mg/mL aprotinin, and 100 mg/mL lysozyme). Cells were disrupted by sonication (30 × 3 s) with ice bath. Supernatant was separated from the sonication homogenate by centrifugation (10,000 ×g; 10 min; 4°C) and passed through Ni-NTA His-Bind™ Resin (Novagen, Madison, WI, USA) column containing Ni2+ as an affinity ligand. After washing with elution buffer (20 mM Tris, pH7.5, 300 mM NaCl, and 20 mM imidazole), unbound contaminant proteins were removed. His-tagged proteins were eluted with washing buffer (20 mM Tris, pH7.5, 300 mM NaCl, and 300 mM imidazole) and purified using a PD-10 column (Amersham Pharmacia Biotech, Uppsala, Sweden) and then concentrated by a centrifugal filter unit (YM-50 kD, Millipore). The efficiency of purification was monitored by SDS-PAGE analysis. The concentration of purified UGT72B14 was determined using BCA protein assay kit (Sigma-Aldrich, USA), and then the purified protein yield was assessed by calculating the purified protein content per litre of culture broth. For recombinant E. coli harbouring the wild-type UGT72B14 plasmid, induction was initiated at OD600 = 0.7 ± 0.1 and growth continued for a further 9 h. Cells were then harvested and protein was purified as described above. To measure the kinetic parameters, tyrosol (Sigma-Aldrich, Saint Louis, Missouri, USA) and UDP-glucose substrates were incubated with purified protein at 30°C for 30 min, and the salidroside product was identified using High Performance Liquid Chromatography (HPLC). Enzyme assays were carried out as previously described [17]. 2.5. Measuring Salidroside Production in the Recombinant E. coli Strains Cultures were grown and induced as described above, and tyrosol, glucose, and sodium citrate (all analytical grades) were added in equimolar concentrations and culturing continued at 30°C with shaking at 150 rpm. Samples were harvested at various time-points during fermentation by centrifugation at 12,857 ×g for 10 min. Supernatants were analyzed directly by HPLC or freeze-dried and extracted with an equal volume of ethanol, evaporated, and dissolved in methanol to prepare Liquid Chromatography and Mass Spectrometry (LC-MS) samples as described previously [17]. 3. Results 3.1. Codon Optimization and Its Effect on Protein Expression UGT72B14 was found to have the highest activity for salidroside production and was thus considered the ideal candidate for testing the effect of codon optimization. The full-length gene sequence (1671 bp) has a G+C content of 56.75% and includes an open reading frame (ORF) of 1422 bp that encodes a polypeptide of 473 amino acids [17]. Codons such as AGA (Arg), AGG (Arg), CGA (Arg), CGG (Arg), AUA (Ile), CUA (Leu), CUC (Leu), and CUU (Leu), present in the wild-type plant sequence, are used at less than 10% in E. coli. Bioinformatics analysis was performed to identify codons that could be modified to better match the host codon usage preferences without altering the resulting amino acid sequence. We replaced most of them with those that are more frequently used in E. coli BL21 (DE3). Codons encoding Arg, Gly, and Leu were altered in 93%, 67%, and 81% of cases, respectively. The termination codon was also modified as UAA. It is well documented that ORFs containing high G+C content are often poorly expressed in A+T rich hosts [14, 18]. After altering 278 nucleotides, the G+C content was decreased to 51.05% in the optimized sequence. The codon-optimized UGT72B14 was submitted to NCBI (GenBank Accession Number KU523897). To study the effect of codon optimization on UGT72B14 expression, the optimized gene was synthesized and used to construct the recombinant plasmid that was subsequently transformed into E. coli, and protein expression was assessed by SDS-PAGE. The cell density at the time of IPTG induction is believed to be important for protein expression, and an optical density of 600 nm (OD600) of 0.6–0.8 was previously demonstrated to be optimum for expression of both UGT73B6 and UGT72B14 [16, 17, 19]. This cell density was thus used for induction of expression of the optimized UGT72B14 gene in the present study. Considerable cell density (Figure 2(a)) and maximum accumulation of the target protein 9 h after induction (Figure 2(b)) were observed, and the variation of cell growth and protein expression was consistent with our previous work [17]. However, expression levels of the codon-optimized UGT72B14 were even lower than the wild-type gene when induction was performed at OD600 ~ 0.6 (Figure 2(b)). Induction was thus tested at OD600 ~ 0.1, 0.3, 0.6, and 0.9, and OD600 ~ 0.3 was found to result in the highest expression of the codon-optimized UGT72B14 gene (Figure 3(a)). SDS-PAGE analysis showed that induction at OD600 ~ 0.3 and growth for a further 6–8 h resulted in peak target protein levels (Figure 3(d)), which was overall significantly superior to that induction at OD600 ~ 0.6 (Figure 3(c)), though lower in cell growth (Figure 3(b)). To our best knowledge, this interesting result is firstly observed in Rhodiola UGT expression. However, it is consistent with a previous report of other proteins [20] that IPTG addition decreased cell growth but enhanced expression of the target gene when added at a relatively low cell density (OD600 ~ 0.3). 3.2. Effect of Codon Optimization on Salidroside Production In Vitro To examine whether codon-optimized UGT72B14 are capable of converting tyrosol to salidroside, the His-tagged UGT72B14 recombinant proteins were purified using Ni affinity chromatography. Result of SDS-PAGE analysis indicated that the codon-optimized gene resulted in not only more target protein expression but also more efficient purification (Figure 4(a)). It was 27.55 mg/L of purified protein obtained from the culture of strain harbouring the codon-optimized UGT72B14, which was an increase of 4.8-fold over that from the wild-type gene (Figure 4(b)). To investigate the effect of codon optimization on salidroside production in vitro, the activity of codon-optimized and wild-type UGT72B14 protein was tested using tyrosol and UDP-glucose as substrates, and production of salidroside was monitored by HPLC and LC-MS. There was no significant difference in specific activity between codon-optimized and wild-type enzymes, as demonstrated by comparable kinetic parameters (K M and V max; Figure 4(b)). Wild-type UGT72B14 has been already proved to exhibit highest level of activity for salidroside production, compared to the other two UGTs we have cloned [17]. Here, an equivalently high enzyme activity and a significant improvement in protein yield of codon-optimized UGT72B14 will offer great promise for salidroside production in vitro. 3.3. Effect of Codon Optimization on Salidroside Production In Vivo Utilizing recombinant enzymes to synthesize plant secondary metabolites in E. coli has many advantages, including simple nutritional requirements, easy cultivation, and the ability to prevent enzyme inactivation [6, 7, 21, 22]. The plant secondary metabolite salidroside is synthesized via a one-step glycosylation reaction from tyrosol in Rhodiola [16, 17, 23]. Following confirmation of codon-optimized UGT72B14 expression and catalytic activity in vitro, substrates were added to growing cultures at 6 h after induction with IPTG and conversion of tyrosol to salidroside was investigated. Tyrosol was tested at 0, 5, 50, and 500 mg/L, and most salidroside was observed at 50 mg/L tyrosol (Figure 5). This tyrosol concentration was subsequently used for a time-course experiment on cultures harbouring codon-optimized or wild-type UGT72B14 plasmids. As shown in Figure 6(a), salidroside was first detected at 2 h after addition of tyrosol, and the concentration gradually increased, peaked at 8 h, and then declined over the following 2 h. A similar pattern was observed for both recombinant strains, but the codon-optimized culture exhibited a much wider range. Codon optimization increased the maximum salidroside production by 3-fold. Time-course experiments revealed a tendency for the target protein yield to decline at the end of batch fermentation, which was consistent with previous reports [18, 24]. We tentatively conclude that batch cultivation resulted in substrate inadequate utilization at the early stages of fermentation, and the low product yield encouraged us to investigate other ways of improving salidroside production. Fed-batch cultivation has been shown to be an effective way to enhance the expression of target proteins and the yield of target products in E. coli [25–28]. In the present study, we performed fed-batch experiments by feeding cultures with four additions of 50 mg/L tyrosol (an initial addition of 20 mg/L, and three more additions of 10 mg/L at 2 h intervals). In these experiments, salidroside was first detected in the culture broth at 2 h after the first tyrosol addition and was then maintained at a high level (with slight fluctuations) between 4 and 11 h after the first addition (Figure 6(b)). Up to 6.7 mg/L of salidroside was accumulated at 9 h in the codon-optimized culture, which was an increase of 3.2-fold over the culture harbouring the wild-type UGT72B14 plasmid. Compared with salidroside production in the batch cultivation method, fed-batch fermentation achieved a higher titre, and salidroside production was stable over a longer period. A gradual supplement of tyrosol is thus beneficial for salidroside production, and optimizing the amount of tyrosol added could further increase salidroside yield. This may explain the higher yield of salidroside that was achieved following reconstitution of the tyrosol biosynthetic pathway in E. coli [19]. 4. Discussion Glycosyltransferases have proved to be very important for plant secondary metabolism synthesis and have become an important research field in improving catalytic efficiency [3, 29]. In terms of UGTs, they have emerged as promising catalyst for UDP-sugar based glycosylation focusing on small molecules like salidroside [4]. Although many UGTs have been cloned and expressed in E. coli glycoside production remains hampered by poor expression of plant genes in this prokaryote host [11]. Codon optimization, as an effective approach to improving expression of heterologous protein, has attracted considerable attention and was applied successfully in some cases [12–14, 17]. However, no systematic studies of codon optimization have been reported for plant UGTs. The UGTs of Rhodiola sachalinensis as one of the most important plants for salidroside supply [30, 31] thus aroused our great interest. Three Rhodiola UGTs were firstly cloned and characterized in our previous work [17]. With the sequence of UGT73B6, one of the three UGTs we published, salidroside has been synthesized in vivo by Bai et al. [19]. Here with the sequence of UGT72B14, codon optimization was systematically studied and proved favorable for enhancing salidroside production both in vivo and in vitro. 5. Conclusions The optimized UGT72B14 expression at a high level was benefited from induction with IPTG at a reasonably low cell density (OD600 ~ 0.3). A fed-batch cultivation method proved optimal for salidroside production in terms of overall yield and the duration over which maximum production occurred. As demonstrated favorable for enhancing UGT72B14 expression and salidroside production in a timely manner in E. coli, codon optimization therefore deserved to be used to improve plant UGTs expression in heterologous hosts to enhance their corresponding glycoside production. Acknowledgments This research was financially supported by “Beijing Natural Science Foundation (2164059),” “Beijing Outstanding Talent Project (2013D005021000003),” “Funding Project for Scientific Research Quality Improvement in Beijing University of Agriculture (GJB2013006, GZL2015012),” “Beijing Municipal Education Commission (CEFF-PXM2016_014207_000038),” “National Natural Science Foundation of China (31300620, 31370674),” and “Foundation of Beijing Municipal Education Committee (KM20140020001).” The authors also thank Mr. Mingcheng Zhang and Jianguo Yang for technical support. Competing Interests The authors declare that they have no competing interests. Authors' Contributions Feiyan Xue and Huili Guo contributed equally to this work and performed most of the experiments and drafted the paper. Yingying Hu, Ran Liu, and Lina Huang participated in selection of positive clones, protein expression, and cultivation runs. Heshu Lv and Mingfeng Yang contributed to some detailed experiments designing. Chunmei Liu helped in doing HPLC-MS analysis. Lanqing Ma coordinated the whole project and completed the paper. All authors read and approved the final version of the paper. Figure 1 Reaction catalysed by UGT during salidroside production. Figure 2 Growth profile and protein expression of the recombinant strains harbouring codon-optimized and wild-type UGT72B14 gene. (a) Cell growth of recombinant strains. Data are presented as mean of three biological replicates, and error bar represents the standard deviation (SD). (b) SDS-PAGE analysis of protein expression. Sample of 20 μL was used for SDS-PAGE analysis. UGT72B14-opt = codon-optimized UGT72B14; UGT72B14-wt = wild-type UGT72B14. Lane M: protein molecular weight markers. Lanes 1–3: protein expression at 4, 6, and 9 h after induction. Target protein is indicated by arrows. Figure 3 Optimization of UGT72B14-opt expression. (a) Protein expression at 10 h after induction with IPTG at varying OD600. (b) Growth profile of recombinant strain during induction. (c) Time courses of protein expression with IPTG induction at OD600 = 0.621 ± 0.032. (d) Time courses of protein expression with IPTG induction at OD600 = 0.303 ± 0.011. Lane M: protein molecular weight markers. Lanes I–IV: UGT72B14-opt expression at OD600 ~ 0.1, 0.3, 0.6, and 0.9 with 10 h after induction. Lanes 1–7: UGT72B14-opt expression at 0, 2, 4, 6, 8, 9, and 10 h after induction. Cell pellets were resuspended, the OD600 was adjusted to 1.00 ± 0.02, and 15 μL was used for SDS-PAGE analysis. Target protein is indicated by arrows. Data are presented as mean of three biological replicates, and error bar represents the SD. Figure 4 Result of the recombinant protein yield and salidroside production in vitro. (a) SDS-PAGE analysis of the recombinant protein expression and purification. Lane M: protein molecular weight markers. Lane 1: total protein in uninduced strain. Lane 2: total protein in induced strain. Lane 3: concentrated protein after being purified by Ni affinity chromatography and PD-10 column. Sample of 15 μL was used for SDS-PAGE analysis. Target protein is indicated by arrows. (b) Result of salidroside production catalysed by UGT72B14 in vitro. Protein yield data of the optimized UGT72B14 was obtained by purified protein after induction of 7 h initiated with OD600 = 0.325 ± 0.021 while that of the wild-type UGT72B14 was obtained by purified protein after induction of 9 h initiated with OD600 = 0.632 ± 0.031. The recombinant enzyme catalysis reaction system (100 μL) contained 50 mM Tris-HCl (pH 7.5), 2 mM UDP-glucose, 250 μM tyrosol, and the enzyme protein of 0.2 mg, proceeded for 30 min at 30°C, and terminated with 200 μL MeOH. Data were presented as mean ± SD. Figure 5 Effect of tyrosol concentration on salidroside production in E. coli expressing codon-optimized UGT72B14. Data are presented as mean of three biological replicates, and error bar represents the SD. Different letters above bars indicate a significant difference at p < 0.05 according to Duncan's test. Figure 6 Result of salidroside production in vivo. (a) Batch cultivation. (b) Fed-batch cultivation. Sal-opt and Sal-wt refer to salidroside synthesized by strains harbouring codon-optimized and wild-type UGT72B14 plasmids, respectively. Tyr-opt and Tyr-wt refer to tyrosol concentration in the culturing broth of strains harbouring codon-optimized and wild-type UGT72B14 plasmids, respectively. 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==== Front Obstet Gynecol IntObstet Gynecol IntOGIObstetrics and Gynecology International1687-95891687-9597Hindawi Publishing Corporation 10.1155/2016/8376260Letter to the EditorResponse to: Female Genital Mutilation and Obstetric Outcomes: Flawed Systematic Review and Meta-Analysis Does Not Accurately Reflect the Available Evidence http://orcid.org/0000-0002-9991-4835Berg Rigmor C. * http://orcid.org/0000-0001-6776-1849Odgaard-Jensen Jan http://orcid.org/0000-0002-6268-7966Fretheim Atle Underland Vigdis Vist Gunn Norwegian Knowledge Centre for the Health Services, P.O. Box 7004, St. Olavs Plass, 0130 Oslo, Norway*Rigmor C. Berg: rigmor.berg@fhi.noAcademic Editor: John R. Van Nagell 2016 15 8 2016 2016 837626024 10 2015 5 5 2016 Copyright © 2016 Rigmor C. Berg et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ==== Body This is our response to Meirik, Banks, Farley, Akande, Bathija, and Ali's letter to the editor from March 2015 [1], where they comment on our paper “The Obstetric Consequences of Female Genital Mutilation/Cutting: A Systematic Review and Meta-Analysis” [2] and a linked technical report. Unfortunately, we were not informed about their letter; hence our response was late. We thank Meirik et al. for their careful reading of our paper and technical report. They raise two concerns: (1) our inclusion of results from different research designs (mixing prospective and retrospectively collected data) and (2) our use of unadjusted effect estimates. In fact, we discussed these concerns directly with Meirik et al. after we had published our systematic review. We acknowledged their concerns and subsequently carried out an update of our analyses limited to prospective studies and adjusted estimates. In the reanalysis, published in this journal last year, we found that “meta-analyses based on adjusted estimates, with or without data from retrospective studies, consistently pointed in the same direction as our earlier findings. There were only small differences in the sizes or the level of statistical significance.” That is, these updated results demonstrate, contrary to Meirik and associates' assumptions, that adjustment did not change the conclusions. Thus, we maintain that there is convincing evidence that FGM/C is associated with an increased risk of obstetric complications, but that the available evidence does not allow for firm conclusions about how strong this relationship is. Competing Interests The authors declare that there is no conflict of interests regarding the publication of this paper. ==== Refs 1 Meirik O. Banks E. Farley T. Akande O. Bathija H. Ali M. Female genital mutilation and obstetric outcomes: flawed systematic review and meta-analysis does not accurately reflect the available evidence Obstetrics and Gynecology International 2014 2014 3 205230 10.1155/2014/205230 2 Berg R. C. Underland V. The obstetric consequences of female genital mutilation/cutting: a systematic review and meta-analysis Obstetrics and Gynecology International 2013 2013 15 496564 10.1155/2013/496564

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==== Front Stem Cells IntStem Cells IntSCIStem Cells International1687-966X1687-9678Hindawi Publishing Corporation 10.1155/2016/5785786Review ArticleCancer Stem Cells and Radioresistance: Rho/ROCK Pathway Plea Attention Pranatharthi Annapurna 1 2 Ross Cecil 1 http://orcid.org/0000-0001-6063-0811Srivastava Sweta 1 * 1St. John's Medical College Hospital, Bangalore 560034, India2National Centre for Biological Sciences, Bangalore 560065, India*Sweta Srivastava: sweta.s@stjohns.inAcademic Editor: Zhi Sheng 2016 15 8 2016 2016 578578612 5 2016 20 7 2016 Copyright © 2016 Annapurna Pranatharthi et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Radiation is the most potent mode of cancer therapy; however, resistance to radiation therapy results in tumor relapse and subsequent fatality. The cancer stem cell (CSC), which has better DNA repair capability, has been shown to contribute to tumor resistance and is an important target for treatment. Signaling molecules such as Notch, Wnt, and DNA repair pathways regulate molecular mechanisms in CSCs; however, none of them have been translated into therapeutic targets. The RhoGTPases and their effector ROCK-signaling pathway, though important for tumor progression, have not been well studied in the context of radioresistance. There are reports that implicate RhoA in radioresistance. ROCK2 has also been shown to interact with BRCA2 in the regulation of cell division. Incidentally, statins (drug for cardiovascular ailment) are functional inhibitors of RhoGTPases. Studies suggest that patients on statins have a better prognosis in cancers. Data from our lab suggest that ROCK signaling regulates radioresistance in cervical cancer cells. Collectively, these findings suggest that Rho/ROCK signaling may be important for radiation resistance. In this review, we enumerate the role of Rho/ROCK signaling in stemness and radioresistance and highlight the need to explore these molecules for a better understanding of radioresistance and development of therapeutics. DBTDST-ITS ==== Body 1. Introduction Radioresistance and relapse are a burden for cancer therapy. Despite best efforts and technological advances, there is a considerable percentage of patients who do not respond to therapy. Most importantly, all the therapies currently in practice have severe side effects. As there are no means of treating relapse or metastatic tumors, patients are usually advised on palliative care. The diagnosis and treatment in the advanced stages are costly exercises with poor prognosis and compliance. Similarly, recurrent tumors also demonstrate poor prognosis. It is important to note that these therapies have severe side effects on the patients; thus, it is essential to stratify the patients who are most likely to respond to therapy. The development of cancer therapy is extremely dependent on an understanding of tumor biology and predictive markers. The predictive markers define the population of patients who will respond to therapy. A solid tumor is a highly complex system comprising of proliferating tumor cells, blood vessels, lymphatic vessels, and nontumor cells. The cross talk between these cell types and regulation by microenvironment is essential for tumor progression and also increases the complexity of treatment. Tumor heterogeneity, by virtue of the presence of different cell subpopulations, plays a major role in differential therapy response. One such subset which has garnered the substantial attention of the scientific community is the cancer-initiating cells (CICs) or cancer stem cell (CSC). One of the first observations on CSCs was published by Dr. Richards in 1955. He demonstrated the existence of a stem cell pool using deoxyribose nucleic acid measurements in Ehrlich's and Krebs's ascites tumor. He observed that only a small fraction of cells was capable of normal and regular mitosis which could be due to the division of the stem cells [1]. The cancer stem cell model argues that it is the major contributing factor to disease progression and therapy response as they have the capability to self-renew and generate heterogeneous lineages of cancer cells [2, 3]. One of the most convincing evidences for the existence of CSCs first surfaced from studies by Bonnet and Dick, who suggested that there is a relatively small population (0.01–1%) of cells that have enhanced tumorigenic properties [4]. CD34+/CD38− cells purified from patients with acute myeloid leukaemia were able to generate tumors in NOD/SCID mice that were histologically similar to the donor. The existence of CSCs has also been reported in several tumors including brain [5], breast [6], colon [7], ovary [8], pancreas [9], and prostate [10]. Recently, the presence of CIC marked by expression of Kr19+/Lgr5− has been reported in colon cancer [11]. 2. Radioresistance and Cancer Stem Cells There is increasing evidence that CSCs determine the fate of tumor and its clinical outcome. It is likely that the abolition of this subset of cells may impact the clinical outcomes. Radiation therapy plays a pivotal role in the treatment of several tumors such as head and neck cancer and cervical carcinoma. However, the emergence of resistance to therapy is a major concern in the treatment of carcinomas. Such resistance may be attributed to various mechanisms that exist or remain to be identified in the stem cells. Glioblastoma multiforme (GBM) is one of the most aggressive tumors with poor prognosis and ionizing radiation alone or adjuvant therapy is only palliative and is noncurative. In glioma, CSCs marked by CD133 expression exhibit properties of resistance to radiation [12, 13]. Similarly, Phillips et al. observed that in breast cancers CICs are relatively more radioresistant [14]. Bao et al. observed that in both cell culture and mice brain the CD133 expressing glioma cells survived radiation by activating the DNA damage checkpoint signaling. In a similar study, Gao et al. observed that fractionated radiation of a human glioblastoma cell line, U87-MG, enhanced cell division signaling pathways, which might be leading to repopulation of the cancer stem cell pool. Interestingly, both the CD133 positive and negative cells have been shown to possess the capability of inducing tumors in mice; however, there are reports which show that radiation induces enrichment of the CD133 expressing cells [15, 16]. CSCs also impact therapy outcome in non-small cell lung cancer (NSCLC). Radiotherapy is also used as a palliative care modality for NSCLC. Using NSCLC derived cell lines, A549 and H460, Gomez-Casal and groups show that cells surviving radiation treatment had enhanced stemness and epithelial-mesenchymal transition (EMT) properties. These cells overexpressed CD24, CD44, Sox2, and Oct4 along with EMT markers such as snail1 and N-cadherin [17]. The interaction of CD44 with Tiam1, a guanine exchange factor and regulator of GTPases, is important for Rac1 signaling activation, which supports cell migration, in metastatic breast cancer cells [18]. The presence of CICs with CD49f and CD133 overexpression has also been reported in cervical carcinoma cell lines. Gene profiling analysis of the cells showed transcriptional upregulation of DNA repair machinery proteins. Similarly, dose-dependent irradiation of the cervical carcinoma cell lines resulted in enrichment of the CICs [19]. These and several other such observations vehemently support that CSCs have a major role in radioresistance. 3. CSCs and Molecular Mechanisms Regulating Radiation Response The role of both intrinsic and extrinsic factors in the induction of resistance has been widely debated. There are various studies which support the role of the microenvironment in regulation of CSC and its function. Similarly, there is strong evidence indicating the role of signaling molecules in the maintenance of CSCs and induction of radioresistance. Landmark studies, in the 1990s, demonstrated that Hif-1-alpha, an oxygen sensing signaling pathway, is operational in tumor cells [20–22], and it can regulate the expression of several genes essential for tumor growth and progression [23]. Studies have also supported the role of reactive oxygen species (ROS) in radioresistance in CSCs. Diehn et al. show that CSCs contain lower ROS levels than the bulk of the tumor cells. Since ROS levels are important for radiation-induced cell death, CSCs harbouring lower levels of ROS have lesser DNA damage and thus elicit better survival [24]. DNA damage and repair mechanisms are widely implicated in radiation-induced effects. DNA damage is one of the earliest events after irradiation. It has been demonstrated that radioresistance in glioma cells is induced by activation of DNA repair pathway. Bao et al. show that radiation of glioma cells results in enrichment of CD133+ CSCs. Upon irradiation, these cells have activated DNA damage checkpoints and better survival rates. They demonstrated that inhibition of Chk1 and Chk2 resulted in enhanced cell death in these cells [12]. L1CAM has also been shown to induce radioresistance, by regulating NBS1, via c-Myc [25]. NBS1 is an important component of the MRN complex, involved in DNA repair. C-Myc regulates Chk1 and Chk2 by directly binding to the promoters of the two genes in nasopharyngeal carcinoma (NPC), resultantly regulating radioresistance [26]. These observations, and several others, strongly support the role of altered DNA repair mechanisms in radiation-induced resistance. There are several other signaling pathways which have important contribution to CSC maintenance (schematic representation in Figure 1). The role of Notch [27, 28], TGF-beta [29, 30], and Wnt [31, 32] in CSC maintenance has been extensively reported. These molecules are also in clinical trials to aid in radiation therapy. Hedgehog pathway inhibitors, in combination with PI3K-m-TOR inhibitors like vismodegib, sirolimus, sonidegib, and buparlisib, have worked well, both in vitro and in vivo, and are in clinical trials [33]. Clinical trials are also underway targeting Notch and Wnt pathways together. However, despite several studies, there is still a dearth of understanding of CSC and its unique molecular signatures that can be exploited for therapeutics. The GTPases have also been added to the existing plethora of molecules, contributing to radioresistance [34, 35]. 4. RhoGTPase Signaling in Radioresistance RhoGTPase pathway has been studied extensively in the context of tumor progression, and its effectors are reported in multiple cancers [36]. RhoGTPases are a family of conserved proteins that have been reported to be involved in cytoskeletal organisation, migration, cell division, cell adhesion, and transcriptional regulation, in which they act as a switch controlling the outcomes of these key processes, in a cell. Rho GTPases cycle between an active GTP and inactive GDP bound form, in response to signaling cues. Among them, the RhoA, RhoB, RhoC, Cdc42, and Rac1 are well-studied members of this family. The Rho GTPases require guanine exchange factors (GEFs), GTPase-activating proteins (GAPs), and Guanine Nucleotide Dissociation Inhibitors (GDIs) for the regulation of their activity, which is dependent upon GTP [36]. The Rho GTPases, in their active conformation (GTP-bound), are capable of binding effectors, for the subsequent downstream signaling activation [37, 38]. The GEFs are the proteins that facilitate the exchange of GDP to GTP on the Rho proteins, rendering them functionally active. The Net1-RhoA, ITSN-L-Cdc42, and Tiam1-Rac1 are few examples of GEFs and their respective GTPases [39–41]. GDIs bind to Rho GTPases like Ras, RhoA, and Cdc42 and play a role in the cytosolic maintenance of these Rho proteins, by sequestering the hydrophobic lipids on these molecules, thereby blocking their docking on the membrane. They also affect the binding of the GTPases to GEFs and the effector kinases [42, 43]. These proteins have an important role in tumor progression [44, 45]. RhoA has been shown to regulate tumor progression in several tumors [46–50]. Interestingly, RhoA also regulates radioresistance in glioblastoma, by modulating Survivin activity [51]. RhoA is shown to be expressed at the tumor front and found to be associated with poor prognosis in prostate cancer [48]. Inhibition of RhoA led to decreased proliferation and migration in gastric cancer cell line [49] and reduced migration in colorectal cancers [50]. Observations suggest that activated RhoA is found in the nucleus upon irradiation of tumor cells [52]. The other member, RhoB, has been reported as a tumor suppressor [53]. It has also been shown to mediate resistance in HeLa cells [54]. RhoC has been shown to regulate tumor progression, in several tumors [52, 55–60]. Interestingly, RhoC regulates several tumor phenotypes including angiogenesis, anoikis resistance, migration, invasion, and metastasis [57, 59–61]. It has been reported to induce EMT in ovarian cancer cells in response to VEGF and TGFbeta1 signaling [62]. The EMT mediated by TGFbeta1 signaling is dependent on RhoC overexpression in cervical cancer [56]. It has contributed to cervical cancer progression mediated by Notch signaling [44]. The most interesting observation is its ability to maintain CSC, in breast and head and neck cancer [57, 59]. RhoC knockdown in HNSCC (Head and Neck Squamous Cell Carcinoma) showed a defect in activation of STAT3 in the cells and therefore a reduction in the expression of core stem cell markers like Oct3/4, Sox2, and Nanog [57]. Rosenthal et al. show that RhoC expression impacts the frequency of CSCs in breast cancers. They also show that RhoC expression alone is enough to induce metastasis in even the non-CSC population using mice xenograft [59]. Rho GTPases have also been occasionally implicated in the resistance of tumor cells to therapy. The other RhoGTPases that have been shown to contribute to tumor progression are Cdc42 and Rac1. Cdc42 inhibition using small molecule inhibitor, AZA197, is reported to suppress the growth of colorectal cancers [63]. There are no mutations reported in the Cdc42 itself, but inhibition of Cdc42 leads to regression of tumors in intestinal cancers which harbour mutations in APC/β-Catenin [64], but there is no direct evidence for its involvement in CSC maintenance. However, Rac1 proved important in CSC activity, in both the side population (SP) cells and non-SP cells within the tumor in NSCLA (Non-Small Cell Lung Adenocarcinoma) [65]. In Fanconi Anemia (FA), there are gene mutations that are known to increase tumor invasion in HNSCC via the Rac1 GTPase, acting downstream of the DNA-PK pathway [66]. PREX2, a GEF for RAC1, has been shown to be associated with PTEN pathway, where the suppression of this GEF is necessary for PTEN tumor suppressor activity [67]. This evidence illustrates association of Rho GTPases with CSC and propels the need to study the GTPases in great detail, as they regulate key cellular processes. The effectors of Rho GTPases, Rho kinases (ROCKs) [36, 68], have also been implicated in tumor progression and metastasis. However, there is no data suggesting their involvement in radiation response. One of the early evidences of nuclear localization of ROCK2 comes from Tanaka et al., where ROCK2 is required for the acetyltransferase activity of p300 [69]. In 2006, Ma et al. show that ROCK2 is important for centrosomal duplication and, hence, important for the maintenance of chromosomal integrity. In tumors, ROCK2 functions as the effector of NPM, a known regulator that controls centrosomal duplication [70]. Hence, deregulation of ROCK2 could have consequences, in the tumor scenario. Another interesting observation published by Wang et al. has broadened the scope of current literature and has added a new dimension in the field, where ROCK2 forms a trimeric complex with BRCA2 and NPM in the centrosomes [71]. In this study, ROCK2 and NPM were identified as binding partners of ROCK2, by mass spectrometric analysis. Deregulated expression of Rho kinases in various tumors and the association of ROCK2 with p300 (epigenetic regulator) and its interaction with the DNA repair pathway indicate the importance of this molecule in cancers and reiterates the need for broadening the perspectives of its study, in the context of CSC and radioresistance. These observations and our unpublished data suggest a possible role for ROCK2 in radiation resistance. Despite convincing reports on their role in various tumor phenotypes, Rho GTPases and their effectors have not been developed further as prognostic markers or therapeutic targets. 5. Conclusion The accumulated data and experimental evidence suggest a role of CSC in therapy resistance, relapse, and metastasis. Their survival and adaptive skills are the factors regulating their maintenance and hence influencing the tumor survival and relapse. In the context of tumor heterogeneity and advent of personalised medicine, it is essential that tumor-specific CSC should be well understood to develop new therapeutic targets. In addition to enhanced DNA repair activity [12, 14, 19], the CSCs have an expression of efflux proteins which endow protective properties to these cells [72]. The defence mechanisms mostly operative in radiation resistance include DNA damage repair and salvage pathways. It is thus essential to understand the various signaling networks and cross talk that have a protective role in the context of radiation-induced DNA repair. There are several molecular pathways which contribute to the properties of radioresistance in CSC that need to be understood and explored in order to identify new druggable targets. The GTPases and their effectors may be one such target. Though these molecules have been well studied in the past in the context of metastatic cancers, there is a dearth of literature clearly indicating their role in the context of radioresistance and absolutely none illustrating the role of the effectors in a similar context. However, there is some evidence to implicate their role in DNA repair and radiation in specific cellular contexts, paving the way for focused research on GTPases and their kinases [51, 52, 68] in radioresistance. Atorvastatin is used as GTPases inhibitor to understand their function. It blocks HMG-CoA reductase pathway required to produce geranylgeranyl pyrophosphate (GGPP) and Farnesyl pyrophosphate (FPP). GGPP and FPP are important modulators of Rho GTPases. Interestingly, there are encouraging reports that the incidence of cancer is reduced in statin users [73, 74]. Study indicates that Simvastatin sensitizes the tumor xenotransplants from FaDu (Hypopharynx Squamous cell carcinoma) cells and A431, a vulvar squamous cell carcinoma derived cell line [75]. In breast cancers, it has been found that the statins reduce the rate of tumor recurrence and act as a neoadjuvant in cases which are difficult to treat like the triple negative and inflammatory breast cancers [76]. However, in another study, the statins have been reported to have radioprotective properties in normal tissues through modulation of production of inflammatory cytokines and enhancing DNA repair in the nontarget tissues [77]. These studies and several others suggest the need to understand the role of Rho GTPases and their effectors in therapy resistance. The hope to find newer and better therapeutic targets, for cancer treatment, nudges us to explore newer signaling pathways. Rho GTPases and their effectors have a significant contribution to cancer progression, and it is only apt that these molecules should be further explored in the context of CSC, to aid in better cancer treatment. Acknowledgments This work has been supported in part by a grant from DBT (Glue grant) awarded to Professor Sudhir Krishna (National Centre for Biological Sciences) and Dr. Cecil Ross. Sweta Srivastava has been supported by DST-ITS travel grant for international conferences. The authors thank Professor Sudhir Krishna for scientific discussions and inputs. Annapurna Pranatharthi has received a fellowship from CSIR-UGC for her Ph.D. work. Competing Interests The authors declare that there is no conflict of interests. Figure 1 Cross talk between multiple signaling pathways involved in tumor progression and resistance. ==== Refs 1 Richards B. M. 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==== Front Psychiatry JPsychiatry JPSYCHIATRYPsychiatry Journal2314-43272314-4335Hindawi Publishing Corporation 10.1155/2016/5480391Research ArticleCognitive Function before and during Treatment with Selective Serotonin Reuptake Inhibitors in Patients with Depression or Obsessive-Compulsive Disorder http://orcid.org/0000-0002-0956-9549Sayyah Mehdi 1 http://orcid.org/0000-0002-0531-7360Eslami Kaveh 2 http://orcid.org/0000-0003-3445-8004AlaiShehni Shabnam 3 http://orcid.org/0000-0002-5695-0596Kouti Leila 2 * 1Education Development Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran2School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz 61357-15794, Iran3Student Research Committee, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran*Leila Kouti: lkouti.pharmacotherapy@gmail.comAcademic Editor: Lenin Pavón 2016 15 8 2016 2016 548039116 4 2016 21 7 2016 Copyright © 2016 Mehdi Sayyah et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objectives. Identification of adverse effects of selective serotonin reuptake inhibitors (SSRIs) is of great importance due to their extensive use in medicine. Some studies have reported the effects of SSRIs on cognitive functions, but the results are conflicting. This study was designed to assess the effect of these drugs on cognition of patients with depression or obsessive-compulsive disorder (OCD). Methods. Patients with depression or OCD, naïve to therapy, and candidates of receiving one drug from SSRI class, voluntarily, entered this study. Mini-Mental State Examination (MMSE) test was the tool to assess their cognitive functions. MMSE scores of each patient were recorded prior to taking SSRIs and at weeks 3, 5, and 8 of drug therapy. Results. 50 patients met our inclusion criteria, with a baseline mean MMSE score of 23.94. At 3, 5, and 8 weeks of treatment, the mean scores were 22.1, 21.4, and 20.66, respectively. With a p value of <0.0001, the gradual decline was statistically significant. Conclusion. The MMSE scores of our patients showed a gradual decline over the consecutive weeks after taking SSRI drugs. It seems that the use of SSRIs in patients with depression or OCD, can cause cognitive dysfunction in the acute phase of treatment. ==== Body 1. Introduction Selective serotonin reuptake inhibitors (SSRIs) are the first-line drug treatment of depression and obsessive-compulsive disorder (OCD). Monotherapy with this class is often recommended for patients primarily diagnosed with these conditions [1, 2]. Despite the potential usefulness of SSRIs, they could cause unwanted side effects such as headaches, weight gain, sexual dysfunction, changes in sleep patterns, and gastrointestinal problems [3]. Originally, SSRIs did not appear to cause cognitive impairment, but nowadays there are conflicting evidences regarding this side effect and many patients complain of memory loss during their course of therapy with SSRIs [4, 5]. The aim of this study is to assess the effect of SSRI drugs on cognitive function of patients with depression or OCD, in acute phase of therapy. In this phase, knowledge and management of side effects are very critical for the patient's compliance to therapy, since these patients might not be motivated enough to cope with adverse effects along with their psychiatric symptoms [6]. In many health problems, drug induced memory loss can be an important cause of medication nonadherence [7, 8]. Current study evaluates the patients' cognitive function, prior to taking SSRIs and throughout the acute course of their therapy. 2. Methods This analytic-descriptive study was conducted in a psychiatric outpatient clinic from April to September 2015. It was permitted by Ahvaz Jundishapur University of Medical Sciences ethics committee. Among patients diagnosed with depression or OCD by a board certified psychiatrist, those who currently were not under any treatment for mental or psychiatric disorders and were candidates of receiving a drug from SSRI class as monotherapy were asked to participate in this study. Inclusion criteria consisted of age between 18 and 60 years, no diagnosed cognitive impairment or mental retardation, no other comorbid disorders in Axis I, the ability to write and read, no visual or auditory impairment, no history of loss of consciousness after head trauma or brain surgery, and no history of using psychoactive drugs such as antidepressants, neuroleptics, benzodiazepines, antiepileptics, or opioids within 14 days prior to the study. Individuals with memory loss complaints in daily functioning would not be included either. If the patient developed any condition in which the use of SSRIs was contraindicated or any disability which interfered with performance of cognitive tests, he/she would be excluded from the study. In addition, patients who stopped their medication for seven consecutive days or did not adhere well with their drug therapy would be excluded. All patients signed a consent form prior to the study. Several approaches have been developed to help with the diagnosis of cognitive impairment. Some of these tests are Montreal Cognitive Assessment, DemTect, Short Test of Mental Status, Clinical Dementia Rating, and Mini-Mental State Examination (MMSE) [9–12]. Among these, MMSE test is the only valid and reliable test in Farsi; thus, we used this tool for our patients [13, 14]. Aspects of cognitive function assessed by this test include orientation, registration, attention, calculation, recent memory, and language skills. Seyedian et al. translated the Farsi version of MMSE. Its reliability based on Cronbach's Alpha is 81% and a cut-off point of 22 showed a sensitivity of 90% and specificity of 93.5% [13, 14]. The patients' demographic data (age, gender, level of education) were collected in the first visit (week 0 of therapy) and MMSE (a 30-point test) was performed at weeks 0, 3, 5, and 8 of drug therapy. Each patient's drug regimen (type of drug and dosage) remained fixed throughout this 8-week period. If for any reason the patient needed a dose reduction or increase or medication discontinuation, he/she would be excluded from the study. The response to therapy and clinical improvement of OCD/depression signs and symptoms would also be evaluated and recorded. 2.1. Statistical Analysis The data were analyzed using repeated measures one-way ANOVA. Results with a p value of less than 0.05 were considered significant. Correlations of MMSE scores and age, sex, and level of education were analyzed by Pearson correlation test, one-way ANOVA test, and paired sample t-test, respectively. 3. Results Fifty patients (28 women and 22 men, ages ranging from 20 to 60 years) participated in this study (50% diagnosed with depression and 50% with OCD). They all completed the study and we had no dropout patients. All of them showed signs of response to treatment of their psychiatric disorder at week 8. In order to analyze the correlation of level of education with MMSE scores, we grouped the patients into six different groups. Nine patients were in the elementary group (under 6 years of education), seven were in the middle school group (7–11 years), and 16 patients had a high school diploma. The number of patients with a bachelor, masters, or Ph.D. degree was 11, 6, and 1, respectively. The prescribed drugs for patients were paroxetine (52%), Zoloft (28%), fluoxetine (12%), and citalopram (8%). Table 1 shows the results of MMSE scores prior to treatment and at weeks 3, 5, and 8. With a p value of less than 0.0001, the patients' MMSE scores lowered significantly from their baseline. Correlation of age and MMSE scores was also assessed and the results showed no significant relationship between them, neither prior to therapy (p value = 0.4) nor during it (p value = 0.12). MMSE scores among male and female patients had a similar decline trend, when week 8 of treatment was compared with the baseline (95% confidence interval of the difference). Level of education showed no significant correlation with decline of cognitive function (p value = 0.14). 4. Discussion Cognitive dysfunction subsequent to SSRI therapy remains controversial and different studies have reported inconsistent results [4]. Memory loss as a side effect can be overwhelming for patients and leads to medication nonadherence [7, 8]. We used MMSE test for the evaluation of cognitive function of such patients. It is widely used and easy to perform and takes only few minutes of patient's time but it has its limitations as well. Some of these psychometric limitations of MMSE test include ceiling effect, floor effect, and poor distinguishing between mild cognitive impairment and Alzheimer's disease [15]. This test might not be the preferred tool for cognitive assessment of our patients but so far it is the only test in Farsi language that has approved validity and reliability [13, 14, 16, 17]. Due to these limitations, we evaluated the correlation of age and level of education with MMSE scores to clarify the possible influence of these factors on the subjects' cognitive function. Also, since the cut-off points of MMSE might not accurately correlate to the patient's cognitive state, we compared the mean scores before and after therapy and did not group the patients according to the cut-offs [15]. Future studies with the help of other cognitive assessment tools can help to have a more accurate identification of this problem. Our findings show that patients taking SSRIs experienced statistically significant memory loss during 8 weeks of treatment; age and gender did not influence this result. In contrast, Levkovitz et al. and Culang-Reinlieb et al. reported that some SSRIs have improved memory function in patients with depression [17, 18]. Herrera-Guzmán and colleagues have investigated the effects of SSRIs on cognitive impairment due to depression, in a series of studies. They performed several cognition tests and evaluated the patients in remission and recovery phase. They reported a positive effect of drug therapy with SSRIs in the long term [19]. Deuschle and colleagues performed California Verbal Learning Test on 24 patients with depression and found a low rate of declarative memory loss after 35 days of treatment with SSRIs (the majority of patients had no change from baseline) but no cognitive impairment at long-term therapy (over 12 months). They concluded that the low-grade impairment is due to depressive symptoms and not a side effect [20]. Nevertheless, some evidences strongly supported our results. The study by Popovic et al. showed that over 20% of patients with depression or anxiety disorders reported memory loss after 6 months of SSRI therapy. This was considered a side effect rather than a symptom of untreated depression/anxiety [21]. Wadsworth et al. and Biringer et al. also reported the probability of memory deficit associated with SSRI use [4, 22]. Two other case reports with memory loss subsequent to fluoxetine use have been published in previous years. In one case report, memory loss was reversed when fluoxetine was changed to another SSRI [23, 24]. Although memory impairments can be due to depression itself, memory loss appears to be more likely due to SSRI therapy rather than depression symptoms. Serretti et al. showed that using SSRIs even in healthy individuals leads to cognitive impairment [25]. The memory loss caused by SSRIs has not yet been convincingly explained; however, serotonin appears to play an important role in learning and memory [26]. Serotonin transporter (SERT) expression has been found to be vital for memory development. Decreased expression of this transporter was found in Alzheimer's disease and other memory impairments and dementia [27]. Benmansour et al. showed a decrease in SERT expression within 21 days (3 weeks) following SSRI use [28]. In order to make a better understanding of this issue, Herzallah et al. studied two groups of patients with major depression (one group naïve to medication (16 patients) and one group under successful treatment with paroxetine (15 patients)) and a control group of 25 healthy volunteers, aged 18–60 years. They performed a task evaluating two different aspects of cognition. The results showed that major depression and SSRIs have different positive and negative effects on cognition. SSRIs may have a positive role in striatal function but can deteriorate hippocampus function, needed for generalization. On the other hand, patients with major depression were normal in the latter function, but slow in learning [29]. Although these findings may partly explain the cause of memory loss due to SSRIs, more studies are needed to discover the exact mechanism responsible for this outcome. We did not evaluate the effect of disease on cognitive function and only targeted the influence of SSRIs on memory. Although all of the patients had higher baseline MMSE scores compared to short-term treatment with SSRIs, their mean score even prior to therapy was lower than normal population (MMSE = 24–26) [30]. 5. Conclusions Our data show that in patients with OCD or depression and those who are naïve to SSRI therapy, a gradual decline in their memory function can develop within the first 8 weeks of initiation of drug treatment with SSRIs. This decline did not have a relationship with the patient's age or gender, nor his/her level of education. We suggest further follow-up studies of these patients to identify the persistence of memory loss. Our results were obtained via MMSE test and regardless of the specific drug or disorder. We hope that soon other cognitive function tests would be valid and reliable for Iranian population. Acknowledgments The authors thank Dr. Ramezani and her kind laboratory staff for their help and support. Competing Interests The authors declare no conflict of interests. Table 1 Results of patients' cognitive assessment prior to and during treatment with SSRs (number of patients = 50). Weeks of treatment Week 0 Week 3 Week 5 Week 8 Mean MMSE score 23.94 22.1 21.4 20.66 p values of comparing MMSE scores of weeks 3, 5, and 8 with baseline score — <0.0001 <0.0001 <0.0001 p values of comparing MMSE scores of weeks 5 and 8 with the results of week 3 — — <0.0001 0.0709 p values of comparing MMSE scores of week 8 with the results of week 5 — — — <0.0001 The trend of decline shows faster decline by 5 weeks and a more subtle decline until week 8. ==== Refs 1 Abramowitz J. S. Treatment of obsessive-compulsive disorder in patients who have comorbid major depression Journal of Clinical Psychology 2004 60 11 1133 1141 10.1002/jclp.20078 2-s2.0-6344275149 15389622 2 Marken P. A. Munro J. S. 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==== Front J OphthalmolJ OphthalmolJOPHJournal of Ophthalmology2090-004X2090-0058Hindawi Publishing Corporation 10.1155/2016/3513794Clinical StudyLow-Fluence Photodynamic Therapy versus Subthreshold Micropulse Yellow Wavelength Laser in the Treatment of Chronic Central Serous Chorioretinopathy http://orcid.org/0000-0001-7561-5075Özmert Emin * Demirel Sibel http://orcid.org/0000-0002-1822-8703Yanık Özge Batıoğlu Figen Department of Ophthalmology, Faculty of Medicine, Ankara University, 06620 Ankara, Turkey*Emin Özmert: eozmert56@gmail.comAcademic Editor: George M. Saleh 2016 15 8 2016 2016 351379415 2 2016 5 7 2016 Copyright © 2016 Emin Özmert et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Purpose. To compare the efficacy and safety of subthreshold micropulse yellow wavelength laser (SMYL) and low-fluence photodynamic therapy (PDT) in the treatment of chronic central serous chorioretinopathy (CSC). Methods. Thirty-three eyes of 30 patients with chronic CSC received either PDT (18 eyes) or SMYL (15 eyes) therapy. Best corrected visual acuity (BCVA), subretinal fluid (SRF) height, and central macular thickness (CMT) were evaluated at the baseline visit and one, three, six, nine, and 12 months after the therapy. Results. After 12 months, mean BCVA improved from 67.3 ± 14.2 to 71.5 ± 21.4 ETDRS letters in SMYL group and from 60.7 ± 16.3 to 64.4 ± 24.9 ETDRS letters in PDT group (p = 0.285 and p = 0.440, resp.). Mean CMT decreased from 242.8 ± 80 μm to 156.9 ± 60 μm in the PDT group and from 287.3 ± 126 μm to 138.0 ± 40 μm in the SMYL group (p = 0.098 and p = 0.003, resp.). SRF resolved completely in 72.2% and 80.0% of the eyes in the PDT and SMYL groups, respectively. Mean SRF height decreased from 117.2 ± 58 μm to 31.3 ± 56 μm in the PDT group and from 130.0 ± 104 μm to 12.5 ± 21 μm in the SMYL group (p = 0.031 and p = 0.014, resp.). Conclusions. Subthreshold micropulse yellow wavelength laser seems to be effective in the treatment of chronic CSC without any side effect and results in the resorption of SRF without causing visible retinal scarring. ==== Body 1. Introduction Central serous chorioretinopathy (CSC) is a disorder of unknown etiology characterized by detachment of the neurosensory retina due to accumulation of serous fluid between the retinal pigment epithelium (RPE) and photoreceptor layers. The disease can present in acute or chronic form. Acute CSC is generally self-limited with spontaneous regression, and it causes minimal sequelae. In chronic CSC, the long-term persistence (longer than six months) of subretinal fluid (SRF) can result in atrophy of the RPE, cystoid retinal degeneration, choroidal neovascularization, and permanent vision loss [1, 2]. Management of chronic CSC includes various options, such as risk factor modification (discontinuation of steroids) [3], medical treatment (carbonic anhydrase inhibitors) [4], conventional focal laser [5], photodynamic therapy (PDT) [6, 7], and intravitreal injection of vascular endothelial growth factor inhibitors [6, 8]. It has been reported that PDT with verteporfin induces the resorption of SRF by reducing choroidal vascular hyperpermeability [9, 10]. However, it has the potential for serious side effects, such as choroidal ischemia, RPE atrophy, and iatrogenic choroidal neovascularization [11, 12]. To avoid these complications, newer PDT protocols, including half-dose [13] and low-fluence [14] applications, have been developed. Nowadays, micropulse laser (MPL) photocoagulation is another possible treatment option for chronic CSC [15, 16]. The mechanism of action depends on targeting a train of ultrashort laser pulses at the particular tissue of interest. These repetitive bursts prevent damage to adjacent tissues from thermal effects, minimize total energy use, and provide time for tissue cooling between pulses [17]. This technology can be paired with either 810 nm or 577 nm wavelength lasers. The first studies on MPL for CSC used an 810 nm diode laser as the laser source [15, 16, 18]. Subthreshold micropulse yellow wavelength (577 nm) laser (SMYL) is a newer technology that offers major advantages such as peak absorption of oxyhemoglobin, minimal xanthophyll absorption in the macula, and better penetration [19]. The aim of this study was to compare the efficacy and safety of SMYL and low-fluence PDT in the treatment of chronic CSC. This is the first study that compares the treatment outcomes of SMYL and PDT in patients with chronic CSC. 2. Methods This retrospective comparative case series included 33 eyes of 30 patients with chronic CSC treated with either low-fluence PDT (18 eyes) or SMYL (15 eyes) therapy at the Ankara University Faculty of Medicine, Department of Ophthalmology, between January 2012 and January 2015. The study was conducted in accordance with the Declaration of Helsinki. The diagnosis of chronic CSC was confirmed by clinical examination, spectral domain optical coherence tomography (SD-OCT) (Spectralis; Heidelberg Engineering, Inc., Heidelberg, Germany), fundus fluorescein angiography (FA), indocyanine green angiography (ICGA), and fundus autofluorescence (FAF) imaging (Heidelberg Retina Angiograph 2; Heidelberg Engineering, Heidelberg, Germany). Inclusion criteria were as follows:Visual impairment history lasting at least six months. CSC with SRF involving the fovea and documented by SD-OCT. Presence of single or multiple active leakage sites and/or RPE changes on baseline FA. Absence of prior history of micropulse laser or PDT therapies. Follow-up period of at least 12 months after both treatment modalities. There were not any criteria such as disease severity for selection of the treatment modalities. Patients with a history of comorbid ocular conditions such as age-related macular degeneration, diabetic macular edema, advanced glaucoma, optic neuropathy, or intraocular surgery within the previous six months were excluded. In the micropulse treatment group (15 eyes), SMYL treatment (Supra Scan 577; Quantel Medical, Clermont-Ferrand, France) had been performed by the same experienced surgeon (EO), using the following parameters: low-intensity (5% duty cycle) and high-density (confluent spots) treatment with 200 msn duration and 160 μm spot size on the slit-lamp adaptor. The power was initially increased upward to the minimum threshold value to cause a barely visible burn on micropulse mode, and then it was adjusted to half of that value. An OCT-guided approach was preferred, and in this manner, an appropriate scan shape was chosen to cover the edematous area entirely on the OCT thickness map. In the low-fluence PDT group (18 eyes), verteporfin (Visudyne; Novartis, Basel, Switzerland) was administered intravenously at a dose of 6 mg/m2 over ten minutes. Fifteen minutes after the start of the infusion, a 689 nm laser was delivered for 83 seconds at a reduced light dose of 25 J/cm2 and an intensity of 300 mW/cm2. The same experienced specialist (FB) performed the procedure on all of the patients in this group. The PDT application location was based on choroidal hyperpermeability identified by ICGA. After the treatment, the patients were instructed to avoid sunlight exposure for five days. All of the patients were examined at the baseline visit and one, three, six, nine, and 12 months after undergoing the SMYL or PDT therapy. At each visit, each participant underwent a complete ophthalmic examination that included best corrected visual acuity (BCVA) with Early Treatment Diabetic Retinopathy Study (ETDRS) charts, slit-lamp biomicroscopy, intraocular pressure measurements, dilated fundus examination, FAF imaging, and OCT; FA was repeated as needed. The morphologic results of the treatment were evaluated with SD-OCT in terms of SRF height and central macular thickness (CMT). Subretinal fluid height was measured manually between the outer segment of the photoreceptor layer and the apical face of the RPE layer. The patients were divided into three groups according to response to treatment: complete response, incomplete response, and unresponsive to therapy. Complete response was defined as complete resolution of SRF in the central 1000 micron area. Any change in SRF within ±100 μm was defined as unresponsive. If the SRF decreased more than 100 μm but was not fully resorbed, the treatment response was defined as incomplete resolution. The main outcome measures were changes in BCVA and SRF height between the baseline and follow-up examinations. The second outcome measure was to compare the complete anatomic resolution, recurrence, and complication rate between treatment modalities at the end of the 1 year. Data are shown as mean and standard deviation for continuous variables. For visual acuity assessment, BCVA was converted to logarithm of the minimum angle of resolution (logMAR) equivalents; in addition, ETDRS letters were used in the statistical analysis. Statistical analysis was performed using SPSS software for Windows version 15.0 (SPSS, Inc., Chicago, IL). A p value <0.05 was considered statistically significant. Specific differences in mean values between different treatment groups were tested for significance using Mann-Whitney U test with Bonferroni's correction of p values for multiple comparison. The changes in the same group throughout the follow-up visits were compared using the Friedman test. Kruskal-Wallis test was used for the evaluation of singly ordered categorical data. 3. Results Thirty-three eyes of 30 patients with chronic CSC were analyzed in this study. The patient group included 22 (73%) male patients and eight (27%) female patients. The mean age (±SD) of the patients at presentation was 49.9 ± 11.1 (31–73) years. The PDT group was older than the SMYL group (52.7 ± 11.2 years versus 44.7 ± 9.5, resp.; p = 0.037), and the duration of chronic CSC prior to treatment was longer in the PDT group (18.8 ± 13.5 months versus 13.0 ± 9.1 months, resp.; p = 0.330). None of the patients in the SMYL group underwent previous therapy. Ten patients in the PDT group had previously received intravitreal ranibizumab injections, but the last injection was performed at least three months prior to PDT. The mean number of ranibizumab injections was 4.7 ± 3.2. Initial mean visual acuity was 0.47 ± 0.32 logMAR units in the PDT group and 0.32 ± 0.29 logMAR units in the SMYL group (p = 0.126). All patients had at least a 12-month follow-up after treatment. In the PDT group, BCVA improved at least 5 ETDRS letters in six eyes (33.3%) and remained stable (within ±4 ETDRS letters) in six eyes (33.3%) (Table 1). In the SMYL group, BCVA improved at least 5 ETDRS letters in ten eyes (66.7%) and remained stable in one eye (6.7%) (p = 0.101). Mean BCVA improved from 67.3 ± 14.2 to 71.5 ± 21.4 ETDRS letters in SMYL group and improved from 60.7 ± 16.3 to 64.4 ± 24.9 ETDRS letters in PDT group (p = 0.285, p = 0.440). The changes in logMAR visual acuity scores and ETDRS letters throughout the follow-up period are shown in Figure 1; there were no statistical differences between the groups (p = 0.079 for both groups). Mean SRF height decreased from 117.2 ± 59 μm to 31.3 ± 57 μm in the PDT group and from 130.0 ± 105 μm to 12.6 ± 21 μm in the SMYL group. The reduction in SRF from baseline to the 12th month was statistically significant in both groups (p = 0.031 and p = 0.014, resp.). The changes in SRF height throughout the follow-up period are shown in Figure 2; there were no statistical differences between the treatment groups (p = 0.735). Mean CMT decreased from 242.9 ± 80 μm to 156.9 ± 61 μm in the PDT group and from 287.3 ± 126 μm to 138.0 ± 41 μm in the SMYL group (p = 0.098 and p = 0.003, resp.). There were no statistical differences between the treatment groups throughout follow-up (p = 0.338). Treatment responses based on SRF resolution were evaluated at the 12-month follow-up (Table 2). In the PDT group, 13 eyes (72.2%) achieved complete anatomic resolution of SRF in the central macula (Figure 3) and one eye (5.6%) had incomplete resolution, confirmed by OCT. Recurrence of SRF after complete resolution occurred in one eye (5.6%); three eyes (16.7%) were unresponsive to PDT. Of these unresponsive eyes, one eye received one more seance of PDT; however, the patient was unresponsive to the treatment again. The other patients were not retreated. In the SMYL group, 12 eyes (80.0%) achieved complete anatomic resolution of SRF in the central macula (Figure 4) and one eye (6.7%) had incomplete resolution. Subthreshold micropulse yellow wavelength laser treatment was repeated in this eye. However, complete anatomic response could not be achieved again. Recurrence of SRF after complete resolution occurred in two eyes (13.3%); no eyes were unresponsive to SMYL. There was no statistical difference between the PDT and SMYL groups in terms of treatment response (p = 0.486). Fundus autofluorescence imaging used to determine the safety profile of the SMYL group revealed hypofluorescent laser spots that were not visible in the fundus examination in one case. There were no significant side effects in the PDT group. 4. Discussion In the present study, the treatment outcomes of chronic CSC patients treated with either low-fluence PDT or SMYL were compared, and changes in logMAR visual acuity scores, ETDRS letters, SRF height, and CMT throughout the follow-up were analyzed. To our knowledge, this is the first study in the literature to compare SMYL and low-fluence PDT treatment results in chronic CRC patients. The results of the study indicate that there were no statistically significant differences in the parameters listed above between the two treatment modalities. Subthreshold micropulse yellow wavelength laser seems to be an effective modality in treatment response, visual acuity improvement, and SRF reduction. The exact pathophysiologic changes that lead to the characteristic RPE leakage associated with CSC remain unclear. However, ICGA has demonstrated the effect of choroidal circulatory abnormalities in the development of CSC, such as hyperpermeability from choriocapillaris [20], venous dilation [21], and vascular congestion [22]. In the treatment of CSC, the aim is to induce absorption of SRF and to improve or stabilize visual acuity. Photodynamic therapy with verteporfin has proven to be effective according to several reports [11, 23, 24]. The mechanism of action of PDT depends on transient choroidal hypoperfusion leading to choroidal vascular remodeling and the reduction of choroidal exudation [6, 23, 25]. However, the conventional PDT protocol has several potential serious adverse effects, including RPE changes, permanent choroidal ischemia, and secondary choroidal neovascularization [11, 12, 23]. To reduce the risk of these potential adverse effects, modified PDT protocols have been developed, such as half-dose verteporfin administration [13, 26, 27] and low-fluence rate [28, 29]. It has been reported that low-fluence PDT reduces choroidal hypoperfusion without causing significant differences in treatment efficacy [28]. Photodynamic therapy is a relatively invasive procedure that requires the intravenous injection of verteporfin. Also, ICG may cause anaphylactic and urticarial reactions in patients both with and without a history of allergy, and hepatic diseases, hemodialysis, and pregnancy are relative contraindications for ICG [30]. A low-fluence PDT protocol was used in the present study, with complete resolution of SFR achieved in 72.2% of the patients. The subthreshold MPL technique introduced the term “photostimulation,” as opposed to “photocoagulation” [18]. The mechanism depends on low-intensity, high-density laser applications in envelopes of repetitive short pulses [31]. These pulses stimulate the production of intracellular antiangiogenic and restorative biological factors without causing visible laser scars [32, 33]. Wavelengths of either 810 or 577 nm can be used as the laser source in micropulse photostimulation. This technique has been used with promising results in the treatment of macular edema in diabetic retinopathy and branch retinal vein occlusion [34–36]. Chen et al. [15] studied subthreshold diode MPL in 26 eyes of 25 CSC patients and reported it to be effective in the presence of point source leakage. However, a less favorable response was noted in eyes with diffuse leakage. Another study reported that, after ICG dye-enhanced subthreshold MPL, neuroepithelial detachment was completely resolved in five of seven patients with CSC within four to eight weeks [16]. In a recent study comparing the outcomes of subthreshold MPL and intravitreal bevacizumab injections, subthreshold MPL photocoagulation was found to be superior to intravitreal injections of 1.25 mg bevacizumab in the treatment of CSC [18]. All of these promising findings define subthreshold MPL as a possible treatment option for chronic CSC. Subthreshold micropulse yellow wavelength laser is a quite different modality than subthreshold micropulse diode laser. The yellow wavelength (577 nm) laser produces combined absorption by both melanin and oxyhemoglobin, which leads to maximum absorption in the pigment epithelium and choriocapillaris [37]. It also has negligible xanthophyll absorption, which allows repeated treatment close to the fovea [19]. While the longer wavelength provides better penetration, it also leads to energy concentration in a smaller volume and allows a shorter pulse duration. The major advantages of the procedure are the tissue-sparing effect and repeatability of the sessions after three to six months in case of recurrence of SRF or unresponsiveness to therapy. Subthreshold micropulse yellow wavelength laser might also lead to an increase in retinal sensitivity in the macular area [38]. In the present study, the effects of the SMYL therapy on macular sensitivity could not be evaluated, due to the lack of availability of a microperimetry device. However, we believe SMYL therapy may improve retinal sensitivity in addition to increasing visual acuity. Only two studies in the literature have reported the efficacy of SMYL in chronic CSC patients. The first study included 15 eyes of 13 patients who had CSC for more than three months. The mean follow-up period was eight weeks, and the researchers reported a reduction in mean SRF height from 232 to 49 μm at the final visit. They determined that the procedure was safe and that it caused no obvious changes in RPE as seen on OCT and FAF [38]. In the second study, the authors reported the results of ten eyes of ten chronic or chronic recurrent CSC patients who received SMYL with a 15% duty cycle. The logMAR visual acuity scores improved from 0.21 ± 0.21 to 0.035 ± 0.063 at the end of the follow-up period (mean, 8 months; range, 3–18 months) [39]. The present study is the first study to compare the treatment outcomes of low-fluence PDT and SMYL, with follow-up period of 12 months. In this study, 80% of the eyes in the SMYL group achieved complete resorption of SRF (72.2% in PDT group), and all of the eyes were responsive to therapy. However, two patients experienced recurrence of CSC, and in another patient, FAF revealed hypofluorescent laser spots that were not visible in the fundus examination. Retinal laser spots on FAF could occur in rare circumstances as a result of RPE changes. The occurrence of retinal burn depends on the pigmentation of the individual and the applied laser power. Several methods were developed for MPL power adjustment. Some authors adjust laser power upward to the minimum threshold value for a visible burn in a continuous wave mode and then switch the apparatus to micropulse model [15, 38, 40]. Other researchers have also used the micropulse mode for power titration and applied 50–80% of the minimum threshold power to cause a barely visible burn [39, 41]; that power adjustment method was used in the present study. However, further studies are needed to determine a standard power titration protocol in SMYL applications. The major limitation of the present study is the retrospective design. A randomized prospective controlled trial will be more rational to compare the treatment modalities. Also the longer duration of the disease and the presence of eyes previously treated with intravitreal anti-VEGF therapy in PDT group are the other main limitations of the study. In conclusion, SMYL seems to be effective in providing resorption of SRF in the treatment of chronic CSC. Although not statistically significant, the rate of complete resolution of SRF and ≥ 5 increase in ETDRS letters were higher in SMYL group. Also, in contrast to the PDT group, no eyes were unresponsive to SMYL. Major advantages of SMYL are having a tissue-sparing effect, being a noninvasive procedure, and allowing safe repetition of the therapy after three to six months. In addition to improving BCVA, SMYL may also improve retinal sensitivity in the macular area without causing collateral chorioretinal damage. Further prospective studies are needed to investigate the correlation between changes in visual acuity and macular sensitivity on microperimetry after SMYL in the treatment of CSC. Competing Interests No conflicting relationship exists for any author. Authors' Contributions All authors conceived the study, participated in its design and coordination, and revised the paper critically for important intellectual content. All authors read and approved the final paper. Figure 1 The changes in logMAR visual acuity scores (a) and ETDRS letters (b) throughout follow-up were shown. Figure 2 The changes in subretinal fluid height throughout follow-up were shown. Figure 3 Spectral domain optical coherence tomography (SD-OCT), fundus autofluorescence (FAF), and fluorescein angiography (FA) images of a 47-year-old female patient with CSC for 7 months. Left eye before photodynamic therapy (PDT): chronic subretinal fluid on SD-OCT (a), hypoautofluorescence on FAF image due to the blockage effect of the subretinal fluid (b), and focal leakage of fluorescein and two small pigment epithelial detachments at the late phase of FA (c). Eight months after PDT treatment: complete resolution of the subretinal fluid (d) and disappearance of hypoautofluorescence on FAF (e). Figure 4 Spectral domain optical coherence tomography (SD-OCT), fundus autofluorescence (FAF), and fluorescein angiography (FA) images of a 42-year-old female patient with CSC for 7 months. Left eye before subthreshold micropulse yellow wavelength laser (SMYL) treatment: chronic subretinal fluid with fibrin accumulation on SD-OCT (a), increased hypoautofluorescence on FAF image due to the blockage effect of the subretinal fluid (b), and focal leakage of fluorescein at the late phase of FA (c). Nine months after SMYL treatment: complete resolution of the subretinal fluid (d) and hypoautofluorescence spots due to RPE atrophies at the previous leakage area on FAF (e). Table 1 Final best corrected visual acuity changes in low-fluence photodynamic therapy and subthreshold micropulse yellow laser therapy groups. BCVA change Low-fluence PDT n (%) SMYL n (%) Increase ≥ 5 ETDRS letters 6 (33.3%) 10 (66.7%) Stable (within ±4 ETDRS letters) 6 (33.3%) 1 (6.7%) Decrease ≥ 5 ETDRS letters 6 (33.3%) 4 (26.7%) Total 18 15     p = 0.101 Table 2 Treatment responses based on SRF resolution in low-fluence photodynamic therapy and subthreshold micropulse yellow laser therapy groups at the end of one-year follow-up period. Treatment response Low-fluence PDT n (%) SMYL n (%) Complete resolution of SRF 13 (72.2%) 12 (80.0%) Incomplete resolution of SRF 1 (5.6%) 1 (6.7%) Unresponsive 3 (16.7%) None Recurrence 1 (5.6%) 2 (13.3%) Total 18 15     p = 0.486 ==== Refs 1 Piccolino F. C. De La Longrais R. R. Ravera G. The foveal photoreceptor layer and visual acuity loss in central serous chorioretinopathy American Journal of Ophthalmology 2005 139 1 87 99 10.1016/j.ajo.2004.08.037 2-s2.0-11844284000 15652832 2 Wang M. S. M. Sander B. Larsen M. 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==== Front Stem Cells IntStem Cells IntSCIStem Cells International1687-966X1687-9678Hindawi Publishing Corporation 10.1155/2016/8281235Research ArticleHair Follicle Morphogenesis in the Treatment of Mouse Full-Thickness Skin Defects Using Composite Human Acellular Amniotic Membrane and Adipose Derived Mesenchymal Stem Cells Minjuan Wu Jun Xiong Shiyun Shao Sha Xu Haitao Ni http://orcid.org/0000-0002-0564-3019Yue Wang * http://orcid.org/0000-0003-2548-9052Kaihong Ji * Department of Histology and Embryology, Second Military Medical University, Shanghai 200433, China*Wang Yue: wangyuesmmu@163.com and *Ji Kaihong: jkhsmmu@163.comAcademic Editor: Giorgio Mori 2016 15 8 2016 2016 828123521 4 2016 24 6 2016 3 7 2016 Copyright © 2016 Wu Minjuan et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Early repair of skin injury and maximal restoration of the function and appearance have become important targets of clinical treatment. In the present study, we observed the healing process of skin defects in nude mice and structural characteristics of the new skin after transplantation of isolated and cultured adipose derived mesenchymal stem cells (ADMSCs) onto the human acellular amniotic membrane (AAM). The result showed that ADMSCs were closely attached to the surface of AAM and grew well 24 h after seeding. Comparison of the wound healing rate at days 7, 14, and 28 after transplantation showed that ADMSCs seeded on AAM facilitated the healing of full-thickness skin wounds more effectively as compared with either hAM or AAM alone, indicating that ADMSCs participated in skin regeneration. More importantly, we noticed a phenomenon of hair follicle development during the process of skin repair. Composite ADMSCs and AAM not only promoted the healing of the mouse full-thickness defects but also facilitated generation of the appendages of the affected skin, thus promoting restoration of the skin function. Our results provide a new possible therapy idea for the treatment of skin wounds with respect to both anatomical regeneration and functional restoration. National Natural Science Foundation of China8130164931101008Natural Science Foundation of Shanghai14ZR1449400 ==== Body 1. Introduction Local or systemic cutaneous lesions arising from skin injury are often related to the loss of barrier function. Early repair of skin injury and maximal restoration of the function and appearance have become important targets of clinical treatment. Autologous free skin grafting, skin flap transplantation, and allogenous or xenogeneic skin transplantation remain the first consideration in conventional clinical treatment of skin injury [1, 2]. Although these techniques are usually effective in most cases, how to solve the problem of covering large wound areas and reduce wound retraction and scar formation in patients with large and life-threatening wounds or those with beauty demands remains a clinical challenge. Construction of an ideal skin substitute has become an inevitable trend in burn and plastic surgery. In 1975, Rheinwald and Green [3] first reported successful treatment of wounds with transplantation of cultured human epidermal cells, which symbolizes a milestone in wound treatment. With the development of modern molecular and cellular biology and tissue engineering, advances in skin substitute research and application have gradually rendered it possible to reduce secondary injury from autologous skin transplantation [4]. Subsequently, researchers have created techniques of autologous epidermal cell culture and transplantation for the treatment of burn and various other acute/chronic wounds, thus providing permanent coverage for large-area wounds. However, the anti-infection ability of these skin substitutes is relatively low, and their functional and appearance degradation is also an unavoidable problem. Epidermal substitutes are mainly used for superficial wounds [5]. The epidermis alone cannot survive long for large, deep, and extensive wounds because it cannot receive nutritional support from the dermis and therefore needs mechanical protection of a dermal substitute. The dermal composition in the skin substitute can prevent the wound from retracting and increase mechanical stability. Knowing that the dermis plays an important role in the regulation of epidermal renewal and reconstruction, accelerating the construction of the dermis is an extremely important link in skin tissue engineering [6]. The human amniotic membrane (hAM) is a natural high-molecular biological material and can express multiple growth factors and mRNA-related proteins including collagen, glycoprotein, protein polysaccharide, integrin, and lamellar body, which are beneficial to cell growth and reproduction. For this reason, hAM is often used as a vector for cell growth and proliferation [7, 8]. Acellular amniotic membrane (AAM) is a natural biologic scaffold and can be used as an extracellular matrix to load cells for the construction of engineered tissues and organs [9]. There have been many reports about the use of AAM for wound coverage [10, 11]. But few studies have reported the use of composite AAM and stem cells for the treatment of skin defects and functional repair. In the present study, we intended to observe the healing of skin defects and histological and structural characteristics of the newborn skin after transplantation of isolated and cultured adipose derived mesenchymal stem cells (ADMSCs) onto AAM and using them to cover the skin defects in nude mice, in an attempt to explore the possibility of seeding ADMSCs on AAM to repair skin defects. 2. Materials and Methods 2.1. Characterization of ADMSCs Fourth-passage ADMSCs stored in our laboratory were characterized for the expression pattern of mesenchymal and pluripotent markers by immunohistochemistry and flow cytometry. P4 ADMSCs were fixed with 4% paraformaldehyde in phosphate buffer for 4 min at room temperature. After being blocked with PBS containing 2% BSA, cells were permeabilized with 0.1% Triton-X 100 for 10 min. Slides were incubated sequentially overnight at 4°C with the following primary antibodies: Oct-4 (goat polyclonal, 1 : 50, Santa Cruz Biotechnology Inc.) and SH-2 (1 : 50, mouse polyclonal, Santa Cruz Biotechnology Inc.). The slides were washed with PBS ± 1% BSA after each step. Finally, cells were incubated for 40 min at room temperature with FITC or Cy3-coupled anti-goat or anti-mouse IgG secondary antibody (1 : 500, Jackson) and observed under a fluorescence microscope (BX41TB, Olympus, Tokyo, Japan). For FACS analysis, cells were trypsinized and spun down by centrifugation for 5 min at 1000 rpm. The cell pellet was resuspended in 100 μL PBS and incubated on ice for 30 min with FITC or PE-conjugated monoclonal antibodies against CD29, CD44, CD105, CD90, CD34, and CD45 (Becton-Dickinson, San Jose, CA). After two washes with cold PBS, the labeled cells were analyzed with a FACStar flow cytometer (Becton-Dickinson, San Jose, CA, USA). 2.2. Preparation and Evaluation of AAM hAM was decellularized using 0.03% (w/v) sodium dodecyl sulfate (SDS), with hypotonic tris buffer and protease inhibitors and nuclease treatment. Both intact hAM and AAM were fixed in 4% paraformaldehyde before HE staining to observe whether the epidermal layer of AAM had been removed completely. 2.2.1. Seeding of ADMSCs on AAM P4 adipose stem cells were seeded on the surface of AAM at a density of 2 × 105 cells/cm2, with the KC-SFM medium replaced on alternative days. After 7-day loading, cell adhesion and growth were observed under an optical microscope by HE staining. 2.3. Preparation of Full-Thickness Skin Defects in Nude Mice and Repair of the Skin Defects in Different Groups After intraperitoneal anesthesia, full-thickness skin defects were made by cutting a 0.8 cm × 0.8 cm wound on the back deep to the fascia in 6-week-old healthy BALB/C-nu mice of either sex weighing 20~30 g. According to the different methods used for wound treatment, the 30 BALB/C-nu mice were equally randomized to three groups: hAM group, where the wounds were treated by native hAM; AAM group, where the wounds were treated with AAM alone; and ADMSCs seeded on AAM group, where the wounds were treated by transplanting composite ADMSCs and AAM to the wounds. Before treatment, the wounds were gently rinsed with gentamycin normal saline (NS). AAM was fixed on the surrounding skin with 1.0-gauge suture and dressed with NS gauze. 2.4. Observation and Measurement Wound contraction: the wound area was measured 7, 14, and 28 days after transplantation in all groups, and the wound healing rate was calculated using the following equation: would healing rate = (original wound area − current wound area)/original wound area × 100%. 2.5. Immunohistofluorescence Analysis and HE Staining Observation Immunohistofluorescence staining was used to examine the expression of cytokeratin 19 (CK19) and human derived mitochondria in the new skin tissue. At day 28 after treatment, OCT-embedded specimens were made into 10 μm thick continuous frozen sections. After being fixed with cold acetone, the slides were preincubated in the working normal goat serum concentration for 10 min and then incubated with a mouse polyclonal anti-CK19-specific antibody (1 : 50, Dako) or human derived mitochondria (1 : 200, Neomarks) at 4°C for 16 h. Then, the slides were incubated for 40 min at room temperature with FITC or Cy3-coupled IgG secondary antibody (1 : 500, Jackson), counterstained with DAPI (Sigma-Aldrich), and observed under a fluorescence microscope (BX41TB, Olympus, Japan). Additionally, part of the skin from the same site was fixed in 4% paraformaldehyde, HE stained, and observed for the skin structure and growth of the skin appendages under the optical microscope. 2.6. Statistical Treatment Statistical analyses were performed using SPSS15.0. Measurement data were expressed as X ± S. Intergroup comparison was performed using t-test. P < 0.05 was considered statistically significant. 3. Results 3.1. Evaluation of Cultured ADMSCs In Vitro Primarily cultured ADMSCs were spindle-shaped, with a relatively large nucleus-cytoplasm ratio. Confluent cells began forming clones at day 4 after seeding. The size of the clonal cells was relatively small, with a large nucleus-cytoplasm ratio, where split kernels were visible. About 80% of the ADMSCs became confluent 12 days after seeding (Figure 1(a) P1). P4 ADMSCs were morphologically uniform (Figure 1(a) P4). Immunofluorescence staining of the fourth-passage adipose stem cells showed strong positive expression for Oct-4 and SH-2, with a positive rate of more than 90% (Figure 1(b)). Flow cytometry showed that P4 ADMSCs were positive for CD29, CD44, CD105, and CD90 and did not express CD45 or CD34 (markers for hematopoietic stem cells) (Figure 1(c)). 3.2. Evaluation of hAM and ADMSCs Seeded on AAM Optical microscopic observation by HE staining: The epidermal layer of the cryopreserved intact hAM was complete and continuous with the nucleus clearly seen (Figure 2(a)) and disappeared after trypsin treatment but the basement membrane kept intact (Figure 2(b)). At day 3 of loading ADMSCs onto AAM, HE staining of optical microscopy showed that cells grew well with large cell bodies protuberating like spindles. At day 7, cells fused into patches covering the surface of AAM cells and turned from a single layer to multiple layers (Figure 2(c)). 3.3. Wound Healing in Different Groups The healed skin was lower than the normal skin in all groups. At day 7 after seeding, AAM was attached onto the wound surface securely, and the wound surface began shrinking; at day 14 after seeding, AAM fell off from the wound surface and the sound surface further shrank and healed; and at day 28, the wound surface was stabilized. Comparison of the wound healing rate at days 7, 14, and 28 after transplantation showed that the wound healing rate in ADMSC-AAM seeding group was significantly higher than that in hAM and AAM groups, and the wound healing rate in AAM group was significantly higher than that in hAM group (Table 1). 3.4. Characteristics of the New Skin Tissue Structure HE staining showed that, at day 28, the hair follicle-like structure appeared in ADMSC-AAM seeding group; the number of epidermal layers in ADMSC-AAM seeding group was greater than that in hAM and AAM groups, and all wounds in ADMSC-AAM seeding group healed completely (Figure 3). The typical hair follicle structure was observed at day 28 day after transplantation, when immunohistochemistry showed that some cells of new hair follicles in the healed epidermis were positive for both keratin 19 and human derived mitochondria. Keratin 19 is a marker when mesenchymal cells are transcribed to epithelial cells, and anti-human-derived mitochondria are often used as a tracing agent to demonstrate cells derived from human ADSCs. Image pro plus showed that the number of hair follicles in ADMSC-AAM seeding group was significantly higher than that in hAM and AAM groups. The process of hair follicle development showed that composite AAM and ADMSCs not only promoted the healing of the full-thickness defects in the mice but also facilitated generation of the appendages of the affected skin, thus promoting restoration of the skin function (Figure 4). HE staining showed that regeneration of skin appendages, especially regeneration of the hair follicle, is very similar to hair follicle formation in the process of normal skin development. 4. Discussion The skin is known as the largest organ of the human integumentary system and is composed of complex tissue structures including hair follicles, sweat glands, sebaceous glands, and other appendages [6]. It plays important roles in barrier protection, thermoregulation, hair generation, and other physiological functions. The skin covers the body externally, making it vulnerable to injuries from various external factors such as burn, trauma, and chronic ulceration. There are about one million burn patients in China who need skin transplantation annually. However, as dermagrafts currently used in clinical practice contain the epidermis alone or epidermis with dermis but without hair follicles, sweat glands, melanin cells, capillaries, fat layers, and other appendages, they lack many physiological functions of the normal skin, thus seriously affecting the patient's quality of life. Since the successful isolation of ADMSCs in vitro, they have become the most extensively used adult stem cells in tissue engineering and regenerative medicine [12–14]. ADMSCs express the mesenchymal and pluripotent markers [15, 16] as we examined, and compared with stem cells derived from other sources, ADMSCs have unique advantages of abundant sources, easy obtainment, high proliferative activity, and multidirectional differentiation potentiality and therefore have become common seed cells in tissue engineering [17, 18]. The use of ADMSCs with an AAM coculture system has been reposted as a stem cell therapy and scaffold transplantation for the treatment and functional repair of skin [16, 19]. Sánchez-Sánchez et al. [19] reported that the radiosterilized hAM and pig skin may prove to be suitable scaffolds for delivery of hADMSCs to promote tissue regeneration in skin injuries. Our present study also demonstrated the feasibility of seeding ADMSC onto AAM for the clinical treatment of skin defects. Both ADMSCs and AAM are easily accessible. Unlike fresh hAM, AAM can be preserved for a relatively long time as a safe and effective scaffold material [20]. Composite culture and transplantation of AAM are relatively easy without special technical requirements. The proliferation and differentiation abilities of hAM epithelial cells are not as good as those of ADMSCs. In addition, ADMSCs can differentiate to skin-related tissues [21]. It was found in the present study that the adipose stem cells were able to closely attach to the surface of AAM and grew well 24 h after seeding. At day 7 after seeding, cells fused to patches and covered the surface of the AAM. But as AAM is easily dried, contraction was observed in 50% of the wound area at day 7 after seeding and also in more than one-third of the wound area in ADMSC-AAM seeding group at day 28 after treatment. If this problem could be solved successfully, AAM's clinical value in skin tissue engineering would be further upgraded. More importantly, we noticed a phenomenon of hair follicle development during the process of using composite ADMSCs and AAM to repair the full-thickness skin defects. Generally, only simple wound coverage is implemented in the process of repairing full-thickness skin defects or deep second-degree burns, without realizing the regeneration of skin appendages and functional recovery. In the present study, we rehearsed the process of skin appendage development and achieved functional recovery of the wounded skin. This result may prove to be clinically significant in the treatment of full-thickness skin defects using skin tissue engineering. Acknowledgments The present study was financed by the National Natural Science Foundation of China (81301649, 31101008) and the National Natural Science Foundation of Shanghai (14ZR1449400). Competing Interests The authors declare that they have no competing interests. Authors' Contributions Wu Minjuan, Xiong Jun, and Shao Shiyun contributed equally to this work. Ji Kaihong is the corresponding author and Wang Yue is the co-corresponding author. Figure 1 Morphology and molecular marker detection of isolated ADMSCs. (a) Phase-contrast micrographs of ADMSCs at P1 and P4. Most ADMSCs were spindle-shaped with scant cytoplasm and with granules around the nuclei (original magnification 100x). (b) Immunofluorescence detection: P4 ADMSCs were positive for Oct-4 and SH-2 (original magnification 100x). (c) FACS analysis: P4 ADMSCs were positive for CD29, CD44, CD105, and CD90 and negative for CD45 and CD34. Figure 2 Morphology of hAM (a), AAM (b), and AAM loaded with ADMSCs (c). The right is the magnification of the left. HE staining showed that the epidermal layer of the intact AM was complete and continuous with the nucleus clearly seen and disappeared in AAM. After loading ADMSCs onto AAM, cells covered the surface of AAM and fused into patches. Figure 3 HE staining showed that, at day 28, a lot of typical hair follicle-like structures appeared in ADMSC-AAM seeding group (c) compared with hAM group (a) and AAM group (b). Immunohistochemistry showed that the healed epidermis was positive for cytokeratin 19. Some cells of new hair follicles were positive for both keratin 19 (green) and human derived mitochondria (red). S: sebaceous; HF: hair follicle. Figure 4 Comparison of hair follicles in different groups, ∗∗ P < 0.05. Image pro plus showed that the number of hair follicles in ADMSC-AAM seeding group was significantly higher than that in hAM and AAM groups. Table 1 Comparison of the wound healing rate between different groups. Groups Day 7 Day 14 Day 28 hAM 33.39 ± 4.7 43.01 ± 2.8 85.42 ± 1.9 AAM 52.68 ± 3.5∗∗ 68.43 ± 2.4∗∗ 79.48 ± 1.6∗∗ ADMSCs seeded on AAM 64.38 ± 3.9∗∗ 79.75 ± 3.8∗∗ 92.02 ± 4.2∗∗   ∗∗ P < 0.05. ==== Refs 1 Markeson D. Pleat J. M. Sharpe J. R. Harris A. L. Seifalian A. M. Watt S. M. Scarring, stem cells, scaffolds and skin repair Journal of Tissue Engineering and Regenerative Medicine 2015 9 6 649 668 10.1002/term.1841 2-s2.0-84931005735 24668923 2 Mueller C. K. Lee S.-Y. Schultze-Mosgau S. 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==== Front J TransplantJ TransplantJTRANSJournal of Transplantation2090-00072090-0015Hindawi Publishing Corporation 10.1155/2016/3454601Research ArticleFor and against Organ Donation and Transplantation: Intricate Facilitators and Barriers in Organ Donation Perceived by German Nurses and Doctors http://orcid.org/0000-0002-6311-9784Hvidt Niels Christian 1 * Mayr Beate 2 3 Paal Piret 4 Frick Eckhard 2 3 Forsberg Anna 5 6 http://orcid.org/0000-0002-5025-7950Büssing Arndt 7 1Research Unit of General Practice, Institute of Public Health, Faculty of Health Sciences, University of Southern Denmark, J. B. Winsløwsvej 9A, 5000 Odense C, Denmark2Forschungsstelle Spiritual Care, Klinik und Poliklinik für Psychosomatische Medizin und Psychotherapie, Munich School of Philosophy, Kaulbachstraße 31, 80539 Munich, Germany3Research Centre Spiritual Care, Department of Psychosomatic Medicine and Psychotherapy, The University Hospital Klinikum rechts der Isar, Langerstraße 3, 81675 Munich, Germany4Hospice Care DaSein, Karlstraße 55, 80333 Munich, Germany5Department of Transplantation and Cardiology, Skåne University Hospital, 221 85 Lund, Sweden6Department of Health Sciences, Lund University, P.O. Box 157, 221 00 Lund, Sweden7Institute of Integrative Medicine, Faculty of Medicine, Witten/Herdecke University, Herdecke, Gerhard-Kienle-Weg 4, 58313 Herdecke, Germany*Niels Christian Hvidt: nchvidt@health.sdu.dkAcademic Editor: Gian Luigi Adani 2016 15 8 2016 2016 345460120 5 2016 25 7 2016 Copyright © 2016 Niels Christian Hvidt et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Significant facilitators and barriers to organ donation and transplantation remain in the general public and even in health professionals. Negative attitudes of HPs have been identified as the most significant barrier to actual ODT. The purpose of this paper was hence to investigate to what extent HPs (physicians and nurses) experience such facilitators and barriers in ODT and to what extent they are intercorrelated. We thus combined single causes to circumscribed factors of respective barriers and facilitators and analyzed them for differences regarding profession, gender, spiritual/religious self-categorization, and self-estimated knowledge of ODT and their mutual interaction. Methods. By the use of questionnaires we investigated intricate facilitators and barriers to organ donation experienced by HPs (n = 175; 73% nurses, 27% physicians) in around ten wards at the University Hospital of Munich. Results. Our study confirms a general high agreement with the importance of ODT. Nevertheless, we identified both facilitators and barriers in the following fields: (1) knowledge of ODT and willingness to donate own organs, (2) ethical delicacies in ODT, (3) stressors to handle ODT in the hospital, and (4) individual beliefs and self-estimated religion/spirituality. Conclusion. Attention to the intricacy of stressors and barriers in HPs continues to be a high priority focus for the availability of donor organs. ==== Body 1. Introduction Alone in the USA over an estimated 120.000 people are waiting for a donor organ with 21 patients dying per day due to the deficit [1]. In Germany around 1.000 persons die per year while awaiting organ transplantation [2], with similar numbers for other developed nations, although most of them implement significant national campaigns and other incentives toward an increase in available organs [3–7]. Despite growing numbers of organ donations and transplantations (ODT), ever more people need donor organs due to the increase in metabolic diseases, for example, diabetes and obesity. The progress of modern medicine enables a growing variety of possible transplantations and hence there is an ever increasing gap between the availability of and the need for donor organs [8]. Despite the flagrant need for donor organs, resistance remains among the general public and even among health professionals to ODT. This should not come as a surprise. ODT constitutes a complex ethical and value laden field of interdisciplinary interventions. It is a surgical and medical field that requires the highest scientific standards, but likewise one, where ethics, values, and personal beliefs play an immense role. Not surprisingly, then, extensive research has been done on attitudes to ODT in the general public, in medical students, and in health professionals (HPs) [9–11] often with the explicit aim of investigating whether they “have the knowledge needed to maximize organ donation rates” [12] or “to inform strategies to improve organ donation rates” [13]. Studying the attitudes of HPs has been found to be of particular relevance. Publications addressing psychosocial and ethical issues have shown that despite the obvious need for organ donation, the most important factor hindering ODT is, despite the will of the deceased, the attitude of intensive care unit members to organ donation [9, 14]. On the basis of existing research literature and an expert focus group study (see the following), it became clear that the barriers and facilitators in ODT medicine are multiple and intricate as they relate to ODT knowledge, ethics, stressors, individual beliefs, and religiosity. Hence, the purpose of this study was to investigate to what extent HPs (physicians and nurses) experience such facilitators and barriers in ODT and to what extent they are intercorrelated. We thus intended to combine single causes to circumscribed factors of respective barriers and facilitators to analyze them for differences regarding profession, gender, spiritual/religious self-categorization, and self-estimated knowledge of ODT and to gauge their interaction. 2. Participants and Methods 2.1. Development of Survey Items We developed a survey in close collaboration with leading staff of the Bavarian branch of the German Organ Transplantation Foundation (DSO) to map personal values and beliefs with attitudes toward ODT. After an extensive literature review, we conducted a multiprofessional focus group discussion with 15 experts in the field of ODT (Mayr et al., in preparation) to identify various facilitators and barriers in ODT. The focus group discussion was recorded and transcribed verbatim and analyzed using Thematic Content Analysis [15], to identify main categories and subthemes, which should be addressed in the intended survey. This review and qualitative development process led to the identification of five relevant themes for the experience of barriers and facilitators in ODT: (1) knowledge of ODT, (2) ethical appraisal of ODT, (3) ethical arguments favoring ODT in dialogue with relatives, (4) stressors, and (5) belief barriers. 2.2. Validation of the Survey Items The above-stated categories were the primary source for the items used in the current survey. The postulated questions were carefully discussed in the preliminary expert rounds and then tested among medical students and professionals. The optimized version was tested among further healthcare professionals by using the think-aloud protocols [16]. The interviews lasted from 30 minutes to an hour and helped us to refine some further misunderstandings and remove unclear statements from the final draft. The saturation was achieved after conducting 9 interviews. The final version of the survey was conducted in summer 2014. Items were scored on a 4-point scale ranging from strong agreement (1) to strong disagreement (4) or similar phrasing, ranging from “applies exactly” (1) to “does not apply at all” (4). Thus, the higher the scores, the stronger the disagreement. During the next step of the validation process, we exploratively tested the factorial structure of the rather heterogeneous item topics and finally the internal reliability (Cronbach's coefficient α) of putatively sound factors in a larger sample of 175 HPs. When such factors were identified (principal component analysis using varimax rotation with Kaiser's normalization), we tested different theoretically plausible structures and subsequently eliminated those items which loaded weekly on the respective factor (<.05), those items which would load strongly on two concurrent factors, or those with a weak item to scale correlation. It was not the intention to design an instrument but to test differences in the attitudes of the health care professionals. In fact, none of the scales were designed as a specific construct (apart from the general topic), and thus some of the identified factors are less balanced with respect to item number. Apart from face validity, for this study we had no external measures to analyze construct validity. 2.3. Statistics Data were entered by scanning the completed paper surveys in the scanning software ZENSUS developed by Blubbsoft. Descriptive statistics, internal consistency (Cronbach's α), and factor analyses as well as analyses of variance and first-order correlations were computed with SPSS 22.0. Due to the exploratory character of the study, the level of significance was set at  .05. The study obtained ethics approval (#383-12/2014) was gotten from the Ethics Committee of Ludwig Maximilian University of Munich. 3. Results A total of 293 paper questionnaires were distributed in around ten wards at University Hospital in Munich to both physicians and nurses working in various ways and to different degrees with ODT in medical and surgical departments. The survey was introduced to the team members of every participating ward by members of the research team. The response rate was 64% (n = 175). Responding HPs were nurses (73%) and physicians (27%); 71% were female. Eleven questionnaires were discarded, because respondents had chosen not to fill in demographic data. Sociodemographic and employment data are presented in Table 1. In total, 45% were Catholics, 21% Protestants, 4% had other affiliations, and 30% were not affiliated. With respect to their religious and/or spiritual self-categorization, 28% regard themselves as both religious and spiritual (R+S+), 12% religious but not spiritual (R+S−), 7% spiritual but not religious (R−S+), and 53% neither religious nor spiritual (R−S−). This quadropartition has been found to be a viable way of identifying different types of R/S [17]. With respect to this self-categorization, there were no significant differences between women and men (data not shown). Within the sample, 41% believed in life after death and 34% did not, whereas 25% were undecided. In trend, more women (47%) than men (27%) were convinced (p = .07), while there were no significant differences with respect to profession (p = .78). Interestingly, a significantly larger percentage of Catholics believed in life after death (54%) than Protestants in the sample among whom only 38% believed in life after death (figures significant). 3.1. Knowledge Barriers and Willingness to Donate Own Organs Within the sample, 92% stated to be adequately informed about the legal regulatory aspects of ODT and 96% about brain death signs. The few who did not consider themselves satisfactorily informed about the regulatory aspects were mainly found in the group of nurses (9.5% of nurses and 2.2% of physicians; p = .092). Further, 67% of HPs agreed with the regulatory aspects of ODT, that is, 54% of physicians and 71% of nurses (p = .030), others obviously not. When asked about their own consent to become an organ donor after death, a vast majority of the HPs agreed to donate their organs (77%) and tissue (such as the cornea or heart valves (71%)). There were no significant differences for gender, profession, and spiritual/religious self-categorization (data not shown). We next intended to combine specific topics (either facilitators or barriers) addressed with different single items to specific factors and tested first their internal reliability before we would address differences between HPs with respect to these topics. 3.2. Reliability of Factors Related to Specific ODT Topics 3.2.1. Ethical Appraisals of ODT To address ethical barriers HPs perceived in ODT, the respective items were condensed to specific factors. However, the internal reliability of these six items was rather weak (Table 2). Exploratory factor analysis pointed to two subconstructs, one with four items (alpha = .67) and one with two items (alpha = .47). Only the first scale (ethical barriers to ODT) might be used for further analyses, while the quality of the second is too weak. Within the first i factor, the lowest scores were found for “justice in the distribution of organs” (indicating agreement) and the highest for “handling of the personal convictions of colleagues” (indicating disagreement). 3.2.2. Ethical Facilitators to ODT in the Dialogue with Relatives In the developmental phase we found that beliefs and values favoring ODT were most clearly formulated as arguments for ODT when HPs conversed respectfully with people who reflected whether they should release the body of their brain dead relative for ODT. Hence, HPs were asked whether they believed it was acceptable to propose arguments to relatives in favor of ODT and what would be viable arguments proposed in such conversations. The facilitating arguments were tested for their reliability. As shown in Table 3, the respective seven items had a satisfactory internal reliability (alpha = .77), with two subconstructs which would explain 61% of variance. The first construct, personal ethical facilitators (alpha = .73), regarded ethical arguments that relatives could see for themselves, whereas the second construct, concrete altruistic effects (alpha = .72), regarded advantages others could have of the act of giving the relatives' organs. The three items addressing concrete altruistic effects scored lower than the personal ethical facilitators (with the highest disagreement score for the item stating that “your consent is an ethical duty”). 3.2.3. Stress Barriers in ODT Next we asked for the stress barriers of HPs which were addressed with six items (Table 4). Exploratory factor analysis pointed to two subconstructs, one with four items and satisfactory internal reliability (medical reasons; alpha = .74) and one with two items and poor internal reliability (team reasons; alpha = .33). Only the first scale might be used for further analyses. Here, the strongest disagreement was found for “care for relatives” as putative stressful barrier, while “acceptance of brain death as death of a human being” was considered less of a stressful barrier. 3.2.4. Belief Barriers in ODT We asked respondents which representations in dying and death could constitute a barrier for ODT from their personal perspective and from the assumed perspective of relatives. Four questions related to the immanent, earthly life, whereas four items related to the transcendent and to the afterlife. We differentiated perceived own ODT barriers (Table 5(a)) and those assumed for the relatives (Table 5(b)). As shown in Table 5(a), the eight items addressing personal perception of ODT barriers had a good internal reliability (alpha = .88) and two subconstructs. Because the item addressing the “wish to be buried as a whole” would load on both factors, it was eliminated from the item pool. Factor one would thus address transcendent barriers: protection of the soul (alpha = .87) and factor two immanent barriers: affection of the physical body (alpha = .79). The mean scores of both subscales are similar. The highest scores (indicating disagreement) were found for the “wish that the body should resurrect integrally.” The same structure was found when HPs considered ODT barriers of relatives (Table 5(b)). Here, the item addressing the “wish to be buried whole” would load best on the factor immanent barriers, but considerably also on the factor transcendent barriers, and was thus eliminated from that item pool, too. 3.3. Correlations between Facilitators and Barriers With these factors we analyzed whether or not the addressed facilitators and barriers were associated in any way. As shown in Table 6, both factors addressing ODT arguments to be communicated to relatives were either not at all or only marginally associated with ODT barriers. Thus, these aspects have to be seen as independent dimensions. Neither the stress barriers nor the ethical issues showed any significant association with the other factors. 3.4. Facilitators and Barriers within the Sample Generally, we can show that, with respect to ODT arguments communicated to relatives, concrete altruistic effects receive higher agreement (lower scores) than personal ethical facilitators (higher scores) (Table 7). Although there were no significant differences with respect to gender, nurses scored higher disagreement for personal ethical facilitators (F = 4.6, p = .034; Cohen's d = 0.40) and concrete altruistic effects (F = 5.9, p = .016; Cohen's d = 0.44). The effects are rather small. However, the SpR self-categorization has a significant effect only on the personal ethical facilitators, which scored higher on disagreement in R−S− HPs (F = 8.5, p = .004; Cohen's d = 0.49). The high scores on the own perception of transcendent and also immanent barriers scales indicate HPs' general disagreement, without any significant difference for gender, profession, or SpR self-categorization. Particularly the assumed immanent ODT barriers of relatives scored lower than transcendent barriers (with similar disagreement level for one's own perception of ODT barriers), suggesting that HPs would particularly disagree with the protection of the soul (transcendent barrier) as an ODT barrier when compared to the affection of the physical body (immanent barrier). Medical reasons as stress barriers are of lower relevance in the sample, particularly for women (F = 11.4, p < .0001; Cohen's d = 0.59) and nurses (F = 16.2, p < .0001; Cohen's d = 0.72) which had the highest scores indicating disagreement. The effect sizes are moderate. HP's SpR attitude had no significant influence. There were no significant differences in the perception of ethical barriers to ODT for gender, profession, or SpR self-categorization. 3.5. Facilitators and Barriers in HPs Who Would Agree to Own ODT When the HPs were categorized for their willingness to serve as an organ donor, we saw significant differences: those who do not wish to donate their own organs showed stronger disagreement to communicate concrete altruistic effects (F = 7.4, p = .007; Cohen's d = 0.55) or personal ethical facilitators (F = 4.6, p = .034; Cohen's d = 0.44) as ODT arguments when compared to those would agree. The effect size is moderate and small, respectively. With respect to the belief barriers, there were no significant differences. Few who do not feel adequately informed about the regulatory aspects of ODT had stronger disagreement for personal ethical facilitators (F = 7.3; p = .008; Cohen's d = 0.78) and concrete altruistic effects (F = 6.6, p = .011; Cohen's d = 0.76) to be communicated to relatives. The effect sizes are moderate. With respect to the belief barriers, there were no significant differences, too. 4. Discussion 4.1. For and against ODT The vast majority of HPs were in favor of ODT. In fact, most consented to donate their own organs (77%), they agreed that the lack of organs is an ethical problem (88%), and they found it acceptable to propose arguments to relatives in favor of ODT (78%). These findings confirm existing research indicating that HPs working in ODT are generally highly motivated [11, 18]. However, the HPs in our study also identified various types of interwoven barriers and facilitators in ODT, confirming the fact that ODT is a difficult medical and ethical field in which to navigate. Interestingly, we saw a tendency that HPs tended to disagree with the barriers that they themselves considered significant factors barring ODT. When communicating with relatives, the majority of HPs found it rather acceptable to propose concrete altruistic arguments that have formerly been found associated with high ODT advocacy [11, 19]. 4.1.1. Knowledge Barriers and Facilitators The HPs in our study reported a high degree of being well informed of legislative aspects of ODT (92%) and the signs of brain death (96%). This does, however, not entail that all HPs agree with the legislative aspects of ODT. In fact, only 67% of HPs in our sample agreed (54% physicians and 71% nurses), suggesting a potential conflict encircling the existing legal practices. The few who did not feel adequately informed about regulatory aspects of ODT tended in general to be less willing to propose arguments in favor of ODT in the dialogue with relatives, suggesting low commitment to ODT advocacy. Our findings thus confirm former research indicating lack of knowledge regarding ODT as one of the primary potential barriers to ODT in the public [20] and in HPs as well [21–25]. It has been found to be of particular importance as HPs approach donor families [26]. The range of barriers in physicians and nurses related to specific knowledge of ODT has been described as perception of organ and tissue transplantation as an experimental procedure, knowledge about criteria for potential donors, request policies and procedures, and understanding and explaining brain death to relatives [25]. As mentioned, most HPs in our sample agreed to donate their own organs, something formerly found to be generally correlated with a high commitment to ODT advocacy [19, 25]. This is confirmed as well in our sample as those who would donate organs tend to be more in favor of providing both above-mentioned types of arguments for ODT in dialogue with relatives than those who do not wish to donate own organs. 4.1.2. Ethical Barriers and Facilitators In our study, HPs saw significant ethical barriers to ODT particularly in the “justice” of the “distribution of organs.” This confirms existing research in the field suggesting that HPs consider legal, ethical, and value laden questions in ODT to constitute significant barriers in HPs to ODT [27, 28]. With regard to the ethical facilitators in ODT, the majority of HPs in our study agreed that it was acceptable to propose ethical arguments in the dialogue with relatives of potential donors that as mentioned has been found associated with high ODT advocacy. We found two ethical constructs that HPs considered important in the dialogue with relatives: personal ethical facilitators regarded arguments that relatives could relate as relevant to themselves, whereas concrete altruistic effects entailed advantages other people could gain from the relatives' decision to agree with the donation of their loved one's organs. The HPs generally favored concrete altruistic effects over personal ethical facilitators. Ethical arguments for ODT, such as ODT being an act of charity or responsibility of fellow human beings as found in our study, have been proposed in favor of monetary or legal incentives [29–32]. Thus, a study by Jasper et al. found that the vast majority of HPs preferred the policy of altruistic organ donation from a moral perspective over different types of possible incentives to donor families, although they also agreed that such altruistic policy was not sufficiently effective [33]. 4.1.3. Stress Barriers The HPs in our sample identified differentiated stress barriers to ODT in their daily clinical work. HPs considered “care for the relatives” a lesser stressful barrier than “acceptance of brain death as death of a human being.” The strongest variance was found for nurses (and thus women, too) who disagreed that suggested medical reasons constituted stress barriers. One might assume that they are more involved in the care of donors and their relatives than with making clinical ODT decisions, and thus they do not see medical reasons as strong arguments against ODT. This should come as no surprise on the basis of international research. Important barriers and facilitators are found in the perceived stress and coping resources in handling ODT. Such stress and resources have not only been identified by relatives of potential donors and by those waiting for an organ [34, 35] but are experienced as well by HPs in ODT. For instance, Hibbert identified multiple stressors experienced by nurses, such as the threat of the dying patient and the inconsistent commitment of physicians to organ donation [36], but she also identified their work as meaningful to them and found coping resources for handling such stress in gaining control over emotions, distancing oneself, and taking timeout [37]. 4.1.4. Belief Barriers and Facilitators In our study, we identified two constructs of barriers: first, “transcendent barriers: protection of the soul,” that is, barriers relating to transcendent, spiritual/religious notions working against ODT advocacy; second, “immanent barriers: affection of the physical body.” In general, the HPs tended to see belief barriers to a lesser degree for themselves than for relatives, particularly immanent beliefs (such as “that ODT violates the body”) as an assumed barrier for relatives. HPs tended to rather disagree that transcendent beliefs (such as “the wish that the body should resurrect integrally”) constituted barriers to ODT. As mentioned, HPs tended to feel discomfort proposing personal ethical facilitator arguments; interestingly R−S− HPs were more reluctant to propose such arguments to relatives than their R+S+ counterparts, which might suggest a correlation between R+S+ and ODT advocacy. There was no significant difference between R−S− and R+S+ with respect to concrete altruistic effects as arguments just as personal R+S+ had no significant effect on their own belief, stress, or ethical barriers to ODT. A large bulk of research has centered on how religion/spirituality can entail both barriers and facilitators to ODT [13, 38–40]. Today, there seems to be a growing tendency for religious/spiritual arguments to favor ODT rather than the opposite. Thus, what surprised Jasper et al. in their study was that religion was offered far more often as a rationale for wanting to help sick people through organ donation than it was for not wanting to donate organs [33]. Likewise, more and more religious leaders recommend giving one's organs as an act of charity [41, 42]. Along the same vein, Abidin et al. even propose “increasing (ODT) awareness of the public through religion” [14]. Moreover, it has been shown that religious beliefs impact the concrete practice of various fields of medicine such as general practice [43], psychiatry [44, 45], gynaecology [46], and end-of-life-care [47]. This research documents how such beliefs may have significant impact on the practice of medicine, including ODT. In our study, we find hints that spiritual/religious attitudes were partly and specifically associated with ODT advocacy. 4.2. Multiplicity and Intricacy of Facilitators and Barriers International research indicates strong correlation between the various facilitators and barriers in ODT. Thus, Irving and coworkers point to the intricacy of multiple barriers and facilitators and write that “intractable factors, such as religion and culture, are often tied in with more complex issues such as a distrust of the medical system, misunderstandings about religious stances and ignorance about the donation process” [13]. Our study lends limited evidence to this insight, as the interdependency of ODT factors (Table 6) and different ODT variables (Table 7) only show some interactions. Such multiplicity and intricacy have been found important in other studies and are evident in the professional setting, even from a structural, systemic perspective, as ODT entails a complex multiprofessional, ethical interaction: (1) it generally depends on the cooperation of various hospitals, departments, professions, and organ allocation institutions; (2) organ donation depends on HP interaction with patients, potential organ donors, and their relatives. This multidimensional interaction holds many inherent barriers against successful ODT. In such a complex field, there are no singular causes. In line with system, process, and force field theory, causes are interdependent and act upon each other [48]. The same goes for psychological behaviour [49] and social interaction [48, 50]. Thus, in a “force field” as complex as ODT, it is important to study and clinically to consider not merely the impact of single but rather numerous intricate causes for the lack of organs today in order to select adequate strategies to enhance the availability of organs for ODT. 5. Limitations The study population is small and HPs were recruited in a few departments of the University Hospital in Munich only and with a response rate of 64%. Although we do not assume the data as representative of ODT HPs in general, we at least can add further important aspects to the general discussion. For future studies larger sample sizes and inclusion of other regions of Germany (with their specific cultural settings) should be included. Due to the cross-sectional design of this exploratory study, causal interpretations are not possible. 6. Conclusion This study confirms a general high agreement with the importance of ODT among ODT HPs. Nevertheless, we identified both facilitators and barriers in the following fields that impact each other: (1) knowledge of ODT and willingness to donate own organs, (2) ethical delicacies in ODT, (3) stressors to handle ODT in the hospital, and (4) individual beliefs and self-estimated religion/spirituality. Thus we found that ODT constitutes a medically and ethically complex and intricate field of medical intervention and that continuous optimization of HPs' knowledge of ODT is of relevance for their own perception of barriers and facilitators through education and continued learning. Continued learning concerning specific knowledge of brain death has decreased the experienced ethical and practical barriers that the notion of brain death constitutes ODT [51]. Trials on the efficiency of increasing knowledge on the facts and needs of ODT in the general public and in health professionals through public campaigns and HPs continued learning raises positive attitudes to ODT, including the will to become a donor [52]. Recognition and articulation of personal beliefs and convictions in both relatives and HPs are likewise of high relevance for ODT, although often considered a personal matter and not one of medical discourse. Insights and experiences could be brought to ODT from the palliative field, where the actual handling of such intricate ethical and spiritual values and beliefs is very much part of medical attention, even in a rather secularized European setting. Our study suggests that actively addressing the perceived belief barriers in ODT through interdisciplinary teamwork including both HPs but also psychologists and chaplains may continue to enhance a favourable ODT culture. Finally, recognizing the intricacy of barriers and facilitators in ODT may contribute to the facilitation of ODT in avoiding blind spots in the continued efforts to help more people survive due to better availability of organs for transplantation. Acknowledgments The authors wish to thank for fruitful collaboration Dr. Thomas Breidenbach, Nicole Erbe, and Dorothee Seidel of German Organ Transplantation Foundation (DSO) as well as the experts of their focus group. Abbreviations ODT:Organ donation and transplantation HP:Health professionals. Competing Interests The authors declare that they have no competing interests. Authors' Contributions Niels Christian Hvidt contributed to design of questionnaire on basis of expert focus group and interpretation of data (primary author). Beate Mayr and Eckhard Frick offered design of questionnaire on basis of expert focus group and major contributions to writing of paper. Piret Paal offered design of questionnaire on basis of expert focus group, interpretation of data, and major contributions to writing of paper. Anna Forsberg offered major contributions to writing of paper. Arndt Büssing offered statistical analysis, interpretation of data, and major contributions to writing of paper. Table 1 Characterization of enrolled persons (n = 175). Age (years) 33.9 ± 11.1 Gender (%)   Women 71 Men 29 Family status (%)   With partner 55 Single 42 Divorced/widowed 3 Confession (%)   Catholic 45 Protestant 21 Other 4 None 30 SpR self-categorization (%)   R+S+ 28 R+S− 12 R−S+ 7 R−S− 53 Profession (%)   Physicians 27 Nurses 73 Employment (%)   Full time 86 Part time 14 Hospital (%)   With TX unit 92.5 Without Tx unit/other 7.5 Working area (%)   Donors 26 Donees 46 Both 20 Neither nor 8 Perceived health impairment (1–11)   Physical 4.0 ± 2.8 Mental 3.8 ± 2.7 Table 2 Mean values, reliability, and factor analysis of item addressing the perception of ethical issues. Factors and items Mean value (score 1–4)∗ SD Corrected item-total correlation Alpha if item is deleted (α = .683) Loading factor 1 Loading factor 2 Factor 1: ethical barriers to ODT (eigenvalue 2.2; 38% explained variance; alpha = .67)             Handling of the personal convictions of colleagues 2.40 0.83 .395 .648 .781   Respect for the individual problems of patients/relatives 2.16 0.76 .426 .638 .726   Transparency of the system 2.01 0.89 .548 .591 .634 .406 Justice in the distribution of organs 1.87 0.83 .445 .631 .533 .403 Factor 2: external ethical issues in ODT (eigenvalue 1.0; 17% explained variance; alpha = .47∗∗)             Scandals in transplantation medicine 1.71 0.77 .335 .667   .773 Lack of organs 1.70 0.76 .326 .669   .737 Extraction of the main components (eigenvalue > 1); varimax rotation with Kaiser's normalization. Rotation is converged in 3 iterations. Both factors explain 55% of variance. ∗Scores range from 1 (agreement) to 4 (disagreement). ∗∗Scale is not suited to be used. Table 3 Mean values, reliability, and factor analysis of item addressing the agreement to consider facilitating ODT arguments with relatives. Factors and items Mean value (score 1–4)∗ SD Corrected item-total correlation Alpha if item is deleted (α = .774) Loading factor 1 Loading factor 2 Factor 1: personal ethical facilitators (eigenvalue 2.9; 42% explained variance; alpha = .73)             Your consent could be a source of meaning in your own life 2.78 0.94 .603 .723 .841   Your consent would be an act of charity 2.75 0.99 .569 .730 .721   Your consent is an ethical duty 3.50 0.77 .355 .771 .710   The death of the diseased would have a purpose 2.70 1.03 .491 .749 .555 .323 Factor 2: concrete altruistic effects (eigenvalue 1.3; 18% explained variance; alpha = .72)             Your consent can save the life of another person 1.47 0.74 .462 .754   .880 Your consent can do good 1.69 0.85 .509 .744   .833 You might come to a point where you yourself could be in need of a transplantation 1.98 0.99 .498 .746 .333 .610 Extraction of the main components (eigenvalue > 1); varimax rotation with Kaiser's normalization. Rotation is converged in 3 iterations. Both factors explain 61% of variance. ∗Scores range from 1 (agreement) to 4 (disagreement). Table 4 Mean values, reliability, and factor analysis of item addressing stress barriers in the care of potential donors with brain death. Factors and items Mean value (score 1–4)∗ SD Corrected item-total correlation Alpha if item is deleted (α = .696) Loading factor 1 Loading factor 2   Factor 1: stress barriers: medical reasons (eigenvalue 2.4; 41% explained variance; alpha = .74)             Spinal or vegetative reflexes, such as lazarus signs 2.56 0.87 .473 .640 .801   Continuation of intensive care, despite established brain death 2.29 0.92 .559 .607 .709 .326 Acceptance of brain death as death of a human being 2.02 0.83 .607 .595 .686 .427 Care for relatives 3.03 0.81 .376 .671 .654   Factor 2: stress barriers: team reasons (eigenvalue 1.0; 17% explained variance; alpha = .33∗∗)             Overwork/having to take the position of a colleague who does not take part in ODT 2.12 0.82 2.74 .703   .867 Overwork/having to take the position of a colleague who does not take part in ODT 2.42 0.86 .285 .699   .565 Extraction of the main components (eigenvalue > 1); varimax rotation with Kaiser's normalization. Rotation is converged in 3 iterations. Both factors explain 57% of variance. ∗Scores range from 1 (agreement) to 4 (disagreement). ∗∗Scale is not suited to be used. Table 5 (a) Mean values, reliability, and factor analysis of item addressing own perception of ODT barriers. (b) Mean values, reliability, and factor analysis of item addressing assumed ODT barriers of relatives. (a) Factors and items Mean value (score 1–4)∗ SD Corrected item-total correlation Alpha if item is deleted (α = .878) Loading factor 1 Loading factor 2 Factor 1: transcendent barriers: protection of the soul (eigenvalue 4.3; 53% explained variance; alpha = .87)             The wish that the body should resurrect integrally 3.07 0.99 .686 .858 .885   Belief in reincarnation, rebirth, karma, or similar 2.96 0.99 .635 .863 .821   The wish to arrive intact in the afterlife 2.67 1.06 .705 .855 .783 .310 That the soul prevails in the body beyond established death 2.92 1.00 .694 .857 .749   The wish to be buried whole∗∗ 2.21 1.02 .666 .859 .527 .512 Factor 2: immanent barriers: affection of the physical body (eigenvalue 1.2; 15% explained variance; alpha = .79)             That ODT violates the body 2.75 1.02 .556 .871   .873 That the corpse would be blemished 2.30 0.99 .663 .860   .832 That the process of death is not complete with brain death 2.21 1.13 .521 .867   .680 Extraction of the main components (eigenvalue > 1); varimax rotation with Kaiser's normalization. Rotation is converged in 3 iterations. Both factors explain 68% of variance. ∗Scores range from 1 (agreement) to 4 (disagreement). ∗∗Without item “wish to be buried whole.” (b) Factors and items Mean value (score 1–4)∗ SD Corrected item-total correlation Alpha if item is deleted (α = .874) Loading factor 1 Loading factor 2 Factor 1: transcendent barriers: protection of the soul (eigenvalue 4.3; 54% explained variance; alpha = .93)             The wish that the body should resurrect integrally 2.34 0.89 .796 .840 .896   Belief in reincarnation, rebirth, karma, or similar 2.38 0.90 .731 .848 .878   The wish to arrive intact in the afterlife 2.22 0.83 .776 .843 .875   That the soul prevails in the body beyond established death 2.35 0.90 .758 .844 .853   Factor 2: immanent barriers: affection of the physical body (eigenvalue 1.4; 18% explained variance; alpha = .75)             That the corpse would be blemished 1.71 0.67 .585 .865   .845 That the process of death is not complete with brain death 1.49 0.66 .321 .886   .770 That ODT violates the body 2.11 0.91 .535 .871   .752 The wish to be buried whole∗∗ 1.87 0.75 .563 .866 .466 .516 Extraction of the main components (eigenvalue > 1); varimax rotation with Kaiser's normalization. Rotation is converged in 3 iterations. Both factors explain 71% of variance. ∗Scores range from 1 (agreement) to 4 (disagreement). ∗∗Without item “wish to be buried whole.” Table 6 Correlations between the tested factors.   ODT arguments to be communicated to relatives Own perception of ODT barriers Putative ODT barriers of relatives Stress barriers: medical reasons Ethical barriers to ODT   Personal ethical facilitators Concrete altruistic effects Transcendent barriers Immanent barriers Transcendent barriers Immanent barriers ODT arguments to be communicated to relatives                 Personal ethical facilitators 1,000 ,463∗∗ −,029 ,142 ,054 ,223 −,003 .012 Concrete altruistic effects   1,000 −,131 ,059 ,003 ,219 ,015 −,013 Putative own ODT barriers                 Transcendent barriers: protection of the soul     1,000 ,512∗∗ ,376∗∗ ,151 −,091 −,025 Immanent barriers: affection of the physical body       1,000 ,193 ,487∗∗ −,148 −,004 Putative ODT barriers of relatives                 Transcendent barriers: protection of the soul         1,000 ,381∗∗ −,023 ,117 Immanent barriers: affection of the physical body           1,000 −,110 ,166 Stress barriers: medical reasons               .010   ∗∗ p < .001 (Spearman rho). Table 7 Mean values.   ODT arguments to be communicated to relatives Own perception of ODT barriers Assumed ODT barriers of relatives Stress barriers: medical reasons Ethical barriers to ODT   Personal ethical facilitators Concrete altruistic effects Transcendent barriers Immanent barriers Transcendent barriers Immanent barriers All Mean 2.92 1.72 2.89 2.41 2.33 1.76 2.46 2.10 SD 0.69 0.72 0.87 0.89 0.80 0.62 0.67 0.59 Adequately informed about regulatory aspects of ODT                   Yes (92%) Mean 2.87 1.68 2.91 2.41 2.32 1.76 2.43 2.12 SD 0.68 0.67 0.86 0.89 0.79 0.59 0.66 0.56 No (8%) Mean 3.40 2.21 2.63 2.46 2.46 1.74 2.79 1.90 SD 0.65 0.98 0.97 0.98 0.97 0.94 0.73 0.92 F value   7,3 6,6 1,2 0,0 0,3 0,0 3,5 1,6 p value   .008 1 .0112 n.s. n.s. n.s. n.s. .064 n.s. Agreement to own ODT                   Yes (78%) Mean 2.86 1.64 2.91 2.44 2.29 1.73 2.44 2.15 SD 0.71 0.67 0.89 0.93 0.82 0.60 0.65 0.57 No (22%) Mean 3.16 2.03 2.86 2.34 2.43 1.80 2.53 1.94 SD 0.59 0.82 0.80 0.81 0.77 0.68 0.73 0.66 F value   4.6 7.4 0.1 0.3 0.7 0.4 0.5 3.5 p value   .0343 .007 4 n.s. n.s. n.s. n.s. n.s. .062 SpR self-categorization                   R−S− (53%) Mean 3.07 1.79 2.91 2.44 2.21 1.75 2.47 2.15 SD 0.68 0.71 0.96 0.94 0.83 0.67 0.63 0.62 R+S+ (47%) Mean 2.74 1.63 2.90 2.39 2.48 1.78 2.45 2.05 SD 0.68 0.68 0.74 0.84 0.73 0.56 0.70 0.55 F value   8.5 1.9 0.0 0.1 4.4 0.1 0.0 1.3 p value   .004 5 n.s. n.s. n.s. .0376 n.s. n.s. n.s. Gender                   Women (71%) Mean 2.89 1.71 2.87 2.41 2.29 1.72 2.58 2.07 SD 0.69 0.72 0.87 0.89 0.78 0.60 0.63 0.56 Men (29%) Mean 2.98 1.76 2.95 2.43 2.43 1.84 2.20 2.20 SD 0.70 0.72 0.88 0.92 0.86 0.66 0.68 0.66 F value   0.5 0.2 0.3 0.0 1.0 1.1 11.4 1.7 p value   n.s. n.s. n.s. n.s. n.s. n.s. <.0001 7 n.s. Profession                   Physicians (26%) Mean 2.73 1.50 2.93 2.36 2.33 1.64 2.13 2.22 SD 0.66 0.62 0.85 0.91 0.85 0.55 0.65 0.65 Nurses (73%) Mean 3.00 1.81 2.88 2.43 2.33 1.80 2.59 2.06 0.60 SD 0.69 0.73 0.88 0.89 0.78 0.64 0.64 0.57 F value   4.6 5.9 1.6 2.0 0.0 2.0 16.2 2.3 p value   .0348 .0169 n.s. n.s. n.s. n.s. <.0001 10 n.s. ∗Scores range from 1 (agreement) to 4 (disagreement). Thus, the higher the scores are, the more the HPs would disagree. 1 Cohen's d = 0.78; 2 Cohen's d = 0.76; 3Cohen's d = 0.44; 4 Cohen's d = 0.55; 5Cohen's d = 0.49; 6Cohen's d = 0.34; 7 Cohen's d = 0.59; 8Cohen's d = 0.40; 9Cohen's d = 0.44; 10 Cohen's d = 0.72. ==== Refs 1 Organ Procurement and Transplantation Network The Need Is Real: Data 2015 Washington, DC, USA Health Resources and Services Administration http://www.organdonor.gov/about/data.html 2 Nationaler Ethikrat Die Zahl der Organspenden erhöhen-Zu einem drängenden Problem der Transplantationsmedizin in Deutschland, Berlin 2007 3 Salim A. 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PMC005xxxxxx/PMC5002485.txt

==== Front Parkinsons DisParkinsons DisPDParkinson's Disease2090-80832042-0080Hindawi Publishing Corporation 10.1155/2016/6827085Research ArticleThe Cognition of Maximal Reach Distance in Parkinson's Disease http://orcid.org/0000-0001-9436-9017Otsuki Satoru 1 2 * Nagaoka Masanori 2 1Department of Rehabilitation, Juntendo University Nerima Hospital, 3-1-10 Takanodai, Nerima-ku, Tokyo 177-8521, Japan2Department of Rehabilitation Medicine, Juntendo University Graduate School, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan*Satoru Otsuki: sootsuki@juntendo.ac.jpAcademic Editor: Cristine Alves da Costa 2016 15 8 2016 2016 682708513 5 2016 10 7 2016 20 7 2016 Copyright © 2016 S. Otsuki and M. Nagaoka.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.This study aimed to investigate whether the cognition of spatial distance in reaching movements was decreased in patients with Parkinson's disease (PD) and whether this cognition was associated with various symptoms of PD. Estimated and actual maximal reaching distances were measured in three directions in PD patients and healthy elderly volunteers. Differences between estimated and actual measurements were compared within each group. In the PD patients, the associations between “error in cognition” of reaching distance and “clinical findings” were also examined. The results showed that no differences were observed in any values regardless of dominance of hand and severity of symptoms. The differences between the estimated and actual measurements were negatively deviated in the PD patients, indicating that they tended to underestimate reaching distance. “Error in cognition” of reaching distance correlated with the items of posture in the motor section of the Unified Parkinson's Disease Rating Scale. This suggests that, in PD patients, postural deviation and postural instability might affect the cognition of the distance from a target object. ==== Body 1. Introduction In daily life, we frequently perform movements such as extending a hand toward an object. Such movements are expressed as reaching movements. The reaching movement is composed of postural control and transport of the upper limbs and hands. These elements are considered to be automatic processes. Regarding the association between the reaching movement and postural control, the functional reach test developed by Duncan et al. [1] determines the maximal reaching distance and uses postural stability as an indicator. Before the reaching movement is actually performed, there is also an essential cognitive process in which people visually perceive a target object and decide how to reach it. Presumably, people estimate how far they can spatially reach on the basis of information from the visual, auditory, and somatic senses and their past experiences and then start movement on the basis of this estimation [2, 3]. The distances that study participants estimate to be reachable through this cognitive process can be measured, and there are several reports on this subject [4–10]. This reaching movement is limited by diseases affecting the movement of the upper limbs and postural control. The reaching movement of patients with Parkinson's disease (PD) has also been described in many reports, and the reported characteristics of their reaching movement include the delay of the start of movement [11], slow speed of movement [12], and impaired coordination between the arm and the trunk [13]. Moreover, in PD patients, the reaching distance determined by the functional reach test is shorter in those with a history of falls than in those without [10, 14, 15], and the reaching distance is suggested to be associated with decreased postural stability and falls. It is assumed that, in PD patients, reaching movement is limited by decreased movement of the upper limbs due to akinesia or bradykinesia, decreased flexibility due to rigidity of the four limbs and trunk, and the impairment of postural reflexes associated with these decreases. Moreover, it has been reported that PD patients experience disorders in eye movement [16–18] and coordination between the eyes and head with reaching movements [19–21]. Presumably, decreased motility of the eyes, head, and neck due to PD may affect the ability to detect the accurate position of a target object in space. Regarding the estimated reaching distance, in studies on PD patients, Kamata et al. [9] reported that overestimation of reaching distance due to progression of symptoms is associated with falls. On the other hand, some reports showed PD patients tend to underestimate spatial distance [10, 22, 23]. Factors affecting the cognition of spatial distance have considered physical factors and environment factors [4–6]. Ehgoetz Martens et al. [24] reported that the cognition of spatial distance differs in experimental conditions in PD patients. But the association with detailed motor symptoms of PD has not yet been clarified. These issues of underestimation or overestimation are related factors that need more investigation and are still open to discuss. The difference in the right and left sides of the spatial cognition has been discussed in PD patients [22, 23, 25, 26]. In PD patients, although estimated reaching distance was analyzed only front reach [9, 10], the differences in the right and left sides need more investigation. Thus, regarding the distance estimated in reaching movement by PD patients, we evaluated distance cognition in a more spatial manner by measuring estimated and actual maximal reaching distance in the right and left directions, in addition to the front direction. Furthermore, the association with the symptoms of PD was also investigated. While various terms are used to indicate estimated reaching distance, we use the term estimated reaching distance (ED) in the present study. Correspondingly, the term actual reaching distance (AD) is used to indicate the actually reachable distance. 2. Subjects and Methods 2.1. Subjects The subjects were 27 PD patients (8 men and 19 women; mean age ± standard deviation (SD): 71.9 ± 5.3 years, mean height ± SD: 155.5 ± 7.6 cm) and a control group of 28 healthy elderly volunteers matched for age and height (10 men and 18 women with a mean age ± SD: 73.6 ± 5.2 years and mean height ± SD: 157.1 ± 8.7 cm). All participants were right-handed. In the case of PD patients, those who provided consent for the objectives and contents of the study were included if they could remain standing for long enough to perform the tasks and had cognitive function sufficient to understand the tasks (24 or higher on the Mini-Mental State Exam [MMSE]). The present study was conducted with the approval of the ethics committee of our institution (Ethics Approval Number 13-29). 2.2. Methods In the PD patients, the following data were collected: disease duration, Hoehn-Yahr scale, the motor section of the Unified Parkinson's Disease Rating Scale (UPDRS3), and history of falls. Moreover, the medications that the PD patients were receiving were identified. All patients were treated with levodopa and measurements were taken under the effects of the medications (on-stage). No patients showed dyskinesia during measurement. In the experiments, both ED and AD were measured by the following method. Regarding the AD measurement, functional reach test was verified as reliable [1]. The reliability of ED measurement was verified by Robinovitch and Cronin [7] but was not sufficient. The measurement of ED was performed three times and an Intraclass Correlation Coefficient (ICC) was used for the intrarater reliability. According to the method developed by Fischer [5], ED was measured under the following conditions. The participants estimated the range in which they could grasp a 3 cm square wooden block on a plate at the level of the shoulder in the standing position without taking a step, in the front, right, and left directions. In order to investigate differences in spatial cognition due to directional effects, ED was measured in not only the front direction but also the right and left directions. During measurement, the participants were prohibited from extending their arms or tilting their bodies and measured the distance only by sight. For estimation of the front reaching distance, the participants stood in front of the block placed at the level of their right acromion. This position was regarded as the starting position. They were instructed to slowly step backward in a straight line from any point where they could grasp the block with certainty and to stop at the farthest point where they decided that they could grasp it with both feet touching the floor. The point at the tip of the right big toe was marked, and the distance from the intersection point between the vertical line from the block and the floor to the marked point was measured and regarded as the front ED (Figure 1(a)). For estimation of the right reaching distance, the participants stood in a way that the block placed at the same position as in estimation of the front ED was positioned immediately lateral to the right acromion. This position was regarded as the starting position. They were instructed to slowly step leftward in straight line from any point where they could grasp the block with certainty and to stop at the farthest point where they decided that they could grasp it with both feet touching the floor. The point at the distal end of the right fifth metatarsal bone was marked, and the distance from the intersection point between the vertical line from the block and the floor to the marked point was measured and regarded as the right ED. Estimation of the left reaching distance was performed in the same manner, and the results were regarded as the left ED. After ED was measured in the three directions, the farthest range in which the participants could actually grasp the block was measured in the three directions in the order of the front, right, and left directions by the following method based on the multidirectional reach test, which Newton [27] had developed by modifying the functional reach test for multidirectional measurement. The participants were instructed to stand at the points marked during measurement of ED and to grasp the block without moving their feet (Figure 1(b)). The block was moved by 1 cm at a time away when they could grasp it or closer when they could not until the farthest point where they could grasp it without stepping out was determined. In this manner, AD was measured in the front direction (front AD), the right direction (right AD), and the left direction (left AD). 2.3. Data Analysis The measurements obtained from the functional reach test have been shown to correlate with body height [1]. Thus, in data analysis, each measurement (i.e., ED and AD) was divided by body height, and values were adjusted for individual differences in body constitution. In order to analyze the differences between estimated and actual measurements, the differences between ED and AD (ED−AD) were calculated. In the present study, these differences are expressed as the difference between ED and AD (DEA). Absolute values of DEA were used for analysis to assess the degree of the differences between ED and AD. In the control group, the right and left sides were regarded as the dominant and nondominant sides, respectively, to perform data analysis. In the PD patients, the symptomatically milder side (MS) and severer side (SS) were determined according to the total scores obtained from the scores on the items of UPDRS3 for the right and left sides. When the total scores were the same, the affected side at the time of onset was determined as the SS. Each measurement (i.e., right ED, left ED, right AD, and left AD) was classified by MS and SS into MS-ED, SS-ED, MS-AD, and SS-AD. In order to assess the association between each measurement and its directionality, Pearson correlation coefficients were calculated for each measurement obtained from the dominant and nondominant sides in the control group and the MS and SS in the PD patients (p values were corrected by the Bonferroni correction, p < 0.012). Moreover, a one-way analysis of variance (ANOVA) was performed with ED, AD, DEA, and absolute values of DEA in each direction (the Bonferroni correction was used for the post hoc test). In both groups, total values of measurement in the three directions were calculated for ED (total ED) and AD (total AD), and their mean values were compared between the two groups by unpaired t-test. In order to evaluate errors in cognition, the mean value was compared between total ED and total AD within each group by paired t-test. Initial significance level was set at 0.05 (p values were corrected by the Bonferroni correction, p < 0.012). As for PD patients, a stepwise linear regression analysis was performed to identify clinical symptoms associated with errors in cognition. Total DEA obtained from DEA in the three directions and absolute values of total DEA were used as target variables. The following items were used as explanatory variables: MMSE, disease duration, Hoehn-Yahr scale, history of falls, UPDRS3 total score, and individual items of UPDRS3 (tremor: 20, 21; rigidity: 22; diadochokinesis: 23–26; posture: 28, 30; walking: 29; and akinesia: 31). Significance level was set at 0.05. For all statistical analyses, SPSS version 21 was used. 3. Results 3.1. Characteristics of PD Mean and standard deviation (SD) of each item were 27.6 ± 1.8 for MMSE, 5.9 ± 3.3 years for disease duration, 2.6 ± 0.7 for Hoehn-Yahr scale, and 16.0 ± 9.5 for UPDRS3. The number of falling subjects was 12 and the number of nonfalling subjects was 15. 3.2. Reliability of ED Measurement A result of the reliability verification of ED measurement showed ICC (1,3) = 0.953; therefore it is considered as reliable method. 3.3. Validity of the Methods and Measurements In the control group, the correlation coefficients for each value obtained from the dominant and nondominant sides were r = 0.907 (p < 0.001) for ED, r = 0.620 (p < 0.001) for AD, r = 0.930 (p < 0.001) for DEA, and r = 0.792 (p < 0.001) for the absolute values of DEA. All variables showed significantly positive correlation. The PD group included 9 patients with SS at the right side and MS at the left side and 18 patients with SS at the left side and MS at the right side. In the PD group, the correlation coefficients for each value obtained at the MS and SS sides were r = 0.820 (p < 0.001) for ED, r = 0.704 (p < 0.001) for AD, r = 0.860 (p < 0.001) for DEA, and r = 0.695 (p < 0.001) for the absolute values of DEA. All variables showed significantly positive correlation (Figure 2). According to directions, no significant differences in ED, AD, and DEA or in the absolute values of DEA were observed either between the dominant and nondominant sides in the control group or between the MS and SS sides in the PD group. Moreover, the values obtained from measurement in the front direction did not significantly differ from those obtained from measurement in the right and left directions (Table 1). 3.4. Relationship between the AD and the ED in the Two Groups The mean total AD (1.57 ± 0.07 cm/cm in the control group and 1.50 ± 0.09 cm/cm in the PD group) showed a significant difference between the two groups (total AD: t(53) = 3.246, p = 0.002) (Figure 3(a)). The mean total ED (1.55 ± 0.17 cm/cm in the control group and 1.39 ± 0.20 cm/cm in the PD group) showed a significant difference between the two groups (total ED: t(53) = 3.337, p = 0.002) (Figure 3(a)). No significant differences between the total AD and the total ED were observed in control group (t(27) = 0.722, p = 0.476) (Figure 3(b)). A significant difference between the total AD and the total ED was observed in PD group (t(26) = 3.165, p = 0.004) (Figure 3(b)). 3.5. Association with Clinical Symptoms of PD Although clinical features associated with total DEA were investigated, all items were not included as significant factors for the total DEA in the PD group. On the other hand, the result of the stepwise multiple regression analysis for the absolute value of total DEA, posture, was shown to be the only significant factor (R 2 = 0.268, β = 0.518, p = 0.006). A relationship between the posture and the absolute value of total DEA is shown in Figure 4. MMSE, disease duration, Hoehn-Yahr scale, history of falls, UPDRS3 total score, tremor, rigidity, diadochokinesis, walking, and akinesia were not included as significant factors for the absolute value of total DEA. 4. Discussion 4.1. What Does DEA Indicate? To evaluate distance cognition in a more actual milieu, we measured ED and AD in three directions. ED and DEA are values determined through cognitive processing and may be affected by hand dominance and the order of measurement. Thus, we first measured ED and AD in not only the front direction but also the right and left directions. Then, we examined whether there was any variance in the measurements with the dominant hand, nondominant hand, severity of symptoms, and order of measurement. All values obtained in each reaching direction showed correlations between the right and left sides (Figure 2), and no difference was observed in the mean values of each variable (Table 1). Thus, ED and DEA are assumed to be free from the effects of reaching direction and hand dominance. In the PD patients, these values were not affected by the differences in the severity of motor dysfunction between the right and left sides. This result may support the report that spatial distance was not affected by the difference between right and left symptoms [22, 25]. Furthermore, as Table 1 shows that the values obtained in the front direction do not differ from those obtained in the right or left directions (the dominant and nondominant sides in the controls and the symptomatically milder and severer sides in the PD patients [right SS : left SS = 9 : 18]), the values are also assumed to be free from the effects of the order of measurement. ED, which is considered to be determined on the basis of sensory information and past experience, is affected by environmental factors, such as position of a target object, and physical factors, such as postural stability, body constitution, and flexibility [4–6]. While information on the spatial and positional relationship based on interactions between the environment and the motor system of the body is stored in the brain, this spatial and positional information is considered to be necessary for reaching movement [3]. ED indicates perception of such spatial and positional relationships, and it is assumed that the accuracy of perception can be determined by DEA and absolute value of DEA. Thus, as described in the following section, the characteristics of the PD patients were assessed with regard to cognition of spatial distance. 4.2. Stability Limits and Cognition of the Limits in the PD Patients According to comparisons of total AD, the reaching distance is shorter in the PD patients than in the controls, and the range where the point vertically projected from the center of gravity can be kept within the base of support (stability limit) is shown to be small in the PD patients. This result is consistent with the results reported in many studies [10, 28–30]. In PD, it is considered that the stability limit becomes smaller because of mobility being reduced by rigidity, impaired postural reaction, impaired motion perception, and so forth [28–30]. Moreover, total ED was also smaller in the PD patients than in the controls, showing that the range of stability limit recognized by the PD patients was small. As described above, it is assumed that ED is affected by environmental and physical factors. However, the present study focused on the differences in ED due to physical factors while the environmental factors, such as direction of reaching movement and height of a target object, were kept constant. In the present study, the physical factors include the cognitive process in which people perceive the distance to a target object, compare this distance to their physical status, and determine whether they can reach the target object. ED obtained in the present study might have been affected by the physical characteristics of the participants under these conditions. It can be assumed that the range of stability limit recognized by PD patients on the basis of their physical characteristics is small. 4.3. Do PD Patients Underestimate Spatial Distance? In the PD group, if both AD and ED have decreased equally, it can be assumed that there is no error in cognition of distance. In order to evaluate error in cognition, AD and ED were compared within each group. The results showed that ED tended to be negative compared to AD in the PD group, in other words, showing that the PD group tended to underestimate reaching distance. While people presumably estimate reaching distance on the basis of information from the visual, auditory, and somatic senses and their past experiences in the cognitive process before executing reaching movement [2, 3], PD patients underestimate reaching distance in this process. In PD patients, because it has been reported that their perception of the range of motion of the upper limbs [31] and joint motion angle [32] is lower than the actual values, underestimation of motion perception may affect estimation of spatial distance. Moreover, other studies [33, 34] reported that the decreased range of motion in PD patients is attributable to the lack of perception of their limited motion. Underestimation by the PD group as observed in the present study suggests that PD patients cannot perceive that the distance recognized by them is smaller than actual distance. Regarding the error in cognition of reaching distance that has been described above, Kamata et al. [9] reported that PD patients tend to overestimate reaching distance. Their result contradicts that of the present study. The reasons for this may be the following 2 factors. One factor is the differences in patient groups. Our results show that although some PD patients overall underestimated the reaching distance, some patients overestimated it. Distribution of MS-DEA and SS-DEA shows the same tendency (Figure 2; DEA, underestimation: the third quadrant, overestimation: the first quadrant). Similarly, the results obtained by Kamata et al. also included overestimation and underestimation values. Depending on the distribution of patients participating in a study, results may show either tendency. The other possible factor is the differences in methods to measure ED. A major difference involves whether the target object is moved or whether participants move during measurement of ED. Ehgoetz Martens et al. [24] investigated the differences in perception of spatial distance by PD patients between the static condition in which they perceived distance from only visual information without moving and the dynamic condition in which they moved to perceive distance. On the basis of these results, Ehgoetz Martens et al. reported that distance is more likely to be underestimated under the dynamic condition than under the static condition. Similarly, Kabasakalian et al. [22] reported hypometric estimates of distance under the dynamic condition. Although the static condition is used in many studies [4–10] including that of Kamata et al., the dynamic condition was used in the present study. This difference in the conditions may cause an underestimation of the reaching distance. Because reaching movements performed in daily life are based on the dynamic condition, the present study may provide important findings with regard to risk factors for falls. 4.4. Error in Cognition of Reaching Distance in PD Patients and Causes of the Error We investigated the factors affecting impaired cognition of distance in PD patients. Because UPDRS3 total scores or Hoehn-Yahr scale were not included as significant factors for both DEA and absolute values of DEA, impaired cognition of reaching distance is not assumed to be necessarily associated with progression of the disease, because all items were not included as significant factors for DEA, which means the factors that affect underestimation of spatial distance were not identified. The tendency of underestimation seems to be affected by the conditions discussed in the previous section rather than clinical symptoms of PD. Meanwhile, it was suggested that postural factors (i.e., posture and postural stability) greatly contributed to the large absolute values of DEA. In PD patients, it has been reported that their perception of body axis is impaired [35, 36]. Moreover the other study reported that impaired spatial cognition correlates with postural factors of UPDRS [37]. Changes in the body axis due to the abnormally flexed posture of PD patients may affect errors in cognition of distance. Moreover, one of the reported factors affecting error in cognition of reaching distance is postural stability. It has been reported that, without the certainty of a stable posture, the error increases [4, 5, 38]. Thus, abnormal posture and impaired postural stability in PD patients appear to be associated with perception of spatial information on the position of their bodies and affect their decisions regarding distance. In this study, measurements were taken only in on-stage. Levodopa may have affected the results of this study because levodopa improves motor functions of PD [35, 39, 40]. Moreover, some researchers reported that levodopa affects perception of movement and space [24, 41–43]. The results of this study showed that spatial cognition of PD patients was impaired even under the effects of levodopa. 4.5. Limitations While the participants in the present study appear to be adequately distributed between mild and moderate severity of PD, the inclusion criteria have been set to ensure the reliability and validity of measurement methods. Although the clinical symptoms of the PD patients widely varied, it was difficult to examine all symptoms and their severity in the present study. Thus, the abnormal cognition of reaching distance observed in the present study may not be applicable to all PD patients. Acknowledgments The authors want to thank members of the Department of Neurology and the Department of Rehabilitation in Juntendo University Nerima Hospital for their assistance with the present study. Disclosure The present affiliation of Satoru Otsuki is as follows: Department of Rehabilitation, Juntendo Tokyo Koto Geriatric Medical Center, 3-3-20 Shinsuna, Koto-ku, Tokyo 136-0075, Japan. Competing Interests The authors declare that there are no competing interests regarding the publication of this paper. Figure 1 (a) Measurement of front ED. Participants stood on the starting position (dotted line). They stepped backward and stopped at the farthest point where they decided that they could grasp target (solid line). The distance from target to stopping point was regarded as ED. (b) Measurement of front AD. Participants reach out and grasp the target. The distance from target to farthest standing position was regarded as AD. Figure 2 Distribution of each value in PD. The vertical axis shows severer side (SS) [cm/cm], and the horizontal axis shows milder side (MS) [cm/cm]. Dotted line shows x = y line. The value distributed on the top left (above x = y line) shows SS is larger than MS. In contrast, the value distributed on the bottom right (below x = y line) shows MS is larger than SS. Figure 3 (a) Means and standard deviations demonstrated by each group during total ED and total AD. The PD group showed small value compared with control in both total ED and total AD. (b) Comparison between total ED and total AD within each group is shown. Only the PD group showed a significant difference (∗ p < 0.012). Figure 4 This figure indicates a regression line obtained by multiple regression analysis for the absolute value of total DEA. Postural score was shown to be the only significant factor (p = 0.006). Table 1 Means and standard deviations of ED, AD, DEA, and absolute value of DEA in each direction. (a)     Front Dominant Nondominant     ED cm/cm 0.51 ± 0.05 0.52 ± 0.06 0.52 ± 0.06 n.s. Healthy elderly AD cm/cm 0.53 ± 0.03 0.52 ± 0.03 0.52 ± 0.02 n.s. n = 28 DEA cm/cm −0.02 ± 0.04 0.00 ± 0.05 −0.01 ± 0.06 n.s.   |DEA| cm/cm 0.04 ± 0.03 0.04 ± 0.03 0.05 ± 0.03 n.s. (b)     Front Milder side: MS Severer side: SS     ED cm/cm 0.47 ± 0.08 0.47 ± 0.08 0.47 ± 0.09 n.s. PD AD cm/cm 0.50 ± 0.03 0.50 ± 0.03 0.50 ± 0.03 n.s. n = 27 DEA cm/cm −0.04 ± 0.06 −0.04 ± 0.07 −0.04 ± 0.07 n.s.   |DEA| cm/cm 0.07 ± 0.05 0.06 ± 0.05 0.07 ± 0.05 n.s. Mean ± SD [cm/cm]. The one-way ANOVA was performed with each item. ED: estimated distance, AD: actual distance, DEA: difference between ED and AD, |DEA|: absolute value of DEA, and n.s.: not significant. ==== Refs 1 Duncan P. W. Weiner D. K. Chandler J. Studenski S. Functional reach: a new clinical measure of balance Journals of Gerontology 1990 45 6 M192 M197 2-s2.0-0025260020 2229941 2 Andersen R. A. Buneo C. A. 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==== Front ScientificWorldJournalScientificWorldJournalTSWJThe Scientific World Journal2356-61401537-744XHindawi Publishing Corporation 10.1155/2016/6382467Research ArticleElevation of Glucose 6-Phosphate Dehydrogenase Activity Induced by Amplified Insulin Response in Low Glutathione Levels in Rat Liver Taniguchi Misako 1 Mori Nobuko 2 Iramina Chizuru 1 http://orcid.org/0000-0003-0686-6860Yasutake Akira 3 * 1Department of Nutrition Sciences, Nakamura Gakuen University, Fukuoka 814-0198, Japan 2Department of Biomedical Laboratory Sciences, Faculty of Life Sciences, Kumamoto University, 4-24-1 Kuhonji, Kumamoto 862-0976, Japan 3Kumamoto University Graduate School of Science and Technology, 2-39-1 Kurokami, Kumamoto 860-8555, Japan*Akira Yasutake: nimdyasutake@yahoo.co.jpAcademic Editor: João Batista T. Da Rocha 2016 15 8 2016 2016 638246715 2 2016 8 7 2016 10 7 2016 Copyright © 2016 Misako Taniguchi et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Weanling male Wistar rats were fed on a 10% soybean protein isolate (SPI) diet for 3 weeks with or without supplementing 0.3% sulfur-containing amino acids (SAA; methionine or cystine) to examine relationship between glutathione (GSH) levels and activities of NADPH-producing enzymes, glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme (ME), in the liver. Of rats on the 10% SPI diet, GSH levels were lower and the enzyme activities were higher than of those fed on an SAA-supplemented diet. Despite the lower GSH level, γ-glutamylcysteine synthetase (γ-GCS) activity was higher in the 10% SPI group than other groups. Examination of mRNAs of G6PD and ME suggested that the GSH-suppressing effect on enzyme induction occurred prior to and/or at transcriptional levels. Gel electrophoresis of G6PD indicated that low GSH status caused a decrease in reduced form and an increase in oxidized form of the enzyme, suggesting an accelerated turnover rate of the enzyme. In primary cultured hepatocytes, insulin response to induce G6PD activity was augmented in low GSH levels manipulated in the presence of buthionine sulfoximine. These findings indicated that elevation of the G6PD activity in low GSH levels was caused by amplified insulin response for expression of the enzyme and accelerated turnover rate of the enzyme molecule. ==== Body 1. Introduction As a protection mechanism against oxidative damage, tissues are equipped with anti-oxidative-reducing thiol compounds such as glutathione (GSH) and metallothionein (MT), and their concentrations are elevated under oxidative stress. In fact, after treating rats with N-nitrosodimethylamine, which forms reactive oxygen species, GSH and MT levels elevated significantly [1, 2]. Concentrations of these substances in tissues are readily affected by dietary conditions, which then affect the protective ability against oxidative damage. GSH levels in the liver were regulated by the amount of sulfur-containing amino acids (SAA, methionine and cystine) in a diet [1], while MT levels were induced by heavy metals [3]. In addition to GSH and MT, NADPH plays an important role as a defense mechanism by providing a reducing-radical against oxidative stress. NADPH is produced primarily by glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme. G6PD is a key enzyme of the pentose phosphate pathway, and its activity is altered by complex interactions of dietary components and hormones. A high carbohydrate or low fat diet increased hepatic stimulated induction of G6PD activity in rat liver [4, 5]. Insulin [6–9] and thyroid hormone [10] elevated induction of G6PD, while glucagon suppressed it [11]. G6PD activity varied by dietary protein type [12], but the mechanism that modulates G6PD activity by the protein has not been elucidated. In Hela cell clones, G6PD expression was enhanced under GSH restricted states [13]. GSH is known as a cysteine reservoir in the liver [14], and intracellular GSH levels readily declined when rats received an SAA-deficient diet [1, 2]. In intact tissues, G6PD molecules exist in three major dimeric forms: fully reduced, partly oxidized, and fully oxidized forms, and different turnover rates were indicated for the three forms [15–17]. In this study, we attempted to elucidate the mechanism that regulates G6PD activity linked with hepatic GSH levels, using a diet with 10% soybean protein isolate (SPI) that has a low SAA content. To amplify SAA content, a 10% SPI diet was supplemented with 0.3% cystine or methionine. Effects of a low SAA diet on ME activity were also examined in comparison to G6PD. In addition, the direct effect of insulin response on G6PD induction at different GSH concentrations was examined using isolated primary cultured hepatocytes. 2. Materials and Methods 2.1. Chemicals Glutathione (both reduced and oxidized forms) and glucose 6-phosphate were purchased from Boehringer Mannheim GmbH (Mannheim, Germany). NADP and NADPH were purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). γ-Glutamylglutamic acid (used as an internal standard in HPLC analysis), γ-glutamylcysteine, and insulin were purchased from Sigma Chemical Co. (St Louis, MO, USA). The materials used for isolation and cell culture were as reported previously by Yoshimoto et al. [8]. All other chemicals were of reagent grade. 2.2. Animals and Diet As protein sources, SPI, provided from Fuji Oil Co., Ltd. (Osaka, Japan), was used. SPI consisted of 91.1% crude protein, 4% salts, and 3.5% carbohydrates. Amino acid composition of SPI was analyzed in the research center of Fuji Oil Co., and 1.4 g of methionine and 1.3 g of cystine were contained per 16 g of nitrogen as described in our previous paper [18]. Diet was prepared according to the recommendation of AIN 93 [19] as shown in Table 1. To examine the effects of SAA supplementation, 0.3% cystine or methionine was added in 10% SPI. As a whole, 3 dietary groups were used in the present study: 10% SPI, 10% SPI + 0.3% cystine, and 10% SPI + 0.3% methionine. Weanling male Wistar rats (Charles River, Japan; body weight of 55 ± 3 g, 6 in each group) were housed in individual cages and maintained in a temperature-controlled room at 20–22°C with a controlled 12 h cycle of light and darkness. Diets and drinking water were given ad libitum during the experiment. After the feeding period for 3 weeks, rats were sacrificed from 09:00 to 10:30 under deep anesthetization with diethylether. Four pieces of liver samples were obtained from each rat for the assays of GSH and MT, for the determination of enzyme activities and mRNA levels. All procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of Nakamura Gakuen University. Note. The above animal experiment was carried out in 1998 when diethylether anesthesia could be used without any regulation. 2.3. Assays of GSH and MT For glutathione assay, a portion (ca. 0.5 g) of the liver was immediately homogenized in ice-cold 5% perchloric acid (PCA) containing 1 mM EDTA. After centrifugation at 2,500 rpm for 10 min, reduced (GSH) and oxidized glutathione (GSSG) in the supernatant fraction were determined by HPLC after derivatizing with dinitrobenzene fluoride [20]. For MT analysis, portions of liver (0.5 g) were kept at −80°C until use. The liver sample was homogenized (20%, w/v) in ice-cold 1.15% KCl using a Polytron homogenizer (Kinematica GmbH, Littau, Switzerland) under N2 atmosphere. An aliquot of the homogenate was subjected to MT assay according to the procedure of Naganuma et al. [21] with a slight modification using nonradioactive HgCl2 [22]. Briefly, the homogenate was treated successively with diethyl maleate and 10 mM CdCl2 and then heated at 95°C for 5 min to precipitate high-molecular weight proteins. Following cooling and centrifugation, the supernatant was successively treated with 5 mM HgCl2, 1 mM ovalbumin, and 12.5% trichloroacetic acid. After centrifugation, the supernatant was filtered through a membrane of 0.22 μm pore diameter (Ultrafree C3, Merck Millipore, Darmstadt, Germany) to afford Hg-MT samples. MT levels were expressed as the amount of mercury bound to thionein molecules after Hg analysis of the Hg-MT samples. Hg levels in the final preparations were determined by the oxygen combustion-gold amalgamation method [23] using an atomic absorption mercury detector MD-A (Nippon Instruments Co., Ltd., Osaka, Japan). 2.4. Assay of Enzyme Activity A portion of the liver (1-2 g) was homogenized in 5 vol. of an ice-cold homogenizing buffer, 0.25 M sucrose solution containing 5 mM Tris-HCl buffer (pH 7.4), and 0.1 mM EDTA. After centrifugation, the upper layer was used for determination of enzyme activities. G6PD and ME activities were determined according to the method of Lee [24] and Ochoa [25], respectively. One unit of G6PD or ME activity was defined as the amount of enzyme that catalyzed oxidation of 1 μmol of glucose 6-phosphate or malate per min at 25°C. γ-Glutamylcysteine synthetase (γ-GCS) activity was determined by the method of Nardi et al. [26] with a slight modification. The supernatant described above was added to the reaction mixture consisting of 6 mM ATP, 50 mM KCl, 6 mM dithiothreitol, 20 mM MgCl2, 3 mM L-cystine, and 15 mM glutamic acid in 0.1 M Tris-HCl buffer (pH 8.2) and then incubated at 37°C for 10 min. The reaction was terminated by adding 110 μL of 5% PCA and centrifuged to afford a supernatant fraction. γ-Glutamylcysteine formed in the mixture was derivatized with dinitrofluorobenzene and analyzed as described above for GSH determination using HPLC [20]. One unit of γ-GCS activity was defined as the amount of enzyme that catalyzes to form 1 μmol of γ-glutamylcysteine per min at 37°C. Protein was determined by the method of Lowry et al. [27], and the enzyme activities were expressed as mU/mg protein. 2.5. mRNA Total RNA was isolated from fresh liver sample (ca. 0.5 g) obtained above by a modified acid guanidinium-phenol extraction method of Chomczynski and Sacchi [28], using a commercially available RNA isolation reagent ISOGEN (Nippon Gene, Tokyo, Japan) according to the protocol recommended by the manufacturer. Yield and purity were determined by absorbance at 260 nm and absorbance ratio A 260/A 280 (1.7–2.0). RNA samples (10 μg each in 0.2 mL of H2O/20x SSC/37% formaldehyde, 2 : 1 : 1) were incubated at 65°C for 15 min and then loaded in the slot-blot apparatus (Bio-Rad Japan, Tokyo) onto a Clear Blot Membrane-N (ATTO Co., Tokyo, Japan). G6PD and ME mRNAs on the membrane were analyzed by a solution hybridization procedure described by Hamilton et al. [29] using α-32P-dCTP and DNA probes for each enzyme. The probes were prepared by RT-PCR method using rat liver RNA and the following primer pairs:  G6PD   sense primer: 5′-GTCCTCTATGTGGAGAATGA-3′   antisense primer: 5′-TCTTGGTCATCATCTTGGTA-3′   ME   sense primer: 5′-TCCAGGTCCTTAGAGTAATT;   antisense primer: 5′-CATGCGTTAAGAACTGAAGA-3′  The mRNA levels were estimated by comparison with β-actin mRNA level. 2.6. Gel Electrophoresis Distribution of fully reduced, partly oxidized, and fully oxidized forms of G6PD in rat liver was determined using electrophoresis according to the method by Martins et al. [17]. Cytosol samples from the respective dietary groups containing equivalent G6PD activity (5 mU) were applied on a polyacrylamide gel and electrophoresed within 3 hr of preparation using a 1.5 mm thick slab gel containing 10% (w/v) acrylamide and 0.26% (w/v) bisacrylamide polymerized in the presence of 0.017% (w/v) ammonium persulphate and 0.033% (v/v) tetramethylene diamine. The gel was stained for G6PD activity in 50 mM Tris-HCl (pH 8.0) containing 0.58 mM glucose 6-phosphate, 0.13 mM NADP+, 10 mM MgCl2, 33 μM phenazine methosulphate, and 0.1 mM nitroblue tetrazolium. After incubation at 4°C for 16 hr, the reaction was terminated by immersing the gel in 7% acetic acid. The gel was dried, and intensities of the enzyme fractions were scanned using computing densitometer (ACD-18; Gelman Science, Inc., Pensacola, FL, USA). 2.7. Cell Culture Liver parenchymal cells were isolated from male Wistar rat (6 weeks of age) fed with laboratory chow diet (CE-2) by in situ perfusion of the liver with collagenase under pentobarbital anesthesia, according to Seglen's method, with a modification by Tanaka et al. [30]. The cells were plated on a 60 mm plastic dish in Williams medium E (WE medium) containing 5% newborn calf serum at 2 × 106 cells/dish and were cultured as monolayers at 37°C in 95% air and 5% CO2. After a one-day culture, the cells were incubated for 48 hr in WE medium containing 10−6 M of dexamethasone, 0.1 μg/mL aprotinin, 30 μg/mL kanamycin, and insulin at 10−10 to 10−6 M. To examine glutathione concentration- dependent alteration in G6PD activity, buthionine sulfoximine (BSO, a specific inhibitor for glutathione synthesis) was added to the basal incubation medium at a series of concentrations up to 0.5 mM in the presence of 10−7 M insulin. For insulin response to G6PD induction under a restricted glutathione concentration, 0.1 mM BSO was added to the basal medium, and G6PD activity was determined at 10−10 to 10−6 M of insulin concentration. 3. Results Liver samples were obtained from rats fed on each of three low protein diets, a 10% SPI, 10% SPI + 3% cystine, and 10% SPI + 3% methionine. Reduced and oxidized glutathione and metallothionein (MT) levels and γ-GCS activity in the liver were shown in Table 2. Selective analysis of reduced (GSH) and oxidized (GSSG) forms of glutathione revealed that the SAA-induced increase in the reduced form was prominent and that the GSSG level changed only slightly (Table 2). γ-GCS activity, a rate-limiting enzyme of glutathione biosynthesis, was lowered when GSH levels were elevated. Elevation of γ-GCS activity in restricted tissue glutathione levels might be a compensative action to raise the levels. In contrast to the increase in GSH levels, G6PD and ME activities were lowered by supplementing SAA (Figure 1). SAA-induced alterations of G6PD and ME activities were considered to have occurred at the transcriptional levels, since the enzyme activities and their mRNA levels showed reliable correlations (Figure 2). G6PD in the liver cytosol is known to exist in 3 dimeric molecular forms: fully reduced, partly oxidized, and fully oxidized, and they can be separated on the polyacrylamide gel electrophoresis as reported by Martins et al. [17]. Liver cytosol samples prepared from rats fed on 10% SPI, 10% SPI + cystine, and 10% SPI + methionine diet were analyzed using a gel electrophoresis, and 3 major bands were obtained as shown in Figure 3. Bands l, 2, and 3 are different molecular forms and are named fully oxidized, partly oxidized, and fully reduced form, respectively [15]. Distribution of the multiple molecular forms of the enzyme was found to be notably different among the diet groups (Figure 3). Although the major components in adult female rats were reported to bands 2 and 3 [17], bands 1 and 2 became the major forms in the 10% SPI group. However, supplementation of SAA to 10% SPI caused significant reduction of band 1, bringing about a distribution similar to the normal pattern reported by Martins et al. [17]. The distribution pattern of the G6PD molecular forms agreed to GSH/GSSG ratio rather than GSSG concentration (Table 2). Insulin's ability to induce G6PD was examined at different concentrations of GSH using primary culture of the hepatocytes. Cells were incubated with 0.01–0.5 mM BSO, a specific inhibitor of glutathione biosynthesis, in the presence of 10−7 M insulin for 48 hr. The GSH concentration-dependent alteration in G6PD activity was depicted in Figure 4. With the increase in the BSO dose, G6PD activity reached its maximum (3-fold of the initial activity) at 0.1 mM BSO with the cellular GSH concentration of less than 5 nmol/mg protein (2.8% of the initial level). It began then to decrease, although the GSH level was kept decreased. Decreased enzyme activity with the higher BSO concentrations would be due to BSO-induced cellular damage, as was made evident from morphological observation using phase contrast microscopy and lactate dehydrogenase activity in the medium (data not shown). Accordingly, a BSO dose level of 0.1 mM was found to be most effective for inducing G6PD activity with minimum cell damage. G6PD activity in the presence of insulin at a series of concentrations (10−10 to 10−6 M) was determined with and without addition of 0.1 mM BSO. As shown in Figure 5, G6PD activity was elevated with the increased insulin concentration, and the response was remarkably amplified in the presence of BSO. The present results with the isolated hepatocytes came about with the notable increase in G6PD mRNA expression found with Hela cells when an intracellular pool of GSH was reduced [13]. 4. Discussion Laboratory rodents fed on a low protein diet having low SAA, such as 10% SPI, are a useful model for producing low hepatic GSH levels. GSH is an intracellular reducing agent, and low GSH levels eventually caused the greater animal susceptibility to oxidative damage [1, 17]. NADPH, another reducing reagent, is supplied by the action of G6PD and ME. In the present study, we researched the alteration of these enzyme activities in the liver under low GSH levels by feeding rats on a 10% SPI diet. SAA supplementation to 10% SPI caused decreases in G6PD and ME activities concomitantly with increase in GSH level. These results suggested that NADPH production was stimulated under low GSH levels and that NADPH could compensate with GSH as a reducing agent in protecting cells from oxidative stress. A reverse relation between G6PD activity and GSH levels was confirmed also using isolated hepatocytes in the presence of BSO, a specific inhibitor for GSH synthesis. G6PD was reported to be induced several fold in the presence of 10−8 M insulin in primary cultured hepatocytes [7]. Using cultured hepatocytes in the present study, G6PD activity was amplified through a range of insulin concentrations from 10−10 to 10−6 M, and the activity was further augmented by GSH deficiency caused by BSO. mRNA analysis suggested that the induction of G6PD to amplify insulin response by GSH suppression existed prior to and/or at the transcriptional event. G6PD in rat liver was shown to be separated into 3 dimeric molecular forms on polyacrylamide gel electrophoresis [11]. Bands 1, 2, and 3 shown in Figure 3 represented fully oxidized, partly oxidized, and fully reduced forms, respectively. It was proposed that a shift towards band 1 could be an early event in the degradation of this enzyme, since this form was the most rapidly inactivated by chymotrypsin or microsomes, possibly leading to faster turnover of this enzyme. Although high ratio of band 1 in the 10% SPI group was considered to be due to high oxidative state, GSSG levels were not significantly different among the dietary groups. The GSH concentration and/or GSH/GSSG ratio would have an important role when determining the proportion of the three molecular forms. Under oxidative stress, a considerable portion of an intracellular oxidized form of glutathione was found in protein-bound mixed disulphides, as can be determined after borohydride treatment [31]. In the present experiment, GSH and GSSG were extracted with perchloric acid, but the protein-bound mixed disulfide could possibly have escaped the extraction. Accordingly, the real amount of the oxidized form of glutathione, GSSG plus protein-bound mixed disulfide, would be higher than the level obtained in the present study, especially in 10% SPI-fed rat liver. Faster degradation of G6PD by forming a fully oxidized form would accelerate the turnover rate of the enzyme in rats fed on 10% SPI diet. Despite the accelerated turnover rate, G6PD activity would have been elevated due to increased transcription under restricted glutathione levels in the liver. A reverse correlation with glutathione levels was also observed in the activity of γ-GCS, a rate-limiting enzyme of glutathione synthesis (Table 2). γ-GCS activity is well-documented to be induced by various impacts [32–34], but it is feedback inhibited by glutathione itself [35]. Thus, activities of the above two enzymes, G6PD and γ-GCS, were regulated by GSH levels but in different manners. G6PD was amplified under low GSH levels via insulin interaction, while γ-GCS was controlled by product-binding inhibition. Rise in G6PD activity would allow increase in NADPH to compensate GSH as a reducing agent under increased oxidative status. The inverse correlation between MT, another cytosolic reducing agent, and GSH was found also in this study (Table 2). In GSH depletion, MT levels are elevated to function as a biological defense system against oxidative stress, together with NADPH produced by G6PD. In conclusion, through animal experiments using rats fed on low protein diet and primary cultured hepatocytes, we found that low GSH levels caused elevation of the hepatic G6PD activity by amplified insulin response for the enzyme expression and accelerated turnover rate of the enzyme molecules by increasing a portion of the fully oxidized form. Competing Interests There is no conflict of interests. Figure 1 Effects of sulfur-amino acids supplemented to a 10% SPI diet on glutathione concentration and activities of glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme (ME) in rat liver. Values are mean ± SD of 6 rats in each diet group. Different letters show significant difference (p < 0.05). Figure 2 Correlations of liver G6PD and ME activities with their mRNA levels in 10% SPI, 10% SPI + cystine, and 10% SPI + methionine groups. mRNA levels were shown as relative amount to β-actin mRNA. Figure 3 Molecular diversity of liver G6PD separated after electrophoresis on 7.5% polyacrylamide slab gel. Cytosol samples with 5 mU equivalent G6PD activity were applied on the gels and stained for G6PD activity after electrophoresis. Lanes (1), (2), and (3) represent 10% SPI, 10% SPI + cystine, and 10% SPI + methionine, respectively. Bands 1, 2, and 3 represent fully oxidized, partially oxidized, and fully reduced forms. Different letters in bands show significant difference (p < 0.05). Figure 4 Effect of BSO on glutathione concentration and G6PD activity of isolated cultured hepatocytes. Isolated rat hepatocytes were incubated for 48 h in WE medium containing 10−6 M dexamethasone, 0.1 μg/mL aprotinin, 30 μg/mL kanamycin, and 10−7 M insulin in the presence of 0–0.5 mM BSO. Glutathione level (closed circle) and G6PD activity (open circle) were determined at each BSO concentration. Figure 5 Amplifying insulin response for G6PD activity and effect of BSO. Precultured isolated hepatocytes were incubated for 48 h in the basal WE medium as described in Figure 4. To the basal medium, insulin at a series of concentrations (10−10 to 10−6 M) was added, and G6PD activity was determined in the presence (closed circle) and absence (open circle) of 0.1 mM BSO. Table 1 Compositions of the diets. Component g/100 g diet Protein 10.0 Dextrin 15.5 Sucrose 10.0 Soybean oil 5.0 Cellulose 5.0 Mineral mixture (AIN 93) 3.5 Vitamin mixture (AIN 93) 1.0 Choline bitartrate 0.25 ∗Cystine or methionine 0.3 Corn starch To 100.0 ∗Supplemented to a diet at 0.3%. Table 2 Effect of diet on concentration of glutathione (reduced and oxidized forms), metallothionein (MT), and γ-glutamylcysteine synthetase (γ-GCS) activity in rat liver. Diet GSH (μmol/g liver) GSSG (nmol/g liver) GSH/GSSG MT (nmol Hg/g liver) γ-GCS (mU/mg protein) 10% SPI 1.2 ± 0.2a 53 ± 14a 22 244 ± 70a 9.2 ± 2.4a 10% SPI + cystine 5.9 ± 0.4b 43 ± 26a 137 66 ± 14b 5.4 ± 0.9b 10% SPI + methionine 7.0 ± 0.6c 39 ± 10a 179 104 ± 31b,c 6.7 ± 1.0b Values are mean ± SD of 6 rats fed on respective diets for 3 weeks. Activity was expressed as mU/mg protein. Amount of mRNA was expressed as relative intensity. Different letters in the same column denote significant difference (p < 0.05). ==== Refs 1 Taniguchi M. Yasutake A. Takedomi K. Effects of dietary sulfur-containing amino acids on oxidative damage in rat liver caused by N -nitrosodimethylamine administration British Journal of Nutrition 2000 84 2 211 217 10.1079/096582197388563 2-s2.0-0033848142 11029972 2 Taniguchi M. Yasutake A. Takedomi K. Inoue K. 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==== Front J OphthalmolJ OphthalmolJOPHJournal of Ophthalmology2090-004X2090-0058Hindawi Publishing Corporation 10.1155/2016/7472542Clinical StudyNovel Technique of Transepithelial Corneal Cross-Linking Using Iontophoresis in Progressive Keratoconus http://orcid.org/0000-0001-8842-7102Lombardo Marco 1 * http://orcid.org/0000-0002-4141-6462Serrao Sebastiano 1 Raffa Paolo 2 Rosati Marianna 1 http://orcid.org/0000-0002-9416-967XLombardo Giuseppe 3 4 1Fondazione G.B. Bietti, IRCCS, Via Livenza 3, 00198 Roma, Italy2Dipartimento di Medicina Molecolare, Università di Padova, Via A. Gabelli 63, 35121 Padova, Italy3Consiglio Nazionale delle Ricerche, Istituto per i Processi Chimico-Fisici, Viale Stagno d'Alcontres 37, 98158 Messina, Italy4Vision Engineering Italy srl, Via Adda 7, 00198 Rome, Italy*Marco Lombardo: mlombardo@visioeng.itAcademic Editor: Flavio Mantelli 2016 15 8 2016 2016 74725425 6 2016 14 7 2016 Copyright © 2016 Marco Lombardo et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.In this work, the authors presented the techniques and the preliminary results at 6 months of a randomized controlled trial (NCT02117999) comparing a novel transepithelial corneal cross-linking protocol using iontophoresis with the Dresden protocol for the treatment of progressive keratoconus. At 6 months, there was a significant average improvement with an average flattening of the maximum simulated keratometry reading of 0.72 ± 1.20 D (P = 0.01); in addition, corrected distance visual acuity improved significantly (P = 0.08) and spherical equivalent refraction was significantly less myopic (P = 0.02) 6 months after transepithelial corneal cross-linking with iontophoresis. The novel protocol using iontophoresis showed comparable results with standard corneal cross-linking to halt progression of keratoconus during 6-month follow-up. Investigation of the long-term RCT outcomes are ongoing to verify the efficacy of this transepithelial corneal cross-linking protocol and to determine if it may be comparable with standard corneal cross-linking in the management of progressive keratoconus. National Framework Program for Research and Innovation PON01_00110Ministero della SaluteFondazione Roma ==== Body 1. Introduction Corneal cross-linking is an established procedure aimed at slowing down or halting keratoconus progression [1]. The efficacy of the conventional riboflavin/UV-A irradiation procedure was primarily demonstrated by laboratory studies suggesting that it increases the biomechanical strength of the treated cornea. In addition, several clinical studies have evidenced how the treatment is effective in slowing down or halting the progression of keratoconus up to 10 years of follow-up [2–9]. The conventional corneal cross-linking procedure includes the removal of corneal epithelium to permit adequate penetration of riboflavin in the stroma. Epithelial removal, however, is responsible for most of the major cross-linking related complications, which include postoperative pain, vision impairment, and risk of infection. Major efforts have been dedicated to overcoming the epithelial barrier to riboflavin penetration and reducing treatment time, with the aim of maintaining efficacy and improving safety of the treatment [10]. On the other hand, there are still controversies on how riboflavin may penetrate in the stroma through an intact epithelium or how the epithelium may limit UV-A irradiation of the stroma soaked by riboflavin. It has been widely shown that dextran-enriched solutions greatly limit the penetration of riboflavin in the stroma through the intact epithelium [10, 11]. Recently, Shalchi et al. [12] have revised a series of peer-reviewed papers comparing the results of standard cross-linking (total of 45 papers) and transepithelial cross-linking (total of 5 papers) in the management of progressive keratoconus. Although transepithelial corneal cross-linking has been shown to be safe without any related epithelial wound healing complication, 75% of the cases have shown a continued keratoconus progression one year after treatment, whereas the majority of studies on standard cross-linking (≥90%) have shown reduction in maximum simulated keratometry in the same period. The only study that has shown comparable results between transepithelial and standard corneal cross-linking did not use dextran-enriched riboflavin solution for the transepithelial treatment [12]. Overall, the limited number of published outcomes from randomized controlled trials (RCTs) makes it challenging to draw clear conclusions on the efficacy of the many techniques used for transepithelial corneal cross-linking [13–20]. Robustly designed controlled trials are required to provide accurate results between techniques. Currently, five RCTs (NCT02117999, NCT02456961, NCT02349165, NCT01181219, and NCT01868620) are aiming to compare the results of transepithelial corneal cross-linking with the standard procedure for stabilization of progressive keratoconus; three of these trials (NCT02117999, NCT02456961, and NCT01868620) are using dextran-free riboflavin solutions to moisten the corneal stroma through the intact epithelium using iontophoresis. Iontophoresis is a noninvasive technique used to deliver a charged substance transcorneally by repulsive electromotive force using a small electrical charge applied to an iontophoretic chamber. In ex vivo studies, we have assessed the diffusion of riboflavin in dextran-free 0.1% hypotonic solution enriched with ethylenediaminetetraacetic acid (EDTA) and trometamol in the corneal stroma of eye bank donor eyes and the effect of rapid UV-A irradiation of the cornea (i.e., 10 mW/cm2 for 9 minutes) after transepithelial soaking using iontophoresis. Experimental work has demonstrated that though the stromal concentration of riboflavin after iontophoresis was lower than conventional soaking, the stiffening effect on the anterior cornea was almost comparable to that of standard cross-linking [21, 22]. Other laboratory studies have shown that iontophoresis is effective to deliver an adequate amount of riboflavin in the stroma through the intact epithelium, as previously discussed [21, 22]. The present RCT with identifier code NCT02117999 was designed to compare the treatment efficacy for progressive keratoconus by transepithelial corneal cross-linking using iontophoresis with the standard corneal cross-linking procedure at 12 months. In this paper, we present the technique and anticipate the outcomes from the complete cohort of 34 eyes after 6 months of follow-up. 2. Patients and Methods 2.1. Study Design This is an ongoing prospective, unmasked, randomized controlled trial (RCT) conducted at the clinical trials center of the IRCCS Fondazione G.B. Bietti, Rome, Italy. The aim of the study is to assess the efficacy and safety of transepithelial corneal cross-linking using iontophoresis (T-ionto CL or study group) in the treatment of progressive keratoconus in comparison with standard corneal cross-linking (standard CL or control group). The primary outcome measure of the study is the maximum simulated keratometry value (K max) at 12 months. Approval was obtained from the IFO-IRCCS Ethical Committee (Rome, Italy), and the conduct of the study adhered to the tenets of the Declaration of Helsinki. The trial is registered with the US National Institutes of Health registry with identifier code NCT02117999 (https://clinicaltrials.gov/ct2/show/NCT02117999). After full explanation of the protocol, written informed consent was obtained from all participants before enrolment in the study. The recruitment started on January 31, 2014, and closed on May 30, 2015. 2.2. Participants Patients with a confirmed diagnosis of progressive keratoconus were invited to participate in this study. Keratoconus was deemed to be progressive if there was an increase of at least 1 diopter (D) in K max derived by computerized Placido disk corneal topography over the preceding 12 months. Exclusion criteria included a minimum corneal thickness of less than 400 μm, K max steeper than 61 D, any corneal scarring, previous refractive or other corneal or ocular surgeries, and other ocular disorders (e.g., cataract, glaucoma, and herpetic keratitis). Patients who were pregnant or breastfeeding at the time of enrolment also were excluded. Only patients aged between 18 and 46 years were included in the study. Eligible patients were randomized after enrolment, with allocation ratio of 2 : 1, into either the study or control group using a computer-generated randomization plan with block randomization in groups of four. Two different blocks were created, which included eyes with K max steeper or flatter than 54 D in order to randomize patients with similar baseline K max values in the study and control groups. If both eyes of a patient qualified for participation in the study, each eye was randomized independently. Second eyes were treated no earlier than 2 months after the first eyes. 2.3. Assessments Contact lens wearers were instructed to discontinue their use for a minimum of 3 weeks before the preoperative eye examination. In addition, we asked all those patients to discontinue the use of contact lens during follow-up in order to avoid bias during the study. At baseline and postoperative visits at 3 and 7 days and 1, 3, and 6 months, all patients underwent slit-lamp examination of the anterior segment of the eye; the haze in the anterior stroma was graded on a scale (grade 0–4) used after photorefractive keratectomy [23]; ocular surface inflammation was graded on signs of bulbar conjunctival hyperemia (grade 0–3) and upper tarsal conjunctival papillae (grade 0–3) according to Akpek et al. [24]. In addition, the following assessments were recorded: best spectacle corrected visual acuity (BSCVA, logMAR units) obtained using Early Treatment Diabetic Retinopathy Study chart at 4 meters, contrast sensitivity function (CSF, log units) evaluated using Pelli-Robson chart, manifest refraction (expressed as spherical equivalent, diopters, D), K max (D) and corneal thickness (micrometers, μm) using combined Placido disk corneal topography and anterior segment optical coherence tomography (Visante, Carl Zeiss Meditec Inc., Dublin, CA, USA), and endothelial cell density (ECD, cells/mm2) measured by no-contact specular microscopy (Perseus, CSO, Italy). All data were acquired and analyzed in an unmasked manner. At each time point, patients received a questionnaire to evaluate symptoms, such as itchiness, tearing, photophobia, and pain (grade 0–3), after treatment. To exclude infection, document epithelial healing, and provide general postoperative care, all patients were assessed also on day 1 after treatment in addition to the described follow-up schedule. To improve the reliability of topography measurements, a minimum of 3 acquisitions were performed for each eye at each time interval. If the value varied by more than 10% between the scans, then a further scan was obtained. The best scan was then selected for analysis. 2.4. Techniques and Treatments For each patient, corneal cross-linking was performed within 4 weeks of the baseline examination. All treatments were performed under topical anaesthesia; anaesthetic eye drops (oxybuprocaine hydrochloride 0.4%, Novesina, Novartis Farma SpA, Italy) were instilled 3 times over a 10-minute period before each treatment. Transepithelial corneal cross-linking using iontophoresis was performed as follows (Figure 1):(1) After a lid speculum was inserted, central corneal thickness was measured by handheld ultrasound pachymeter (Pachmate, DGH, Exton, USA); thereafter, sterile Biopore membrane attached to a plastic cylinder (Millicell, cod. PICM01250, Merck SpA, Italy) was pressed against the central cornea with sufficient pressure to applanate the central cornea for 3 seconds and remove the precorneal mucin layer. (2) Corneal soaking with ETDA and trometamol enriched riboflavin-5-phosphate 0.1% hypotonic solution (Ricrolin+, Sooft Italia SpA, Italy) was performed using a commercial iontophoresis device (Iontophor CXL, Sooft Italia SpA, Italy). The passive electrode was applied to the forefront of the eye to be treated. The active electrode, a bath tube made of plastic, was applied to the corneal surface. After suctioning of the tube to the corneal epithelium, it was filled with riboflavin solution. The current intensity was set at 1.0 mA for 5 minutes. After iontophoresis, the corneal surface was gently washed with chilled 0.9% sodium chloride solution. (3) Immediately after iontophoresis, the central corneal thickness was again measured by handheld ultrasound pachymeter. (4) Corneal UV-A irradiation was then applied using 10 mW/cm2 device (370 ± 8 nm; Vega 10 mW, CSO, Italy) at 56 mm distance for 9 minutes. One drop of chilled 0.9% sodium chloride solution was applied over the corneal epithelium every 3 minutes during irradiation. The control group received conventional corneal cross-linking according to the “Dresden protocol” [3, 4]. The central 10 mm corneal epithelium was removed using Amoils' brush (Innovative Excimer Solutions Inc., Toronto, ON); central stromal thickness was then measured by handheld ultrasound pachymeter. A solution containing 20% dextran-enriched 0.1% riboflavin (Ricrolin, Sooft Italia SpA, Italy) was instilled every 3 minutes for 30 minutes before UV-A irradiation. After corneal soaking, the stromal surface of each tissue was gently washed using chilled 0.9% sodium chloride solution; thereafter, the central stromal thickness was measured by handheld ultrasound pachymeter. The corneal stroma was then irradiated with a UV-A device (Vega 3 mW, 370 ± 8 nm) with an irradiance of 3 mW/cm2 for 30 minutes. The UV-Adelivery system was located 56 mm from the cornea. Diluted riboflavin (0.025%) drops were instilled over the stromal surface every 3 minutes during UV-A irradiation. The UV-A devices were calibrated with a power meter before corneal irradiation and an irradiation area of 9.00 mm diameter was used in all cases. At the end of treatments, 2 drops of ofloxacin 0.3% (Monofloxofta, Sooft Italia SpA, Italy) were applied in all cases. A bandage contact lens was applied only to patients treated by conventional corneal cross-linking; it remained in place until epithelial closure was confirmed. After surgery, all patients continued taking ofloxacin 0.3% 5 times daily for 6 days, sodium hyaluronate 0.10% (Ribolisin, Sooft Italia SpA, Italy) 6 times daily for 3 months, and fluorometholone acetate 0.1% (Fluaton, Bausch & Lomb, Rochester, NY) 2 times daily from day 7 to day 21. 2.5. Immunofluorescence Microscopy Imaging Immediately after the applanation, the Biopore membranes were fixed in 4% paraformaldehyde and shipped to the laboratory. Each membrane was gently removed from the plastic cylinder and placed into the wells of a multiwell plate. The samples were blocked in 1x phosphate buffered saline containing 1% bovine serum albumin (BSA; Sigma-Aldrich, Saint Louis, MO) and then incubated with antibody against mucin-4 (MUC4, goat polyclonal, 1 : 100; Santa Cruz Biotechnology, Santa Cruz, CA) for 2 hours and with rhodamine-conjugated secondary antibodies (anti-goat produced in donkey, 1 : 200; Sigma-Aldrich, Saint Louis, MO) for 1 hour at room temperature. In addition, the cell nuclei were stained with far-red fluorescent DNA dye (DRAQ5®, 1 : 2000; Cell Signaling Technology, Boston, MA) for 10 minutes at room temperature. Specimens were then mounted in Dako Glycergel mounting medium (Dako, Glostrup, Denmark) for immunofluorescence microscopy imaging. Images were acquired using a Nikon A1Rsi+ confocal laser scanning microscope equipped with NIS-Elements Advanced Research software (Nikon Instruments Inc., Melville, NY). 2.6. Statistical Analysis Statistical analysis was performed using SPSS (version 17, IBM Corp., NY). All data are reported as the mean ± standard deviation. Normal data distribution was tested by using the one-sample Kolmogorov-Smirnov test. Sample size calculation was performed to detect a difference of 0.95 D between the mean K max changes for the T-ionto CL and standard CL groups at 12 months, at a significance level of 5% and power of > 80%, assuming a standard deviation of 1.20 D. The sample size of the study was 34 cases (allocation ratio of 2 : 1). In this work, the difference from baseline for each parameter was calculated at each time point (3 days, 7 days, and 1, 3, and 6 months) for each eye. The differences within each group were compared using paired Student's t-test. These changes were also compared between the study and control group using unpaired Student's t-test. 3. Results Thirty-four eyes of 25 patients were randomized to T-ionto CL (20 patients, 22 eyes) and standard CL (10 patients, 12 eyes) treatments. The demographic data showed a strong skew toward male patients (20; 80%); the mean age was 31.05 ± 6.64 years and 29.40 ± 5.60 years in the study and control group (P = 0.55), respectively. Eleven right eyes (50%) and eleven left eyes (50%) were treated by T-ionto CL; seven right eyes (60%) and five left eyes (40%) were treated by standard CL (Table 1). Nine patients were treated in both eyes: two patients underwent T-ionto CL in both eyes (A100 and A500; A1600 and A2000); two patients underwent standard CL in both eyes (B200 and B500; B300 and B400); and five patients received T-ionto CL in one eye and standard CL in the fellow eye (A900 and B1100; A1300 and B1200; A1400 and B600; A1500 and B700; A1800 and B900). All patients completed the 6-month follow-up. 3.1. Technique Immediately after corneal applanation with a Biopore membrane, the corneal epithelium was moderately stained with fluorescein dye (Figures 1(A) and 1(B)). The Biopore membranes, which were applanated to the cornea, showed confluent staining for MUC4 mucin and the presence of scattered epithelial cells in all cases (Figures 1(C) and 1(D)). In the study group, two eyes (10%) had perilimbal haemorrhage after removing the iontophoresis tube. This was likely related to peripheral corneal suctioning of the tube. One eye had a small central epithelial defect (<2 mm) at the end of the procedure. This was the first case in the series (A100). A bandage contact lens was applied for 24 hours and then removed once the epithelium was intact. In controls, epithelial closure was completed and the bandage contact lens was removed at day 3 in all eyes. In the study group, central corneal thickness ranged between 491 ± 35 μm and 492 ± 40 μm (0.2% average change) before and after iontophoresis, respectively. In the control group, central stromal thickness decreased from 464 ± 34 μm to 332 ± 27 μm (28% average change) from before to after soaking with 20% dextran-enriched riboflavin solution. After UV-A irradiation of the cornea, the stromal thickness returned back toward baseline values (445 ± 16 μm). 3.2. Symptoms At day 1, controls complained of more tearing than those with eyes treated by T-ionto CL (P = 0.04); no significant differences for other symptoms were found between groups at day 1 postoperatively. At day 3, tearing was again greater in patients treated by standard CL than in those treated by T-ionto CL (P = 0.006); photophobia was also greater in patients treated by standard CL than in those treated by T-ionto CL (P = 0.001). No differences in pain score were found between groups at day 1 (1.9 ± 1.0 and 2.1 ± 0.9 in T-ionto CL and standard CL groups, resp., P = 0.71) and day 3 (0.6 ± 0.5 and 0.5 ± 0.7, resp., P = 0.65) postoperatively. After 1 week, there was no significant symptom complained of by any patient and no difference was found between groups. 3.3. Objective Evaluation At day 1, bulbar conjunctival hyperaemia was greater after standard CL than after T-ionto CL, though without reaching statistical significance (P = 0.08); however, it was significantly greater 3 days (P < 0.001) and 1 week (P = 0.03) after standard CL than after T-ionto CL. Thereafter, no difference was found between groups. Upper tarsal conjunctival papillae were greater at days 1 (P = 0.004) and 3 (P < 0.001) after standard CL than after T-ionto CL. No difference was found between groups afterwards. Two eyes (10%) and six eyes (50%) showed stromal edema in the T-ionto CL and control groups, respectively, both at 3 and at 7 days postoperatively. Thereafter, no edema was evidenced in the study group. Corneal edema, which was mostly confined to the posterior stroma, was still found in 4 eyes (33%) and 1 eye (8%) at 3 and 6 months after standard CL, respectively. At 3 months, two eyes (10%) in the T-ionto CL showed faint corneal haze (grade: ≤0.50); at 6 months, all the eyes in the T-ionto CL group had clear cornea. In the control group, at 3 months, haze graded 0.50 in four eyes (33%) and 1.00 in two eyes (17%); at 6 months, haze graded 0.5 and 1.00 in five eyes (42%) and one eye (8%), respectively. Representative pictures of study and control eyes over 6 months of follow-up are shown in Figure 2. 3.4. Topography Measurements The mean increase of K max was 2.88 ± 2.20 D and 2.78 ± 1.87 D in the T-ionto CL and standard CL groups in the preceding 12 months, respectively. At baseline, the difference in K max between groups was not statistically significant (T-ionto CL: 54.7 ± 4.0 D; standard CL: 54.7 ± 4.3 D; P = 0.87). At 6 months, there was a significant average improvement in both groups with a flattening of K max by 0.72 ± 1.20 D (P = 0.01) and 0.86 ± 0.89 D (P = 0.006) in the T-ionto CL and standard CL groups, respectively (Figure 3). Flattening of more than 1.00 D occurred in 7 eyes (32%) and 5 eyes (42%) in the T-ionto CL and control group, respectively. Two eyes (9%; A1000 = 1.2 D and A2200 = 1.2 D) of the T-ionto CL group showed progression of K max more than 1.00 D during the same period; no eye showed progression in the standard CL group. Comparing the K max changes between groups revealed no statistically significant differences (P = 0.72) at 6 months (Figure 4). 3.5. Visual Acuity and Refractive Outcomes At baseline, the difference in BSCVA between groups was not statistically significant (P = 0.29). On average, BSCVA improved compared with baseline at 6 months in the T-ionto CL group (from 0.12 ± 0.20 logMAR to 0.01 ± 0.10 logMAR; P = 0.001). In the control group, the 6-month improvement of BSCVA compared with baseline approached statistical significance (from 0.06 ± 0.10 logMAR to 0.01 ± 0.07 logMAR; P = 0.08; Figure 5(a)). At 6 months, BSCVA improved by 1 or more ETDRS line in fourteen eyes (64%) and four eyes (33%) in the T-ionto CL and control group, respectively. The changes of BSCVA between the two groups did not show statistical significance (P = 0.19) at 6 months. The contrast sensitivity function recovery was slower in the control than in study group (P < 0.001 at day 3). In the study group, CSF did not change during follow-up (preoperative range: 1.20–1.65 log units; 6-month range: 1.35–1.65 log units); in the control group, the postoperative values decreased immediately after treatment (from 1.59 ± 0.12 to 1.36 ± 0.22 log units at day 3, P < 0.001), approaching baseline values at 1 week (1.54 ± 0.14 log units; Figure 5(b)). After 6 months, CSF improved by one triplet in seven eyes (32%) and three eyes (25%) in the T-ionto CL and control group, respectively. The manifest spherical equivalent refraction changed averagely by +0.65 ± 1.20 D (P = 0.02) and +0.24 ± 0.77 D (P = 0.32) in the T-ionto CL and standard CL groups, respectively, at 6 months. No significant difference in the change of manifest refraction was found between groups (P = 0.30) during 6 months of follow-up (Figure 5(c)). 3.6. Corneal Thickness Measurements At baseline, the mean central corneal thickness (CCT) was 484 ± 37 μm and 494 ± 34 μm in the T-ionto CL and standard CL group (P = 0.44), respectively. At 6 months, no significant CCT differences were found in the T-ionto CL (480 ± 33 μm; P = 0.50), while significant corneal thinning was found in the control group (481 ± 29 μm; P = 0.03) with respect to baseline (Figure 6(a)). On average, corneal thickness significantly increased at 3 days (P = 0.001) after standard CL, approaching baseline values at 1 week and slightly progressing to decrease over 6 months. 3.7. Endothelial Cell Density The ECD ranged from 2635 ± 387 cells/mm2 and 2625 ± 281 cells/mm2 preoperatively to 2666 ± 235 cells/mm2 and 2647 ± 351 cells/mm2 6 months postoperatively in the T-ionto CL (P = 0.66) and standard CL (P = 0.68) group, respectively. In the control group, ECD dropped at 3 days postoperatively (P = 0.03; Figure 6(b)), likely related to the loss of corneal transparency due to stromal edema found in 50% of eyes at the same visit. 3.8. Adverse Events One eye in the standard CL group (B600; left eye) showed two small peripheral subepithelial infiltrates at day 3, which did not delay epithelial wounding and did not affect visual acuity. The anterior chamber was clear. The eye was treated with application of netilmicin sulfate 0.3% (Nettacin, Sifi SpA, Italy) and ciprofloxacin chlorhydrate 0.3% (Oftacilox, Alcon SA, Puurs, Belgium) drops 5 times daily each for 7 days. Fluorometholone acetate 0.1% was initiated 1 week later than scheduled in the study protocol. By 3 months, there were only two faint corneal scars, with visual performance being stable (BSCVA = 20/20 and CSF = 1.65 log units). No postoperative complications occurred in the right eye of the same subject (A1400) that underwent T-ionto CL. 4. Discussion In this work, we reported the surgical techniques and preliminary clinical findings of the RTC with code identifier NCT02117999 comparing transepithelial corneal cross-linking with iontophoresis and conventional corneal cross-linking on all enrolled patients having completed 6 months of follow-up. The eyes recruited in both arms of the present study showed similar progression of K max in the preceding 12 months, with mean steepening of 2.88 ± 2.20 D and 2.78 ± 1.87 D in the T-ionto CL and standard CL groups, respectively. Because the main treatment objective is to stabilize the underlying disease process, corneal topography (K max) at 12 months was considered the key outcome measure of the study [1, 2]. Due to the inherent clinical interest in novel surgical techniques for treating progressive keratoconus, we are anticipating the 6-month clinical outcome. At 6 months, we found statistically significant flattening of K max by 0.72 ± 1.20 D (P = 0.01) and 0.86 ± 0.89 D (P = 0.006) in the T-ionto CL and standard CL groups, respectively, with no difference between groups, though two eyes in the study group (9%) showed progression of K max of 1.2 D during the same period. Less variability and more favorable outcomes have been in general reported for standard cross-linking from the 6-month follow-up onwards, due to epithelial-stromal remodelling [2, 3, 8, 12, 25, 26]; for this reason, the design of the present RCT did not include any further treatment before completing the 1-year follow-up. Retreatment of an ectatic cornea has been previously indicated if the K max value increased by at least 1.0 D over 2 consecutive follow-up visits compared with its value during the steady-state period after the first treatment [3]. Visual performance was not affected by T-ionto CL in the first days after surgery; at 3 days postoperatively, all eyes except for two (90%) had the same or improved BSCVA and CSF with respect to baseline. The same result was found in 50% of cases treated by standard CL in the same period. The differences between treatments were mostly related to epithelial debridement and wound healing. Six months after T-ionto CL, we found a significant average improvement in BSCVA (−0.11 logMAR; P = 0.001), which was not found after standard CL. No change in CSF was found after T-ionto CL (mean changes lower than +0.08 log units during a period of 6 months), whereas a decrease in CSF was measured 3 days after standard CL (on average, −0.23 log units), which was likely caused by epithelial debridement and wound healing. The manifest refraction showed a statistically significant average reduction of myopia (+0.65 ± 1.20 D; P = 0.02) only after T-ionto CL treatment at 6 months. No changes in CCT and ECD were measured during 6 months of follow-up after T-ionto CL treatment. After standard CL, corneal thickness increased, due to stromal edema, during the first week postoperatively (on average, +34 μm), decreasing during 6-month follow-up. A drop in ECD count, which was related to decreased transparency and more corneal scattering caused by stromal swelling in 50% of eyes, was measured at day 3 after standard CL (on average, −337 cells/mm2). The use of hypotonic riboflavin solution to promote stromal swelling in order to achieve a minimum thickness of 400 μm before UV-A irradiation has been shown to minimize the risk of early postoperative stromal swelling [27]. Although there was no difference in pain score between groups at any time during follow-up, symptoms of ocular discomfort were greater after standard CL than after T-ionto CL during the first 3 days postoperatively. The eyes treated by standard CL showed greater conjunctival hyperaemia and tarsal conjunctival papillae than eyes treated by T-ionto CL during the first week [24]. At 3 months, 2 eyes (10%) in the T-ionto CL group showed faint corneal haze (grade ≤ 0.5), which disappeared in all cases at 6 months. In the control group, six eyes (50%) showed mild corneal haze (grade between 0.5 and 1.0) during the same period [23]. We had one case in the control group that showed peripheral sterile corneal infiltrates at day 3, which did not delay corneal wound healing or affect visual performance or topography outcome (K max flattened by −0.8 D at 6 months). No adverse events were recorded in the study group. Data from the control group are consistent with those already published in the literature showing the time course of clinical and instrumental measures after standard CL [2–5, 9, 25]. In general, visual acuity and corneal steepness worsen in the first month postoperatively; resolution to baseline occurs by approximately 3 months, with improvement thereafter. Improvement includes flattening of K max (on average between 1 and 2 D in averagely 50% of cases), reduction in myopic spherical equivalent (averagely between +0.2 and +0.7 D), and increase of BSCVA (≥1 Snellen line in 50% of cases), which occur during a period of 1 year after treatment [2, 3, 8, 10, 25, 26]. Central corneal thickness remains slightly decreased from baseline to 12 months after standard CL and then recovers to baseline thickness after more than 18 months [8]. Early postoperative complications after standard CL include stromal swelling (averagely 70% of cases) and sterile infiltrates (averagely 8% of cases), which however have been shown to resolve in the vast majority of cases within 3 months after treatment [2, 6, 28]. Stromal swelling may be due to epithelial debridement, excessive stromal thinning due to the hyperosmolar riboflavin solution, and the direct UV-A irradiation of the corneal stroma. The mechanism underlying sterile infiltration is still unknown but may relate to an altered immune response to antigens in areas of static tear pooling beneath the bandage contact lens or may be a direct result of the phototoxic effect of corneal cross-linking [28, 29]. Corneal haze is also a common adverse event in the early postoperative period after standard CL (up to 70% of cases), which however resolves in the vast majority of cases by 1 year after treatment [28, 30, 31]. In this RCT, the major technical differences between the T-ionto CL and standard CL treatments include the epithelial debridement in the latter case, the use of two different riboflavin solutions, and the total UV-A energy dose delivered to corneal stroma, which is on average 20% higher in standard CL than in T-ionto CL, due to the UV-A filtering effect of the epithelium [10]. Three previous uncontrolled studies [32–34] have reported the clinical outcome of transepithelial corneal cross-linking with iontophoresis for the management of keratoconus in adults. In general, the authors have reported an improvement in BSCVA, stable or decreased K max, and no changes in CCT and ECD count during 1-year follow-up. In two studies [33, 34], iontophoresis has been performed with the same iontophoresis device used in the present RCT. Recently, the 1-year clinical outcome of transepithelial corneal cross-linking with iontophoresis (20 eyes) to treat progressive keratoconus has been compared to the standard corneal cross-linking protocol (20 eyes). The authors have shown a significant reduction of K max by −0.31 ± 1.87 D and −1.05 ± 1.51 D, respectively, with no patient eye showing continuous progression of keratoconus at the end of follow-up [35]. In this trial, iontophoresis was preceded by removal of precorneal mucin layer with the intent to increase epithelial permeability to riboflavin (molecular weight of 340 Da) [36–39]. At physiologic pH, the intact corneal epithelial surface acts as a chemophysical barrier due to the function of the intercellular tight junctions and the epithelium-associated mucins, which regulate paracellular transport of compounds [22, 40]. By removing the epithelium-associated mucins, we aimed to decrease the epithelial barrier function in order to ensure enough bioavailability of riboflavin in the stroma using iontophoresis, as found in laboratory studies [21, 22]. Gentle applanation of the central corneal surface with Biopore membrane for 3 seconds was effective to remove the central precorneal tear film, as shown by confocal immunofluorescence analysis of the membranes containing MUC4 mucin, which is the predominant mucus moiety in the precorneal tear film [38, 39], and by corneal imaging using slit-lamp biomicroscopy and fluorescein dye (molecular weight of 376 Da). No damage to the corneal epithelium was made in any case and this clinical observation was supported by the presence of only scattered superficial epithelial cells on the Biopore membranes. In conclusion, the preliminary outcome from the present RCT provides evidence that transepithelial corneal cross-linking with iontophoresis using 10 mW/cm2 UV-A device is safe for the treatment of progressive keratoconus in adults and improves keratometry readings in 90% of cases over 6 months postoperatively. Investigation of the long-term RCT outcomes is warranted to verify the efficacy of transepithelial corneal cross-linking and determine whether it may be comparable with standard corneal cross-linking in the management of progressive keratoconus. Acknowledgments Research for this work was supported by the National Framework Program for Research and Innovation PON (Grant no. 01_00110), the Italian Ministry of Health, and Fondazione Roma. Competing Interests No competing interests exist for any author. Figure 1 (A) Slit-lamp photograph of the cornea 1 minute after the applanation with the Biopore membrane. (B) The corneal epithelium showed moderate punctate staining with fluorescein dye (molecular weight: 376 Da). (C) Homogeneous immunofluorescent staining for MUC4 mucin (false color red) was found on Biopore membranes where the cornea was applanated. (D) A small number of scattered epithelial cells (false color blue) were observed on the membranes. In (C) and (D), scale bars are 100 μm. (E) Iontophoresis was performed with the current intensity set at 1.0 mA for 5 minutes using a commercial device. (F) After iontophoresis, the cornea was irradiated using 10 mW/cm2 UV-A device for 9 minutes. The arrow indicates the mark of the suction tube on the corneal epithelium. Strong fluorescence was emitted by stromal riboflavin inside the area of iontophoresis delivery. Figure 2 (a) Slit-lamp photographs of a representative case (A1200) after T-ionto CL. One day after treatment, moderate epithelial haze of the central cornea (arrow), with no fluorescein staining, was noted in 5 cases (23%); this superficial haze regressed in all cases within 1 week after treatment. Six months after T-ionto CL, the cornea was clear in all cases. (b) Slit-lamp photographs of a representative case (B700) after standard CL. Six months after treatment, anterior corneal haze and mild stromal edema, which was confined to the posterior stroma, were still observed in 50% and 8% of controls, respectively. Figure 3 Graph showing K max readings (D) compared with baseline at 3 days, 1 week, and 1, 3, and 6 months after T-ionto CL (black curve) and standard CL (grey curve). Bars indicate ± standard deviation. On average, both procedures halted progression of keratoconus during 6 months of follow-up. Figure 4 Tangential corneal topography maps in a patient who underwent transepithelial corneal cross-linking in the right eye (A1500) and standard cross-linking in the left eye (B700). The preoperative, 1-week, and 1-, 3-, and 6-month postoperative maps are shown from the left to right, respectively. At 6 months, the flattening of K max was 1.74 D (from 59.46 D to 57.72 D) and 0.92 D (from 50.55 D to 49.63 D) in the right and left eye, respectively. Scale bars are normalized and shown in diopters. RE: right eye; LE: left eye; K steep and K flat represent the simulated keratometry values. Figure 5 ((a), (b), and (c)) Graphs showing best spectacle corrected visual acuity (BSCVA, LogMAR), contrast sensitivity function (CSF, log units), and spherical equivalent refraction (D) compared with baseline at 3 days, 1 week, and 1, 3, and 6 months after T-ionto CL (black curve) and standard CL (grey curve), respectively. Bars indicate ± standard deviation. BSCVA improved significantly (P = 0.08) and spherical equivalent refraction was significantly less myopic (P = 0.02) after T-ionto CL. On average, visual performance decreased in the first week after standard CL and reached preoperative values at 3 months postoperatively. Figure 6 ((a) and (b)) Graphs showing central corneal thickness (CCT, μm) and endothelial cell density (ECD, cells/mm2) compared with baseline at 3 days, 1 week, and 1, 3, and 6 months after T-ionto CL (black curve) and standard CL (grey curve), respectively. Bars indicate ± standard deviation. No significant changes of CCT and ECD were found after T-ionto CL during 6 months of follow-up. Major differences between treatments in the early postoperative period are due to consequences of epithelial debridement and direct UV-A irradiation of the stroma thinned by hypertonic riboflavin solution. Table 1 Baseline demographics and clinical characteristics of eyes in the study (T-ionto CL) and control (standard CL) groups (M ± SD).   T-ionto CL Standard CL P value Age (years) 31.0 ± 6.6 29.4 ± 5.6 P = 0.55 Male/female gender (%) 18/3 (86%) 8/4 (67%)   K max (D) 54.74 ± 4.01 54.76 ± 4.30 P = 0.87 BSCVA (logMAR) 0.12 ± 0.20 0.06 ± 0.10 P = 0.29 Central corneal thickness point on AS-OCT (µm) 484 ± 37 494 ± 34 P = 0.44 Spherical equivalent refraction (D) −2.64 ± 2.41 −1.75 ± 2.12 P = 0.29 Endothelial cell density (cells/mm2) 2635 ± 387 2625 ± 281 P = 0.93 K max: maximum simulated keratometry (D: diopters); BSCVA: best spectacle corrected visual acuity (logMAR). ==== Refs 1 Raiskup F. Spoerl E. Corneal crosslinking with riboflavin and ultraviolet A. Part II. clinical indications and results Ocular Surface 2013 11 2 93 108 10.1016/j.jtos.2013.01.003 2-s2.0-84876512051 23583044 2 Wittig-Silva C. Chan E. Islam F. M. A. Wu T. Whiting M. Snibson G. R. 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==== Front Tuberc Res TreatTuberc Res TreatTRTTuberculosis Research and Treatment2090-150X2090-1518Hindawi Publishing Corporation 10.1155/2016/1354356Research ArticleTreatment Outcome and Associated Factors among Tuberculosis Patients in Debre Tabor, Northwestern Ethiopia: A Retrospective Study http://orcid.org/0000-0002-2362-5500Melese Addisu 1 * Zeleke Balew 2 Ewnete Biniam 3 1Department of Medical Laboratory Science, College of Health Sciences, Debre Tabor University, P.O. Box 272, Debre Tabor, Ethiopia2Department of Nursing, College of Medicine and Health Science, Bahir Dar University, Bahir Dar, Ethiopia3Department of Medicine, College of Health Sciences, Debre Tabor University, P.O. Box 272, Debre Tabor, Ethiopia*Addisu Melese: addisum22@gmail.comAcademic Editor: Alexander S. Apt 2016 15 8 2016 2016 135435610 3 2016 17 7 2016 Copyright © 2016 Addisu Melese et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Assessing the outcomes of tuberculosis (TB) treatment is an important indicator for evaluation of the effectiveness of tuberculosis control programs. In Ethiopia, directly observed treatment short course (DOTS) was included in the national tuberculosis control program as a strategy but little is known about its effectiveness in the study area. Therefore, this study was aimed at assessing the treatment outcomes of TB patients and associated factors in Debre Tabor, northwest Ethiopia. Methods. A retrospective study was conducted among TB patients for the period from May 2008 to April 2013 at Debre Tabor Health Center, northwest Ethiopia. Data were entered and analyzed using SPSS version 20.0. Descriptive statistics were used to generate frequency tables and figures. Logistic regressions were used to identify factors associated with treatment outcomes at P value ≤ 0.05. Results. Out of 339 patients (197 males and 142 females) registered for antituberculosis treatment in Debre Tabor Health Center, only 303 patients were included in the treatment outcome analysis and 87.1% had successful treatment outcome while 12.9% had unsuccessful treatment outcome. In the multivariate logistic regression analysis, the odds of successful treatment outcome were higher among patients ≥45 years of age (AOR = 3.807, 95% CI: 1.155–12.544) and lower among females (AOR = 0.347, 95% CI: 0.132–0.917), rural residents (AOR = 0.342, 95% CI: 0.118–0.986), and negative smear result at the second month of treatment (AOR = 0.056, 95% CI: 0.005–0.577) as compared to their counterparts. Conclusion. The treatment outcome of all forms of tuberculosis patients in Debre Tabor health center was satisfactory as expected from effective implementation of DOTS. Although the observed successful treatment outcome was in agreement with the national target, follow-up of patients during the course of treatment to trace the treatment outcomes of transferred-out patients and assessment of other potential sociodemographic factors that could affect the treatment outcomes of TB patients were also recommended. ==== Body 1. Introduction Tuberculosis has long been recognized as a major public health problem and leading cause of death alongside with HIV/AIDS [1]. Since then, control efforts including directly observed treatment short course (DOTS) have been initiated by WHO as a strategy [2]. After its launch, DOTS was recommended by international tuberculosis authorities. DOTS has shown to be effective in achieving a high successful treatment outcome [3] and became an important indicator to evaluate the effectiveness of tuberculosis control programs [4]. Early diagnosis and appropriate treatment of TB are among the cornerstones of the DOTS strategy being implemented globally. The targets to control the global epidemic, as recognized by the WHO/Stop TB Partnership and included in the Millennium Development Goals (MDGs), are to diagnose at least 70% of infectious cases and successfully treat at least 85% of them [2]. TB fueled by HIV/AIDS epidemic still remained a major health problem in Ethiopia [5]. According to the 2010/11 national population based survey of Ethiopia, the prevalence of all forms of TB was 224 per 100,000 population [6]. With this prevalence, Ethiopia ranked 7th among the 22 high TB burden countries. In response to this burden, Ethiopia has adopted DOTS as a strategy for TB prevention and control program in the early 1990s [5]. Although DOTS has been estimated to have 100% geographical coverage, it is found only at 95% at health facilities [7]. Studies conducted in southern Ethiopia indicated that DOTS improved the treatment outcomes of tuberculosis and service coverage [8], hence preventing new infections and emergence of drug resistance, but, in Debre Tabor, even though DOTS has been implemented two decades before, the outcomes of treatment and associated factors were not yet assessed. Therefore, this study was aimed to assess the treatment outcomes and associated factors among tuberculosis patients in Debre Tabor, northwest Ethiopia, for the period covering from May 2008 to April 2013. 2. Methods 2.1. Study Design and Setting A retrospective study was employed to assess the treatment outcome and associated factors among all forms of TB patients in Debre Tabor. Debre Tabor is a zonal town, 667 kilometers away from the capital, Addis Ababa. The health service delivering institutions are comprised of one general hospital, three health centers, and two private clinics during data collection. Debre Tabor Health Center was randomly selected for this study. Health centers are primary healthcare units capable of serving 15,000–25,000 population. 2.2. Data Collection Data were collected by nurses using a structured sheet specially designed for this study. Patients diagnosed for TB were transferred to DOTS clinic and started treatment according to the national guideline. Patient data on age, sex, residence, type of TB, smear results at baseline and 2nd, 5th, and 7th months, HIV status, year of treatment, patient category during the start of treatment, and the outcomes of treatment were extracted from Debre Tabor Health Center, DOTS clinic. 2.3. Inclusion and Exclusion Criteria Patients diagnosed for any form of TB and started treatment from May 2008 to April 2013 were included while patients under treatment and with incomplete sociodemographic information were excluded. 2.4. Laboratory Diagnosis of TB In Debre Tabor Health Center, pulmonary TB was diagnosed using suggestive clinical signs and symptoms in combination with Ziehl-Neelsen staining and/or chest X-ray. Spot-morning-spot sputum was the specimen used to diagnose PTB. EPTB was diagnosed using clinical information supported with radiography, ultrasound, and cytology and/or pathological procedures through patient referral system with nearby hospitals (Debre Tabor, Felege Hiwot, Gamby, and Gondar) and private clinics. 2.5. Laboratory Diagnosis of HIV For the screening of HIV, the nationally recognized test algorithm was employed in the health center according to the manufacturer's instructions and HIV results were obtained from patient registers. KHB (Shanghai Kehua Bio-Engineering Co., Ltd., China) was used as screening and positive test results were repeated with STAT-PACK (Chembio HIV-1/2 STATPAK™ Assay, CHEMBIO DIAGNOSTIC SYSTEMS, Inc., Medford, NY, USA). Discordant results from KHB and STAT-PACK were defined by the tie-breaker (UNI-GOLD, HIV, Trinity Biotech PLC, Co., Wicklow, Ireland). 2.6. Data Analysis Data were entered and analyzed using Statistical Package for Social Sciences (SPSS) Version 20.0 (IBM SPSS Statistics for Windows, Armonk, NY: IBM Corp., 2011). Descriptive statistics were used to generate and summarize frequencies. Bivariate and multivariate logistic regressions were used to assess the relationship between treatment outcome and independent variables. 3. Case and Treatment Outcome Definitions Patient category, type of TB, and treatment outcome definitions were used according the National Tuberculosis and Leprosy Control Program Guideline (NTBLCP) [5]: 3.1. Patient Category New Case. It is a patient who has never had treatment for TB before or has been on anti-TB treatment less than four weeks. Relapse. It is a patient who has been declared cured or has completed treatment of any form of TB in the past but who reports back and was found to be smear positive. Treatment Failure. It is a patient who while on treatment remained smear positive or became again smear positive at the end of the five months or later, after commencing treatment. Default. It is a patient who had previously registered as defaulted from treatment and returns to the health facility and found to be smear positive sputum. Transfer In. It is a patient who started treatment in one health facility (reporting unit) and transferred to another health facility (receiving unit) to continue treatment. Unknown. It is a patient whose category at the start of treatment was not mentioned or neither of the above. 3.2. Type of TB Smear Positive Pulmonary TB. It is a patient with at least two sputum specimens positive for AFB by microscopy or one positive sputum specimen for AFB by microscopy and a positive culture or a patient with one positive sputum specimen by microscopy and abnormal chest X-ray indicative of active TB as decided by a clinician. Smear Negative Pulmonary TB. It is a patient with symptoms that are suggestive of TB with three negative sputum smear results by direct microscopy and that do not respond to courses of broad-spectrum antibiotics or three negative sputum smear results by direct microscopy and radiographic abnormalities indicative for pulmonary TB or a patient with three negative sputum smear results by direct microscopy and positive sputum culture for MTB. Extrapulmonary TB (EPTB). It is tuberculosis of organs/tissues other than lungs proven by culture, histopathology, and symptoms suggestive of active extrapulmonary TB and decisions made by clinicians to treat with anti-TB drugs. Sputum examination and chest radiographs were used to evaluate the involvement of lungs. 3.3. Treatment Outcome Cured. They are patients who completed treatment with negative bacteriology result at the end of treatment. Completed. They are patients who finished treatment, but without bacteriology result at the end of treatment. Failure. They are patients who remained smear positive at five months/later despite correct intake of medication. Defaulted. They are patients who interrupted their treatment after registration for treatment. Died. They are patients who died from any cause during the course of treatment. Transferred Out. They are patients in whom information on treatment outcome cannot be obtained due to transfer to another health facility. Successful Treatment. It is a patient who was cured or has completed treatment. Treatment Success Rate (TSR). It is the sum of the percentages of cured and patients who completed treatment. 4. Results 4.1. Sociodemographic Characteristics of Patients Out of 339 TB patients included in this study, 197 (58.1%) were males and 142 (41.9%) were females. Majority (60.8%) of the patients were urban residents. Most cases reported were from 25 to 44 years of age. The mean age and standard deviation (SD) of the patients were 34.9 ± 17.4 (range 1–78) years (Table 1). 4.2. Category of Patients Among TB patients at the start of treatment, 89.1% were new cases, 3.8% were relapsed, 0.6% were failed, 5.3% were transferred in, and 1.2% were unknown cases. Based on the type of TB, 38.9% of the patients were diagnosed as EPTB, 33.3% smear negative PTB, and 27.7% smear positive PTB. Females, patients ≤ 14 years of age, and new TB patients had higher rate of EPTB than smear negative and smear positive PTB. HIV test was done for 71.68% of patients and 12.7% were positive. The rate of TB-HIV coinfection was 16.28% among smear positive PTB, 32.56% among smear negative PTB, and 51.16% among EPTB (Table 2). Over the five years, the types of TB showed a different trending pattern from one year to another as shown in Figure 1. 4.3. Treatment Outcome Among the TB patients included in this study, 67 patients (19.8%) were cured, 197 (58.1%) completed the treatment, 12 (3.5%) failed, 8 (2.4%) defaulted, 19 (5.6%) died, and 36 (10.6%) patients were transferred out (transferred to another health facility). The rate of cure among all forms TB cases was 19.8% while the rate of treatment completion was 58.1%. The rate of treatment failure, default, and death was 3.5%, 2.4%, and 5.6%, respectively. As age of the patient increased, the trend of completing treatment showed a decreasing pattern while death rate showed an increasing pattern (Table 3). The trend of transferred-out TB patients has showed a similar pattern while other treatment outcomes had different trends over the course of five years (Figure 2). Logistic regression was employed to assess sociodemographic variables including age, sex, place of residence, HIV status, baseline smear result, smear result at the 2nd, 5th, and 7th months of treatment, type of TB, year of treatment, and patient category at start of treatment. In the multivariate analysis, the treatment outcome was varied with age, sex, place of resident, and smear result at 2nd month and year of treatment. The odds of successful treatment outcome was 3.807 (95% CI: 1.155–12.544) times higher among patients older than 45 years of age compared to patients younger than 14 years and 25–44 years of age. Females had lower rates of successful treatment (AOR: 0.347, 95% CI: 0.132–0.917). Patients were less likely to have successful treatment if they were rural residents (AOR: 0.342, 95% CI: 0.118–0.986) compared to urban residents. Successful treatment outcome was less frequent (AOR: 0.056, 95% CI: 0.005–0.577) among smear negative patients than smear positive patients at the 2nd month of treatment. 5. Discussion Evaluation of the treatment outcome and associated factors for TB patients has the greatest importance in assessing the effectiveness of DOTS program in a country (Table 4). Since the treatment outcome of transferred-out cases was unknown, they were excluded from the final evaluation. Of the 303 patients assessed for their treatment outcomes at Debre Tabor Health Center under DOTS clinic, 264 (87.1%) had successful treatment outcome. This overall treatment success rate for all cases of tuberculosis in our study was supported by various studies conducted in Ethiopia with success rates of 86.2% in northeastern Ethiopia [9] and 85% at Kola Diba [10] while it was higher than studies conducted at Felege Hiwot Referral Hospital (26%) [11], in southern region (49.5%) [8], in Addis Ababa (82.7%) [12], and lower than studies conducted at Enfranz (94.8%) [13]. This satisfactory treatment success rate might be attributable to relatively lower transferred-out rates (10.62%), failures (3.5%), default rate (2.4%), and death rate (5.6%). Our study revealed that males were more likely to default, to fail, to die, and to transfer out than females and this was consistent with a study conducted in southern Ethiopia [14]. Literatures showed that poor treatment outcomes are associated with inadequate treatment adherence. A study conducted somewhere else in Ethiopia reported that patient behavior and attitude about the disease are major factors affecting adherence to TB treatment [15]. The higher social interaction outside home by males, social isolation associated with TB leading to treatment rejection, alcoholism, and other related behaviors among males might contribute to their higher default, failure, death, and transfer-out rates. From May 2008 to April 2011, the trends of smear negative PTB showed a decreasing pattern while it showed an increasing trend in later years. Smear negative PTB patients had high treatment success rates compared to EPTB and smear positive PTB patients with treatment success rate of 89.5%, 87.2%, and 84.0%, respectively. The treatment success of smear positive pulmonary TB patients in this study was slightly lower than the 87% WHO international target, 89.0% in Tigray region [16], and 89.3% in the southern region of Ethiopia [17] but higher than studies conducted in Ethiopia including 72.2% in Gambela region [18], 74.8% in southern region [14], and 29.5% in Gondar [3]. The relatively lower overall successful treatment outcome of smear positive PTB in our study when compared to WHO target and other studies conducted in Ethiopia could be attributed to poor smear microscopy resulting in false smear negative PTB. Another possible reason for the relatively lower successful treatment outcome could be lack of tracing out of the treatment outcomes of defaulted patients. The overall TB-HIV coinfection rate at Debre Tabor Health Center was 12.7%. This figure was lower than previous studies conducted at Gondar University Hospital and northeastern parts of Ethiopia showing high proportions (52.1% and 24.3%, resp.) of coinfection [9, 19]. The lower rate in this study might be explained in terms of unavailability of HIV counseling and testing services in earlier years in the study area or refusal of patients to be tested for HIV (83 patients were registered with their HIV status unknown). Majority of study participants showing TB-HIV coinfection were those with EPTB (51.2%) followed by smear negative pulmonary (32.6%) and the least coinfection was reported among smear positive pulmonary TB patients (16.3%). The rate of occurrence of TB-HIV coinfection among EPTB patients was three times higher than smear positive pulmonary patients and 1.5 times higher than smear negative PTB. This finding was in agreement with studies conducted in Ethiopia and India [19–21] and evidenced by other literatures as EPTB and smear negative PTB are HIV associated infections. Contrary to our findings, studies conducted in northeastern part of Ethiopia showed that TB-HIV coinfection was higher among smear positive PTB than smear negative PTB and EPTB [9]. Smear positive PTB and HIV positive patients had experienced higher rate of treatment failure than their counterparts. HIV status, baseline smear result, smear result at the 5th and 7th months of treatment, type of TB, year of treatment, and patient category at start of treatment had no significant association with treatment outcome. This study also compared the treatment success rates at Debre Tabor Health Center with the national success rates. To this end, there was a relatively similar treatment success rate among patients in Debre Tabor Health Center compared to the national treatment success rates (83.3% versus 84% and 80.9% versus 84%, resp.) in the first two years of treatment while relatively higher success rates were observed in the last three years of treatment (87.8% versus 83%, 91.5% versus 86% and 93.4% versus 91%), respectively (Table 5). 6. Conclusion The treatment outcome of all forms of tuberculosis patients in Debre Tabor was satisfactory as expected from effective implementation of DOTS. Although the observed successful treatment outcome was in agreement with the national target, follow-up of patients during the course of treatment to trace the treatment outcomes of transferred out patients and assessment of other potential sociodemographic factors that could affect the treatment outcomes of TB patients were also recommended. Acknowledgments The authors would like to thank staff of Debre Tabor Health Center, DOTS clinic, for their unreserved cooperation throughout the course of data collection. Additional Points Limitations of the Study. Patient registration books reviewed lacked data about the HIV status of all TB patients for the year 2008/2009 and few patients for the year 2009/2010 in which patients HIV status was not reported. This will affect the TB-HIV coinfection rate and their treatment outcomes. The retrospective nature of the study could not identify sociodemographic factors that could affect the treatment outcomes of patients that were not included during registration of patients for treatment. Ethical Approval Ethical clearance was obtained from Ethical Review Committee of College of Medicine and Health Science, Bahir Dar University, and the Amhara Regional Health Bureau. Letter of permission was given to South Gondar zone health department and Debre Tabor town health office. Permission was also sought from Debre Tabor Health Center to access patient data. To ensure confidentiality, patient identifiers were not included and data were used only for the intended study. Competing Interests The authors declare that they have no competing interest. Authors' Contributions Addisu Melese conceived and designed the study, participated in data collection, entered and analyze data, interpreted the results, and drafted the paper. Balew Zeleke participated in the design, data collection, and analysis. Biniam Ewnete participated in data collection and interpretation. All authors reviewed and approved final drafts of the paper. Figure 1 Trends of types of TB over the course of five years at Debre Tabor Health Center, 2008–2013. Figure 2 Trends of treatment outcomes of TB over the course of five years at Debre Tabor Health Center. Table 1 Characteristics of TB patients (N = 339), Debre Tabor Health Center, 2008–2013. Characteristics Frequency Percentage Sex      Male 197 58.1  Female 142 41.9 Residence      Urban 206 60.8  Rural 133 39.2 Age      ≤24 102 30.1  25–44 140 41.3  ≥45 97 28.6 HIV status      Positive 43 12.7  Negative 200 59.0  Unknown 96 28.3 Patient category      New 302 89.1  Relapse 13 3.8  Failure 2 0.6  Transfer in 18 5.3  Unknown 4 1.2 Baseline smear result      Smear positive 94 27.7  Smear negative 285 72.3 Table 2 Patients characteristics and type of TB at Debre Tabor Health Center, 2008–2013. Variables Type of TB Smear positive PTB (%) Smear negative PTB (%) EPTB (%) Total (%) Sex          Male 55 (28.0) 71 (36.0) 71 (36.0) 197 (58.1)  Female 39 (27.5) 42 (29.6) 61 (43.0) 142 (41.9) Residence          Urban 52 (25.2) 70 (34) 84 (40.8) 206 (60.8)  Rural 42 (31.6) 43 (32.3) 48 (36.1) 133 (39.2) Age group          ≤24 24 (23.5) 31 (30.4) 47 (46.1) 102 (30.1)  25–44 49 (35.0) 42 (30.0) 49 (35.0) 140 (41.3)  ≥45 21 (21.6) 40 (41.2) 36 (37.1) 97 (28.6) HIV status          Positive 7 (16.3) 14 (32.6) 22 (51.2) 43 (12.7)  Negative 60 (30.0) 63 (31.5) 77 (38.5) 200 (59.0)  Unknown 27 (28.1) 36 (37.5) 33 (34.4) 96 (28.3) Patient category          New 80 (26.5) 101 (33.4) 121 (40.1) 302 (89.1)  Relapse 11 (84.6) 2 (15.4) 0 (00.0) 13 (3.8)  Failure 2 (100%) 0 (00.0) 0 (00.0) 2 (0.6)  Transferred in 1 (5.6) 9 (50.0) 8 (44.4) 18 (5.3)  Unknown 0 (00.0) 1 (25.0) 3 (75.0) 4 (1.2)  Total 94 (27.7) 113 (33.3) 132 (38.9) 339 (100) Table 3 Characteristics and treatment outcome of TB patients at Debre Tabor, 2008–2013. Variables Treatment outcome Cured (%) Completed (%) Failed (%) Defaulted (%) Died (%) Transferred out (%) Sex              Male 37 (18.8) 103 (52.3) 9 (4.6) 6 (3.0) 14 (7.1) 28 (14.2)  Female 30 (21.1) 94 (66.2) 3 (2.1) 2 (1.4) 5 (3.5) 8 (5.6) Residence              Urban 37 (18.0) 121 (58.7) 8 (3.9) 6 (3.0) 15 (7.3) 19 (9.2)  Rural 30 (22.6) 76 (57.1) 4 (3.0) 2 (1.5) 4 (3.0) 17 (12.8) Age              ≤24 17 (16.7) 63 (61.8) 4 (3.9) 3 (2.9) 1 (1.0) 14 (13.7)  25–44 38 (27.1) 80 (57.1) 3 (2.1) 2 (1.4) 7 (5.0) 10 (7.1)  ≥45 12 (12.4) 54 (55.7) 5 (5.2) 3 (3.1) 11 (11.3) 12 (12.4) Type of TB              Smear positive PTB 65 (69.1) 3 (3.2) 10 (10.6) 1 (1.1) 2 (2.1) 13 (13.8)  Smear negative PTB 1 (0.9) 93 (82.3) 1 (0.9) 0 (0.0) 10 (8.8) 8 (7.1)  Extrapulmonary TB 1 (0.8) 101 (76.5) 1 (0.8) 7 (5.3) 7 (5.3) 15 (11.4) HIV status              Positive 5 (11.6) 30 (69.8) 2 (4.7) 2 (4.7) 2 (4.7) 2 (4.7)  Negative 43 (21.5) 117 (58.5) 9 (4.5) 3 (1.5) 7 (3.5) 21 (10.5)  Unknown 19 (19.8) 50 (52.1) 1 (1.0) 3 (3.1) 10 (10.4) 13 (13.5) Patient category at start              New 59 (19.5) 175 (57.9) 8 (2.6) 8 (2.6) 17 (5.6) 35 (11.6)  Relapse 6 (46.2) 2 (15.4) 3 (23.1) 0 (0.0) 2 (15.4) 0 (0.0)  Failure 1 (50.0) 0 (0.0) 1 (50.0) 0 (0.0) 0 (0.0) 0 (0.0)  Transferred in 1 (5.6) 16 (88.9) 0 (0.0) 0 (0.0) 0 (0.0) 1 (5.6)  Unknown 0 (0.0) 4 (100.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)  Total 67 (19.8) 197 (58.1) 12 (3.5) 8 (2.4) 19 (5.6) 36 (10.6) Table 4 Factors associated with treatment outcome of TB patients in Debre Tabor, 2008–2013, N = 303. Variables Treatment outcome COR (95% CI) P value AOR (95% CI) P value Successful (%) Unsuccessful (%) Sex              Male 144 (83.2) 29 (16.8) 1.00   1.00    Female 120 (92.3) 10 (7.7) 0.414 (0.194–0.883) 0.023 0.347 (0.132–0.917) 0.033 Residence              Urban 158 (85.0) 29 (15.0) 1.00   1.00    Rural 106 (84.1) 10 (7.9) 0.514 (0.240–1.099) 0.086 0.342 (0.118–0.986) 0.047 Age in groups              ≤24 80 (90.9) 8 (9.1) 1.00   1.00    25–44 118 (90.8) 12 (9.2) 1.017 (0.398–2.600) 0.972 1.041 (0.298–3.634) 0.949  ≥45 66 (77.6) 19 (22.4) 2.879 (1.185–6.996) 0.020 3.807 (1.155–12.544) 0.028 Type of TB              Smear positive PTB 68 (84.0) 13 (16.0) 1.00   1.00    Smear negative PTB 94 (89.5) 11 (10.5) 0.612 (0.259–1.449) 0.264 0.240 (0.034–1.724) 0.156  Extrapulmonary TB 102 (87.2) 15 (12.8) 0.796 (0.344–1.718) 0.522 0.355 (0.049–2.603) 0.308 HIV status              Negative 160 (89.4) 19 (10.6) 1.00   1.00    Positive 35 (85.4) 6 (14.6) 1.444 (0.537–3.877) 0.466 1.155 (0.322–4.146) 0.825  Unknown 69 (83.1) 14 (16.9) 1.709 (0.810–3.602) 0.159 1.558 (0.312–7.769 0.589 Patient category              New 234 (87.6) 33 (12.4) 1.00   1.00    Relapse 8 (61.5) 5 (38.5) 4.432 (1.368–14.355) 0.013 3.906 (0.470–32.483) 0.207  Failure 1 (50.0) 1 (50.0) 7.091 (0.433–116.103) 0.170      Transferred in 17 (100.0) 0 (0.00)          Unknown 4 (100.0) 0 (0.00)         Baseline smear result              Smear positive 68 (84.0) 13 (16.0) 1.0        Smear negative 196 (88.3) 26 (12.9) 0.694 (0.338–1.426) 0.320 0.460 (0.071–2.963) 0.414 Smear result at 2nd month              Positive 3 (30.0) 7 (70.0) 1.00   1.00    Negative 63 (94.0) 4 (6.0) 0.027 (0.005–0.147) 0.000 0.056 (0.005–0.577) 0.015  Not done 198 (87.6) 28 (12.4) 0.061 (0.015–0.248) 0.000 0.140 (0.004–5.559) 0.296 Year of treatment              May 2008–April 2009 55 (83.3) 11 (16.7) 2.850 (0.856–9.489) 0.088 2.726 (0.319–23.266) 0.359  May 2009–April 2010 55 (80.9) 13 (19.1) 3.368 (1.035–10.965) 0.044 3.650 (0.806–16.522) 0.093  May 2010–April 2011 43 (87.8) 6 (12.2) 1.988 (0.528–7.485) 0.310 3.624 (0.701–18.727) 0.124  May 2011–April 2012 54 (91.5) 5 (8.5) 1.319 (0.336–5.174) 0.691 1.637 (0.302–8.880) 0.568  May 2012–April 2013 57 (93.4) 4 (6.6) 1.00   1.00    Total 264 (87.1) 39 (12.9)         Table 5 Comparison of TSR in Debre Tabor with the national TSR, 2008–2013, N = 303. Year of treatments Treatment success TSR in DTHC National TSR Success (N) Unsuccessful (N) May 2008–April 2009 55 11 83.3 84a May 2009–April 2010 55 13 80.9 84b May 2010–April 2011 43 6 87.8 83c May 2011–April 2012 54 5 91.5 86d May 2012–April 2013 57 4 93.4 91e Total 264 39 87.1   a: WHO 2010, b: WHO 11, c: WHO 2012, d: WHO 2013, and e: WHO 2014. ==== Refs 1 World Health Organization (WHO) Global Tuberculosis Report, 2015 2 World Health Organization (WHO) Global tuberculosis control: surveillance, planning and financing WHO Report 2008 WHO/HTM/TB/2008.393 Geneva, Switzerland WHO 3 Tessema B. Muche A. Bekele A. Reissig D. Emmrich F. Sack U. Treatment outcome of tuberculosis patients at Gondar University Teaching Hospital, Northwest Ethiopia. A five-year retrospective study BMC Public Health 2009 9, article 371 10.1186/1471-2458-9-371 2-s2.0-70449360341 4 Tessema B. Beer J. Emmrich F. Sack U. Rodloff A. C. Analysis of gene mutations associated with isoniazid, rifampicin and ethambutol resistance among Mycobacterium tuberculosis isolates from Ethiopia BMC Infectious Diseases 2012 12, article 37 10.1186/1471-2334-12-37 2-s2.0-84856771328 5 Ministry of Health of Ethiopia (MOH) Guidelines for Clinical and Programmatic Management of TB, TB/HIV and Leprosy 2013 5th 6 Alebachew Z. Kebede A. Tsegaye F. First Ethiopian National Population Based Tuberculosis Prevalence Survey July 2011 7 Federal Ministry of Health: overview of national TB control implementation status 2011 Proceedings of the 6th National TB Research Workshop June 2011 Gondar, Ethiopia Gondar University 8 Shargie E. B. Lindtjørn B. DOTS improves treatment outcomes and service coverage for tuberculosis in South Ethiopia: a retrospective trend analysis BMC Public Health 2005 5, article 62 10.1186/1471-2458-5-62 2-s2.0-23044442161 9 Mekonnen D. Derbie A. Desalegn E. TB/HIV co-infections and associated factors among patients on directly observed treatment short course in Northeastern Ethiopia: a 4 years retrospective study BMC Research Notes 2015 8 1, article 666 10.1186/s13104-015-1664-0 2-s2.0-84947869468 10 Beza M. G. Wubie M. T. Teferi M. D. A five years tuberculosis treatment outcome at kolla diba health center, dembia district, northwest ethiopia: a retrospective cross-sectional analysis Journal of Infectious Diseases and Therapy 2013 1, article 101 10.4172/2332-0877.1000101 11 Biadglegne F. Anagaw B. Debebe T. A retrospective study on the outcomes of tuberculosis treatment in Felege Hiwot referral hospital, Northwest Ethiopia International Journal of Medicine and Medical Sciences 2013 5 2 85 91 12 Getahun B. Ameni G. Medhin G. Biadgilign S. Treatment outcome of tuberculosis patients under directly observed treatment in Addis Ababa, Ethiopia Brazilian Journal of Infectious Diseases 2013 17 5 521 528 10.1016/j.bjid.2012.12.010 2-s2.0-84884710331 23830051 13 Endris M. Moges F. Belyhun Y. Woldehana E. Esmael A. Unakal C. Treatment outcome of tuberculosis patients at enfraz health center, Northwest Ethiopia: a five-year retrospective study Tuberculosis Research and Treatment 2014 2014 7 726193 10.1155/2014/726193 14 Muñoz-Sellart M. Cuevas L. E. Tumato M. Merid Y. Yassin M. A. Factors associated with poor tuberculosis treatment outcome in the Southern Region of Ethiopia International Journal of Tuberculosis and Lung Disease 2010 14 8 973 979 2-s2.0-77954726957 20626941 15 Gelaw M. Genebo T. Dejene A. Lemma E. Eyob G. Attitude and social consequences of tuberculosis in Addis Ababa, Ethiopia East African Medical Journal 2001 78 7 382 388 10.4314/eamj.v78i7.9014 2-s2.0-0035411256 11957266 16 Berhe G. Enquselassie F. Aseffa A. 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==== Front Oxid Med Cell LongevOxid Med Cell LongevOMCLOxidative Medicine and Cellular Longevity1942-09001942-0994Hindawi Publishing Corporation 10.1155/2016/7813072Review ArticleRole of NADPH Oxidase in Metabolic Disease-Related Renal Injury: An Update http://orcid.org/0000-0002-4129-6413Wan Cheng Su Hua http://orcid.org/0000-0003-3565-8024Zhang Chun * Department of Nephrology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430022, China*Chun Zhang: drzhangchun@hust.edu.cnAcademic Editor: Juan F. Santibanez 2016 15 8 2016 2016 78130722 6 2016 17 7 2016 Copyright © 2016 Cheng Wan et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Metabolic syndrome has been linked to an increased risk of chronic kidney disease. The underlying pathogenesis of metabolic disease-related renal injury remains obscure. Accumulating evidence has shown that NADPH oxidase is a major source of intrarenal oxidative stress and is upregulated by metabolic factors leading to overproduction of ROS in podocytes, endothelial cells, and mesangial cells in glomeruli, which is closely associated with the initiation and progression of glomerular diseases. This review focuses on the role of NADPH oxidase-induced oxidative stress in the pathogenesis of metabolic disease-related renal injury. Understanding of the mechanism may help find potential therapeutic strategies. National Natural Science Foundation of China8117066231200872814709648157067181522010Wuhan Science and Technology Bureau2015060101010039Specialized Research Fund for the Doctoral Program of Higher Education of China20130142110064 ==== Body 1. Introduction Metabolic syndrome is a constellation of interconnected risk factors for cardiovascular diseases and type 2 diabetes, including dyslipidemia, hypertension, hyperglycemia, abdominal obesity, and insulin resistance [1, 2]. Along with cardiovascular diseases and type 2 diabetes, accumulating evidence shows that metabolic syndrome contributes to an increased risk of microalbuminuria and/or chronic kidney disease (CKD) [3–7]. However, it remains unclear whether there is a definitive cause-and-effect relationship between metabolic syndrome and renal injury. Research on the underlying pathogenesis of metabolic disease-related renal injury has suggested an important role of oxidative stress, which is a result of reactive oxygen species (ROS) overproduction, mitochondrial dysfunction, and/or impaired antioxidant system [8]. There are numerous intrarenal sources of ROS, such as mitochondrial electron transport chain, xanthine oxidase, and uncoupled nitric oxide (NO) synthase, while nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is generally accepted as the major producer [9–13]. NADPH oxidases are multisubunit enzymes composing membrane and cytosolic components that transfer electrons across biological membranes. There are seven members in the Nox family of NADPH oxidase, including Nox1–Nox5 and dual oxidases, Duox1 and Duox2, with different activation mechanisms and tissue distribution [13–16]. The Nox homologues are widely expressed throughout the kidney. Nox1, Nox2, Nox4, and Nox5 are predominantly expressed in glomerular endothelial cells, tubulointerstitial cells, and glomerular cells, that is, mesangial cells and glomerular epithelial cells [17]. Various homologue-specific mechanisms regulate the activity of the Nox family involving a complex series of protein/protein interactions, phosphorylation and translocation of its subunits, and Rac activation. Numerous stimuli and agonists like transforming growth factor-β (TGF-β), angiotensin II (Ang II), hyperglycemia, oxidized low density lipoprotein (oxLDL), insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), and aldosterone are capable of upregulating the activity and/or the expression of NADPH oxidases, subsequently leading to overproduction of ROS including the immediate product superoxide and the following hydrogen peroxide. The proposed functions of NADPH oxidase-derived ROS in the kidney are mainly regulation of renal blood flow, alteration of cell fate, and regulation of gene expression. Superoxide avidly reacts with nitric oxide (NO) limiting its relaxing effect on afferent arterioles and mediates the activation of inflammasome, while hydrogen peroxide is involved in the activation of protein tyrosine kinases, phospholipases, serine/threonine kinases, and so forth, resulting in enhanced epithelial-to-mesenchymal transition (EMT), apoptosis of podocytes, and promotion of cellular hypertrophy [18–25]. The present review will focus on the role of NADPH oxidase-induced oxidative stress in the pathogenesis of metabolic disease-related renal injury. 2. NADPH Oxidase and Diabetic Nephropathy Diabetic nephropathy (DN) is the major complication of type 1 and type 2 diabetes and is one of the leading causes of end-stage renal disease (ESRD) [26]. It is characterized by functional deficits with proteinuria and decreased glomerular filtration, as well as structural changes, such as loss of podocytes, proliferation and expansion of mesangial cells and matrix, thickening of glomerular and tubular basement membranes, tubular atrophy, interstitial fibrosis, and arteriosclerosis. Increasing evidence has demonstrated that NADPH oxidase-induced oxidative stress plays a pivotal role in the initiation and development of DN [11, 27]. Blockade of NADPH oxidase-derived ROS generation ameliorates diabetes-induced glomerular injury via reducing podocyte loss, proteinuria, glomerular hypertrophy, and mesangial matrix expansion [28–33]. Damage and depletion of podocytes due to apoptosis occur during early DN, presented as actin cytoskeleton rearrangement, podocyte foot process effacement, and slit diaphragm disruption [34]. Studies have highlighted the role of podocytes in DN pathogenesis and revealed the upregulation of the NADPH oxidase subunits expression, predominantly Nox4 and Nox1, in type 1 diabetic OVE26 mice and type 2 diabetic db/db mice, following excessive ROS generation and podocytes apoptosis which contributes to albuminuria [20, 35–37]. In vitro studies also have shown that high glucose induced the upregulation of NADPH oxidase expression, enhancement of NADPH oxidase activity, and apoptosis induction in podocytes at later time points [35, 37–39]. Eid et al. found that the increase of Nox4 expression was attributed to the inactivation of AMP-activated protein kinase (AMPK), and Nox4 promoted podocyte apoptosis via p53- and PUMA-dependent apoptotic pathway in high glucose condition [35, 40]. Other NADPH oxidase subunits, such as Nox2, p22phox, and p67phox, are also expressed on podocytes. However, very little is known concerning the regulation of these subunits in the presence of high glucose [11, 41–43]. Besides podocyte injury, two other morphological alterations during early DN are mesangial matrix accumulation and cell hypertrophy leading to thickening of glomerular basement membrane [27, 44]. The important role of NADPH oxidase in mesangial cell injury has been demonstrated in experimental models of diabetes as well as in cultured cells exposed to high glucose, while the molecular mechanisms remain speculative. High glucose induces upregulation of Nox4 and p22phox expression in mesangial cells as well as in diabetic kidney, and Nox4 and p22phox mediate cell hypertrophy and fibronectin expression [12, 45–48]. Since p22phox interacts with Nox4 and enhances its activity, Gorin and Wauquier suggested that p22phox and Nox4 might form a complex that contributed to high glucose-dependent oxidative stress and the subsequent fibrotic processes [13]. The role of other NADPH oxidase subunits in mesangial cell injury has been less studied and the findings are controversial. Furthermore, NADPH oxidase also mediates the ROS generation induced by other mediators in DN such as Ang II and TGF-β [42, 49–51]. Induced by Ang II, an acute increase and prolonged upregulation of Nox4 expression both take place in mesangial cells, and Nox4 mediates ROS generation leading to activation of signalling, for instance, extracellular signal-regulated kinase-1/2 (ERK1/2) [52], Akt/protein kinase B (Akt/PKB) [50], and proline-rich tyrosine kinase-2 (Pyk-2)/Src/3-phosphoinositide-dependent protein kinase-1 (PDK-1) [22], which results in hypertrophy and increased fibronectin expression. Induced by TGF-β, Nox4 expression within mitochondria in podocytes is upregulated via the Sma and Mad homologue (Smad) 2/3 pathway and ultimately results in ROS overproduction, mitochondrial dysfunction, and podocyte apoptosis [53, 54]. 3. NADPH Oxidase and Hyperhomocysteinemia-Associated Glomerular Injury Hyperhomocysteinemia (hHcys) is defined as a pathological condition characterized by abnormal elevation of homocysteine (Hcys) plasma concentration and has been considered as a pivotal independent risk factor in the development of progressive glomerulosclerosis and/or ESRD [55–57]. Previous evidence has revealed that Hcys induces endothelial injury, vascular smooth muscle cells proliferation, and extracellular matrix (ECM) metabolism disturbance [58–61]. Considering the similarity of pathological alterations between Hcys-induced arterial injury and glomerular injury, the role of hHcys in glomerulosclerosis has been verified. Although the mechanism by which Hcys induces glomerular injury remains poorly understood, there is evidence that NADPH oxidase-derived oxidative stress is involved in the development of glomerular injury induced by Hcys [62–65]. An experimental model of hHcys was reported to develop glomerulosclerosis, characterized by local oxidative stress, podocyte dysfunction, mesangial expansion, and fibrosis, which could be significantly attenuated by treatment of NADPH oxidase inhibitors [64]. Podocyte injury is a critical early event leading to glomerulosclerosis. It has been revealed that Hcys induces podocyte damage and slit diaphragm disruption, causing proteinuria and glomerular sclerosis [66]. Zhang et al. [67] found that, in mice lacking Nox2 gene, hHcys induced by folate-free diet led to less severe podocyte injury and glomerulosclerosis, as shown by attenuated foot process effacement and podocyte loss, lower proteinuria, and glomerular damage index, as well as higher glomerular filtration rate. Thus, NADPH oxidase is suggested to be essential for Hcys-induced podocyte injury and glomerulosclerosis. Furthermore, Hcys stimulation was documented to upregulate NOX2 and p47phox expression and induce their aggregation in lipid raft (LR) clusters in podocytes, while disrupting LR clustering markedly blocked the enrichment of the NADPH oxidase subunits, decreased the enzyme activity, and functionally attenuated Hcys-induced podocyte injury. These findings indicate that NADPH oxidase subunits aggregation and activation through LR clustering are important molecular mechanisms in Hcys-induced podocytes injury [68]. Hcys is also confirmed to induce podocytes to undergo EMT and inflammasome activation through NADPH oxidase-derived oxidative stress, which consequently leads to glomerular injury and sclerosis [69–71]. Ingram's research group and others also have clarified that Hcys induces alterations of ECM metabolism in mesangial cells, another important event leading to glomerulosclerosis and loss of renal function [72]. Hcys was reported to upregulate tissue inhibitor of metalloproteinase-1 and induce collagen type I accumulation, accompanied by enhanced cell proliferation and NADPH oxidase activity in rat mesangial cells [73]. Hcys-induced activation of NADPH oxidase is suggested to be mediated by enhanced ceramide synthesis and the subsequent increase of Rac GTPase activity [74]. There is also evidence showing that the N-methyl-D-aspartate (NMDA) receptor may mediate activation of NADPH oxidase in hHcys-associated glomerular injury [75]. In addition, Hcys has been found to cause mesangial apoptosis via oxidative stress and p38-mitogen-activated protein kinase activation, thereby suggesting another underlying mechanism of hHcys-associated glomerular injury [63]. 4. NADPH Oxidase and Hyperlipidemia-Associated Glomerular Injury The concern of the association between hyperlipidemia and renal diseases may date back to the 19th century. Since then, accumulating evidence in experimental findings and clinical observations has suggested an important role of hyperlipidemia in the progression of glomerulosclerosis [76–81]. Hyperlipidemia-associated glomerular injury is mainly characterized by lipid or lipoprotein deposition, macrophage infiltration, and mesangial expansion. As with other metabolic factors, such as hyperglycemia and hHcys, oxidative stress is proved to contribute to the deleterious effects of hyperlipidemia on renal injury. In high-fat diet fed mice, the expression of NADPH oxidase subunits, including p47phox, Nox2, and p67phox, was significantly upregulated, and the inhibitor could ameliorate hyperlipidemia-induced endothelial dysfunction via inhibition of NADPH oxidase expression [82]. However, in the study of Scheuer et al., it is reported that xanthine oxidoreductase rather than NADPH oxidase mainly accounted for the generation of ROS in glomeruli and tubulointerstitium induced by hyperlipidemia [83]. In addition, Joles et al. clarified that both hypercholesterolemia and hypertriglyceridemia aggravated renal injury predominantly via podocytes, accompanied by activation and injury of tubulointerstitial cells, lacking evidence of mesangial activation, proliferation, or matrix accumulation [80]. Furthermore, hyperlipidemia often coexists with other metabolic syndrome components and accelerates the progression of glomerular injury together [84, 85]. 5. NADPH Oxidase and Hyperuricemia-Related Kidney Disease Uric acid (UA) is an intermediate product in the purine degradation pathway in cells but is the final product of purine catabolism in humans, due to the loss of uricase activity during hominoid evolution [86]. The role of UA in CKD remains controversial, and the “UA debate” has been going on for decades [87]. UA has been considered as a major antioxidant in protecting cells from oxidative injury proved by abundant experimental and clinical evidence [88]. On the other hand, epidemiologic evidence and experimental models also have shown that hyperuricemia may impose detrimental effects as a prooxidant [89–92]. UA is often associated with other risk factors of CKD, including diabetes, hypertension, and inflammation [93], which makes it difficult to dissect the role of UA itself in the progression of CKD. However, a recent study showed an association between hyperuricemia and renal damage independently of hypertension and intrarenal renin-angiotensin system (RAS) activation [94]. In the past, hyperuricemia was thought to cause kidney disease by a crystal-dependent mechanism. The crystal of monosodium urate may induce potassium efflux, lysosomal rupture, and mitochondrial ROS production, which provoke inflammasome and induce the secretion of proinflammatory cytokines, eventually causing inflammation and renal injury. The crystal-independent mechanism of hyperuricemia-related kidney disease remains poorly understood. The main pathophysiological mechanisms of hyperuricemia-related kidney disease include endothelial dysfunction, activation of local RAS, oxidative stress, and proinflammatory and proliferative effects. NADPH oxidase is suggested to play a role in the pathogenesis of hyperuricemia-related kidney disease, as with other metabolic disease-related renal injuries. It has been revealed that hyperuricemia is associated with endothelial dysfunction, due to oxidative stress with activation of RAS and a decrease of NO bioavailability [95, 96]. In an experimental model of hyperuricemia, enhanced intrarenal oxidative stress, increased expression of NOX-4 and Ang II, and decreased NO bioavailability were observed [97]. The aging and apoptosis of endothelial cells induced by hyperuricemia were ameliorated by antioxidants [98]. Furthermore, there is evidence that mitochondrial alterations and decreased intracellular ATP are implicated in UA-induced endothelial dysfunction [99]. In cultured renal tubular cells, it has been shown that UA induces EMT and apoptosis of renal tubular cells which is ameliorated by antioxidants, suggesting a detrimental role of oxidative stress [100]. 6. NADPH Oxidase and Obesity-Related Kidney Disease Oxidative stress is also associated with other metabolic kidney diseases such as obesity-related kidney disease [101]. It is well documented that the glomerular scarring in obesity-associated focal segmental glomerulosclerosis is driven by podocyte injury, which may partly be a result of the NADPH oxidase-derived oxidative stress induced by upregulated Ang II and TGF-β [102]. There is supplemental data supporting the fact that NADPH oxidase-mediated oxidative injury to the proximal tubule contributes to proteinuria in obese rats [103]. In addition, oxidative stress is demonstrated to play a role in the pathogenesis of renal injury through its contribution to progressive vascular dysfunction and remodeling [104, 105]. Collectively, NADPH oxidase-derived oxidative stress is suggested to trigger the progression of obesity-related kidney disease. 7. Conclusion The NADPH oxidase is widely expressed throughout the kidney and is a major source of intrarenal oxidative stress. Metabolic stimuli elicit the upregulation of NADPH oxidase expression and the enhancement of NADPH oxidase activity. As depicted in Figure 1, ROS generated by NADPH oxidase plays a pivotal role in the pathogenesis of glomerular diseases related to metabolic diseases. Hence, approaches to reduce oxidative stress by antioxidants may be potential therapies to prevent and treat metabolic disease-related renal injury. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (no. 81170662, no. 31200872, no. 81470964, no. 81570671, and no. 81522010), a grant from Wuhan Science and Technology Bureau (no. 2015060101010039), and Specialized Research Fund for the Doctoral Program of Higher Education of China (no. 20130142110064). Competing Interests The authors declare that they have no competing interests. Figure 1 NADPH oxidase-derived ROS in the pathogenesis of metabolic disease-related renal injury. Metabolic stimuli may upregulate the expression of NADPH oxidase and enhance the activity of NADPH oxidase, which subsequently leads to overproduction of ROS. NADPH oxidase-derived oxidative stress is involved in podocyte injury, endothelial dysfunction, mesangial proliferation, and so forth, eventually resulting in renal injury. NADPH: nicotinamide adenine dinucleotide phosphate; ROS: reactive oxygen species; NO: nitric oxide. ==== Refs 1 Kassi E. 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==== Front Mediators InflammMediators InflammMIMediators of Inflammation0962-93511466-1861Hindawi Publishing Corporation 10.1155/2016/8675905Research ArticleNovel Role of Endogenous Catalase in Macrophage Polarization in Adipose Tissue http://orcid.org/0000-0001-6718-5482Park Ye Seul http://orcid.org/0000-0003-2911-3255Uddin Md Jamal http://orcid.org/0000-0002-3781-875XPiao Lingjuan http://orcid.org/0000-0002-4479-9374Hwang Inah http://orcid.org/0000-0003-1423-3895Lee Jung Hwa http://orcid.org/0000-0002-5601-1265Ha Hunjoo * Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 120-750, Republic of Korea*Hunjoo Ha: hha@ewha.ac.krAcademic Editor: Wilco de Jager 2016 15 8 2016 2016 867590528 3 2016 11 6 2016 29 6 2016 Copyright © 2016 Ye Seul Park et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Macrophages are important components of adipose tissue inflammation, which results in metabolic diseases such as insulin resistance. Notably, obesity induces a proinflammatory phenotypic switch in adipose tissue macrophages, and oxidative stress facilitates this switch. Thus, we examined the role of endogenous catalase, a key regulator of oxidative stress, in the activity of adipose tissue macrophages in obese mice. Catalase knockout (CKO) exacerbated insulin resistance, amplified oxidative stress, and accelerated macrophage infiltration into epididymal white adipose tissue in mice on normal or high-fat diet. Interestingly, catalase deficiency also enhanced classical macrophage activation (M1) and inflammation but suppressed alternative activation (M2) regardless of diet. Similarly, pharmacological inhibition of catalase activity using 3-aminotriazole induced the same phenotypic switch and inflammatory response in RAW264.7 macrophages. Finally, the same phenotypic switch and inflammatory responses were observed in primary bone marrow-derived macrophages from CKO mice. Taken together, the data indicate that endogenous catalase regulates the polarization of adipose tissue macrophages and thereby inhibits inflammation and insulin resistance. National Research Foundation of Korea2012R1A2A1A03006922015H1D3A1062189 ==== Body 1. Introduction Excess body weight and obesity have become worldwide epidemics, resulting in detrimental health problems including hypertension, atherosclerosis, cancer, type 2 diabetes mellitus, and insulin resistance [1]. The development and progression of these diseases are linked to chronic and low-grade inflammation in adipose tissue [2–5]. In turn, inflammation is promoted or resolved by macrophages, which polarize into classically (M1) or alternatively (M2) activated macrophages depending on environmental stimuli [5–7]. Accordingly, macrophages have been proposed as components of adipose tissue inflammation [8, 9]. Obesity not only increases the number of adipose tissue macrophages but also induces a phenotypic switch [10]. In lean adipose tissue, resident macrophages are polarized toward M2 and thus express F4/80, CD301, CD206, interleukin-10 (IL-10), and arginase-1 [11, 12]. M2 macrophages help maintain adipose tissue function by preventing inflammation and promoting insulin sensitivity [13]. In contrast, macrophages in obese adipose tissue are predominantly M1 and express F4/80, CD11c, tumor necrosis factor-α (TNF-α), IL-6, and inducible nitric oxide synthase (iNOS) [11]. These M1 macrophages also secrete proinflammatory cytokines that interfere with insulin signaling and form crown-like structures to phagocytize dead adipocytes [14–19]. In addition, excess glucose and fatty acids in obese adipose tissue may increase the mitochondrial electron flux and impair mitochondrial respiratory capacity, resulting in an overload of reactive oxygen species (ROS) [20]. Oxidative stress due to these species then accelerates insulin resistance, promotes the development metabolic disorders [21], and induces the phenotypic switch in adipose tissue macrophages [22]. Catalase is a well-recognized antioxidant enzyme, and overexpression protects transgenic mice against inflammation-associated injury, including atherosclerosis [23] and diabetic kidney disease [24, 25]. In addition, forced overexpression of catalase in mitochondria protects insulin-producing β-cells against injury from ROS and against toxicity from proinflammatory cytokines [26]. On the other hand, catalase deficiency induces oxidant-mediated tissue injury [27], increases mitochondrial ROS in mice with excess body weight, and accelerates diabetic kidney injury [28]. However, the relationship between catalase and macrophage activity is not well characterized. In J774.16 macrophages, exogenous catalase reduces nitric oxide production in response to lipopolysaccharides (LPS) and interferon-γ [29]. In human alveolar macrophages, exposure to 3-aminotriazole, a catalase inhibitor, increases basal H2O2, activates p38 MAP kinase, and elicits inflammatory responses [30]. However, the role of macrophage catalase in obesity-induced inflammation of the adipose tissue remains to be studied. Thus, we used macrophage cultures and a mouse model to examine the effects of catalase deficiency on the polarization of adipose tissue macrophages. We found that catalase deficiency increases the M1/M2 ratio in mice on both normal diet (ND) and high-fat diet (HFD) and thereby enhances inflammation and insulin resistance in adipose tissue. 2. Materials and Methods 2.1. Reagents and Chemicals Dulbecco's Modified Eagle Medium (DMEM) and penicillin-streptomycin were purchased from Life Technologies (Carlsbad, CA, USA). Fetal bovine serum (FBS) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). All other chemicals were obtained from Sigma-Aldrich unless otherwise noted. 2.2. Animals Animals were housed at 22 ± 2°C on a 12-hour dark/12-hour light cycle and were provided with tap water ad libitum unless indicated otherwise. Male catalase wild type (WT) and catalase knockout (CKO) C57BL/6 J mice [27] were provided by Professors Rhee and Woo at Ewha Womans University. Mice were fed for 21 weeks with either ND or HFD. In ND, 28%, 54%, and 18% of calories are derived from protein, carbohydrate, and fat, respectively (Harlan Teklad 2018S, Indianapolis, IN, USA), while 18.4%, 21.3%, and 60.3% of calories in HFD are derived from protein, carbohydrate, and fat, respectively (Harlan TD06414). Plasma insulin was measured in blood collected from the orbital sinus a day before sacrifice at 21 weeks. For in vivo insulin stimulation and analysis of insulin signaling in adipose tissue, mice were fasted overnight and anesthetized with 16.5% urethane (10 mL/kg). Abdominal cavities were opened, and the right section of epididymal white adipose tissue (WAT) was rapidly excised for analysis of insulin signaling. Mice were then injected via the inferior vena cava with 10 U/kg Humulin® R (Eli Lilly, Indianapolis, IN, USA). All animal experiments were approved by the Institutional Animal Care and Use Committee at Ewha Womans University (number 2013-01-011). 2.3. Blood Parameters Blood samples were centrifuged at 3,000 rpm for 15 min at 4°C, and plasma in the supernatant was collected. Plasma triglycerides, low-density lipoprotein (LDL)/very low-density lipoprotein (VLDL), high-density lipoprotein (HDL), and free fatty acids were measured using EnzyChrom™ colorimetric assay kits (BioAssay Systems, Hayward, CA, USA) according to the manufacturer's instructions. Commercial ELISA kits (R&D Systems) were used to measure plasma insulin following the manufacturer's protocol. 2.4. Isolation of Stromal Vascular Fraction Epididymal fat pads were collected and minced in phosphate-buffered saline (PBS) supplemented with 1% bovine serum albumin and 0.2 mg/mL DNase I. Tissues were then digested with 2 mg/mL collagenase for 20 min at 37°C and 400 rpm shaking, filtered through 100 μm, and centrifuged at 300 ×g for 5 min to separate floating adipocytes from the pellet [11]. 2.5. Preparation of PA PA was dissolved in 50% ethanol, clarified by heating to 60°C, and added dropwise to warmed 10% fatty acid-free bovine serum albumin (45~52°C) dissolved in PBS. The pH of the mixture was adjusted to 7.0~7.4 with NaOH, and aliquots were frozen and stored at −20°C. 2.6. Bone Marrow-Derived Macrophages After euthanasia, 8-week-old C57BL/6 mice were sprayed with 70% ethanol, and femurs were dissected using scissors, cutting through the tibia below the knee joints, as well as through the pelvic bone close to the hip joint. Muscles connected to the bone were removed using clean gauze, and femurs were placed on ice in polypropylene tubes containing sterile PBS. Fresh bone marrow cells [31] were obtained by aseptically flushing bones with a syringe filled with RPMI1640 medium. Cells were resuspended in 10 mL RPMI1640 medium supplemented with 10% fetal bovine serum, 20 ng/mL macrophage colony stimulating factor, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were then seeded in non-tissue culture treated Petri dishes and incubated at 37°C in 5% CO2. Media were refreshed four days later, and cultures were incubated for an additional three days. Bone marrow-derived macrophages attached to plates were harvested with trypsin (Gibco) and treated with or without PA or IL-4 for 6 h. 2.7. Culture of RAW264.7 Macrophages RAW264.7 cells were procured from American Type Culture Collection (Manassas, VA, USA) and maintained at 37°C in humidified air with 5% CO2 and in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 44 mM NaHCO3. Cells were then grown to 60–70% confluence in 6-well plates, incubated with low-serum media for 24 h, and treated with 3-aminotriazole (3-AT) for 6 h or 24 h to inactivate catalase. The inhibitor was preincubated for 1 h in LPS or for 6 h or 24 h in PA and bovine serum albumin (BSA). Pure BSA at 0.3% was used to control for the effects of PA. 2.8. Immunohistochemistry A commercially available kit based on immunoperoxidase was used for immunohistochemistry (Dako, Glostrup, Denmark). Briefly, epididymal adipose tissues from WT and CKO mice on ND or HFD were fixed in 10% formalin, dehydrated, embedded in paraffin wax, and subsequently deparaffinized. Endogenous peroxidase was quenched for 30 min using Dako peroxidase solution, and tissue sections were then washed and blocked with Dako serum-free blocking solution. Sections were subsequently probed overnight at 4°C with antibodies against F4/80 (1 : 100, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), CD11c (1 : 200, Santa Cruz Biotechnology Inc.), CD206 (1 : 50, Santa Cruz Biotechnology Inc.), and nitrotyrosine (1 : 200, Santa Cruz Biotechnology Inc.). After washing in phosphate-buffered saline, sections were processed with a LSAB2 Kit (Dako) and labeled with 3,3′-diaminobenzidine for 4 min. Digital images were captured on a Zeiss microscope equipped with an Axio Cam HRC digital camera and software (Carl Zeiss, Thornwood, NY, USA) and analyzed with the open-source program ImageJ v1.34s (Rasband, WS, ImageJ, US National Institutes of Health, Bethesda, MD, USA). 2.9. Western Blot Adipose tissue homogenates or harvested cells were analyzed by western blot to assess phosphorylation of c-Jun N-terminal kinase (JNK) at Thr183 and Tyr185, as well as expression of t-JNK and iNOS [28]. Briefly, adipose tissues were lysed in TGN buffer and centrifuged at 13,000 rpm and 4°C for 15 min. The protein concentration in lysates was determined by Bradford method (Bio-Rad Laboratories, Hercules, CA, USA). Samples of tissues and cell extracts were mixed with buffer containing SDS and mercaptoethanol, heated at 95°C for 5 min, resolved by SDS-PAGE, and transferred onto a polyvinylidene fluoride membrane (GE Healthcare BioSciences Co., Piscataway, NJ, USA) in a transblot chamber with Tris buffer. The membrane was blocked for 1 h at room temperature with 5% skim milk in TBS buffer supplemented with Tween 20 and probed overnight at 4°C with polyclonal antibodies against phosphorylated JNK (1 : 1000, Cell Signaling Technology), t-JNK (1 : 2000, Cell Signaling Technology), iNOS (1 : 2000, Santa Cruz), and β-actin (1 : 2000, Santa Cruz). Membranes were then washed, labeled for 1 h at room temperature with peroxidase-conjugated secondary antibody, washed another few times, and visualized with enhanced chemiluminescence detection reagent (GE Healthcare BioSciences Co.) according to the manufacturer's instructions. Immunoreactive bands were quantified by densitometry using ImageJ software and normalized to t-JNK and β-actin. 2.10. Real-Time RT-PCR Real-time RT-PCR was used to measure expression of arginase-1, CD11c, CD206, F4/80, IL-1β, IL-6, IL-10, plasminogen activator inhibitor (PAI-1), and TNF-α. Total RNA was extracted from tissues and cells using TRIzol (Life Technologies), and cDNA was synthesized as previously described [28]. mRNA expression was assessed in duplicate on an ABI 7300 real-time PCR thermal cycler (Applied Biosystems, Foster City, CA, USA) using SYBR Green PCR Master Mix Kit (Applied Biosystems) in a final volume of 20 μL. Amplified products were then quantified from standard curves using Applied Biosystems software, and expression was normalized to 18S mRNA. Primer sequences are listed in Table 1. 2.11. Statistical Analysis Data are reported as mean ± standard error. Analysis of variance was used to compare multiple groups. If the F statistic was significant, means were compared by Fisher's least significant difference method. A p value < 0.05 was considered to indicate significance. 3. Results 3.1. Metabolic Characteristics Are Altered in CKO Mice As expected, HFD increased most metabolic parameters in both WT and CKO mice, including body weight, epididymal fat mass, plasma triglycerides, LDL/VLDL, fed and fasting insulin, and homeostatic model assessment-insulin resistance (HOMA-IR) (Table 2). Surprisingly, plasma free fatty acids and triglycerides were significantly higher in CKO mice than in WT mice, even on ND (Table 2). Further, catalase deficiency accelerated insulin resistance due to HFD (Table 2). These results demonstrate that catalase deficiency causes systemic dyslipidemia in mice regardless of diet. Macrophages are not the only cells regulated by catalase, but other types of cell are also regulated by catalase. In fact, mammalian catalase is the peroxisomal protein and abundantly expressed in liver and kidney [27]. In addition, we observed catalase expression in WAT (see Supplementary Figure 1 in Supplementary Material available online at http://dx.doi.org/10.1155/2016/8675905). Thus, these organs which express catalase will be affected by catalase knockdown. Since liver and WAT play key roles in lipid metabolism and insulin resistance [32–34], the functions of these organs in CKO mice must be affected as reflected by systemic dyslipidemia and insulin resistance (Table 2). 3.2. Catalase Deficiency Accelerates Oxidative Stress, Macrophage Infiltration, and Formation of Crown-Like Structures in Epididymal WAT Oxidative stress in epididymal WAT dramatically increased in CKO mice on ND (Figure 1(a)), as measured by nitrotyrosine staining [35]. HFD further amplified this effect (Figure 1(a)). Similar trends were observed in the abundance of F4/80 mRNA, a measure of macrophage infiltration into epididymal WAT (Figure 1(b)). Accordingly, F4/80 immunostaining was higher in CKO mice than in WT mice regardless of diet. In addition, F4/80 immunostaining was stronger in mice on HFD than those on ND (Figure 1(c)). Notably, few crown-like structures, which are formed by M1 macrophages around dead adipocytes, were observed on ND. However, the number of crown-like structures per 100 adipocytes increased over 2-fold in CKO mice on HFD compared to that in WT mice on the same diet (Figure 1(d)). These findings suggest that catalase inhibits macrophage infiltration and formation of crown-like structures in epididymal WAT. 3.3. Catalase Deficiency Alters Inflammation and Macrophage Activation in Epididymal WAT In epididymal WAT, immunostaining for CD11c, a marker of classically activated M1 macrophages, was stronger in CKO mice on HFD than in WT mice (Figure 2(a)). To characterize macrophages more specifically, the stromal vascular fraction was obtained. This fraction contains macrophages, preadipocytes, and other immune cells, but not adipocytes. Expression of CD11c, TNF-α, and PAI-1 mRNA in this fraction was significantly higher in WT mice on HFD than in mice on ND (Figures 2(b)–2(d)). These markers were also more abundantly expressed in CKO mice on ND than in WT mice on the same diet (Figures 2(b)–2(d)). Further, HFD amplified expression of CD11c and PAI-1 mRNA in CKO mice (Figures 2(b) and 2(d)). On the other hand, catalase deficiency decreased the abundance of alternatively activated M2 macrophages, as assessed by CD206 staining of epididymal WAT from mice on HFD (Figure 3(a)). Accordingly, CKO significantly reduced CD206 mRNA by 0.45- and 0.58-fold in mice on ND and HFD, respectively, in comparison to WT (Figure 3(b)). Additionally, mRNA expression of arginase-1, an enzyme released from M2 macrophages, was decreased in CKO mice on ND compared to that in WT mice on the same diet (Figure 3(c)). However, expression of IL-10 mRNA was comparable among strains regardless of diet (Figure 3(d)). Collectively, the data indicate that catalase is a key regulator of inflammation and macrophage phenotype. 3.4. Genetic Knockdown of Catalase Exacerbates Palmitate-Induced Inflammation Bone marrow-derived macrophages were isolated from WT and CKO mice and exposed to 500 μM PA. In comparison to WT, CKO upregulated JNK phosphorylation with or without exposure to PA (Figure 4(a)). Accordingly, mRNA levels of IL-1β, IL-6, and TNF-α also increased in CKO cells regardless of exposure to PA (Figures 4(b)–4(d)). These data demonstrate that catalase knockdown induces basal inflammation, an effect enhanced by exposure to PA. 3.5. Pharmacological Inhibition of Catalase Induces Inflammation in RAW264.7 Macrophages To support the genetic intervention employed in vivo, RAW264.7 macrophages were treated with 3-AT, a known pharmacological inhibitor of catalase. The inhibitor boosted the abundance of phosphorylated JNK in dose-dependent manner (Figure 5(a)). Notably, we found that 5 and 10 mM 3-AT upregulated the basal mRNA expression of the proinflammatory cytokines IL-1β, IL-6, and TNF-α (Figures 5(b)–5(d)). These results suggest that inhibition of catalase induces basal inflammation. Further, western blotting showed that exposure to 200 μM PA induced phosphorylation (activation) of JNK. This effect was further enhanced by pretreatment with 2 or 5 mM 3-AT for 1 h (Figure 5(e)). Accordingly, pretreatment with 3-AT significantly accelerated PA-induced production of proinflammatory cytokines (Figures 5(f)–5(h)). These data indicate that catalase suppresses basal and PA-induced inflammation in murine macrophages. 3.6. Pharmacological or Genetic Inhibition of Catalase Alters Activation of Macrophages and Inflammation In RAW264.7 macrophages exposed to 3-AT and in primary bone marrow-derived macrophages from CKO mice, classical macrophage activation was enhanced under basal (Figures 5(a)–5(d) and 6(c)) and LPS-induced conditions, as measured by JNK phosphorylation (Figure 6(a)) and iNOS expression (Figures 6(b)–6(d)). On the other hand, 2 and 5 mM 3-AT reduced alternative activation of RAW264.7 macrophages in response to IL-4, as measured by CD206 and arginase-1 mRNA (Figures 6(e) and 6(f)). Similarly, IL-4-induced alternative activation was repressed in bone marrow-derived macrophages from CKO mice compared to that in WT macrophages (Figures 6(g) and 6(h)). Notably, basal levels of CD206 mRNA were significantly reduced in CKO macrophages compared to those in WT (Figure 3(b)), in agreement with in vivo data. Taken together, the data imply that pharmacological or genetic inhibition of catalase activity favors classical activation of macrophages and thus promotes inflammation. 4. Discussion As expected, HFD increased most metabolic parameters in both WT and CKO mice. Notably, plasma free fatty acids and triglycerides were significantly increased in CKO mice on ND compared to those in WT mice on ND, supporting the notion that endogenous catalase may play an important role in basal lipid metabolism. Considering that adipose tissue and liver are the most important organs for lipid metabolism [36] and that catalase is highly expressed in WAT as well as liver, it is speculated that catalase deficiency in WAT and liver may play critical roles in dyslipidemia in CKO mice. CKO mice on HFD presented significantly increased markers of insulin resistance, such as fed and fasting plasma insulin and HOMA-IR. In line with our data, repeated administration of catalase conjugated to polyethylene glycol improves glucose tolerance and insulin sensitivity in obese mice [37]. On the other hand, triglycerides are esters of glycerol with three fatty acid groups and constitute the main chemical form by which vertebrates store and transport lipids in the body [38]. Increased level of triglycerides is the main feature of insulin resistance which results from increased oxidative stress in WAT and liver [32–34]. While the effect of catalase deficiency on the function of adipocytes and hepatocytes remains to be studied, our preliminary data suggest that catalase deficiency also accelerates high-fat diet-induced insulin resistance in WAT and liver (data not shown). In adipose tissue, macrophages are major components of inflammation and insulin resistance [39]. Therefore, macrophage infiltration in epididymal WAT was estimated by mRNA and protein expression of the macrophage marker F4/80. Increased macrophage infiltration was observed in CKO mice compared to that in WT mice, regardless of diet. In addition, the number of crown-like structures, which are characteristic of M1 macrophages, was higher in CKO mice on HFD than in WT mice on the same diet, suggesting that endogenous catalase is key regulator of polarization in adipose tissue macrophages. Therefore, we further investigated macrophage polarization in WAT, especially in the stromal vascular fraction, which contains preadipocytes, macrophages, T-cells, mast cells, and granulocytes, but not adipocytes. We found that protein and mRNA expression of CD11c, a major surface receptor marker of M1 macrophages [40], was significantly elevated in CKO mice on ND and HFD, along with levels of the proinflammatory cytokines iNOS and TNF-α, which are secreted from M1 macrophages [40]. In addition, PAI-1 mRNA levels were significantly increased in CKO mice. We note that PAI-1 has been suggested to facilitate the development of obesity and insulin resistance [41] and is upregulated especially in WAT macrophages of mice on HFD [42]. Indeed, PAI-1 has been used as an M1 marker in some studies, although it is not a specific M1 macrophage marker [43]. In contrast, CKO reduced protein and mRNA expression of CD206, a major receptor surface marker of M2 macrophages, regardless of diet. These results suggest that catalase deficiency induces a switch in macrophage polarization regardless of diet. Confirming the reversal of CKO-mediated inflammation by catalase overexpression is a necessary strategy to provide proof-of-concept of direct role of catalase. The pharmacological and genetic approaches to study the role of catalase will complement each other. Due to our limitations in performing catalase overexpression into CKO macrophages, we have utilized 3-AT, a pharmacological inhibitor of catalase on the inflammatory response in RAW264.7 macrophages. Notably, the inhibitor induced inflammation, as assessed by increased JNK phosphorylation and mRNA expression of proinflammatory cytokines. We note that macrophages promote obesity-induced insulin resistance and inflammation through JNK expression [40]. In line with this result, siRNA knockdown of catalase also induced JNK phosphorylation (data not shown). Further, reduced catalase activity enhanced PA-induced inflammation and M1 polarization but suppressed M2 activation. Similarly, classically activated M1 macrophages were more abundant than M2 in primary bone marrow-derived macrophages genetically deficient in catalase, regardless of exposure to PA. Altogether, these data suggest that catalase regulates macrophage activation and preserves the balance between M1 and M2 macrophages. Catalase is associated with various human diseases, including osteoarthritis, cancer, psoriasis and other skin disorders, ischemia-reperfusion injury, neurodegenerative disorders, and type 2 diabetes [44]. Catalase deficiency also stimulates fibronectin expression, accelerates diabetic kidney injury [27, 28], and increases mitochondrial reactive oxygen species, especially in Hs27 human diploid fibroblasts [45], presumably by increasing β-oxidation. Although we did not measure mitochondrial reactive oxygen species, these molecules are known to enhance classical M1 activation [46]. In addition, 3-AT elicits inflammatory responses in human alveolar macrophages by increasing basal H2O2 [30], highlighting the role of catalase in altering macrophage polarization to promote inflammation in adipose tissue. In summary, we suggest that endogenous catalase plays an important role in the polarization of adipose tissue macrophages, both in basal conditions and under metabolic stress. Consequently, the enzyme inhibits inflammation and insulin resistance, as measured by fed and fasting insulin and by HOMA-IR. Thus, strategies based on catalase may be therapeutic against metabolic diseases. Supplementary Material Supplementary figure 1. Catalase expresses in adipose tissue along with liver and kidney. Liver, kidney, heart, lung, brown adipose tissue (BAT), white adipose tissue (WAT), pancreas, spleen, testis and thyroid from wild type C57BL/6 J mice were subjected for catalase protein (obtained from Young In Frontier, Seoul, Korea) expression using western blotting analysis as described in method section. Acknowledgments This work was supported by a grant from the National Research Foundation of Korea (nos. 2012R1A2A1A0300692 and 2015H1D3A1062189). Competing Interests The authors declare that they have no competing interests regarding the publication of this paper. Authors' Contributions Inah Hwang and Hunjoo Ha conceived and designed experiments. Ye Seul Park, Lingjuan Piao, and Jung Hwa Lee executed experiments. Md Jamal Uddin and Ye Seul Park analyzed data. Md Jamal Uddin, Ye Seul Park, and Hunjoo Ha wrote and critically revised the paper for important intellectual content. Hunjoo Ha provided final approval of the version to be published. Ye Seul Park and Md Jamal Uddin contributed equally to this work. Figure 1 Catalase deficiency accelerates oxidative stress, macrophage infiltration, and crown-like structures in epididymal WAT. (a) Immunohistochemistry for nitrotyrosine (1 : 200), a marker of oxidative stress. Scale bar, 50 μm; magnification, 100x. (b) F4/80 was assessed by real-time PCR and (c) immunohistochemistry (1 : 100) to measure macrophage infiltration. Immunostaining was quantified in Image Pro. Scale bar, 100 μm; magnification, 200x. (d) Crown-like structures in M1 macrophages were also quantified by immunohistochemistry for F4/80 (1 : 100, brown) and image analysis in Image Pro. Scale bar, 100 μm; magnification, 100x. Tissues were costained with hematoxylin (blue). Data are mean ± SE of 6–8 mice, and representative immunohistochemistry images are shown. ∗ p < 0.05. Figure 2 Catalase deficiency favors M1 activation and inflammation in the stromal vascular fraction of epididymal WAT. (a) To measure M1 activation, CD11c (1 : 200) was assessed by immunohistochemistry (brown) and quantified in Image Pro. Scale bar, 50 μm; magnification, 200x. Samples were costained with hematoxylin (blue). (b) CD11c, (c) TNF-α, and (d) PAI-1 mRNA levels were determined by real-time PCR. Data are mean ± SE of 6–8 mice. ∗ p < 0.05. Figure 3 Catalase deficiency suppresses M2 macrophages in the stromal vascular fraction of epididymal WAT. (a) CD206 (1 : 20, brown) was assessed by immunohistochemistry to measure M2 activation, using hematoxylin as counterstain (blue). Images were analyzed in Image Pro. Scale bar, 50 μm; magnification, 200x. (b) CD206, (c) arginase-1, and (d) IL-10 mRNA levels were determined by real-time PCR. Data are mean ± SE of 6–8 mice. ∗ p < 0.05. Figure 4 Catalase knockdown increases palmitate-induced inflammation in bone marrow-derived macrophages. Bone marrow-derived macrophages from WT and CKO mice were treated with 500 μM PA for 6 h, using BSA as control. (a) JNK phosphorylation was measured by western blot (upper panel) and densitometry (lower panel). (b) IL-1β, (c) IL-6, and (d) TNF-α mRNA expression was measured by real-time PCR. Data are mean ± SE of four experiments. ∗ p < 0.05 versus WT macrophages exposed to BSA; † p < 0.05 versus CKO macrophages exposed to BSA; § p < 0.05 versus WT macrophages exposed to PA. Figure 5 Catalase inhibition accelerates inflammation in RAW264.7 macrophages. RAW264.7 cells were treated for 6 h with 0, 1, 2, 5, and 10 mM 3-AT. (a) JNK phosphorylation was quantified by western blot (upper panel) and densitometry (lower panel). (b) IL-1β, (c) IL-6, and (d) TNF-α mRNA levels were measured by real-time PCR. (e) JNK phosphorylation was also determined by western blot (upper panel) and densitometry (lower panel) in cells treated with 0, 1, 2, 5, and 10 mM 3-AT 1 h prior to stimulation with 200 μM PA for 6 h. (f) IL-1β, (g) IL-6, and (h) TNF-α mRNA levels were also measured by real-time PCR in these cells. Data are mean ± SE of four experiments. BSA was used as control for the effects of PA. ∗ p < 0.05 versus control; † p < 0.05 versus PA. Figure 6 Pharmacological or genetic inhibition of catalase alters macrophage activation in response to LPS or IL-4. RAW264.7 cells were treated with 0, 1, 2, 5, and 10 mM 3-AT and stimulated with 1 ng/mL LPS for 6 h or 24 h. (a) JNK phosphorylation at 6 h and (b) iNOS levels at 24 h were quantified by western blot (upper panel) and densitometry (lower panel). ∗ p < 0.05 versus control; † p < 0.05 versus LPS. (c) JNK phosphorylation at 6 h and (d) iNOS abundance at 24 h were also measured by western blot (upper panel) and densitometry (lower panel) in WT and CKO bone marrow-derived macrophages treated with 1 ng/mL LPS for 6 h or 24 h. ∗ p < 0.05 versus unstimulated WT macrophages; † p < 0.05 versus unstimulated CKO macrophages; § p < 0.05 versus WT macrophages exposed to LPS. All data are mean ± SE of four experiments. (e) CD206 and (f) arginase-1 mRNA levels were measured in RAW264.7 cells treated with 0, 2, and 5 mM 3-AT 1 h prior to stimulation with 10 ng/mL IL-4 for 6 h. ∗ p < 0.05 versus control; † p < 0.05 versus IL-4. (g) CD206 and (h) arginase-1 mRNA levels were also measured in WT and CKO bone marrow-derived macrophages treated with 10 ng/mL IL-4 for 6 h. ∗ p < 0.05 versus unstimulated WT macrophages; † p < 0.05 versus unstimulated CKO macrophages; § p < 0.05 versus WT macrophages exposed to IL-4. Data are from real-time PCR and are reported as mean ± SE of four experiments. Table 1 Primer sequences of gene used in this study. Gene name Forward Reverse Arginase-1 5′-TGGCTTGCGAGACGTAGAC-3′ 5′-CGTCAGGTGAATCGGCCTTT-3′ CD11c 5′-TGGCTTCAACTTGGATGCAG-3′ 5′-CAACCACCACCCAGGAACTA-3′ CD206 5′-GTGGAGTGATGGAACCCCAG-3′ 5′-CTGTCCGCCCAGTATCCATC-3′ F4/80 5′-CTGTAACCGGATGGCAAACT-3′ 5′-ATGGCCAAGGCAAGACATAC-3′ IL-1β 5′-GTCAACGTGTGGGGGATGAA-3′ 5′-AAGCAATGTGCTGGTGCTTC-3′ IL-6 5′-AGTTGCCTTCTTGGGACTGA-3′ 5′-TCCACGATTTCCCAGAGAAC-3′ IL-10 5′-GCTCTTACTGACTGGCATGA-3′ 5′-CGCAGCTCTAGCATGTG-3′ PAI-1 5′-AGGGCTTCATGCCCCACTTC-3′ 5′-AGTAGAGGGCATTCACCAGC-3′ TNF-α 5′-CGTCAGCCGATTTGCTATCT-3′ 5′-CGGACTCCGCAAAGTCTAAG-3′ 18S 5′-CGAAAGCATTTGCCAAGAAT-3′ 5′-AGTCGGCATCGTTTATGGTC-3′ Table 2 Metabolic characteristics of experimental mice. Parameters ND HFD WT CKO WT CKO Body weight (g) 32.2 ± 0.7 31.5 ± 0.9 45.1 ± 1.5∗ 47.8 ± 2.2# Epididymal fat mass (g) 0.38 ± 0.06 0.41 ± 0.10 1.94 ± 0.13∗ 1.66 ± 0.13# Plasma free fatty acids (µM) 0.47 ± 0.02 0.6 ± 0.04∗ 0.52 ± 0.07 0.77 ± 0.13$ Plasma triglycerides (mM) 0.72 ± 0.04 0.93 ± 0.07∗ 0.97 ± 0.07∗ 1.04 ± 0.11 Plasma LDL/VLDL (mg/dL) 21.5 ± 4.0 30.7 ± 1.5 70.9 ± 5.0∗ 62.2 ± 8.6# Plasma HDL (mg/dL) 89.7 ± 6.0 95.7 ± 4.9 90.7 ± 8.0 101.5 ± 5.5 Fed plasma insulin (ng/mL) 1.54 ± 0.38 2.22 ± 0.51 6.16 ± 1.11∗ 10.08 ± 1.83#,$ Fasting plasma insulin (ng/mL) 0.21 ± 0.02 0.24 ± 0.06 0.50 ± 0.09∗ 1.20 ± 0.29#,$ HOMA-IR 2.44 ± 0.34 2.22 ± 0.69 7.39 ± 1.24∗ 14.17 ± 4.04#,$ WT and CKO C57BL/6 J mice were divided into four groups: WT ND, CKO ND, WT HFD, and CKO HFD. They were fed either ND (18% fat-derived calories) or HFD (60.3% fat-derived calories) for 21 weeks. 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==== Front Oxid Med Cell LongevOxid Med Cell LongevOMCLOxidative Medicine and Cellular Longevity1942-09001942-0994Hindawi Publishing Corporation 10.1155/2016/7530853Research ArticleIdentification of MicroRNAs Involved in Growth Arrest and Apoptosis in Hydrogen Peroxide-Treated Human Hepatocellular Carcinoma Cell Line HepG2 http://orcid.org/0000-0001-8081-7237Luo Yuan 1 2 Wen Xinyu 3 Wang Ling 3 Gao Jing 3 Wang Zi 1 Zhang Chunyan 1 Zhang Pengjun 1 Lu Chengrong 2 http://orcid.org/0000-0003-1187-8655Duan Lianning 2 * http://orcid.org/0000-0001-9169-5095Tian Yaping 1 3 * 1Core Laboratory of Translational Medicine, State Key Laboratory of Kidney Disease, Chinese PLA General Hospital, Beijing 100853, China2Aviation Medicine Research Laboratory, Air Force General Hospital, PLA, Beijing 100142, China3Department of Clinical Biochemistry, Chinese PLA General Hospital, Beijing 100853, China*Lianning Duan: duan_ln@126.com and *Yaping Tian: tianyp61@gmail.comAcademic Editor: Kum Kum Khanna 2016 15 8 2016 2016 753085318 3 2016 1 7 2016 13 7 2016 Copyright © 2016 Yuan Luo et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Although both oxidative stress and microRNAs (miRNAs) play vital roles in physiological and pathological processes, little is known about the interactions between them. In this study, we first described the regulation of H2O2 in cell viability, proliferation, cycle, and apoptosis of human hepatocellular carcinoma cell line HepG2. Then, miRNAs expression was profiled after H2O2 treatment. The results showed that high concentration of H2O2 (600 μM) could decrease cell viability, inhibit cell proliferation, induce cell cycle arrest, and finally promote cell apoptosis. Conversely, no significant effects could be found under treatment with low concentration (30 μM). miRNAs array analysis identified 131 differentially expressed miRNAs (125 were upregulated and 6 were downregulated) and predicted 13504 putative target genes of the deregulated miRNAs. Gene ontology (GO) analysis revealed that the putative target genes were associated with H2O2-induced cell growth arrest and apoptosis. The subsequent bioinformatics analysis indicated that H2O2-response pathways, including MAPK signaling pathway, apoptosis, and pathways in cancer and cell cycle, were significantly affected. Overall, these results provided comprehensive information on the biological function of H2O2 treatment in HepG2 cells. The identification of miRNAs and their putative targets may offer new diagnostic and therapeutic strategies for liver cancer. National High Technology Research and Development Program of China2011AA02A111National Natural Science Foundation of China8137190621375133 ==== Body 1. Introduction Hepatocellular carcinoma (HCC) is the most common primary liver cancer and accounts for about 80% of all cases of this disease. Globally, HCC is the fifth most common cancer, but also the third cause of cancer-related mortality [1, 2]. It is established that various factors are associated with the development of HCC, including hepatitis virus infection, alcoholic liver damage, ingestion of aflatoxin B1, nonalcoholic fatty liver disease, obesity, diabetes mellitus, and iron accumulation. Although the dominant role of these risk factors in different countries or regions might vary, the occurrence and development of HCC are a multistep process associated with years of chronic inflammation of liver [3–5]. Inflammatory cells, including neutrophils, macrophages, mast cells, and dendritic and natural killer cells, are recruited within inflammatory sites to release chemical mediators, such as cytokines, chemokines, and reactive oxygen species (ROS). The latter, in turn, might play a vital pathogenic role in the long-term progression, originating from chronic inflammation to hepatitis and ultimately leading to cancer [6–8]. ROS are chemically reactive molecules containing oxygen. The group includes hydrogen peroxide (H2O2), superoxide anion (O2 −), hydroxyl radical (OH•), singlet oxygen (1O2), ozone (O3), and some other small molecules. In a biological context, ROS are mainly deprived from oxidative metabolism as natural byproducts and play important roles in cell signaling and homeostasis. However, in some pathological conditions or during environmental stress, ROS levels can increase dramatically and lead to the disruption of the prooxidant/antioxidant equilibrium. This is known as oxidative stress and it could lead to damaging of many intracellular molecules, including DNA, RNA, lipids, and proteins [9–11]. Specifically, ROS can promote many aspects of tumor development and progression either directly by activating proinflammatory transcriptional factors such as NF-κB and AP-1 or indirectly by inducing DNA damage and oncogene activation [12]. Meanwhile, some latest studies have cast new light on the complicated interplay network between inflammation, oxidative stress, cancer, and microRNAs (miRNAs) [13, 14]. The latter, which are a series of small noncoding RNAs (containing about 22 nucleotides), could modulate gene expression through canonical base pairing between the seed sequences of themselves and 3′ untranslated region (3′ UTR) of target mRNAs. In general, miRNAs downregulate gene expression by inhibiting the translation and/or reducing the stability of target mRNAs and therefore provide a novel level of posttranscriptional regulation [15, 16]. Although publications in the past decade have described the involvement of miRNAs in almost all kinds of physiological and pathological processes, little is known about the specific mechanisms of the interaction between oxidative stress and miRNAs. To the best of our knowledge, no previous researches have reported the miRNAs expression profile in H2O2 treated human hepatocellular carcinoma cell lines. Therefore, in this study, we examined the changes of the miRNAs expression profiles in HepG2 cells upon treatment with H2O2. The target genes of significantly changed miRNAs were predicted by in silico prediction algorithms. Combined with biological experiments, we present a more holistic view indicating that H2O2-sensitive miRNAs regulate cell proliferation inhibition, cycle arrest, and apoptosis promotion. 2. Materials and Methods 2.1. Cell Culture HepG2 cells were obtained from China infrastructure of cell line resources. The cells were maintained as a monolayer culture in Dulbecco's modified Eagle's medium (DMEM; Gibco, Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Invitrogen Life Technologies, Carlsbad, CA, USA), 100 IU/mL penicillin, and 100 μg/mL streptomycin. HepG2 cells were cultured in a humidified incubator with 5% CO2 at 37°C. 2.2. Cell Viability Assay Cell viability and proliferation were quantified by Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer's protocol. To assess the cell viability, cells were seeded into 96-well plates at a concentration of 8 ∗ 103 HepG2 cells/well and each group was repeated in 5 wells. After the cells were treated with various concentrations of H2O2 for 24 hours (h), 10 μL CCK-8 was added into each well. After mixing, the cells were incubated for an additional 2 h, and then a microplate reader (RT-6000; Rayto, Rayto Life and Analytical Sciences Co., Ltd., Guangdong, China) was used to measure the absorbance at 450 nm (OD450) for each well. The cell viability rates were calculated according to the following formula: the cell viability ratio (%) = [(As − Ab)/(Ac − Ab)]∗100%, where As is the OD450 of H2O2 experiment group, Ab is the OD450 of blank control group, and Ac is the OD450 of non-H2O2 control group. The cell viability graph was drawn in which the viability ratios were plotted at the vertical axis and H2O2 concentration was plotted at the horizontal axis. The IC50 (half maximal inhibitory concentration) value, which represents the concentration of H2O2 in determining 50% of cell viability, was calculated by nonlinear regression analysis using GraphPad Prism software (San Diego, USA). 2.3. Cell Proliferation Assay To assess the cell proliferation, cells were seeded into 96-well plates at a concentration of 2 ∗ 103 HepG2 cells/well and each group was repeated in 5 wells. After cells were treated with 0–100 μM of H2O2 for 24 h, the growth medium was replaced by fresh medium. At the following 4 detection time points (0, 24, 48, and 72 h), 10 μL CCK-8 was added into each well. After mixing, cells were incubated for an additional 2 h before measuring the OD450. The cell proliferation graph was drawn, with OD450 plotted at the vertical axis and incubation time in hours at the horizontal axis. 2.4. Cell Cycle Assay The cell cycle and apoptosis were analyzed by flow cytometry (FCM). To assess the cell cycle, HepG2 cells in logarithmic phase were harvested and plated in 24-well culture plates at a concentration of 2 ∗ 105 cells/mL. Cells were starved overnight to achieve synchronization and then treated with 0–200 μM of H2O2 for 24 h. Using FCM instrument (FACS Calibur, BD Biosciences, San Jose, CA, USA), cell cycle was detected with cycle test Plus DNA reagent kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions. 2.5. Cell Apoptosis Assay To assess the level of apoptosis, HepG2 cells were plated in 24-well culture plates in the same concentration as mentioned above. After cell adhering, 0–800 μM of H2O2 were added and cells were incubated for 24 h. The Annexin V-PE/7-AAD Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA) was used to detect cell apoptosis according to the manufacturer's instructions. 2.6. Determination of Total ROS Intracellular ROS levels were also determined by FCM. HepG2 cells were plated in 12-well culture plates at a concentration of 2 ∗ 105 HepG2 cells/well. After cell adhering, 0–600 μM of H2O2 was added and cells were incubated for 24 h. Cells were harvested and then incubated with 10 μM 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-DCHF-DA, Invitrogen Life Technologies, Carlsbad, CA, USA), which could be cleaved by intracellular esterases and transformed into a fluorescent dye when oxidized at 37°C for 30 min. The samples were analyzed by FCM instrument with CellQuest software. For each sample, 10,000 cells were analyzed. 2.7. RNA Preparation and miRNAs Microarray HepG2 cells were seeded in 10 cm dishes and cultured as usual until they reached 80% confluence. The cells were treated with H2O2 (0, 30, and 600 μM) for 24 h and each concentration was repeated in 3 dishes. Total RNA was extracted from the treated cells by using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and then purified with a QIAGEN RNeasy Mini Kit (Qiagen, Valencia, CA, USA). After assessing the RNA's quality and quantity, the miRNAs microarray analysis (Affymetrix microRNA 4.0 Array, Santa Clara, CA, USA) was performed according to the manufacturer's instructions. Briefly, 1 μg of total RNA was labeled with Biotin using the FlashTag™ Biotin HSR RNA Labeling Kit (Genisphere, Hatfield, PA, USA) and then hybridized overnight with the array, which was washed, stained, and read by an GeneChip Scanner 3000 7G (Affymetrix). 2.8. Data Analysis of miRNAs Microarray CEL-files of the raw data were first exported by Affymetrix GeneChip Command Console Software Version 4.0 (Affymetrix) and then uploaded to the website of Gminix-Cloud Biotechnology Information (GCBI, http://www.gcbi.com.cn/gclib/html/index, Genminix Informatics Co., Ltd., Shanghai, China) for further analysis, including difference analysis of miRNAs profiles, prediction of miRNAs target genes, GO/pathway enrichment analysis, and miRNAs-gene-network and miRNAs-GO-network analysis. The miRNAs array data discussed in this paper has been uploaded to the NCBI Gene Expression Omnibus and is accessible through GEO series accession number GSE84406 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE84406). 2.9. Statistical Analysis Each experiment was performed independently at least 3 times with similar results. Student's t-test (two-tailed) was used to determine the statistical significance of quantitative data and Chi-square test was used for the statistical analysis of constituent ratio. P < 0.05 was considered to be statistically significant. 3. Results 3.1. H2O2 Treatment Decreases Cell Viability and Inhibits Proliferation of HepG2 Cells To determine the cytotoxicity of H2O2, we comprehensively detected the changes of viability, proliferation, cell cycle, and apoptosis in HepG2 cells. After exposure of HepG2 cells to H2O2 for 24 h, CCK-8 assay was firstly performed to determine the cell viability and proliferation. As shown in Figure 1, although 30 μM H2O2 seems to increase the cell viability and even proliferation, there were no statistically significant differences between them and control groups (P > 0.05). Conversely, in the presence of higher concentrations of H2O2, both cell viability and proliferation decreased significantly (P < 0.05). The IC50 value calculated through nonlinear regression analysis was 70.3 μM. These data suggest that H2O2 could cause an obvious dose-dependent reduction of cell viability and proliferation, while no significant effect could be found at low concentrations of this compound. 3.2. H2O2 Treatment Induces Cell Cycle Arrest and Apoptosis of HepG2 Cells FCM was utilized to detect the cell cycle and apoptosis of HepG2 cells after H2O2 treatment. As shown in Figure 2, lower concentrations of H2O2 did not affect the cell cycle in HepG2 cells (P > 0.05). However, once the concentration of H2O2 was higher than 100 μM, HepG2 cells exhibited significant increase in G2/M phase but obvious reduction in S phase and even G0/G1 phase (P < 0.05). Figure 3 showed that H2O2 could cause a dose-dependent increase of cell apoptosis, especially early apoptosis. In the presence of 600 and 800 μM H2O2, a significant (P < 0.001) increase in cell early apoptosis of 20.9% and 58.0%, respectively, was observed. These data suggested that H2O2 could induce cell cycle arrest in G2/M phase and promote a dose-dependent cell apoptosis of HepG2 cells, which might directly cause the reduction of cell viability and proliferation. 3.3. H2O2 Treatment Stimulates ROS in a Dose-Dependent Manner To confirm the ROS induction, FCM was used to detect the total ROS. As shown in Figure 4, 30 μM of H2O2 did not increase ROS in HepG2 cells (P > 0.05). However, once the concentration of H2O2 was higher than 100 μM, ROS levels were significantly increased (P < 0.05). These data showed that H2O2 could also stimulate ROS in a dose-dependent manner, which was consistent with the changes in cell biological functions. 3.4. Identification of miRNAs Modulated by Oxidative Stress To verify whether miRNAs could be modulated by oxidative stress, we stimulated HepG2 cells with H2O2 to develop a cell model of oxidative stress. Taking into account the results listed above, we chose 30 and 600 μM of H2O2 as low and high concentrations for miRNAs profiling, respectively. After stimulating HepG2 cells for 24 h, H2O2-induced changes in the miRNAs expression profiles were analyzed by the Affymetrix microRNA 4.0 Array, which contains 2578 probes and can interrogate all mature miRNAs sequences in miRBase Release 20. The results revealed that 131 miRNAs were deregulated in high concentration group under the condition of “Q < 0.05 and fold change > 2,” compared with normal control. Among them, 125 miRNAs were upregulated and 6 were downregulated (Figure 5(a)). However, there were no statistically significant differences between low concentration group and normal control under the same condition. After adjusting the condition to “Q < 0.5 and fold change > 1.2,” 16 deregulated miRNAs were determined in low concentration group, all of which were downregulated (Figure 5(b)). All of the deregulated miRNAs were listed in Supplementary Table 1 in Supplementary Material available online at http://dx.doi.org/10.1155/2016/7530853. Evidently, no deregulated miRNAs were overlapping between the two concentration groups. These data suggest that higher concentration of H2O2 mainly upregulated the expression of miRNAs, while lower concentration of H2O2 might have no effects and might even play the opposite role in the miRNAs expression. In view of no statistical differences between the low concentration group and normal control, we did not pursue the subsequent bioinformatics analysis of that group. 3.5. Identification of H2O2-Sensitive miRNAs Putative Target Genes and GO/Pathway Enrichment Analysis Our study has identified 131 miRNAs that were significantly deregulated in response to 600 μM H2O2, compared with normal control. As miRNAs play their biological roles through regulating target genes expression at the posttranscriptional level, we firstly predicted the target genes of H2O2-sensitive miRNAs by GCBI online tools, which are mainly based on the algorithms of miRanda and TargetScan. As a result, 13504 genes were predicted as putative target genes of high concentration H2O2-sensitive miRNAs. To identify the biological functions of these genes, GO and pathway enrichment analysis were performed, respectively. As illustrated in Figure 6(a), the top ten deregulated GOs sensitive to high concentration of H2O2 were “transcription, DNA-dependent,” positive regulation of transcription from RNA polymerase II promoter, signal transduction, negative regulation of transcription from RNA polymerase II promoter, axon guidance, nervous system development, “regulation of transcription, DNA-dependent,” apoptotic process, and synaptic transmission. GO analysis obviously suggests that high concentration of H2O2 could affect expression of many miRNAs, through which many important functions such as transcription regulation, signal transduction, and apoptotic process are involved in apoptotic regulation of HepG2 cells. Combining with KEGG database, we analyzed the pathways in which the putative target genes were involved. As illustrated in Figure 6(b), the top ten deregulated pathways sensitive to high concentration of H2O2 were pathways in cancer, MAPK signaling pathway, axon guidance, endocytosis, focal adhesion, HTLV-I infection, regulation of actin cytoskeleton, proteoglycans in cancer, Wnt signaling pathway, and PI3K-Akt signaling pathway. Then, we performed Path-net analysis to draw an interaction network covering 61 significantly changed pathways (Figure 7). Among them, MAPK signaling pathway (degree = 44), apoptosis (degree = 29), pathways in cancer (degree = 28), and cell cycle (degree = 24) showed the highest degree, suggesting that these four pathways might play a core role in apoptosis induced by H2O2 treatment. Based on the significantly regulated GOs and pathways, we selected intersected genes and further constructed miRNAs-gene-networks and miRNAs-GO-networks to screen the key regulatory functions of the identified miRNAs and their target genes, respectively. As shown in Figures 8 and 9 and Table 1, the top rated six miRNAs from the two analyses were the same, including hsa-miR-4763-3p, hsa-miR-149-3p, hsa-miR-762, hsa-miR-5001-5p, hsa-miR-5787, and hsa-miR-6791-5p. All of these miRNAs were upregulated by H2O2 treatment in HepG2 cells during apoptosis. The top six target genes were CPLX2 (complexin 2), ZNF385A (zinc finger protein 385A), NFIX (nuclear factor I/X (CCAAT-binding transcription factor)), CNIH2 (cornichon family AMPA receptor auxiliary protein 2), SOX12 (SRY- (sex determining region Y-) box 12), and WDTC1 (WD and tetratricopeptide repeats 1). Our analysis also showed that the deregulated miRNAs mainly play vital roles in various biological processes, including transcription regulation, apoptotic process, gene expression, and signal transduction. Taken together, these results represented comprehensive information on the biological function of H2O2 treatment in HepG2 cells. Through deregulation of certain miRNAs and several important pathways, elevated concentrations of H2O2 could decrease cell viability, inhibit cell proliferation, induce cell cycle arrest, and finally promote cell apoptosis in HepG2 cells. 4. Discussion H2O2, one of the most studied ROS, is a protonated form of O2 − and can produce OH• in the presence of transition metals like cupper or iron. H2O2 is also a hypochlorous acid precursor [17, 18]. As a soluble lipid, this strong oxidizing agent has been found to diffuse throughout the cell membrane via some aquaporins and can cause damage of cellular membranes, proteins, and DNA [19, 20]. Although H2O2 is widely used as oxidative stress stimuli, little is known about the biological functions of it on HepG2 cells. To investigate the role of H2O2 on HepG2 cells, we comprehensively examined the changes in cell biological functions, including cell viability, proliferation, cycle, and apoptosis. Specifically, H2O2 could significantly decrease cell viability and inhibit proliferation around 70.3 μM, which was calculated as the IC50 value. Once the concentration of H2O2 is higher than 100 μM, it can significantly induce cell cycle arrest in G2/M phase (P < 0.05). Meanwhile, if the concentration of H2O2 is higher than 200 μM, it can obviously promote cell early apoptosis (P < 0.05). In contrast, HepG2 cells exhibit no statistically significant changes under the treatment of low concentration of H2O2. In general, H2O2 could decrease cell viability, inhibit cell proliferation, induce cell cycle arrest, and finally promote cell apoptosis in a dose-dependent manner. These results suggested that H2O2 may be used as an anticancer agent if used at appropriate concentrations [21, 22]. Indeed, many chemotherapy drugs play their roles in killing tumor cells by producing ROS [10]. It could be confirmed from another perspective that several clinical trials based on the concept that oxidants were toxic and antioxidants were favorable for cancer prevention were largely unsuccessful [23]. As mentioned above, DNA damage responses induced by H2O2 usually end up with the decrease of cell viability and promotion of apoptosis. In addition to its effects on apoptosis, H2O2 could also induce necrosis or autophagy depending on the stimulus intensity. Thus, H2O2 leads to cell death in different manners [24–27]. In this study, we found that 100 μM H2O2 could decrease the cell viability to nearly 10% and induce obvious cell proliferation inhibition and cycle arrest, but there are no statistically significant differences in apoptosis under the same condition. This discrepancy might be caused by the following reasons. Firstly, different methodologies may lead to the inconsistent results. Specifically, cell viability and proliferation were detected by CCK-8 assay, while cell cycle and apoptosis were analyzed by FCM. Secondly, under the treatment of 100 μM H2O2, nearly 90% reduction of cell viability was caused by multiple mechanisms, including cell apoptosis, necrosis, and autophagy. Finally, although H2O2 could induce biological changes in a dose-dependent manner, the potentially different sensitivity or reactivity of various biological functions, such as cell viability, cell proliferation, cell cycle, and cell apoptosis, might lead to the discrepancy. In other words, these biological functions react or adapt differently to 100 μM H2O2 treatment. Furthermore, to confirm the induction of ROS by H2O2 treatment, we used FCM to detect the total ROS. Our results showed that 100 μM of H2O2 could significantly induce ROS (P < 0.05) and it also stimulates ROS in a dose-dependent manner. Although oxidative stress is closely related with liver cancer, further and detailed investigation is required to elucidate the molecular mechanisms of the interaction among oxidative stress, miRNAs, and liver cancer. By comparing the miRNAs expression profiles of control and high concentration group (600 μM), we identified 131 differentially expressed miRNAs (Q < 0.05, fold change > 2), of which 125 were upregulated and 6 were downregulated (Figure 5(a)). However, only 16 downregulated miRNAs could be determined in low concentration group after adjusting the condition to “Q < 0.5 and fold change > 1.2” (Figure 5(b)). These data suggest that higher concentration of H2O2 mainly upregulated the expression of miRNAs, while lower concentration of H2O2 might play the opposite role on the miRNAs expression. In view of the fact that miRNAs generally inhibit translation of their target genes, we could easily deduce that higher concentration of H2O2 mainly downregulates the translation of target proteins, while lower concentration of H2O2 might play the opposite role. Among the top ten fold changed miRNAs (Supplementary Table 1), half of them (hsa-miR-371b-5p, hsa-miR-663a, hsa-miR-1225-5p, hsa-miR-1202, and hsa-miR-572) were closely linked to the occurrence and development of tumors and might play vital roles as oncogenes or tumor suppressor genes [28–31]. These data confirmed that oxidative stress could interplay with cancer biology through modulating specific miRNAs expression. However, the rest of the top changed miRNAs were relatively novel miRNAs, which are rarely studied before and their functions are mostly unknown. Then, 13504 genes were predicted as putative target genes of high concentration H2O2-sensitive miRNAs using target prediction methods. According to the result of GO analysis, the predicted target genes were mainly involved in transcription, signal transduction, regulation of transcription, apoptotic process, and synaptic transmission (Figure 6(a)). These biological processes are reported to be crucial in the regulation of apoptosis. As for biological pathways, pathways in cancer, MAPK signaling pathway, endocytosis, focal adhesion, proteoglycans in cancer, Wnt signaling pathway, and PI3K-Akt signaling pathway were the top enriched pathways of the predicted target genes (Figure 6(b)). It is well known that these pathways play important roles in regulation of cell apoptosis and survival outcome. The Path-net analysis covering 61 significantly changed pathways showed that MAPK signaling pathway, apoptosis, pathways in cancer, and cell cycle play a more important role in apoptosis induced by H2O2 treatment (Figure 7). To screen for the important genes involved in apoptosis regulated by H2O2 in HepG2 cells, we performed regulatory network analysis by overlapping the significantly regulated miRNAs, GOs, and pathways. The results showed that the top six miRNAs were hsa-miR-4763-3p, hsa-miR-149-3p, hsa-miR-762, hsa-miR-5001-5p, hsa-miR-5787, and hsa-miR-6791-5p, while the top six target genes were CPLX2, ZNF385A, NFIX, CNIH2, SOX12, and WDTC1 (Figures 8 and 9). Taken together, these data suggest that H2O2 treatment may regulate biological functions of HepG2 cells through the changes in expression of specific miRNAs. However, it is well acknowledged that predicting the miRNAs targets merely by the means of bioinformatics is not sufficient. miRNAs targets should be strictly verified by further functional experiments, such as gain or loss of miRNAs function, luciferase report assay, and western blot confirmation [32, 33]. Therefore, our future investigations will be aimed at picking up some significantly deregulated miRNAs and carrying out experiments to confirm their targets and then unveil the mechanisms involved in the interplay among oxidative stress, miRNAs, and cancer. 5. Conclusions Since H2O2 could decrease cell viability, inhibit proliferation, induce cycle arrest, and promote apoptosis in a dose-dependent manner, this ROS is a very promising potential therapeutic tool to fight cancer. The proper and cautious use of H2O2 in combination with significantly deregulated miRNAs' mimics or antagomirs may have synergistic effects on increasing liver cancer cell death. Supplementary Material To verify whether miRNAs could be modulated by oxidative stress, we utilize miRNAs array to profile their expression changes after H2O2 treatment of 30 and 600 μM, respectively. The results revealed that 131 miRNAs were deregulated in high concentration group under the condition of “Q < 0.05 and Fold change > 2”, compared with normal control. Among them, 125 miRNAs were upregulated and 6 were downregulated. However, there were no statistically significant differences between low concentration group and normal control under the same condition. After adjusting the condition to “Q < 0.5 and Fold change >1.2”, 16 deregulated miRNAs were determined in low concentration group, all of which were downregulated. Acknowledgments This study was supported by the National High Technology Research and Development Program of China (863 Program, no. 2011AA02A111) and the National Natural Science Foundation of China (no. 81371906; no. 21375133). The authors wish to thank Dr. Wenjing Zhao (Genminix Informatics Co., Ltd., Shanghai, China) for providing help in bioinformatics analysis of miRNAs array data. Competing Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Figure 1 H2O2 treatment decreases cell viability and inhibits cell proliferation of HepG2 cells. (a) HepG2 cell viability was measured by CCK-8 assay after H2O2 treatment for 24 h. (b) After H2O2 treatment for 24 h, the cell proliferation was quantified at different detection periods. Error bars denote mean ± SD. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001. Figure 2 H2O2 treatment induces cell cycle arrest in G2/M phase. (a) The cell cycle was analyzed by flow cytometry. After synchronization induced by serum starvation overnight, HepG2 cells were treated with H2O2 for 24 h. (b) The histogram shows the cell cycle percentage detected by FCM. ∗ P < 0.05; ∗∗∗ P < 0.001. Figure 3 H2O2 treatment induces cell apoptosis in a dose-dependent manner. (a) The cell apoptosis was analyzed by FCM assay. HepG2 cells were treated with H2O2 for 24 h. (b) The histogram shows the early apoptotic cell percentage detected by FCM. ∗ P < 0.05; ∗∗∗ P < 0.001. Figure 4 H2O2 treatment stimulates ROS in a dose-dependent manner. (a) The total ROS was determined through FCM assay. HepG2 cells were treated with H2O2 for 24 h. (b) The histogram shows the ROS positive cell percentage detected by FCM. ∗ P < 0.05; ∗∗∗ P < 0.001. Figure 5 Changes in miRNAs expression profiles in H2O2 treated HepG2 cells. Total RNA was extracted from control group and HepG2 cells treated with 600 or 30 μM H2O2 for 24 h. miRNAs microarray was performed as described in Materials and Methods. (a) High concentration versus control group, changes in miRNAs expression > 2-fold, and Q < 0.05 are illustrated by heat map. Green indicates a relatively low expression and red indicates a relatively high expression. (b) Low concentration versus control group, changes in miRNAs expression > 1.2-fold, and Q < 0.5 are illustrated by heat map. High: high concentration of H2O2 group, 600 μM; Con: control group; Low: low concentration of H2O2 group, 30 μM. Figure 6 Significantly changed GO/pathways of predicted target genes of deregulated miRNAs after H2O2 treatment (600 μM). (a) Significantly changed GOs of predicted target genes. The y-axis shows GO category and the x-axis shows −lg P. The larger −lg P indicated a smaller P value. (b) Significantly changed pathways of predicted target genes. The y-axis shows significantly changed pathways. −lg P: negative logarithm of the P value. Figure 7 Pathway network (Path-net). Significantly changed pathways were connected in a Path-net to show the interaction network among these pathways. Each pathway in the network was measured by counting the upstream and downstream pathways. The red circle represents pathways involving upregulated miRNAs, while the yellow circle represents pathways involving both upregulated and downregulated miRNAs. The size of the circle represents the degree value and the lines show the interaction between pathways. A higher degree of pathway indicates that it plays a more important role in the signaling network. Figure 8 miRNAs-gene-network. According to the interactions between miRNAs and the intersected target genes, miRNAs-gene-network was constructed to illustrate the key regulatory functions of the identified miRNAs and their target genes. The red circles represent genes, while red square nodes represent upregulated miRNAs. The size of the circle or square node represents the degree value. A higher degree of gene/miRNAs indicates that it plays a more important role in the signaling network. Figure 9 miRNAs-GO-network. The miRNAs-GO-network was generated according to the relationship of significant biological functions and miRNAs. The yellow circles represent GOs, red square nodes represent upregulated miRNAs, and blue square nodes represent downregulated miRNAs. The size of the circle or square node represents the degree value. A higher degree of GO/miRNAs indicates that it plays a more important role in the signaling network. Table 1 The top 10 miRNAs with high degrees of miRNAs-gene-networks and miRNAs-GO-networks. Rank miRNAs-gene-networks miRNAs-GO-networks miRNAs Degree Feature miRNAs Degree Feature 1 hsa-miR-4763-3p 265 Up hsa-miR-4763-3p 436 Up 2 hsa-miR-149-3p 251 Up hsa-miR-149-3p 408 Up 3 hsa-miR-762 211 Up hsa-miR-762 346 Up 4 hsa-miR-5001-5p 207 Up hsa-miR-6791-5p 337 Up 5 hsa-miR-5787 195 Up hsa-miR-5001-5p 336 Up 6 hsa-miR-6791-5p 188 Up hsa-miR-5787 333 Up 7 hsa-miR-4498 180 Up hsa-miR-2861 332 Up 8 hsa-miR-4651 164 Up hsa-miR-4505 322 Up 9 hsa-miR-4505 159 Up hsa-miR-4498 320 Up 10 hsa-miR-4632-5p 150 Up hsa-miR-665 303 Up ==== Refs 1 Addissie B. D. Roberts L. R. 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==== Front Front Plant SciFront Plant SciFront. Plant Sci.Frontiers in Plant Science1664-462XFrontiers Media S.A. 10.3389/fpls.2016.01252Plant ScienceOriginal ResearchGuava Waste to Sustain Guava (Psidium guajava) Agroecosystem: Nutrient “Balance” Concepts Souza Henrique A. 1Parent Serge-Étienne 2Rozane Danilo E. 3Amorim Daniel A. 4Modesto Viviane C. 5Natale William 6Parent Leon E. 2*1Empresa Brasileira de Pesquisa Agropecuária - Embrapa Meio-NorteTeresina, Brazil2Department of Soils and Agrifood Engineering, Université Laval, QuébecCA, Canada3Cursos de Engenharia Agronômica e Engenharia de Pesca, Universidade Estadual Paulista “Júlio de Mesquita Filho,”Registro, Brazil4Empresa de Pesquisa Agropecuária de Minas Gerais - EPAMIG OesteUberaba, Brazil5Faculdade de Engenharia, Universidade Estadual Paulista “Júlio de Mesquita Filho,”Ilha Solteira, Brazil6Departamento de Fitotecnia, Universidade Federal do CearáFortaleza, BrazilEdited by: B. Mohan Kumar, Nalanda University, India Reviewed by: Lukas Van Zwieten, New South Wales Department of Primary Industries, Australia; Simon Bilodeau-Gauthier, Université du Québec à Montréal, Canada; Jiri Tuma, University of Hradec Králové, Czech Republic *Correspondence: Leon E. Parent leon-etienne.parent@fsaa.ulaval.caThis article was submitted to Agroecology and Land Use Systems, a section of the journal Frontiers in Plant Science 29 8 2016 2016 7 125216 3 2016 08 8 2016 Copyright © 2016 Souza, Parent, Rozane, Amorim, Modesto, Natale and Parent.2016Souza, Parent, Rozane, Amorim, Modesto, Natale and ParentThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.The Brazilian guava processing industry generates 5.5 M Mg guava waste year−1 that could be recycled sustainably in guava agro-ecosystems as slow-release fertilizer. Our objectives were to elaborate nutrient budgets and to diagnose soil, foliar, and fruit nutrient balances in guava orchards fertilized with guava waste. We hypothesized that (1) guava waste are balanced fertilizer sources that can sustain crop yield and soil nutrient stocks, and (2) guava agroecosystems remain productive within narrow ranges of nutrient balances. A 6-year experiment was conducted in 8-year old guava orchard applying 0–9–18–27–36 Mg ha−1 guava waste (dry mass basis) and the locally recommended mineral fertilization. Nutrient budgets were compiled as balance sheets. Foliar and fruit nutrient balances were computed as isometric log ratios to avoid data redundancy or resonance due to nutrient interactions and the closure to measurement unit. The N, P, and several other nutrients were applied in excess of crop removal while K was in deficit whatever the guava waste treatment. The foliar diagnostic accuracy reached 93% using isometric log ratios and knn classification, generating reliable foliar nutrient and concentration ranges at high yield level. The plant mined the soil K reserves without any significant effect on fruit yield and foliar nutrient balances involving K. High guava productivity can be reached at lower soil test K and P values than thought before. Parsimonious dosage of fresh guava waste should be supplemented with mineral K fertilizers to recycle guava waste sustainably in guava agroecosystems. Brazilian growers can benefit from this research by lowering soil test P and K threshold values to avoid over-fertilization and using fresh guava waste supplemented with mineral fertilizers, especially K. Because yield was negatively correlated with fruit acidity and Brix index, balanced plant nutrition and fertilization diagnosis will have to consider not only fruit yield targets but also fruit quality to meet requirements for guava processing. nutrient budgetisometric log rationutrient balanceplant nutritionguavaFundação de Amparo à Pesquisa do Estado de São Paulo10.13039/501100001807Conselho Nacional de Desenvolvimento Científico e Tecnológico10.13039/501100003593Natural Sciences and Engineering Research Council of Canada10.13039/5011000000382254 ==== Body Introduction Guava (Psidium guajava) is a tropical tree grown on Brazilian Oxisols and Ultisols and reaching high productivity 3 years after establishment (Hernandes et al., 2012). Brazil is the world leader in red guava production with 16,000 ha producing 342,000 Mg of fresh fruits annually (IBGE, 2012). The state of São Paulo accounts for 36% of total Brazilian guava production and 55% of the industrially processed production. “Paluma” is the main red guava cultivar (Natale et al., 2009). The typical guava orchard in São Paulo state is 5.6 ha in size. Crop performance for processing is measured in terms of yield, sugar content (“Brix” index) and acidity. Compared to yield, fruit sweetness and acidity may be more influenced by annual climate variations than crop management (Le Bourvellec et al., 2015). Nutrient transfers from soil to plant are influenced by climate variables and plant nutrient availability (Barber, 1995). Mineral fertilization is thus required to sustain guava yield and quality (Natale et al., 1995, 1996, 2001). Guava processing into juice and jelly generates large amounts of waste with great potential for recycling as slow-release nitrogen fertilizers (Mantovani et al., 2004; de Souza et al., 2011). Considering that waste production is 80 kg waste Mg−1 of fresh fruits, the state of São Paulo generates 5.5 × 109 kg of guava waste per year, most often discarded in landfills because their fertilizer value is little documented (de Souza et al., 2014a,b). In an agroecosystem approach, guava waste should be recycled to sustain plant nutrition over several guava production cycles. In order to recycle nutrient sustainably in guava agroecosystems, guava waste additions can be guided by fertilization concepts such as “balance sheets,” “balanced” fertilization, “balanced” plant nutrition, and “balanced” nutrient ratios (Roy et al., 2006). Nutrient balance sheets report on inputs and outputs expressed in kilograms of nutrient per hectare of agricultural land (Organization for Economic Cooperation and Development, 2016). Variations in soil stocks (Kremer, 2013; Morel et al., 2014) are then interpreted using concepts such as soil nutrient buildup and maintenance to maintain soil nutrient levels close to some optimum (Dahnke and Olson, 1990), sufficiency levels of available nutrients or SLAN and basic cation saturation ratios or BCSR (McLean et al., 1983). On the other hand, misbalanced plant nutrition can be quantified using tissue tests (Jones and Case, 1990), especially for deep-rooted plants such as fruit trees (Smith, 1985), then interpreted against isolated nutrients or ratios of nutrients at high yield level (Walworth and Sumner, 1987). Nutrient dual ratios (Walworth and Sumner, 1987) and ternary diagrams (Lagatu and Maume, 1934) have long been used to represent nutrient interactions in plant tissues (Wilkinson, 2000). However, dual ratios are asymmetrical and difficult to analyze statistically (van Kempen and Van Vliet, 2000). In addition, changing the scale of measurement for concentrations, such as dry of fresh mass basis (Walworth and Sumner, 1987) or ratios scaled on N (Ingestad, 1987), P (Güsewell, 2004), or other nutrients (Walworth and Sumner, 1987), may lead to different conclusions due to spurious correlations and sub-compositional incoherence (Aitchison, 1986). Like other compositions, soil and tissue compositions form compositional vectors constrained to some whole (Aitchison, 1986). The SLAN, BCSR, concentration ranges and DRIS were developed before compositional data analysis techniques were found to improve the soundness and robustness of soil nutrient and plant ionome diagnoses across several agroecosystems worldwide (e.g., Parent and Dafir, 1992; Raghupathi and Bhargava, 1998; García-Hernández et al., 2004; Hernandes et al., 2012; Parent et al., 2012a,b, 2013a,b,c; Xu et al., 2015; Barłóg, 2016). Compositional vectors are made of D parts and return D-1 degrees of freedom for modeling purposes because one component can be computed by difference due to closure to measurement unit or scale (Aitchison and Greenacre, 2002). In contrast, there are D separate diagnoses for concentrations and D(D–1)/2 dual ratios and D indexes in DRIS, denying closure. Compositional nutrient diagnosis standards has been elaborated for several crops at regional scale, e.g., Rozane (2016). Our objectives were to elaborate nutrient budgets and diagnose soil, foliar, and fruit nutrient balances in guava orchards fertilized with guava waste. We hypothesized that (1) guava waste are balanced fertilizer sources that can sustain crop yield and soil nutrient stocks under local climate conditions, and (2) guava agroecosystems are productive within narrow ranges of nutrient balances. To verify hypothesis (1), nutrient budgets were elaborated across several fertilization treatments during six guava cropping cycles where annual climate conditions and soil stocks varied. Hypothesis (2) was verified using locally derived foliar nutrient balance standards for “Paluma” guava at high yield level under the ceteris paribus assumption. Materials and methods Experimental site and design Perennial fruit-bearing plants such as guava require conducting long-term experiments to document nutrient issues due to the continuous nutrient supply and removal and to internal nutrient cycling (Natale et al., 2012). The trial was conducted between 2006 and 2010 in 8 year old “Paluma” guava orchard at Vista Alegre do Alto, Sao Paulo state, 21 08′S and 48 30′W and 603 m in altitude. Climatic data were provided by a local meteorological station (Table 1). Plants were irrigated at need with artesian water at a rate of 31 L h−1 plant−1 using micro-sprinklers whenever the tensiometer installed 0.20 m below surface indicated that soil water content reached 60% of field capacity. Table 1 Average climatic conditions during the experimental period. Month Year Mean Year Mean Year Mean 2006 2007 2008 2009 2010 2006 2007 2008 2009 2010 2006 2007 2008 2009 2010 Total precipitations (mm) Average temperature (°C) January 237.9 430.8 355.9 239.7 287.9 310.4 19.9 20.9 19.8 19.6 20.6 20.2 30.5 28.7 28.7 29.3 30.0 29.4 February 316.6 209.6 322.0 334.9 99.2 256.5 20.0 19.9 19.2 20.1 20.4 19.9 30.1 30.4 30.7 30.7 31.6 30.7 March 193.9 128.7 182.0 172.5 180.5 171.5 20.2 19.9 17.6 19.8 19.9 19.5 30.4 31.4 29.6 30.2 30.9 30.5 April 25.9 27.2 98.3 98.6 60.7 62.1 17.2 18.8 16.4 17.0 17.0 17.3 29.2 30.6 28.8 28.8 28.9 29.3 May 13.0 71.1 32.6 21.7 19.1 31.5 12.7 14.3 12.5 15.3 13.7 13.7 26.4 26.6 26.4 27.8 26.8 26.8 June 9.8 4.9 5.5 19.4 8.9 9.7 12.8 13.5 14.1 12.0 11.9 12.9 26.8 27.4 27.2 24.7 26.8 26.6 July 5.5 65.0 0.0 19.7 3.8 18.8 12.9 12.9 12.9 14.3 13.8 13.4 28.3 26.4 28.0 27.6 28.5 27.8 August 10.1 0.0 22.2 84.8 0 23.4 14.4 14.2 15.1 14.6 13.2 14.3 27.0 27.2 30.5 28.0 30.0 28.5 September 34.7 4.3 11.9 203.4 69.2 64.7 15.8 17.5 14.7 17.9 16.9 16.6 27.7 30.8 28.4 29.2 30.9 29.4 October 110.9 60.6 47.9 68.9 125.2 82.7 18.5 19.6 19.4 18.3 17.2 18.6 30.1 33.0 31.7 30.4 30.0 31.0 November 278.3 187.3 93.7 120.2 96.7 155.2 18.8 18.6 18.9 20.8 18.5 19.1 30.3 29.6 31.5 31.7 30.5 30.7 December 343.1 120.0 212.4 257.5 187.5 224.1 20.5 19.8 19.0 20.3 20.4 20.0 29.5 30.9 30.1 29.3 30.7 30.1 Industrial guava waste consist of seeds and fruit skin and pulp. Guava waste provided each year by a nearby guava-processing plant was air-dried for 1 month from 25–30 to 5–7% moisture content at 20–30°C on a concrete floor in a shelter with sides opened for ventilation. The 50-cm layer was turned over once a week. The air-dried waste was ground mechanically (26% of dry mass in the range of 0.6–2 mm, ~61% of dry mass between 0.3 and 0.6 mm, and 13% < 0.3 mm) to facilitate handling, and stored in the shelter. Fresh residues were collected yearly from the same process and transported to the field; the 3–5 cm thick layers were exposed to relatively fast air drying (within 1–2 weeks). The trial comprised 28 permanent plots consisting of four randomized blocks and seven treatments annually applied on the same plots during five consecutive years as mineral fertilization or guava waste. Treatments were applied manually onto soil surface without incorporation. Trees were 5 m apart on the row and row spacing was 7 m (35 m2 per tree) for a total of 140 trees for the 4900 m2 experimental orchard. There were five doses of waste (0, 9, 18, 27, and 36 Mg dry matter ha−1 year−1) applied as dried-and-ground material to facilitate manual handling, a dosage of 18 Mg dry waste-equivalent ha−1 year−1 of fresh waste as would be machine-applied in practice, and a standard treatment of locally recommended mineral fertilization. Mineral fertilizers and guava waste were spread manually each year the same day within crown projection area. The sources of mineral fertilizers were urea (45% N), ordinary superphosphate (18% P2O5, 19% Ca, 12% S), and KCl (60% K2O, 47.5% Cl), supplying 229 kg N ha−1, 12.5 kg P ha−1, 71.4 kg K ha−1, 31.5 kg Ca ha−1, and 19 kg S ha−1. Treatments started in March 2006 and continued yearly in January after soil sampling (Table 2). Table 2 Milestones of the long-term guava experiment. Date Fertilization Soil sampling Tissue sampling Harvest March 2006 X X June 2006 X December 2006 X X January 2007 X May 2007 X July–September 2007 X November 2007 X December 2007 X January 2008 X February–April 2008 X September 2008 X December 2008 X January 2009 X January–March 2009 X July 2009 X December 2009 X January 2010 X November 2009–January 2010 X March 2010 X August–October 2010 X December 2010 X January 2011 X February 2011 X April–March 2011 X Chemical analyses Waste and plant analyses Moisture content in guava waste was determined after drying at 67 ± 2°C in a forced air oven before chemical analysis (Bataglia et al., 1983; Abreu et al., 2006). The waste was ground (<2 mm) then analyzed for macro- and micro-nutrients according to Bataglia et al. (1983). Total carbon was determined by dichromate oxidation (Abreu et al., 2006). The pH was measured in 0.01 M CaCl2 (1:1 volumetric ratio). The N was determined by micro-Kjeldahl including nitrate. The P and S were quantified by colorimetry and cations (K, Ca, Mg, Cu, Fe, Mn, Zn) by atomic absorption spectrophotometry after digestion in a mixture of nitric and perchloric acids. Boron was determined by colorimetry after ashing waste for 3 h in a muffle furnace at 550°C. The average nutrient composition of guava waste presented in Table 3 met the Brazilian regulation for fertilizer class “A” (Ministerio da Agricultura, 2009). The C/N ratios averaged 23.8 ± 1.4 in the dry and 23.0 ± 1.4 in the fresh waste over the 2006–2011 period. The dose of 18 Mg dry guava waste ha−1 provided 202–237 kg N ha−1 and that of fresh guava waste, 289–310 kg N ha−1. Table 3 Chemical analysis of the dry and fresh guava waste (mean ± standard deviation) during the 2006–2011 period. Element Dry guava waste Fresh guava waste 2006–2011 2006–2011 Mantovani et al. (2004) g kg−1 (DRY MASS BASIS AT 65°C) C 289.0 ± 15.2 382.0 ± 5.09 355 N 12.2 ± 1.02 16.5 ± 0.58 16 P 2.20 ± 0.19 2.20 ± 0.16 2 K 2.42 ± 0.22 2.88 ± 0.08 3 Ca 0.86 ± 0.09 0.94 ± 0.13 0.5 Mg 0.96 ± 0.05 0.96 ± 0.11 0.5 S 1.26 ± 0.11 1.30 ± 0.07 − mg kg−1 (DRY MASS BASIS AT 65°C) B 10.8 ± 1.84 6.7 ± 1.8 − Cu 10.6 ± 1.14 10.5 ± 1.1 9 Fe 145.7 ± 9.5 101.7 ± 5.5 39 Mn 12.2 ± 1.3 9.7 ± 1.1 8 Zn 28.6 ± 1.8 24.7 ± 1.8 18 Twelve pairs of the first mature leaves were collected in each plot during full bloom at tree mid-height (Natale et al., 1996), then composited for tissue analysis. Foliar tissues were gently washed with distilled water, dried in a forced air oven at 65–70°C to constant weight, ground to <1 mm and analyzed for macro- and micro-nutrients as above (Bataglia et al., 1983). Fruit sugar content was measured as the “Brix” refractive index and fruit acidity was determined as 0.1 N NaOH titratable acidity (De Mello Prado et al., 2005). Soil analysis The soil was classified as dystrophic red-yellow Ultisol (EMBRAPA, 2006). Soil surface (0–0.2 m) contained 177 g clay kg−1, 50 g silt kg−1, 130 g very fine sand kg−1, 450 g fine sand kg−1, 190 g medium sand kg−1, and 10 g coarse sand kg−1. The sublayer (0.2–0.4 m) contained 260 g clay kg−1, 60 g silt kg−1, 100 g very fine sand kg−1, 390 g fine sand kg−1, 180 g medium sand kg−1, and 10 g coarse sand kg−1. Soil available nutrient levels and pH were determined yearly each December in the 0–0.20 m and 0.20–0.40 m layers where the bulk of guava roots is located (Fracaro and Pereira, 2004). In March 2006 before treatment applications, 20 soil subsamples were collected below tree crown using a Dutch sampler. Subsamples were composited, air dried, ground and sieved to <2 mm, then analyzed for pH (0.01 M CaCl2), organic matter content, K, Ca, Mg, and (H + Al) according to van Raij et al. (2001). The Cu, Fe, Mn, and Zn were extracted using DTPA and quantified by atomic absorption spectrophotometry. The B was extracted using the hot water method and quantified by colorimetry. The P was extracted using an exchange resin Amberlite IRA-400 (20–50 mesh), quantified by colorimetry using the ascorbic acid method, and reported as mg dm−3. The K, Ca and Mg were extracted by exchange resin Amberlite IRA-120 (20–50 mesh), quantified by flame photometry (K) or atomic absorption spectrophotometry (Ca, Mg), and reported as mmolc dm−3. Potential acidity (H+Al) was quantified by the SMP pH buffer method (Shoemaker et al., 1961) and using the equation of Quaggio et al. (1985) to convert buffer pH into mmolc (H+Al) dm−3. Cation exchange capacity (CEC) was calculated as the sum of cationic species. Base saturation was computed as the sum of molar concentrations of K, Ca, and Mg divided by CEC, to conduct BCSR diagnosis. Other nutrients were diagnosed in isolation using SLAN. Budgeted balance sheets Severe pruning, irrigation, and fertilization allowed fruit harvesting thrice per 2 years (Rozane et al., 2009; de Souza et al., 2012). Nutrient input by guava waste was obtained by multiplying waste dosage (Mg ha−1) on dry mass basis by nutrient content in kg Mg−1 dry mass. Nutrient removal was quantified by multiplying fruit yield in Mg fruit ha−1 (mean of 11% dry matter in fruit at harvest) by fruit nutrient concentration in kg nutrient Mg−1. Because fruit analysis was conducted at three occasions only, nutrient concentrations by treatment (four replicates times three periods, hence 12 values per treatment) were obtained averaging the ilr values then transforming them back to concentrations. Starting 90–100 days after fruit set, guava fruits were harvested 1–3 times per week during 2 months from three representative central trees per plot at proper fruit ripening, commercial size, yellow skin, and characteristic aroma for industrial processing (Salazar et al., 2006). About 60% of the crown was pruned after each harvest. Pruning residues were shredded mechanically and left on soil as mulch. Pruning residues were not analyzed hence not budgeted, assuming internal cycling. Nutrient budgets were computed between the first soil sampling in December 2006 and the last one in January 2011. Tissue nutrient balances The simplest function to remove one degree of freedom without losing any information from a composition closed to 100% is the logistic variable, i.e., log[x/(1−x)] where one proportion “x” is expressed relatively to its complement “(1 − x).” The closing element (1 − x) to 100% may be defined as a filling value (Fv) computed as the difference between the unit or scale of measurement and the analyzed nutrient, generally expressed on a dry mass basis. After statistical analysis, the x and (1 − x) centroids are recovered by back transforming the mean of the logistic variable to original units. In many areas of natural sciences, two entities X and Y are often reduced to a single one as dual log ratio reflecting the interaction between X and Y. Using Fv as basis, the log of X/Fv, and Y/Fv are called additive log ratios or alr (Aitchison, 1986). For three components (X, Y, and Fv), there are two alrs in the compositional vector, hence removing one degree of freedom. Again, results of statistical analysis can be back-transformed to original units from three equations: the values of log[X/Fv]and log[X/Fv], and [X+Y+Fv = 100%]. However, the alrs are not independent (i.e., orthogonal) from one another: the angle of 60° between alrs makes it difficult to project them into the Euclidean space (Pawlowsky-Glahn and Egozcue, 2006). Orthogonality is a special case of linear independence where orthogonal vectors fall perfectly at right angle to each other (Rodgers et al., 1984). Egozcue et al. (2003) were the first to derive D - 1 linearly independent variables from compositional vectors containing more than two parts. They constrained (D > 2)-parts to D - 1 isometric log ratios (ilr) as orthogonally arranged “balances” from binary partitions between two subsets of non-overlapping, strictly positive, components. The ilr data transformation can project compositions into the Euclidean space of D - 1 Cartesian coordinates. Orthogonality qualifies the isometric log ratio as the most appropriate data-transformation technique to conduct multivariate and univariate statistical analyses on compositional data (Filzmoser et al., 2009). In this paper, we defined nutrient balances as a system of nutrient relationships using a sequential binary partition (SBP) in which nutrients labeled “+1” (group numerator) are contrasted with nutrients labeled “-1” (group denominator) in each row. A part labeled “0” is excluded. The composition is partitioned sequentially at every ordered row into two contrasts until the (+1) and (−1) subsets each contain a single part. Balances are computed as follows (Egozcue et al., 2003): (1) ilrj=nj+nj−nj++nj−lng(cj+)g(cj−), where, in the jth row of the SBP, nj+ and nj− are numbers of components at numerator (symbol +, plus) and denominator (symbol -, minus), respectively, g(cj+) and g(cj−) are geometric means of components in the + and – groups, respectively. Coefficient nj+nj−/(nj++nj−) is a normalization coefficient used to obtain unitary vectors on the basis. We conventionally represented balances as [-1 group denominator as components of g(cj−) |+1 group numerator as components of g(cj+)] because, in algebra, the minus sign is located on the left-hand side of the vector, hence the log ratio gets more negative as the weight of the geometric mean at denominator increases and conversely (Parent et al., 2013c). Although there are D! × (D - 1)!/2D−1 possible sequential binary partitions (SBP) for the D - 1 balances derivable from D-parts compositions (Pawlowsky-Glahn et al., 2011), ad hoc SBP can be defined to facilitate interpreting meaningful balances in relation to specific issues (Parent et al., 2013c). However, because balances are orthogonal to each other, linear, or distance-based multivariate analyses return consistent results whatever the choice of the SBP. In the SBP elaborated in Table 4 the filling value between measurement unit and the sum of determined elements is contrasted with nutrients as measure of nutrient dilution or accumulation (Jarrell and Beverly, 1981), macro-nutrients and boron interact, mobile macro-nutrients are contrasted with immobile nutrients Ca and B, N, and P reflect the relationship between protein synthesis and energy requirements known as Redfield or N/P ratio, and K and Mg interact as competing cationic species (Marschner, 1986). Because cationic micronutrients depend largely on the frequency and the moment of fungicide sprays (Cu, Zn, Mn) they were excluded from the SBP to avoid this source of high variation in the compositional vector. Table 4 Sequential binary partition of six tissue nutrients and the filling value (Fv) to compute the six ilr coordinates as contrasts between orthogonally arranged subsets of components. ilr N P K Ca Mg S B Fv Notation [denominator | numerator] 1 1 −1 0 0 0 0 0 0 [P |N] 2 0 0 1 0 −1 0 0 0 [Mg |K] 3 −1 −1 1 0 1 0 0 0 [N, P |K, Mg] 4 1 1 1 0 1 −1 0 0 [S |N, P, K, Mg] 5 0 0 0 1 0 0 −1 0 [B |Ca] 6 1 1 1 1 1 1 1 −1 [Fv |N, P, K, Mg, S, Ca, B] Subsets are assigned −1 when at denominator, and +1 when at numerator, of the log ratios. Statistical analysis and nutrient diagnosis Statistical computations were conducted in the R statistical environment (R Core Team, 2015). Compositional data transformations were computed using the R “compositions” package (van den Boogaart et al., 2014). Confidence intervals were computed at P = 0.05 for means comparison. We discretized yield between low and high yielders in order to predict the yield class using a k nearest neighbors (knn) classification algorithm, appropriate for the Euclidean space of ilrs, as the “kknn” package launched with the R “caret” package (Kuhn, 2016). We optimized the parameters of the knn model based on the accuracy using 10-fold cross-validations through a grid of parameters, then derived sub-populations of true negative (TN), false negative (FN), true positive (TP), and false positive (FP) plant specimens. The parameters of the model were optimized with a number of 10 nearest neighbors, a distance of four in the ilr, space and a kernel set at optimum (Schliep et al., 2016). The TN specimens are considered balanced and high-yielding; FP specimens represent cases of sub-optimal nutrient concentrations, luxury consumption, or contamination; TP specimens are misbalanced due to nutrient deficiency or excess; FN specimens are balanced but show lower yield limited by other growth factors. The TN ilrs were compared to the TP ilrs using a mobile design where statistics are presented in the balance domain at fulcrums and nutrient concentrations are appreciated in buckets (Parent et al., 2013c). Accuracy was computed as (TN+TP)/(TN+TP+FN+FP). To compute critical concentration ranges, we generated a large number (100,000) of uniformly distributed values between the ranges of the confidence intervals (P = 0.05) about the TN ilrs. We then back-transformed each row of randomly generated ilrs to concentrations, and finally extracted the minimum and maximum univariate concentrations across the TN ilr confidence ranges. Results Nutrient budgets and soil stocks Nutrient budgets except the K were positive across guava waste treatments (Figure 1). The nutrient surpluses and K deficits increased with the dose of guava waste. As a result, nutrient use efficiency was increasingly lower for treatments exceeding 9 Mg dry guava waste ha−1. Ordinary superphosphate provided Ca and S in mineral fertilization in amounts comparable to or higher than guava waste, but Mg and micronutrients were in deficit with the mineral fertilization (RM) and the control, hence potentially affecting soil Mg stocks. The pH increased from 5.0 in 2006 to 5.4–6.2 in 2010 in the 0–20 cm layer but was stationary in the 0.2–0.4 m layer, indicating reduction of potential aluminum toxicity (Natale et al., 2012). Figure 1 Nutrient budgets computed by difference between nutrient inputs from fertilization and nutrient removal through harvest. Treatments 9–36 are guava waste additions on dry mass basis (Mg DGW ha−1), SF represents fresh guava waste, and RM is current fertilizer recommendation as mineral fertilizers. Bold segments show dispersion of balance sheets data (P = 0.05 confidence intervals, four replications indicated by symbol X). Soil analyses in the 0–0.2 and 0.2–0.4 m layers and the corresponding recommended optimum soil test values to conduct SLAN and BCSR diagnoses are presented in Table 5. In the 0–0.2 m layer, soil pH was within optimum ranges. Compared to initial conditions, soil pH increased to pH values close to 6.0, except for the mineral fertilizer treatment due likely to the acidifying action of urea. Soil test SO4-S increased in the mineral fertilization treatment due to gypsum (CaSO4.2H2O) in simple superphosphate. Soil test SO4-S did not vary across guava waste treatments despite additions of organic S. Soil tests Cu and Mn increased significantly due likely to fungicide applications while B decreased significantly in the control treatment only. Soil tests Ca and Mg appeared low, leading to base saturation near the lower limit suggested for guava (Natale et al., 1996; van Raij et al., 1997). Soil tests P and K were well below the Brazilian standards (Natale et al., 1996; van Raij et al., 1997). Soil test K was the only soil fertility index decreasing systematically across treatments in both the 0–0.2 and 0.2–0.4 m layers. The decline in soil test K confirmed the substantial mining of soil K reserves resulting from cumulated K deficits. Table 5 Soil properties in the 0–0.20 and 0.20–0.40 m soil layers under tree crown (mean ± standard error) at the onset of the experiment and gain (+) or loss (–). Property Suggested targets Initial soil properties Dried-and-ground guava waste Fresh guava waste Mineral fertilization Mg dry matter ha−1 0 9 18 27 36 18 – Gain (+) or loss (–) compared to initial conditions (confidence intervals about mean, P = 0.05) 0–0.2 m SOIL LAYER pH (CaCl2) 4.5–7.0£ 5.01 ± 0.03 1.15 ± 0.54 0.83 ± 0.13 1.23 ± 0.33 1.00 ± 0.22 0.40 ± 0.16 0.98 ± 0.33 0.50 ± 0.59 5.1–5.5§ mg dm−3 Resin P 13–30§ 10.2 ± 0.8 −1.0 ± 2.8 2.0 ± 4.1 6.8 ± 2.5 10.5 ± 9.3 12.3 ± 4.0 10.0 ± 5.0 8.8 ± 5.7 B – 0.24 ± 0.004 −0.12 ± 0.08 −0.04 ± 0.10 −0.08 ± 0.18 −0.05 ± 0.21 −0.14 ± 0.14 −0.11 ± 0.14 −0.11 ± 0.14 Cu – 9.8 ± 0.50 3.2 ± 2.6 3.3 ± 2.3 3.2 ± 2.1 5.1 ± 3.3 5.2 ± 4.3 5.6 ± 3.2 3.1 ± 2.0 Fe – 17.2 ± 0.52 −4.5 ± 4.5 −4.5 ± 1.7 −5.0 ± 1.8 0.5 ± 6.1 7.0 ± 10.7 −1.3 ± 2.4 1.5 ± 1.7 Mn – 17.5 ± 0.57 1.6 ± 5.4 7.3 ± 2.0 8.4 ± 2.9 13.9 ± 3.6 12.8 ± 3.7 13.9 ± 5.8 18.5 ± 6.6 Zn – 0.48 ± 0.020 0.08 ± 0.38 0.13 ± 0.54 −0.03 ± 0.31 0.03 ± 0.25 0.18 ± 0.42 0.23 ± 0.15 −0.05 ± 0.16 SO4-S – 2.8 ± 0.29 2.0 ± 1.4 1.3 ± 1.9 0.5 ± 1.0 1.5 ± 1.7 2.0 ± 2.6 0.3 ± 2.1 7.5 ± 4.0 mmolc dm−3 K 1.6–3.0§ 2.3 ± 0.065 −1.9 ± 0.7 −1.2 ± 0.8 −1.1 ± 0.4 −1.1 ± 0.4 −1.2 ± 0.4 −1.3 ± 0.7 −1.8 ± 0.3 Ca 27§ 15.3 ± 0.49 10.3 ± 11.9 7.3 ± 2.6 4.8 ± 2.8 5.8 ± 1.7 2.3 ± 3.8 3.8 ± 2.1 4.5 ± 7.6 Mg 9§ 6.4 ± 0.27 3.8 ± 4.6 4.5 ± 3.9 3.5 ± 1.3 5.0 ± 0.8 3.8 ± 1.5 4.8 ± 2.5 2.8 ± 2.6 Σ bases – 24.1 ± 0.7 12.2 ± 15.9 9.8 ± 3.6 7.2 ± 4.1 9.6 ± 2.3 4.8 ± 5.1 7.2 ± 3.7 5.5 ± 10.3 CEC – 43.8 ± 0.7 6.4 ± 13.3 6.3 ± 4.6 3.9 ± 4.6 6.1 ± 3.0 6.1 ± 3.9 3.2 ± 3.4 7.3 ± 6.4 % Base saturation 51–70§ 54.5 ± 1.65 14.8 ± 9.7 21.6 ± 13.7 13.2 ± 9.6 11.9 ± 11.0 −0.8 ± 6.5 9.3 ± 5.2 −3.4 ± 18.1 g dm−3 OM 13.3 ± 0.2 −1.5 ± 4.8 2.0 ± 1.8 −1.3 ± 3.3 1.8 ± 1.5 −0.3 ± 1.0 1.0 ± 4.7 0.8 ± 4.3 0.2–0.4 m SOIL LAYER pH (CaCl2) 4.5–7.0£ 5.11 ± 0.06 0.70 ± 0.35 0.45 ± 0.55 0.55 ± 0.42 0.68 ± 0.42 0.48 ± 0.51 0.58 ± 0.27 0.30 ± 0.39 5.1–5.5§ mg dm−3 Resin P 13–30§ 5.36 ± 0.32 −1.3 ± 2.1 −0.3 ± 2.3 1.3 ± 5.8 3.3 ± 2.5 3.0 ± 1.0 3.3 ± 4.8 1.0 ± 2.0 B – 0.18 ± 0.018 −0.09 ± 0.14 −0.05 ± 0.07 −0.06 ± 0.14 −0.02 ± 0.19 0.10 ± 0.24 −0.08 ± 0.03 −0.05 ± 0.08 Cu – 2.8 ± 0.30 0.65 ± 0.76 0.65 ± 0.38 0.93 ± 1.70 1.45 ± 1.80 1.70 ± 2.92 0.55 ± 1.88 0.73 ± 1.48 Fe – 11.5 ± 0.29 −2.8 ± 1.8 −2.5 ± 1.2 −1.8 ± 0.6 −2.0 ± 1.7 −0.3 ± 1.2 −2.0 ± 1.4 0.3 ± 1.2 Mn – 14.8 ± 0.99 7.4 ± 9.8 7.2 ± 3.6 9.0 ± 2.1 9.8 ± 2.3 15.1 ± 0.6 10.8 ± 5.4 16.3 ± 8.1 Zn – 0.25 ± 0.015 −0.13 ± 0.06 0.10 ± 0.10 −0.05 ± 0.16 −0.05 ± 0.12 0.03 ± 0.12 −0.15 ± 0.07 −0.10 ± 0.10 SO4-S – 6.7 ± 0.45 −3.3. ± 1.5 −0.5 ± 5.1 −1.5 ± 4.1 −4.0 ± 3.0 −1.3 ± 2.5 −1.3 ± 1.5 1.0 ± 4.6 mmolc dm−3 K 1.6–3.0§ 1.8 ± 0.06 −1.2 ± 0.9 −1.0 ± 0.9 −1.2 ± 0.5 −1.3 ± 0.4 −0.9 ± 0.9 −0.9 ± 0.9 −0.9 ± 0.4 Ca 27§ 13.0 ± 0.51 2.5 ± 4.2 1.0 ± 2.6 3.3 ± 5.6 1.8 ± 2.5 3.5 ± 4.5 4.0 ± 3.9 −0.5 ± 1.6 Mg 9§ 5.5 ± 0.30 1.8 ± 2.7 0.8 ± 0.6 1.8 ± 2.3 1.8 ± 1.2 2.8 ± 0.6 3.0 ± 2.2 1.0 ± 2.0 Σ bases – 20.4 ± 0.7 3.1 ± 6.0 0.8 ± 2.3 3.8 ± 5.6 2.2 ± 3.4 5.4 ± 4.6 6.1 ± 6.2 −0.4 ± 3.4 CEC – 38.1 ± 0.8 1.6 ± 5.9 1.3 ± 6.7 3.3 ± 3.9 1.2 ± 4.4 7.9 ± 3.8 5.8 ± 7.6 −0.2 ± 4.0   % Base saturation 51–70§ 53.8 ± 0.9 6.2 ± 9.4 −0.1 ± 5.7 4.6 ± 8.1 3.8 ± 9.3 2.5 ± 10.7 5.5 ± 2.2 −4.6 ± 9.5 g dm−3 OM 8.6 ± 0.4 −0.5 ± 1.6 −0.3 ± 2.3 1.3 ± 5.8 −0.3 ± 3.2 1.0 ± 4.0 0.8 ± 4.0 0.3 ± 2.1 £ Crane and Balerdi (2015). § Natale et al. (1996), van Raij et al. (1997). Climate and nutrient balances impact on fruit yield and quality Increased precipitations and the minimum and maximum monthly temperature were correlated positively with fruit yield but negatively with fruit acidity (Figure 2). Precipitations and temperature influenced both the Brix index and yield but in different directions. The Brix index significantly decreased with maximum temperature, indicating an upper temperature limit for fruit sweetness that was exceeded during the experiment. Because temperature varied little across the experimental period while total precipitations varied widely, 2009 being the wettest year and 2010, the driest, subtle variations in temperature as well as large variations in precipitations could impact on fruit yield and quality. Climate thus nurtured the ties between high fruit yields and fruit characteristics important to the processing industry. Figure 2 Correlations between fruit and foliar nutrient balances, climate conditions, and crop performance. *0.01 < p-value ≤ 0.05; **0.001 < p-value ≤ 0.01; *** p-value ≤ 0.001. Foliar nutrient balances were also influenced by climate conditions. Higher maximum and minimum temperatures and precipitations increased foliar nutrient accumulation vs. the filling value. The leaf [B |Ca] balance increased significantly with maximum temperature while the [P |N] balance decreased with minimum temperature and precipitations that may slow down organic N mineralization. The leaf [Mg |K] balance increased with maximum temperature, indicating a prominent role of K for osmotic adjustment to maintain higher cell turgor pressure (Wang et al., 2013). The balance between relatively immobile (B, Ca) and relatively mobile (S, Mg, K, P, N) nutrients increased with precipitations and minimum temperature. In the present experiment, maximum temperature apparently hastened convective flow of Ca and B to the leaf, hence increasing the foliar [B,Ca |S,Mg,K,P,N] balance. Positive correlations between fruit and foliar nutrient balances were found to be significant only for the [B |Ca] and [P |N] balances, indicating proportionate supply of those nutrients to leaf and fruit between blooming and harvest and little proportionality for other nutrients. Fruit yield was correlated positively with the fruit [Fv |B,Ca,S,Mg,K,P,N], [P |N], [B,Ca |S,Mg,K,P,N], and [B |Ca] balances (Figure 2). In contrast, fruit acidity was correlated negatively with the fruit [Fv |B,Ca,S,Mg,K,P,N] balance, indicating that fruit acidity increased with lesser nutrient accumulation in the fruit. The Brix index was correlated negatively with the fruit [Fv |B,Ca,S,Mg,K,P,N] and [B |Ca] balances, and positively with the fruit [Mg |K] balance, indicating that fruit sweetness increased with fruit B and K concentrations in particular but with nutrient dilution in general. Although different fruit nutrient balances can lead to different levels of fruit yield and quality, plant nutrient status is most commonly diagnosed using interpretation methods that relate foliar tissue analysis to crop yield to secure the yield. Nutrient standards at high yield level using k nearest neighbors classification The accuracy of the partition using the k nearest neighbors (knn) classification across ilrs was 82% in cross-validation and 93% on the whole data set, with 96 true negatives, 85 true positives, 4 false negatives, and 10 false positives (negative predictive value = 0.96; positive predictive value = 0.89; specificity = 0.91; sensitivity = 0.96). Confidence intervals (P < 0.05) about ilrs and the corresponding minimum and maximum concentration values for TN and TP specimens are presented in the mobile design in Figure 3 where statistical analyses are conducted at fulcrums in the balance domain and nutrient concentrations are appreciated in buckets using minimum and maximum values. Despite large K deficits, the K concentration ranges and the K balances involving K did not differ between TN and TP specimens, because the soil K stocks declined markedly to support plant K nutrition. Concentration ranges for TN and TP specimens did not overlap for P and S, and nearly so for Ca and B. Apparent critical values were 1.77 g P kg−1, 2.81 g S kg−1, 9.00 g Ca kg−1, and 30 mg B kg−1. Because other ranges overlapped between TN and TP specimens, apparent critical values could not be computed for that guava agroecosystem. Figure 3 Mobile design showing the domain of nutrient balances with confidence intervals (P = 0.05) at fulcrums and the nutrient concentration domain in buckets showing minimum, mean, and maximum concentration values for true negative (TN) and true positive (TP) specimens. Discussion Nutrient budgets Despite small annual variations in compositions between batches of guava waste, the differences between nutrient inputs and outputs remained linear (Figure 1). At maximum fruit yield of 60–61 Mg fruit ha−1 (de Souza et al., 2014b), all nutrients but K were found to be in excess of removal through harvest, indicating potential soil accumulation of all nutrients but K. While guava waste has been classified as a slow-released N fertilizer (de Souza et al., 2011), indicating potential residual effects of guava waste on the following crop as was the case for manure and compost (Eghball et al., 2004), there was no N shortage even at low dosage. Although guava yield response was found to be linearly related to added guava waste (de Souza et al., 2014b), a closer examination of the data showed that yield response apparently plateaued at 9 Mg ha−1 where added N was 101–118 kg N ha−1. de Souza et al. (2011) found that 30% of total N from 9 Mg guava waste ha−1 was mineralized after 126 days in a laboratory incubation experiment. The first-order kinetics models returned maximum mineralizable N of 69 mg N kg−1 for the control without amendment and 167 mg N kg−1 added as 9 Mg guava waste ha−1. The difference was 98 mg mineralized N kg−1 compared to 52.2 mg total N kg−1 added to soil as guava waste, indicating priming effect of guava waste on soil organic matter decomposition. Indeed, organic matter decomposition is mediated by fast- and slow-growing microbial communities specialized for utilizing various sources of organic matter (Chen et al., 2014). The first-order models showed that an amount of 52.2 mg total N kg−1 from guava waste was mineralized after 100 days, well within the 10 months period required to complete the guava cycle. Irrigation and heavy rainfall may have further stimulated biological activity and organic matter mineralization under field conditions (Dersch and Böhm, 2001; Calderón and Jackson, 2002; Sainju et al., 2010; Condron et al., 2014). While K is extracted in large amounts by guava fruits (Natale et al., 1994), the K deficit did not affect crop performance during the 6 years of experimentation. Indeed, fruit species can consume internal nutrient reserves to supply the demand for fruit production (Adrian et al., 2015). It is often assumed that the change in plant nutrient reserves at steady state is approximately equal to change in the pruned nutrient biomass that is left at soil surface (Tagliavini and Scandellari, 2012). The contribution of pruning residues to nutrient cycling was not quantified in this study but can be assessed from literature. Guava pruning residues may contain 1.9–8.2% ash, of which 25–52% was K, 20–37% Ca, 7–16% Mg, 5–9% P, and 3–5% S, while a guava tree returned 9–12 kg of pruning biomass after fruit harvesting (Camarena-Tello et al., 2015). Assuming concentration averages and 10.5 kg pruning residues tree−1, pruning residues could have contributed 152 kg K ha−1, 45 kg Mg ha−1, and 112 kg Ca ha−1, i.e., 3.89 mmolc K dm−3, 3.77 mmolc Mg dm−3, and 5.61 mmolc Ca dm−3 to soil reserves. Pruning residues apparently sustained soil Ca and Mg during the experimental period but not the K reserves (Table 5). The K deficit could be managed using the nutrient buildup and maintenance concept based on nutrient balance sheets to avoid long-term soil K depletion below optimum soil test K. While soil test K threshold may depend on the chemistry of pH-dependent charges (Levy et al., 1988; Melo et al., 2002) and the yield level (Parent et al., 2012b), the optimum soil test K of 1.6 mmolc K dm−3 commonly used in Brazil for SLAN diagnosis appeared to be too high because no K shortage was found in the guava diagnostic leaf despite cumulated K deficits. To avoid excessive K deficits and excess of other nutrients on the long run in this guava agroecosystem, the parsimonious dosage of guava waste (e.g., 9 Mg ha−1 on dry mass basis) could be supplemented by mineral K fertilizers to maintain soil test K above 0.7 mmolc K dm−3 at productivity levels of 60–61 Mg ha−1 or 1.0–1.2 mmolc K dm−3 at productivity levels of 68–77 Mg ha−1 (Parent et al., 2012b). Soil test P appeared to be adequate at 10 mg P dm−3, as shown by excessive P supply to the leaf in TP specimens, indicating that the optimum soil test P range of 13–30 mg P dm−3 commonly used in Brazil was also too high. Nevertheless, being not compositional, the SLAN, and BCSR interpretation methods used in this paper to interpret the results of soil analysis could be revisited using compositional methods. Nutrient balances Data mining and compositional data analysis techniques were used to estimate nutrient concentration ranges at high yield level. The balance concept provided a data transformation technique that reduced D parts to D - 1 orthogonal variables that accounted for nutrient interactions hidden in concentration values likely affecting concentration ranges (Bates, 1971). The accuracy of the knn classification across ilr values was 0.93, higher than 80% or more obtained in other studies using balances (Marchand et al., 2013; Parent et al., 2013a,c; Parent et al., 2015; Modesto et al., 2014) and up to 73% obtained in DRIS studies (Wadt et al., 2016). Concentration ranges obtained from 100 000 Monte Carlo simulations can be compared to the literature. Natale et al. (2002) proposed ranges of 20–23 g N kg−1, 1.4–1.8 g P kg−1, 12–17 g K kg−1, 7–11 g Ca kg−1, 3.4–4.0 g Mg kg−1, 2.5–3.5 g S kg−1, and 20–25 mg B kg−1. Maia et al. (2007) suggested the following ranges: 20.2–25.3 g N kg−1, 1.4–1.5 g P kg−1, 19.0–21.7 g K kg−1, 7.7–8.3 g Ca kg−1, and 2.7–2.8 g Mg kg−1. Brazilian nutrient ranges thus appeared too high for N and Mg and too wide for K at lower bound for this guava agroecosystem. Different upper bound concentration values were also found for S and B. The N/P (Redfield) ratio ranged from 11.6 and 12.2 for TN specimens and 10.4–11.3 for TP specimens due apparently to excessive P levels in TP specimens. The lower critical value for the N/P ratio was 11.45. Other estimates of N/P ranges for guava at high yield level in Brazil were 11.5–13.1 (Parent et al., 2012a), 11.0–16.4 (Natale et al., 2002), 13.5–18.2 (Maia et al., 2007), and 10.3–13.7 (Hernandes et al., 2012), within the wide range of 10–20 as reported by Güsewell (2004). Hence, the soil test P of 10 mg P dm−3 appeared to be adequate to sustain the productivity of this guava orchard. Conclusion Fertilization treatments with guava waste produced large cumulated K deficits during the 6 years of experimentation. However, the leaf and fruit tissues did not show any K shortage despite soil K mining. Guava waste could thus be recycled in guava orchard at parsimonious dosage to avoid N and P excess on the long run and supplemented with K to avoid K deficiency. On the other hand, depending on subtle climate change, imbalanced fertilization with guava waste affected to different degrees the nutrient budgets of the agroecosystem, the nutrient balances in fruit and foliar tissues, and fruit yield, and quality. Foliar nutrient balances could be monitored and diagnosed accurately against standards developed using tools of data mining and compositional data analysis. Brazilian guava growers could benefit from this research by (1) revisiting optimum soil test K and P thresholds as well as the SLAN and BCSR interpretation models used in Brazil to achieve parsimonious nutrient management in guava agroecosystems where guava waste is recycled, and (2) monitoring the nutrient balance of fruit and foliar tissues to reach balanced fertilization in relation with targeted fruit yield and quality standards, and variations in climate conditions. Author contributions HS: Field work, data collection and acquisition, statistical analyses and interpretation, literature review. SP: Data modeling (data mining, compositional data analysis, statistical analyses), graphics and tables, co-writer of the paper. DR, DA, VM: Field work, data collection, statistical analyses, and data set organization. WN: Conception and design of the experiment, laboratory methods, relation with the industrial partner, data interpretation, paper review, literature review. LP: Data set, literature review, co-writer of the paper. Funding We are grateful to Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq), Indústria de Polpas e Conservas VAL Ltda., and the Natural Sciences and Engineering Research Council of Canada (NSERC-DG 2254) for financial support. Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Abbreviations BCSRbasic cation saturation ratio CECcation exchange capacity DGWdry guava waste DRISdiagnosis and recommendation integrated system FNfalse negative FPfalse positive ilrisometric log ratio knnk nearest neighbors RMrecommended mineral fertilization SFfresh guava waste SBPsequential binary partition SLANsufficiency level of available nutrient TNtrue negative TPtrue positive. ==== Refs References Abreu M. F. 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==== Front Comput Math Methods MedComput Math Methods MedCMMMComputational and Mathematical Methods in Medicine1748-670X1748-6718Hindawi Publishing Corporation 10.1155/2016/2564584Research ArticleA Numerical Simulation of Cell Separation by Simplified Asymmetric Pinched Flow Fractionation Ma Jing-Tao 1 2 http://orcid.org/0000-0002-4482-893XXu Yuan-Qing 1 3 * Tang Xiao-Ying 1 3 1School of Life Science, Beijing Institute of Technology, Beijing 100081, China2School of Engineering and Information Technology, University of New South Wales, Canberra, ACT 2600, Australia3Key Laboratory of Convergence Medical Engineering System and Healthcare Technology, The Ministry of Industry and Information Technology, Beijing Institute of Technology, Beijing 100081, China*Yuan-Qing Xu: bitxyq@bit.edu.cnAcademic Editor: Yi Sui 2016 15 8 2016 2016 256458415 4 2016 11 7 2016 Copyright © 2016 Jing-Tao Ma et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.As a typical microfluidic cell sorting technique, the size-dependent cell sorting has attracted much interest in recent years. In this paper, a size-dependent cell sorting scheme is presented based on a controllable asymmetric pinched flow by employing an immersed boundary-lattice Boltzmann method (IB-LBM). The geometry of channels consists of 2 upstream branches, 1 transitional channel, and 4 downstream branches (D-branches). Simulations are conducted by varying inlet flow ratio, the cell size, and the ratio of flux of outlet 4 to the total flux. It is found that, after being randomly released in one upstream branch, the cells are aligned in a line close to one sidewall of the transitional channel due to the hydrodynamic forces of the asymmetric pinched flow. Cells with different sizes can be fed into different downstream D-branches just by regulating the flux of one D-branch. A principle governing D-branch choice of a cell is obtained, with which a series of numerical cases are performed to sort the cell mixture involving two, three, or four classes of diameters. Results show that, for each case, an adaptive regulating flux can be determined to sort the cell mixture effectively. National Natural Science Foundation of China81301291Beijing Higher Education Young Elite Teacher ProjectYETP1208 ==== Body 1. Introduction Sorting various categories of particles from the mixture to achieve pure sample is of great importance in biological and medical engineering. With the rapid development of micro total analysis systems, small sample volume, high throughput sample processing, high efficiency, and precise particle fractionation are several representative requirements to guide the design of sorting scheme [1]. And correspondingly, a host of particle sorting techniques have been developed in these years: for example, the fluorescence-activated cell sorting [2–4], magnetic-activated cell sorting [5–7], dielectrophoresis sorting [8, 9], and size-dependent sorting [10–12]. The last one has received a remarkable attention attributing to its promising advantages of low cost, high efficiency, and being label-free. There are four typical size-dependent sorting methods that are generally reported, the deterministic lateral displacement [10, 13], the pinched flow fractionation (PFF) [14–16], the cross-flow filtering [17], and the inertial focusing sorting [18]. PFF is relatively simple because there is no extra and specific microstructure needed in the channel, and it has been used to sort polymer beads [14], microparticles [19], and emulsion droplets [20] and for blood cells [21] in recent years. In these above researches, an asymmetric pinched flow fractionation scheme (AsPFF) proposed experimentally first by Takagi et al. [19] is reported to perform a continuous separation and collection for 1.5~5 μm particles; it bettered the traditional PFF remarkably, while there are still some aspects that could be improved, for example, to perform a hydrodynamic analysis and further develop an active and controllable cell or particle sorter. In the present study, a numerical AsPFF cell sorter model is established with an immersed boundary-lattice Boltzmann method (IB-LBM), where the channel structure, the flow, the multiple sizes of cells, and their interactions are considered. Based on the model, cells with a prescribed size can be manipulated to enter a desired D-branch simply by regulating the flux of one D-branch (or the pressure of one outlet). The numerical results demonstrate that the numerical cell sorter is effective to perform an active and controllable cell sorting, which suggests an improved scheme of AsPFF and is valuable for guiding the experimental design of cell sorter on microfluidic chips. 2. Models and Methods 2.1. Mathematical Models In the numerical model, the fluid motion is solved by LBM with D2Q9 lattice model. The discrete lattice Boltzmann equation of a single relaxation time model is [26–28] (1) gix+eiΔt,t+Δt−gix,t=−1τgix,t−gieqx,t+ΔtGi, where g i(x, t) is the distribution function for particles of velocity e i at position x and time t, Δt is the time step, g i eq(x, t) is the equilibrium distribution function, τ is the nondimensional relaxation time, and G i is the body force term. In the two-dimensional nine-speed (D2Q9) model [29], e i are given as follows: (2) e0=0,0,ei=cos⁡πi−12,sin⁡πi−12hΔt,for  i=1  to  4,ei=cos⁡πi−9/22,sin⁡πi−9/222hΔt,for  i=5  to  8, where h is the lattice spacing. In (1), g i eq and G i are calculated by [26, 30] (3) gieq=ωiρ1+ei·ucs2+uu:eiei−cs2I2cs4,Gi=1−12τωiei−ucs2+ei·ucs4ei·f, where ω i are the weights defined by ω 0 = 4/9, ω i = 1/9 for i = 1 to 4, and ω i = 1/36 for i = 5 to 8, u is the velocity of the fluid, c s is the speed of sound defined by cs=h/3Δt, and  f is the body force acting on the fluid. The relaxation time related to the kinematic viscosity of the fluid is in terms of (4) ν=τ−0.5cs2Δt. Once the particle density distribution is known, the macroscopical quantities, including the fluid density, velocity, and pressure, are then computed from (5) ρ=∑igi,u=∑ieigi+0.5fΔtρ,p=ρcs2. Although the lattice Boltzmann method is original from a microscopic description of the fluid behavior, the macroscopic continuity (6) and momentum equations (7) can be recovered from it through the Chapman-Enskog multiscale analysis [31]. Then the LBM maybe can be viewed as a way of solving the macroscopic Navier-Stokes equations: (6) ∂ρ∂t+∇·ρu=0, (7) ∂u∂t+u·∇u=−1ρ∇p+ν∇2u+f. For the IB-LBM frame, the fluid motion is first solved by LBM; then the position of immersed boundary can be updated within one-time step of Δt through [32] (8) Us,t=∫Ωux,tDx−Xs,tdx,∂X∂t=Us,t, where X(s, t) is the position of the cell membrane s at time t. U(s, t) is the membrane velocity and u(x, t) is the fluid velocity. d x is the lattice side length; Ω is the nearby area of the membrane defined by the Delta function D(x − X) [33–35]: (9) Dx−X=∏i=1nδxi−Xi, where (10) δxi−Xi=3−2xi−Xi+1+4xi−Xi−4xi−Xi28,xi−Xi≤1,5−2xi−Xi−−7+12xi−Xi−4xi−Xi28,1<xi−Xi≤2,0,xi−Xi>2. In (9) and (10), n denotes the total dimension of the model. The fluid-structure-interaction is enforced by the following equation [27, 32, 33, 36]: (11) fx,t=∫ΓFs,tDx−Xs,tds, where F(s, t) is Lagrangian force acting on the ambient fluid by the cell membrane. In the present study, the cell model is proposed as (12) F=Fl−Fb+Fs+Fe, where F l is the tensile force, F b is the bending force, F s is the normal force on the membrane which controls the cell incompressibility, and F e is the membrane-wall extrusion acting on the cell. The four force components are [33, 37–39] (13) Fl=∂∂sKl∂Xs,t∂s−1∂Xs,t∂s, (14) Fb=Kb∂4Xs,t∂s4, (15) Fs=KsS−S0S0n, (16) Fe=KeXs,t−XwXs,t−Xw3,Xs,t−Xw≤rc,0,Xs,t−Xw>rc, where K l, K b, K s, and K e are the constant coefficients for the corresponding force components. In (15), S is the evolving cell area, S 0 is the reference cell area, and n is unit normal vector pointing to fluid. In (16), X w is the position of the vessel wall, and r c is the cut-off distance of the effective scope in the membrane-wall interaction. 2.2. Physical Model and Simulation Setup The geometry model of for cell sorting is illustrated in Figure 1, which consists of 2 upstream branches (U-branches), 1 transitional channel, and 4 downstream branches (D-branches). The U-branches and D-branches branches are labeled with the numbers, as well as the corresponding inlets and outlets. The two U-branches are perpendicular and symmetrical about the center line of the transitional channel. The transitional channel connects the U-branches and a circular buffer area which assembles the entrances of the four D-branches. The D-branches 1 and 4 are straight, while 2 and 3 are folded for the convenience to conduct the boundary condition of outlets; 1 and 4 are also symmetrical about the center line of the transitional channel, as well as 2 and 3. The entire length x 0 and width y 0 of device are 458 μm and 400 μm, respectively. The width of inlet 1 and inlet 2 w i is 70.71 μm. The width of pinched segment w 0 is 30 μm. The width of outlet 1, outlet 4, and unfolded part of outlets 2 and 3 w b is 26 μm. The width of folded part of outlets 2 and 3 w e is 23 μm. Q = Δp/R is defined as [19], where Q is the flux of a D-branch, Δp is the pressure difference between the buffer center and the outlet, and R is the flow resistance produced by the microchannel. In order to allocate the flow averagely for all the D-branches under the same pressure boundary conditions, Rs in all D-branches should be equal. A way to make R be equal is described as two steps. First, set the pressure of all outlet to be the same. Second, change the length of the folded part of D-branches 2 and 3 until the stable flows of all outlets are equal. When sorting different size of cells, set the pressure of outlets 1, 2, and 3 to be the same, while the pressure of outlet 4 is regulatable, and the flows of D-branches can be reallocated by altering the outlet pressure. To quantify the the capacity of the reallocation of flow by regulating the flow of outlet 4, we define β = Q out4/(∑i=1 4 Q outi), where bigger β means bigger flow through outlet 4 and smaller flow through 1, 2, and 3. In addition, since the flow resistance R in each D-branch is the same, the flow Q is in proportion to ΔP; that is, regulation of flow can be simply realized by regulating the pressure difference; this means that β also can be defined as ΔP 4/(∑i=1 4ΔP i). 3. Results and Discussion 3.1. Validation The method and model are validated carefully here by performing a simulation of flow past a stationary circular cylinder. This simulation is carried out by employing IB-LBM model. The computational domain is shown in Figure 2. The length L and width H of the computational domain are 1000 and 800, respectively. The center point of cylinder is located at x = 301 and y = 401 and the diameter of cylinder D = 40. The cylinder is discretized into a series of points, and the spacing between two adjacent points is 0.6. The cylinder is handled by utilizing immersed boundary method (IB), and the feedback-force principle is adopted to compute the force density on the cylinder, which is described as [22, 40] (17) Fxs,t=α1∫0tuxs,t−Uxs,tdt+α2uxs,t−Uxs,t, where F(x s, t) denotes the interaction force between the fluid and the immersed boundary (cylinder), α 1 and α 2 are large negative free constants, u(x s, t) is the fluid velocity obtained by interpolation at the IB, and U(x s, t) is the velocity of the cylinder expressed by U(x s, t) = d x s/dt. Here, U(x s, t) equals 0 because cylinder is stationary. In this case, the ratio of length of the recirculation zone and cylinder diameter L w, the drag force coefficient C d (18), the lift force coefficient C l (19), and the Strouhal number S t are calculated at Reynolds numbers 40 and 100: (18) Cd=FD0.5ρU∞2D, (19) Cl=FL0.5ρU∞2D. The results are shown in Table 1. As shown in Table 1, the present results show close agreements with the general results reported by other literatures. This means the IB-LBM model adopted in present paper is accurate enough. 3.2. Determination of the Inlet Flow Ratio α In order to actualize the pinched flow to sort cells, it is necessary to establish an appropriate pinched segment in the transitional channel, which is able to lead all cells to move along with the lower sidewall of the transitional channel. There are three aspects for establishing the pinched segment. First, the width w 0 of the transitional channel is better to set as 1.3~1.5 times as the largest diameter of the cells, since it has been proved that a wider w 0 can reduce the fraction effect of pinched flow [14]. Second, the length of the transitional channel is suggested to set as 2 times as w 0; a too long transitional channel may result in central tendency of the flexible cells, which is unfavourable to control the cells to move along with the lower sidewall. Finally, the inlet flow ratio α = Q in1 : Q in2 is also important to achieve the effective cell sorting. To get a proper α, a set of numerical cases are performed by setting α = 1/8,1/6,1/4,1/2,1, 2,4, 6,8, and 10, where 20 cells with 8 μm diameter (the smallest size) are initialized and randomly placed in the U-branch 2 to test the function of the pinched flow. The cell center positions at the end of the transitional channel are recorded and shown in Figure 3. As shown in Figure 3, the cell center positions when leaving the pinched segment drop with the increase of α, and finally they reach a relatively steady state when α > 6. Although α = 8 and α = 10 seem to be much better, this means much higher shear stress, which may do damage to the cells. Therefore, α = 6 is the choice for the present study. 3.3. Effect of β and Cell Size on D-Branch Choice In our consideration, specific, multiple classes of cells with different sizes can be sorted if every class enters a D-branch. In this section, the parameter β and the cell size are regulated to manipulate a specific-diameter cell to enter one D-branch, and a series of numerical cases are performed to exhibit the relation of β, the cell size, and the choice of D-branch. To set up the numerical model, β is regulated from 0.1 to 0.9 with an increment of 0.1. Cells with the same initial diameter are released into U-branch 2. For each case of β, four sizes of cell diameter are chosen as 8 μm, 16 μm, 20 μm, and 24 μm to make clear which D-branch a specific diameter of cells prefers to enter. In order to eliminate the possible effect of the initial position of the cell to the D-branch choice, in each case, three randomly placed cells are released into the U-branch, and all the D-branch choices are taken into account. A D-branch choice for a rigid circular particle can be predicted by the following experimental equations [19]: (20) w0∗1−βNB−1N−1<D2<w0∗1−βNB−1NN=1,2,3, (21) D2>w0∗1−βNB−1N−1N=4, where w 0 is the width of pinched segment as marked in Figure 1, β is the outflow ratio at outlet 4, N B is the total number of outlets, and D is the particle diameter. According to the above two equations, the particle will enter the Nth (N = 1,2, 3,4) D-branch if D ranges in the scope which can be described with (20) or (21), where (20) is for N = 1,2, or 3, and (21) is only for N = 4. The predicted and numerical results of the choice of D-branch which is related to the cell diameter and β are exhibited in Figure 4. In these results, 11 numerical results out of 68 are found not to be consistent to the predicted results, which generally occur at the transition where the cell has approximate probability to enter two neighbouring D-branches. A most possible reason to result in the 11 differences is the predicted results are for rigid particles while cells are flexible. According to the results, by regulating β, the 8 μm and 16 μm cells can be sent into any one of all four D-branches, and some snapshots of the D-branch choice of 16 μm cell are displayed in Figure 5. By contrast, the 20 μm and 24 μm cells can select one of three D-branches labeled 2, 3, and 4, and the 20 μm cell snapshots are shown in Figure 6. The results indicate that, by simply regulating the flux of one D-branch, cells with the diameters ranging from 8 to 24 μm can be manipulated to enter different D-branches, which gives us an inspiration to sort cells with different sizes if they enter different D-branches at a given β. 3.4. Size-Dependent Cell Sorting As discussed in Section 3.3, cells with different diameters can be manipulated to choose a desired D-branch at a proper β; this gives us a potential scheme for sorting cell mixture with different sizes if the cell-cell interaction is not present; that is, all cells in mixture are discrete. In this section, a continuous size-dependent cell sorting is proposed based on the regulation of β. According to Figure 4, it is clear which D-branch a certain cell will enter at a specific β; therefore, two sizes of cells are sorted once they enter different D-branches. For example, at β = 0.1, the 8 μm cell can be sorted from the 20 or 24 μm cell since 8 μm will enter D-branch 1 while the latter two will enter D-branch 2, and the same result will happen if the 8 μm cell is replaced by 16 μm cell. Some corresponding snapshots are shown as in Figures 7(a) and 7(b). By this means, at β = 0.4, it can be predicted that three sizes of cell can be sorted, they are 8, 16, and 20 μm cells or 8, 16, and 24 μm cells. Two snapshots of the two cases are displayed as in Figures 7(c) and 7(d), respectively. Especially at β = 0.6, the 8, 16, 20, and 24 μm are predicted to enter four different D-branches, and the numerical experiment result validates this actually as exhibited in Figure 7(e). 4. Summary and Conclusion A size-dependent cell sorting model with an asymmetric pinched flow is investigated numerically by immersed boundary-lattice Boltzmann method. In the model, three aspects are summarized as the following. First, the geometry of the channels is designed specially according to the effective cell sorting, where the size of the transitional channel for controlling the pinched segment is discussed in detail. Second, the parameters α and β are defined, respectively, for the flux ratio of the two inlets and the flux proportion of outlet 4 in all outlets. α = 6 is considered as a proper value to prepare for the cell sorting, based on which the regulation of β can manipulate cells with different diameters to enter different D-branches. Finally, four sizes of cells are taken into account to exhibit the capacity of cell sorting, and the relations of the regulation flux, the cell size, and the choice of D-branch are analyzed systematically. The simulation results indicate that cells with different diameters can be successfully sorted into different D-branches, this evinces that the model we established is effective, which can provide a directive reference for the design of microfluidic chip for sorting multiple sizes of cells or particles. Acknowledgments This work is supported by the National Natural Science Foundation of China (no. 81301291) and the Beijing Higher Education Young Elite Teacher Project (no. YETP1208). Competing Interests The authors declare that they have no competing interests. Figure 1 The basic schematic structure of the simulated device. Figure 2 The computational domain for flow past a stationary circular cylinder. Figure 3 8 μm cell positions in pinched segment at different inlet flow ratio. Figure 4 Comparison of simulation and predicted outflow position. Figure 5 The 16 μm cell outflow positions at different outflow ratios: (a) 0.1, (b) 0.3, (c) 0.7, and (d) 0.9. Figure 6 The 20 μm cell outflow positions at different outflow ratios: (a) 0.1, (b) 0.5, (c) 0.6, and (d) 0.8. Figure 7 Separation of different-sized cells: (a) separation of 8 μm and 20 μm cells at β = 0.1, (b) separation of 16 μm and 24 μm cells at β = 0.2, (c) separation of 8 μm, 16 μm, and 20 μm cells at β = 0.4, (d) separation of 8 μm, 16 μm, and 24 μm cells at β = 0.5, and (e) separation of 8 μm, 16 μm, 20 μm, and 24 μm cells at β = 0.6. Table 1 Comparison of the flow characteristics. Literatures Re = 40 Re = 100 L w C d C d C l S t Present 2.40 1.57 1.39 ±0.35 0.160 Reference [22] 2.59 1.58 1.39 ±0.35 0.160 Reference [23] 2.31 1.57 1.36 ±0.34 0.163 Reference [24] 2.35 1.66 1.38 ±0.34 0.170 Reference [25] 2.40 1.57 1.40 ±0.34 0.162 ==== Refs 1 Autebert J. Coudert B. Bidard F.-C. Microfluidic: an innovative tool for efficient cell sorting Methods 2012 57 3 297 307 10.1016/j.ymeth.2012.07.002 2-s2.0-84865599598 22796377 2 Cho S. H. Chen C. H. Tsai F. S. Godin J. M. Lo Y.-H. 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==== Front Biomed Res IntBiomed Res IntBMRIBioMed Research International2314-61332314-6141Hindawi Publishing Corporation 10.1155/2016/8209453Research ArticleHigh Dimensional Variable Selection with Error Control http://orcid.org/0000-0002-2496-7211Kim Sangjin http://orcid.org/0000-0003-4135-2777Halabi Susan * Department of Biostatistics and Bioinformatics, Duke University Medical Center, Box 2717, Durham, NC 27710, USA*Susan Halabi: susan.halabi@duke.eduAcademic Editor: Weiwei Zhai 2016 15 8 2016 2016 82094533 4 2016 25 5 2016 Copyright © 2016 S. Kim and S. Halabi.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. The iterative sure independence screening (ISIS) is a popular method in selecting important variables while maintaining most of the informative variables relevant to the outcome in high throughput data. However, it not only is computationally intensive but also may cause high false discovery rate (FDR). We propose to use the FDR as a screening method to reduce the high dimension to a lower dimension as well as controlling the FDR with three popular variable selection methods: LASSO, SCAD, and MCP. Method. The three methods with the proposed screenings were applied to prostate cancer data with presence of metastasis as the outcome. Results. Simulations showed that the three variable selection methods with the proposed screenings controlled the predefined FDR and produced high area under the receiver operating characteristic curve (AUROC) scores. In applying these methods to the prostate cancer example, LASSO and MCP selected 12 and 8 genes and produced AUROC scores of 0.746 and 0.764, respectively. Conclusions. We demonstrated that the variable selection methods with the sequential use of FDR and ISIS not only controlled the predefined FDR in the final models but also had relatively high AUROC scores. National Institutes of HealthR01CA155296U01CA157703United States Army Medical ResearchW81XWH-15-1-0467 ==== Body 1. Introduction Prognosis will continue to play a critical role in patient management and decision making in 21st century medicine. Advanced technologies for genomic profiling are now available and they include millions of sets of molecular data in these assays. A critical element of personalized medicine is utilizing and implementing validated diagnostic signatures (or classifiers) for diagnosing or treating cancer patients. These signatures are built and validated utilizing common statistical methods and machine learning tools. For example, the Decipher signature has been developed as a prognostic model to predict metastasis after radical prostatectomy in patients with prostate cancer [1]. The Decipher score is a 22-feature genomic classifier that has been used to predict metastasis and has been independently validated for prediction of prostate metastasis [2–5]. Another example is oncotypeDx that has been used to stratify randomization and guide treatment in women with breast cancer [6]. A vital step in model building is data reduction. It is assumed that there are several variables that are associated with the clinical outcome in the large dimensional data. The main purpose of the variable selection is to detect only those variables related to the response. Variable selection is composed of two steps: screening and model building. The screening step is to reduce the large number of variables into moderate size while maintaining most of the informative variables relevant to the clinical response. In contrast, in the model building step, investigators develop a single best model utilizing a proper evaluation criterion. Penalized variable selection methods have played a key role in identifying important prognostic models in several areas in oncology [7–9]. Many articles focused on the development of methodologies related to “small N and large P” with the advent of high throughput technology in cancer. The sure independence screening (SIS) was introduced to reduce the high dimension to below the sample size to efficiently select the best subset of variables to predict clinical responses [10]. Although this approach is popular, it does not perform well under some situations. First, unimportant variables that are heavily correlated with important variables are more highly likely to be selected than important variables that are weakly associated with the response. Second, important variables that are not marginally significantly related to the response are screened out. Finally, there may be collinearity between variables that may impact the calculations of the individual predictors. The iterative sure independence screening (ISIS) was proposed to overcome the above issues. The procedure is to apply iteratively high dimensional variable screening followed by the proper scale of variable selection until the best subset of variables with high predictive accuracy is obtained. ISIS screening, however, is also computationally intensive and leads to high false discovery rate (FDR) in ultra-high dimensional setting (P ≫ 1 mils). The oncology literature is rich in articles related to the use of validated signatures. Despite their abundance, comparisons and the performance of these various methods have not been studied. We propose to use the false discovery rate (FDR) of the multiple testing correction methods as a screening method to reduce the high dimension to lower dimension as well as controlling the false discovery rate in the final model. We investigate the feasibility of the sequential use of FDR screening method with the ISIS and utilize three popular variable selection methods: LASSO [11], SCAD [12, 13], and MCP [14], through the extensive simulation studies. To the best of our knowledge, this is the first paper that thoroughly analyzes and compares the performance of the variable selection methods with the sequential use of FDR and ISIS screening methods. We use a prostate cancer signature as an example [1] where the number of probes is around 1.4 million and the clinical outcome is binary in nature: presence of metastasis (presence of metastasis = 1, no metastasis = 0) by fitting models based on the simulation results. In addition, we provide a broad review of the existing penalized variable selection methods with screening methods. The remainder of this paper is organized as follows. In Section 2, we provide general details of the screening methods of FDR [15] and ISIS [10] and the variable selection methods with the penalized logistic regression. In Section 3, we describe the simulation studies and in Section 4, we summarize the results of the simulations. We then apply the best screening methods from the simulation studies to the real data in Section 5. Finally in Section 6, we discuss our findings. 2. Methods We divide this section into several subsections describing the methods used in our paper. The screening section briefly discusses commonly used methods that reduce high dimensionality: false discovery rate (FDR) and iterative sure independence screening (ISIS). We then describe the methods needed to assess variable selection models. The final section considers three existing popular variable selection methods with the logistic regression. All simulations and calculations were carried out using glmnet and ISIS packages in the R library, and the code is available at https://www.duke.edu/halab001/FDR. 2.1. Benjamini and Hochberg False Discovery Rate (FDR) The false discovery rate is defined as the expected proportion of incorrectly rejected null hypotheses. That is, (1) EVR ∣ R>0, where V is the number of falsely rejected hypotheses and R is the total number of rejected hypotheses. We focus on the Benjamini and Hochberg FDR [15] method as a screening method in the simulation studies and application. Briefly, the procedure works as follows. Let q denote the FDR, where q ∈ (0,1).(1) Let p 1,…, p m be the p values of the m hypothesis tests and sort them from smallest to largest. Denote these ordered p values by p (1),…, p (m). (2) Let k^=maxk:p(k)≤(k×q)/m,  k = 1,2,…, m.   If k^>1, then reject p (1),…, p (k) and if k^=0, then there is no rejection of the m hypothesis. 2.2. Iterative Sure Independence Screening (ISIS) The ISIS method was proposed to overcome the difficulties caused by the sure independence screening [16]. Briefly, the algorithm works in the following way:(1) The likelihood of marginal logistic regression (LMLR) is computed for every j ∈ S = {1,2,…, p}. Then d which is N/4log⁡(N) of the top ranked variables of the descending order list of the LMLR is selected to obtain the index set I^1. (2) Apply those variables in I^1 to the penalized logistic models to obtain a subset of indices M^1. (3) For every variable j∈{S-M^1}, the likelihood of the marginal logistic regression condition on the variables in M^1 is solved. Then the likelihood estimators are sorted in descending order and then the d top ranked variables are selected to get the index set I^2. (4) Apply those variables in I^2∪M^1 to the penalized logistic models to obtain a new index set M^2. (5) Steps (3) and (4) are repeated until M^l = d or M^l=M^l-1. 2.3. Regularizing Methods with Penalized Logistic Regression The logistic regression is one of the most commonly used methods for assessing the relationship between a binary outcome and a set of covariates and building prognostic models of clinical outcomes. In addition, it is widely used in the classification of two classes such as the development of metastasis in prostate cancer [1]. The purpose of variable selection with the logistic regression model in high dimensional setting is to select the optimal subset of variables that will improve the prediction accuracy [17]. Variable selection in high dimensional setting is composed of two components: a likelihood function and a penalty function in order to obtain better estimates for prediction. Let the covariates of ith individual be denoted as x i = (x i1,…,x ip)T  for  i = 1,…, N and p is the total number of covariates. The penalized logistic regression is as follows: (2) −1N∑i=1Nyilog⁡pi+1−yilog⁡1−pi+pβ,i=1,2,…,N, where p(β), a penalty, is function and y i is 1 for cases and 0 for controls. The probability that ith individual is a case based on covariates' information is expressed as (3) pi=exp⁡xi′β1+exp⁡xi′β,i=1,2,…,N. The regression coefficients are obtained by minimizing the objective function (2). One of the most popular penalty functions is the least absolute shrinkage and selection operator (LASSO) [11]. It forces the coefficients of unimportant variables to be set to 0 and then the LASSO has sparsity property. The LASSO estimates are obtained by minimizing the above penalized logistic regression form (2). It has a satisfactory performance in identifying a small number of representative variables. Though LASSO is widely used in most applications [18–21], its robustness is open to question as it has the tendency to randomly select one of the variables with high correlation and exclude the rest of the predictors [22]. Another disadvantage of LASSO is that it always chooses at most N (sample size) number of predictors even though there are more than N variables with true nonzero coefficients [23]. The coefficients estimates are obtained by minimizing the following objective function based on the likelihood function of logistic regression: (4) β^lasso=argminβ⁡−1N·∑i=1Nyilog⁡pi+1−yilog⁡1−pi+λ∑j=1pβj. Another method commonly employed is the smoothly clipped absolute deviation (SCAD) with a concave penalty function that overcomes some of the limitations of the LASSO [12]. The coefficients from SCAD are solved by minimizing the following objective function: (5) β^scad=argminβ⁡−1N·∑i=1Nyilog⁡pi+1−yilog⁡1−pi+∑i=1pfλ,γβj. The SCAD penalty function, f λ,γ(β j), is defined by (6) fλ,γβj=λγβjI0≤βj≤λ+λγβj−λ−βj2−λ2/2a−1+λ2·Iλ<βj≤λγ+γ+1λ22 with λ ≥ 0 and γ > 2. The minimum concave penalty (MCP) is also a recognized method with SCAD, where the coefficients are estimated via minimization of the following objective function: (7) β^mcp=argminβ⁡−1N·∑i=1Nyilog⁡pi+1−yilog⁡1−pi+∑i=1pfλ,γβj. The MCP penalty function, f λ,γ(β), is defined by (8) fλ,γβ=2λγβj−βj22γIβj≤λγ+λ2γ2Iβj>λγ, for λ ≥ 0 and γ > 1. 3. Simulation Studies 3.1. Simulation Setup We performed extensive simulation studies to explore the performance of three popular variable selection methods: LASSO, SCAD, and MCP in high dimensional setting. We employed 10-fold cross validation to tune the regularization parameter for the methods. Figure 1 describes the schema of the simulation procedures. Based on the logistic regression model, we generated the binary outcome and covariates for each simulation as follows. First, we generated z 1, z 2,…, z p independently from N(0,1), and each of z i is an N × 1 vector. We defined x 1 = z 1, xi=ρxi-1+(1-ρ2)zi, where i = 2,…, p, so that correlation of x k and x l was ρ |k−l| for some ρ ∈ [0,1). That is, the covariates were generated with serialized correlation structure (AR (1)). Next, we specified the true regression coefficients β. We fixed all of β's except the first 25 β's to be 0. The true nonzero β's were sampled independently from uniform distribution [−1.5,2]. We considered 25 true effects of the regression coefficients since several classifiers including the Decipher score had selected 20–25 genes [1, 2] and because that number predicted reasonably well the outcome. The number of variables was fixed at P = 100,000, and the sample size was set at N = 900. Finally, the corresponding binary response y i was simulated based on the Bernoulli distribution with the following: (9) yi~Bernpxi,pxi=exiβ1+exiβ, where x i = (x i,1, x i,2,…, x i,p) and β = (β 1, β 2, …,β p)′. Covariates were generated until the target number of 450 cases and 450 controls was reached. We considered different simulation scenarios for the correlation matrix ΣP×P, ρ = {0, 0.1, 0.4} among variables. Each simulation scenario was composed of the nine different models with the combination of the FDR, the ISIS, and the random filtering (RF1000). The RF1000 selected 1,000 variables with the smallest unadjusted p values obtained from the marginal logistic regression with the three variable selection methods (LASSO, SCAD, and MCP). The reason we used RF1000 from the 100,000 potential variables was that the number of false discovery rates is low relative to the other random filtering (such as 2,000 or higher). Therefore, we considered the top 1,000 variables to be a reasonable number of variables screened as reference to be compared with our proposed methods. We then simulated the data 500 times because of computational intensity. In each simulation, we randomly divided the data into two parts: the training set (N = 600) for model selection and the testing set (N = 300) for validation. 3.2. Metrics of Performance We calculated the true positive rate (TP), the false positive rate (FP), the false discovery rate (FDR), the average number of false positives in the final model, the average model size, the average of the area under receiver operating characteristic (AUROC), and the number of screened true important variables through the FDR and the RF1000 to assess the impact of the FDR-ISIS screening method with the three variable selection methods. The true positive rate (TP), also called sensitivity, is the proportion of positives that are identified correctly given true positives: (10) True  Positive  rate  TP=TPTP+FN, where TP is the number of the true positives and FN is the number of false negatives. The false positive rate is the proportion of incorrect identification as a true positive given true negatives. That is, (11) False  Positive  rate  FP=FPTN+FP, where the FP is the number of false positives and the TN is the number of true negatives. In addition, the average number of false positives (ANFP) was computed as the number of false positives that were selected in the final model out of 500 simulations. Furthermore, the average model size was computed as the number of variables selected in the final model out of 500 simulations. Finally, the AUROC was utilized as a measure of the performance of the logistic regression and is the proportion of the time which a model predicts correctly given observations of a random positive and negative. A perfect model produces an AUROC = 1 whereas a random model has an AUROC = 0.5. 4. Simulation Results We summarized the simulation results for ρ = 0.1, one of three correlation structures in Table 1, where all 25 important covariates were assumed to have linear effects. Table 1 presents the performance of the nine different models with the FDR, ISIS, and random filtering based on 500 simulations. The average true positive rates (TP) were 0.223 and 0.268 for the three variable selection methods using FDR.05 − ISIS and FDR.20 − ISIS. The average true positive rates (TP) of the LASSO, SCAD, and MCP with RF1000 − ISIS were 0.46013, 0.46365, and 0.46739, respectively. These values were much higher than the two FDR screening methods which were below 0.30. On the other hand, the three variable selection methods with RF1000 − ISIS selected several of the false positive variables that consequently increased the false positive rate (FP). LASSO, SCAD, and MCP with RF1000 − ISIS included a higher average number of the false positives of 12.364, 12.260, and 12.170, respectively. Although the FDR filtering method did not select a higher number of true important variables, this screening method reduced the false positive rates below the predefined target α. The average numbers of the false positives in the final models with the FDR − ISIS methods were much smaller than that of using RF1000 − ISIS (Table 1). Specifically, the average numbers of the false positives in the final models with the LASSO, SCAD, and MCP with FDR.05 − ISIS were 0.216, 0.214, and 0.214 with the corresponding standard deviations 0.0219, 0.0215, and 0.0215. As expected, the three variable selection methods with RF1000 − ISIS had selected a higher average model size of 22.8 than the FDR methods. Similar results were observed for FDR.20 − ISIS. We also calculated the false discovery rate. The variable selection models with the FDR at the target α = 0.05 and α = 0.20 controlled the false discovery rate below α whereas over 40% of the finally selected variables were incorrectly selected using the random filtering methods. The average AUROC scores with RF1000 − ISIS were relatively higher than the FDR.05 − ISIS and FDR.20 − ISIS. Similar results were noted for independent and moderate correlation ρ = {0,0.4} as presented in Tables S1 and S2 in Supplementary Material available online at http://dx.doi.org/10.1155/2016/8209453. Figure 2 presents the selection frequency for the 25 important variables under the three different screening methods. The x-axis denotes the variable name and the y-axis represents the frequency of selection out of 500 simulations. The variables not depicted on the x-axis in Figure 2 did not have any counts and thus were not selected in the simulation. The variables with the highest selection frequencies had true regression coefficients that were strongly associated with the clinical response. These variables were g07, g03, g19, g23, g01, g24, g12, and g06 and were selected over 100 times out of 500 simulations with average corresponding regression coefficients of 1.65, −1.45, 1.57, −1.17, 1.12, 0.968, 0.963, and −1.01 (see Table S3 in Supplementary File). The coefficients of the eight variables were ranked the highest among the 25 absolute values of the true regression coefficients which had strong effects on the response. There were no differences in selecting the important variables by the variable selection methods (LASSO, SCAD, and MCP). In addition, similar patterns of the selection frequencies were observed for both FDR.05 − ISIS and FDR.20 − ISIS as shown in Figures 2(a) and 2(b) while Figure 2(c) showed a little variation with RF1000 − ISIS. The results were similar for independent and moderate correlation ρ = {0,0.4} (Figures S1 and S2 in Supplementary File). To gain more insights into the comparisons of the methods, we present the plots of the AUROC scores and the corresponding false discovery rate under ρ = 0.1 in Figure 3. (a), (c), and (e) in Figure 3 represent the AUROC scores whereas (b), (d), and (f) represent the false discovery rates using three different screening methods. The variable selection methods with random filtering screening had relatively higher AUROC scores compared to the FDR methods. However, there were a number of false positive in the final models as seen in Figure 3(f). It is noteworthy that the variable selection methods using the FDR not only controlled the FDR below the target α = 0.05 and α = 0.20 but also had AUROC scores that were relatively high (Figures 3(d) and 3(e)). Similar patterns were observed for independent and moderate correlation ρ = {0,0.4} (Figures S3 and S4 in Supplementary File). Therefore, the FDR − ISIS screening method is preferred to RF1000 − ISIS since it allowed the variable selection methods to obtain the proper AUROC scores while controlling the false discovery rate at the nominal level of α. As a result of the simulation studies, we applied the three variable selection methods with the FDR and ISIS screening to the high dimensional data of the prostate cancer in the following section. 5. Real Data Analysis We analyzed the prostate cancer data from the public domain (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE46691: GSE46691). The dataset has 1.4 million probes and the primary outcome is presence of metastasis (yes or no) by fitting the LASSO, SCAD, and MCP methods using the FDR and ISIS screenings suggested from the simulation studies with the sequential filtering of both FDR and ISIS. In the prostate cancer application, we considered the false discovery rate (FDR) at α = 0.01 as the screening method. Figure 4 describes the schema of the prognostic model building for the prostate cancer. We utilized the training set that was obtained from the random split and was composed of 359 individuals (140 cases and 219 controls) with 1.4 million probes to build each of the three models. We then estimated the AUROC scores with the validation set with 186 individuals (72 cases and 114 controls). We used 10-fold cross validation for each of the variable selection models to tune the parameters after the screening. We obtained 39 variables with FDR at α = 0.01. We repeated each of the three models 100 times to improve the AUROC with those screened variables. Figure 5 shows the AUROC plots of the three models. Based on FDR at α = 0.01, the LASSO, SCAD, and MCP identified 12 genes (CAMK2N1, AN07, RPL7A, MALAT1, MYBPC1, TMP0, UBE2C, DID01, RAB25, LOC728875, FTH1, and MKI67), 11 genes (CAMK2N1, AN07, RPL7A, MALAT1, MYBPC1, TMP0, UBE2C, DID01, RAB25, LOC728875, and FTH1), and 8 genes (CAMK2N1, AN07, RPL7A, MALAT1, MYBPC1, TMP0, UBE2C, and DID01) gene models out of 39 potential variables with AUROC scores of 0.746 (95% CI = 0.675–0.818), 0.746 (95% CI = 0.674–0.817), and 0.764 (95% CI = 0.695–0.834), respectively (refer to Table S4 for more details in Supplementary File). It is noteworthy to note that MCP selected the same set of genes as SCAD and LASSO and the 95% confidence intervals were overlapping. On the other hand, using the FDR at α = 0.05, LASSO, SCAD, and MCP selected 15, 13, and 15 gene models out of 565 potential genes with corresponding AUROC scores of 0.697 (95% CI = 0.619–0.775), 0.714 (95% CI = 0.637–0.791), and 0.683 (95% CI = 0.603–0.763), respectively. It is worthwhile to note that MCP had the highest AUROC score (FDR-ISIS at α = 0.01 and AUROC = 0.764) followed by LASSO (FDR-ISIS at α = 0.01 and AUROC = 0.746) although the results were not consistent with the FDR at α = 0.05. This could be due to the larger number of potential variables (565 variables) when using FDR at a higher level. Nevertheless because our interest was to use FDR at α = 0.01, MCP and LASSO methods were used for the variable selection in our real example. Table 2 presents the selected probes and their corresponding genes from the two models that had two highest AUROC scores among the six models. LASSO and MCP identified each of the 12 and 8 genes that were associated with developing prostate cancer metastasis. The four genes (ANO7, UBE2C, MYBPC1, and CAM2KN1) associated with developing prostate cancer metastasis were detected in both models. These four genes were a subset of the 22 biomarkers for the Decipher PCa classifier [1]. MYBPC1 (Myosin-Binding Protein C) on chromosome 12 and ANO7 (Anoctamin 7) on chromosome 2 were only downregulated genes whereas the other 10 genes including UBE2C (Ubiquitin-Conjugating Enzyme E2C) on chromosome 20 and CAMK2N1 (Calcium/Calmodulin-Dependent Protein Kinase II Inhibitor 1) on chromosome 1 were the top upregulated genes as presented in Figure S5. 6. Discussion This paper explored the feasibility of using the false discovery rate (FDR) followed by ISIS as screening methods in conjunction with three popular variable selection methods in ultra-high dimensional data for the purpose of controlling FDR and improving AUROC scores. Our simulation studies demonstrated that the variable selection methods with FDR − ISIS not only controlled the false discovery rate below the target α, but also produced high AUROC scores. Furthermore, the results showed that the false discovery rate was controlled conservatively even with the increased correlation structures. As demonstrated in the simulation studies, if the truly prominent variables have not passed through the screening, they would lose the opportunity to be selected to the final model. Thus, the prediction accuracy may be relatively reduced. Currently, most multiple testing correction methods underscore the priority of identifying prominent variables. Therefore, effective filtering techniques are ultimately needed for the situation when there are weak effects among the important variables. Although RF1000 − ISIS produced the highest AUROC through the simulation studies, it also had the highest false discovery rate. It is reasonable to expect that if one variable is not selected during the screening step, then the other variables that were correlated with the unselected variable have a tendency not to be chosen in the final model. As expected, the true positive rates of RF1000 − ISIS were relatively higher than those of using FDR − ISIS. This is because random filtering had more opportunity to select the true important variables. Due to the relatively high number of true important variables selected, the number of unimportant variables highly correlated with the true important variables was also high. This may explain why the average AUROC scores were highest in our simulation studies for RF1000 − ISIS. There are some caveats to the sequential use of FDR with ISIS. First, the total number of true important variables was restricted to 25 variables in the simulation studies. This may explain why the three variable selection methods, LASSO, SCAD, and MCP, performed similarly in the simulations. Second, the computational time for 500 simulations with ISIS alone was 52,500 minutes (around 36.45 days). Although ISIS is computationally intensive, FDR with ISIS performed very well and it took 9,625 minutes to perform the 500 simulations (7 days). Turning back to our motivation example of prostate cancer, LASSO and MCP under the FDR0.01 − ISIS screening methods produced the best AUROC scores. We also present the results for other LASSO and MCP methods selected 12 and 8 genes out of 39 screened probes using the FDR at the target α = 0.01 and had the AUROC scores of 0.7462 and 0.7644, respectively. The AUROC score of the MCP was 0.144 points higher than what was reported by Erho et al. ([1]; AUC = 0.75). Although the authors did not report a 95% confidence interval for the AUROC scores, it is most likely that the 95% confidence intervals for the AUROC scores of Erho et al. and the MCP were overlapping. In summary, based on our extensive simulations, FDR with ISIS seems to be superior to random filtering in terms of error control and is less computationally intensive compared with ISIS only. We also showed that the classifier based on 8 genes detected by the MCP had similar performance to the prognosis for early clinical metastasis prostate cancer model. To our knowledge, this is the first paper that systematically compared the performance of high dimensional methods with screening methods. Based on the extensive simulation studies, effective screening procedures with penalized logistic regression methods would not only lead to controlling the FDR but also produce high area under receiver operating characteristic curve. Supplementary Material Supplementary file is composed of the six parts of additional information that were not explained in the manuscript. Table S1 and S2 are the comparison of the performance for random filtering screening and FDR screenings with independent and moderate correlation ρ = {0, 0.4}, respectively in the simulation studies. Table S3 represents average true regression coefficients for the 25 variables through 500 replicates. Table S4 is comparison of area under the curve (AUC) and the corresponding 95% confidence interval with three popular variable selection methods (LASSO, SCAD, and MCP) for random screenings (1000,2000, and 4000) and FDR screenings at α = 0.01,0.05, and 0.20. Figure S1 and S2 shows the selection frequencies of each of the 25 variables across the LASSO, SCAD, and the MCP during 500 simulations with independent and moderate correlation ρ = {0, 0.4}. Figure S3 and S4 present AUC scores and the corresponding mean false discovery rate during the simulations with ρ = {0, 0.4}. Finally, Figure S5 shows boxplots of the expressions of 12 identified genes on metastasis in the prostate cancer. Acknowledgments This research was supported in part by National Institutes of Health Grants R01CA155296 and U01CA157703 and United States Army Medical Research W81XWH-15-1-0467. The authors thank Lira Pi for her help and would like to acknowledge the contribution of the Duke University Compact for Open-Access Publishing Equity fund for its support of this paper. Competing Interests The authors declare that they have no competing interests. Figure 1 Diagram showing simulation procedures. Figure 2 Selection frequencies of each of the 25 variables across the LASSO, the SCAD, and the MCP during 500 simulations with ρ = 0.1. The x-axis depicts the names of the variables, and the y-axis is the frequency of variables selected out of 500 simulations. The variables not depicted on the x-axis in Figure 2 did not have any counts. Each of the three methods is identified by color in legend. Figure 3 (a, c, e) The AUROC scores under ρ = 0.1. The x-axis is the name of methods and y-axis is AUROC scores. (b, d, f) The corresponding mean proportion of falsely selected variables in the model. The x-axis is the name of methods and the y-axis is the false discovery rate. Figure 4 The schema of prognostic model building for the prostate cancer. Figure 5 AUROC plots of using the LASSO, SCAD, and MCP with the screenings of both FDR at 0.01 and ISIS. (a), (b), and (c) are for the LASSO, the SCAD, and the MCP variable selection methods, respectively. Table 1 The true positive rate (TP), the false positive rate (FP), and the false discovery rate (FDR), the average number of false positives (ANFP) in the final models, the final model size (size), the area under the curve (AUROC), and the number of filtered truly important variables from FDR and RF (# filter) under the low correlation coefficients, ρ = 0.1 among variables. Screening Method TP FP FDR ANFP Size AUROC # filter FDR.05-ISIS LASSO 0.22336  (0.00203) 2.2e − 06  (0) 0.03086  (0.00303) 0.216  (0.02199) 5.8  (0.05816) 0.80285  (0.00157) 5.826 (0.05985) SCAD 0.22336  (0.00203) 2.1e − 06  (0) 0.03071  (0.003) 0.214  (0.02157) 5.798  (0.05791) 0.80286  (0.00157) MCP 0.22336  (0.00203) 2.1e − 06  (0) 0.03071  (0.003) 0.214  (0.02157) 5.798  (0.05791) 0.80286  (0.00157) FDR.20-ISIS LASSO 0.2676  (0.00229) 1.17e − 05  (0) 0.12618  (0.0055) 1.17  (0.05914) 7.86  (0.09344) 0.81965  (0.00154) 7.904 (0.09499) SCAD 0.2676  (0.00229) 1.16e − 05  (0) 0.12522  (0.00547) 1.158  (0.05866) 7.848  (0.09288) 0.81964  (0.00154) MCP 0.2676  (0.00229) 1.13e − 05  (0) 0.12355  (0.00541) 1.134  (0.05698) 7.824  (0.09096) 0.81967  (0.00154) RF1000-ISIS LASSO 0.42112  (0.00236) 0.0001237  (0) 0.53987  (0.00265) 12.364  (0.06298) 22.892  (0.01389) 0.83244  (0.00149) 13.286 (0.06807) SCAD 0.42336  (0.00243) 0.0001226  (0) 0.53635  (0.00276) 12.26  (0.06622) 22.844  (0.01624) 0.83196  (0.00147) MCP 0.42656  (0.00249) 0.0001217  (0) 0.53261  (0.00283) 12.17  (0.06797) 22.834  (0.01666) 0.83191  (0.00147) ( ): standard deviation. Table 2 Probes and corresponding genes identified by the LASSO and MCP methods with FDR at α = 0.01 for association with the prostate cancer metastases. The Adj. p is based on the marginally adjusted p values by the BH-FDR method. Gene Probe ID Ch Start Stop Adj. p LASSO MCP RAB25 2361272 chr1 156041891 156042035 0.003122859   ∗   CAMK2N1 2400181 chr1 20810150 20810212 0.003122859   ∗   ∗ LOC728875 2432120 chr1 143692898 143692956 0.007211879   ∗   AN07 2536262 chr2 242163962 242164581 0.003122859   ∗   ∗ FTH1 2590344 chr2 181551038 181551091 0.009379258   ∗   RPL7A 3284321 chr10 33483529 33483624 0.007451575   ∗   ∗ MKI67 3312502 chr10 129899547 129899701 0.003122859   ∗   MALAT1 3377635 chr11 65206468 65206658 0.009379258   ∗   ∗ MYBPC1 3428626 chr12 102030464 102030494 0.009379258   ∗   ∗ TMP0 3467302 chr12 98943231 98943926 0.008090076   ∗   ∗ UBE2C 3887068 chr20 44445472 44445507 0.001041641   ∗   ∗ DID01 3913561 chr20 156041891 156042035 0.003122859   ∗   ∗ ∗Each gene is identified by the variable selection method. ==== Refs 1 Erho N. Crisan A. Vergara I. A. 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==== Front Biomed Res IntBiomed Res IntBMRIBioMed Research International2314-61332314-6141Hindawi Publishing Corporation 10.1155/2016/6294098Research ArticleInduction of Osmoregulation and Modulation of Salt Stress in Acacia gerrardii Benth. by Arbuscular Mycorrhizal Fungi and Bacillus subtilis (BERA 71) Hashem Abeer 1 2 http://orcid.org/0000-0002-8509-8953Abd_Allah E. F. 3 4 * Alqarawi A. A. 3 Al-Huqail A. A. 1 Shah M. A. 5 1Department of Botany and Microbiology, Faculty of Science, King Saud University, Riyadh 11451, Saudi Arabia2Mycology & Plant Disease Survey Department, Plant Pathology Research Institute, ARC, Giza 12511, Egypt3Department of Plant Production, Faculty of Food & Agricultural Sciences, P.O. Box 2460, Riyadh 11451, Saudi Arabia4Seed Pathology Department, Plant Pathology Research Institute, ARC, Giza 12511, Egypt5Department of Botany, University of Kashmir, Srinagar, Jammu and Kashmir 190001, India*E. F. Abd_Allah: eabdallah@ksu.edu.saAcademic Editor: Diby Paul 2016 15 8 2016 2016 629409811 5 2016 27 6 2016 Copyright © 2016 Abeer Hashem et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.The role of soil microbiota in plant stress management, though speculated a lot, is still far from being completely understood. We conducted a greenhouse experiment to examine synergistic impact of plant growth promoting rhizobacterium, Bacillus subtilis (BERA 71), and arbuscular mycorrhizal fungi (AMF) (Claroideoglomus etunicatum; Rhizophagus intraradices; and Funneliformis mosseae) to induce acquired systemic resistance in Talh tree (Acacia gerrardii Benth.) against adverse impact of salt stress. Compared to the control, the BERA 71 treatment significantly enhanced root colonization intensity by AMF, in both presence and absence of salt. We also found positive synergistic interaction between B. subtilis and AMF vis-a-vis improvement in the nutritional value in terms of increase in total lipids, phenols, and fiber content. The AMF and BERA 71 inoculated plants showed increased content of osmoprotectants such as glycine, betaine, and proline, though lipid peroxidation was reduced probably as a mechanism of salt tolerance. Furthermore, the application of bioinoculants to Talh tree turned out to be potentially beneficial in ameliorating the deleterious impact of salinity on plant metabolism, probably by modulating the osmoregulatory system (glycine betaine, proline, and phenols) and antioxidant enzymes system (SOD, CAT, POD, GR, APX, DHAR, MDAHR, and GSNOR). King Saud UniversityRGP-271 ==== Body 1. Introduction Salinity is a common environmental stress that has adverse impacts on crop growth and development [1, 2], reduces yield of crops, and disrupts the local ecological balance [3]. Plants have been shown to combat the salinity stress through the enzymatic and nonenzymatic antioxidant defense systems that allow the scavenging of reactive oxygen species (ROS) for dynamic equilibrium [4]. Nonenzymatic antioxidant defense systems in plants include glutathione (GSH), phenolic compounds, and alkaloids [5]. Furthermore, the antioxidants act as a substrate for the antioxidant enzymes in the removal of ROS and hence play a direct role in the elimination of ROS [6]. On the other hand, the enzymatic defense system scavenges ROS and repairs the damage caused by ROS [7, 8]. The main antioxidant enzymes are superoxide dismutase, guaiacol peroxidase, glutathione peroxidase, catalase, glutathione reductase, ascorbate peroxidase, monodehydroascorbate reductase, and dehydroascorbate reductase [9]. Recent studies have shown that the colonization of plant roots with PGPR and AMF increases plants tolerance to salinity [10] through induction of osmoregulation and modulation of the impact of salt stress [2, 11, 12]. Plants inoculated with AMF show lower lipid peroxidation and enhanced antioxidant enzyme activities in common bean [13] as systemic resistance tool against salt stress. Similarly, PGPRs have been reported to alleviate salt stress in host plants through multiple mechanisms, including the rapid upregulation of conserved salt stress responsive signaling pathways [14]. Encouraging the vegetation to grow in hot desert conditions is a challenge which becomes even more important when the target plants are economically significant. The genus Acacia generally, and Acacia gerrardii particularly, is considered one of the most important tree groups in Saudi Arabia because the species is a good source of gums and tannins, besides being used as wood and forage [15]. However, this plant species has been found to be negatively influenced by salt stress conditions which tells upon its population size and distribution status [16]. Hence, we aimed to elucidate the salt stress defense and tolerance mechanisms of A. gerrardii by mutualistic interaction with endophytic B. subtilis and AMF. 2. Materials and Methods 2.1. Plants, Soil, and Endophytic Microorganisms Seeds of Talh tree (Acacia gerrardii Benth.) were provided by a nursery in Alghat city, Riyadh, that produces tree seedlings. The soil used was loamy sand type with the following properties (%): sand (87.6); clay (7.2); silt (5.2); organic carbon (0.12); total nitrogen (0.005); pH 7.5. The surface of seeds was sterilized with concentrated sulfuric acid followed by 70% (v/v) ethanol for 3 min and rinsed several times with sterile water. The sand was washed with 1.0 N H2SO4 for one hour, followed by 1.0 N Ca carbonate, and was washed using distilled water. The surface-sterilized seeds were sown in acid-washed sterile sand and kept in a plant growth chamber under the trap culture protocol for one month. The endophytic bacterium (Bacillus subtilis BERA 71) isolated from roots of Talh trees was collected from the top 20 cm of soil in a natural meadow at Khuraim in Riyadh, Saudi Arabia. The 16S rDNA sequence of Bacillus subtilis BERA 71 was submitted to the GenBank nucleotide sequence database and the accession number is KX090253 (http://www.ncbi.nlm.nih.gov/nuccore/KX090253?report=GenBank). Bacillus subtilis BERA 71 (Bs) was formulated as described in detail by Hashem et al. [17]. The lyophilized bacterial growth was mixed with talc powder (1.0% carboxy methyl cellulose as an adhesive agent) to give a final concentration of 3.6 × 109 cfu (colony forming units) of Bs/gram of formulated material. The bacterial formulated material was suspended in phosphate buffered saline solution and diluted to a cell density of 108 cells/mL. The AMF (Claroideoglomus etunicatum [syn. Glomus etunicatum]; Rhizophagus intraradices [syn. Glomus intraradices]; and Funneliformis mosseae [syn. Glomus mosseae]) used in the present study were extracted from soil samples surrounding the roots of A. gerrardii by wet sieving, decanting, and sucrose density gradient centrifugation as reported by Daniels and Skipper [18] and modified by Utobo et al. [19]. AMF species were identified based on subcellular structures (spore color, shape, surface ornamentation, spore contents, and wall structures) of asexual spores provided by the international collection of vesicular and arbuscular mycorrhizal fungi [20] and other descriptive protocols [21–23]. The trap culture protocol suggested by Stutz and Morton [24] was used in the current study to propagate the selected mycorrhizal isolates. The trap culture was used as mycorrhizal inoculum and was added to the experimental soil as 25 g of trap soil culture (approx. 100 spores/g trap soil, M = 80%)/pot. Soils not inoculated with mycorrhiza and PGPR served as the control. 2.2. Plant Growth Condition The pot experiment was carried out in the growth chamber of the Plant Production Department, Faculty of Food & Agricultural Sciences, King Saud University, Riyadh, Saudi Arabia. The soil used was loamy sand soil with the following properties (%): sand (87.6); clay (7.2); silt (5.2); organic carbon (0.12); total nitrogen (0.005); pH 7.5. Talh tree seeds were provided by a personal nursery in Alghat city, Riyadh, that produces tree seedlings. The seedlings were transplanted in plastic pots (2 Kg in capacity, one seedling/pot) kept in a plant growth chamber at 25 ± 2°C with an 18 h photoperiod, 750 μmol m−2 s−1 photosynthetic photon flux density, and 70–75% relative humidity for three months. Half-strength Hoagland's solution was used to irrigate the pots. The experiment was a completely randomized design with five replicates for each treatment: (i) control without microbes, (ii) Bs, (iii) AMF, and (iv) Bs + AMF. Similar treatments with salt were used as references. Salinity was established by adding NaCl to the irrigation solution to obtain a constant concentration of 250 mM. Plants were grown in a greenhouse for 12 weeks after transplantation. At harvest, plants were harvested carefully, and roots were separated from shoots to estimate the intensity of structural colonization. The shoot was dried to constant weight at 100°C to estimate the tools of nutritional value. Fresh leaf samples were used to assess the content of other chemical and biochemical analyses. 2.3. Determination of Arbuscular Mycorrhizal Colonization The fine roots were collected from the lateral root system of each treatment and were fixed in FAA solution, until their further processing. Roots were stained with trypan blue in lactophenol [25] and assessed for mycorrhizal infection. More pigmented roots, after clearing, were bleached in alkaline hydrogen peroxide (0.5% NH4OH and 0.5% H2O2 v/v in water) to remove any phenolic compounds [26] before acidification. For the assessment of mycorrhizal colonization, stained root segments were mounted on glass slides with lactophenol and observed under digital microscope (Model DP-72, Olympus) at 20x magnification. A minimum of 50 segments for each replicate sample were observed for the assessment of structural colonization of AM fungi associated with roots. Twenty or more segments (depending on the slide size) were mounted on each slide and examined under microscope. Presence of mycelium, vesicles, and arbuscules was recorded and analyzed for determining the structural colonization. The intensity of colonization was measured as poor (P), moderate (M), and abundant (A) type of colonization with each of the individual structures as described by Kormanik and McGraw [26] and McGonigle et al. [27]. Intensity of AMF colonization (mycelium, vesicles, and arbuscules) in Talh roots was calculated according to the following formula: (1) %  Colonization=Total number of AM positive segmentsTotal number of segments studied×100. 2.4. Estimation of Nutritional Value Fine powdered shoot samples were used for estimation of total nitrogen, ash content, and total lipids according to the protocol of Association of Official Agricultural Chemists [28]. Neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) were determined according to the method of Van Soest and Robertson [29]. The determination of condensed tannins (HCl-Butanol) was carried out spectrophotometrically (550 nm) according to the method described in detail by Makkar [30]. Spectrophotometer (T80 UV/VIS Spectrometer, PG Instruments Ltd., USA) was used for estimation of color intensity. 2.5. Estimation of Lipid Peroxidation (Malondialdehyde, MDA) 0.1 g leaves were homogenized with 1 mL of 10% trichloroacetic acid and centrifuged at 10000 g for 10 minutes. 1 mL supernatant was mixed with 20% trichloroacetic acid containing 0.25% thiobarbituric acid and was heated at 95°C for 30 min, quickly cooled in an ice bath, and then centrifuged again at 10,000 ×g for 10 min in a refrigerated centrifuge at 4°C. The absorbance of the supernatant was read at 532 and 600 nm [31]. Calculations were done using an extinction coefficient of 155 mM cm−1. 2.6. Estimation of Hydrogen Peroxide (H2O2) Content H2O2 content of leaf samples was calorimetrically measured as described by Mukherjee and Choudhuri [32]. Leaf samples were extracted with cold acetone. 200 μL aliquot was mixed with 0.04 mL of 0.1% TiO2 and 0.2 mL NH4OH (20%). The pellet was decollated with acetone and resuspended in 0.8 mL H2SO4. The mixture was then centrifuged at 6,000 ×g for 15 min and supernatant was read at 415 nm. 2.7. Estimation of Total Soluble Phenolics Total phenolics were extracted with 80% (v/v) acetone and estimated using Folin and Ciocalteu's phenol reagent following Slinkard and Singleton [33]. Optical density of the mixture was read at 750 nm. Computation was done from standard curve of pyrogallol. Total soluble phenolics were expressed as mg/g on fresh weight basis. 2.8. Glycine Betaine (GB) Content Dry powdered leaf material (0.5 g) was shaken in 10 mL toluene (0.5%) and was left overnight at 4°C. After centrifugation, one mL of the filtrate was added to 1 mL of sulfuric acid (2 N) and from this 0.5 mL was taken in a test tube and potassium tri-iodide (200 μL) solution was added. The contents were cooled in a chiller and 2.8 mL of ice cooled deionized H2O was added followed by addition of 5 mL of 1-2 dichloroethane. Thereafter the absorbance of the organic layer (lower layer) was recorded spectrophotometrically (T80 UV/VIS Spectrometer, PG Instruments Ltd., USA) at 365 nm [34]. Concentrations of GB were calculated from a standard curve of GB. 2.9. Estimation of Proline Free proline was estimated following the method of Bates et al. [35]. 0.5 gm leaf was extracted in sulfosalicylic acid (3%) followed by centrifugation at 3000 g for 30 minutes. 2.0 mL supernatant was mixed with equal volume of acid ninhydrin solution [1.25 g ninhydrin dissolved in 30 mL glacial acetic acid and 20 mL of 6 M phosphoric acid] and glacial acetic acid. The samples were then incubated at 100°C for 10 min and reaction was terminated by keeping the tubes in ice container. After cooling, proline was separated with 4 mL toluene and optical density was measured at 520 nm. 2.10. Extraction and Estimation of Enzymatic Antioxidants The method of Malik and Singh [36], described in detail by Alqarawi et al. [37], was used for extraction of antioxidant enzymes from fresh plant leaves. Protein in the enzyme extract was estimated according to Lowry et al. [38]. The method of Giannopolitis and Ries [39] was used to estimate superoxide dismutase (SOD, EC 1.15.1.1) activity measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium. The reaction mixture was incubated under light (20 W fluorescent lamps) for 20 min. The absorbance was measured spectrophotometrically at 540 nm. The results were expressed as EU mg−1 protein. The method of Chance and Maehly [40] was used for estimation of catalase (CAT, EC 1.11.1.6) activity following the decrease in optical density of the reaction mixture every 20 seconds for 2 min spectrophotometrically at 240 nm and the activity was expressed as EU mg−1 protein. Peroxidase (POD, EC 1.11.1.7) was estimated according to the method described by Kar and Mishra [41]. The assay mixture comprised phosphate buffer (125 μM, pH 6.8), pyrogallol (50 μM), H2SO4 (50 μM), and 50 μL of enzyme extract. The reaction mixture was incubated at 25°C for 5 min; thereafter 0.5 mL of 5% (v/v) H2SO4 was added to stop the enzyme reaction. The end product (purpurogallin) was determined spectrophotometrically at 420 nm and activity was expressed as EU mg−1 protein. The method of Nakano and Asada [42] was followed to estimate the activity of ascorbate peroxidase (APX, EC 1.11.1.11) by monitoring change in absorbance spectrophotometrically at 290 nm and activity was expressed as EU mg−1 protein. The activity of glutathione reductase (GR, EC 1.6.4.2) was assayed as described by Carlberg and Mannervik [43] spectrophotometrically at 340 nm. The activity of GR was calculated using the extinction coefficient of 0.12 mM NADPH of 6.2 mM−1 cm−1 and expressed as EU mg−1 protein as described by Ahmad et al. [44]. For extraction nitrosoglutathione reductase (GSNOR, EC 1.1.1.284) enzyme, fresh leaves were homogenized in an assay mixture containing Tris-HCl (20 mM, pH 8.0), NADH (0.2 mM), and EDTA (0.5 mM) at 4°C [45]. The homogenate was centrifuged for 10 min at 3000 g. The supernatant was used as enzyme source. The method of Bai et al. [46], described in detail in our previous work by Cheng et al. [45], was used to estimate the activity of GSNOR spectrophotometrically at 340 nm at 25°C by monitoring the oxidation of NADH nm. The reaction was initiated by adding S-nitrosoglutathione (Calbiochem, San Diego, CA, USA) to the supernatants at a final concentration of 400 mM. The enzyme activity was expressed as nanomoles NADH consumed per minute per milligram of protein as described by Cheng et al. [45]. The method of Nakano and Asada [42] was used for determination of dehydroascorbate reductase (DHAR, EC: 1.8.5.1) activity and the absorbance was read at 265 nm for 1 min spectrophotometrically using extinction coefficient of 14 mM−1 cm−1 as described by Ahmad et al. [44]. The method of Miyake and Asada [47] was employed for the estimation of monodehydroascorbate reductase (MDHAR, EC 1.6.5.4). MDAR activity was expressed as μmol NADPH oxidized/(EU mg−1 protein) as described by Cheng et al. [45]. 2.11. Statistical Analyses Duncan's multiple range test was performed using one-way analysis of variance (ANOVA) for a completely randomized design by SPSS-21 software and significant differences in means were determined by the least significant differences (LSD) (p = 0.05) test. p ≤ 0.05 was considered as significant [48]. Data presented are the means ± SE (n = 5). 3. Results 3.1. Host Plant-Soil Biota Interactions AMF colonized roots of A. gerrardii with good intensity revealed by the presence of mycelia, vesicles, and arbuscules (Figures 1(a)–1(f)). Generally, the incidence of root length colonization (mycelia and arbuscules) by AMF was found to be relatively poor (P) with percent colonization of 51.2 and 63.6, respectively. However, the incidence of dormant status (vesicles) was moderate (M) to the tune of 50.4%. The application of Bs stimulated the intensity AMF colonization in the order of 120.7%, 48.5%, and 383.7% for mycelia, vesicles, and arbuscules, respectively, as compared to control. The intensity of abundant and moderate colorization of AMF as mycelium, vesicles, and arbuscules was negatively affected by salinity but inoculation with Bs significantly reduced the adverse impacts of salinity on the AMF structures (mycelium and arbuscules) in root of A. gerrardii (Table 1). The application of Bs significantly stimulated the structural colorization of AMF under saline and nonsaline conditions in terms of visible increase in the incidence of mycelia, vesicles, and arbuscules. The inoculated roots, however, showed significant decrease in mycelium, vesicles, and arbuscules, respectively, both in saline (34.4%, 52.9%, and 13.6%) and in nonsaline conditions (81.6%, 42.8%, and 64.77%), compared to the respective control (Table 1). 3.2. Nutritive Value In absence of salt stress (control), inoculation of A. gerrardii with AMF and Bs in combination showed the highest impact on nutritive value compared to their individual treatments. Plants treated with salt suffered a loss in nutritive value as indicated by an increase in ash, tannin, and lignin content by 40.1%, 261.8%, and 72.6%, respectively, though total lipids were reduced by 65.4% compared to the control (Table 2). Also, neutral detergent fiber (NDF) decreased by 46.1% and acid detergent fiber (ADF) increased by 29.8% in salt treated plants (Table 2). The loss of nutritive value was lower in presence of AMF and Bs under salt stress compared to the corresponding controls. It was observed that the combination between AMF and Bs was more positively effective to improve the nutritive value in absence (control) of salt stress. However, the salinity caused slight inhibition in the positive impact of AMF and Bs. 3.3. Hydrogen Peroxide (H2O2) and Malonaldehyde (MDA) As shown in Figures 2(a) and 2(b), H2O2 Figure 2(a) and MDA Figure 2(b) were negatively affected by salinity and significant increase by 45.6% and 234.1% was, respectively, observed as compared to the nonsaline control. But inoculation with AMF and Bs significantly reduced the adverse effects of salinity on both H2O2 and MDA. It is evident that inoculation with AMF or Bs only (in absence of salt stress) caused slight increase in H2O2 and MDA, though inoculation with AMF and Bs together caused more increase compared to individual inoculation and nontreated control plants. 3.4. Glycine Betaine and Proline Effect of salinity, AMF, and Bs on glycine betaine and proline is depicted in Figures 3(a) and 3(b). Glycine, betaine, and proline increased by 100% and 125.1% in salinity stressed plants (Figures 3(a) and 3(b)). However, in salt stressed AMF inoculated (AMF + 250 mM NaCl) plants showed an increase of 54.07% and 82.05% while as in salt stressed Bs inoculated (250 mM NaCl + Bs) treatment showed 38.4% and 56.7% increase in glycine betaine and proline, respectively (Figures 2(a) and 2(b)). In salt stressed conditions inoculation with AMF and Bs (250 mM NaCl + AMF + Bs) showed only 31.1% and 54.5% increase in H2O2 and MDA production (Figures 2(a) and 2(b)). Individually inoculation of AMF induced 2.9% and 16.55% increase while Bs induced 9.6% and 33.1% increase in glycine betaine and proline, respectively (Figures 3(a) and 3(b)). 3.5. Total Phenol Salinity stress increased phenol content by 69.6% while AMF and Bs inoculated plants showed an increase of 8.3% and 13.8%, respectively (Figure 3(c)). AMF and Bs inoculated salt stressed (AMF + Bs + 250 mM NaCl) treatments showed an increase of the order of 27.7%. 3.6. Antioxidant Enzyme Activity Results depicting the effect of salinity, AMF, and Bs alone, as well as in combination, on the activities of antioxidant enzymes are shown in Figures 4(a)–4(g). Activities of the antioxidants studied (SOD, CAT, POD, GR, APX, DHAR, MDAHR, and GSNOR) increased with AMF, Bs, and salinity stress. Relative to control, percent increase in SOD, CAT, POD, GR, APX, DHAR, MDAHR, and GSNOR for AMF inoculated was 23.5%, 6.8%, 24.2%, 17.6%, 33.5%, 50.1%, 20.6%, and 6.8%, respectively. On the other hand, in case of AMF and Bs inoculated plants enhancement was much more pronounced with percent increase of 41.1%, 22.9%, 66.3%, 45.3%, 63.3%, 69.4%, 55.1%, and 16.2% with respect to SOD, CAT, POD, GR, APX, DHAR, MDAHR, and GSNOR, respectively. Salt stressed plants showed an enhancement of 152.9%, 98.2%, 166.6%, 169.5%, 598.3%, 169.1%, 350.5%, and 75.6% in SOD, CAT, POD, GR, APX, DHAR, MDAHR, and GSNOR activity, respectively (Figures 4(a)–4(g)). However, inoculation of AMF and Bs to salt stressed plants (250 mM NaCl + AMF + Bs) resulted in marked enhancement in activities of antioxidant enzymes studied and the percent increase being 374.7%, 210.3%, 263.6%, 251.8%, 656.4%, 178.1%, 401.1%, and 124.7% for SOD, CAT, POD, GR, APX, DHAR, and MDAHR, respectively, which is more as compared to the control as well as salt stressed plants and the AMF or Bs inoculated plants (Figures 4(a)–4(g)). 4. Discussion Inoculation by AMF and Bs increased the tissue water content and the nutrient uptake and caused hormonal balance in the treated plants. This resulted in optimal activity of metabolic processes to meet the growing needs of host plants for coping with the stress as reflected by other studies [4, 49]. Our results of reduction in percent AMF colonization and related attributes such as spore density due to salinity support the findings of Aroca et al. [2] and Alqarawi et al. [37]. The synergistic interactions of AMF and PGPR were evidenced by the fact that Bs supported significant increase in incidence of AMF colonization either in saline or in nonsaline conditions. The production of plant growth hormones by Bs plays direct and indirect role in promotion of mycorrhizal colonization. On the other hand, Bs may have more essential role in management of ROS [50] which in turn tends to decrease the affinity between AMF and host plant [14]. Salt stressed plants showed higher production of MDA, a product of lipid peroxidation, reflecting thereby the reduced membrane stability and impaired membrane functioning. By and large similar results have been obtained by Ahmad et al. [44] for Brassica juncea. In Phaseolus vulgaris [13] and Ephedra alata [51] it has been demonstrated that colonization by AMF reduces the peroxidation of membrane lipids, thereby providing stability to membranes against salt stress. Improved stability to lipids from the oxidative degradation of toxic reactive oxygen species due to AMF and PGPR in salt stressed plants is due to the enhancement in the free radical scavenging mechanisms [13, 51]. Tuna et al. [52] have confirmed that higher salinity enhances the membrane leakage through increased peroxidation of lipids. Harsh environmental conditions induce excessive production as well as accumulation of reactive oxygen species (ROS) and ultimately triggering the peroxidation of unsaturated membrane lipids thereby resulting in the loss of membrane integrity which ultimately leads to leakage and desiccation [44, 53]. Recently, Ahmad et al. [44] and Abd Allah et al. [13] observed increased production of free radicals like H2O2 in salt stressed plants and subsequent reversal of their damaging effects by AMF and PGPR to some extent. Improvement in membrane stability reduced MDA content in AMF inoculated plants may be due to the substantial increase in antioxidant activities and phosphate metabolism [13, 37]. In addition to reducing the cell membrane index and cell caspases, PGPR inoculation provides salt tolerance through downregulating the protease activity and programmed cell death resulting in enhanced cell viability [54]. Stressed environmental conditions trigger the accumulation of proline, glycine betaine, and other compatible osmolytes in plants. Accumulation of organic solutes including proline, sugars, and glycine betaine is one of the important tolerance strategies adapted by plants during stressful conditions [4]. In the present study, increase in content of proline and glycine betaine under salinity stress is in corroboration with Ahmad et al. [9, 44]. In Ephedra aphylla [37] and Sesbania sesban [13] accumulation of proline leads to salinity stress amelioration through better extraction of water from the soil solution by its active role in osmotic adjustment. They also reported that, under both normal and salt stressed conditions, inoculation of AMF induced enhanced synthesis of proline which helps in stress adaptation. Under salt stress, accumulation of proline results from the upregulation of the enzymes involved in proline synthesis which is concomitant with the downregulation of the catabolizing enzymes [55]. Proline accumulation can also result from the reduction in its incorporation within the proteins during protein synthesis or degradation of the existing proteins [56]. Among the important roles of proline and glycine betaine is the maintenance of tissue water balance of plants so that the stress induced ravage gets insulated, in addition to the antioxidant potential to mediate scavenging of the toxic radicals. Besides, optimal concentration of proline and glycine betaine positively influences the protein turnover besides being implicated in the regulation of several stress protective proteins [4, 55]. Under normal as well as salt stressed conditions AMF and PGPR inoculated plants showed enhanced proline accumulation as compared to control and similar results have been reported by Ghorbanpour et al. [57] in Hyoscyamus niger. In Triticum aestivum introgression of glycine betaine synthesizing gene increases tolerance to multiple stresses like drought, salinity, and cold through the maintained membrane integrity, activity of enzymes, photosynthetic efficiency, and scavenging of ROS [4, 45, 50, 58]. Glycine betaine also stabilizes the constituent proteins of PSII photosynthetic complex and also helps to maintain the order of membranes at extreme temperatures and salinity levels [4]. Accumulation of higher content of phenolic compounds such as lignins, tannins, and fibers is another important strategy for avoiding the stress induced changes. Tannins and phenols are the group of secondary metabolites implicated in plant protection and have been recognized for their antioxidant property. They are involved in eliciting the proper response in plants during biotic and abiotic factors [4, 11, 58]. Improved phenol and tannin content support better growth and also mediate the radical scavenging. In our target plant species inoculation with AMF and Bs enhanced accumulation of phenols and tannin as reflected in enhanced membrane stability in such plants. Among the antioxidants, SOD has the distinction of scavenging the superoxide radicals. In salt stressed Acacia gerrardii increase in activity of SOD was observed which supports the findings of Rasool et al. [59] for Cicer arietinum and Alqarawi et al. [37] for Ephedra aphylla. Enhanced activity of SOD causes quick conversion of superoxide into H2O2 through Haber-Weiss reaction and thereby reduces the chances of hydroxyl (OH−) radical formation. Efficient removal of free radicals facilitates normal membrane functioning. Higher POD activity in salinity stressed plants corroborates with the results of Abd Allah et al. [13]. Enhanced expression of the POD enzymes results in the increased production of lignins and the associated protective compounds like quinones which ultimately work for reducing the damage induced by oxidative stress. Efficient detoxifications of ROS help plants in maintenance of growth and the physiological activity of plant [4, 12, 13]. Increase in the activity of antioxidant enzymes (SOD and POD) as a result of AMF colonization coincides with the results of Abd Allah et al. [13] for Phaseolus vulgaris and Alqarawi et al. [37] for Ephedra aphylla. Catalase (CAT) is another important antioxidant enzyme having indispensable role in stress amelioration. In concurrence with our results, increased activity of CAT in salt stressed plants is the result of several workers [13, 51, 59]. Inoculation of AMF and PGPR further enhanced the activities of antioxidant enzymes thereby strengthening the antioxidant defense system. Greater activity of CAT allays the stress damage by mediating the quick removal of H2O2 [58]. Enhancement in CAT activity as a result of AMF has been reported in soybean [11, 60] and Ephedra aphylla [37]. Acacia gerrardii plants subjected to salt stress showed increased activities of the enzymes of ascorbate-glutathione cycle (APX, MDHAR, DHAR, and GR) resulting in quick generation of redox buffer components, glutathione, and ascorbate, for scavenging of H2O2. Our results of increased activities of APX, MDHAR, DHAR, and GR support the findings of Ahmad et al. [44]. Ascorbate-glutathione cycle is an important ROS scavenging pathway that involves sequential redox reactions in which both enzymatic and nonenzymatic components are actively engaged for removal of ROS. In this pathway, ascorbate, glutathione, and NADPH serve as electron donors for the efficient activity of enzymatic components, APX, MDHAR, DHAR, and GR [13]. In our study, activity of APX, MDHAR, DHAR, and GR was further increased by the inoculation of AMF and Bs resulting in amelioration of oxidative stress effects of salinity. In Ocimum basilicum, Heidari et al. [61] and Heidari and Golpayegani [62] have also demonstrated the upregulation of antioxidant enzyme activities due to inoculation of different PGPR species. Upregulation of antioxidant system and scavenging of ROS go hand in hand and are often correlated with stress tolerance [13, 44, 51]. AMF inoculation induced a significant increment in the antioxidant enzyme activities under normal as well as salt stressed condition. These results are in confirmation with the results of Alqarawi et al. [51] and Sirajuddin et al. [63]. Plants with increased glutathione reductase activity maintain optimal ratio of NADP+/NADPH thereby receiving electrons of photosynthetic electron transport chain by molecular oxygen and reducing the formation of superoxide radical [4, 11, 31, 53, 58]. 5. Conclusion In conclusion, our results indicate that endophytic bacteria and AMF that live entirely within plant tissues in A. gerrardii are jointly involved in ameliorating its tolerance to salt stress. Synergistic interaction of B. subtilis and AMF enhanced activities of antioxidant enzymes thereby preventing ROS induced oxidative damage. Increase in proline and glycine betaine in AMF and B. subtilis inoculated plants supports their role for growth improvement of Acacia gerrardii under salinity. Hence, a microbial consortium based on these species in combination with other beneficial soil microbes can potentially be used for large scale growth of A. gerrardii under salt stressed conditions. Acknowledgments The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding this research group no. RGP-271. Competing Interests The authors declare that they have no competing interests. Figure 1 Illustration of different arbuscular mycorrhizal fungi colonization in root of Acacia gerrardii. (a) Interadical spores (IS), coiled hyphae (CH), and vesicles (V) [20x]. (b) Intact interadical spores (IIS) and subtending hyphae (SH) [40x]. (c) Arbuscules (A) and hyphae (H) [10x]. (d) Arbuscules (A), hyphae (H), and coiled hyphae (CH) [10x]. (e) Arbuscules (A) and trunk (T) [40x]. (f) Vesicle (V), intact interadical spores (IIS), and subtending hyphae (SH) [40x]. Figure 2 Effect of salinity stress (250 mM NaCl) on (a) hydrogen peroxide [H2O2] (LSD at 0.05 : 0.025; coefficient of variance: 1.867) and (b) malonaldehyde [MDA] (LSD at 0.05 : 0.567; coefficient of variance: 3.518) in Acacia gerrardii with and without AMF and Bacillus subtilis (Bs). Figure 3 Effect of salinity stress (250 mM NaCl) on (a) glycine betaine (LSD at 0.05 : 0.162; coefficient of variance: 10.567), (b) proline (LSD at 0.05 : 0.026; coefficient of variance: 5.023), and (c) total phenol (LSD at 0.05 : 0.109; coefficient of variance: 2.705) in Acacia gerrardii with and without AMF and Bacillus subtilis (Bs). Figure 4 Effect of salinity stress (250 mM NaCl) on (a) superoxide dismutase [SOD, EC 1.15.1.1] (LSD at 0.05 : 0.069; coefficient of variance: 1.910); (b) catalase [CAT, EC 1.11.1.6] (LSD at 0.05 : 0.173; coefficient of variance: 1.830); (c) peroxidase [POD, EC 1.11.1.7] (LSD at 0.05 : 0.238; coefficient of variance: 3.597); (d) glutathione reductase [GR, EC 1.6.4.2 (nmol/min/g FW)] (LSD at 0.05 : 4.056; coefficient of variance: 1.903); (e) ascorbate peroxidase [APX, EC 1.11.1.11, nmol/min/g FW] (LSD at 0.05 : 6.907; coefficient of variance: 1.394); (f) dehydroascorbate reductase (DHAR, EC: 1.8.5.1, nmol/g FW) (LSD at 0.05 : 2.492; coefficient of variance: 1.751); (g) monodehydroascorbate reductase [MDHAR, EC 1.6.5.4, nmol/min/g FW] (LSD at 0.05 : 4.374; coefficient of variance: 1.805); and (h) nitrosoglutathione reductase [GSNOR, EC 1.1.1.284, nmol/g FW] (LSD at 0.05 : 4.335; coefficient of variance: 1.108) in Acacia gerrardii with and without AMF and Bacillus subtilis (Bs). Table 1 Effect of salinity stress (250 mM NaCl) on intensity of structural colonization (%) of arbuscular mycorrhizal fungi (AMF) as mycelia (M), vesicles (V), and arbuscules (A) in Acacia  gerrardii in response to endophytic B. subtilis (Bs). Data presented are the means ± SE (n = 5). Treatments Intensity of structural colonization (%) Mycelium Vesicles Arbuscules P M A P M A P M A Control AMF 51.2 27.6 21.2 22.4 50.4 27.2 63.6 29.0 7.4 AMF + Bs 9.4 43.8 46.8 12.8 34.4 52.8 22.4 41.8 35.8 250 mM NaCl AMF 77.2 16.8 6 71.4 19.4 9.2 80.8 14.4 4.8 AMF + Bs 50.4 34.4 15.2 33.6 41.2 25.2 69.8 19.4 10.8 LSD at 0.05% 14. 51 8.71 7.04 8.09 9.74 5.11 7.23 5.24 2.07 Coefficient of variance 4.229 3.127 2.531 2.065 4.235 3.011 4.512 3.234 2.107 P = poor; M = moderate; A = abundant. Table 2 Effect of salinity stress (250 mM NaCl) on ash content, total lipids, lignin, tannin, and fiber content (neutral detergent fiber (NDF) and acid detergent fiber (ADF)) in Acacia gerrardii with and without arbuscular mycorrhizal fungi (AMF) and Bacillus subtilis (Bs). Data presented are the means ± SE (n = 5). Treatments Nutritional value Ash content (%) Total lipid content (%) Lignin Tannin  Fiber content (%) ADF NDF Control Control 45.0 2.75 46.9 1.65 119.7 277.0 Bs 32.3 3.52 40.6 1.98 145.7 299.4 AMF 36.6 2.90 52.4 1.75 132.8 282.0 AMF + Bs 30.6 3.53 49.6 2.26 154.5 310.7 250 mM NaCl Control 63.1 0.95 81.0 5.97 148.9 149.1 Bs 50.1 1.93 61.7 3.14 212.9 213.6 AMF 57.2 1.37 77.5 4.01 192.4 194.2 AMF + Bs 48.6 2.23 71.6 2.79 221.1 219.4 LSD at 0.05% 1.62 0.08 1.56 0.16 2.38 4.11 Coefficient of variance 4.136 4.311 2.997 6.305 1.659 1.955 ==== Refs 1 Shekoofeh E. Sepideh H. Roya R. 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==== Front Biomed Res IntBiomed Res IntBMRIBioMed Research International2314-61332314-6141Hindawi Publishing Corporation 10.1155/2016/7247983Research ArticleAnalysis of Spatiotemporal Characteristics of Pandemic SARS Spread in Mainland China Cao Chunxiang 1 http://orcid.org/0000-0003-0303-3978Chen Wei 1 * Zheng Sheng 1 Zhao Jian 1 Wang Jinfeng 2 Cao Wuchun 3 1State Key Laboratory of Remote Sensing Science, Beijing 100101, China2Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences, Beijing 100101, China3Beijing Institute of Microbiology and Epidemiology, Beijing 100101, China*Wei Chen: chenwei@radi.ac.cnAcademic Editor: Francesco Dondero 2016 15 8 2016 2016 724798329 3 2016 23 6 2016 29 6 2016 Copyright © 2016 Chunxiang Cao et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Severe acute respiratory syndrome (SARS) is one of the most severe emerging infectious diseases of the 21st century so far. SARS caused a pandemic that spread throughout mainland China for 7 months, infecting 5318 persons in 194 administrative regions. Using detailed mainland China epidemiological data, we study spatiotemporal aspects of this person-to-person contagious disease and simulate its spatiotemporal transmission dynamics via the Bayesian Maximum Entropy (BME) method. The BME reveals that SARS outbreaks show autocorrelation within certain spatial and temporal distances. We use BME to fit a theoretical covariance model that has a sine hole spatial component and exponential temporal component and obtain the weights of geographical and temporal autocorrelation factors. Using the covariance model, SARS dynamics were estimated and simulated under the most probable conditions. Our study suggests that SARS transmission varies in its epidemiological characteristics and SARS outbreak distributions exhibit palpable clusters on both spatial and temporal scales. In addition, the BME modelling demonstrates that SARS transmission features are affected by spatial heterogeneity, so we analyze potential causes. This may benefit epidemiological control of pandemic infectious diseases. Special Fund for Forest Scientific Research in the Public Welfare201504323National High Technology Research and Development Program of China2013AA12A302Young Talent Project of the State Key Laboratory of Remote Sensing Science15RC-09Special Grant for Prevention and Treatment of Infectious Diseases2008ZX10004-012 ==== Body 1. Introduction In recent years, emerging infectious disease of severe acute respiratory syndrome (SARS) has become one of the most egregious public health problems in 21st-century China. SARS is caused by a new pathogen that was finally identified as a coronavirus (SARS-Cov) [1, 2]. SARS-Cov is regarded as a person-to-person infectious disease that infects suspected individuals through droplet transmission [3]. After infection, patients go through a 2-to-10-day incubation period before typical symptoms (fever, cough, and body aches) appear. During the incubation period, the patients are contagious to surrounding people [4]. To date, there is no vaccine for SARS, no reliable diagnostic test, and no specific treatment. This leads to the need for refinement of public health controls to be effective [5, 6]. Much loss of life, a high mortality rate, and substantial wealth damage were associated with SARS throughout a wide part of China in 2002 and 2003. The first case of SARS was identified and confirmed by the National Laboratory of China in Foshan, a city in Guangdong Province, South China, on November 16, 2002. With rapid development over 3 months, SARS became a disease that was difficult to control, and it spread to other cities through regular population movement [7]. According to the World Health Organization (WHO), after nearly 9 months of spread (November 16, 2002, through July 13, 2003), 29 countries had been affected by SARS. In this period, 8096 persons were infected and 774 died, which caused domestic panic in countries of East and Southeast Asia [8, 9]. The evidence shows patterns of vast geographic transmission that distinguish SARS from other epidemic infectious diseases. Traditionally, epidemiologists focus on the temporal epidemic evolution and describe it through various types of mechanical models [4, 10, 11]. Riley et al. admitted the spatial impact, but their model lacked detailed spatial analysis. However, because of the wide spread of SARS, typical temporal models have been unable to effectively explain its dynamics. As a result, the pandemic cannot be explained as a combination of endemics. Complex spatial transmission networks [12], compound period epidemics, and overwhelming government controls inspired epidemiologists to research this type of person-to-person infectious disease more profoundly with respect to its geographic features. Dye and Gay [13] suggested that doing so could lead to variations in average contact rate. Lloyd-Smith et al. simulated SARS dynamics in a complex network based on an epidemiological mechanism with full consideration of population, contact rate, control strategies, and spatial diffusion [14, 15]. However, their approach requires the assumption of numerous parameters, which in turn require the support of detailed data. The approach makes it difficult to explain overall spatial transmission without geographic epidemic analysis. Wang et al. [7] unveiled the broad existence of spatial clusters during the Beijing epidemic periods, influencing government intervention [16]. This work inspired us to investigate spatial principles in SARS transmission, which could include significant indications for pandemics within a more general perspective. A study that combines both a temporal epidemic study and spatial transmission is required to understand SARS dynamics. Recent advances in geostatistical analysis, which have been effectively used in infectious disease mapping, health risk assessment, and epidemiological studies, could be applied in SARS research for spatiotemporal analysis [17, 18]. The Bayesian Maximum Entropy (BME) approach of modern geostatistics incorporates higher-order statistical estimation for space-time epidemic phenomena and has shown more accurate mapping results than those derived from linear kriging geostatistics [19]. We benefited from such advantages in investigating statistical features of the SARS epidemic and in simulating geographic transmission aspects on a day-by-day basis. We focused on exploring and understanding spatial heterogeneity impacts of SARS transmission and simulating and mapping the disease temporally. Under the increasing threat of pandemics in 21st century, the present study illustrates actual epidemic spread and provides useful information toward establishing government intervention to preventing pandemics. 2. Materials The surveillance sanitary system of the Chinese Centre for Disease Control and Prevention (Chinese CDC) monitored all SARS infections in mainland China and confirmed them in the national microbe laboratory. The infections were recorded cautiously and totaled 5318 cases. The data were updated and include cases from November 2002 through June 2003. A confirmation system slightly different from that of WHO statistics raised the number of cases to 5327. The recorded information includes basic patient information (e.g., name, sex, age, and occupation; Figure 1), spatial information (including registered permanent residence location, company, first symptom onset location, current resident location, and hospital location), and temporal information (e.g., dates of first symptom onset, hospital treatment, recovery, or death). The daily number of SARS infections and their temporal characteristics were analyzed and are shown in Figure 2. In mapping all cases, the location of infection onset is an elaborate process. Even though some cases were recorded at locations using various scales, the records were eventually sorted at county level to facilitate processing. Considering that population movement is one of the effects on SARS diffusion, the following order was observed in the geocoding process: (1) registered permanent residence; (2) temporary residence (work address); (3) onset location; and (4) reporting unit location. To maintain rigorous containment, sanitary departments collected minute information for spatial indexing. Thus, the registered permanent residence was selected as the first index for the benefit of data integrity and for monitoring the impact of mass population movement. On February 1, 2003, during the period of SARS outbreaks, the Spring Festival took place. People commonly travel to join family for this traditional Chinese festival, returning to their permanent residence after 1–4 weeks. This population movement increased the probability of cross infection among family members and travellers. Consequently, with some exceptions, the temporary residence, onset, and reporting unit locations could be additional spatial information in geocoding the infections. Some cases lacked records of both permanent residence and workplace, and the only available addresses for these were syndrome onset locations. If all the above addresses were void, the hospital address was used as the only thread to locate the patient, although this was rare in our research. We focused on outbreak regions for convenience and clarity in the process. There are 194 counties in the focus area, which is described in greater detail in subsequent sections. Among them, Beijing, Guangzhou, and Taiyuan experienced the most severe SARS outbreaks. We used a 2002 county-level digital map to illustrate the epidemic (Figure 3). In temporal mapping, we also sorted the cases chronologically. All records were sorted at daily level. The onset date of patients was set as the optimal time index. Prior to this date, there was a 2–8-day incubation period [8, 20], which is generally intractable. The period from onset to hospitalization, including the incubation period, was considered the contagion period. Because it is impossible to know the infection date accurately for a patient, incubation period data are unavailable. Therefore, onset date was the first temporal index in our research. If this date was unavailable, then the hospitalization date was used as a secondary index. 3. Methods 3.1. BME Model Typical modes of investigation in health research focus on discovering spatial patterns or establishing a time series to study the principles that govern health phenomena. Separation of the space-time domain into spatial and temporal components permits analysis of those subdomains individually but ignores principles and correlations that may exist because of the composite spatiotemporal structure [21]. In infectious disease research, and especially the study of person-to-person contagious diseases, epidemic conditions are closely related to both spatial and temporal dimensions. Thus, proper relationships must be constructed to combine and account for the space-time continuum. BME provides an effective stochastic method, based on a cogent theoretical and technological strategy, to analyze relationships of SARS outbreaks in the composite space-time domain. It comprises not only epidemiological knowledge bases but also spatiotemporal statistics and dynamic modelling aspects for study. It also offers a software framework for modelling and prediction of epidemic conditions across space-time [22, 23]. Details include the following.BME integrates knowledge from epidemiology into Geographic Information System science by assimilating epidemic laws, empirical relations, and statistical calculations in SARS studies. BME considers spatial heterogeneity in SARS outbreaks, which broadens the traditional epidemic research field from the temporal to space-time domain. BME simulates SARS transmission and predicts diffusion tendency. The resultant study of these processes can reflect interesting underlying principles behind SARS spread. For stochastic representation of the outbreaks, we considered the daily number of outbreaks as a random variable within a three-dimensional space-time random field (S/TRF) [24]. Then each of the SARS records is a single realization out of all possible values that can be observed at a specific space-time location. Two of the S/TRF dimensions correspond to geographic coordinates of the records, and the third dimension axis represents the temporal dimension. In this sense, we studied the SARS outbreaks in a composite spatiotemporal domain, in which each observed record is uniquely represented by the space-time vector p = (s, t). BME takes these records as individual S/TRF points, to which each is assigned a spatial location and temporal instance. To illustrate the composite spatiotemporal approach, consider a spatial-only map at the time instance t 0, represented by (1) xmap0=x1,…,xm,xk1,…,xkn, where [x 1,…, x m] denotes observation points at t 0 and [x k1,…, x kn] represents the estimation points at the same t 0. We computed SARS distribution maps across a time interval of such n instances [t 1,…, t n], and we eventually obtained a joint set of maps p map = [x map(1),…, x map(n)]. The BME method operates in three successive stages to analyze the stochastic epidemic process. In the first stage, structural characteristics of a space-time random field are incorporated in the analysis by means of all available epistemic information about the S/TRF. This information comes from theoretical or empirical sources related to the procedure and is known as the general knowledge base or G-KB. At the end of the first stage, the input allows the computation of probability density functions (PDFs) that describe the S/TRF based on the G-KB. In the case of the SARS outbreaks, we used the observations to explore general structural characteristics of the SARS S/TRF, that is, the existence of mean (or surface) trends in the space-time domain, and to explore the underlying temporal and spatial structure of the S/TRF with suitable covariance functions. The second stage relates to the selection of case-specific information so that BME can assess characteristics and perform inference given the particular S/TRF realization facilitated by the recorded information. This information is known as the specific knowledge base or S-KB. The SARS sampling dataset that was described in the previous section consists of m sampling points (p 1, p 2,…, p m) and comprised the S-KB in this study. In the final stage, BME integrates the G-KB and S-KB to compute updated prediction PDFs at selected space-time locations. The prediction or posterior PDFs provide a complete statistical description of the health attribute distribution in space-time, and they enable selection of a predictor of choice for assessment of the SARS outbreak distribution. In traditional analysis, health attributes exhibit higher similarity for closer occurrences. Consequently, spatial distance is typically considered as the sole substantial factor that describes and demonstrates autocorrelation and the underlying disease field structure. However, the epidemic time is also important in the study of disease spread, which could extend further with rapid transportation and population movement. The previous discussion leads us to define a composite space-time distance, dp, as (2) dp=Gds,dt, which depends on both spatial and temporal distances (ds and dt, resp.), connected through an appropriate spatiotemporal metric G. Hence, the distance combined with temporal effects is more representative in epidemic analysis. In our research, ds = s 1 − s 2 and dt = t 1 − t 2 for two S/TRF points p 1(s 1, t 1) and p 2(s 2, t 2). Once distances are defined within the S/TRF, permissible functions can be used to describe correlations in space-time. The covariance function that yields spatial and temporal autocorrelation between two points, p and p′, is given by (3) cxp,p′=Xp−Xp¯Xp′−Xp′¯¯. In this function, X(p) and X(p′) represent a pair of realizations (possibilities) at p and p′ in the SARS outbreak S/TRF. It is expected that the observation data sample statistics should be the same as the S/TRF. For example, the data mean model should coincide with the average statistic, and the empirical covariance model should follow the covariance statistic of the field. Let c x be the arithmetic covariance. c x is presumed to be a theoretical function that is the same as the traditional covariance models in kriging analysis, such as the spherical, exponential, and nugget models. Following this initial setup, the classic Bayesian model is then incorporated, as shown by (4) fXk ∣ Xdata=A−1∫DdXfGXmap, where (5) A=∫DdXfGXdata is a normalization constant and D is the domain field of X k. In (4), note that the posterior PDF on the left side is conditioned upon the BME prior PDF, which is given in more detail by (6) fGXmap=eμ0+μTgXmap=eμ0+∑a=1NμagXa. With the support of BME, we can compute various statistical quantities, such as the mean, covariance, semivariogram, and higher-order statistics, because BME estimates the posterior PDF according to the constraints. For instance, we sought the most probable value for X k and so computed the differential coefficient for X k that gives the prediction PDF mode, that is, (7) ∂fXk ∣ Xdata∂Xk=A−1B∫DdX∂∑a=1nμagXa∂Xk, where B = e μ0 is a constant, g(X a), a = 0,…, n are functions representing the stochastic G-KB information, and μ a, a = 0,…, n are the so-called Lagrange coefficients that are in spatiotemporal coordinates. The latter serve as weights for the corresponding g(X a) functions in (7). BME theory develops a system of equations for each value of g(X a), from which the μ a coefficients are calculated. Subsequently, their values are inserted in (4) and (6) to evaluate the posterior PDF, whose maximum gives the prediction mode estimate. A cross-validation procedure is followed to assess BME mapping accuracy. Specifically, we use a set of n ref validation data that were randomly selected as references for accurate estimation. We take turns to exclude each validation datum and calculate its value at the corresponding spatiotemporal coordinates, and then we compute the mean square error (MSE), given by (8) MSE=1nref∑i=1nrefXref,i−Xi2. Thus, the MSE provides a measure of the uncertainty of BME estimation. In (8), X ref,i is the BME estimate and X i is the ith validation observation value. 3.2. Model Implementation BME computations were performed with the specialized MATLAB-based interactive software interface known as the Spatiotemporal Epistemic Knowledge Synthesis Graphical User Interface, or SEKS-GUI [21]. SEKS-GUI is a rich-featured intuitive visual interface that provides a variety of G-KB and S-KB information types, adjusts model parameters for BME mapping, performs the analysis, and visualizes the predictions [25]. All types of data were input to the SEKS-GUI software. The original observations were sorted in a file, in which each row corresponds to a certain spatial location, date, and outbreak condition. For computing, outbreak cases recorded at county level had their spatial index converted to longitude and latitude of the corresponding county centroid. This conversion typically introduces additional uncertainty into the result, because we effectively approximate the spatial reference for each observation. But it was used in our case because these uncertainties were considered acceptable, for the following two reasons. First, all counties with SARS outbreaks were in the south and east of mainland China. These locations have high population densities and relatively small county areas. Second, the SARS infections were overwhelmingly observed in towns near their county centroids. There were 950 outbreak cases observed in 194 locations. In addition to the previous activities, we had to define the study area. West China has only ~1% of the total national population but covers nearly half the country's area. For this reason, we refined the research area and focused on the regions 102.04°–126.56°E and 20.90°–44.12°N. The study period was November 16, 2002, to May 21, 2003—a total of 186 days. 4. Results For statistical homogeneity, the data of outbreak numbers should have a normal distribution. In our study, the observation cumulative density function (CDF) showed as much as 36.99% deviation from the normal CDF distribution (Figure 4(a)). Thus, the original data were transformed using the normal scores method, which forces the original data distribution into the shape of the normal distribution (Figure 4(b)). Following estimation, the data are back-transformed to the original value space. For structural correlation analysis of the spatiotemporal epidemic spread, we incorporated information from the empirical second statistical moment of the SARS S/TRF. Specifically, we computed the empirical spatiotemporal covariance and then fit a suitable permissible mathematical model to describe it for BME computation. Figure 5 depicts both the empirical and fitted theoretical spatiotemporal covariances characterizing the SARS S/TRF. Figure 5 shows normalized covariance values, in which darker colors correspond to higher correlation. Note that spatial correlation has some fluctuation close to zero distance. That is, covariance in space decreased rapidly and then briefly rebounded. This behavior indicates a potentially smaller-scale correlation that we could not detect with our method. Correlation in the temporal axis decreases more smoothly from its maximum value to a minimum point far from the temporal origin. Given this behavior of empirical covariance, we fit the following theoretical covariance model, which has a sine hole spatial component and exponential temporal component: (9) cxp,p=c0exp⁡−3ras−sin⁡rbt, where r is distance and sin⁡(r) indicates a sine hole spatial component; the sill coefficient c 0 is estimated at ~1.0068; the spatial lag coefficient a = 0.45 and the temporal lag coefficient b = 60. Using the aforementioned covariance model, we obtained BME estimates representative of the pandemic-spread behavior. The approach above allowed for a different possible explanation for the rapid decline and recovery in spatial correlation in Figure 5, as follows. A spatial lag coefficient a = 0.45 is in good agreement with the first geographical law; that is, the numbers of outbreak events that were <0.45 degrees in longitude (~50 km) apart had greater similarity to corresponding numbers of closer events. Inversely, there was little to no spread relationship between numbers of outbreak events >2 degrees (~220 km) apart. However, from a different perspective, two locations >220 km apart possibly showed increased correlation because of population movement between more remote locations. The accurate fit of the exponential model to empirical temporal covariance suggests that SARS outbreak events belong in the category of traditional person-to-person epidemics. Accordingly, the specified temporal coefficient b of 60 days is based primarily on the duration observed in the most severely affected cities, such as Beijing, Guangzhou, and Taiyuan. It is technically possible to limit the number of observations that are simultaneously used for BME estimation at each output grid. This is a necessary step to balance between adequate numbers of neighbors that should be used for estimation at a specific point. This is based on the assumed spatiotemporal correlation ranges on the one hand and the pragmatic need to maintain reasonable computation time for estimation, on the other. In our case study, we used up to 50 of the closest pieces of data for each estimation location. Based on the spatial lag and temporal coefficients in (9) and considerations in the previous subsection, we defined the maximum spatial and temporal ranges for neighbor search to be 15 degrees in longitude and 20 days, respectively. The parameters were set to refine calculations within reasonable estimation ranges, as follows. First, given the limited period of spread, the SARS infection presumably could not reach a location 15 degrees away within mainland China, even considering rapid population transportation by air. Then, the observed data suggest that taking the 50 pieces of data nearest an estimation point essentially considers average temporal ranges of 5 days and distances ~10 degrees. The MSE of 0.69 indicates that the BME estimation in this case was effective and the accuracy was acceptable. Finally, we estimated the outbreaks by choosing a spatiotemporal metric parameter value of 0.3. This parameter was used to convert the space-time coordinates of any location into a common spatiotemporal distance in the continuum of SARS outbreak S/TRFs. For instance, with the above choice, the spatiotemporal distance of two outbreaks occurring at the same location 10 days apart is roughly equal to that of two simultaneous outbreaks three degrees apart. The aforementioned parameter value was based on correlation considerations and the spatiotemporal covariance in (9). 5. Discussion According to our analysis, the SARS epidemic was divided into two distinct phases, namely, endemic spread and pandemic spread. The result, refined to 220 km and 60 days, shows that the SARS outbreaks were effectively captured by spatiotemporal autocorrelation. SARS is a typical person-to-person, rapidly spreading infectious disease, and it takes about 60 days to spread from an affected area to adjacent ones. The process of outbreak spread to the adjacent areas is a stochastic one, owing to the synergistic effect of three factors. First, the SARS outbreaks were related to population density. Urban areas have higher population density than rural and residential areas, so for an outbreak in a city, it is more likely that it will spread within the urban limits than outside those limits. For example, among the total 5318 cases, 1934 were recorded in Beijing. This number is much higher than the number of outbreak cases observed in nearby cities and entire counties. Second, cities typically have convenient transportation to nearby cities and counties. Thus, commuting and population exchange between cities and nearby locations is likely to be much greater than corresponding activity between those cities and more distant areas. Consequently, suspected individuals from the nearby countryside are more likely to have travelled to a city and contacted infected people, and vice versa. This would increase the probability that the disease spreads in urban areas and their environs. The third and final factor is that control measures limit population transport, so the disease is likelier to have greater spread over nearby areas. Following the SARS outbreaks, the Chinese government established strict control measures for quarantine of the suspect population. People travelling from affected areas were mandated to undergo several diagnostic tests during their trip. As a result, the entire transportation system worked as a rigorous diagnostic tool for potential SARS cases, and all train, airplane, and long-distance bus passengers were surveilled for the epidemic. Among the measures, individual travellers had their temperature taken. If this was above 37.5°C, the individual would be forced into a state of quarantine in isolation wards until the temperature recovered to normal levels. Such strict measures were effective in restricting outbreak spread to limited areas for each city [26, 27]. BME estimation led to informative results, shown in the plot of Figure 6 and the maps of Figure 7. This output reveals higher temporal correlation and relatively low spatial correlation for the SARS outbreaks and could be summarized in the following three priority rules of spreading: first, spreading occurs within the city; second, stochastic spreading occurs between the city and the towns closest to it; finally, stochastic spreading takes place across different cities through quick transportation. The population density is an explanatory factor for the fact that SARS spread is mostly in urban areas. Approximately 19% of people working in health services were infected by their patients. These professionals were able to obtain rapid treatment, which means that it was unlikely that they would contribute to SARS spread outside their areas of residence. Housewives and retired people are groups that were extremely susceptible to infection by their families. However, these population groups typically have no desire to move to distant places. The most probable suspects for disease spread were labourers and businessmen, but these groups constituted only ~7% of total infection cases. The randomness of SARS spread among various cities is a characteristic that has been explained via the theory of super spreaders. The starting point of this theory is that the SARS virus always evolves and mutates. Most virus strings evolve slowly with similar infectivity, but a small fraction evolve into extremely infective virus types. People affected by such virus strings are so-called super spreaders. These individuals were comparatively very contagious, and they transmitted the virus to dozens of other people. For instance, five super spreaders in Singapore were found to have infected 103 people [28, 29]. The randomness of virus mutation to high infectivity indicates considerable randomness in the way people became infected. This randomness also extended to the relatively random directions in which the epidemic spread among cities. 6. Conclusions SARS generated one of the most egregious public health events in China of the beginning of 21st century, causing great loss of life and a grave threat to human survival and development. In the present study, spatial and temporal aspects of this person-to-person contagious disease were explored, and its spatial and temporal transmission dynamics were simulated through the BME method. Based on these analyses, it is concluded that SARS transmission varies in its epidemiological characteristics. Moreover, there was a high temporal correlation and relatively low spatial correlation of SARS outbreaks. In addition, the BME modelling demonstrated that SARS transmission features are affected by spatial heterogeneity. Therefore, we analyzed potential causes, including infected populations and transportation modes. Our findings can benefit epidemiological control of pandemic infectious diseases and public health protection in the future. Acknowledgments This paper has been supported by the Special Fund for Forest Scientific Research in the Public Welfare (no. 201504323), the National High Technology Research and Development Program of China (2013AA12A302), the Young Talent Project of the State Key Laboratory of Remote Sensing Science (no. 15RC-09), and Special Grant for Prevention and Treatment of Infectious Diseases (2008ZX10004-012). The authors thank all the people who have given help on this paper. Disclosure State Key Laboratory of Remote Sensing Science is jointly sponsored by the Institute of Remote Sensing and Digital Earth of Chinese Academy of Sciences and Beijing Normal University. Competing Interests The authors declare that they have no competing interests. Authors' Contributions Chunxiang Cao and Wei Chen were the main researchers and responsible for the data analysis and paper writing. Wei Chen and Sheng Zheng performed data processing and model implementation. Jinfeng Wang commented on idea selection. Wuchun Cao provided SARS data and supervised the modelling details. Jian Zhao and Chaoyi Chang greatly helped in result analysis and gave excellent advice on paper preparation. Figure 1 Basic patient information. (a) The occupation distribution as a percentage of all SARS infections. (b) The age percentage of SARS infections. Figure 2 Daily number of SARS infections and temporal characteristics. (a) Number of SARS infections each day. The two outbreak peaks caused by disease spreading in Guangzhou and Beijing are clearly distinguishable in the plot. (b) Histogram of period from SARS onset to treatment (unit: day). The average period is 3.96 days, which indicates fast and rigorous health service for SARS treatment. (c) Histogram of period from treatment to recovery (unit: day). The average treatment takes 25.19 days. This period corresponds exactly to half the size of an outbreak circle. Figure 3 Spatial pattern of the SARS outbreaks. Figure 4 SARS data transformation. (a) Comparison of the SARS raw dataset cumulative density function (CDF) and the corresponding one of the normal distribution. The maximum observed deviation between those two CDF is about 28.88% at data value 5. (b) N-score transformed CDF of the SARS study data and the normal distribution. The maximum observed deviation between those two CDF has dropped down, compared to panel (a), to about 2.26% at data value −0.63853. Figure 5 Spatiotemporal covariance used in the BME modelling. (a) Plot of the empirical spatiotemporal covariance; the covariance was estimated at the nodes shown with small red circles connected with coarse surface plates (the S-lag is in degrees and the T-lag is in days). (b) Spatial cross-section at t = 0 of the estimated covariance surface and the empirical covariance. (c) Temporal cross-section at s = 0 of the estimated covariance surface and the empirical covariance. (d) Fitted theoretical covariance plot (densely gridded surface) superimposed on the empirical covariance. Figure 6 Contrast between observed SARS outbreak number and BME estimation number. The green line represents actual SARS infections on a daily basis from November 16, 2002, to May 20, 2003. The blue line represents the corresponding daily number of cases according to the BME estimates. BME reflects relatively accurately the temporal pattern in the daily number of infections, although it overestimates systematically the number values. Figure 7 BME estimation results at selected instances. 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==== Front Front PsycholFront PsycholFront. Psychol.Frontiers in Psychology1664-1078Frontiers Media S.A. 10.3389/fpsyg.2016.01320PsychologyGeneral CommentaryCommentary: Effects of Age and Initial Risk Perception on Balloon Analog Risk Task: The Mediating Role of Processing Speed and Need for Cognitive Closure Walasek Lukasz *Psychology, University of WarwickCoventry, UKEdited by: Emma V. Ward, Middlesex University, UK Reviewed by: Johannes Schiebener, University of Duisburg-Essen, Germany *Correspondence: Lukasz Walasek l.walasek@warwick.ac.ukThis article was submitted to Cognition, a section of the journal Frontiers in Psychology 29 8 2016 2016 7 132011 7 2016 18 8 2016 Copyright © 2016 Walasek.2016WalasekThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.A commentary on Effects of Age and Initial Risk Perception on Balloon Analog Risk Task: The Mediating Role of Processing Speed and Need for Cognitive Closure by Koscielniak, M., Rydzewska, K., and Sedek, G. (2016). Front. Psychol. 7:659. doi: 10.3389/fpsyg.2016.00659agingrisk-takingballoon analog risk task (BART)cognitive modelingdual-system theoriesLeverhulme Trust10.13039/501100000275RP2012-V-022 ==== Body Existing research strongly suggests that age-related changes in the cognitive system influence preferential choice. While the reduction of fluid cognitive ability can lead to sub-optimal decision outcomes (Finucane et al., 2000), experience garnered during one's lifespan can also improve one's decision making (Mata et al., 2007; Bruine de Bruin et al., 2014). How can research on aging and decision making explain such mixed results? A reasonable approach is to adhere to a clear definition of optimality in choice behavior, which must be grounded in principles of cognitive psychology. Indeed, this approach has led many researchers to identify distinct cognitive processes that may be responsible for suboptimal decisions among older adults. Among many, these include memory (Buckner, 2004), perception (Schneider and Pichora-Fuller, 2000), and executive functions (Schiebener and Brand, 2015). In this special issue, Koscielniak et al. (2016) focus on the effect of aging on risky choice. Comparing the performance of two age groups, they found that older female adults exhibit a lower propensity to take risks on the Balloon Analog Risk Task (BART). At the same time, their results showed that both younger and older females adapt to initial failures and successes and are capable of adjusting their risk taking behavior over the course of repeated trials. Koscielniak et al. (2016) positioned their findings within the broader framework of the dual-system reasoning, attributing the overall poor performance of older adults to a “decline in deliberative processes” (p. 6). In this commentary, I offer a cautionary note about the reliance on such theoretical frameworks to make predictions about the cognitive underpinnings of preferential choice. While the conclusions drawn by the authors are supported by their data, the use of verbal (i.e., not quantitative, Lewandowsky and Farrell, 2011) theories can lead to erroneous inferences. Instead, future advances should rely on cognitive modeling to decouple competing mechanisms that can give rise to differences in choices between younger and older adults. In challenging verbal and descriptive (i.e., statistical) models in study of risky choice and aging, many now argue that cognitive modeling removes ambiguity associated with the interpretation of cognitive processes (Lewandowsky and Farrell, 2011). In the case of the BART itself, several models have been proposed, each specifying specific cognitive mechanisms behind decisions to either pump or to secure one's earnings (Wallsten et al., 2005; Schmitz et al., 2016). In a 4-parameter version of the model by Wallsten et al. (2005), for example, α and μ parameters control the learning rate at which one's belief that a balloon will burst on a given trial is updated, γ+ represents the general propensity to take risks, and β captures the behavioral consistency of the agents. Crucially, Wallsten and colleagues showed that parameters recovered in their study correlated with self-reported indices of risky behaviors, supporting the view that their specification of the model captures the cognitive components of risk taking in BART (but see a discussion of alternative models by van Ravenzwaaij et al., 2011). In fact, research has found that the differences between young and old on BART performance can be attributed to heightened reward-sensitivity and the initial perception of risk (Cavanagh et al., 2012), as opposed to differences in the ability to update beliefs based on observed outcomes (Rolison et al., 2012). These results are consistent with the efforts of Koscielniak et al. (2016), but the results of model fitting present a clear advantage over verbal theories for a number of reasons. First, interpretable parameters of the model applied to BART can be used to draw parallels with other areas in which the effect of aging has been studied. Comparisons with performance on other risk tasks (and of other populations) can produce converging evidence about the cognitive processes involved. This is particularly important as it reduces the chance that a particular paradigm (e.g., BART) becomes the subject of empirical research in itself. Second, cognitive modeling can lead to alternative interpretations concerning risky choice. To illustrate this point, consider a widely held belief about the negative correlation between cognitive ability and risk aversion (e.g., Dohmen et al., 2010). Recent findings by Andersson et al. (2013) showed that this association is in fact spurious and simply reflects an increased rate of random choice among those with lower cognitive ability. Although the authors did not use cognitive modeling to illustrate their finding, it is easy to see that in many cases such mistakes could be avoided if a correctly specified model with a noise parameter was fit to the data. In the context of BART and aging, existing models can discern between noisiness of responding and risk preferences, and they are therefore well equipped to recover such patterns. Indeed, previous studies have found that older adults are less consistent in their decisions (Finucane et al., 2005). Finally, cognitive models can be extended to study dynamic aspects of cognition. Much work has applied drift diffusion models to understand cognitive performance of younger and older adults on a range of tasks (Ratcliff et al., 2006; McKoon and Ratcliff, 2013). Such models take into account response times and can therefore tell us more about the time-accuracy tradeoffs involved in a decision process. This modeling approach is particularly suitable for studying the effects of aging, as older participants often adapt their strategies to account for the decline in their fluid cognitive ability (Smith and Brewer, 1995). In sum, Koscielniak et al. (2016) contribute to the understanding of how cognitive and motivational factors influence preferential choice at different ages. This commentary highlights the fact that future research can build on these findings using cognitive modeling techniques to identify specific aspects of the cognitive process that impact risk preferences among young and old adults. Such efforts correspond to a shift from verbal and descriptive models toward quantitative models of cognition. Author contributions The author confirms being the sole contributor of this work and approved it for publication. Funding This work was supported by Leverhulme Trust Grant RP2012-V-022. Conflict of interest statement The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. ==== Refs References Andersson O. Tyran J.-R. Wengström E. Holm H. J. (2013 ). 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==== Front Comput Math Methods MedComput Math Methods MedCMMMComputational and Mathematical Methods in Medicine1748-670X1748-6718Hindawi Publishing Corporation 10.1155/2016/6472397Research ArticleAn Active Contour Model Based on Adaptive Threshold for Extraction of Cerebral Vascular Structures Wang Jiaxin 1 2 Zhao Shifeng 1 2 Liu Zifeng 1 2 http://orcid.org/0000-0001-5574-2325Tian Yun 1 2 * Duan Fuqing 1 2 Pan Yutong 1 2 1College of Information Science and Technology, Beijing Normal University, Beijing 100875, China2Beijing Key Laboratory of Digital Preservation and Virtual Reality for Cultural Heritage, Beijing 100875, China*Yun Tian: tianyun@bnu.edu.cnAcademic Editor: Giancarlo Ferrigno 2016 15 8 2016 2016 647239726 4 2016 8 7 2016 17 7 2016 Copyright © 2016 Jiaxin Wang et al.2016This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Cerebral vessel segmentation is essential and helpful for the clinical diagnosis and the related research. However, automatic segmentation of brain vessels remains challenging because of the variable vessel shape and high complex of vessel geometry. This study proposes a new active contour model (ACM) implemented by the level-set method for segmenting vessels from TOF-MRA data. The energy function of the new model, combining both region intensity and boundary information, is composed of two region terms, one boundary term and one penalty term. The global threshold representing the lower gray boundary of the target object by maximum intensity projection (MIP) is defined in the first-region term, and it is used to guide the segmentation of the thick vessels. In the second term, a dynamic intensity threshold is employed to extract the tiny vessels. The boundary term is used to drive the contours to evolve towards the boundaries with high gradients. The penalty term is used to avoid reinitialization of the level-set function. Experimental results on 10 clinical brain data sets demonstrate that our method is not only able to achieve better Dice Similarity Coefficient than the global threshold based method and localized hybrid level-set method but also able to extract whole cerebral vessel trees, including the thin vessels. National Science Foundation of China61472042Beijing Natural Science Foundation41520274152028Fundamental Research Funds for the Central Universities2015KJJCB25Program for New Century Excellent Talents in UniversityNCET-13-0051 ==== Body 1. Introduction Cerebral vascular diseases have become the main incentives to dizziness, disability, and even death in many countries around the world, and the research for vessels arouses concern. The segmentation of cerebral vascular structures is important for the clinical diagnosis and analysis. In medical image processing field, segmentation means the extraction of anatomical structures of interest from original data [1, 2]. Because of low contrast of images, edge blur, and structure complexity of cerebral vessels, the accurate segmentation is still a challenging task and deserves to be researched [3, 4]. Over the past few decades, a large number of methods for vessel segmentation have been proposed, including atlas-based techniques [5–10], machine learning techniques [11–14], and active contour model (ACM) [15, 16]. A comprehensive review can be referred to in Lesage et al. work [17]. Among these techniques, the ACM has been widely applied in medical image segmentation because of its easy extensibility. The ACM is based on geometric curve evolution theory and the essential idea of that technique is to evolve the initial curve or surface to the boundaries of target objects driven by internal forces and external forces [18]. Active contours can be implicitly presented by the level-set methods, which put original curves into higher dimensional spaces to research and are achieved in numerical computations by the Eulerian approach [19]. ACMs using level-set formulation have various forms of expression, and they are divided into three major categories: edge-based, region-based, and hybrid level-set models. In edge-based models, edges are usually generated first by an edge-detection algorithm and then using postprocessing to adjust to the final boundaries [20]. The typical edge-based model is the geodesic active contour model [21]. The model combines active contours with the computation of geodesic distance curve, and it allows to associate classical snakes based on energy minimization with geometric active contours based on the theory of curve evolution. A method using a new indicator (i.e., salient edge energy) to guide a given contour robustly and accurately towards the target object boundary was proposed by W. Kim and C. Kim [22]. They defined the salient edge energy by exploiting the higher order statistics on the diffusion space and embedded it into a variational level-set function. But the edge-based models are sensitive to noise and seek to oversegment an image. Region-based models are built on using the similarity among pixels to form homogeneous regions in an image [20]. The Mumford-Shah (M-S) model is the typical technique of that, and it depends on the defined edge function based on image gradient to stop the evolution process of active contour curves. When the contour curve is closer to the boundary of a target object, the value of gradient is higher, which causes the edge function to be closer to zero, and the evolutionary curve stops at the location of boundaries [23]. Based on the M-S model, Chan and Vese proposed the famous C-V model [24]. The C-V model can detect objects whose boundaries are not necessarily defined by gradient, because the stopping term defined in the energy function depends on the gradient of an image and is instead associated with particular segmentation of the image. In addition, the authors give a numerical algorithm using finite differences. Based on the C-V model, Tian et al. [25] proposed embedding local intensity weighting and a vessel vector field into the vessel active contour model. However, the model needed to be improved to better match segment 3D vessels. In these methods, it is essential to reinitialize the level-set function to make it close to the signed distance function [26]. However, the periodic reinitialization is time-consuming, and it is difficult to prevent the level-set function from being too steep or flat during the evolution [27]. To solve the problem, Li et al. [28] proposed a method through embedding the penalty term for penalizing the deviation of the level-set function from a signed distance function into the energy function. Combining region-based methods with other information, Said [29] proposed a robust level-set-based multiregion and texture image segmentation approach. Zhang et al. [30] proposed a method to associate interactively specified regions of interest with the active contour model while keeping the user interaction to the minimum. Sciolla et al. [31] proposed a multigrid level-set segmentation method based on a region-based function, the Hellinger distance. Jiang et al. [32] used the hybrid level-set method with a nonlinear speed function to extract brain from cerebral MRI volume. Zhao et al. [33] developed a MIP-guided approach for brain vessel segmentation. They first projected the volume onto the 2D plane, applied an integrated active contour model to extract blood vessels from MIP images, and then projected back to the 3D volume. The proposed method showed satisfying segmenting results. However, their method is a little complicated with several projection and back projection operations. In this study, we propose an ACM implemented by the level-set method in order to segment cerebral vascular structures from TOF-MRA data. We consider both the region information and edge information and combine them to characterize the energy function. A fixed gray threshold is used into the region term to represent the global information. In addition, we embed the adaptively dynamic threshold into our model to depict local region information, which is helpful for segment more integrated vessels. To avoid reinitializing level-set function in every evolution, the penalty term proposed by Li et al. is extended to 3D and applied into our model. The organization of this study is as follows. In Section 2, we will introduce the related works. In Section 3, the proposed segmentation methodology is depicted. Experimental validations and discussion are given in Section 4. Section 5 concludes the paper. 2. Related Works Considering both region information and boundary information and combining them to characterize the energy function is a good idea for segmenting the complex objects. Zhang et al. [34] proposed a hybrid level-set (i.e., HLS) method for segmentation of medical images. They use preset μ value which represents the lower gray-level of target object to replace μ in and μ out in the region term of traditional C-V model, and the geodesic active contour model is applied to represent the edge term. The definition of energy function to be minimized is defined as (1) εϕ=−α∫ΩI−μHϕdΩ+β∫Ωg∇HϕdΩ, where I is the image to be segmented, Ω is the image domain, α and β are weighting factors to balance the first-region term and the second-boundary term, and the zero level set of level-set function ϕ represents the active contour. H(ϕ) is the Heaviside function defined as (2) Hϕ=1,if  ϕ≥00,if  ϕ<0. Parameter μ is preseted representing the lower gray boundary of the object to be segmented, which means it will extract the object with gray higher than μ. However, since the value of μ is fixed, it cannot fit the wide intensity distribution of vessels well, especially for those small thin ones. To solve the problem, Hong et al. [35] proposed a localized hybrid level-set (i.e., LLS) method for the segmentation of 3D vessel images, and they calculated locally specified dynamic threshold μ(u) to indicate the lower bound of target object and embedded the local gray information into the region term. Defined function μ(u) is (3) μu=k∗Kσu∗HϕuIuKσu∗Hϕu, where u ∈ Ω, k ∈ [0.5,1] is an adjusting coefficient for preventing the active contours stopping evolution inside the target areas before reaching the boundary, and K σ is the Gaussian kernel function characterizing the intensity profile of a blood vessel cross section, such as (4) Kσu=12πσ2e−u2/2σ2 which is used to convolve with the image in order to detect the main vessels. By the use of dynamic threshold μ(u) defined in (3), the method can segment the tiny vessels better, but it may lose some intensity information of thick parts due to dynamic μ(u) formulation limitations. In any way, the above two methods both have respective pros and cons. Former preseted μ value drives the contours to enclose thick vessel boundaries with gray-levels greater than μ, but it can not perform well on tiny vessels. The latter with dynamic μ(u) value can deal with the tiny vessels better but does not extract the thick vessels completely. Thus, in our study, we take full advantage of these two methods. Meanwhile, we also extend the penalty term proposed by Li et al. [28] from 2D segmentation to 3D application and embed it into the energy function to keep the characteristic of a signed distance function. 3. Proposed Methodology 3.1. Definition of the Energy Function To tackle 3D cerebral vessel segmentation, we propose a new hybrid level-set model (i.e., NHLS model) inspired by models in [34, 35]. To segment more integral vessels, we incorporate dynamic μ(u) value to the original hybrid model, and the proposed energy function is defined as follows: (5) εϕ=−α1∫ΩI−μ0HϕdΩ−α2∫ΩI−μuHϕdΩ+β∫Ωg∇HϕdΩ+γPϕ, where I is the 3D volume data to be segmented, Ω is data domain, u ∈ Ω, μ 0 is set based on the lower gray-level boundary of the target object, and μ(u) is calculated according to (3) representing the local threshold. In (5), the four terms play different roles. The first term represents the global region information which drives the active contour curve to get close to the regions with bigger intensity value than μ 0. The second term is used to represent local region information which adaptively adjust to the threshold to segment the local tiny parts. The role of the third-boundary term is equivalent to the geodesic active contour model, and it encourages the contour curve to enclose the regions with high image gradient. Parameters α 1, α 2 and β are used to balance the two region terms and one boundary term. And the fourth term is the penalty term, in which γ is a preseted parameter controlling the effect of penalizing the deviation of ϕ from a signed distance function, and P(ϕ) is the penalty term to avoid reinitializing ϕ in evolution, which is defined as (6) Pϕ=∫Ω12∇ϕ−12dΩ. The related PDE can be derived from the gradient decent flow applied to functional (5): (7) ∂ϕ∂t=δϕ·α1I−μ0+α2I−μu+β div⁡g∇ϕ∇ϕ+γΔϕ−div⁡∇ϕ∇ϕ. 3.2. Implementation Edge function g(·) represents the regularized gradient map used for geodesic active contour and nonlinear diffusion related to boundary feature of the image. In this study, g(·) is defined as (8) gx=11+x2. Function H(·) is the Heaviside function and the original function is not continuous; therefore, it cannot fit the smooth boundary curve of the practical object. To solve this problem, it is usual to use a kind of smooth function to replace the original one. There are various proposed smooth types of the Heaviside function. We adopt the smooth Heaviside function H(ϕ) as follow: (9) Hϕ=121+2πarctan⁡ϕε. And the definition of corresponding Dirac function δ(ϕ) is (10) δϕ=1πεε2+ϕ2. We use the above computations H(ϕ) and δ(ϕ) to replace original H(·) and δ(·) in (2) and (7), respectively. Considering the penalty term in (7), Δ is the Laplacian operator, and (11) Δϕ−div⁡∇ϕ∇ϕ=div⁡1−1∇ϕ∇ϕ has factor 1 − 1/|∇ϕ| as diffusion rate. If |∇ϕ | > 1, the diffusion rate is positive. If |∇ϕ | < 1, the diffusion rate is negative. Equation (7) can be simply written as (12) ϕk+1−ϕkΔt=α1M1+α2M2+βN+γP, where ϕ k+1 and ϕ k denote the level-set function ϕ in (k + 1)th and kth iterations, respectively, Δt is the preset time step, M 1 is the global region term, M 2 is the local term, N is the edge term, and P is the penalty term. It is required to illustrate that M 1 is a fixed value decided by I and μ 0 is not related to ϕ. M 2, N, and P can be also expressed as M 2(ϕ k), N(ϕ k), and P(ϕ k), and they are affected by ϕ k. Difference equation (12) can be represented as follows: (13) ϕk+1=ϕk+Δtα1M1+α2M2+βN+γP. Iteration from ϕ k to ϕ k+1 includes five steps:(i) Compute dynamic localized threshold μ(u) according to (3). (ii) Compute penalty term P in terms of (6). (iii) Calculate α 1 M 1 + α 2 M 2 + γP. (iv) Update ϕ k to ϕ k′ using ϕ k′ = ϕ k + Δt(α 1 M 1 + α 2 M 2 + γP). (v) Update ϕ k′ to ϕ k+1 using ϕ k+1 = ϕ k′ + ΔtβN, which is achieved by the semiexplicit method. In fact, we can also use the explicit method to get ϕ k+1 directly, which plays the same role with the explicit method. However, explicit methods have limitations in time steps, and they need to set time steps small to keep methods stable. If we use explicit methods, the time steps maybe set small to make sure that the process of evolution maintains stability, which leads to time-consuming process [36]. Thus, we choose the semiexplicit method. There is an additional problem to address that is to set the initial curve of level set. In this study, we apply Frangi's vessel enhancement algorithm into the original data and then implement the canny detection to get the fuzzy boundary of vessel. That boundary curve is used as the initial curve. By this method, it can make sure that every evolution is around the vessel region, which improves efficiency. 3.3. Outlier Removement Since the intensity value of some nonvessel points are very close to those of vessel points, some nonvessel points (i.e., outlier) exist in the segmentation results. In order to remove the outlier points around vessels as much as possible, we need to consider the shape feature of vessels. Eigenvalues of the Hessian matrix have been successfully used in blood vessel enhancement [37]. For a 3D volume, we assume that the eigenvalues of the Hessian matrix are sorted as |λ 1| ≥ |λ 2| ≥ |λ 3|. The ideal tubular structure in a 3D volume would have (14) λ3≈0,λ3≪λ2,λ1≈λ2. Furthermore, in MRA images, the fact is that vessel structures are brighter than the background and the Frangi's vessel enhancement algorithm makes use of all the eigenvalues of Hessian matrix, and it can consider fully the geometric feature that the eigenvalues represent and suppress the impact of irrelevant points on vessels. They define two geometric ratios R A,  R B, and S, respectively, as (15) RB=λ1λ2λ3,RA=λ2λ3,S=λ12+λ22+λ32, where R B gets maximum for a blob structure, R A differentiates plane structures from line structures because in the latter situation it will be zero, and S is the measure to distinguish background which will be slow because the eigenvalues are small in the background. On the basis of the three parameters, Frangi et al. define a vesselness function combining those components as follows: (16) where α, β, and c are thresholds of 3D vesselness function which is used to control the sensitivity of vessel enhancement filter to parameters R A,  R B, and S. As multiscale eigenanalysis of local Hessian operators can enhance local rod-like shapes of varying radii. The value of vesselness function is between 0 and 1. If objects are tubular structures, vesselness function V σ(S) is close to 1. For an ideal tubular structure, R A ≈ 1, R B ≈ 0. It is noticed that when R B ≈ 0, the second term in (16) is approximately equal to 1. However, when R A ≈ 1, the value of the first term in (16) has slight gap with 1. In order to make V σ(S) approximately equal 1, we take advantage of function tan⁡(π/2)x. When x ≈ 1, tan⁡(π/2)x approaches positive infinity, and exp⁡(−tan⁡(π/2)(x)) is approximately equal to 0, so (1 − exp⁡(−tan⁡(π/2)(x))) is closer to 1 than before. Thus, in order to enhance the tubular structures to a larger extent, we modified the vesselness function as follows: (17) Considering some available information that can be used to help remove noise may be lost during the process of segmentation; therefore, the algorithm is first used onto the original data to obtain the enhancement vessel structure instead of being applied directly onto the segmentation result. Then, we use the vessel structure to guide the elimination process of nonvessel outlier points in the segmentation result. 4. Experimental Results and Discussion Experiments in extracting cerebral vessels have been conducted on 10 TOF-MRA data sets which were acquired from Navy General Hospital. The 4 sets of data (Data 1, Data 2, Data 3, and Data 4) analyzed in this paper are with the size of 512 × 512 × 216 voxels, the resolution of 0.39 × 0.39 mm2, and a slice thickness of 1.2 mm. The experiments are implemented on a computer with Intel® Core™ i5-4590 CPU 3.30 GHZ CPU, 12.0 G RAM, and Windows 7 operating system. The parameters used are as follows: Δt = 2.0, α 1 = α 2 = 0.003, β = 0.02, γ = 1.0, ε = 1.0. 4.1. Comparisons with the HLS and LLS Model As Figures 1(a) and 1(c) suggest, TOF-MRA is sensitive to fat tissues which would shutter the blood vessels. A circle of points on the top of the head is introduced in the segmented volume due to similar intensity value between tissues and blood vessels. To eliminate them, the volume is processed with an automatic connectivity filter. We first perform the Frangi's vessel enhancement method onto the original MRA data. Then, we preserve the points in our segmentation result that the first step obtained and then start regional growth algorithm using vessel connectivity. Figures 1(b) and 1(d) present the results after applying such a filter to LLS model and our NHLS model. All the three methods have been experimented with 10 data sets. Results of the three tests are depicted in Figure 2. The first column of Figure 2 shows the MIP images. The second column shows the segmentation results by HLS model. The third column shows the segmentation results by LLS model. The last column shows the results segmented by our NHLS model. As for HLS model, through analyzing the histogram of the data set, we notice that the intensity value of cerebral vascular structures is approximately higher than 200. But, there exist differences among different parts of vessels, and for some of them the intensity value may be between 150 and 200. In our experiments, we set low intensity value μ 0 of the three level-set method to be 200; it will extract the vessels with intensity higher than 200 and those lower than 200 will not be extracted as well. The segmentation result is shown in the second column. As we can see, the segmentation result of cerebral vascular structures is not ideal that it only extracts the large artery structures of vessels but loses many tiny vessel branches. In that method, key parameter μ 0 is predefined to be 200 which means it is unable to extract the small branches with intensity lower than 200. On the other hand, predefined μ 0 is a global threshold; however, the intensity value of different vessel branches is inhomogeneous and has some differences. Therefore, it is essential to consider the local features. As for LLS model, which is an improved method of HLS model replacing predefined μ 0 with dynamic μ(u), dynamic μ(u) is the automatically computed local threshold. The definition of μ(u) is achieved by the Gaussian kernel function modeling the intensity of a blood vessel cross section. Deviation σ of Gaussian kernel is 3.24 in our experiment. The segmentation result is in the third column. It is noticed that the segmentation result of the second method can extract not only the thick artery structures of vessels but also the tiny branches. On one hand, because the vessel branches are very complex and intensity inhomogeneity occurs in vessel structures, threshold μ(u) dynamically calculated can characterize the local information of vessels better. On the other hand, using dynamic thresholds representing the lower bound of vessels can consider the regional information better and the segmentation results are more integral than the original one. However, compared with HLS, the regions of the thick vessels extracted by LLS are not brighter, which means the segmentation result of the thick vessels is not integrated. The defect is caused since the LLS model pays more attention to local and tiny information and neglects some global information, and the segmentation result includes some irrelevant points with the similar intensity value to vessels around the vessels. The proposed method in this paper is inspired by the HLS model and the LLS model. The result is in the last column. Our model combines the global threshold information with the localized threshold information. We analyze the histogram of data and find that the intensity value of vessels is approximately higher than 200; therefore we set global threshold μ 0 to be 200 which means it will extract the target regions with intensity value larger than 200. By embedding the global threshold into the energy function, we define and extract the thick main artery structures of vessels better. In addition, we conceive a dynamic threshold through the role of the Gaussian kernel function, which is used to characterize the local intensity information of vessels. The local thresholds segment the tiny vessels from background more completely. To highlight advantages of our approach, Figure 3 presents some details of Data 1. The first row is the segmentation result, and the second row and third row are the amplified spatial details corresponding to the local regions (marked with the blue boxes). The details show that the result of the HLS model loses many surrounding branches, the LLS model segments tiny branches, but branches are not continuous. The last column is the result segmented by NHLS which extracts those branches, at the same time, it is more integrated. In addition, the thick structures are hollow segmented by LLS with dynamic threshold, and our method solves the defect. Besides the visual inspection, we also evaluate the segmentation accuracy using the Dice Similarity Coefficient (DSC), a widely used metric to evaluate segmentation algorithms for different medical image modalities [38]. Radiologists are invited to segment four sets of MRA data, and the segmentation results are as the ground truth. We, respectively, count the voxel numbers of results segmented by HLS, LLS, and our NHLS model and the voxel numbers of corresponding ground truths. The DSC is defined as (18) DSC=2×NM∩GNM+NG×100, where M and G are the segmentation results and ground truths and N is the voxel number. And ∩ denotes the set cardinality. It has value 1 when M and G are equal and 0 when they do not share any voxel. Figure 4 summarizes the average DSC for three methods. Three observations can be made from Figure 4. First, the DSC achieved by our method is over 80% for most cases. This might be because parts of the vessel were not highlighted due to the vascular disease causing disconnection among voxels in the spatial domain. Second, the average DSC of our method is 29.7%~44.8% higher than that of LLS. We think that is mainly due to not ideal segmentation of main thick vessels. Third, the average DSC of our method is 22.1%~33.9% higher than that of HLS method. We believe HLS model's poor performance is mainly due to the static intensity threshold. Although we could manually select the most suitable threshold value for evolution, it remains challenging to distinguish low contrast vessel from background. 4.2. Sensitivity Analysis for the Parameters The corresponding parameters of the above experiments are α 1, α 2, β, Δt, γ, and ε. Among them, three parameters α 1, α 2, and Δt have more effects on the segmentation results. Parameters α 1 and α 2 are the weight coefficients of the two region terms, and they balance the roles between the global grayscale and the local information. By our test, when α 1 equals α 2, which means the two region terms play the same role, our segmentation results are better. The test on Data 1 is as shown in Table 1. About Δt, we reference the selection of time step in [28], which considers both the speed of evolution and the error in the boundary location, and it concludes that time step Δt usually is set smaller than 10. In our experiments, if the time step is bigger, the evolution can be speeded up; however, there exist more nonvessel points in the segmentation result, which affects the accuracy. Δt = 2.0 is a tradeoff and suitable for this study. 5. Conclusions We have introduced a new hybrid method for the automatic segmentation of cerebral vessels based on an active contour model. The joint energy terms of static and adaptive dynamic kernel within the level-set framework allow for the extraction of thick and thin vessels as well. We have evaluated our method on 10 data sets showing that approximately 80% of DSC are required, and the method performs comparably better than the other two algorithms. Our future work includes acceleration of the current method and further accuracy improvement through vascular compartment recognition. Acknowledgments This work is supported by the National Science Foundation of China (Grant no. 61472042), the Beijing Natural Science Foundation (Grant nos. 4152027 and 4152028), the Fundamental Research Funds for the Central Universities (no. 2015KJJCB25), and the Program for New Century Excellent Talents in University (NCET-13-0051). Competing Interests The authors declare that they have no competing interests. Figure 1 Outlier removement. NHLS segmentation before (a) and after (b) outlier voxels are eliminated with the connectivity filter and LLS segmentation before (c) and after (d) the connectivity filter. Figure 2 The result of cerebral vascular structures segmented by three models. Each row relates to one patient: the first column represents the MIP images. The second column shows the segmentation result of HLS model; the third column shows the segmentation result of LLS model after noise voxels are eliminated with the connectivity filter; the last column shows the segmentation of NHLS model after noise voxels are eliminated with the connectivity. Figure 3 Some details. The first row represents the segmentation results by HLS, LLS, and NHLS. The second and third row are the amplified spatial details corresponding to the local region, respectively (marked with the blue boxes). Figure 4 The DSC of four sets of data using three methods. 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==== Front Front Public HealthFront Public HealthFront. Public HealthFrontiers in Public Health2296-2565Frontiers Media S.A. 10.3389/fpubh.2016.00176Public HealthOriginal ResearchA Content Analysis of Arabic and English Newspapers before, during, and after the Human Papillomavirus Vaccination Campaign in the United Arab Emirates Elbarazi Iffat 1Raheel Hina 2Cummings Kim 1Loney Tom 1*1Institute of Public Health, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, Abu Dhabi, United Arab Emirates2Department of Obstetrics and Gynaecology, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, Abu Dhabi, United Arab EmiratesEdited by: Harshad Thakur, Tata Institute of Social Sciences, India Reviewed by: William Augustine Toscano, University of Minnesota, USA; Caroline Payne-Purvis, University of Florida, USA *Correspondence: Tom Loney, tom.loney@uaeu.ac.aeSpecialty section: This article was submitted to Public Health Education and Promotion, a section of the journal Frontiers in Public Health 29 8 2016 2016 4 17628 6 2016 09 8 2016 Copyright © 2016 Elbarazi, Raheel, Cummings and Loney.2016Elbarazi, Raheel, Cummings and LoneyThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Background Cervical cancer is the fourth most common cancer among females in the United Arab Emirates (UAE) with an estimated incidence of 7.4 per 100,000 persons per year. In March 2008, the Health Authority of Abu Dhabi launched a free school-based campaign to provide all female Emirati students aged 15–17 years in the emirate of Abu Dhabi with the human papillomavirus vaccine (HPVV). Despite the proven efficacy of the HPVV in clinical trials, there has been limited research exploring the acceptance of this vaccine within a conservative Islamic society. The media plays a key role in changing beliefs and attitudes toward specific public health initiatives, such as vaccination programs. The primary aim of this study was to explore the content and communication style of the UAE newspapers (both Arabic and English) before, during, and after the HPV vaccination program. Methods A systematic literature search was conducted on six national newspapers with the highest circulation figures in the UAE (Arabic: Al Ittihad, Al Khaleej, and Emarat El Youm; English: Khaleej Times, The National, and Gulf News) to retrieve articles related to cervical cancer prevention from January 2000 to May 2013. One bilingual researcher (Arabic–English) utilized content analysis to study the subject matter of communication in each article. Results A total of 79 newspaper articles (N = 31 Arabic) were included in the study. Content analysis coding revealed five main themes: (i) “HPV Screening or Vaccination Programmes in the UAE” (N = 30); (ii) “Cervical Cancer Statistics in the UAE” (N = 22); (iii) “Aetiology of Cervical Cancer and HPVV Efficacy” (N = 12); (iv) “Cultural Sensitivity and Misconceptions Surrounding HPVV in School-Aged Females” (e.g., promoting promiscuity) (N = 8); and (v) “Cost-Effectiveness, Efficacy, and Safety” (N = 7). Conclusion The UAE media is playing an important role in raising public awareness about cervical cancer and specific governmental health initiatives such as the HPVV program. Governmental health authorities may want to consider collaborating with the UAE media to develop a communication strategy to reduce the fears and misconceptions surrounding HPVV. Improved parental and adolescent knowledge on the HPVV may lead to increased acceptance and uptake in the UAE society. health communicationhealth promotionhuman papillomavirushuman papillomavirus vaccineUnited Arab Emiratesuterine cervical neoplasmsschool health services ==== Body Introduction Human papillomavirus (HPV) is a common sexually transmitted disease with ~40 different strains that can lead to genital warts and cervical, anal, penile, and vulvar cancers (1). Genotypes 16 and 18 of the virus cause ~70% of all cervical cancers and precancerous cervical lesions worldwide (1, 2). Cervical cancer is the fourth most common cancer in women, and the seventh overall, with an estimated 528,000 new cases and 266,000 deaths in 2012 (1, 3). Although the United Arab Emirates (UAE) has a relatively young national population structure, cancer is the third leading cause of mortality, and cervical cancer is the fourth most common cancer among females in the UAE with an estimated age-standardized incidence of 9.5 and mortality of 4.4 per 100 000 persons per year (4, 5). Approximately, half of all cases of cervical cancer occur in women between 35 and 55 years of age and 70% of cervical cancer cases are diagnosed at a late stage due to the lack of awareness on cervical cancer screening and the cost of screening in the UAE (6). As such, there is an urgent need to increase rates of cervical cancer prevention and early detection in the UAE. In 2006, the United States (US) Food and Drug Administration approved the first preventive HPV vaccine (HPVV) Gardasil and in 2007 the US Advisory Committee on Immunization Practices recommended routine vaccination of females 11–12 years of age (7, 8). Previous studies have shown that the HPVV is safe and highly efficacious in reducing the risk of acquiring HPV infection of genotypes 16 and 18 (9–12). As such, a well-implemented HPVV program with good coverage and uptake is expected to be a more cost-effective measure to reduce the public health and economic burden of cervical cancer within a specific population compared with treatment and screening costs (11, 13). According to the WHO (1), 88% of HPV cases occur in low-income countries, largely due to the lack of regular screening (1, 3). However, in a high income country like the UAE, the major barrier to an effective cervical screening program is not economic but cultural as women are less likely to seek preventive internal examinations (14, 15). Therefore, vaccination campaigns in Islamic countries may be more effective than traditional screening programs in Westernized countries due to cultural barriers related to health education on sexually transmitted diseases and a reluctance to participate in a cervical screening program. Human Papillomavirus Virus Vaccination in the UAE Abu Dhabi is the largest of the seven UAE emirates in terms of land mass and population [2012 mid-year estimates ≈2.3 million; (16)]. In March 2008, the Health Authority Abu Dhabi (HAAD) launched a campaign to vaccinate all girls in Abu Dhabi public schools in the first high-school years. The annual uptake rates of the Abu Dhabi school-based HPVV program ranged from 60 to 80% between 2007 and 2011 with higher uptake rates in public government schools compared with private schools (17). Many factors can affect the acceptability and hence uptake of the HPVV. These factors range from beliefs and perceptions of vaccine effectiveness and susceptibility, parental attitudes, sexual and cultural practices, provider attitudes, setting, and cost and availability of the vaccine (18). Vaccination against cervical cancer can be a controversial issue in many countries, as its opponents claim that it encourages promiscuity (19, 20). A challenging obstacle that can affect the acceptance of the vaccine in UAE is that the vaccine targets a sexually transmitted disease that is still a taboo subject that can lead to stereotypes and stigmatization when this issue is discussed. The UAE is a conservative Muslim society, and numerous factors can play a role in deterring women and guardians from consenting for the vaccine. These factors include certain beliefs such as that the vaccine might encourage girls to become promiscuous and sexually active before marriage. As a Muslim country, it is illegal to commit adultery, and women are supposed to start their sexual activity after marriage; therefore, the UAE HPVV campaign targets females above the age of 15 years and also during their premarital processing tests. The UAE HPVV vaccination program has faced many challenges; among them, the acceptability of the vaccine amid parents, spouses, and providers due to its cultural sensitivity. Ortashi and colleagues conducted a series of studies to explore the knowledge, attitudes, and behaviors of these groups with regard to the HPVV (21–24). Ortashi et al. (21) conducted a cross-sectional study to assess the knowledge and attitude of school nurses (n = 125) about the HPVV in the emirate of Abu Dhabi, and 48% of participants reported cultural or religious unacceptability and 21% cited women’s lack of concern about their own health as major barriers toward 100% vaccination uptake in the UAE. Moreover, the majority (87%) of the nurses reported that they would not feel comfortable counseling high-school-aged females or parents about HPVV (21). Ortashi et al. (22) also conducted a questionnaire-based cross-sectional study to assess the knowledge and acceptability of the HPVV among male University students (n = 356) in the UAE. Less than one-third (32%) of male University students had ever heard of HPV and only 24% indicated that they would definitely accept the HPVV (22). The factors rated most likely to prevent students from using the vaccine were fear of the side effects (85%), absence of clear benefits (38%), and objections from a religious authority (25%). Interestingly, marital status and sexual activity were associated with greater knowledge of HPV but not with greater acceptance of vaccination (22). Finally, Ortashi and colleagues conducted a cross-sectional study utilizing a convenient sample of women (both UAE nationals and non-nationals) aged 18–50 years (n = 640) from the emirate of Abu Dhabi to assess their knowledge of HPV infection and HPVV in the UAE (23, 24). Less than one-third (29%) of the sample had ever heard of HPV infection, only 15% recognized it as a sexually transmitted infection, and less than one-quarter (22%) had heard of the HPVV (23, 24). Husband’s level of education was positively associated with better knowledge of HPV infection (23, 24). Overall, the knowledge of HPV infection and vaccine is low in the UAE. Numerous researchers have suggested that designing effective vaccine-delivery strategies maximizes the potential of vaccines to decrease the heath and economic cancer burden of HPV (9, 11). A successful vaccination program will require the support of public health authorities, the coordination of health workers from different areas, and increased public awareness, acceptance, and uptake (9). Specifically, mass media communication is usually employed in health promotion to raise public health awareness, to create a climate of opinion conducive to policy change, and to shift attitudes and prompt behavior change through stressing the negative effects of health damaging behaviors and the benefits of health-conducive behaviors (25). Therefore, the media has the potential to reach and deliver targeted and tailored health messages based on current scientific evidence to large proportions of the population who may benefit from the HPVV. Several researchers have conducted content analysis of national newspapers articles in Canada and the US to examine the news information presented related to HPVV and concluded that the media, particularly newspapers, can be a powerful tool to promote vaccine uptake or to induce fear among the public against the use of HPVV (26–28). To the best of our knowledge, there have been no studies exploring the framing of public health messages related to cervical cancer and HPVV in the newspapers of an Islamic country such as the UAE. The primary aim of this study was to address this gap by analyzing the content and communication style of UAE newspaper coverage on cervical cancer and HPVV over a 13-year period from 2000 to 2013, before and after the HPV vaccination program was implemented in the emirate of Abu Dhabi. Materials and Methods Sample Selection and Data Sources Arabic is the official language in the UAE, and English is also widely spoken due to the diversity of the expatriates living in the UAE. A census of all newspaper articles reporting on cervical cancer, HPV, and HPVV over a 13-year period (January 2000 to May 2013) was collected from six national newspapers (three Arabic and three English) with the highest circulation figures in the UAE (29). The print and online news outlets included in the study were Al Ittihad, Al Khaleej, Emarat El Youm (all Arabic), Al Khaleej Times, Gulf News, and The National (all English). These newspapers were selected based on their popularity and their distribution. According to the Arab Media Outlook Report 2011–2015 (29), Gulf News is the most popular and widely read newspaper in the UAE, followed by Al Khaleej, Al Emarat Al Youm, and Khaleej Times. The Arab Media Outlook Report conducted market research on the UAE population and 48% of the sample reported reading the Gulf News, 39% the Al Khaleej, 32% the Al Emarat Al Youm, 28% the Khaleej Times, and 27% the Al Ittihad newspaper (note: respondents reported reading multiple newspapers) (29). In terms of reported subscription rates, 34% of respondents reported subscribing to Gulf News, 17% to Al Ittihad, 14% Khaleej Times, and 12% the Al Khaleej newspaper. News Coverage Identification The systematic literature search included searching the six UAE newspapers with the highest circulation figures for articles about cervical cancer screening and HPVV (29). Internet search engines and individual newspaper search engines were used to investigate the period from January 2000 to May 2013 employing a combination of MESH terms, free-text words, and entry terms “(HPV OR human papillomavirus) AND cervical cancer AND (Gardasil OR Cervarix OR vaccine).” This specific time period was before and after the implementation of the HPVV program in the emirate of Abu Dhabi. Only articles that discussed cervical cancer screening, prevention, and vaccination were included in the content analysis. Articles that discussed cancer in general including cervical cancer were excluded to avoid bias. Data Coding Process and Analysis The unit of analysis was each newspaper article. Descriptive statistics were calculated to determine the proportion of news coverage in different newspapers and languages. Each article was examined to inquire about the frequency of general information about the HPV vaccine, the types of sources referenced in articles, and headlines and message frames coded using emergent themes. One bilingual researcher (Arabic–English) utilized content analysis to study the subject matter of communication in each article. Specifically, the researcher attempted to gain familiarity with the article by re-reading and reviewing them to gain a deeper understanding of the health message framing while bracketing their own preconceived beliefs about cervical cancer or HPVV. To determine the overall content and framing of the article, the researcher annotated the article text by attaching key words to segments of text. An “emergent” coding procedure was used in contrast to “priori” codes as this method allows the codes to reflect the views of the journalist rather than limiting the analysis to “prefigured” codes dictated by theory. Results Media Coverage by Language and Newspaper A total of 79 articles were identified about the HPVV from 6 national newspapers in the UAE (Arabic: Al Ittihad, Al Khaleej, and Emarat El Youm; English: Al Khaleej, The National, and Gulf News) for the period from January 1, 2000 to June 30, 2013. Overall, 59.5% of articles were published in English newspapers, and 40.5% were published in Arabic newspapers. The distribution of articles in the English newspapers was 46.8% in Khaleej Times, 31.9% in Gulf News, and 21.3% in The National newspaper. In the Arabic newspapers, the distribution of the articles was 53.1% in Al Emarat Elyoum, 37.5% in Al Ittihad, and 9.4% in the Al Khaleej newspaper (Table 1). Table 1 Breakdown of newspaper articles by year and newspaper. Year The national Gulf news Khaleej times Al Iitihad Al Khaleej Al Emarat Elyoum No. of articles 2001 0 1 0 0 0 0 1 2006 0 3 1 0 0 0 4 2007 0 3 4 0 0 0 7 Launch of Abu Dhabi HPV vaccination campaign 2008 0 2 4 1 0 4 11 2009 0 2 3 0 0 4 9 2010 0 0 2 0 0 0 2 2011 3 1 3 1 0 3 11 2012 2 2 4 2 1 3 14 2013 4 1 2 8 2 3 20 Total 9 15 23 12 3 17 79 Coverage by Month and Year In the English newspapers, there was a fairly constant number of articles published about HPV, cervical cancer, or HPVV across the time period with small peaks in March (6.4%), April (6.4%), and May 2012 (6.4%; Table 1; Figure 1). However, there were distinct spikes in the number of articles published in the Arabic newspapers in January (25.0%) and June 2013 (9.4%). Figure 1 Graph showing distribution of HPVV articles in Arabic and in English newspaper between April 2006 and May 2013. Dotted line represents the launch of Health Authority Abu Dhabi’s HPV campaign in March 2008 to vaccinate all UAE National girls in Abu Dhabi public high schools in the first high-school years. Article Frames and Themes Five major article content themes were identified: (i) “HPV Screening or Vaccination Programmes in the UAE” (N = 30); (ii) “Cervical Cancer Statistics in the UAE” (N = 22); (iii) “Aetiology of Cervical Cancer and HPVV Efficacy” (N = 12); (iv) “Cultural Sensitivity and Misconceptions Surrounding HPVV in School-Aged Females” (e.g., promoting promiscuity) (N = 8); (v) “Cost-Effectiveness, Safety, and Side Effects of HPVV” (N = 7) (Table 2). Majority of articles (N = 30) discussed HAAD and other authorities’ campaigns and activities related to HPV screening and awareness programs (Table 2; Table S1 in Supplementary Material). Only eight articles discussed the cultural sensitivity and misconceptions issues related to HPVV in the UAE, and seven articles addressed the cost of the HPVV in general and the importance of including non-national citizens within the vaccination program (Table 2; Table S1 in Supplementary Material). Finally, 22 articles discussed cervical cancer statistics vaccination and screening programs, and 12 articles discussed the etiology of the vaccine and its efficacy (Table 2; Table S1 in Supplementary Material). Newspapers articles are listed in Table S1 in Supplementary Material. Overall, articles showed a general trend toward advocating for the vaccine and screening program in a non-direct manner by communicating data and statistics in an informative rather than persuasive style. Table 2 Number (%) of articles published related to each content theme. Theme Number of articles (%) HPV Screening or Vaccination Programmes in the UAE 30 (38) Cervical Cancer Statistics in the UAE 22 (28) Aetiology of Cervical Cancer and HPVV Efficacy 12 (15) Cultural Sensitivity and Misconceptions Surrounding HPVV 8 (10) Cost-Effectiveness, Safety, and Side Effects of HPVV 7 (9) HAAD, Health Authority Abu Dhabi. Discussion All selected Arabic and English newspapers highlighted the efforts of the government and local health authorities to reduce cervical cancer cases in the country and described the programs (e.g., screening and vaccination) implemented in the country to achieve their goal. According to Kelly et al. (30), media coverage around the cervical cancer vaccine increased significantly after the initial approval of the vaccine in 2006 in many countries. This trend was also observed in the UAE. Newspapers in the UAE started communicating data on the prevalence and incidence of cervical cancer globally and in the UAE from as early as 2006. The volume of articles increased significantly from 2008 the year of the launch of the vaccination program (Table 1; Figure 1). The distinct spikes in the number of articles published in the Arabic newspapers in January (25.0%) and June 2013 (9.4%) may potentially be explained by the announcement of the intention of health authorities in other Emirates, particularly Dubai, to introduce HPV immunization as part of the authority’s public health initiatives. However, the limited number of articles published in the past 10 years suggests that cervical cancer is perceived as an emerging public health issue in the UAE. Overall, the UAE newspaper articles provided superficial coverage of all of the issues surrounding the HPVV and focused on advertising and highlighting the cervical cancer-related activities of local UAE health authorities. The majority of articles described and discussed the burden of cervical cancer and the importance of screening and vaccination programs. For example, the UAE media discussed the need for vaccinations, the importance of reducing the number of women with cervical cancer, and the programs initiated by the authorities and policy makers to overcome this issue. However, the communication style of these articles was to inform the public about HPV and the vaccination program rather than to persuade the public, particularly parents, about the benefits of HPVV and overcome the misconceptions and stigma associated with the vaccine. The academic literature is concerned with vaccine acceptance and strategies to change knowledge and attitudes toward any beneficial vaccine to maximize uptake. However, the newspaper articles reviewed in this study did not seem to focus on this important factor as a high priority. Rather, the media failed to discuss important matters such as strategies to increase the number of women completing timely pap smears and screening tests, encourage parents to vaccinate their daughters, and how to convince adolescents to receive the vaccination. This review of UAE newspapers mirrored the findings of that of Kelly et al. (30) and Penţa and Băban (31). Specifically, the vaccine efficacy and the Emirati societal need for the vaccine were highlighted using scientific evidence as there were recurrent references to the academic literature when the media discussed the efficacy of the vaccine and the impact of cervical cancer on the UAE society. A deeper discussion of HPVV acceptability and the burden of the disease and its effects on the UAE society were lacking. The English media highlighted the fact that non-nationals (expatriates constituting >85% of the UAE population) are not provided with the vaccine free of charge. The media content stressed the importance of introducing free vaccination for everyone to reduce the number of cases of cervical cancer and to improve the prevention program efficacy. In 2006, the Government of Abu Dhabi introduced Law No. 23 (of 20058) which outlined mandatory private health insurance for all expatriate residents. However, the range of services offered and treatments available for expatriate residents varies depending on their monthly salary, and the majority of health insurance schemes do not cover HPVV meaning expatriates would be required to pay out of pocket. Therefore, numerous newspaper articles highlighted the role of private insurance companies calling for full coverage for the vaccine and for cervical cancer screening tests by these companies to the entire UAE population included expatriates. To modify the general public’s attitude toward a culturally sensitive topic in a conservative Islamic country such as the UAE, it is very important to use all available sources to reach the public. Mass media represented by printed and audio–visual means can reach a large proportion of the public especially parents and teachers who can disseminate the correct message and information. Printed and web-based newspaper media can be a useful vehicle for policy makers to improve people’s knowledge, positively influence their attitudes, and potentially enhance their health through the promotion of healthy lifestyle behaviors or engagement in specific public health programs such as vaccinations. In relation to HPVV, it is important to change people’s attitude and knowledge, especially when the issue can be culturally sensitive (30). The HPVV has been controversial as there are fears that it encourages teenagers to participate in pre-marriage sexual activities (19, 20) which are not culturally acceptable and illegal in the UAE. Our review of the UAE newspaper articles published around cervical cancer screening and vaccination showed that the discussion around socio-cultural issues related to HPVV in the UAE was superficial and shallow. It seems that the primary role of the UAE media was to provide information about the availability and efficacy of HPVV without discussing issues related to the acceptance and uptake of the vaccine, safety concerns, or cultural sensitivity. Kelly et al. (30) suggest that one factor contributing to the lack of knowledge about HPVV may be related to the lack of news coverage which may negatively impact people’s adoption of healthy behaviors like vaccination and screening. Naidoo and Wills (25) list four different models on how the media can affect audiences and influence public health. The first model is the direct effect that manipulates the passive audience. The second model is the two-step model where mass communication influences key opinion leaders who are active members of the audience and who will spread ideas to other people through interpersonal communication. The third model suggests that people use the media to meet their needs by reinforcing existing beliefs or rejecting new ones. The last model is the cultural effects which indicate that media has a key role in creating beliefs and values about health, illnesses, and diseases. Our review revealed that Arabic and English newspapers in the UAE do not seem to follow any of these models as there is no obvious use for an approach to manipulate passive audience or to influence key opinion leaders. Also, the existing media do not reinforce existing beliefs or reject new ones and do not have much influence on beliefs and values illnesses and diseases. Upon reviewing the newspaper articles included in this study, it is clear that the power of mass communication is not well used in UAE context to promote HPVV uptake and to increase education and awareness. Although there were some timid efforts to discuss this topic, the media is required to be more proactive in terms of raising awareness and educating the public. The analysis of the media that discusses HPV, HPVV, and cervical cancer shows that there is still missing information on HPV prevention, transmission, symptoms, screening, and the importance of the vaccine. There is a dearth of discussion on the commonly held parental misconceptions (e.g., HPVV encourages promiscuous behavior) with the existing media preferring to concentrate on publicizing the local health authorities’ efforts, programs, and future plans. One of the major strengths of this study is the inclusion of newspapers in two different languages to ensure a comprehensive coverage of newspaper articles as the UAE is a multicultural country with Arabic as the official language and English widely spoken. A weakness of the study was the exclusion of other forms of media (e.g., websites, radio, and television) and social media (e.g., twitter) that may have covered cervical cancer and HPVV and reached the younger segments of the UAE population. In addition, newspapers targeting specific nationalities within the UAE population (e.g., The Indian Times) were not included; rather, we focused on the Arabic and English national newspapers with the highest circulation and/or subscriptions rates that are most likely to be read by UAE nationals. Conclusion Printed and web-based newspapers have the potential to act as a powerful persuader or discourager of public health behaviors in any country. Newspapers can play a positive role in changing people’s attitude toward HPVV and promoting vaccine acceptance by community groups, particularly middle-aged and older male adults who may be responsible for deciding whether or not a female family member receives the HPVV. The UAE media can potentially accelerate the acceptance and uptake of the vaccine which may help to reduce cervical cancer-related morbidity and mortality in the UAE. Governmental health authorities may want to consider collaborating with public health researchers, communication specialists, and the UAE media to develop a campaign strategy to reduce the fears and misconceptions surrounding HPVV. Author Contributions IE conceived the idea. IE, HR, KC, and TL developed the protocol and conducted the literature search for newspaper articles. IE and HR reviewed the articles and conducted the thematic analysis. IE drafted the manuscript, and HR, KC, and TL revised the manuscript. All authors have critically reviewed, provided intellectual input to the manuscript, and approved the final version of the manuscript. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Supplementary Material The Supplementary Material for this article can be found online at http://journal.frontiersin.org/article/10.3389/fpubh.2016.00176 Click here for additional data file. ==== Refs References 1 World Health Organization . Human Papillomavirus (HPV) and Cervical Cancer . (2013 ). Available from: http://www.who.int/mediacentre/factsheets/fs380/en/ 2 Muñoz N Bosch FX Castellsagué X Díaz M de Sanjose S Hammouda D Against which human papillomavirus types shall we vaccinate and screen? The international perspective . Int J Cancer (2004 ) 111 (2 ):278 –85 .10.1002/ijc.20244 15197783 3 International Agency for Research on Cancer . 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==== Front Front PsychiatryFront PsychiatryFront. PsychiatryFrontiers in Psychiatry1664-0640Frontiers Media S.A. 10.3389/fpsyt.2016.00151PsychiatryOriginal ResearchThe Brain-Derived Neurotrophic Factor Val66Met Polymorphism Moderates the Effects of Childhood Abuse on Severity of Depressive Symptoms in a Time-Dependent Manner Webb Caitlin 1Gunn Jane M. 2Potiriadis Maria 2Everall Ian P. 134Bousman Chad A. 1245*1Department of Psychiatry, The University of Melbourne, Parkville, VIC, Australia2Department of General Practice, The University of Melbourne, Parkville, VIC, Australia3NorthWestern Mental Health, Melbourne, VIC, Australia4Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, VIC, Australia5Centre for Human Psychopharmacology, Swinburne University of Technology, Hawthorne, VIC, AustraliaEdited by: Ming D. Li, Seton Hall University, USA Reviewed by: Carlos M. Opazo, The University of Melbourne, Australia; Zhongli Yang, Zhejiang University, China *Correspondence: Chad A. Bousman, cbousman@unimelb.edu.auSpecialty section: This article was submitted to Molecular Psychiatry, a section of the journal Frontiers in Psychiatry 29 8 2016 2016 7 15111 3 2016 17 8 2016 Copyright © 2016 Webb, Gunn, Potiriadis, Everall and Bousman.2016Webb, Gunn, Potiriadis, Everall and BousmanThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Cross-sectional studies have demonstrated that the brain-derived neurotrophic factor (BDNF) Val66Met single-nucleotide polymorphism moderates the association between exposure to negative life events and depression outcomes. Yet, it is currently unclear whether this moderating effect is applicable to positive life events and if the moderating effect is stable over time. To address these gaps in the literature, we examined clinical and BDNF genotypic data from a 5-year prospective cohort of 310 primary care attendees. Primary care attendees were selected based on existence of depressive symptoms at screening. Depressive symptoms were assessed at baseline and annually for 5 years post-baseline using the Primary Care Evaluation of Mental Disorders Patient Health Questionnaire-9 (PHQ-9). Linear mixed models assessed differences in depressive symptom severity over the 5-year follow-up period by BDNF Val66Met and history of life events, both negative and positive. Analysis identified a novel three-way interaction between the BDNF Val66Met polymorphism, history of severe childhood abuse, and time. Post hoc analysis stratified by time showed a two-way interaction between Val66Met and severe childhood abuse at baseline that was not detectable at any other time point. An interaction between Val66Met and positive life events was not detected. Our longitudinal results suggest that the BDNF Val66Met polymorphism moderates the depressive symptom severity experienced by those with a history of severe childhood abuse but does so in a time-dependent manner. Our results further support the notion that gene–environment–depression interactions are dynamic and highlight the importance of longitudinal assessment of these interactions. Given these novel longitudinal findings; replication is required. depressiongene–environmentbrain-derived neurotrophic factorchildhood adversitystressful life eventslongitudinal cohortNational Health and Medical Research Council10.13039/501100000925299869, 454463, 566511, 1002908 ==== Body Introduction The etiologies of depression are poorly understood, with the most recent etiological theories focusing on gene–environment interactions. Depressive phenotypes have long been associated with exposure to negative events, yet phenotypic variation is evident, with individuals exposed to the same environmental stress either not developing depressive symptoms or developing a range of symptom severity (1). This phenotypic variation is the basis of the diathesis–stress psychological framework (2), which suggests individuals possess “risk” factors (e.g., genetic variants), which make them more vulnerable to developing psychological symptoms when exposed to an adverse event. Historically, the diathesis–stress framework has guided research into gene–environment interactions. However, an emerging alternative framework is that of differential susceptibility. The differential susceptibility framework postulates that traditionally defined “risk” factors may better be defined as “phenotypic plasticity” factors in that individuals carrying these factors are more sensitive to both positive and negative environmental influences (3). Environmental influences consistently linked to depression include adverse childhood events (4, 5) and current domestic abuse among women (6). However, environmental stress alone has been identified as insufficient to cause depression (7), emphasizing the gene–environment hypothesis. Current literature clearly identifies numerous genetic variants associated with mediating the differential response to negative life events in the development of depression (8). Among these genes is the brain-derived neurotrophic factor (BDNF), specifically the Met allele in the Val66Met single-nucleotide polymorphism located in the coding region of exon 2 (rs6265 – A66G) (8). BDNF is a pro-survival factor, involved in brain cell survival and proliferation (8) and has also been suggested to affect neuronal plasticity (9). Depressed individuals are shown to have decreased serum BDNF levels compared to controls (10–12) and carriers of the BDNF rs6265 Met allele show a reduction in BDNF activity (13). Studies examining the interaction between BDNF genetic variation and stressful life events have reported conflicting results. A number of studies identified an association between the BDNF Met allele and greater depressive symptom severity in the context of childhood abuse or adult abuse (14). Other studies reported that BDNF moderates the response to some, but not all, forms of abuse (15, 16). While two large studies, one a cross-sectional study (17) and another a case–control study (18) reported no moderating effect of BDNF genetic variation on the association between stressful life events and depressive symptoms. In light of these conflicting results, the role of BDNF genetic variation in moderating depressive symptoms in the context of stressful life events remains unclear. In addition, the role BDNF plays in moderating depressive symptom severity in the context of life events over time is not clear. As such, we examined whether the BDNF Val66Met polymorphism moderated the relationship between life events (negative and positive) and depressive symptom severity in a 5-year prospective cohort of 310 primary care attendees. Materials and Methods Participants Participants were recruited from the Diagnosis, Management and Outcomes of Depression in Primary Care (diamond) study, an ongoing prospective cohort that commenced in 2005 (19). The diamond study aims to document the experiences, health outcomes, treatment, and service usage of primary care attendees identified as having clinically relevant depressed mood at screening; with patients recruited from 30 rural and metropolitan general practices randomly recruited in Victoria, Australia (19). Primary care patients were eligible for the diamond cohort if they were: (a) aged 18–75 years, (b) able to read English, (c) not terminally ill, (d) did not reside in a nursing home, and (e) scored 16 or higher on the Center for Epidemiologic Studies Depression Scale (CES-D). Participants were assessed annually using postal surveys as well as computer-assisted telephone interviews. In 2011 (cohort year 6), participants enrolled in the cohort were invited to provide a saliva sample for DNA extraction and genotyping. All procedures were conducted in accord with principles expressed in the Declaration of Helsinki and obtained approval from the University of Melbourne Human Research Ethics Committee (Ethics ID 1135247.1). Depressive Symptom Measures Longitudinal depressive symptoms were assessed at baseline and annually for 5 years thereafter using the self-administered Primary Care Evaluation of Mental Disorders Patient Health Questionnaire-9 (PHQ-9) (20). The PHQ-9 is based directly on the nine signs and symptoms of major depressive disorder as described in the Diagnostic and Statistical Manual of Mental Disorders-IV (DSM-IV) (21) and has been validated to screen and monitor depression severity in the primary care setting (22). The PHQ-9 asks respondents to rate their symptoms over the past 2 weeks and is scored on a scale of zero (“not at all”) to three (“nearly every day”) for each item with a range of 0–27 (20). Scores of 5, 10, 15, and 20 on the PHQ-9 represent cut-points for mild, moderate, moderately severe, and severe depression, respectively (20). DSM-IV criteria (23) for major depressive disorder was assessed using the Composite International Diagnostic Interview (CIDI) Auto version 2.1 (24) by a trained research assistant. Childhood Abuse At baseline exposure to physical and sexual childhood abuse was measured using the Child Maltreatment History Self-Report (CMHSR) (25). The CMHSR consists of 11 items, 7 pertaining to physical abuse and 4 to sexual abuse. The physical abuse questions have a four-point response option (never, rarely, sometimes, often), whereas the sexual abuse questions have a yes/no response option. Severe abuse was defined using the scoring algorithm previously described by Macmillan and associates (25). Partner Abuse History of partner abuse in adulthood was measured using a history of partner fear as a proxy marker. This was done because the available composite abuse scale (CAS) in our cohort has not been validated in males as well as to minimize the risk of under-reporting associated with the CAS and to reflect the importance on an individual’s perception in the experience of abuse (26). Life Events At baseline, participants completed a 13-item life events questionnaire adapted from the Life Experiences Scale and Life Events Questionnaire (27, 28). A full list of events measured is displayed in Table S1 in Supplementary Material. For each life event, participants were asked to indicate whether or not they experienced the life event in the past 12 months and if so, what impact the event had on them. There were six possible responses for each life event: (1) No, I have not experienced this event in the past 12 months; (2) Yes, and it has had an extremely negative impact on me; (3) Yes and it has had a slightly negative impact on me; (4) Yes but it has had no impact on me; (5) Yes and it has had a slightly positive impact on me; (6) Yes and it has had an extremely positive impact on me. In this study, presence of a positive event was defined as one or more events reported as slightly or extremely positive. Presence of a negative event was defined as one or more events reported as slightly or extremely negative. Potential Confounding Variables At baseline, potential confounding factors, including demographics, smoking status, quality of life (29), and self-rated health status (30) were assessed. Panic and other anxiety syndromes were also assessed using the anxiety module of the PHQ (22). Alcohol and drug abuse/dependence (i.e., cannabis, opioid, sedative, cocaine, amphetamine, hallucinogens, inhalants) was assessed using the CIDI Auto version 2.1 (24). At each assessment point, the use of antidepressants, anxiolytics, and antipsychotics, as well as self-reported visits in the past 12 months to a psychiatrist and/or psychologist were also assessed. DNA Extraction and Genotyping DNA extraction and genotyping details have previously been published (31). In brief, DNA was recovered from stabilized saliva samples and the rs6265 polymorphism was genotyped as part of a larger genotyping project with the Sequenom MassARRAY MALDI-TOF genotyping system (Sequenom Inc., San Diego, CA, USA). To detect for the presence of population stratification, 60 unlinked ancestry-informative markers (AIMs; Table S2 in Supplementary Material) representing the three HapMap phase III populations (Northern/Western European, Han Chinese, and Yoruba in Nigeria) were also genotyped (32). Statistical Analysis Chi-square analysis of BDNF genotype was used to detect departures from Hardy–Weinberg equilibrium. Due to the low number of Met/Met carriers (n = 9), individuals who carried the Met allele (Val/Met or Met/Met) were compared to homozygous Val carriers. To estimate the presence of population stratification, the 60 AIMs were used to assign each participant to the HapMap ancestral group (Northern/Western European, Han Chinese, and Yoruba in Nigeria) for which they carried the greatest proportion of that population’s AIMs. Linear mixed models were used to determine trajectory differences in PHQ-9 depressive symptom severity over the 5-year follow-up period by genotype and abuse/life event type. The mixed models approach enables use of all repeated measurements, accounts for clustering of participants within primary care sites, and provides unbiased estimates in the presence of missing data. Prior to creation of interaction terms and modeling, genotypes, abuse, and life event variables as well as covariates were centered (33). Potential covariates were assessed for their association with each of the abuse/life event types and genotypes using chi-square, Fisher’s exact, or analysis of variance tests, depending on the variable structure. Covariates with p-values ≤0.05 were retained for adjusted analysis (Table S3 in Supplementary Material). The unadjusted model included fixed effects of time, genotype, abuse/event, and a time × genotype, time × abuse/event, genotype × abuse/event, and time × genotype × abuse/event interaction terms. Random effects included individual, primary care site, intercept (PHQ baseline score), and slope (time). Unadjusted models that returned significant main effects (p < 0.05) were then adjusted for covariates to account for any bias from baseline characteristics. Adjusted models included relevant covariates as well as covariate × time, covariate × genotype, and covariate × abuse/event type interaction terms as previously recommended (34). Covariance models used for the random and repeated effects were unstructured and first-order autoregressive, respectively. Adjusted models were then refined using a backward stepwise penalized likelihood model selection strategy by starting with the most complex model and removing the term with the largest p-value above 0.05 using unrestricted maximum likelihood estimations. If removal of a covariate term increased the Bayesian information criterion (BIC) compared to previous more complex model the covariate was retained. Missing data of longitudinal measurements were assumed missing at random. All analyses were performed using SPSS 21.0 (IBM, Armonk, NY, USA). Results Sample Characteristics A total of 789 participants were recruited into the diamond cohort of whom 498 were enrolled at the time of DNA collection (cohort year 6) and 344 (69%) consented and returned a DNA sample. Individuals missing genotype data (n = 22) or abuse data (n = 13) were excluded. A total sample of 310 participants was included in the analysis (Table 1). Genotype frequencies for BDNF rs6265 were 67% Val/Val, 33% Met carriers (30% Val/Met and 3% Met/Met) and were in Hardy–Weinberg equilibrium (p = 0.768). All participants were of Northern/Western European (CEU) ancestry based on 60 unlinked AIMs (Figure 1). Table 1 Participant characteristics (n = 310). Baseline variables Demographics  Age, mean (SD) years 48.9 (11.9)  Sex, % (n) female 71 (220)  English as first language, % (n) 99 (307)  Northern European genetic ancestry, % (n) 100 (310) Depression history  DSM-IV major depressive disorder, % (n)a 49.2 (149)  Relative with a history of depression, % (n)b 75.4 (193) Co-morbid substance use  Smoker, % (n)c 23.9 (74)  Alcohol abuse/dependence, % (n)a 13 (39)  Substance abuse/dependence (excludes alcohol), % (n) – 301 6.3 (19) Medication use  Antidepressant, % (n) 40.6 (126)  Anxiolytic, % (n) 9.7 (30)  Antipsychotic, % (n) 5.5 (17)  Sedative, % (n)c 31.1 (96)  St John’s Wort, % (n)c 9.4 (29) Visited counselor/psychologist/psychiatrist past 12 months, % (n) 33.2 (103) Self-rated health, % (n) good to excellent 63.5 (197) Education, % (n)c  Completed year 12 or less 47.3 (147)  Diploma or certificate 25.2 (78)  Bachelor degree or higher 27.2 (84) Managing on available income, % (n)d  Easily/not too bad 48.1 (148)  Difficult some of the time 35.7 (110)  Difficult all of the time/impossible 16.2 (50) WHO Quality of Life and Functioning  Environmental context, mean (SD) 64.2 (13.1)  Social context, mean (SD) 49.7 (23.8) Severe childhood abuse, % (n)  Sexual 12.9 (40)  Physical 12.9 (40)  Either or both – sexual and/or physical 39 (121) History of partner fear, % (n) 33.2 (103) Life events in past 12 months, % (n)  At least one positive 25.8 (80)  At least one negative 81.9 (254) an = 301. bn = 256. cn = 309. dn = 308. WHO, World Health Organization. Figure 1 Ancestry estimation of study sample. Participants were assigned to the HapMap ancestral group (Northern/Western European, CEU; Han Chinese, CHB; or Yoruba in Nigeria, YRI) for which they carried the greatest proportion of that population’s ancestry-informative markers. Unadjusted Findings Unadjusted linear mixed models for the BDNF rs6265 genotype across the abuse and life event types are summarized in Table 2. A two-way interaction was not found between BDNF rs6265 and positive events (F1, 307 = 0.12, punadjusted = 0.728) or BDNF rs6265 and negative events (F1, 307 = 0.04, punadjusted = 0.844). However, a two-way interaction was detected between BDNF rs6265 and severe childhood abuse (F1, 307 = 4.6, punadjusted = 0.031) and a three-way interaction was observed between the BDNF rs6265 genotype, severe childhood abuse, and time (F1, 299 = 5.4, punadjusted = 0.021). Table 2 General linear mixed model parameter p-values for main and interaction effects of BDNF rs6265 genotype, abusive experiences, and life events on 60-month depressive symptom trajectories (n = 310). Unadjusted p-values for Type III fixed effects* Event type in model BDNF rs6265 Event type Time rs6265 × event type rs6265 × time Event type × time rs6265 × event type × time History of severe child abuse 0.757 0.001 0.001 0.031 0.084 0.482 0.021 History of partner fear 0.777 0.001 0.001 0.090 0.332 0.104 0.636 Life events  Positive 0.899 0.147 0.001 0.728 0.541 0.026 0.473  Negative 0.920 0.008 0.001 0.844 0.316 0.395 0.894 Bolded values indicate p < 0.05. Covariate-Adjusted Findings Only the three-way interaction survived adjustment for covariates (F1, 292 = 4.9, pcovariate adjusted = 0.027) (Table 3; Table S4 in Supplementary Material). Figure 2 shows Met allele carriers with a history of severe childhood abuse had a significantly greater reduction in depressive symptom severity over the 5-year follow-up period compared to their Val/Val carrying counterparts. Post hoc analysis stratified by time showed the two-way interaction between BDNF rs6265 and severe childhood abuse was present at baseline (F1, 297 = 5.4, punadjusted = 0.021) but no other time points (Figure 3). Importantly, further post hoc analyses suggested this time-dependent effect was not due to differential receipt of psychosocial (i.e., visits to counselor, psychologist, or psychiatrist) or pharmacological (i.e., antidepressant, anxiolytic, or antipsychotic) therapy over the 5-year study period (Figures 4 and 5). Table 3 Type III fixed effectsa for terms included in the final covariate-adjusted linear mixed model. Parameter Degrees of Freedom F p Numerator Denominator Intercept 1 310.128 356.731 0.000 Time 1 291.276 16.695 0.000 Genotype – rs6265 1 333.991 0.804 0.371 Severe child abuse 1 332.223 1.897 0.169 rs6265 × time 1 291.919 1.753 0.187 Severe child abuse × time 1 292.359 0.018 0.895 rs6265 × severe child abuse 1 305.182 2.786 0.096 rs6265 × severe child abuse × Time 1 292.215 4.925 0.027 DSM-IV Depression diagnosis 1 317.757 33.355 0.000 DSM-IV Depression diagnosis × Time 1 290.903 5.853 0.016 DSM-IV Depression diagnosis × rs6265 1 297.564 0.032 0.858 DSM-IV Depression diagnosis × severe child abuse 1 297.116 0.205 0.651 Self-rated health 1 307.656 38.684 0.000 Self-rated health × time 1 291.349 5.061 0.025 Self-rated health × rs6265 1 296.148 0.334 0.564 Self-rated health × severe child abuse 1 296.054 0.136 0.712 DSM IV substance abuse/addiction 1 306.156 4.014 0.046 DSM IV substance abuse/addiction × time 1 289.680 0.041 0.839 DSM IV substance abuse/addiction × rs6265 1 294.717 3.176 0.076 DSM IV substance abuse/addiction × severe child abuse 1 294.647 0.470 0.493 WHOQOL Social 1 300.581 18.361 0.000 WHOQOL Social × rs6265 1 299.536 3.026 0.083 WHOQOL Social × severe child abuse 1 297.433 1.061 0.304 aDependent variable: PHQ depressive symptom severity. Bolded values indicate p < 0.05. Figure 2 Interaction effect of severe childhood abuse and BDNF Val66Met genotype on 5-year depressive symptom trajectories. Baseline represented by time point 0, annual assessment thereafter. Points and associated SE bars represent predicted values based on the final covariate-adjusted model. PHQ-9 = Primary Care Evaluation of Mental Disorders Patient Health Questionnaire-9. Figure 3 Interaction effect of severe child abuse and BDNF rs6265 genotype by time point. Time point zero represents baseline, measured annually from baseline. Points and associated SE bars represent predicted values based on the final covariate-adjusted model. PHQ-9 = Primary Care Evaluation of Mental Disorders Patient Health Questionnaire-9. Figure 4 Longitudinal measurements of medication usage by percent of individuals reportedly taking the medication by genotype and history of severe child abuse. Figure 5 Longitudinal measurements of psychosocial treatment by percent of individuals reported as having seen a psychiatrist, psychologist, or counselor once or more in the preceding 12 months, by genotype and history of severe child abuse. Discussion We demonstrated a novel three-way interaction between the BDNF Val66Met polymorphism, a history of severe childhood abuse and time. This interaction withstood adjustment for key covariates and could not be attributed to differential receipt of psychosocial or pharmacological therapy. Previous studies have showed the BDNF Val66Met polymorphism moderates the relationship between history of childhood adversity and depression outcomes (15, 35, 36). Our longitudinal results not only support these previous findings but also suggest the moderating effect conferred by the BDNF Val66Met polymorphism may not be stable over time. In fact, we showed the presence of the interaction between BDNF rs6265 and severe childhood abuse was dependent on the time at which it was assessed; suggesting time as a key factor when testing gene–environment interactions. The stability of gene–environment–depression interactions over time has only recently been described (37) but to our knowledge the current study is the first to demonstrate that these interactions may not be stable over time. Demonstrating the stability of a gene–environment interaction is critical to our conceptual and clinical understanding of depression and related outcomes. For example, at baseline, our analysis detected an interaction that is consistent with the differential susceptibility framework (3) in that Met allele carriers reported fewer depressive symptoms in the absence of severe childhood abuse and greater depressive symptoms in the presence of a history of severe childhood abuse, compared to Val/Val individuals who had similar depressive symptom severity regardless of their history of childhood abuse. However, over the course of 5 years, this interaction was gradually attenuated and support for the differential susceptibility framework was diminished. Furthermore, the presence of a time-dependent interaction may in part explain the sub-optimal reproducibility of BDNF by environment interactions in previous cross-sectional studies. Although other explanations for this interaction effect instability likely exist, we ruled out many of these explanations (e.g., therapy exposure) in our analyses. Nevertheless, further longitudinal examinations of BDNF by environment interactions are required to validate our findings. Potential Mechanism(s) The mechanism(s) by which the BDNF rs6265 polymorphism moderates the relationship between severe childhood abuse and depressive symptom severity in adulthood is unclear and is beyond the scope of this study. However, previous studies have shown BDNF gene expression and serum protein levels are decreased in depressed individuals compared to healthy controls (10–12) and are also decreased in individuals with the Met allele (13), albeit in healthy individuals Val/Val carriers had decreased BDNF levels (38). Nevertheless, there is modest evidence for a link between peripheral BDNF levels and depressive symptoms. Importantly, a number of studies have shown that serum BDNF levels increase with administration of antidepressant medication (39), which is negatively correlated with depressive symptom severity (10, 11). Additionally, Alder and Thakker-Varia (9) suggest that BDNF plays a role in neuronal plasticity, particularly emotional processing networks that are compromised in depression. However, further research into the effects of BDNF on neuroplasticity and neurodevelopment using post-mortem brain tissue and neuroimaging cohorts with childhood abuse exposure and depressive symptom data are needed before firm conclusions can be made on the moderating mechanisms by which BDNF genotypic variation acts. Strengths and Limitations This study has several notable strengths, including the longitudinal study design, comprehensive covariate adjustment, and centering of data. However, a number of limitations should be acknowledged. First, the assessment of environmental exposures and self-reporting of abuse is limited and subject to bias. Specifically, within the childhood abuse group, only those who had experienced severe childhood abuse either physical or sexual were included in analysis. Emotional abuse or neglect experienced during childhood has also been associated with depressive symptoms (40) yet were not considered in this study. Validity of self-reporting has also been questioned due to significant recall bias, often resulting in under-reporting of abuse (41) in both reporting stressful life events and recalling adverse childhood experiences (41, 42). Thus, our findings may represent a conservative estimate, which would dilute the interaction effect within the sample population and could contribute to negative results. Second, our measures of positive environmental exposure (e.g., positive life events) were sub-optimal and only capture adulthood experiences. Measures of “positive” exposures in childhood, such as maternal attachment or parental engagement, would be ideal but were not available. Third, BDNF serum levels were not measured in our study. Given that BDNF serum levels have been negatively correlated with depressive symptoms (39) and carriage of the Met allele (13), future study into BDNF genetic variation as a moderator of life events on depressive symptoms should include measurement of BDNF serum levels. Finally, rather than diagnosis of major depressive disorder, depressive symptom severity was the primary outcome measure limiting extrapolation and comparison to similar studies. However, the PHQ-9 scale consists of the nine diagnostic criteria outlined in DSM-IV and has been validated as a tool for assessing depressive symptom trajectories (20) and may translate better into the primary care setting. Conclusion We have identified a novel three-way interaction between BDNF genetic variation, a history of severe childhood abuse, and time that was associated with depressive symptom severity. This interaction highlights the dynamic nature of gene–environment–depression interactions and the importance of longitudinal assessment of these interactions. Discrepancies among previous study results could be associated with not just study design, but also time of analysis. Given this is the first longitudinal study of its kind; replication of results is needed. Author Contributions JG conceived and established the diamond cohort. CB, IE, and JG conceived the current study design and analysis. MP project managed the data collection. CW and CB conducted the analysis and wrote the first draft of the manuscript. All authors contributed to further drafts of the manuscript. All authors have read and approve the current version of the manuscript. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer CO declared a shared affiliation, though no collaboration or input in the work presented in this article, with the authors, to the handling Editor, who ensured that the process nevertheless met the standards of a fair and objective review. The diamond study is funded by the National Health and Medical Research Council (IDs 299869, 454463, 566511, and 1002908) and the Victorian Centre for Excellence in Depression and Related Disorders, an initiative between beyondblue and the Victorian Government. The collection of DNA and genotyping was funded by the LEW Carty Chartable Fund (ID 7284). No funding body had a role in the study design; the collection, analysis, and interpretation of data; or the writing of the manuscript for publication. We acknowledge the 30 dedicated GPs, their patients, and practice staff for making this research possible. We thank the diamond project team, including associate investigators and researchers involved in the diamond study: Ms. Aves Middleton, Ms. Konstancja Densley, Professor Helen Herrman, Professor Christopher Dowrick, Dr. Gursharan Chana, and casual research staff. Supplementary Material The Supplementary Material for this article can be found online at http://journal.frontiersin.org/article/10.3389/fpsyt.2016.00151 Click here for additional data file. ==== Refs References 1 Heim C Binder EB . Current research trends in early life stress and depression: review of human studies on sensitive periods, gene-environment interactions, and epigenetics . Exp Neurol (2012 ) 233 (1 ):102 –11 .10.1016/j.expneurol.2011.10.032 22101006 2 Monroe SM Simons AD . Diathesis-stress theories in the context of life stress research: implications for the depressive disorders . 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==== Front Health Promot PerspectHealth Promot PerspectHealth Promot PerspectTBZMEDHealth Promotion Perspectives2228-6497Tabriz University of Medical Sciences 10.15171/hpp.2016.19Review ArticleAssessing environmental assets for health promotion program planning: a practical framework for health promotion practitioners Springer Andrew E. 1 *Evans Alexandra E. 2 1Assistant Professor of Health Promotion and Behavioral Sciences, Michael & Susan Dell Center for Healthy Living, University of Texas Health Science Center at Houston School of Public Health, Austin, TX, USA2Associate Professor of Health Promotion and Behavioral Sciences, Michael & Susan Dell Center for Healthy Living, University of Texas Health Science Center at Houston School of Public Health, Austin, TX, USA* Corresponding Author: Andrew E. Springer; 1616 Guadalupe Street, Suite 6.300, Austin, TX 78701. Tell: (512)391-2523 Andrew.E.Springer@uth.tmc.edu2016 10 8 2016 6 3 111 118 01 7 2016 24 7 2016 © 2016 The Author(s).2016This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.http://journals.tbzmed.ac.ir/HPPConducting a health needs assessment is an important if not essential first step for health promotion planning. This paper explores how health needs assessments may be further strengthened for health promotion planning via an assessment of environmental assets rooted in the multiple environments (policy, information, social and physical environments) that shape health and behavior. Guided by a behavioral-ecological perspective- one that seeks to identify environmental assets that can influence health behavior, and an implementation science perspective- one that seeks to interweave health promotion strategies into existing environmental assets, we present a basic framework for assessing environmental assets and review examples from the literature to illustrate the incorporation of environmental assets into health program design. Health promotion practitioners and researchers implicitly identify and apply environmental assets in the design and implementation of health promotion interventions;this paper provides foundation for greater intentionality in assessing environmental assets for health promotion planning. Health needs assessmentProgram planningHealth promotionHealth behaviorEnvironmentEcological modelsCitation: Springer AE, Evans AE. Assessing environmental assets for health promotion program planning: a practical framework for health promotion practitioners. Health Promot Perspect. 2016;6(3):111-118. doi: 10.15171/hpp.2016.19. ==== Body Introduction In planning health promotion interventions, conducting a health needs assessment is an important if not essential first step.1-4 While the reasons for conducting a health needs assessment vary- from identifying and prioritizing health needs and population groups most at risk as part of a community health assessment (CHA) or community health needs assessment (CHNA),2-4 to designing a health intervention to address a specific health problem for a given population,1 to other reasons such as policy development, public health assurance (e.g., enforcement of sanitary codes), and community engagement,2,3 central to current health needs assessment approaches has been a focus on assessing both the health needs as well as the assets or resources of a given community. This combined focus on needs and assets is reflected in current definitions of CHAs and CHNAs2 and reflects the WHO Health Promotion Glossary’s general definition of health needs assessment as “a systematic procedure for determining the nature and extent of health needs in a population, the causes and contributing factors to those needs, and the human, organizational and community resources which are available to respond to these.”5 The incorporation of an asset assessment holds great potential to enhance the health needs assessment process as well as program efficacy and sustainability, and as such, merits increased attention in the field of health promotion planning. The movement toward an asset-based assessment approach was spearheaded in part by Kretzman and McKnight’s6 seminal work on asset mapping in the field of community organizing, which reframed community assessment from a deficit approach focused purely on needs of a community, to an approach and philosophy that promotes harnessing existing local resources and capacities for community development. An asset-based approach to community development includes identifying individual-level assets such as skills, talents and knowledge of people within a given community; organizational assets existing within the community such as community associations, local businesses, and religious organizations; organizational assets controlled from outside of the community, such as hospitals, schools and financial institutions; and physical resources such as land use.7 In the field of health promotion, a similar movement toward an asset-based approach took place during the same period of the 1990s, exemplified in part by a heightened focus on constructs such as community capacity,8 social capital,9 and developmental assets.10,11 Currently, the incorporation of an assessment of community assets – in addition to health needs - has become a standard best practice promoted by organizations such as the National Association of County and City Health Officials (NACCHO) and the Public Health Accreditation Board in the United States.3 Despite the movement toward the identification of community assets in the health needs assessment process, the application of an asset assessment approach guided by theory has received little attention in the health promotion literature to date. As health behavior is at the core of disease prevention and health promotion, comprising various types of behaviors that include health promoting behaviors (e.g., physical activity and fruit and vegetable consumption), risk behaviors (e.g., substance use), self-management behaviors (e.g., asthma management), and other preventive and compliance behaviors (e.g., screening, medical visits, and medication adherence),1 we posit that asset assessment can be further enhanced for health promotion planning by explicitly identifying assets rooted in environments and settings that hold potential to influence the ultimate targets of health behavior and health outcomes. With the overarching aim of contributing to the science and practice of health promotion asset assessment, the purpose of this paper is threefold: (1) to describe the rationale for balancing a needs assessment with an assessment of the assets of a priority population, their environments, and the settings that surround them; (2) to explore a basic environmental asset assessment framework guided by ecological theories of health behavior and principles from the field of implementation science; and (3) to illustrate the application of an environmental asset approach within health promotion planning using examples from the scientific literature and the health promotion practice field. Balancing the needs assessment with an assessment of assets: Why health asset assessment matters for health promotion Several reasons exist for incorporating an assessment of assets and capacities of the priority population and their environments, including the importance of a community empowerment vs. needs-based approach, the opportunity to enhance intervention effectiveness, and the potential to increase implementation and sustainability of health promotion interventions. In the following section we explore why the incorporation of an asset assessment approach in the health needs assessment process is essential and merits heightened emphasis in CHA and intervention planning. Asset assessment for community empowerment In following the credo in the medical field of “first, do no harm”, in the field of health promotion practice, we must also take caution against victimizing a priority population or community by focusing solely on the multiple problems and deficits that confront them. McKnight and Kretzman7 caution against the potentially adverse consequences of needs-based solutions to community development in which low-income neighborhoods may become environments of services where “…residents come to believe that their well-being depends upon being a client.” Given that all populations and communities have strengths and assets, health promotion program planners are in a unique position to co-learn and plan with communities to harness and activate these capacities and assets for health promotion planning and intervention. Asset assessment for enhancing intervention effectiveness Beyond the philosophical shift of an asset assessment approach, identifying the assets and capacities in a given community holds potential to broaden our understanding of potential factors that influence and promote positive health outcomes. An asset-based approach to program planning builds from the strengths of individuals, communities and their environments and is supported by theoretical-based concepts of positive deviance12 and resiliency,13 which focus on uncovering the factors associated with “positive” health and development, and not just the factors associated with the problem. Positive social interpersonal relationships and social cohesion14,15 are examples of positive assets that hold potential for reducing exposure to youth risk behavior. Asset assessment for implementation and sustainability An important focus of the growing field of implementation research is on understanding the context in which health interventions are delivered,16 which includes understanding how an intervention “couples” with the intended setting in order to increase the likelihood that programs will be effectively implemented and “stick” with the setting over time.17,18 Balancing the needs assessment with an assessment of a community’s assets – with specific attention to uncovering factors that can support and deliver an intervention within a given context or setting - holds potential to enhance a health program’s implementation and sustainability by allowing for the identification of potential opportunities to couple or weave intervention efforts into existing settings – including organizations and communities. Exploring a basic environmental asset assessment framework for health promotion planning Several definitions and conceptualizations have been cited in the literature to describe the general concept of a community asset, which is often used synonymously with the term resource. In the field of community organizing, Kretzman and colleagues19 propose five key types of community assets: local residents’ skills, passions, capacities, and willingness to contribute to a given project; local voluntary organizations, clubs and networks; local institutions such as schools and businesses; physical assets such as land and infrastructure; and economic assets. In the context of healthy adolescent development, the Search Institute defines assets as “…important relationships, skills, opportunities and values that help guide adolescents away from risk behaviors, foster resilience, and promote thriving”, with a framework that includes both internal assets and external assets.10 In the field of CHNA, The Community Tool Box20 provides a robust definition of a community asset as “…anything that can be used to improve the quality of community life”, which may range from individuals, to physical structures, to community services such as public transportation. Applying ecological theory to asset assessment for health promotion planning While the definitions of assets listed above share similarities, their differences underscore how asset assessment is often bound and shaped by the field for which it is being applied. In identifying assets for the specific purpose of health promotion planning and health behavior change, ecological models of health behavior provide a robust framework.21-23 Ecological models of behavior stem from the premise that individuals are a ‘product of their environment,’ and that environments hold the potential to directly and indirectly shape individuals’ health and health behavior,22 with pathways of influence that include social norms, social comparison and role models, social support, and other forms of social influence; opportunities or barriers to engage in a given behavior; information transfer; and incentive motivation via rewards or punishment.21-27 Central to ecological models of health behavior are the principles of: multiple levels of influence (e.g., societal, organizational, interpersonal and individual) and multiple environments (e.g., policy, information, social, physical) that shape behavior; behavioral settings - such as schools, workplaces, and church - which can serve to both reach populations and influence behavior; and interactions of influence, which refers to the interaction between and among levels or environments that may enhance or inhibit a given health outcome.22,27 These principles direct our attention to identifying the facets of environments and settings that can be harnessed for influencing health and health behavior. In defining the concept of environment, we adhere to Albert Einstein’s broad conceptualization of environment as ‘everything that isn’t me’.28 In following such a broad definition, we open up the environmental space for further conceptualization and identification of assets “outside the individual” that may be harnessed toward health promotion. Although there is great potential to further conceptualize environment, we build from existing social-ecological theory22,27 and propose an initial framework for asset assessment guided by four key environments: policy environment, information environment, social/organizational environment, and the physical environment. The Policy Environment: While it is common to separate “policy” from “environment” (e.g., “policy and environmental interventions”), we share Sallis et al’s27 conceptualization of policy as a representation of a specific kind of environment. By stating “policy environment”, we anchor policy to an environmental space, which may include a household setting (e.g., a family rule on TV watching), a classroom (e.g., classroom policy for earning more recess time for good behavior), a school (e.g., policy on only serving water at school events), a school district (e.g., daily PE class), or a state or nation (e.g., 30 minutes of physical activity a day for school children). Assessing existing policies and practices within different settings at the asset assessment phase allows for uncovering gaps in policy and practice, identifying policies that may adversely affect health, identifying existing policies that may not be fully implemented yet may provide an anchor for proposed health program efforts, and identifying existing policies in which a health focus can be interwoven, building off the “health in all policies” approach.29 The Information Environment: The information environment broadly refers to the messaging within a given setting that holds potential to positively or negatively influence health or health behavior. This messaging can take many forms (e.g., verbal, nonverbal, written, symbolic) and be delivered via diverse communication channels (e.g., posters, newsletter, art, social media, marketing). Numerous examples of how the information environment can influence health and behavior are provided in the literature, ranging from national campaigns such as the VERB campaign and its influence on physical activity in middle school students,30 to mass media campaigns for child survival,31 to the role of menu labeling and promotion of healthier eating,32 among others. As we discuss below, an important aspect of asset assessment is the exploration and identification of existing channels of communication (e.g., office newsletter, school marquee, church bulletin board) that can be incorporated into health intervention design. The Social, Cultural & Organizational Environment: This environment broadly refers to the types of social and cultural organization that exist within a given setting (e.g., neighborhood organizations, parent-teacher associations, cultural centers, etc.) as well as the specific types of social, cultural or organizational factors that relate to health and health behavior (e.g., family meals and their effect on childhood obesity33; social support and physical activity in adolescents34). In our asset assessment phase with the CATCH Middle School Program,35 for example, we learned of existing social organizational activities, such as the literacy night in one school district, which then became the platform for incorporating a CATCH family health night. Asset assessment should include identifying the social organizations that exist within a given setting or community along with specific social, cultural and organizational factors and activities that can be incorporated into health promotion program planning. The Physical Environment: Moos,36 in his work on social ecology, defines the physical environment as encompassing both features of the built environment (e.g., how we construct our buildings or neighborhoods) and features of the natural environment (e.g., green space). In recent years, we have seen an explosion of research on facets of the built environment in topic areas that range from physical activity,37 to the food environment,38 to mental health,39 among others. Beyond actual ‘space’, the built environment may also encompass aspects such as children’s access to physical activity equipment40 and access to outlets that sell fruits and vegetables.41,42 The natural environment includes access to green space- with emerging evidence that includes the positive effects of outdoors on physical activity and reduction of stress and depression.43,44 An assessment of the physical environment holds potential to expand intervention opportunities for a given health problem. Toward a conceptualization of environmental assets for health promotion With guidance from ecological models of health behavior21-23 and the premise that individuals and their behavior are shaped by their environment, we propose a refined conceptualization of community asset rooted in the construct of environmental asset. The focus on environment aims to direct health planners to an intentional assessment of assets rooted in the multiple environments (e.g., policy, social, information and physical) and settings (e.g., home, school, workplace, and neighborhood) that hold potential to shape health and health behavior. In building from previous definitions of a community asset,20 we define an environmental asset as any aspect of the multiple environments that surround individuals that can be harnessed toward promoting the health of individuals and populations. In Figure 1, we present a basic framework for conducting an environmental asset assessment for a health promotion and health behavior change intervention, guided by ecological models of health behavior. This framework begins with first identifying the settings where priority populations can be reached, which may include neighborhoods, schools, afterschool programs, and worksites, among other settings. The second step involves exploring the specific environmental assets within those settings that are posited to influence behavior, including the policy environment, the information environment, the social/cultural/organizational environment, and the physical environment. We offer this framework not as a recipe for environmental asset assessment, but rather as a practical and theoretical framework to complement existing health needs assessment approaches and for program planners and researchers to build from, modify, and enhance. As a brief example, we share findings in Figure 1 from a recent asset assessment workshop with afterschool program leaders (n=24) conducted as part of the Central Texas Afterschool Network BOOST Initiative, an initiative funded by the St. David’s Foundation aimed at enhancing child health in out-of-school-time programs.45 Figure 1 Basic environmental asset assessment framework for health promotion planning. Applying environmental assets to health promotion In applying an environmental asset assessment approach to the field of health promotion, we take heart in the saying ‘old wine in new bottles’, as the general environmental asset assessment framework we propose represents a common practice health promotion practitioners and researchers have long embraced. An early and brilliant example in the field of public health of using environmental assets is the fortification of salt with iodine, in which a natural environmental asset (salt) was harnessed for delivering a health intervention (iodine) to large masses of people, and thus reducing iodine-deficiency-related disease.46 In the section below, we illustrate how health promotion practitioners and researchers have incorporated existing environmental assets into the design of health promotion programs and interventions. In doing so, we aim to highlight how the basic environmental asset framework described above can provide a new bottle of wine lens through which program planners can enhance asset assessment with greater intentionality for health promotion intervention design. Policy environment School schedules as an environmental asset for physical activity: In our formative work with the CATCH Middle School Project,35 we learned that middle school students were being dropped off at school in the morning and being directed to sit and wait in the cafeteria. Recognizing this opportunity to incorporate more physical activity opportunities, we partnered with school staff and principals to create an “Open Gym” policy/school practice in which schools opened their gyms and/or playfields for a free-play activity time with teacher supervision. Campus Improvement Plans as a vehicle for child health policy: The nonprofit organization Texas Action for Healthy Kids worked with central Texas middle schools to take advantage of the Campus Improvement Plan, a document that describes the goals, practices and activities of a given campus for enhancing the student educational experience, to interweave child health policy. Campus Improvement Plans of schools participating in the initiative resulted in increased written policy language related to coordinated school health, such as scheduling of structured activity time.35 Information environment Electric bills and physical activity in Brazil. In Sao Paulo, Brazil, the Agita Sao Paulo Program delivered physical activity messaging via an existing communication channel with widespread reach: residents’ electric bills.47 According to the authors, this approach required no funding from the program and reached 7 million residents. Soap operas and HIV prevention. Soap operas and other entertainment media represent a powerful communication channel for interweaving health messaging, with a growing body of evidence on their effectiveness in promoting health-related knowledge, attitudes, intentions and behavior.48 In an episode of the soap opera The Bold and the Beautiful, the insertion of an HIV/AIDS subplot along with displaying a national AIDS and STD hotline resulted in dramatic increases in hotline calls.49 2-1-1 Information System and Community Health Promotion. In the United States, the 2-1-1 system is a 3-digit phone number designated by the Federal Communications Commission as a free information resource to connect callers with health and social services in their community.50 An emerging body of research provides evidence on the application of this community information resource as a promising communication channel for increasing health screening and delivery of health interventions.50-52 Social/cultural/organizational environment Harnessing cafeteria workers for fruit and vegetable consumption. In addition to an array of other environmental strategies, the 5-A-Day Cafeteria Power Plus project harnessed an existing school social environmental asset to positively encourage fruit and vegetable consumption in elementary school children: cafeteria workers working on the serving line.53 Verbal encouragement from cafeteria staff was found to be associated with increased child fruit and vegetable intake.53 Cultural organizations as a vehicle for healthy lifestyle promotion in Filipino-Americans. Nutrition and physical activity were promoted via Filipino-American social clubs in San Diego, California by forming health committees and training 2-3 members of each social club in health education, behavior change skills development, and organizational policy change.54 The 18-month intervention resulted in significant increases in physical activity and selected dietary outcomes among study participants.54 Physical environment WIC clinics, farm stands and family fruit and vegetable promotion. In order to increase access to fresh produce in central Texas, produce stands were placed at Women, Infants, and Children (WIC) clinics located in food desert type communities, which resulted in greater fruit and vegetable consumption among WIC recipients and residents living within a half mile of the farm stand.55 Schoolyards and physical activity. In exploring opportunities to support children’s physical activity in low income communities in New Orleans, Louisiana, Farley and colleagues56 took advantage of an existing built environmental asset: schoolyards that were locked after school. With limited resources, schoolyards were activated for children’s afterschool play by incorporating adult supervisors and a parent permission process.56 School design and healthy eating. A recent review by Frerichs et al57 provides evidence for the influence of school design on healthy eating, which includes quasi-experimental evidence that increased access to healthy items and decreased access to unhealthy items (e.g., access to healthier foods in vending machines and healthier foods on cafeteria serving lines) improves student dietary behaviors. Barbershops as settings for health promotion. A systematic review of the literature shows that beauty salons and barbershops are both feasible and effective settings for health promotion, with topics that include cancer screening, hypertension, and diabetes.58 Salons and barbershops are an example of a physical setting with wide reach of specific subgroups. Discussion Beyond the multiple benefits of conducting a CHNA for planning interventions aimed at advancing a population’s health, conducting a CHNA has received heightened attention in recent years in countries such as the United States, where current health policy under the Patient Protection and Affordable Care Act now requires federally funded hospitals to conduct a CHNA every three years.59 In this paper, we provide a basic conceptual framework for enhancing the health needs assessment process for health promotion planning via an assessment of environmental assets (e.g., policy, information, social, and physical environment) of a given community, organization or setting. A strength of this paper is the application of ecological theory of health behavior and principles of implementation science for identifying environmental assets that hold potential to enhance the design, implementation and sustainability of interventions directed at health behavior change. In providing the basic conceptual framework for environmental asset assessment described in this paper, we recognize that there are other promising frameworks that hold value in guiding asset assessment for different fields and different purposes. For example, Green andHaines60 conceptualize asset assessment for community development in terms of seven types of capital: human, social, physical, financial, environmental, political and cultural capital. As we describe in this paper, we posit that asset assessment can be enhanced via the lens of the field for which it is being applied. The basic environmental asset framework proposed in this paper for the field of health promotion planning differs from others by specifically applying both a behavioral-ecological perspective- one that seeks to identify environmental assets that can shape behavior, and an implementation science perspective- one that seeks to ‘couple’ and ‘interweave’ health promotion intervention strategies into existing environmental assets. This approach aims to increase health promotion intervention effectiveness while increasing implementation and sustainability of health promotion intervention initiatives. Although we describe in this paper various examples of how environmental assets have been applied in health promotion research and practice, we recognize that the conceptual framework proposed here is basic, theory-based, and merits further empirical evaluation. This limitation notwithstanding, we hope that this paper serves as a catalyst to continue to grow the science and practice around environmental asset assessment for health promotion planning. Conclusion and future directions While many health promotion practitioners and researchers implicitly identify and apply environmental assets in the design and implementation of health promotion interventions, this paper provides a foundation for greater intentionality in assessing environmental assets that hold potential to directly shape health and health behavior. As described herein, the concept of environmental asset assessment holds great potential for furthering the field of health needs assessment. Future directions of this work include the development of a common vocabulary and constructs, further conceptualization of additional ‘environments’ important for health promotion- such as the arts and aesthetic environment (see Semenza and Krishnasamy61 for inspiring examples), identification of methods, and attention to process for conducting an environmental asset assessment. Acknowledgements We gratefully acknowledge Dr. Chris Markham and Dr. Kay Bartholomew, lead editors and authors of Planning health promotion programs: An Intervention Mapping Approach, who provided initial encouragement for this paper. We also express our gratitude to Dr. Cheryl Perry and Dr. Chris Markham from the Department of Health Promotion and Behavioral Sciences at the University of Texas Health Science Center at Houston (UTHealth) School of Public Health for their insightful and constructive comments of earlier drafts. Lastly, we greatly value the editorial and research support provided by Ms. Sarah Bentley, Graduate Research Assistant, and Mr. Tim Cooley, Dell Undergraduate Scholar, at the Michael & Susan Dell Center for Healthy Living at the University of Texas School of Public Health-Austin Regional Campus. Funding This paper was supported in part by a community health grant from the Michael & Susan Dell Foundation, which provides funding for the Michael & Susan Dell Center for Healthy Living/UTHealth School of Public Health-Austin where the authors are based. Ethics approval This paper is based primarily on a review of existing literature and does not report any individual data. The example cited on our work with the CTAN BOOST Initiative is based on study protocols for that project that were reviewed and approved by the University of Texas Health Science Center Committee for the Protection of Human Subjects (IRB #HSC-SPH-13-0190). Competing interests The authors formally declare that we have no financial interest in the research or conflict of interest. Authors’ contributions AS and AE jointly conceived this paper and prepared and edited the manuscript. ==== Refs References 1 Bartholomew Eldredge LK, Markham CM, Ruiter RA, Fernández ME, Kok G, Parcel GS, editors. Planning health promotion programs: An Intervention Mapping approach. 4th ed. San Francisco, CA: Jossey-Bass; 2016. 2 U.S. Centers for Disease Control and Prevention. Community Health Assessment for Population Health Improvement: Resource of Most Frequently Recommended Health Outcomes and Determinants. Atlanta, GA: Office of Surveillance, Epidemiology, and Laboratory Services; 2013. 3 National Association of County and City Health Officials (NACCHO). Community Health Assessment and Improvement Planning. http://www.naccho.org/programs/public-health-infrastructure/community-health-assessment. 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==== Front Health Promot PerspectHealth Promot PerspectHealth Promot PerspectTBZMEDHealth Promotion Perspectives2228-6497Tabriz University of Medical Sciences 10.15171/hpp.2016.20Systematic ReviewAn assessment of maternal, newborn and child health implementation studies in Nigeria: implications for evidence informed policymaking and practice Uneke Chigozie Jesse 1 *Sombie Issiaka 2 Keita Namoudou 2 Lokossou Virgil 2 Johnson Ermel 2 Ongolo-Zogo Pierre 3 1Knowledge Translation Platform, African Institute for Health Policy & Health Systems Studies, Ebonyi State University, PMB 053 Abakaliki Nigeria2Organisation Ouest Africaine de la Santé, 175, avenue Ouezzin Coulibaly, 01 BP 153 Bobo-Dioulasso 01, Burkina Faso3Hospital Central Yaounde, CDBPH Lawrence VERGNE Building 2nd Floor, Avenue Henry Dunant, Messa, Yaoundé, Cameroon* Corresponding Author: Chigozie Jesse Uneke; Tel: +234 08038928597; unekecj@yahoo.com2016 10 8 2016 6 3 119 127 13 5 2016 27 6 2016 © 2016 The Author(s).2016This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.http://journals.tbzmed.ac.ir/HPP Background: The introduction of implementation science into maternal, newborn and child health (MNCH) research has facilitated better methods to improve uptake of research findings into practices. With increase in implementation research related to MNCH world-wide, stronger scientific evidence are now available and have improved MNCH policies in many countries including Nigeria. The purpose of this study was to review MNCH implementation studies undertaken in Nigeria in order to understand the extent the evidence generated informed better policy. Methods: This study was a systematic review. A MEDLINE Entrez PubMed search was performed in August 2015 and implementation studies that investigated MNCH in Nigeria from 1966 to 2015 in relation to health policy were sought. Search key words included Nigeria, health policy, maternal, newborn, and child health. Only policy relevant studies that were implementation or intervention research which generated evidence to improve MNCH in Nigeria were eligible and were selected. Results: A total of 18 relevant studies that fulfilled the study inclusion criteria were identified out of 471 studies found. These studies generated high quality policy relevance evidence relating to task shifting, breastfeeding practices, maternal nutrition, childhood immunization, kangaroo mother care (KMC), prevention of maternal to child transmission of HIV, etc. These indicated significant improvements in maternal health outcomes in localities and health facilities where the studies were undertaken. Conclusion: There is a dire need for more implementation research related to MNCH in low income settings because the priority for improved MNCH outcome is not so much the development of new technologies but solving implementation issues, such as how to scale up and evaluate interventions within complex health systems. MaternalNewbornChildImplementation studiesEvidence informedPolicymakingCitation: Uneke CJ, Sombie I, Keita N, Lokossou V, Johnson E, Ongolo-Zogo P. An assessment of maternal, newborn and child health implementation studies in Nigeria: implications for evidence informed policymaking and practice. Health Promot Perspect. 2016;6(3):119-127. doi: 10.15171/hpp.2016.20. ==== Body Introduction With the introduction of implementation science into maternal, newborn and child health (MNCH) research, better methods to improve the uptake, implementation, and translation of research findings into routine and common practices have evolved.1-4 Findings from sufficient number of studies have indicated that research evidence can enhance health policy process and development by informing decisions about policy content and direction and evaluating the impact of policy.5-8 The World Health Organization (WHO), noted that better use of research evidence in development policy making can save lives through more effective policies that respond to scientific and technological advances.9 However, according to Peters and colleagues,10 one of the greatest challenges facing the global health community is how to take proven interventions and implement them in the real world. They therefore noted that implementation research, is crucial to meeting that challenge, providing a basis for the context-specific, evidence-informed decision-making needed to make what is possible in theory a reality in practice.10 In a recent USAIDS report, it was noted that implementation science is the use of strategies to adopt, adapt, and integrate evidence-based health interventions and policies, changing practice patterns within specific settings.11 Implementation research on MNCH therefore, has the potential of generating high quality research evidence that can improve MNCH policy and practice globally. This is why Whitworth and colleagues2 argued that the priority for maternal and child survival is not so much the development of new technologies but solving implementation issues. According to them, such implementation research should not only focus the attention of policy makers and implementers, but also improve decision making.2 Although implementation research is a relatively new and somewhat neglected field, interest in it is growing, largely in recognition of the contribution it can make to maximizing the beneficial impact of health interventions.10 With the increase in implementation research related to MNCH world-wide, stronger scientific evidence are now available and have helped to improved MNCH policies in many countries. Consequently, the total number of maternal deaths worldwide has reportedly dropped by a third within the last decade, although there is yet to be any significant reduction in maternal mortality in most low- and middle-income countries (LMICs).12 In Nigeria, with a population of over 160 million and weak health systems, health outcomes especially those related to maternal and child health remains poor. With approximately 2.5% of the world’s population, the country is reportedly having more than 10% of all under-5 and maternal deaths – more than 1 million newborn, infant, and child deaths and more than 50 000 maternal deaths every year.13-15 However, there has been some level of reduction in maternal and child mortality with the last few years. The national maternal mortality rate (MMR) reduced from 800/100 000 in 200516,17 to 545/100 000 in 200818 and to 110/100 000 according to the recent Nigeria Demographic and Health Survey (NDHS) 2013.19 The 2008 NDHS reported an under five mortality rate (U5MR) of 157 deaths per 1000 live births, suggesting a 22% decline from the NDHS report of 2003 which had shown an U5MR of 201 per 1000 live births.18,20 According to the World Bank recent report, the Nigeria U5MR further declined to 117 per 1000 live births in 2013.21 Although this reduction in maternal and child mortality in Nigeria could be attributed to implementation of various intervention policies, it is however not very clear as to what extent the policies were informed by research evidence from implementation research. Some implementation research undertaken have attempted to evaluate policy recommendation regarding MNCH to generate more evidence to either sustain or revise existing policy, while others have attempted to implement new recommendations to generate more evidence that will translate into MNCH policies. Till date no attempt has been made to review these implementation studies in order to understand the extent the evidence generated informed better health policy. The objectives of this study are as follows: first, to gain a better understanding of how implementation research has demonstrated the efficacy of available interventions in Nigeria warranting scaling-up, secondly, to review the various MNCH health issue of intervention assessed using implementation research and their implication for policy and practice. Materials and Methods A MEDLINE Entrez PubMed search was performed in August 2015 and studies published in English that investigated maternal and child health in Nigeria in relation to health policy were sought. We used mainly the PubMed database for extraction of relevant publications principally because in Nigeria and possibly world-wide, studies indexed in PubMed are regarded to have undergone highest form of peer review process and are hence regarded as scientifically reliable evidence. We searched the PubMed for studies from Nigeria undertaken from 1966 to 2015. The following were the categories and search strategies/key words used and the publications yielded. Category 1: Nigeria, health policy, maternal health= 146 publications; Category 2: Nigeria, health policy, newborn health= 76 publications; Category 3: Nigeria, health policy, child health= 249 publications. No software was employed for the search; however the references of all the resulting publications were hand searched for additional studies and information relevant to the review. Publications that did not completely fulfill the study inclusion criteria but adjudged by the authors to contain vital information necessary for narrative aspect of the review were selected and used accordingly. The principal author performed independent data extraction using the predetermined review criteria while two other authors validated extracted data. Authors were satisfied with amount and quality of available information from selected publications and needed not to contact their respective authors/investigators for data confirmation or additional information. All the publications in the three categories were subjected to the following study inclusion criteria: (i) Must be an implementation or intervention research conducted in Nigeria; (ii) Must either have reported the implementation of an intervention to generate evidence to improve MNCH or evaluated an interventions on MNCH already operational in Nigeria; (iii) Must have produced evidence that is relevant to MNCH policy; Of the 146 publication yielded in category 1, 8 (5.5%)22-29 fulfilled the study inclusion criteria regarding maternal health and were selected (Table 1). Out of the 76 publications yielded in category 2, 7(9.2%)30-36 satisfied the study inclusion criteria regarding newborn health and were selected (Table 2), while of the 249 publication from category 3, only 3 (1.2%)37-39 met the study inclusion criteria regarding child health and were selected (Table 3). The flowchart of the study selection procedure is shown in Figure 1. The selected publications were then grouped according to the following: Author/year of publication; Category of intervention; Health issue of intervention; Evidence-generated; Policy relevant conclusion. Table 1 Profile and characteristics of scientific publications reporting the implementation of an intervention to generate evidence to improve maternal health or evaluation of interventions on maternal health already operational in Nigeria Author/year of publication/reference Category of intervention Health issue of intervention Evidence-generated Implication for policy and practice Deller et al 201522 Evaluated MCNH intervention already operational Task shifting in maternal and newborn health care Key components that facilitate effective task shifting including policy & regulatory support, education & training, service delivery support & referral systems Task shifting should be considered as a part of the larger health system that needs to be designed to equitably meet the needs of mothers, newborns, children, and families. Oseji and Ogu 201423 Evaluated MCNH intervention already operational Contributions of professional bodies & stakeholders in implementing community-based interventions for MNCH Reduction of maternal mortality through advocacy, awareness creation, and sensitisation programmes Health stakeholders impacted positively in the quest for reduction of maternal mortality. Findley et al 201524 Implemented an intervention to generate evidence to improve MCNH Integrated MNCH program to improve maternal health outcomes Integrated MNCH program increased health care utilization Significant improvements in communities show importance of integrated approach blending supply- and demand-side IMNCH interventions. Senbanjo et al 201425 Evaluated MCNH intervention already operational Breastfeeding policy and practices Breastfeeding practices and policy implementation were suboptimal There is need for interventions to increase knowledge of the benefits of breastfeeding and to provide support for its longer term duration. Findley et al 201326 Implemented an intervention to generate evidence to improve MCNH Integrated MNCH program to improve maternal health outcomes Infant and child mortality declined greatest in intervention communities. Participatory and community-based interventions focusing on improved newborn and infant care were effective at improving outcomes in intervention communities Girard et al 201227 Evaluated MCNH intervention already operational Maternal nutrition policy and programming Perceived weak advocacy for nutrition and its role in economic development and the lack of coordination among actors contribute to low prioritization Advocacy for maternal nutrition could hasten prioritization, coordination, and investment in maternal nutrition Erim et al 201228 Evaluated MCNH intervention already operational Use of obstetric care in healthcare facilities Most of the primary healthcare facilities were unable to provide all basic Emergency Obstetric Care (bEmOC) services Reducing maternal deaths will require attention to increasing the number of facilities with high-quality EmOC capability Prata et al 201229 Implemented an intervention to generate evidence to improve MCNH Community mobilization to reduce postpartum hemorrhage in home births High level community participation in health care interventions reduce postpartum hemorrhage in home births Community mobilization can have a significant impact on the successful distribution and uptake of a potentially life-saving health intervention Table 2 Profile and characteristics of scientific publications reporting the implementation of an intervention to generate evidence to improve newborn health or evaluation of interventions on newborn health already operational in Nigeria Author/year of publication/reference Category of intervention Health issue of intervention Evidence-generated Implication for policy and practice Ado et al 201430 Evaluated MCNH intervention already operational National emergency action plan to eradicate polio Significant improvements in the management of polio eradication initiative (PEI) activities with marked improvement in the quality of supplemental immunization activities (SIAs), as measured by lot quality assurance sampling (LQAS). Sustained improvement in SIA quality, surveillance, and outbreak response and special strategies in security-compromised areas are needed to interrupt wild poliovirus (WPV) transmission Ogbolu et al 201331 Evaluated MCNH intervention already operational Prevention of Maternal to Child Transmission of HIV Key PMTCT practices are not being adequately translated from research into practice Strategies derived from an implementation science perspective are suggested as a means to improve the translation of PMTCT research into practice Adegboye et al 201432 Evaluated MCNH intervention already operational Childhood immunization uptake and coverage Statistical significance of the community-survey year interaction term suggests an increase in the odds of a child being immunized over the years and spread over communities Evidence-based policy should lay more emphasis on mother- and community-level risk factors in order to increase immunization coverage Okonofua et al 201133 Evaluated MCNH intervention already operational Advocacy program implementing a policy of free maternal and child health (MCH) services Advocacy consisted of public presentation on MCH to high-level policymakers, dissemination of situational analysis report, and media publicity Advocacy and public health education is effective in increasing the commitment of policymakers to provide resources for implementing evidence-based maternal and child health services Okafor et al 200934 Implemented an intervention to generate evidence to improve MCNH Risk factors for perinatal mortality associated with anaesthesia for caesarean delivery in patients with pre-eclampsia/eclampsia Pre-eclampsia/eclampsia continues to be a cause of foetal loss even where essential obstetric services are available. Early onset management of severe pre-eclampsia with maintenance of adequate placental perfusion during anaesthesia may result in lower perinatal deaths. Nwogu et al 200835 Evaluated MCNH intervention already operational Outcome of the Expanded Program on Immunization (EPI) Results showed that the EPI program had little effect on under-five mortality rate (UFMR) Nigeria needs a unified approach to healthcare delivery, rather than fragmented programs, to overcome cultural and political divisions in society Ibe et al 200436 Implemented an intervention to generate evidence to improve MCNH Comparison of kangaroo mother care (KMC) and conventional incubator care (CC) for thermal regulation of infants <2000 g Mothers felt that KMC was safe, and preferred the method to CC because it did not separate them from their infants Where equipment for thermal regulation is lacking or unreliable, KMC is a preferable method for managing stable low birthweight infants. Table 3 Profile and characteristics of scientific publications reporting the implementation of an intervention to generate evidence to improve child health or evaluation of interventions on child health already operational in Nigeria Author/year of publication/reference Category of intervention Health issue of intervention Evidence-generated Implication for policy and practice Rowe et al 201237 Evaluated MCNH intervention already operational Health worker performance after implementing the Integrated Management of Childhood Illness strategy Performance trends were essentially flat for nearly all outcomes and absolute levels of performance revealed substantial performance gaps. No evidence that performance declined over 3 years after IMCI training. However, important performance gaps found immediately after IMCI training persisted and should be addressed. Mafe et al 200538 Implemented an intervention to generate evidence to improve MCNH Mass delivery of praziquantel among school-aged children in rural communities Community channel of delivery achieved the best coverage compared to the health facility and school channels Community channel of praziquantel delivery ensures good coverage of both in and out-of-school children. Omotade et al 200039 Evaluated MCNH intervention already operational Treatment regimens for acute diarrhoea in children Not all types of diarrhoea were recognized as illnesses, and only those considered to be illnesses were treated The need to adapt health policy and programmes to cultural norms should be addressed to improve the impact of programmes Figure 1 Flowchart of publication identification and selection process. Results Regarding maternal health, five identified scientific publications22-25,27,28 reported the evaluation of interventions on maternal health already operational in Nigeria while three24,26,29 reported the implemented an intervention to generate evidence to improve maternal health (Table 1). The evaluated MCNH intervention already operational included task shifting in maternal and newborn health care; contributions of professional bodies and stakeholders in implementing community-based interventions. Others included breast feeding policy and practices; maternal nutrition policy and programming and use of obstetric care in healthcare facilities. The implemented intervention to generate evidence to improve maternal health outcomes included integrated MNCH, and community mobilization to reduce postpartum hemorrhage in home births (Table 1). These studies generated high quality policy relevance evidence which indicated significant improvements in maternal health outcomes where the studies were undertaken. Table 2 presents five identified scientific publications30-33,35 reporting the evaluation of interventions on newborn health already operational in Nigeria. There were also two publications34,36 that implemented an intervention to generate evidence to improve newborn health. The existing interventions evaluated included: national emergency action plan to eradicate polio and prevention of maternal to child transmission of HIV. Others included childhood immunization uptake/coverage; and free maternal/child health services. The implemented intervention to generate evidence to improve newborn health outcomes included, early onset management of severe preeclampsia with maintenance of adequate placental perfusion; and comparison of kangaroo mother care (KMC) and conventional incubator care (CC) for thermal regulation of infants < 2000 g (Table 2). The results of these studies indicated that the interventions gave rise to significant improvements in newborn health outcomes. Of the three scientific publications reporting interventions to improve child health in Nigeria, two37,39 evaluated intervention programmes already operational while one38 implemented an intervention to generate policy relevant evidence (Table 3). The existing interventions evaluated included health worker performance after implementing the integrated management of childhood illness strategy and treatment regimens for acute diarrhea in children. The implemented intervention to generate evidence to improve child health outcome was on mass delivery of praziquantel among school-aged children. These studies generated high quality policy relevance evidence which indicated significant improvements in child health outcomes (Table 3). Discussion This review was designed to provide more insight into the process of evidence-informed policymaking and knowledge transfer/exchange based on implementation research regarding MNCH in Nigeria. The findings of this review showed that implementation research still remains one of the most effective processes of demonstrating the efficacy of available or new health interventions in order to improve access to, and the use of, these interventions to improve MNCH. All the studies reviewed reported tremendous improvements in maternal and child health outcomes as a result of the interventions implemented. There is therefore the need for scaling up of the intervention at country level. In recent times there is a world-wide consensus regarding the dire need for scaling up life-saving interventions as a way to reduce maternal and infant mortality and morbidity.2,4 Implementation research is therefore gaining global attention due to the large underuse of several life-saving interventions in LMICs.10 Because scaling up proven MNCH interventions are inevitable, this review makes a case for more implementation studies that will identify and address implementation bottle-necks and the major barriers that hamper access to interventions. Out of the combined total of 471 publications found from the MEDLINE search related to MNCH and policy in Nigeria, only 18 (3.8%) fulfilled the inclusion criteria and found to be implementation research with policy relevant outcomes. The implication of this finding suggests that inadequate number of implementation research relevant to MNCH policy has been undertaken in Nigeria. According to Averting Maternal Death and Disability Program (AMDD),40 one reason for insufficient MNCH implementation research in LMICs is because it is still relatively new to the field of public health. Even though it offers multiple approaches to building the more detailed and specific evidence base needed to answer critical questions about how to promote equitable access to maternal healthcare.40 Peters and colleagues10 added that implementation research, continues to be a neglected field of study, partly because of a lack of understanding regarding what it is. They also noted that it is due to a lack of investment in it.10 In Nigeria, there is grossly inadequate funding support for implementation research and this appears to be a common scenario in most LMICs. In a recent report, Dean and colleague41 noted that although a substantial proportion of maternal and child deaths in LMICs are preventable, progress in reducing these deaths is far too slow due to the bias that remains in health care and research investment. Dean and colleagues41 further cited an instance where 7.6 million children died worldwide in 2010 which is equivalent to global deaths due to cancer and slightly higher than deaths due to heart disease.42,43 Yet funding favors breakthrough research for cancer and heart disease, while implementation research and delivery for maternal/child health is sidelined.42,43 It is of interest to note from this review that of the 18 selected studies, 12 (66.7%) were published between years 2012-2015. The finding suggests that there is increasing interest in implementation research related to MNCH in Nigeria within the last few years. This is consistent with what is obtainable in parts of Africa and other LMICs as attested by some recent studies which proved that there is increasing recognition of the importance and value of implementation research to improve MNCH policy and practice.4,44-47 According to WHO,48 implementation research is increasingly being recognized as one of the most important interfaces between the availability of tools, strategies and interventions and their use within health systems and control programmes. This is because limited uptake of research findings and innovations in real-world settings has led to mounting interest in implementation research for public health.48 Furthermore, in a very recent study on critical maternal health knowledge gaps in LMICs, Kendall and Langer49 noted that the global maternal health researchers consulted placed high priority on implementation research to improve the delivery of existing evidence-based maternal health interventions. This is consistent with the results of a recently published international survey on priorities for maternal and perinatal health research.50 The studies reviewed in this report evaluated more that 14 MNCH policy issues of high priority in Nigeria including: task shifting, community-based interventions, breastfeeding policy and practices, maternal nutrition policy, use of obstetric care in healthcare facilities, community mobilization to reduce postpartum hemorrhage in home births, emergency action plan to eradicate polio, prevention of maternal to child transmission of HIV, childhood immunization uptake and coverage, free maternal and child health services, management of severe preeclampsia, KMC, integrated management of childhood illness strategy, and treatment regimens for acute diarrhea in children. One very interesting feature of all the studies is that they all generated high quality policy relevance evidence which indicated significant improvements in MNCH outcomes in localities and health facilities where the studies were undertaken. These findings further provide a confirmation of the value and necessity of implementation or intervention studies in strengthening the provision of research evidence that will not only produce strong policies but effective healthcare practice. The findings from these studies provided evidence which informed three important policy documents in Nigeria including the Nigeria’s Call to Action to Save Newborn Lives,51 the Saving One Million Lives Initiative of Nigeria52 and The National Health Bill, 2014.53 This is because the findings of the studies were part of the evidence incorporated in the recommendations in these three important documents. It is therefore pertinent to state that more implementation research in MNCH is needed not only in Nigeria but in other LMICs where health systems are weak and where maternal and child health outcomes are poor. The Centre for Population and Environmental Development,54 noted in their policy brief on the improvement of MNCH in Nigeria that less attention has been paid to implementation research in Nigeria. This entails the production of evidence on the best ways to support the adoption of, and optimize use of innovations in MNCH care. Therefore the ability to test diverse MNCH implementation pathways and to identify what works in rural community settings is critical to the improvement of MNCH care. According to WHO,48 implementation research will provide evidence on the best ways to support the adoption of, and optimize use of innovations and holds promise for scale-up and for greater commitment and investment. Study limitations This study had two main limitations. First, we used only the PubMed for data extraction. Although PubMed is regarded as one of the most outstanding and globally recognized easily assessable databases for health sciences publications. Our inability to search other databases may have resulted in missing additional relevant publications. We advocate the inclusion of other databases in future studies. Another limitation to this study has to do with the scope of the reviewed publications. Every study reviewed was conducted only in as section of Nigeria. Consequently it may be inappropriate to generalize the findings because of the diverse socio-economic and cultural settings of Nigeria. There may be a need to repeat some of the studies in other parts of the country to see if there will be similar or contrary outcomes. Conclusion There is no doubt that there is a dire need for more implementation research related to MNCH in LMICs including Nigeria. This is because the priority for maternal and child survival is not so much the development of new technologies but solving implementation issues, such as how to scale up and evaluate interventions within complex health systems.2 Not only do we need to identify the most effective ways to deliver, scale up and sustain both basic and comprehensive emergency obstetric care, especially for postpartum hemorrhage and preeclampsia, but implementation research is needed to ensure we deliver the right packages of care at the right levels of care.3 However, it must be noted that generating the necessary robust evidence for improved MNCH outcome is not easy. The reason is because implementation research is not free from limitations and challenges. It will certainly present the same problems as other types of research. Most obviously, findings of implementation research need to be taken up by the implementers to close the gap between evidence generated by the implementation researchers and practices.55 Furthermore, evaluation and implementation research of the delivery of MNCH interventions that focus on impact, specifically those assessing changes in morbidity or mortality, is advocated since they have been deemed critical to determine the effectiveness of programs being implemented.56 Finally, in the Federal Ministry of Health of Nigeria publication on saving newborn lives,57 stakeholders are encouraged to conduct locally driven implementation research and act on the results as well as prioritize use of local data for decision making and implementation research to fill knowledge gaps for maternal, newborn, and child health. Acknowledgements This study was one of the outcomes of the “Moving Maternal, Neonatal and Child Health Evidence into Policy in West Africa” ​​(MEP) project undertaken by West African Health organization (WAHO) funded by International Development Research Centre (IDRC) Canada (Reference: IDRC 107892_001). Ethical approval This review was a key component of the “Moving Maternal, Neonatal and Child Health Evidence into Policy in West Africa” ​​(MEP) project undertaken by West African Health Organization (WAHO). Ethical clearance was obtained from the University Research Ethics Committee of Ebonyi State University Nigeria (the institution of the principal author). Competing interests The authors declare no competing interest. Authors’ contributions All authors participated in the design and development of the study. CJU, IS and PO reviewed the studies selected and agreed on the studies included. CJU drafted the manuscript, with contributions from IS, PO and NK. 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The World Bank Group; 2015. http://data.worldbank.org/indicator/SH.DYN.MORT. 22 Deller B Tripathi V Stender S Otolorin E Johnson P Carr C Task shifting in maternal and newborn health care: key components from policy to implementation Int J Gynaecol Obstet 2015 130 Suppl 2 S25 31 10.1016/j.ijgo.2015.03.005 26115853 23 Oseji M Ogu R Community based interventions for the reduction of maternal mortality - the role of professional health associations, non-governmental organisations and community-based organisations in delta state, Nigeria Niger Postgrad Med J 2014 21 4 343 9 25633455 24 Findley SE Doctor HV Ashir GM Kana MA Mani AS Green C Reinvigorating health systems and community-based services to improve maternal health outcomes: case study from northern Nigeria J Prim Care Community Health 2015 6 2 88 99 10.1177/2150131914549383 25217416 25 Senbanjo IO Oshikoya KA Ogbera OA Wright KO Anga AL Breastfeeding policy and practices at the general paediatric outpatient clinic of a teaching hospital in Lagos, Nigeria Int Breastfeed J 2014 9 10 10.1186/1746-4358-9-10 25018776 26 Findley SE Uwemedimo OT Doctor HV Green C Adamu F Afenyadu GY Early results of an integrated maternal, newborn, and child health program, Northern Nigeria, 2009 to 2011 BMC Public Health 2013 13 1034 10.1186/1471-2458-13-1034 24175944 27 Girard AW Dzingina C Akogun O Mason JB McFarland DA Public health interventions, barriers, and opportunities for improving maternal nutrition in Northeast Nigeria Food Nutr Bull 2012 33 2 Suppl S51 70 22913107 28 Erim DO Kolapo UM Resch SC A rapid assessment of the availability and use of obstetric care in Nigerian healthcare facilities PLoS One 2012 7 6 e39555 10.1371/journal.pone.0039555 22745784 29 Prata N Ejembi C Fraser A Shittu O Minkler M Community mobilization to reduce postpartum hemorrhage in home births in northern Nigeria Soc Sci Med 2012 74 8 1288 96 22377106 30 Ado JM Etsano A Shuaib F Damisa E Mkanda P Gasasira A Progress toward poliomyelitis eradication in Nigeria J Infect Dis 2014 210 Suppl 1 S40 9 10.1093/infdis/jiu318 25316862 31 Ibe OE Austin T Sullivan K Fabanwo O Disu E Costello AM A comparison of kangaroo mother care and conventional incubator care for thermal regulation of infants <2000 g in Nigeria using continuous ambulatory temperature monitoring Ann Trop Paediatr 2004 24 3 245 51 15479575 32 Adegboye OA Kotze D Adegboye OA Multi-year trend analysis of childhood immunization uptake and coverage in Nigeria J Biosoc Sci 2014 46 2 225 39 10.1017/S0021932013000254 23710666 33 Okonofua F Lambo E Okeibunor J Agholor K Advocacy for free maternal and child health care in Nigeria--Results and outcomes Health Policy 2011 99 2 131 8 10.1016/j.healthpol.2010.07.013 20727612 34 Okafor UV Efetie ER Igwe W Okezie O Anaesthetic management of patients with pre-eclampsia/ eclampsia and perinatal outcome J Matern Fetal Neonatal Med 2009 22 8 688 92 10.1080/14767050902994473 19504406 35 Nwogu R Larson JS Kim MS Reducing child mortality in Nigeria: a case study of immunization and systemic factors Soc Sci Med 2008 67 1 161 4 18420324 36 Ogbolu Y Iwu EN Zhu S Johnson JV Translating research into practice in low-resource countries: progress in prevention of maternal to child transmission of HIV in Nigeria Nurs Res Pract 2013 2013 848567 10.1017/S0021932013000254 23738064 37 Rowe AK Osterholt DM Kouamé J Piercefield E Herman KM Onikpo F Trends in health worker performance after implementing the integrated management of childhood illness strategy in Benin Trop Med Int Health 2012 17 4 438 46 10.1111/j.1365-3156.2012.02976.x 22950471 38 Mafe MA Appelt B Adewale B Idowu ET Akinwale OP Adeneye AK Effectiveness of different approaches to mass delivery of praziquantel among school-aged children in rural communities in Nigeria Acta Trop 2005 93 2 181 90 15652332 39 Omotade OO Adeyemo AA Kayode CM Oladepo O Treatment of childhood diarrhoea in Nigeria: need for adaptation of health policy and programmes to cultural norms J Health Popul Nutr 2000 18 3 139 44 11262766 40 Averting Maternal Death and Disability Program (AMDD). 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==== Front Health Promot PerspectHealth Promot PerspectHealth Promot PerspectTBZMEDHealth Promotion Perspectives2228-6497Tabriz University of Medical Sciences 10.15171/hpp.2016.21Original ArticleThe association of physical activity and cholesterol concentrations across different combinations of central adiposity and body mass index Loprinzi Paul D. 1 *Addoh Ovuokerie 2 1Director of Research Engagement – Jackson Heart Study Vanguard Center of Oxford, Physical Activity Epidemiology Laboratory, Department of Health, Exercise Science and Recreation Management, The University of Mississippi, University, MS 38677, USA2Physical Activity Epidemiology Laboratory, Department of Health, Exercise Science and Recreation Management, The University of Mississippi, University, MS 38677, USA* Corresponding Author: Paul D. Loprinzi, PhD; Tell: 662-915-5521; Fax: 662-915-5525; pdloprin@olemiss.edu2016 10 8 2016 6 3 128 136 01 5 2016 30 5 2016 © 2016 The Author(s).2016This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.http://journals.tbzmed.ac.ir/HPP Background: The purpose of this study was to investigate if those who are physically active,compared to physically inactive, have better cholesterol profiles across different combinations of body mass index (BMI) and waist circumference (WC). Methods: Data from the 1999-2006 National Health and Nutrition Examination Survey (NHANES) were used (N = 16 095). Cholesterol parameters included total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), TC/HDL-C ratio, triglycerides and at herogenic index(Log10 [triglycerides/HDL-C]). Physical activity (PA) was assessed via self-report, with BMI and WC objectively measured. Cholesterol concentrations of 6 combinations of BMI and WC were evaluated among active and inactive participants. Multivariable linear regression analysis was utilized. Results: Findings were not consistent across sex. There was little evidence to suggest an association of PA on TC across varying BMI and WC combinations. For example, among those who had an obese BMI and high WC, inactive participants did not have different TC level when compared to active participants (β = -1.2; 95% CI: -3.9-1.5, P = 0.38). There was evidence to suggest a favorable association of PA on HDL-C, triglycerides and at herogenic index across varying BMI and WC combinations. For example, among those who had an obese BMI and high WC, inactive (vs. active) participants had a lower HDL-C (βadjusted = -1.6, P < 0.01). When considering either gender, there was sufficient evidence to suggest a favorable association of PA on at least one of the evaluated cholesterol parameters for each of the BMI/WC combinations with the exception of normal BMI and high WC. Conclusion: Except for those having normal weight central obesity, PA is favorably associated with cholesterol parameters across various combinations of BMI and WC. Body mass indexEpidemiologyExerciseNHANESWaist circumferenceCitation: Loprinzi PD, Addoh O. The association of physical activity and cholesterol concentrations across different combinations of centraladiposity and body mass index. Health Promot Perspect. 2016;6(3):128-136. doi: 10.15171/hpp.2016.21. ==== Body Introduction Obesity is an important risk factor for various chronic diseases, such as diabetes, hypertension, dyslipidemia, coronary heart disease and mortality.1 Due to the convenience of measuring body mass index (BMI), coupled with epidemiological findings that BMI is associated with morbidity and mortality,1 BMI is a clinical standard for the identification of patients at risk for adiposity-related morbidities. In addition to the important clinical implications of BMI assessment, body fat distribution is also an important risk factor for various obesity-related chronic diseases. As such, waist circumference (WC) is an often considered surrogate marker of central fat mass2 and it associates with morbidity and mortality to a similar extent to waist-to-hip ratio (see Table 4.1 in the WHO expert consultation3). In fact, WC, even across various ranges of BMI, spanning from normal BMI to obese BMI, predicts worse health outcomes, such as early mortality4 and dyslipidemia.5 Further, both WC and BMI are independently associated with dyslipidemia,5,6 underscoring the potential clinical importance of considering both BMI and WC. In addition to the observed independent associations of BMI and WC on health,5,6 emerging work suggests that varied combinations of BMI and WC may have differential effects on a patient’s health.7 For example, Sharma et al7 demonstrated that patients with normal weight central obesity had the highest mortality risk when compared to other BMI and WC combinations. Considerations of characteristics that influence cholesterol concentrations among patients with varied BMI and WC combinations is important as cholesterol profile is a well-established indicator of chronic disease and early mortality.8,9 One such health characteristic to consider is regular participation in physical activity (PA). Research demonstrates that regular participation in PA is inversely associated with BMI, WC and cholesterol.10 However, the combined associations of BMI, WC and PA on dyslipidemia, is less clear. Thus, the purpose of this study was to address this PA-related obesity risk classification model (i.e., combinations of PA, BMI and WC) as it relates to patient cholesterol concentrations among a national sample (National Health and Nutrition Examination Survey, NHANES) of adults in the United States. Materials and Methods Study design The NHANES is an ongoing survey conducted by the Center for Disease Control and Prevention (CDC) designed to evaluate the health status of US adults through a complex, multistage, stratified clustered probability design. Participants are interviewed in their homes and then subsequently examined in a mobile examination center (MEC). Further information on NHANES methodology and data collection is available on the NHANES website (http://www.cdc.gov/nchs/nhanes.htm). Procedures were approved by the National Center for Health Statistics review board. Consent was obtained from all participants prior to data collection. In the 1999-2000, 2001-2002, 2003-2004, and 2005-2006 cycles, the respective NHANES response rates for adults (20+ years) in the MEC were 68.3%, 71.6%, 68.1%, and 69.8%. These response rates are relatively high for epidemiological studies. Participant data from the 1999-2006 NHANES were utilized. Analyses are based on data from 16 095 adults (20-85 years) who provided complete data for the study variables, inclusive of PA, BMI, WC, cholesterol, age, gender, race-ethnicity, cholesterol medication use, mean arterial pressure, C-reactive protein (CRP), previous year changes in PA, congestive heart failure, coronary artery disease, heart attack, emphysema, chronic bronchitis, stroke, diabetes and smoking status; a flow description of the participant sample size after exclusions is described in the results section. In the 1999-2006 NHANES, 20 311 adults 20-85 years were enrolled. Among these, 2472 had missing PA, BMI or WC data (Nresultant=17 839), as not all of the enrolled participants completed the examinations in the MEC. Among these 17839 participants, a further 292 were excluded because of having a BMI <18.5 kg/m2 (Nresultant=17 547). Among these, a further 1452 participants were excluded because of missing data on the cholesterol concentrations or the covariates (Nresultant=16 095). These 16 095 adult participants constituted the analytic sample. Obesity risk classification model BMI was calculated from measured mass in kg divided by squared height in meters. Weight status was defined as BMI-determined normal weight (18.5-24.9 kg/m2), overweight (25-29.9 kg/m2) or obese (30+ kg/m2).11,12 WC was measured with a tape measure at the (uppermost) lateral border of the hip crest (ilium bone).13,14 High WC was defined as ≥102 cm for men and ≥88 cm for women.15,16 Consistent with current US government PA guidelines,17 participants were defined as “active” based on at least 2000 moderate-to-vigorous PA (MVPA) MET (metabolic equivalent of task)-min-month (assessment of MVPA described below). Based on these three parameters (BMI, WC and PA), participants were classified into 6 mutually exclusive combinations of BMI and WC (normal BMI and normal WC; normal BMI and high WC; overweight BMI and normal WC; overweight BMI and high WC; obese BMI and normal WC; and obese BMI and high WC) among active and inactive participants (see Tables 1 and 2). Cholesterol assessment High-density lipoprotein cholesterol (HDL-C), triglycerides, and total cholesterol (TC) were assessed enzymatically in serum or plasma via a blood sample. Notably, triglyceride data was only evaluated among ½ of the NHANES sample. The Hitachi 704 analyzer was used to calculate these cholesterol parameters. Specific details on the assessment of these cholesterol parameters is discussed thoroughly in a previous publication.18 Physical activity As described elsewhere,19 participants were asked open-ended questions about participation in leisure-time PA over the past 30 days. Data were coded into 48 activities, including 16 sports-related activities, 14 exercise-related activities, and 18 recreational-related activities. For each of the 48 activities where participants reported moderate or vigorous-intensity for the respective activity, they were asked to report the number of times they engaged in that activity over the past 30 days and the average duration they engaged in that activity. For each activity, MET-min-month was calculated by multiplying the number of days, by the mean duration, by the respective MET level (MET-min-month = days*duration*MET level). The MET levels for each activity are provided elsewhere.20 As described elsewhere, this PA assessment has demonstrated evidence of convergent validity by positively associating with accelerometer-assessed PA.19 Statistical analysis and covariates Statistical analyses were performed via procedures from survey data using Stata (v.12; Stata Corp, College Station, TX). Due to multiple comparisons, statistical significance was set at P≤0.01. Analyses accounted for the complex survey design employed in NHANES by utilizing sample weights, primary sampling units and strata via the Taylor series (linearization) method. Sample weights were re-weighted to account for the use of combined NHANES cycles.21 Information on the use of sample weights to generate population weighted estimates is available elsewhere.22 Linear regression analysis was used to examine cholesterol (TC, HDL-C, TC/HDL-C and triglyceride) differences across the respective activity status groups (i.e., inactive vs. active across the different BMI/WC combinations), with results stratified by gender. For the multivariable linear regression models, covariates included cholesterol medication use (yes/no), age (years; continuous), gender (male/female), race-ethnicity (Mexican American, non-Hispanic white, non-Hispanic black, and other), mean arterial pressure (mm Hg; continuous), CRP (mg/dL; continuous), previous year changes in PA (categorical; activity increased, decreased or stayed the same), congestive heart failure (yes/no), coronary artery disease (yes/no), heart attack (yes/no), emphysema (yes/no), chronic bronchitis (yes/no), stroke (yes/no), diabetes (yes/no) and smoking status (categorical; smokes every day, smokes some days, former smoker, never smoker). These covariates were selected based on previous research demonstrating their association with obesity, PA and cholesterol.23 Notably, results were similar when including all of these covariates versus a minimally adjusted model that did not include certain covariates (e.g., CRP) that may be involved in the mechanistic pathway between PA, obesity and cholesterol. Thus, each of these covariates were included in the adjusted models. Age, gender, race-ethnicity, changes in PA, and smoking status were assessed via a self-report questionnaire. With regard to changes in PA, participants were asked, “How does the amount of activity that you reported for the past 30 days compare with your PA for the past 12 months? (Response options: more active, less active or about the same”.) Regarding the chronic diseases (congestive heart failure, coronary artery disease, heart attack, emphysema, chronic bronchitis, stroke, and diabetes), participants were asked if they had ever been told by a physician or other health professional that they had this disease. In addition to diabetes being assessed via self-report of physician diagnosis, here we also defined individuals as having diabetes if they had a fasting plasma glucose ≥126 mg/dL or an A1C ≥6.5%. Using the average of up to four manually assessed blood pressure measurements, mean arterial pressure was calculated using the following formula: ([(diastolic blood pressure × 2) + systolic blood pressure]/3). Lastly, high sensitivity CRP concentration was quantified using latex-enhanced nephelometry. Further details on the assessment of these laboratory-based parameters can be found elsewhere.18 Results Table 1 displays the study variable characteristics among these 16 095 analyzed participants, with results stratified by the obesity risk classification groups. Across these 12 groups, the sample size ranged from 90 participants (Group 9; obese BMI, normal WC and active) to 3442 participants (Group 12; obese BMI, high WC and inactive). Generally, those with a high WC were older than those with a normal WC. Women, compared to men, were more likely to have a high WC with a normal BMI. The active group compared to the inactive group, across all combinations of BMI and WC, generally had more favorable levels for all chronic diseases and biomarkers. Table 2 displays the weighted regression associations (β, P value) examining the inactive vs. active groups for each evaluated cholesterol concentrations, with results presented for the entire sample and stratified by sex. Generally, unadjusted and adjusted results were similar. Across all BMI and WC combinations, there was no statistically significant difference in TC levels across activity status, with the exception of inactive women classified as obese BMI and normal WC having higher TC (35.5 mg/dL higher) than their active counterparts. With regard to HDL-C, those who were inactive (vs. active) had lower levels of HDL-C for all the BMI and WC combinations with the exception of normal BMI and high WC and obese BMI and normal WC. Results, however, were not consistent across sex, and results were similar for TC/HDL-C. For triglycerides, higher PA (vs. not meeting guidelines) was only associated with lower levels of triglycerides among three BMI/WC combinations: normal BMI and WC; overweight BMI and high WC; and obese BMI and high WC. For the atherogenic index, lower PA (vs. higher PA) was associated with a higher atherogenic index score for men with a normal BMI and normal WC, for women with an overweight BMI and a high WC, and for both men and women with an obese BMI and high WC. Collectively, and when considering either sex, meeting PA guidelines (vs. not) was favorably associated with at least one of the evaluated cholesterol concentrations for each of the BMI/WC combinations with the exception of normal BMI and high WC. Table 1 Characteristics of the study variables (mean [95% CI]), 1999-2006 NHANES (N= 16095)a Variable Normal BMI and normal WC Normal BMI and High WC Overweight BMI and normal WC Overweight BMI and high WC Obese BMI and normal WC Obese BMI and high WC Group number 1 2 3 4 5 6 7 8 9 10 11 12 Active Inactive Active Inactive Active Inactive Active Inactive Active Inactive Active Inactive Sample size (n) 1971 2406 170 355 1237 1460 1102 2092 90 101 1669 3442 TC, mean mg/dL 191.8 (189-194) 195.5 (193-197) 210.7 (202-218) 211.1 (202-220) 200.2 (196-203) 204.9 (202-207) 207.7 (204-211) 213.1 (210-216) 201.2 (190-212) 200.2 (190-209) 204.4 (201-207) 205.5 (203-207) HDL, mean mg/dL 60.4 (59-61) 57.7 (56-58) 60.6 (57-63) 58.8 (56-60) 49.9 (48-51) 48.9 (47-49) 54.7 (53-56) 52.2 (51-53) 45.8 (42-48) 47.2 (43-51) 47.2 (46-48) 47.4 (46-48) Ratio of total/HDL 3.37 (3.3-3.4) 3.63 (3.5-3.7) 3.69 (3.4-3.9) 3.83 (3.5-4.0) 4.26 (4.1-4.3) 4.53 (4.4-4.6) 4.1 (3.9-4.2) 4.39 (4.3-4.5) 4.65 (4.2-5.0) 4.60 (4.1-5.0) 4.60 (4.4-4.7) 4.60 (4.5-4.7) Triglyceride, mean mg/dLb 98.6 (95-102) 116.0 (109-122) 150.5 (130-171) 182.2 (103-261) 145.1 (132-157) 150.3 (139-161) 163.2 (135-191) 163.8 (155-171) 213.2 (112-313) 154.9 105204) 158.8(148-168) 181.2 (169-192) Atherogenic index, mean mmol/Lc -.17 (-.19 to -.16) -.11 (-.13 to -.08) .009 (-.06-.08) .03 (-.06-.13) .03 (.004-.06) .05 (.02-.09) .03 (-.006-.06) .09 (.07-.11) .17 (.03-.31) .03 (-.10-.16) .10 (.07-.12) .15 (.13-.17) % Cholesterol medication 4.8 6.6 16.6 18.9 8.9 7.2 17.7 14.9 8.4 5.2 15.2 15.2 Age, mean yrs 40.8 (39-41) 44.3 (43-45) 54.6 (51-57) 56.5 (53-59) 41.7 (40-42) 43.4 (42-44) 51.1 (49-52) 53.0 (52-54) 36.1 33-38) 38.4 (35-41) 45.0 (43-46) 48.6 (47-49) Male, % 45.7 46.5 7.9 8.4 80.0 78.2 40.4 35.1 87.6 83.7 53.5 38.5 Non-Hispanic white, % 78.9 69.5 85.6 79.3 74.1 58.7 84.0 72.6 53.9 40.6 73.8 69.9 Non-Hispanic black, % 6.8 10.1 3.4 6.3 9.0 12.6 5.1 8.7 18.9 24.0 12.0 13.7 Mexican American, % 4.3 8.6 2.9 4.6 7.0 14.3 4.7 8.2 10.0 23.7 5.5 8.5 MAP, mean mm Hg 85.2 (84-86) 86.0 (85-87) 88.1 (86-89) 88.5 (86-90) 88.0 (87-89) 89.0 (89-90) 89.3 (88-90) 90.1 (89-91) 89.6 (87-92) 88.9 (86-91) 90.8 (90-92) 91.4 (90-92) CRP, mean mg/dL 0.21 (.19-.24) 0.30 (.26-.33) 0.28 (.21-.34) 0.38 (.32-.44) 0.27 (.22-.32) 0.36 (.29-.43) 0.38 (.33-.44) 0.47 (.44-.50) 0.29 (.22-.35) 0.40 (.15-.64) 0.51 (.48-.54) 0.70 (.66-.74) MET-min-month, mean 10295.2 341.3 9402.2 260.1 10889.1 386.9 8326.5 329.8 12681.8 259.8 9079.1 327.7 Become less active in past year, % 18.0 22.2 20.9 28.7 16.8 26.3 15.3 24.7 19.1 20.1 17.5 24.8 Waist circumference, mean cm 81.1 82.3 92.2 92.5 92.6 93.0 99.5 99.9 97.1 96.9 112.4 113.7 BMI, mean kg/m2 22.3 22.3 23.9 23.6 26.9 26.8 27.8 27.8 31.2 30.9 34.8 35.7 CHF, % 0.6 1.8 0.2 3.0 0.7 1.7 1.7 4.2 0 1.4 1.4 3.8 CAD, % 1.2 2.7 3.4 4.0 2.9 1.9 6.4 4.7 0 2.8 3.5 4.5 Heart attack, % 1.1 3.2 2.3 3.9 2.3 2.7 4.2 5.1 0 4.5 3.0 4.8 Stroke, % 0.8 1.7 4.3 2.8 0.4 1.3 3.0 4.1 0 0.3 1.7 3.8 Emphysema, % 1.0 2.3 1.4 3.5 0.5 1.2 0.7 3.0 0 0 0.7 2.0 Chronic bronchitis, % 4.0 6.4 9.2 11.6 2.7 4.3 5.7 7.7 5.1 3.7 7.6 10.1 Diabetes, % 2.3 4.3 11.3 12.3 3.7 6.4 9.3 12.2 4.2 9.5 12.9 17.8 Daily smoker, % 17.6 33.6 13.9 25.7 14.9 22.7 13.7 21.3 19.9 23.8 15.3 19.0 Abbreviations: BMI, body mass index; CAD, coronary artery disease; CHF, congestive heart failure; CRP, C-reactive protein; HDL, high-density lipoprotein cholesterol; MAP, mean arterial pressure; MET, metabolic equivalent of task; WC, waist circumference. aFor categorical variables, percentages are reported. For continuous variables, means (95% CI) are reported. Normal BMI (18.5-24.9 kg/m2); Overweight BMI (25.0-29.9 kg/m2); Obese BMI (30+ kg/m2); Normal Waist Circumference (<102 cm for men and <88 cm for women); High Waist Circumference (≥102 cm for men and ≥88 cm for women); Active (≥ 2000 MET-min-month); Inactive (<2000 MET-min-month). b Triglyceride levels were only assessed among a ½ subsample of NHANES participants. In these analyses, 7899 participants had triglyceride data. The sample sizes across the 12 respective groups were: 966, 1211, 86, 176, 589, 701, 551, 1039, 46, 45, 816, and 1673. c Atherogenic index (expressed in mmol/L) calculated as follows: (log10(triglycerides/HDL-C). To convert HDL-C from mg/dL to mmol/L, divide by 38.67; to convert triglycerides from mg/dL to mmol/L, divide by 88.57. Table 2 Weighted regression sex-specific associations (β, p-value) examining the inactive vs. active groups for each evaluated cholesterol parameters, 1999-2006 NHANES (N=16095)a Variable Normal BMI and normal WC Normal BMI and high WC Overweight BMI and normal WC Overweight BMI and high WC Obese BMI and normal WC Obese BMI and high WC Group Number 1 2 3 4 5 6 7 8 9 10 11 12 Active Inactive Active Inactive Active Inactive Active Inactive Active Inactive Active Inactive TC Entire Sample Unadjusted Referent 3.7 (P=0.01) Referent 0.33 (P=0.95) Referent 4.7 (P=0.02) Referent 5.4 (P=0.01) Referent -1.1 (P=0.86) Referent 1.0 (P=0.48) Adjustedb Referent 1.9 (P=0.17) Referent -0.63 (P=0.92) Referent 2.9 (P=0.15) Referent 3.3 (P=0.11) Referent -1.2 (P=0.84) Referent -1.2 (P=0.38) Men Unadjusted Referent 4.9 (P=0.03) Referent -9.8 (P=0.65) Referent 5.0 (P=0.03) Referent 3.7 (P=0.25) Referent -6.1 (P=0.40) Referent -0.65 (P=0.76) Adjustedb Referent 3.3 (P=0.14) Referent -18.7 (P=0.41) Referent 3.2 (P=0.16) Referent 1.5 (P=0.65) Referent -7.2 (P=0.31) Referent -1.2 (P=0.57) Women Unadjusted Referent 2.8 (P=0.06) Referent 1.3 (P=0.83) Referent 4.4 (P=0.26) Referent 6.0 (P=0.01) Referent 29.9 (P=0.05) Referent 1.6 (P=0.36) Adjustedb Referent 0.8 (P=0.55) Referent 0.7 (P=0.90) Referent 2.1 (P=0.56) Referent 3.5 (P=0.13) Referent 35.5 (P=0.01) Referent -1.5 (P=0.37) HDL Cholesterol Entire Sample Unadjusted Referent -2.6 (P<0.01) Referent -0.26 (P=0.85) Referent -1.0 (P=0.14) Referent -2.5 (P<0.01) Referent 1.3 (P=0.60) Referent 0.24 (P=0.61) Adjustedb Referent -2.4 (P<0.01) Referent -1.7 (P=0.25) Referent -1.2 (P=0.04) Referent -3.2 (P<0.01) Referent 0.64 (P=0.76) Referent -1.6 (P<0.01) Men Unadjusted Referent -1.4 (P=0.05) Referent 2.0 (P=0.75) Referent -1.4 (P=0.01) Referent -2.1 (P=0.04) Referent 0.02 (P=0.99) Referent -1.3 (P=0.01) Adjustedb Referent -1.8 (P=0.01) Referent 1.5 (P=0.81) Referent -1.6 (P=0.01) Referent -2.1 (P=0.05) Referent -0.33 (P=0.88) Referent -1.4 (P=0.01) Women Unadjusted Referent -3.4 (P<0.01) Referent -2.1 (P=0.15) Referent -0.39 (P=0.80) Referent -3.7 (p<0.01) Referent 4.5 (P=0.45) Referent -1.1 (P=0.10) Adjustedb Referent -3.1 (P<0.01) Referent -1.9 (P=0.16) Referent -1.0 (P=0.50) Referent -4.2 (p<0.01) Referent 4.6 (P=0.43) Referent -1.5 (P=0.04) Total/HDL Ratio Entire Sample Unadjusted Referent 0.26 (P<0.01) Referent 0.13 (P=0.48) Referent 0.26 (P<0.01) Referent 0.29 (P<0.01) Referent -0.05 (P=0.86) Referent 0.01 (P=0.93) Adjustedb Referent 0.19 (P<0.01) Referent 0.10 (P=0.61) Referent 0.23 (P<0.01) Referent 0.29 (P<0.01) Referent -0.01 (P=0.98) Referent 0.09 (P=0.06) Men Unadjusted Referent 0.27 (P<0.01) Referent 0.04 (P=0.94) Referent 0.31 (P<0.01) Referent 0.32 (P<0.01) Referent -0.05 (P=0.87) Referent 0.10 (P=0.20) Adjustedb Referent 0.24 (P<0.01) Referent -0.18 (P=0.78) Referent 0.26 (P<0.01) Referent 0.23 (P=0.07) Referent -0.04 (P=0.88) Referent 0.09 (P=0.27) Women Unadjusted Referent 0.24 (P<0.01) Referent 0.13 (P=0.41) Referent 0.17 (P=0.09) Referent 0.35 (P<0.01) Referent 0.35 (P=0.28) Referent 0.16 (P=0.01) Adjustedb Referent 0.17 (P<0.01) Referent 0.10 (P=0.52) Referent 0.17 (P=0.09) Referent 0.32 (P<0.01) Referent 0.41 (P=0.19) Referent 0.11 (P=0.10) Triglycerides Entire Sample Unadjusted Referent 17.3 (P<0.01) Referent 31.7 (P<0.01) Referent 5.2 (P=0.57) Referent 0.53 (P=0.97) Referent -58.3 (P=0.30) Referent 22.4 (P<0.01) Adjustedb Referent 15.1 (P<0.01) Referent 29.5 (P=0.45) Referent 2.9 (P=0.75) Referent 0.97 (P=0.94) Referent -58.8 (P=0.30) Referent 24.4 (P<0.01) Men Unadjusted Referent 19.5 (P<0.01) Referent -7.3 (P=0.73) Referent 9.3 (P=0.40) Referent -29.0 (P=0.41) Referent -59.5 (P=0.32) Referent 34.0 (P=0.01) Adjustedb Referent 19.8 (P<0.01) Referent -20.0 (P=0.39) Referent 5.8 (P=0.62) Referent -33.1 (P=0.33) Referent -61.4 (P=0.30) Referent 32.0 (P=0.03) Women Unadjusted Referent 15.8 (P<0.01) Referent 36.7 (P=0.39) Referent -3.2 (P=0.74) Referent 23.0 (P<0.01) Referent 34.5 (P=0.12) Referent 23.8 (P<0.01) Adjustedb Referent 11.0 (P=0.04) Referent 33.2 (P=0.40) Referent -2.9 (P=0.74) Referent 20.6 (P<0.01) Referent 23.4 (P=0.45) Referent 19.6 (P<0.01) Atherogenic Index c Entire Sample Unadjusted Referent 0.06 (P<0.01) Referent 0.02 (P=0.71) Referent 0.02 (P=0.30) Referent 0.06 (P=0.009) Referent -0.13 (P=0.16) Referent 0.04 (P=0.002) Adjustedb Referent 0.05 (P<0.01) Referent 0.01 (P=0.84) Referent 0.01 (P=0.39) Referent 0.06 (P<0.01) Referent -0.13 (P=0.15) Referent 0.06 (P<0.001) Men Unadjusted Referent 0.07 (P=0.002) Referent -0.04 (P=0.75) Referent 0.04 (P=0.11) Referent 0.02 (P=0.53) Referent -0.13 (P=0.19) Referent 0.07 (P=0.002) Adjustedb Referent 0.06 (P=0.002) Referent -0.08 (P=0.59) Referent 0.03 (P=0.23) Referent 0.01 (P=0.65) Referent -0.13 (P=0.14) Referent 0.06 (P<0.01) Women Unadjusted Referent 0.06 (P=0.002) Referent 0.03 (P=0.63) Referent -0.01 (P=0.70) Referent 0.10 (P<0.001) Referent 0.10 (P=0.56) Referent 0.07 (P=0.002) Adjustedb Referent 0.05 (P=0.02) Referent 0.01 (P=0.76) Referent -0.01 (P=0.68) Referent 0.10 (P<0.001) Referent 0.10 (P=0.58) Referent 0.06 (P<0.01) Abbreviations: BMI, body mass index; CRP, C-reactive protein; HDL, high-density lipoprotein cholesterol; WC, waist circumference. aNormal BMI (18.5-24.9 kg/m2); Overweight BMI (25.0-29.9 kg/m2); Obese BMI (30+ kg/m2); Normal WC (<102 cm for men and <88 cm for women); High WC (≥102 cm for men and ≥88 cm for women); Active (≥ 2000 MET-min-month); Inactive (<2000 MET-min-month). b Adjusted regression models controlled for cholesterol medication use (yes/no), age (years; continuous), gender (male/female), race-ethnicity (Mexican American, non-Hispanic white, non-Hispanic black, and other), mean arterial pressure (mm Hg; continuous), CRP (mg/dL; continuous), previous year changes in physical activity (categorical; activity increased, decreased or stayed the same), congestive heart failure (yes/no), coronary artery disease (yes/no), heart attack (yes/no), emphysema (yes/no), chronic bronchitis (yes/no), stroke (yes/no), diabetes (yes/no) and smoking status (categorical; smokes every day, smokes some days, former smoker, never smoker). cAtherogenic index (expressed in mmol/L) calculated as follows: (log10 (triglycerides/HDL-C). To convert HDL-C from mg/dL to mmol/L, divide by 38.67; to convert triglycerides from mg/dL to mmol/L, divide by 88.57. Bold values indicate statistical significance (P≤0.01) for the adjusted models. TC, triglycerides and HDL-C are expressed in mg/dL units. Discussion High BMI and WC are independently associated with cholesterol profile,5,6 and PA is favorably associated with BMI, WC and cholesterol.10 However, the extent to which PA is associated with cholesterol concentrations across varying combinations of BMI and WC, is less clear. Thus, a novelty of this study is addressing the association between PA and cholesterol concentrations across various combinations of BMI and WC. The main findings for this study were that, after adjustments (including adjustment for age, gender, race-ethnicity and cholesterol medication use): Findings were not consistent across sex. There was little evidence to suggest a favorable association of PA on TC across varying BMI and WC combinations. There was sufficient evidence to suggest a favorable association of PA on HDL-C across varying BMI and WC combinations. Although not to the extent of HDL-C, there was some evidence to suggest a favorable association of PA on triglycerides and the atherogenic index across several BMI and WC combinations. When considering either sex, there was sufficient evidence to suggest a favorable association of PA on at least one of the evaluated cholesterol concentrations for each of the BMI/WC combinations with the exception of normal BMI and high WC. Given that PA did not appear to have a beneficial or protective effect on TC for the different BMI/WC combinations, but did for HDL-C, the protective effects observed for the TC/HDL-C ratio is likely driven from the PA-HDL-C findings. These findings are in general agreement with other related research demonstrating stronger associations for PA and HDL-C when compared to TC.24,25 There is also other evidence demonstrating favorable effects of PA on triglyceride levels.26 Our non-consistent findings across sex are important to consider. Fewer studies on this topic have evaluated sex-specific associations regarding the relationship between PA and lipid profile. Using data from the Atherosclerosis Risk in Communities (ARIC) study, Monda et al26 also showed sex-varying associations. Other research has also demonstrated sex differences in the response of lipids to exercise,27 which is thought to be a result of menopausal status and use of hormone replacement therapy.26 At this point, explanation of such sex-specific lipid responses to exercise is not clear and worthy of future investigations. Another important consideration in this study is the notable observation that PA had a favorable association with at least one of the evaluated lipid concentrations across all BMI/WC combinations with the exception of those with a normal BMI but high WC. This observation may be a result of several factors observable from data in Table 1. Those in this group tended to have higher TC concentrations, were older and were predominately female (whom were less active). Additionally, recent work demonstrates that this group (normal BMI but high WC), compared to other BMI/WC combinations, has the highest mortality risk.7 Thus, PA may have less of an effect on this vulnerable at-risk group. Alternatively, this group may be less inclined to engage in PA. Limitations of this study include the subjective assessment of PA and the cross-sectional study design, rendering causality not possible. Major strengths include the study’s novelty and national sample. The major finding of this study is that across most BMI and WC combinations, active individuals, compared to inactive individuals, have higher levels of HDL-C and lower triglyceride levels. Thus, these findings have several important clinical implications. These findings underscore the importance of clinician promotion of central adiposity control strategies such as directed PA considering that the beneficial effects of PA (regarding cholesterol) appears to be attenuated by central adiposity. Additionally, given the varied cholesterol profile across BMI and WC combinations, when feasible, clinicians may wish to integrate WC measurements into their practice. Further, given the null associations between PA and those with normal weight central obesity, coupled with recent work suggesting that this group is at the highest mortality risk, clinicians may wish to identify and implement health-enhancing strategies among this vulnerable group. Ethical approval The study procedures were approved by the ethics committee of the National Center for Health Statistics. Competing interests None. Authors’ contributions PDL and OA conceptualized the study and contributed to writing the manuscript. PDL conducted the analyses. ==== Refs References 1 Calle EE Thun MJ Petrelli JM Rodriguez C Heath CW Jr ody-mass index and mortality in a prospective cohort of U.S. adults N Engl J Med 1999 341 1097 105 10.1056/NEJM199910073411501 10511607 2 Pouliot MC Despres JP Lemieux S Moorjani S Bouchard C Tremblay A Waist circumference and abdominal sagittal diameter: best simple anthropometric indexes of abdominal visceral adipose tissue accumulation and related cardiovascular risk in men and women Am J Cardiol 1994 73 460 8 8141087 3 Yau JW Rogers SL Kawasaki R Lamoureux EL Kowalski JW Bek T Global prevalence and major risk factors of diabetic retinopathy Diabetes Care 2012 35 556 64 10.2337/dc11-1909 22301125 4 Cerhan JR Moore SC Jacobs EJ Kitahara CM Rosenberg PS Adami HO A pooled analysis of waist circumference and mortality in 650,000 adults Mayo Clin Proc 2014 89 335 45 10.1016/j.mayocp.2013.11.011 24582192 5 Janssen I Katzmarzyk PT Ross R Waist circumference and not body mass index explains obesity-related health risk Am J Clin Nutr 2004 79 379 84 14985210 6 Bekkers MB Brunekreef B Koppelman GH Kerkhof M de Jongste JC Smit HA BMI and waist circumference; cross-sectional and prospective associations with blood pressure and cholesterol in 12-year-olds PLoS One 2012 7 e51801 10.1371/journal.pone.0051801 23251628 7 Sharma S Batsis JA Coutinho T Somers VK Hodge DO Carter RE Normal-weight central obesity and mortality risk in older adults with coronary artery disease Mayo Clin Proc 2016 91 3 343 51 10.1016/j.mayocp.2015.12.007 26860580 8 Goldschmid MG Barrett-Connor E Edelstein SL Wingard DL Cohn BA Herman WH Dyslipidemia and ischemic heart disease mortality among men and women with diabetes Circulation 1994 89 991 7 8124839 9 Miselli MA Nora ED Passaro A Tomasi F Zuliani G Plasma triglycerides predict ten-years all-cause mortality in outpatients with type 2 diabetes mellitus: a longitudinal observational study Cardiovasc Diabetol 2014 13 135 10.1186/s12933-014-0135-6 25301574 10 Kodama S Tanaka S Saito K Shu M Sone Y Onitake F Effect of aerobic exercise training on serum levels of high-density lipoprotein cholesterol: a meta-analysis Arch Intern Med 2007 167 999 1008 10.1001/archinte.167.10.999 17533202 11 Fitzpatrick AL Kronmal RA Gardner JP Psaty BM Jenny NS Tracy RP Leukocyte telomere length and cardiovascular disease in the cardiovascular health study Am J Epidemiol 2007 165 14 21 10.1093/aje/kwj346 17043079 12 Cherkas LF Hunkin JL Kato BS Richards JB Gardner JP Surdulescu GL The association between physical activity in leisure time and leukocyte telomere length Arch Intern Med 2008 168 154 8 10.1001/archinternmed.2007.39 18227361 13 Simpson RJ Cosgrove C Chee MM McFarlin BK Bartlett DB Spielmann G Senescent phenotypes and telomere lengths of peripheral blood T-cells mobilized by acute exercise in humans Exerc Immunol Rev 2010 16 40 55 20839490 14 Kim JH Ko JH Lee DC Lim I Bang H Habitual physical exercise has beneficial effects on telomere length in postmenopausal women Menopause 2012 19 1109 15 10.1097/gme.0b013e3182503e97 22668817 15 Grundy SM Cleeman JI Daniels SR Donato KA Eckel RH Franklin BA Diagnosis and management of the metabolic syndrome: an American Heart Association/National Heart, Lung, and Blood Institute scientific statement: Executive Summary Crit Pathw Cardiol 2005 4 198 203 18340209 16 Klein S Allison DB Heymsfield SB Kelley DE Leibel RL Nonas C Waist circumference and cardiometabolic risk: a consensus statement from Shaping America’s Health: Association for Weight Management and Obesity Prevention; NAASO, The Obesity Society; the American Society for Nutrition; and the American Diabetes Association Am J Clin Nutr 2007 85 1197 202 17490953 17 Du M Prescott J Kraft P Han J Giovannucci E Hankinson SE Physical activity, sedentary behavior, and leukocyte telomere length in women Am J Epidemiol 2012 175 414 22 10.1093/aje/kwr330 22302075 18 Marzetti E Calvani R Cesari M Buford TW Lorenzi M Behnke BJ Mitochondrial dysfunction and sarcopenia of aging: from signaling pathways to clinical trials Int J Biochem Cell Biol 2013 45 2288 301 10.1016/j.biocel.2013.06.024 23845738 19 Loprinzi PD Dose-response association of moderate-to-vigorous physical activity with cardiovascular biomarkers and all-cause mortality: considerations by individual sports, exercise and recreational physical activities Prev Med 2015 81 73 7 10.1016/j.ypmed.2015.08.014 26307435 20 Ainsworth BE Haskell WL Whitt MC Irwin ML Swartz AM Strath SJ Compendium of physical activities: an update of activity codes and MET intensities Med Sci Sports Exerc 2000 32 S498 504 10993420 21 Collins M Renault V Grobler LA St Clair Gibson A Lambert MI Wayne Derman E Athletes with exercise-associated fatigue have abnormally short muscle DNA telomeres Med Sci Sports Exerc 2003 35 1524 8 10.1249/01.MSS.0000084522.14168.49 12972872 22 Centers for Disease Control and Prevention. National Health and Nutrition Examination Survey, 2010. Overview of NHANES Survey Design and Weights. Available from: http://www.cdc.gov/nchs/tutorials/environmental/orientation/sample_design/. 23 Vischer UM Safar ME Safar H Iaria P Le Dudal K Henry O Cardiometabolic determinants of mortality in a geriatric population: is there a “reverse metabolic syndrome”? Diabetes Metab 2009 35 108 14 10.1016/j.diabet.2008.08.006 19237305 24 Pelletier DL Baker PT Physical activity and plasma total- and HDL-cholesterol levels in Western Samoan men Am J Clin Nutr 1987 46 577 85 3661475 25 Skoumas J Pitsavos C Panagiotakos DB Chrysohoou C Zeimbekis A Papaioannou I Physical activity, high density lipoprotein cholesterol and other lipids levels, in men and women from the ATTICA study Lipids Health Dis 2003 2 3 10.1186/1476-511X-2-3 12852790 26 Monda KL Ballantyne CM North KE Longitudinal impact of physical activity on lipid profiles in middle-aged adults: the Atherosclerosis Risk in Communities Study J Lipid Res 2009 50 1685 91 10.1194/jlr.P900029-JLR200 19346332 27 Katzmarzyk PT Leon AS Rankinen T Gagnon J Skinner JS Wilmore JH Changes in blood lipids consequent to aerobic exercise training related to changes in body fatness and aerobic fitness Metabolism 2001 50 841 848 10.1053/meta.2001.24190 11436192

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==== Front Health Promot PerspectHealth Promot PerspectHealth Promot PerspectTBZMEDHealth Promotion Perspectives2228-6497Tabriz University of Medical Sciences 10.15171/hpp.2016.22Original ArticleUsing multi-theory model to predict initiation and sustenance of small portion size consumption among college students Sharma Manoj 1 Catalano Hannah Priest 2 Nahar Vinayak K. 3 4 *Lingam Vimala 1 Johnson Paul 5 Ford M. Allison 4 1Behavioral & Environmental Health, School of Public Health, Jackson State University, MS, USA2Public Health Studies, School of Health and Applied Human Sciences, University of North Carolina Wilmington, NC, USA3Department of Health, Physical Education, and Exercise Science, School of Allied Health Sciences, Lincoln Memorial University, Harrogate, TN, USA4Department of Health, Exercise Science & Recreation Management, School of Applied Sciences, University of Mississippi, MS, USA5Department of Management, School of Business Administration, University of Mississippi, MS, USA* Corresponding Author: Vinayak K. Nahar, MD, MS; Lincoln Memorial University, Mary Mars, 6965 Cumberland Gap Parkway, Harrogate, TN 37752, USA. Tell: (662) 638-5126; vinayak.nahar@LMUnet.edu2016 10 8 2016 6 3 137 144 15 6 2016 23 6 2016 © 2016 The Author(s).2016This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.http://journals.tbzmed.ac.ir/HPP Background: Consumption of large portion sizes is contributing to overweight and obesity.College students are a vulnerable group in this regard. The purpose of this study was to use multi-theory model (MTM) to predict initiation and sustenance of small portion size consumption in college students. Methods: A total of 135 students at a large Southern US University completed a 35-item valid (face, content, and construct) and reliable (internally consistent) survey electronically in a cross-sectional design. The main outcome measures were intention to start eating small portion sizes and continuing to eat small portion sizes. Only those students who ate large portion sizes during the past 24 hours were included. Results: Step wise multiple regression showed that initiation of small portion size consumption was explained by participatory dialogue (advantages outweighing disadvantages), behavioral confidence, age, and gender (adjusted R2 = 0.37, P < 0.001). Males were less likely to initiate small portion size consumption than females (β = -0.185, 95% CI = -0.71– -0.11). Regarding sustenance, emotional transformation, changes in social environment, and race were the significant predictors (adjusted R2 = 0.20, P < 0.001). Whites were less likely to sustain small portion size change than other races (β = -0.269, 95% CI = -0.97 – -0.26). Conclusion: Based on this study’s findings, MTM appears to be a robust theoretical framework for predicting small portion size consumption behavior change. Interventions in this regard need to be designed. ObesityOverweightPortion sizeDietNutritionCitation: Sharma M, Catalano HP, Nahar KV, Lingam V, Johnson P, Ford MA. Using multi-theory model to predict initiation and sustenanceof small portion size consumption among college students. Health Promot Perspect. 2016;6(3):137-144. doi: 10.15171/hpp.2016.22. ==== Body Introduction Obesity is a public health crisis in the United States with more than one third (34.9%) of the adult population classified as obese.1 Obesity increases the risk for coronary heart disease, stroke, cancer, hypertension, type 2 diabetes, gallbladder disease, osteoarthritis, and sleep apnea.2 Food portion sizes are a major contributing factor to the obesity epidemic in the United States.3 Findings from research studies provide evidence that increases in food portion size have been directly proportional to increases in obesity rates.4,5 The concept of supersizing portion sizes is practiced almost all over the world with the greatest increases in the United States.6 Consumption of healthy portion sizes, and subsequently reduced caloric intake, is an essential strategy for obesity prevention.7 However, few educational interventions have been conducted to promote healthy portion size consumption.8 Portion size interventions have had mixed results, though none have explicitly applied a theoretical framework. However, one intervention9 applied the self-regulation construct from social cognitive theory.10 Most of the interventions targeted improving participants’ portion size estimation skills and increasing awareness and knowledge of appropriate portion sizes.11-13 College is a critical period in which individuals establish lifestyle behaviors, including dietary behavior, which impacts weight and long-term health outcomes.14,15 College students often have poor dietary habits, including low intake of fruits and vegetables, skipping meals, inadequate consumption of a variety of foods, consuming large portion sizes, snacking, frequent consumption of fast food, and high intake of high energy-dense foods.14 Only 3.8% of college students consume the recommended five servings of fruits and vegetables per day.16 Furthermore, several studies have reported substantial weight gain among college students throughout their college experience.17,18 Approximately one quarter (23.3%) of college students nationwide are overweight and an additional 16.3% of college students are obese. One approach to reducing obesity problem is to reduce portion sizes which will be the focus of this study. Sharma conceptualized the multi-theory model (MTM) to predict one-time and continuous health behaviors. The MTM proposes that health behavior change occurs through two components: initiation of the behavior change and sustenance of the behavior change. Existing health behavior theories have not considered this distinction with health behavior, which has often resulted in poor predictive power when operationalizing those constructs.19 The MTM proposes that three constructs predict the initiation of behavior change, namely, participatory dialogue (in which advantages outweigh disadvantages), behavioral confidence, and changes in physical environment. Participatory dialogue is similar to the perceived benefits and perceived barriers constructs within the health belief model (HBM) and pros and cons within the trans-theoretical model (TTM).20,21 However, the construct of participatory dialogue is unique because it requires participation and mutual exploration, which is a process that Freire emphasized but is disregarded by the HBM and TTM.22 Behavioral confidence is derived from Ajzen’s 23 perceived behavioral control construct and Bandura’s10 self-efficacy construct. However, behavioral confidence is distinct from these constructs in that the target is on changing behavior in the future rather than at the present time.19 Thus, behavioral confidence is defined as how sure an individual is that he/she can perform a behavior change in the future. Furthermore, behavioral confidence acknowledges that an individual’s source of confidence is not exclusively internal. Thus, behavioral confidence may be derived from external sources such as important people in life, higher being, health educator, etc.19 The changes in physical environment construct only pertains to the physical environment and entails modifying the “obtainability, availability, accessibility, convenience, and readiness of resources.”24 Figure 1 presents constructs in initiation of health behavior change in MTM. Within the MTM, three constructs are posited to influence the sustenance of health behavior change or modification for health behavior change in the long-term. According to the MTM, emotional transformation, practice for change, and change in social environment explain and predict the sustenance of health behavior change.19 Emotional transformation involves collecting one’s own emotions and directing and transforming those emotions toward the health behavior change. Practice for change is derived from the praxis construct within Freire’s 22 adult education model, which refers to dynamic reflection and reflective behavior. The practice for change construct entails continually ruminating the health behavior change, integrating modifications to existing behavior change strategies, managing barriers, and maintaining focus on the health behavior change. The final MTM construct is change in social environment. The change in the social environment construct entails developing social support within the environment. A variety of professionals including health educators, nurse educators, health coaches, dieticians, etc. may help facilitate transformations in the social environment, and this change may be artificial or natural.24 Figure 2 presents constructs in sustenance of health behavior change in MTM. Research suggests that public health and health promotion interventions that employ theoretical models rooted in the social and behavioral sciences are more effective than a theoretical interventions.25,26 However, theories should be empirically tested prior to being utilized for intervention development, implementation, or evaluation.27 Therefore, the purpose of this study was to test the utility of the MTM in predicting initiation and sustenance of small portion size consumption among college students. This study offers theoretical evidence regarding the efficacy of the MTM, which will guide the development of healthy portion size interventions targeting college students. Materials and Methods Study design, population and sampling The present study utilized a cross-sectional design. The population for the study was college students, more specifically college students at a large size Southern public University in United States. The G*Power sample size calculation for regression modeling showed that a minimum of 114 participants were required to achieve a statistical power of 0.80 at an alpha level of 0.05 with 0.10 (medium) effect size and three predictors in the equation.28 This sample size was inflated by 15% for any missing values to arrive at a sample size of 131. While not ideal for structure equation modeling needed for confirmatory factor analysis, previous Monte Carlo studies suggest that this sample size was sufficiently powered to evaluate the hypothesized measurement models.29 This study utilized online quota sampling procedures. Participants were eligible to participate in this study if they were undergraduate or graduate students over the age of 18 who self-reported consuming a large portion size at a meal within the past 24 hours. The independent variables were constructs of MTM and the dependent variables were intention to initiate behavior change of eating small portion size and intention of continuing to eat small portion sizes. Instrumentation The instrument consisted of 35 items, of which, seven asked respondents about their standard socio-demographic information (gender, age, ethnicity, class level, current grade point average, location of living, and work status). An additional 28 items measured the following MTM constructs for the two models: Initiation model Advantages (participatory dialogue) were measured with five items (i.e., if you consume a small portion size at every meal you will… “be healthy,” “feel relaxed,” “manage your weight,” “have more energy,” and “enjoy life more”). Each item was scored on a five-point scale (0 = never to 4 = always). Responses for individual items were added together for maximum possible score (ranging from 0–20). Disadvantages (participatory dialogue) were measured with five items (i.e., if you consume a small portion size at every meal you will … “feel tired,” “be hungry most of the time,” “have less energy,” “get sick,” and “have less enjoyment”). Each item was scored on a five-point scale (0 = never to 4 = always). Responses for individual items were added together for maximum possible score (ranging from 0–20). In order to achieve the score on participatory dialogue (ranging from -20 – + 20), the total possible score of disadvantages was subtracted from the total possible score of advantages. Behavioral confidence was assessed using five items. Participants were asked about their level of certainty to consume a small portion size in every meal “this week,” “this week while finding time to complete all academic/work-related task,” “this week while finding time for leisure,” “this week without feeling tired,” and “this week without feeling hungry.” Each item was scored on a five-point scale (0 = not at all sure to 4 = completely sure). Responses for individual items were added together for maximum possible score (ranging from 0–20). Changes in physical environment was assessed using two items that asked participants about their level of certainty to “be able to eat a small portion size at a restaurant” and “be able to refuse a large portion size at a meal.” Each item was scored on a five-point scale (0 = not at all sure to 4 = completely sure). Responses for individual items were added together for maximum possible score (ranging from 0–8). To measure initiation, participants were asked “how likely is it that you will eat small portion sizes at every meal in the upcoming week?” This item was followed by five-point response scale (not at all likely = 0 to completely likely = 4). Sustenance model Emotional transformation was assessed using three items that asked participants about their level of certainty of “directing feelings/emotions,” “motivating themselves,” and “overcoming self-doubt” to eat small portion sizes at every meal. Each item was scored on a five-point scale (0 = not at all sure to 4 = completely sure). Responses for individual items were added together for maximum possible score (ranging from 0–12). Practice for change was assessed using three items that asked participants about their level of surety of “keeping a self-diary to monitor eating small portion sizes at every meal,” “be able to eat small portion sizes at every meal if you encounter barriers,” and “change your plan for eating small portion sizes at every meal if you face difficulties.” Each item was scored on a five-point scale (0 = not at all sure to 4 = completely sure). Responses for individual items were added together for maximum possible score (ranging from 0–12). Changes in social environment was assessed using three items that asked participants about their level of surety of asking help from “family member,” “friend,” and “health professional” to support you eating small portion sizes at every meal. Each item was scored on a five-point scale (0 = not at all sure to 4 = completely sure). Responses for individual items were added together for maximum possible score (ranging from 0–12). To measure sustenance, participants were asked “how likely is it that you will eat small portion sizes at every meal from now on?” This item was followed by five-point response scale (not at all likely = 0 to completely likely = 4). Face and content validity A total of six experts in the area of health behavior research were selected from multiple institutions to establish face and content validity of the instrument. Experts were requested to provide qualitative evaluation of the items. Based on experts’ recommendations, instrument was revised over a two-round process. The Flesch Kincaid Reading Ease of the instrument was 47.4 and Flesch-Kincaid Grade level of the instrument was 8.5 and thus acceptable for administration to college students.30 Construct validity In order to determine the factor structure, we performed a confirmatory factor analysis (CFA) in which we analyzed covariance matrices applying maximum-likelihood estimation using Mplus version 7.28. We used four indices to determine how well our models fit the data:31 chi-square, root mean square error of approximation (RMSEA), comparative fit index (CFI), and standardized root mean square residual (SRMR). RMSEA values of 0.06 or less, in conjunction with CFI values of 0.95 or greater were considered indicative of good fit. Models were considered to have adequate fit if they met the less stringent, but traditionally accepted, values of 0.90 or greater for CFI, and values less than 0.08 for RMSEA. We also included SRMR because it has been identified as the index that is most sensitive to miss-specified factor covariances or latent structures. For SRMR, values less than 0.10 are acceptable, with values less than 0.08 being preferred. Reliability Cronbach’s alpha was calculated to determine internal consistency reliability. The results of Cronbach’s alpha for all subscales are reported in the Results section. Data analyses Descriptive statistics were performed on all study variables. Data were checked for the assumptions of multiple regression (i.e., homoscedasticity, existence, independence, linearity, and normality). Stepwise multiple regression modeling for initiation and sustenance were conducted separately to determine best possible predictors of portion size behavior change while controlling for the socio-demographic variables namely age, race, and gender. For stepwise multiple regression procedure, the a priori probability levels for F to enter the predictor in the model and F to remove the predictor from the model were chosen as ≤ 0.05 and ≥ 0.10, respectively. All statistical analyses were conducted using SPSS (version 20.0). For gender the reference category was females and for race the reference category was other races as compared to Whites. Results A total of 135 participants completed this study. The mean age of the study sample was 23.27 (SD: 6.11) years. The majority of participants (62.2%) were women. Whites represented 77.8% of the sample. Participants’ characteristics are presented in Table 1. The path diagram in Figure 3 presents the findings for the CFA in Model 1. Fit for the model was good: χ2=239.40 (df=126), P<0.001, χ2/df=1.90, CFI=0.90, RMSEA=0.08 (90% CI=0.07-0.10), SRMR=0.06. Additionally, all item loadings were significant at P<0.001. Latent covariances ranged from −0.75 between advantages and disadvantages, to 0.53 between initiation and behavioral confidence. Chi-square difference tests showed that an alternative one-factor model achieved poorer fit (χ2=625.29 (df=135), P<0.001, CFI=0.58, RMSEA=0.16, SRMR=0.12). The path diagram in Figure 4 presents the findings for the CFA in Model 2. Fit for the model was good: χ2=47.51 (df=30), P<.05, χ2/df=1.58, CFI=0.97, RMSEA=0.07 (90% CI=0.25-0.10), SRMR=0.04. Additionally, all item loadings were significant at P<0.001. Latent covariances ranged from 0.20 between emotional transformation and sustenance, to 0.64 between practice for change and changes in social environment. Chi-square difference tests showed that an alternative one-factor model achieved poorer fit: χ2=243.60 (df=35), P<0.001, χ2/df=6.96, CFI=0.68, RMSEA=0.21 (90% CI=0.19-0.24), SRMR=0.12. In sum, the analyses for both models support the hypothesized factor structure of the variables. Table 2 presents reliability coefficient of the subscales and the scale as a whole. As shown in Table 2, the Cronbach’s alpha for all subscales were over 0.70 except for the subscale on physical environment which was close to 0.65. In behavioral and social sciences, scales with Cronbach’s alpha greater than 0.70 are considered respectable and those around 0.65 are considered minimally acceptable. From Table 2 it is also evident that the mean score for the construct advantages was 10.25 units (SD: 3.71) which indicated that the participants’ sometimes view eating small portion sizes as beneficial. For the disadvantages construct, the mean score was 9.31 units (SD: 3.73) which showed that participants’ sometimes view eating small portion sizes as disadvantageous. With regard to behavioral confidence, the mean of 5.25 units (SD: 4.79, median 5, range 0-20) indicated that the participants were less sure to eat small portion sizes. The mean score for changes in physical environment was 2.47 units (SD: 1.95) which demonstrated that participants were less sure to make changes in physical environment to eat small portion sizes. Finally, the participants had a mean of 1.01 units (SD: 1.08) which represented that participants were less likely to eat small portion sizes at every meal in the upcoming week. For the construct of emotional transformation, the mean score was 4.28 units (SD: 3.47) which indicated that participants were less sure in converting their emotions toward engagement in eating small portion sizes. The mean score for the practice for change construct was 3.68 units (SD: 2.86) which showed that participants were less sure to prepare themselves to eat small portion sizes. With regard to changes in social environment, the mean of 3.68 units (SD: 2.86) indicated that participants were less likely to take help of family member or friend to eat small portion sizes. Finally, the participants had a mean of 0.63 units (SD: 0.95) which represented that participants were less likely to eat small portion sizes at every meal from now on. Table 3 depicts the results of stepwise multiple regression analysis for initiation model. It indicated that 37.1% of the variance in the initiation of small portion size consumption was explained by participatory dialogue (advantages outweighing disadvantages), behavioral confidence, age, and gender, F (4, 130) = 20.773, adjusted R2= 0.37, P<0.001. For gender, males were less likely to initiate small portion size consumption than females. The construct of physical environment was not significant. Table 4 depicts the results of stepwise multiple regression analysis for sustenance model. It indicated that 20.5% of the variance in the sustenance of small portion size consumption was explained by emotional transformation, changes in social environment, and race, F (3, 131) = 12.535, adjusted R2= 0.20, P<0.001. For race, Whites were less likely to sustain small portion size change than other races. The construct of practice for change was not significant. Discussion The purpose of this article was to use MTM of health behavior change to predict small portion size consumption in college students. The study found that for intention to initiate small portion size consumption the significant predictors were participatory dialogue (advantages outweighing disadvantages), behavioral confidence, age, and being female. Participatory dialogue that underscores the advantages outweighing the disadvantages and behavioral confidence have been found to be beneficial in other behaviors as well such as physical activity behavior in college students.32 A variant of behavioral confidence has also been used by Poelman and colleagues in an intervention aimed at altering portion control behavior.9 While no studies have been conducted on portion size and age, it seems logical to propose as age increases, weight increases, and people would be more inclined to employ weight management strategies such as initiating portion size control. Likewise, this study found that women were more likely to initiate small portion size consumption. This also makes intuitive sense as women are generally more diet conscious and likely to engage in reducing their portion sizes. Further, Gans and colleagues found that portion sizes of Black women were large for most food items and were keen to reduce those.33 In this study the construct of physical environment from MTM was not found to be significant in initiation of small portion size behavior. It seems the role of physical environment is limited. For example, even if a person is served a large portion size he or she has the choice to leave the food, thereby diminishing the role of physical environment on this behavior. On the whole, the initiation model predicted 37.1% variance in the intention to initiate small portion size consumption which is substantial for behavioral and social science studies. For intention to sustain small portion size consumption, this study found that it was explained by emotional transformation, changes in social environment, and race, F (3, 131)=12.535, P<0.001. For race, Whites were less likely to sustain small portion size change than other races. This study showed that being White decreases the chances of sustaining the intention of small portion size consumption. The constructs of emotional transformation and changes in social environment were significant; these constructs have been found to be significant in other behaviors as well such as physical activity behavior in college students.32 The construct of practice for change was not found to be significant in this study. This could be due to the fact that perhaps the respondents felt that keeping a diary to monitor portion sizes was too cumbersome. On the whole, the sustenance model predicted 20.5% variance in the intention to sustain small portion size consumption which is moderately substantial for behavioral and social science studies. Thus, the MTM appears to be a useful model for explaining both the initiation and sustenance of behavior change to small portion size consumption, and may be used in designing and evaluating health promotion interventions. Regression analyses also show that the constructs do not have much shared variance; hence, the constructs are independent of each other and are mutually exclusive, providing support for the application of the MTM to other health behaviors. The participants reported very low intention to initiate small portion size consumption behavior change (mean of 1.01 [SD: 1.08] units) and low intention to sustain change for small portion size consumption (mean 0.63 [SD: 0.95] units). This finding underscores the need for developing interventions to promote small portion size consumption in this target population. Also evident from the low scores is that such interventions may be difficult as the motivation to change in the target population is very low in this regard. However, MTM offers a robust framework to design such interventions. Limitations First, this research had a cross-sectional design which looks at all the variables at one time. As a result, temporality of association of variables cannot be established. Therefore, we cannot say that the MTM constructs occur before the portion size behavior. However, all the previous theories have indicated that the attitudinal and environmental constructs like the ones in MTM precede the behavior; consequently, we can also assume the same for portion size behavior in college students. Future studies need to utilize more robust study designs. Second, the real behavior has not been measured in this study; intention for initiation of behavior change and sustenance of behavior change served as proxies for the behavior, which can be considered a limitation of this study. However, previous theories, particularly theory of reasoned action and theory of planned behavior have used intentions as proxies for behavior and shown that intentions precede behavior.30 Hence, the operationalization of behavior the way it was done in this study is justified. Future studies can operationalize behavior more objectively. Third, the instrument utilized in this study was based on self-report which is subject to measurement bias. Self-report, especially when it comes to assessing one’s portion size, can lead to recall bias, dishonesty, false reporting, under reporting, extreme reporting and other biases. However, there are no other methods to assess attitudes, therefore, this limitation must be considered within that context. Fourth, since this was a convenience quota sample the results for this study are only applicable to this sample and strictly speaking cannot be generalized or are not externally valid. However, sample size estimation was performed and the purpose of the study was model testing for which the methods were appropriate. Finally, the test-retest (stability) reliability of the instrument was not done in this study. Hence, it cannot be concluded that the constructs measured in this study are indeed reliable over time. Test-retest reliability assessment should be mandatory for replication studies. Implications for practice It is clear from this study that there is a definitive need for designing and evaluating interventions to change portion size consumption behavior in college students. MTM offers a robust framework to design such interventions and evaluate them for efficacy and effectiveness. Such interventions can consist of one-on-one counseling, group interventions or campus wide campaigns. In order to impact initiation of small portion size consumption behavior the two constructs that this study found to be significant were participatory dialogue, which underscores the importance of advantages exceeding disadvantages, and behavioral confidence. Participatory dialogue is easy to implement in one-on-one counseling and group interventions where the facilitator (i.e., counselor, health educator, health education specialist, physician, other health care provider) can promote an open, two-way discussion of the advantages and disadvantages of the behavior, and swing the discussion in favor of the advantages. In a campus wide campaign, one would need to be innovative with regard to participatory dialogue where use of social media (i.e., Facebook, Twitter etc.) or emails may have to be employed in facilitating a two-way dialogue with a large audience. To build behavioral confidence, the behavior of small portion size consumption could be broken down into small steps, confidence may be built to perform the behavior in near future, and the person’s motivation be strengthened to reduce the portion size. This can be accomplished at the individual level through one-on-one counseling and at the group level by group discussion or other affective strategies such as role play. At the campus level, practices such as psychodrama can be utilized. In order to impact sustenance of small portion size consumption behavior, emotional transformation and changes in social environment should be targeted. For modifying emotional transformation, the participants should be trained to direct their emotions such as anger, frustration, anxiety, etc. toward a goal of consuming small portion sizes. The skills to continually self-motivate oneself and overcome self-doubt in achieving this goal must also be taught. This may be accomplished through one-on-one counseling or group dialogue, or for campus wide campaigns, in the form of campus-wide contests or interaction via social media. Finally, in order to influence the construct of social environment, support from family, friends, and health professionals should be mobilized for interventions at all three levels. Acknowledgements We would like to thank all the participants who participated in this research study. Ethical approval This research study was approved by the University Institutional Review Board (IRB). After IRB approval, an online questionnaire was sent out to students who had been enrolled in the spring semester of 2016. All research participants provided informed consent electronically. The data for the present study were collected over a three-week period. Two reminder emails were sent to students in the second and third week. Competing interests None to declare. Authors’ contributions Manuscript conceptualization: MS, HPC, VL, and VKN; Manuscript writing: MS, HPC, VKN, VL, PJ, and MAF; Literature review: HPC and VL; Instrument development: MS; Data collection: VKN and MAF; Data analysis: VKN, MS, and PJ; Data interpretation: MS, VKN, HPC, VL, PJ, and MAF. Table 1 Socio-demographic characteristics of the participants (n = 135) Summary statistics Age (years) 23.27 (6.11) Gender Male 51 (37.8%) Female 84 (62.2%) Race/Ethnicity White/Caucasian 105 (77.8%) African American 12 (8.9%) Asian American 7 (5.2%) American Indian 2 (1.5%) Hispanic American 2 (1.5%) Other 7 (5.2%) Class level Freshmen 21 (15.6%) Sophomore 24 (17.8%) Junior 25 (18.5%) Senior 26 (19.3%) Graduate 39 (28.9%) Current overall GPA Less than 1.99 1 (0.7%) 2.00–2.49 5 (3.7%) 2.50–2.99 20 (14.8%) 3.00–3.49 40 (29.6%) 3.50–4.00 69 (51.1%) Living arrangements On campus 36 (26.7%) Off-campus 99 (73.3%) Work Status Yes 72 (53.3%) No 63 (46.7%) Mean (SD) is presented for age and n(%) for other variables. Table 2 Descriptive statistics of study variables (n=135) Constructs Possible Range Observed Range Mean (SD) Cronbach’s alpha Initiation 0–4 0–4 1.01 (1.08) – Participatory dialogue: advantages 0–20 0–20 10.25 (3.71) 0.84 Participatory dialogue: disadvantages 0–20 0–20 9.31 (3.73) 0.84 Participatory dialogue: advantages – disadvantages score -20 – +20 -20 – +17 0.94 (6.68) – Behavioral confidence 0–20 0–20 5.25 (4.79) 0.90 Changes in physical environment 0–8 0–8 2.47 (1.95) 0.63 Sustenance 0–4 0–4 0.63 (0.95) – Emotional transformation 0–12 0–12 4.28 (3.47) 0.90 Practice for change 0–12 0–12 3.68 (2.86) 0.73 Changes in social environment 0–12 0–12 3.68 (2.86) 0.76 Entire scale – – – 0.81 Table 3 Parameter estimates based on stepwise regression analysis to predict initiation of portion size consumption behavior change (n = 135) Variables B SE B β 95% CI P value Participatory dialogue (advantages outweighing disadvantages) 0.035 0.012 0.214 0.010–0.059 0.006 Behavioral confidence 0.101 0.017 0.447 0.067–0.135 <0.001 Age 0.035 0.012 0.200 0.011–0.060 0.005 Gender (males) -0.411 0.153 -0.185 -0.714 – -0.108 0.008 F(4, 130) = 20.773, P < 0.001, R2(Adjusted R2) = 0.390 (0.371). Dependent variable is initiation of physical activity behavior change; B = unstandardized coefficient; SEB = standard error of the coefficient; β = standardized coefficient; P = level of significance; CI = confidence interval. Table 4 Parameter estimates based on stepwise regression analysis to predict sustenance of portion size consumption behavior change (n=135) Variables B SE B β 95% CI P value Emotional transformation 0.074 0.022 0.272 0.030–0.119 0.001 Changes in social environment 0.050 0.023 0.174 0.004–0.096 0.033 Race (Whites) -0.614 0.178 -0.269 -0.967 – -0.261 0.001 F(3, 131) = 12.535, P < 0.001, R2(Adjusted R2) = 0.223 (0.205). Dependent variable is sustenance of physical activity behavior change; B = unstandardized coefficient; SEB= standard error of the coefficient; β = standardized coefficient; P = level of significance; CI = confidence interval. Figure 1 Constructs in initiation of health behavior change in multi-theory model (MTM) of health behavior change. Figure 2 Constructs in sustenance of health behavior change in multi-theory model (MTM) of health behavior change. Figure 3 Confirmatory factor analysis for initiation model. Abbreviations: adv, advantages; dis, disadvantages; behcon, behavioral confidence; phys, changes in physical environment; init, initiation. All item loadings are significant to P<0.001. Figure 4 Confirmatory factor analysis for sustenance model. emot, emotional transformation; prac, practice for change; chng, changes in social environment; sus, sustenance. All item loadings are significant to P<0.001. ==== Refs References 1 Ogden CL Carroll MD Kit BK Flegal KM Prevalence of childhood and adult obesity in the United States, 2011-2014 JAMA 2014 311 8 806 14 10.1001/jama.2014.732 24570244 2 U.S. Department of Health and Human Services. Managing overweight and obesity in adults systematic evidence review from the obesity expert panel, 2013. http://www.nhlbi.nih.gov/sites/www.nhlbi.nih.gov/files/obesity-evidence-review.pdf. Accessed May 20, 2016. 3 Ledikwe JH Ello-Martin JA Rolls BJ Portion sizes and the obesity epidemic J Nutr 2005 135 4 905 9 15795457 4 Cohen DA Story M Mitigating the health risks of dining out: The need for standardized portion sizes in restaurants Am J Public Health 2014 104 4 586 90 10.2105/AJPH.2013.301692 24524513 5 Collins CE Collins K Watson JF Food and beverage portion sizes in Australian children: a secondary analysis of 1995 and 2007 national data BMC Public Health 2014 14 517 10.1186/1471-2458-14-517 24886094 6 Vermeer WM Steenhuis I Poelman MP Small, medium, large or supersize? The development and evaluation of interventions targeted at portion size Int J Obes 2014 38 S13 8 10.1038/ijo.2014.84 7 Bryant R Dunde L Portion distortion: a study of college students J Consum Aff 2005 39 2 399 408 8 Steenhuis IH Vermeer WM Portion size: review and framework for interventions Int J Behav Nutr Phys Act 2009 6 58 10.1186/1479-5868-6-58 19698102 9 Poelman MP de Vet E Velema E de Boer MR Seidell JC Steenhuis IHM PortionControl@HOME: Results of a randomized controlled trial evaluating the effect of a multi-component portion size intervention on portion control behavior and body mass index Ann Behav Med 2015 49 1 18 28 10.1007/s12160-014-9637-4 25142041 10 Bandura A. Social Foundations of Thought and Action: A Social Cognitive Theory. Englewood Cliffs, NJ: Prentice-Hall, Inc; 1986. 11 Ayala GX An experimental evaluation of a group-versus computer-based intervention to improve food portion size estimation skills Health Educ Res 2006 21 1 133 145 10.1093/her/cyh049 16100228 12 Brown RM Oler CH A food display assignment and handling food models improves accuracy of college students’ estimates of food portions J Am Diet Assoc 2000 100 9 1063 4 11019355 13 Byrd-Bredbenner C Schwartz J The effect of practical portion size measurement aids on the accuracy of portion size estimates made by young adults J Hum Nutr Diet 2004 17 4 351 7 15250844 14 Plotnikoff RC Costigan SA Williams RL Hutchesson MJ Kennedy SG Robards SL Effectiveness of interventions targeting physical activity, nutrition and healthy weight for university and college students: a systematic review and meta-analysis Int J Behav Nutr Phys Act 2015 12 45 10.1186/s12966-015-0203-7 25890337 15 Small M Bailey-Davis L Morgan N Maggs J Changes in eating and physical activity behaviors across seven semesters of college: Living on or off campus matters Health Educ Behav 2013 40 4 435 41 10.1177/1090198112467801 23232092 16 American College Health Association. American College Health Association-National College Health Assessment (ACHA-NCHA II) Reference Group Executive Summary – Fall 2015. Hanover, MD: American College Health Association; 2015. 17 Fedewa MV Das BM Evans EM Dishma RK Change in weight and adiposity in college students: A systematic review and meta-analysis Am J Prev Med 2014 47 5 641 52 10.1016/j.amepre.2014.07.035 25241201 18 Gropper SS Simmons KP Connell LJ Ulrich PV Changes in body weight, composition, and shape: A 4-year study of college students Appl Physiol Nutr Metab 2012 37 6 1118 23 10.1139/h2012-139 22978391 19 Sharma M Multi-theory model (MTM) for health behavior change WebmedCentral Behaviour 2015 6 9 WMC004982 20 Prochaska JO. Systems of Psychotherapy: A Transtheoretical Analysis. Homewood, IL: Dorsey Press; 1976. 21 Rosenstock IM Historical origins of the health belief model Health Educ Behav 1974 2 4 328 35 22 Friere P. Pedagogy of the Oppressed. New York: Herder and Herder; 1970. 23 Ajzen I The theory of planned behavior Organ Behav Hum Decis Process 1991 50 179 211 24 Sharma M. Theoretical Foundations of Health Education and Health Promotion. Sudbury: Jones & Bartlett Learning; 2017. 25 Ammerman AS Lindquist CH Lohr KN Hersey J The efficacy of behavioral interventions to modify dietary fat and fruit and vegetable intake: a review of the evidence Prev Med 2002 35 1 25 41 12079438 26 Glanz K Bishop DB The role of behavioral science theory in development and implementation of public health interventions Annu Rev Public Health 2010 31 399 418 10.1146/annurev.publhealth.012809.103604 20070207 27 Plotnikoff RC Lippke S Courtneya KS Birkett N Sigal RJ Physical activity and social cognitive theory: A test in a population sample of adults with type 1 or type 2 diabetes Appl Psychol 2008 57 4 628 43 10.1111/j.1464-0597.2008.00344.x 28 Faul F Erdfelder E Lang AG Buchner A G* Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences Behav Res Methods 2007 39 2 175 91 17695343 29 Wolf EJ Harrington KM Clark SL Miller MW Sample size requirements for structural equation models an evaluation of power, bias, and solution propriety Educ Psychol Meas 2013 73 6 913 34 25705052 30 Sharma M, Petosa RL. Measurement and Evaluation for Health Educators. Burlington, MA: Jones & Bartlett Learning; 2014. 31 Hu LT Bentler PM Cutoff criteria for fit indexes in covariance structure analysis: conventional criteria versus new alternatives Struct Equ Modeling 1999 6 1 1 55 32 Nahar VK Sharma M Catalano HP Ickes M Johnson P Ford MA Testing multi-theory model (MTM) in predicting initiation and sustenance of physical activity behavior among college students Health Promot Perspect 2016 6 2 58 65 10.15171/hpp.2016.11 27386419 33 Gans KM Risica PM Kirtania U Jennings A Strolla LO Steiner-Asiedu M Dietary behaviors and portion sizes of black women who enrolled in SisterTalk and variation by demographic characteristics J Nutr Educ Behav 2009 41 1 32 40 10.1016/j.jneb.2008.05.013 19161918

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==== Front Health Promot PerspectHealth Promot PerspectHealth Promot PerspectTBZMEDHealth Promotion Perspectives2228-6497Tabriz University of Medical Sciences 10.15171/hpp.2016.23Original ArticleRasch analysis of the Persian version of PedsQLTM Oral Health Scale: further psychometric evaluation on item validity including differential item functioning Lin Chung-Ying 1 Kumar Santhosh 2 Pakpour Amir H. 3 *1The Hong Kong Polytechnic University, Hung Hom, Hong Kong2Griffith University, Gold Coast, Australia3Social Determinants of Health Research Center, Qazvin University of Medical Sciences, Shahid Bahounar BLV, Qazvin, Iran* Corresponding Author: Amir H Pakpour; Tell: +98-28-33239259; Pakpour_Amir@yahoo.com, apakpour@qums.ac.ir2016 10 8 2016 6 3 145 151 04 4 2016 10 5 2016 © 2016 The Author(s).2016This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.http://journals.tbzmed.ac.ir/HPP Background: The study aimed to further evaluate the psychometric properties of one recently developed oral health related quality of life (OHRQoL) instrument (PedsQL Oral Health Scale), including student self-report and parent-proxy report. Specifically, we tested the item validity,threshold order, local dependency, and differential item functioning (DIF) across gender and rater. Methods: This is a cross-sectional study, and study population was recruited in Qazvin, Iran using one-stage sampling with the unit of school. Students and their parents (1529 dyads) separately completed the Persian version of PedsQL Oral Health Scale. The psychometric properties were analyzed using Rasch rating scale model, including item validity, threshold order for response categories, and DIF across gender (boys vs. girls in student self-report) and rater (student self report vs. parent-proxy report). Results: All items had satisfactory in fit and outfit mean square error. One disordering category (the response of often) was found in parent-proxy report, while all categories were ordered in student self-report. All items were DIF-trivial across gender and rater. Conclusion: PedsQL Oral Health Scale is a valid instrument to measure OHRQoL. However, our results indicated that the parent-proxy report was inferior to the student self-report, and healthcare providers should primarily use the student self-report. ChildrenConstruct validityDifferential item functioningOral healthQuality of lifeRaschCitation: Lin CY, Kumar S, Pakpour AH. Rasch analysis of the Persian version of PedsQLTM Oral Health Scale: further psychometric evaluationon item validity including differential item functioning. Health Promot Perspect. 2016;6(3):145-151. doi: 10.15171/hpp.2016.23. ==== Body Introduction Over the last decades, the concept of quality of life (QoL) in relation to general and oral health has received increased attention,1 and many instruments have been developed because of this growing importance.2 However, the development of valid self-reported measures of oral health related QoL (OHRQoL) in children has occurred only in the last decade, prior to which parents were used as proxy.3 Using child self-report to accurately measure the QoL for children is a trend,4,5 and several child self-reported OHRQoL instruments have been proposed and found to be valid, viz., Child Oral Health-related Quality of Life,6 Child Oral Health Impact Profile,7 Child Oral Impacts on Daily Performances8 and Scale of Oral Health Outcomes for 5-year-old children.9 In addition to the above mentioned OHRQoL instrument, a feasible and efficient measure called Pediatric Quality of Life InventoryTM (PedsQL) Oral Health Scale has been developed very recently in 2009. The PedsQL Oral Health Scale has been designed as a 5-item questionnaire including child self-reports and parent-proxy reports. In addition, the PedsQL Oral Health Scale can be used along with PedsQL 4.0 Generic Core Scales and disease-specific modules because oral health stands as a specific condition which is not measured by generic and disease-specific instruments.10 Furthermore, the PedsQL Oral Health Scale has been translated to Brazilian Portuguese11 and Persian1 versions with acceptable validity and reliability. Although PedsQL Oral Health Scale has satisfactory psychometric properties among different language versions,1,10,11 we considered its evaluation is still underdeveloped. In order to well understand the properties of the QoL instruments, including the strengths and drawbacks, psychometric theories involved in developing the rating scales are suggested.12 Classical test theory (CTT) and item response theory (IRT) are two types of analytical strategies which can be used for this purpose.13 CTT emphasizes on the total summated scores in contrast to IRT which measures the properties of each individual item in reference to the data related to the latent trait.12 IRT allows analyzing scoring data by modeling both item and respondent characteristics concurrently and thus is advantageous over CTT.14 Therefore, IRT has become a method of choice and state of art in psychometric evaluation. There are three types of IRT model based on the estimated parameters, and the simplest model is the one-parameter logistic model, which is well-known as the Rasch model.15 Rasch analysis is based on assumptions that the scale is unidimensional and a person’s response to each item is independent of their response on other items.12 The Rasch model is based on the concept put forward by a Danish mathematician, Georg Rasch,16 and it involves testing the summated ordinal score obtained from multi-item instruments against the Rasch measurement model.17 The fit statistics obtained from Rasch analysis demonstrate the extent to which various items in the instrument describe the group and that how well the subjects fit the group.16,17 Rasch analysis also helps in evaluating the psychometric properties, such as item difficulty hierarchy and person separation statistics.18 The Rasch rating scale model has been widely used in psychometric analysis of various OHRQoL questionnaires19,20 and various modules of PedsQL.12,17 However, to the best of our knowledge, PedsQL Oral Health Scale has never been examined using Rasch analysis. In this study, therefore, we aimed to evaluate the validity of Persian PedsQL Oral Health Scale using Rasch analysis. Materials and Methods The study was a cross-sectional study that conducted in Qazvin (a city near Tehran with a population of about 453554 inhabitants.) between May to September 2014. The socioeconomic status in Qazvin is comparable to the average of Iranian. Participants were secondary school students of ages 13-18 years. The students were approached through a list of high schools in the Qazvin city, and the potential candidates were public schools. A one-stage sampling procedure was used: The stage unit was school. Eight schools were randomly selected from 47 high schools in the Qazvin city. Letters detailed describing the project and informed consents were given to all students of the eight schools. Students with intellectual disability (as assessed by a trained psychiatrist) were excluded from the study. The students took the letters and consents home to inform their parents, and those who returned their written informed consents were included in the study (n=1529). The PedsQL Oral Health Scale was completed by the students in their classroom under supervision while the parents filled the forms at home. Measures Demographic characteristics A questionnaire was used to gather information on the socioeconomic characteristics, including age, gender, frequency of dental brushing and dental flossing, parents’ educational level and household income. Oral health related quality of life The OHRQoL was assessed by the PedsQL Oral Health Scale.10 The PedsQL Oral Health Scale is a self-reported measure with five items (Appendix A). There are two parallel forms for the PedsQL Oral Health Scale: A child self-report form and a parent-proxy report form. Because our participants were all students, the child self-report in our study was defined as student self-report. All scores are rated on a 5-point Likert scale ranging from 0 (never a problem) to 4 (almost always a problem) and each item score is linearly transformed into a 0-100 scale. The average score of the 5 item can then be calculated to represent the total score of PedsQL oral health scale, and a higher score indicates a higher OHRQoL. The Persian version of the PedsQL Oral Health Scale was found to be highly valid and reliable for using in Iranian children and adolescents.1 For example, the test-retest reliability for 1 month is high (intraclass correlation coefficient=0.86 and 0.81), the internal consistency is excellent (α=0.79 and 0.89), construct validity is supported (comparative fit index=0.99; root mean square error of approximation=0.028 and 0.052), and known-group validity is good (children with decayed, missing and filled teeth have significantly lower score in PedsQL Oral Health Scale than those without decayed, missing and filled teeth do). Data analysis Sample size for Rasch analysis should be at least of 25 × numbers of categories in the response.27 Because we used 5-point Likert scale, the participants should be more than 25 × 5=125, and our participant number (n=1529) was sufficient. Before testing psychometric properties of each item, we reversely recorded the item scores of the PedsQL Oral Health Scale. That is, we used 0 to represent almost always a problem, and 4 to represent never a problem. All the following Rasch analyses used the recorded scores. We applied the Rasch rating scale model (RSM) to examine the PedsQL Oral Health Scale, respectively for the student self-report and the parent-proxy report, and used infit mean square error (MnSq) and outfit MnSq to test the unidimensionality of each item. The criteria of infit and outfit MnSq were set at 0.6 to 1.4,12 and MnSq> 1.4 suggests an out-of-concept item, while MnSq <0.6 a redundant item.21 Item difficulty with the unit of logit (a standardized score with mean as 0 and SD as 1) was also calculated for each item in both student self-report and parent-proxy report. In addition to testing unidimensionality, we also examined the separation reliability and indices, threshold order, local dependency, and differential item functioning (DIF) for the PedsQL Oral Health Scale. The separation reliability include person separation reliability (measuring the reproducibility of the person ordering) and item separation reliability (detecting the reproducibility of the item difficulty); separation index include person separation index (discriminating the respondents into different clusters based on respondents’ ability) and item separation index (separating the items into different levels based on items’ difficulty).22 The acceptable values were 0.7 for reliability and 2 for index.12,23 The threshold order was examined using average measure, step measure, and fit statistics. Because we anticipated that the response should be located in their expected order (i.e., the response of 0 should be to easier than the response of 1, 1 easier than 2, and so on), both average and step measures should monotonically increase with the responses.24 In addition, some researchers12,23 suggest using the infit and outfit MnSq with the range of 0.6 and 1.4 to additionally examine the threshold order. The local dependency was evaluated using Pearson correlations (r) of the Rasch residuals between every two items, and an r=0 indicates perfect independent for the two items. However, the 0 relationship is practically unrealistic,25 and an alternative is using an absolute r ≤ 0.4.21 The DIF analysis was conducted using only student self-report for gender, and using both student self-report and parent-proxy report for rater. The DIF across gender examined that whether males and females interpret the items of PedsQL Oral Health Scale differently, and DIF across rater investigated that whether students and parents perceive a different OHRQoL for students. Ideally, the items should be DIF-free (i.e., a non-significant t test for two groups), while DIF-trivial (i.e., a DIF contrast < 0.5 logits, which means an odds ratio of 1.65) is also acceptable, for researchers comparing item scores across groups.26 In addition, the missing data did not impact the estimation in Rasch because the expected marginal scores are computed from non-missing observations, and missing data were skipped over in these additions.27 All Rash analyses were done using Winsteps.27 Results In total, 1529 students participated in the study, and their mean (SD) age was 15.05 (3.16) years. Most of the students were female (54.61%). The mean educational years in school for father and mother were 8.35 (5.43) and 6.59 (4.63) years, respectively. Approximately one fifth (n=321) of the students reported that they brushed their teeth twice a day. Only about one fifth (n=321) of students indicated that they brushed their teeth twice a day, and approximately half of the students reported that they never used dental floss (n=706). An average household monthly income was reported as US$ 246.62 with an SD of 178.88. Participant demographic characteristics are shown in Table 1. The performance of the rating scale is shown in Table 2, and the most difficult item was OH2 (Having tooth pain when eating or drinking something hot, cold, or sweet; 0.78 and 0.47 logits) and the easiest item was OH4 (Having gum pain; −1.01 and −1.12 logits). In addition, except for the item OH5 on parent-proxy report (Having blood on toothbrush after brushing teeth) had a slight misfit based on infit MnSq (1.41), all other items fit well in the underlying construct of OHRQoL. The person separation reliability and separation index were slightly low for both the student self-report (reliability=0.63 and index=1.32) and the parent-proxy report (reliability=0.72 and index=1.59). However, the item separation reliability and separation index were excellent for both the student self-report (reliability=0.99 and index=12.80) and parent-proxy report (reliability=0.99 and index=13.72). The performance of threshold order is presented in Table 3, and the average measure was monotonically increased by responses (i.e., the smallest value in 0 and the largest value in 4). Though all infit and outfit MnSq fell in the reasonable range, a disordering category (i.e., the response of 1) was found for parent-proxy report based on the step measure. We further visualized the threshold disorder in Figure 1, and we could clearly see that parents tended not to rate the response of 1 (i.e., often) as indicated by the circle. That is, the probability of rating shifted from 0 to 2. The probability of response 1 was lower than that of response 0 when the underlying ability for children was less than -1; the probability of response 1 was lower than that of response 0 when the underlying ability was greater than -1. No local dependency was found for student self-report (absolute r=0.14 to 0.38). However, two absolute r coefficients were higher than the recommendation though they were slightly higher (0.42 and 0.44; Table 4). In addition, all items were DIF-trivial across gender and rater though three items on parent-proxy report were not DIF-free across rater. Items OH2 (Having tooth pain when eating or drinking something hot, cold, or sweet; P<0.01) and OH4 (Having gum pain; P<0.01) were found to be more difficult for students than for parents; contrarily, item OH5 (Having blood on toothbrush after brushing teeth; P<0.01) was easier for students than for parents (Table 5). Table 1 Demographic characteristics of the sample of Iranian children n (%) Age (year)a 15.1 (3.2) Gender Boys 694 (45.4) Girls 835 (54.6) Father's educational yeara 8.4 (5.4) Mother's educational yeara 6.6 (4.6) Tooth brushing Never 111 (7.3) Less than once a month 78 (5.1) Less than once a week 82 (5.4) Once a week 216 (14.1) Once a day 721 (47.2) Twice a day 321 (21.0) Dental floss Never 706 (46.2) Less than once a month 169 (11.1) Less than once a week 184 (12.1) Once a week 225 (14.7) Once a day 245 (16.0) Monthly family income $0-500 489 (32.0) $500-800 802 (52.4) > $800 238 (15.6) aPresenting as mean (SD). Table 2 Item difficulty and fit statistics for PedsQL Oral Health Scale Scale and item n Mean (SD) Difficulty Infit Outfit Child self-report 1529 80.46 (18.95) OH1: I have tooth pain 1525 79.48 (25.71) 0.17 0.89 0.89 OH2: I have tooth pain when eating or drinking something hot, cold, or sweet 1526 72.26 (28.00) 0.78 0.91 0.88 OH3: I have teeth that are dark in color 1523 80.73 (23.12) 0.05 0.78 0.81 OH4: I have gum pain 1522 89.17 (21.85) -1.01 1.25 1.03 OH5: I have blood on toothbrush after brushing teeth 1527 81.04 (25.06) 0.01 1.35 1.33 Parent-proxy report 1496 74.99 (26.37) OH1: Having tooth pain 1525 74.26 (30.51) 0.15 0.86 0.86 OH2: Having tooth pain when eating or drinking something hot, cold, or sweet 1525 70.64 (31.36) 0.47 0.90 0.92 OH3: Having teeth that are dark in color 1525 75.77 (29.51) 0.02 0.82 0.89 OH4: Having gum pain 1520 85.02 (28.84) -1.12 1.11 0.87 OH5: Having blood on toothbrush after brushing teeth 1517 70.30 (35.80) 0.48 1.41 1.27 Table 3 Threshold disordering tests for PedsQL Oral Health Scale Average measure Step measure Infit MnSq Outfit MnSq Child self-report 0 = almost always -2.82 – 1.22 1.28 1= often -1.27 -1.45 1.05 1.06 2 = sometimes -0.12 -0.73 0.97 0.93 3 = almost never 1.23 0.30 0.95 0.95 4 = never 3.11 1.88 1.00 1.00 Parent-proxy report 0 = almost always -2.29 – 1.19 1.15 1 = often -1.05 -0.71a 0.84 0.79 2 = sometimes -0.19 -0.78 1.01 0.96 3 = almost never 0.94 -0.08 0.96 0.90 4 = never 2.80 1.57 1.06 1.02 a The response of 1 (often) is disordered. Table 4 Test for local dependency No. Item No. Item r Child self-report Parent-proxy report OH1 OH2 -0.24 -0.12 OH3 -0.15 -0.10 OH4 -0.16 -0.29 OH5 -0.38 -0.42a OH2 OH3 -0.24 -0.26 OH4 -0.28 -0.20 OH5 -0.37 -0.44a OH3 OH4 -0.19 -0.12 OH5 -0.29 -0.37 OH4 OH5 -0.14 -0.07 aAbsoluter > 0.4 Table 5 Test for differential item functioning (DIF) No. Item and description Difficulty DIF contrast a SE P Test for gender Male Female OH1: Having tooth pain 0.11 0.22 -0.11 0.08 0.17 OH2: Having tooth pain when eating or drinking something hot, cold, or sweet 0.84 0.74 0.10 0.07 0.16 OH3: Having teeth that are dark in color 0.08 0.02 0.07 0.08 0.42 OH4: Having gum pain -0.96 -1.05 0.09 0.10 0.37 OH5: Having blood on toothbrush after brushing teeth 0.08 0.07 -0.14 0.08 0.08 Test for rater Child Parent OH1: Having tooth pain 0.16 0.16 0.00 0.05 1.00 OH2: Having tooth pain when eating or drinking something hot, cold, or sweet 0.73 0.47 -0.26 0.05 <0.001 OH3: Having teeth that are dark in color 0.05 0.01 -0.04 0.06 0.45 OH4: Having gum pain -0.96 -1.17 -0.22 0.07 0.003 OH5: Having blood on toothbrush after brushing teeth 0.02 0.49 0.47 0.05 <0.001 a DIF contrasts were calculated as logit of Male (Child) minus logit of Female (Parent). For gender, a positive DIF contrast indicates that Male has a higher item score than does Female, and vice versa; for rater, a positive DIF contrast indicates that the item score on children’s oral-related quality of life is higher in Child reports than Parent reports, and vice versa. Figure 1 Probabilities of each response for PedsQL Oral Health Scale. Legends: 0=almost always; 1=often; 2=sometimes; 3=almost never; 4=never. (a) is for student self-report and (b) for parent-proxy report. Discussion To the best of our knowledge, this is the first study using Rasch analysis to examine the psychometric properties of the recently developed PedsQL Oral Health Scale. Generally speaking, PedsQL Oral Health Scale is a promising instrument for healthcare providers to capture the OHRQoL for students. Although our results found that PedsQL Oral Health Scale contained some limitations, of majority was in the parent-proxy report, the limitations were not substantial. The major weakness for parent-proxy report was the disordering response in the category of 1 (which indicates “often”), while other unsatisfactory properties were minor, such as the slightly high fit statistics and local dependency; slightly low person separation index. In contrast, almost all psychometric properties of student self-report were satisfactory, except for the close-to-acceptable person separation reliability and index. No DIF items were found across gender, while three DIF-trivial items were found across student self-report and parent-proxy report, which is reasonable and acceptable. The PedsQL Oral Health Scale has been confirmed as an appropriate OHRQoL instrument in terms of the original English version,10 the Brazilian Portuguese version,11 and the Persian version.1 Although the previous studies1,10,11 applied CTT to test the psychometric properties of PedsQL Oral Health Scale, the psychometric evidences are suggested to be reexamined using IRT or Rasch models.12,23 Using Rasch models helps healthcare providers to understand the psychometric properties that cannot be examined using CTT. As a result, we tried using Rasch analyses to reconfirm the feasibility and validity of PedsQL Oral Health Scale, and the results suggested that all items were embedded in the same construct (i.e., OHRQoL). In addition, our results of the satisfactory item separation reliability (0.99) outperformed the internal consistency (0.68 and 0.84 in English; 0.65 and 0.59 in Brazilian Portuguese; 0.86 and 0.81 in Persian versions) from previous CTT findings.1,10,11 Moreover, our person separation reliability (0.63 and 0.72) was similar to or lower than the above mentioned internal consistency values. Rasch analyses estimate the reliability separately for items and respondents, and CTT does not separate the items and respondents to estimate the reliability.12,16 Therefore, we concluded that the items of PedsQL Oral Health Scale are reliable, while the low internal consistency in previous studies may be attributable to respondents’ characteristics, as confirmed by our person separation reliability. The cognitive ability of children and adolescents are still under development, and parents may not fully understand their children’s oral health. Hence, person separation reliability and internal consistency were found to be low in our results and previous studies.10,11 Although the PedsQL Oral Health Scale exhibited the expected threshold ordering and acceptable item dependence in the student self-report, a disordered category and some high item dependencies were found in the parent-proxy report. Similarly, the parent-proxy of PedsQL Generic Core Scale is found to have two disordered categories.28 In the study of Amin et al,28 they found that the responses of “almost never” and “often” were disordered, while our results only found “often” being disordered. Amin et al28 further combined the response of “almost never” with that of “never”; the response of “almost always” with that of “often”, and found a substantial improve in the Rasch models. Though collapsing two categories into one somewhat can account for the disordered issues, we tended not doing so because we were unsure that whether “often” should be combined with “almost always” or combined with “sometimes”. Instead, we encouraged future empirical studies applying two sets of response (i.e., one set combined the responses of “often” and “almost always”, and the other combined those of “often” and “sometimes”) to further probe this issue. In terms of the DIF items across gender, our results are comparable to the PedsQL Generic Core Scale28 that there were no DIF items. In addition, we found three DIF-trivial items across student self-report and parent-proxy report. However, we considered that this is not a serious problem because of the following reason. The difference between student self-report and parent-proxy report is well documented in many QoL instruments,29-31 and the trend is using the student self-report as the primary measure. Parent-proxy was used for two purposes: one is to substitute the student self-report when the student is too young or too ill to answer a questionnaire5; the other is to understand that whether the parents underestimate or overestimate their students’QoL.32 Because the DIF was not substantial, we considered that the parent-proxy report of PedsQL Oral Health Scale can fulfill the two purposes mentioned above. There are some limitations in this study. First, all participants were recruited in the same city, with culture and socioeconomics might be various in different cities of the same country, generalizing our results to the entire Iran population should be cautioned. Second, students filled out a student self-report under the supervision of a research assistant, while parents completed a parent-proxy report at home. Therefore, we could make sure that students were concentrating while answering the questionnaire. However, we did not know whether parents paid enough attention on the questionnaire. This also somewhat explains that parent-proxy report had inferior properties to student self-report. Third, the parent-proxy reports were answered by heterogeneous raters (e.g., mothers and fathers), and the perspectives on students’ OHRQoL might be differed in different raters. Finally, we did not define the social class when recruited our participants, and this may affect our results. Conclusion In conclusion, PedsQL Oral Health Scale is a promising QoL instrument to help healthcare providers understand the OHRQoL for students. The student self-report demonstrated stronger properties than the parent-proxy report did, and we followed the trend to recommend using student self-report as the primary measure. In addition, future studies are warranted to further examine the disordered issue in the parent-proxy report. Ethical approval Permission for this study was obtained from the Organization for Education at Qazvin involved and the study protocol was approved by the ethics committee in the Qazvin University of Medical Sciences. Each participant was informed about the aims of the study and signed a consent form before participation and their data were kept confidential. Participation in the study was voluntary and the participants were free to leave the study at any stage. Competing interests All the authors declare that there is no conflict of interests. Authors’ contributions CYL and AHP were responsible for designing the study, analyzing and interpreting the data, and drafting the manuscript. AHP was responsible for data collection. CYL and SK interpreted the data, and revised the manuscript. SK participated in study conception and design and critical revision. All authors have read and approved the final manuscript. Appendix A. Pediatric Quality of Life Inventory™ (PedsQL™) Oral Health Scale Items Child Self-Report Item Content 1. I have tooth pain. 2. I have tooth pain when I eat or drink something hot, cold, or sweet. 3. I have teeth that are dark in color. 4. I have gum pain. 5. I have blood on my toothbrush after brushing my teeth. Parent-Proxy Report Item Content 1. Having tooth pain. 2. Having tooth pain when eating or drinking something hot, cold, or sweet. 3. Having teeth that are dark in color. 4. Having gum pain. 5. Having blood on toothbrush after brushing teeth. Reproduced with permission from J.W. Varni, Ph.D. Copyright © 1998. The PedsQL™ is available at http://www.pedsql.org. ==== Refs References 1 Pakpour AH Yekaninejad MS Zarei F Hashemi F Steele MM Varni JW The PedsQL Oral Health Scale in Iranian children: reliability and validity Int J Paediatr Dent 2011 21 342 52 10.1111/j.1365-263X.2011.01130.x 21489002 2 Koposova N Eriksen HM Widstram E Eisemann M Opravin A Koposov R Oral health-related quality of life among 12-year-olds in Northern Norway and North-West Russia Oral Health Dent Manag 2012 11 206 14 23208598 3 Barbosa TS Gaviao MB Oral health-related quality of life in children: part I. How well do children know themselves? A systematic review Int J Dent Hyg 2008 6 93 99 10.1111/j.1601-5037.2007.00276.x 18412720 4 Cheng CP, Luh WM, Yang AL, Su CT, Lin CY. Agreement of children and parents scores on Chinese version of Pediatric Quality of Life Inventory Version 4.0: further psychometric development. Appl Res Qual Life. 2015. doi: 10.1007/s11482-015-9405-z . 5 Limbers CA Newman DA Varni JW Factorial invariance of child self-report across age subgroups: a confirmatory factor analysis of ages 5 to 16 years utilizing the PedsQL 4.0 Generic Core Scales Value Health 2008 11 659 68 10.1111/j.1524-4733.2007.00289.x 18179671 6 Jokovic A Locker D Tompson B Guyatt G Questionnaire for measuring oral health-related quality of life in eight- to ten-year-old children Pediatr Dent 2004 26 512 8 15646914 7 Broder HL Wilson M Reisine S Phillips C Janal M Reliability and validity of the child oral health impact profile (COHIP) J Dental Res 2005 84 2652 8 Gherunpong S Tsakos G Sheiham A Developing and evaluating an oral health-related quality of life index for children; the CHILD-OIDP Community Dent Health 2004 21 161 9 15228206 9 Tsakos G Blair YI Yusuf H Wright W Watt RG Macpherson LM Developing a new self-reported scale of oral health outcomes for 5-year-old children (SOHO-5) Health Qual Life Outcomes 2012 10 62 10.1186/1477-7525-10-62 22676710 10 Steele MM Steele RG Varni JW Reliability and validity of the PedsQL™ oral health scale: measuring the relationship between child oral health and health-related quality of life Child Health Care 2009 38 3 228 44 10.1080/02739610903038818 11 Bendo CB Paiva SM Viegas CM Vale MP Varni JW The PedsQL Oral Health Scale: feasibility, reliability and validity of the Brazilian Portuguese version Health Qual Life Outcomes 2012 10 42 10.1186/1477-7525-10-42 22531004 12 Jafari P Bagheri Z Ayatollahi SM Soltani Z Using Rasch rating scale model to reassess the psychometric properties of the Persian version of the PedsQL 4.0 Generic Core Scales in school children Health Qual Life Outcomes 2012 10 27 10.1186/1477-7525-10-27 22414135 13 Sebille V Hardouin JB Le Neel T Kubis G Boyer F Guillemin F Methodological issues regarding power of classical test theory (CTT) and item response theory (IRT)-based approaches for the comparison of patient-reported outcomes in two groups of patients--a simulation study BMC Med Res Methodol 2010 10 24 10.1186/1471-2288-10-24 20338031 14 Waller J Ostini R Marlow LA McCaffery K Zimet G Validation of a measure of knowledge about human papillomavirus (HPV) using item response theory and classical test theory Prev Med 2013 56 35 40 10.1016/j.ypmed.2012.10.028 23142106 15 Furr RM, Bacharach VR. Psychometrics: An Introduction. Item Response Theory and Models. Thousand Oaks, CA: Sage Publications, Inc; 2008. 16 Prieto L Alonso J Lamarca R Classical test theory versus rasch analysis for quality of life questionnaire reduction Health Qual Life Outcomes 2003 1 27 10.1186/1477-7525-1-27 12952544 17 Pickles T Playle R Hood K Gillard J Robling M Rasch analysis of the PedsQL 3.0 diabetes module Trials 2013 14 O40 10.1186/1745-6215-14-S1-O40 18 Gothwal VK Wright TA Lamoureux EL Pesudovs K Rasch analysis of visual function and quality of life questionnaires Optom Vis Sci 2009 86 1160 8 10.1097/OPX.0b013e3181bab10c 19741562 19 Franchignoni M Giordano A Levrini L Ferriero G Franchignoni F Rasch analysis of the Geriatric Oral Health Assessment Index Eur J Oral Sci 2010 118 278 83 10.1111/j.1600-0722.2010.00735.x 20572862 20 Wong HM McGrath CP King NM Rasch validation of the early childhood oral health impact scale Community Dent Oral Epidemiol 2011 39 449 57 10.1111/j.1600-0528.2011.00614.x 21504439 21 Chang CC Su JA Tsai CS Yen CF Liu JH Lin CY Rasch analysis suggested three unidimensional domains for Affiliate Stigma Scale: additional psychometric evaluation J Clin Epidemiol 2015 68 674 83 10.1016/j.jclinepi.2015.01.018 25748074 22 Lin CY, Yang SC, Lai WW, Su WC, Wang JD. Rasch models suggested the satisfactory psychometric properties of the WHOQOL-BREF among lung cancer patients. J Health Psychol. 2015. doi: 10.1177/1359105315603474 . 23 Kook SH Varni JW Validation of the Korean version of the pediatric quality of life inventory 4.0 (PedsQL) generic core scales in school children and adolescents using the Rasch model Health Qual Life Outcomes 2008 6 41 10.1186/1477-7525-6-41 18518951 24 Jafari P Bagheri Z Safe M Item and response-category functioning of the Persian version of the KIDSCREEN-27: Rasch partial credit model Health Qual Life Outcomes 2012 10 127 10.1186/1477-7525-10-127 23078650 25 Wang WC Wilson M Exploring local item dependence using a random-effects facet model Appl Psychol Meas 2005 4 296 318 10.1177/0146621605276281 26 Shih CL Wang WC Differential item functioning detection using the multiple indicators, multiple causes method with a pure short anchor Appl Psychol Meas 2009 33 184 99 10.1177/0146621608321758 27 Linacre JM, Wright BD. A User’s Guide to WINSTEPS. Chicago: MESA Press; 2009. 28 Amin L Rosenbaum P Barr R Sung L Klaassen RJ Dix DB Rasch analysis of the PedsQL: an increased understanding of the properties of a rating scale J Clin Epidemiol 2012 65 1117 23 10.1016/j.jclinepi.2012.04.014 22910540 29 Cremeens J Eiser C Blades M Factors influencing agreement between child self-report and parent proxy-reports on the Pediatric Quality of Life Inventory 4.0 (PedsQL) generic core scales Health Qual Life Outcomes 2006 4 58 10.1186/1477-7525-4-58 16942613 30 Upton P Lawford J Eiser C Parent-child agreement across child health-related quality of life instruments: a review of the literature Qual Life Res 2008 17 895 913 10.1007/s11136-008-9350-5 18521721 31 Robitail S Simeoni MC Ravens-Sieberer U Bruil J Auquier P Group K Children proxies’ quality-of-life agreement depended on the country using the European KIDSCREEN-52 questionnaire J Clin Epidemiol 2007 60 469 478 10.1016/j.jclinepi.2006.09.007 17419958 32 Su CT Wang JD Lin CY Child-rated versus parent-rated quality of life of community-based obese children across gender and grade Health Qual Life Outcomes 2013 11 206 10.1186/1477-7525-11-206 24325683

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==== Front Health Promot PerspectHealth Promot PerspectHealth Promot PerspectTBZMEDHealth Promotion Perspectives2228-6497Tabriz University of Medical Sciences 10.15171/hpp.2016.24Original ArticlePsychometric evaluation of a self-reported physical activity questionnarie used in the pilot phase of the AZAR Cohort Study Mirzaei Maryam 1 Asghari-Jafarabadi Mohammad 2 Amini-Sani Nayyreh 3 Bakhtari-Aghdam Fatemeh 3 Dastgiri Saeed 4 *1Department of Biostatistics & Epidemiology, Tabriz University of Medical Science, Tabriz, Iran2Road Traffic Injury Research Center, Tabriz University of Medical Science, Tabriz, Iran3Department of Health Education & Promotion, Tabriz University of Medical Science, Tabriz, Iran4Tabriz Health Services Management Research Centre,Tabriz University of Medical Sciences, Tabriz, Iran* Corresponding Author: Saeed Dastgiri; Tell: +98 (41) 33364673; dastgiris@tbzmed.ac.ir2016 10 8 2016 6 3 152 158 18 2 2016 01 6 2016 © 2016 The Author(s).2016This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.http://journals.tbzmed.ac.ir/HPP Background: The purpose of this study was to assess the psychometric properties of a self-reported physical activity (PA) questionnaire based on data from the pilot phase of the AZAR Cohort Study. Methods: In this cross-sectional study, all 35-70 years old people living in Khameneh, a city in East Azarbaijan, Iran were invited to take part in the pilot phase of the AZAR Cohort Study. A total of 952 people completed the self-reported PA questionnaire and the International Physical Activity Questionnaire (IPAQ). Construct validity was evaluated by exploratory and confirmatory factor analyses (EFA and CFA). Spearman’s correlation coefficient between the scores of the two instruments was used to examine the concurrent validity. Reliability was measured using intraclass correlation coefficient (ICC) and Cronbach’s alpha coefficient. Results: In EFA applying principal component analysis with varimax rotation, four factors were identified including recreational leisure time (variance = 52.73%), sedentary leisure time (variance = 38.68%), household/gardening work (variance = 38.66%), and occupation work (variance = 12.67%). The extracted factors were also supported by the CFA (CFI = 0.98, GFI =0.936, RMSEA=0.057). The results indicated moderate concurrent validity (ρ = 0.62, P < 0.001). ICC and Cronbach’s alpha were 0.59 and 0.7, respectively. Conclusion: These results showed acceptable and moderate psychometric properties for the self-reported PA questionnaire to assess PA in this population-based study. Physical activityValidityReliabilityQuestionnaireCitation: Mirzaei M, Asghari-Jafarabadi M, Amini-Sani N, Bakhtari-Aghdam F, Dastgiri S. Psychometric evaluation of a self-reported physical activity questionnarie used in the pilot phase of the AZAR Cohort Study. Health Promot Perspect. 2016;6(3):152-158. doi: 10.15171/hpp.2016.24. ==== Body Introduction The link between physical activity (PA) and health status has been evaluated in various epidemiological studies. Physical inactivity poses a behavioral risk factor for some types of non-communicable diseases (NCDs) including cardiovascular diseases, stroke, high blood pressure, non–insulin-dependent diabetes mellitus, osteoporosis, and certain types of malignancies.1-5 Nevertheless, only a small proportion of individuals gets sufficiently adequate PA.6 PA measurement is hard to quantify due to its complex nature.7,8 In data collection at the population level, selecting appropriate and precise estimation method to measure PA as a variable is a challenging task for investigators.5-9 In comparison, a variety of methods have been used to assess PA, such as self-reports (interviews, diaries, and questionnaires) and doubly-labeled water at population-based studies. Self-reported PA questionnaires are usually chosen as the most feasible methods to assess PA. As such questionnaires are inexpensive, brief, and have general acceptance, they can be applied to measure the frequency, duration, and intensity of PA.10,11 The AZAR Cohort Study, initiated in 2014, is a population-based follow-up study conducted in East Azerbaijan province, Iran. Investigating the level of PA and its effects on the health status of the population in this prospective cohort study has been considered as a crucial task. Hence, a valid and reliable questionnaire was required to determine the habitual PA of the people in this large-scale epidemiological study.5 In general, several questionnaires have been validated to assess PA among adults, but each instrument has some disadvantages. 8 In order to conduct this cohort study, there was a need for a scale developed to measure PA by questionnaire only, which particularly designed for a large epidemiological study.12 This self-reported PA questionnaire was designed, developed, and validated by Aadahl and Jorgensen in 2003, and it is a PA scale with a simple usage to measure the level of PA among sedentary adults. This questionnaire was previously modified and validated among Danish adults.12,13 In different settings, the accuracy of the self-reported PA questionnaires depends on the diversity of the sociocultural and ethnic context. Therefore, in order to assure an accurate measurement of PA level in a specific population there is a need to examine the validation of the tool being used for the study.4 Although the Persian translation of this questionnaire was previously used for the Childhood & Adolescence Surveillance and Prevention of Adult Non-communicable Diseases (CASPIAN) study,14,15 among adult population, no validated Iranian version has been reported to date. Previous studies reported the concurrent validity of this self-report PA questionnaire.12,13 In those studies the findings of the questionnaire were compared with accelerometer, PA dairy questionnaire and Vo2max. Additional studies are needed to validate it among other target populations. In addition, the factor structure of the Persian version of the questionnaire needs to be established. Searching the literature no published study was found on the factor structure of this PA scale among Iranian adult population. The present study aimed to assess the validity of this self-reported PA questionnaire by examining the construct, discriminate and concurrent validities. Temporal reliability and internal consistency were also examined. Therefore, the primary objectives of this study were to explore the dimensionality and evaluate psychometric properties (validity and reliability) of the self-report PA questionnaire in the pilot phase of the AZAR Cohort Study conducted on 35-70 years old population in Khameneh in 2014 Materials and Methods Sample and data collection The AZAR Cohort Study, a state-level of a nationwide cohort study (Persian cohort, http://persiancohort.com) in Iran, is a longitudinal study assessing risk factors related to the most prevalent NCD in East Azarbaijan province. This cohort study has been conducted by Tabriz University of Medical Sciences in Shabestar – a county located in East Azarbaijan province. All the invited people for taking part in this study were 35 to 70 years old and met the inclusion criteria (the permanent resident of this city, ability to response to the questions, Iranian originality). Exclusion criteria were refusal to participate in the study, being in travel out of the area during the study period and being with mental and physical disabilities. The pilot phase of the AZAR Cohort Study was conducted in Khameneh, a small town in Shabestar county between October 2014 and January 2015. The target participants for this investigation were elected from the framework of the pilot phase of the study which included 952 respondents (35-70 years, mean: 49.84, standard deviation: 8.82). During the first questionnaire survey (participation rate = 82%), the participants took part in in-site interviews by trained interviewers. The baseline socio-demographic characteristics including age, education, occupation, nutritional habits, medical history, and anthropometric data such as height and weight, as well as the self-report PA were investigated. Physical activity instrument In order to measure PA in this large sample, the validated self-reported PA questionnaire as a scale to assess PA was utilized, which has previously been shown to be valid and reliable in the Danish population.13 This classified self-reported PA, consisted of 23 items based on nine ranges that have different metabolic equivalent (MET) activities (from sleep/rest [0.9 METs] to high-intensity PA [>6 METs]). The participants had to report all domains of their PA, such as occupational PA (PA at work), recreational PA (leisure time PA), and exercise activity on an average weekday; in all domains, the amount of time spent on sedentary behaviors was also assessed. To estimate the MET-time scores, the times engaging in scales were multiplied by an estimate of the METs of the reported activity. Also, to achieve the same scale, MET-h was calculated from the MET-min in International Physical Activity Questionnaire (IPAQ), by adding the MET-time from all weekday and dividing by 60 minutes. Study procedures for psychometric tests The process of the translation and cultural adaptation was performed in previous studies by Kelishadi et al.14,15 Thus, the content validity using a qualitative manner was assessed by cohort investigators before the commencement of this study and some minor changes were made to revise the wording and structure of some sentences. The psychometric properties of the questionnaire included three specific methodological steps: First step: Reliability was evaluated by determining internal consistency (Cronbach’s α) and temporal stability which was assessed over a period of two weeks of test-retesting. In the first step of this process, 50 participants were recruited based on a list of random numbers for testing the reproducibility of the questionnaire at two time points. Second step: Concurrent form of criterion validity for the self-reported PA questionnaire was evaluated by comparing its total score with IPAQ – as a criterion measure. IPAQ is known as an accurate scale with a confirmed validity and reliability in an Iranian population.16 To answer the research questions concerning the association between the self-reported PA questionnaire and the IPAQ, a subset (n = 50) of the participants was also asked to complete the IPAQ. In addition, the Bland-Altman plot, 95% limits of agreement, was utilized to show graphically the agreement between the self-report PA and IPAQ. Third step: In order to determine the underlined structure of the items and test the hypothesized structures, the construct validity was determined by exploratory factor analysis (EFA) and confirmatory factor analysis (CFA). Furthermore, the construct validity was evaluated by performing the known groups’ comparison - as an additional approach to establishing construct validity. It was hypothesized that known groups (gender, education, and occupation subgroups) would report different total scores. The questionnaire was considered valid based on these criteria. Statistical analyses Temporal stability and internal consistency were measured using intraclass correlation coefficient (ICC) and Cronbach’s alpha coefficient, respectively. In this study, the ICC and Cronbach’s alpha more than 0.7 were considered as acceptable reliabilities.17,18 In order to conduct the known group analyses and the hypothesis that the total scores would be significantly different between the subgroups, the Kruskal-Wallis H test and the Mann–Whitney U test were performed to compare the subgroups. EFA and CFA approaches were implemented to identify the factor structure of the questionnaire. In the first step, principal component analysis (PCA) was used to extract the factors, with the assumption of the abnormality of the data distribution and the optimality of the procedure. Also, due to the independency of the factors, the varimax orthogonal rotation was applied in the EFA.19,20 Factor-item loading values were considered acceptable to offer an item to a factor if the value was equal to or greater than 0.20. The significant eigenvalues was considered equal to or greater than1.0. The Kaiser-Meyer-Olkin (KMO) method and Bartlett’s test of sphericity were performed to test the sampling adequacy. In the second step, the CFA model using the robust maximum likelihood was used to estimate model parameter. The absolute fit of the model to the data was evaluated using the χ2 statistic, root mean square error of approximation (RMSEA), goodness-of-fit index (GFI), adjusted goodness-of-fit index (AGFI), and the comparative fit index (CFI). Values of the GFI, AGFI, and CFI greater than 0.90, and the RMSEA value below 0.08 was acceptable as a good model fit.21All data analyses were performed by SPSS 23.0 (Chicago, IL, USA), also other complementary software according to the objectives presented in the related sections and the statistical significance level was set at P<0.05. Results General characteristics of the study participants Table 1 shows the characteristics of the total sample in the EFA (n = 952) and the sub-sample used to determine CFA (n = 572). The age range of the total sample was 35 to 70 years with a mean of 49.84 (SD = 8.82) years. The most of the participants were married (91.7%), 11.5% were with no formal education, and 50.7% were employed. In addition, the body mass index (BMI) value for 42.7% of the participants was in the range of 25-30 (overweight). Temporal stability and internal consistency The analysis of test-retest reliability with the method of ICC showed moderate temporal stability for self-reported PA items at two time points (0.59; 95% CI: 0.20–0.74). It should be noted that the log transformation improved normality and these values were used throughout the analysis. The internal consistency (coefficient Cronbach’s α) for the scale was 0.7, which indicated satisfactory internal consistency. Factorial (construct) validity EFA was conducted in the scale base to identify the factor model using all the observations. Based on the origin structure and the preliminary analyses of the items, it was found that the extracted factors may be divided into four sub-scales as detailed below: Inactive leisure time, 5 items (PB1-PB2-PA1-PA2-PA3) Household/gardening work, 3 items (PD1-PE1-PG2) Occupation work, 11 items (PC1-PC2-PC3-PD2-PF4-PH1-PE2-PG1-PF2-PF3-PG3). More specifically, the dimension reduction process was implemented for all the domains, separately, to identify underling potential factor (sub-scales). Table 2 shows the results of the scale based test of the item convergence validity. The final model found to be with four factors and 21 items (two items did not load on any factor [factor loadings < 0.2] and was removed): Factor 1 with 4 items and 2 sub-factors (variance = 52.73%), factor 2 with 6 items and 2 sub-factors (variance = 38.68%), factor 3 with 3 items (variance = 38.66%), and factor 4 with 10 items (variance = 12.67%). This model indicated that the extracted factors were suitable for the factor model in the observed dataset. The extracted sub-factors were named as sports (2 items), walking & bicycling (2 items), sitting (3 items), and sleep (3 items). The other recognized factors were given the same names as the basic factors of the underlying domains. In addition, the eigenvalues of all the domains were more than 1. The factor analysis results showed the value of the KMO measure of the sampling adequacy to be 0.69, and Bartlett’s test of sphericity showed the adequacy of the model (P<0.001). In order to achieve a CFA model with a good external validity, it is highly recommended to perform the CFA in a random subsample (a random sample drown from the main sample is normally satisfied).20 To do so, the CFA was conducted on the 21 items of the questionnaire with AMOS 23.0 software to test the fit of the final four-factor model. Sixty percent of the participants were considered as the sub-sample; through random sampling the data of 60% out of all the participants in the SPSS software were included in the CFA analysis. CFA supported the four-factor structure and displayed appropriate good fit to the data (χ2 [163] = 462.139, P<0.001; CFI = 0.98; GFI = 0.936; AGFI = 0.90; RMSEA [90% CI] = 0.057 [(0.51-0.063]). Moreover, all the standardized coefficients in the factor showed moderate correlations between the latent factors (Figure 1). Figure 1 Confirmatory factor analysis. Concurrent validity Spearman’s correlation coefficient between the scores (MET-time) of the self-report PA questionnaire and the IPAQ showed positive and moderate-to-good correlations (r = 0.62) between the factors, which was statistically significant (P<0.001). Also, the Bland-Altman plot indicated moderate agreement between the two instruments (Figure 1). In the Bland-Altman plot, the mean difference was -0.02 with wide 95% limits of agreement (-0.2 to 0.25), but four out of 50 values (8%) were outside the 95% limits of agreement. Known group’s analyses In the known group’s analysis, the item-discriminant validity aspect was tested by the relative test. Significant differences in MET-time score were observed only by job, marital status, and education subgroups. As it was expected, the difference was found in the MET-time score between employed and unemployed. The difference was statistically significant (P<0.05). Unemployed group had lower MET-time score. Also, as hypothesized, the Kruskal-Wallis H test revealed significant difference in the total score in MET-time by education and marital status (Table 3). Table 1 General characteristics of the study participants Variables Total sample for EFA (n=952) Subsample for CFA (n=572) No. (%) No. (%) Gender Male 440 (46.1) 256 (44.8) Female 514 (53.9) 316 (55.2) Age (years) 35-45 313 (32.8) 202 (35.3) 45-55 385 (40.4) 227 (39.7) 55-65 210 (22) 120 (21.0) ≥65 46 (4.8) 23 (4.0) Marital status Unmarried 24 (2.5) 17 (3.0) Married 875 (91.7) 525 (91.8) Divorce/widow 55 (5.8) 30 (5.2) Educational level No formal education 114 (11.9) 66 (11.5) Elementary 281 (29.5) 170 (29.7) Middle school 177 (18.6) 113 (19.8) High school 202 (21.2) 117 (20.5) College/ university and above 180 (18.9) 106 (18.5) Occupation status Employed 484 (50.7) 293 (51.2) None 470 (49.3) 279 (48.4) BMI (kg/m2) Underweight (BMI <18.5) 10 (1.0) 7 (1.2) Normal weight (18.5-25) 241 (25.3) 135 (23.6) Overweight (25-30) 407 (42.7) 235 (41.1) Obese (BMI ≥30) 296 (31.0) 195 (34.1) Abbreviations: BMI, body mass index; EFA, exploratory factor analysis; CFA, confirmatory factor analysis. Table 2 Exploratory factor loading (principal axis factoring extraction with varimax rotation) of the questionnaire itemsa Factor Subfactor Item Loading Factor 1. Recreational leisure time Sport PH2 0.729 PI 0.710 Commuting (Walking/bicycle) PE3 0.781 PF1 0.581 Factor 2. Sedentary leisure time Sitting PC3 0.769 PB2 -0.565 PB1 0.390 Sleep PA1 0.728 PA2 0.617 PA3 0.330 Factor 3. Household/gardening work PG2 -0.687 PD1 0.683 PE1 0.471 Factor 4. Occupation work PC1 -0.720 PD2 0.504 PG1 0.467 PF4 0.338 PH1 0.221 PE2 0.231 PF2 0.212 PC2 -0.211 aAll loadings above 0.20 are presented; the negative values show indirect relation between an item and a scale. Table 3 PA profile (MET-time scores) of the study participants Known groups Median (P25 to P75) N = 952 P value Total PA 35.25 (33.11-38.12) Employment <0.001a Unemployment 34.85 (32.91-37.45) Employment 35.63 (33.35-38.90) Marital status 0.026b Unmarried 34.15 (32.‏22-36.‏70) Married 35.38 (33.18-38.26) Divorce/widow 34.53 (31.40-36.70) Level of education <0.001b No formal education 35.13 (33-38.70) Elementary 35.83 (33.93-38.70) Middle school 34.98 (33.05-37.80) High school 35.48 (32.85-38.45) College/ university and above 34.43 (32.38-36.65) BMI (kg/m2) 0.527b <18.5 36.18 (34.15-39.60) 18.5-25 35.53 (33.43-38.26) 25-30 35.21 (33.08-38.15) >30 35.19 (32.93-37.84) Gender 0.285a Male 35.28 (32.83-39.14) Female 35.25 (33.30-37.63) Abbreviations: BMI, body mass index; PA, Physical activity; MET, metabolic equivalent. Median (Percentile 25 to Percentile 75) was reported. aMann-Whiteny U test; bKruskal-Wallis H test. Discussion Insufficient PA is considered as a behavioral risk factor for NCDs. Without a valid instrument, the associations between PA and health status may not be accurately evaluated and identified.22,23 The present study sought to determine whether the selected self-reported PA questionnaire was a valid scale to assess PA patterns among the adults elected for the AZAR Cohort Study. From the results of the present study confirmed the internal consistency of the scale. Moreover, moderate temporal stability of the questionnaire during two separate occasions (correlation coefficient = 0.60) was found. These findings were in line with those obtained in the study that assessed the PA among Iranian young adults (correlation coefficient = 0.87).14 Although the ICC value showed moderate temporal stability, the 95% CI was wide for the scores. This wide 95% CI for the ICC value could be due to the fact that the PA has a multinomial nature and it is not a stable behavior.7,8 However, it does not seem that the actual changes in the PA pattern of the subjects occurred during 2-weeks interval between the test-retest. The moderate coefficient of the correlation between the two occasions provided evidence for temporal validity of the self-reported PA questionnaire. This is the first investigation to address the EFA and CFA approach of the self-reported PA questionnaire. The results confirmed the factorial structure of the questionnaire in a sample of 952 adults. The sample size was adequate for factor analysis in the present study as the proportion of the sample size was based on more than 5 participants per item.24 The findings of the present study suggested that the questionnaire might have a four factors structure for the instrument, including recreational leisure time, sedentary leisure time, household/gardening work, and occupation work. The results showed that the factor loadings of the items PG3 and PF3 were less than 0.2. As, the considered cut-off value to retain an item in the scale was 0.2, these items were not included in the final model. Also, the CFA provided evidence to support the factor structure represented by these items. Therefore, the questionnaire assessed PA in four factors (domains) and four sub-factors with 21 items. A concern in the analysis of this instrument was its construct validity which had not been investigated previously. So, the identified model should be further assessed. However, the preliminary factor structure found in the present study was not different from those found in the original domains. Previously, some validation studies on the questionnaire have been conducted in Denmark and Iran, but the demographics and geographic contexts of the studies were different.13,14 In the present study, the IPAQ, as a subjective measure, was considered as a criterion measure for concurrent validity. In consistent with the findings of the prior research in Denmark,13 a significant but not so strong correlation was found between the two self-reported questionnaires (r = 0.62, P<0.001). However the significant moderate correlation confirmed the concurrent validity of the PA questionnaire. Concurrent validity of the questionnaire was assessed among Danish adults by Aadahl and Jorgensen in 2003. They found a high correlation between the scale and a PA dairy questionnaire (r = 0.74) and a poor correlation between the scale and an accelerometer (r = 0.20, P = 0.04); so the self-reported PA scale was approved as a valid instrument to assess the PA among the adult with sedentary to moderately active populations.13 In a study to validate the self-report PA questionnaire against maximal oxygen uptake (Vo2max testing), correlation between the two scales was assessed and it was found that the activity scale had an acceptable validity.12 In this study, the total amount of PA was not significantly associated with Vo2max (r2= 0.69, P = 0.098), but the amount of daily vigorous intensity PA and Vo2max had a strong and significant association (r2= 0.76, P<0.001).12 As a matter for validation, using subjective methods to assess criterion validity can be considered as a limitation for the present study. The PA questionnaires as an objective measure are prone to recall and desirability biases. Hence, the result of subjective methods should be paid attention in terms of misclassification while assessing the PA habits.12,25,26 The Bland-Altman plot was used to verify the agreement between the two questionnaires, but a non-constant bias was observed over the whole range of the instruments. Therefore, as recommended by Bland-Altman, log transformation approach was applied in the present study.27Nevertheless this method could not improve the agreement between the two scales. As shown in the Bland-Altman plot (Figure 2), the discrepancy between the two scales was obvious in lower and higher values (a trend line for bias) and it is increased with increasing/decreasing total MET-time values. This discrepancy infers that activities with moderate intensity are being measured more accurately compared to the light/vigorous intensity activities. The results of our study are consistent with those found in the previous study.13 Figure 2 Bland-Altman plot: Difference in MET-time scores from the activity scale versus MET-time scores from the IPAQ (y-axis), plotted against the average MET-time scores of the two measures (x-axis). The central horizontal line represents the mean difference and the flanking lines represent the 95% limits of agreement. Mean difference: -0.02±2, SD = -0.2 to 0.24. In another aspect of the results of the present study, one may note that the mean difference between the two methods was small, but the 95% limits of the agreement in the mean scores of MET-time values between the two self-reported questionnaires were wide and affected by four outliers. This would be an evidence for bias between the activity scales and, therefore, the agreement between the instruments may not be a gratifying result. Although the objective methods may provide more accurate information, it is not feasible to be used in population-base surveys.10,11 Additional validation studies of the self-reported PA questionnaire against objective methods is recommended in pilot studies with smaller sample size. In the rest of the cohort profile, exploring the predictive validity of the self-reported PA questionnaire is suggested. This exploration may be conducted through examining the relationships among self-reported time spent to PA and several health outcomes related to chronic diseases, such as blood pressure (BP) and High-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol. As another finding, when the known group validity analysis was conducted, the significant differences of total MET-time scores were observed in certain subgroups (e.g., marital, education and employment status) suggesting an acceptable achieved discriminative validity for the self-reported PA questionnaire. However, no significant difference was found in the activity score by age, gender and BMI, which was similar with those found in the previous studies.28,29 The known group comparisons were not assessed in the level of subdomains which may be a reason for the non-discrepancy found in the results. As an evidence for this claim, the previous studies showed known groups discrepancy by relating subgroups to each domain of self-report PA.15,30 Despite these issues, it may indicate that the validity of the self-reported PA questionnaire is not influenced by age, gender, and BMI. As the participants were already engaged in the AZAR Cohort study (with Azari culture), this may limit the generalizability of our results to the general Iranian adults. However, the significant association of the MET-time score was not affected by age, gender, and BMI groups in the sample this limitation to be slight. Further research is warranted in a variety of settings, as there was not found any validation studies evaluating the self-reported PA questionnaire among adults with a cultural adaptation in Iran. Conclusion The results of the present study suggested that the self-reported PA questionnaire has adequate psychometric properties for assessing PA in Khameneh adults. The modest reliability found for the instrument suggests that the self-reported PA questionnaire is internally consistent, stable, and valid. Although this instrument was applied in an Azari population in Iran, additional studies will be needed to better comprehend the psychometric properties of the scale among different populations. Acknowledgments This research was conducted under a thesis grant for the Master’s degree from the Department of Statistics & Epidemiology, Tabriz University of Medical Sciences. We gratefully acknowledge the researchers of the AZAR Cohort Study for their close collaboration during the research process. Ethical approval Ethics Committee in Tabriz University of Medical Sciences provided permission to conduct this survey. Signed informed consent was obtained from all participants of the AZAR Cohort Study prior to the data collection. Competing interests The authors declare that there is no conflict of interest. Authors’ contributions SD and NA-S contributed in original idea and protocol, conception of the work, conducting the study, revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work. MA-J contributed in the design of the work, doing the analysis, revising the draft and approval of the final version of the manuscript. FBA contributed in conception of the work and approval of the final version of the manuscript. MM contributed in conception of the work, conducting the study, wrote and editing of this manuscript. ==== Refs References 1 Barwais FA Cuddihy TF Washington T Tomson LM Brymer E Development and validation of a new self-report instrument for measuring sedentary behaviors and light-intensity physical activity in adults J Phys Act Health 2014 1 1097 104 10.1123/jpah.2012-0118 23963650 2 Warburton D Charlesworth S Ivey A Nettlefold L Bredin S A systematic review of the evidence for Canada’s Physical Activity Guidelines for Adults Int J Behav Nutr Phys Act 2010 7 39 10.1186/1479-5868-7-39 20459783 3 Gomersall SR Olds TS Ridley K Development and evaluation of an adult use-of-time instrument with an energy expenditure focus J Sci Med Sport 2011 14 143 8 10.1016/j.jsams.2010.08.006 20932797 4 Jurj AL Wen W Xiang YB Matthews CE Liu D Zheng W Reproducibility and validity of the Shanghai Men’s Health Study physical activity questionnaire Am J Epidemiol 2007 165 1124 33 10.1093/aje/kwk119 17351289 5 Hu B Lin L Zhuang M Yuan Z Li S Yang Y Reliability and relative validity of three physical activity questionnaires in Taizhou population of China: the Taizhou Longitudinal Study Public Health 2015 129 1211 7 10.1016/j.puhe.2015.03.024 25957853 6 Ishii K Shibata A Oka K Environmental, psychological, and social influences on physical activity among Japanese adults: structural equation modelling Int J Behav Nutr Phys Act 2010 7 61 10.1186/1479-5868-7-61 20684794 7 Laporte RE Montoye HJ Caspersen CJ Assessment of physical activity in epidemiologic research: problems and prospects Public Health Rep 1985 100 131 46 3920712 8 Hagströmer M Oja P Sjöström M The International Physical Activity Questionnaire (IPAQ): a study of concurrent and construct validity Public Health Nutr 2006 9 755 62 10.1079/PHN2005898 16925881 9 Pettee GK McClain JJ Lee CD Swan PD Alvar BA Mitros MR Evaluation of physical activity measures used in middle-aged women Med Sci Sport Exer 2009 41 1403 12 10.1249/MSS.0b013e31819b2482 10 Martínez-González MA López-Fontana C Varo JJ Sánchez-Villegas A Martinez JA Validation of the Spanish version of the physical activity questionnaire used in the Nurses’ Health Study and the Health Professionals’ Follow-up Study Public Health Nutr 2005 8 920 7 10.1079/PHN2005745 16277809 11 Prince SA Adamo KB Hamel ME Hardt J Gorber SC Tremblay M A comparison of direct versus self-report measures for assessing physical activity in adults: a systematic review Int J Behav Nutr Phys Act 2008 5 56 10.1186/1479-5868-5-56 18990237 12 Aadahl M Kjær M Kristensen JH Mollerup B Jørgensen T Self-reported physical activity compared with maximal oxygen uptake in adults Eur J Cardiovasc Prev Rehabil 2007 14 422 8 10.1097/HJR.0b013e3280128d00 17568243 13 Aadahl M Jørgensen T Validation of a new self-report instrument for measuring physical activity Med Sci Sports Exer 2003 35 1196 202 10.1249/01.MSS.0000074446.02192.14 14 Kelishadi R, Rabiee K, Khosravi A, Famori F, Sadeghi M, Roohafza H. Assessment of physical activity in adolescents of Isfahan. J Shahrekord Uni Med Sci 2004;3:55-65. 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Methods of Multivariate Analysis. New York: John Wiley & Sons, Inc; 2003. 20 Tinsley HE, Brown SD. Handbook of Applied Multivariate Statistics and Mathematical Modeling. New York: Academic Press; 2000. 21 Dishman RK Saunders RP McIver KL Dowda M Pate RR Construct validity of selected measures of physical activity beliefs and motives in fifth and sixth grade boys and girls J Pediatr Psychol 2013 38 563 76 10.1093/jpepsy/jst013 23459310 22 Oyeyemi AL Bello UM Philemon ST Aliyu HN Majidadi RW Oyeyemi AY Examining the reliability and validity of a modified version of the International Physical Activity Questionnaire, long form (IPAQ-LF) in Nigeria: a cross-sectional study BMJ Open 2014 4 e005820 10.1136/bmjopen-2014-005820 23 Van Holle V De Bourdeaudhuij I Deforche B Van Cauwenberg J Van Dyck D Assessment of physical activity in older Belgian adults: validity and reliability of an adapted interview version of the long International Physical Activity Questionnaire (IPAQ-L) BMC Public Health 2015 15 433 10.1186/s12889-015-1785-3 25928561 24 DeVellis RF. Scale Development: Theory and Applications. 2nd ed. Thousand Oaks, CA: Sage; 2003. 25 Sallis JF Saelens BE Assessment of physical activity by self-report: status, limitations, and future directions Res Q Exercise Sport 2000 71 2 Suppl 1 14 10.1080/02701367.2000.11082780 26 Orsini N Bellocco R Bottai M Hagströmer M Sjöström M Pagano M Validity of self-reported total physical activity questionnaire among older women Eur J Epidemiol 2008 23 661 7 10.1007/s10654-008-9273-z 18704705 27 Bland JM Altman DG Measuring agreement in method comparison studies Stat Methods Med Res 1999 8 2 135 60 10501650 28 Harada ND Chiu V King AC Stewart AL An evaluation of three self-report physical activity instruments for older adults Med Sci Sport Exer 2001 33 962 970 10.1097/00005768-200105001-01412 29 Matthiessen J Biltoft-Jensen A Rasmussen LB Hels O Fagt S Groth MV Comparison of the Danish physical activity questionnaire with a validated position and motion instrument Eur J Epidemiol 2008 23 311 322 10.1007/s10654-008-9228-4 18270796 30 Vasheghani-Farahani A Tahmasbi M Asheri H Ashraf H Nedjat S Kordi R The Persian, last 7-day, long form of the International Physical Activity Questionnaire: translation and validation study Asian J Sports Med 2011 2 106 16 10.5812/asjsm.34781 22375226

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==== Front Health Promot PerspectHealth Promot PerspectHealth Promot PerspectTBZMEDHealth Promotion Perspectives2228-6497Tabriz University of Medical Sciences 10.15171/hpp.2016.25Original ArticleInvestigating incidence of bacterial and fungal contamination in shared cosmetic kits available in the women beauty salons Dadashi Leila Dehghanzadeh Reza * Department of Environmental Health Engineering, Faculty of Health, Tabriz University of Medical Sciences, Tabriz, Iran* Corresponding Author: Reza Dehghanzadeh, Department of Environmental Health Engineering, School of Health, Tabriz University of Medical Sciences, Golgasht St., Azadi Ave., Tabriz, Iran. Tel: +98 9144184167; Fax: +98 41 33344731; dehghanzadehr@tbzmed.ac.ir2016 10 8 2016 6 3 159 163 12 3 2016 26 4 2016 © 2016 The Author(s).2016This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.http://journals.tbzmed.ac.ir/HPP Background: Rich texture of cosmetics can provide a suitable medium for growth of pathogenic microorganisms. In addition, skin microflora of anyone is unique which might be harmful to another person. Skin and eye pathogenicity could be communicated by sharing cosmetics in beauty saloons. The main objective of this study was to evaluate microbial contamination of in-use skin and eye cosmetics which are available as public make-up kits for women in the beauty salons. Methods: Fifty-two in-use skin and eye cosmetics were included in this cross sectional study.The specimens from all the cosmetics were collected following the owner’s informed consent, and then about 1 g of the cosmetics was added to nine ml of liquid Eugon LT100 broth medium,two for each product. Ten beauty salons randomly selected from different regions of Tabriz city between June and August 2016. Cosmetics were sampled and carried to the laboratory in sterile condition and then examined to determine bacterial and fungal species in the samples. Results: All of in-use cosmetic were contaminated with bacteria (95% CI = 93.1%-100.0%) and about 19.2% by fungus and yeast (95% CI = 10.8%-31.9%). Streptococcus spp., Pseudomonas spp., Acinetobacter, Bacillus spp., Staphylococcus spp., Escherichia coli, Salmonella, Klebsiella,Citrobacter, Rhodotorula and Candida were dominant species which were isolated from the cosmetics. Powders with 38.5% (95% CI = 17.7%-64.5%) and eyeliners with 30.0% (95%CI = 6.7%-65.2%) were the most fungal contaminated products. Conclusion: Shared cosmetics in beauty salons are almost contaminated by bacteria and fungus.Therefore, it is suggested to avoid sharing cosmetics by women and prevent use of public cosmetics in toilet saloons. Shared cosmeticBeauty salonsMicrobial contaminationWomenBacteriaFungiCitation: Dadashi L, Dehghanzadeh R. Investigating incidence of bacterial and fungal contamination in shared cosmetic kits available in the women beauty salons. Health Promot Perspect. 2016;6(3):159-163. doi: 10.15171/hpp.2016.25. ==== Body Introduction In recent years, cosmetics are extensively used for beauty purposes. Meanwhile, beauty salons play an important role in possible transfer of skin and eye infections due to the use of public make-up kits by the women.1 Although the microbial standards of cosmetics have been progressively improved by stringent legislations, their contamination has been frequently reported and even in some cases, has generated serious problems for consumers.2 Often production and expiration date are not labeled on the cosmetics, also effectiveness of cosmetic’s preservative decreases with time. In addition, cosmetics comprise essential minerals, growth factors, organic and inorganic compounds and humidity which provide suitable conditions for augmentation of microorganisms.3 Skin microflora of anyone is unique and could be transferred to the others by using common tools such as brushes and pads which could threaten the healthiness of the women.4 Therefore, it is likely that common cosmetics in beauty salons have more diversity and density of microorganisms. Survey on personal toiletries show that Bacillus, Staphylococcus spp., Pseudomonas spp., Enterobacter, Aspergillus, Penicillium and Candida are more predominant species in cosmetics.3,5 Also the most common skin infections are caused by Staphylococcus epidermis and Staphylococcus aureus.6 However, microbial contamination of in-use cosmetics in beauty salons could be hardly found in the literature and often brushes and combs and other similar devices have been surveyed. The most dominant isolated species from in-use tools in beauty salons have been Streptococcus spp., Staphylococcus spp., Escherichia coli, Citrobacter freundi, Klebsiella, Enterobacter and Pseudomonas aeruginosa and also fungus like Aspergillus and Penicillium.1 Cosmetic products can be contaminated by three ways; (1) application of unsterile raw material as ingredients, (2) in the course of production process, or (3) during use of cosmetics.7 On the other hand, trafficking counterfeit cosmetic products is the serious problem in many countries. Consumption of cosmetics is growing in developing countries. Microbial contamination and occurrence of skin contamination due to cosmetics is still one of the major causes for product recalls in the world.8 Then, the main purpose of this study was to evaluate bacterial and fungal contamination of in-use eye and skin cosmetics shared by women in beauty salons. Materials and Methods Sampling To determine the microbial contamination of in-use shared cosmetics available in the beauty salons, about 52 in-use skin (powder and cream) and eye (mascara and eyeliner) cosmetics were included in this cross-sectional study based on sample size calculation for dichotomous variable. The sample size was estimated about 61 (significance level=0.05, population proportion=0.2 and relative error=10%), but nine of the samples were discarded because of probably contamination during handling. The specimens from all the cosmetics were collected following the owner’s informed consent, and then about one gram of the cosmetics was added to nine ml of liquid Eugon LT100 broth medium, two for each product. Ten beauty salons randomly selected from different reign of Tabriz city between June and August 2016. Sampling of cosmetics was conducted in the salons. Microbial survey In sterile conditions, about 1 g of the cosmetics was added to nine ml of liquid Eugon LT100 broth medium to neutralize the growth inhibitors present in the ingredients of the cosmetics. The samples immediately were carried to the laboratory and analyzed in accordance with the standards of Food and Drug Administration (FDA) and Institute of Standards and Industrial Research of Iran.9 First the tubes were incubated for 48-72 hours at 37°C. Then, 1 mL of each culture was removed and transferred to the Cetrimide Agar medium, Levine eosin methylene blue Agar medium, Baird Parker Agar, and Sabouraud Dextrose Chloramphenicol Agar and incubated for 24-48 hours at 37°C. Afterwards, the plates containing growing colonies were isolated and the total count of colony forming unit per gram or milliliter of cosmetics (CFU g-1) was determined by counting the colonies on the medias. Further identification of the isolated bacteria were carried out according to the bacteria’s morphology and biochemical tests using standard bacteriological methods.10 Fungi and molds were identified in terms of appearance. In addition, the relevant test to detecting Candida yeast including culturing in human serum and incubation at 37°C was conducted for 3 hours.11 Statistical analyses Variance between the contamination levels in the in-use cosmetics as well as between different cosmetic types was determined by chi-square k-sample Pearson analysis with significance level of 0.05 using SPSS software (IBM SPSS Statistics 19, SPSS Inc., USA). Confidence intervals (CI) were calculated by Stata MP 14 (Stata Corp LP, USA). Results Table 1 shows that, exactly 100% (95% CI = 93.1%-100%) of the total examined in-use cosmetics in the beauty salons were contaminated by bacteria. However, only 19.2% (95% CI=10.8%-31.9%) of the cosmetic products were contaminated by fungi or yeast. Generally powders demonstrated higher contamination by fungi. The results show that creams did not indicated any contamination by fungi. The number of colony forming units of fungi in cosmetics was between 3.5-200×103 CFU g-1 (Table 2). Also the number of colony forming units of isolated bacteria was 12-960×103 CFU g-1. High levels of Staphylococcus spp. and Escherichia coli counts (>500 CFU g-1) were found in the in-use powders and eyeliners. Figure 1 and 2 demonstrate the diversity and frequency of the isolated bacteria and fungi separately in skin and eye cosmetics obtained from beauty salons. Fungi and bacteria constituted 9.2% (95% CI=5.1%-16.1%) and 90.8% (95% CI=83.9%-94.9%) of the isolates, respectively. Also about 51.5% (95% CI=41.8%-61.1%) of the isolated bacteria were belong to gram-negative group and the remains were gram-positive. Streptococcus spp., Acinetobacter and Pseudomonas spp. were the most dominant in the skin cosmetics. Candida, Rhodotorula and Penicillium were the only isolated yeasts and fungi. Also Bacillus spp., Staphylococcus spp. and Escherichia coli isolated from the skin cosmetics. Streptococcus spp., Pseudomonas spp. and Acinetobacter were the most frequently isolated bacteria from in-use eye cosmetics. Rhodotorula and candida were the only isolated yeasts. Also Bacillus spp, Staphylococcus spp., Escherichia coli, Salmonella, Klebsiella and Citrobacter were isolated from the eye cosmetics. Streptococcus species was the most predominant bacteria that were isolated from the in-use skin and eye cosmetics. Considering the fungi isolated from in-use skin powders, Penicillium was definitely the most predominant fungi genus (6%), which was followed in order by Rhodotorula (6%) and Candida (3%). Furthermore, Rhodotorula (12%) and Candida (4%) were the most isolated fungi from the in-use mascaras and eyeliners. Moreover, the most fungal diversity was observed in the in-use skin powders. Referring to the isolated bacteria from the in-use skin cosmetics, the most predominant bacteria were Streptococcus (32%), Pseudomonas (23%), Acinetobacter (19%), Bacillus (11%), Staphylococcus (6%) and E. coli (4%). Among the in-use eye cosmetics, Streptococcus (25%) and Pseudomonas (24%) were the predominant isolated bacteria, which was followed in a descending order by Acinetobacter and Staphylococcus (10% each), Bacillus and E. coli (8% each), Salmonella and Klebsiella (4% each), and Citrobacter (2%). Table 1 Summery of microbial contamination rate in the sampled cosmetics from women beauty salons Cosmetic type No. of samples Microbial contamination rate Bacteria Fungi n (%) 95% CI P value n (%) 95% CI P value Skin Powder 13 13(100.0 ) 93.12-100.0 NSa 5(38.5) 17.7-64.5 Cream powder 12 12(100.0) 75.8-100.0 NS 0.0 0.0-24.3 Eye 0.063 Mascara 17 17(100.0) 81.6-100.0 NS 2(11.8) 3.3-34.3 Eyeliner 10 10(100.0) 72.5-100.0 NS 3(30.0) 10.8-60.3 aBecause of complete response, no significant diffeence was observed between cosmetic types. Table 2 Microbial Counts (103 CFU g-1) and association between contamination by bacteria and fungi in shared cosmetics available in women beauty salons Microorganisms Powder Cream Mascara Eyeliner P value Bacteria Acinetobacter 300 350 320 NC 0.255 Escherichia coli NCa - - 850 0.008 Bacillus NC 230 320 500 0.802 Pseudomonas 208 195 180 125 0.576 Staphylococcus 960 544 410 144 0.518 Streptococci NC 23 440 684 0.324 Klebsiella - - 21 - 0.233 Citrobacter - - 12 - 0.552 Salmonella - - 32 - 0.233 Alcanigenes - - 20 - 0.383 Fungi Candida - - 30 - 0.662 Rhodotorula 200 - 126 115 0.131 Penicillium 3.5 - - - 0.100 aNon-countable. Figure 1 Microbial contamination rate in the in-use skin cosmetics in women beauty salons. Figure 2 Microbial contamination rate in the in-use eye cosmetics in women beauty salons. Discussion Results showed that all of the sampled cosmetics were contaminated by bacteria which is more than the rate of 63% reported from in-use individual cosmetics.8 Preservatives of the cosmetics remain active on the skin that might alter the skin microflora which are responsible for protection and supplying skin safety.12 Cosmetics are not produced in sterile condition and are often shared in beauty salons which could cause the increase of microbial contamination within cosmetics.13 Contamination level in our study is higher in comparison with a study in the United States that was conducted on 3000 shared cosmetic tester kits available to the public and reported 50% contamination of the products by bacteria.14 Contamination level in the powders was higher than the other cosmetics. It can be deduced that the powders are frequently in contact with air and also the common use of skin powder pads can cause the higher contamination rate. In addition, application of the natural ingredients in the formulation of powders including talc, Fuller’s earth and bentonite might increase the contamination level.15 Contaminated eye cosmetics, particularly mascaras, are associated with ocular infections.5,16 Our result revealed that in all the examined cosmetics, mascaras had a more bacterial diversity, because of its aqueous-based formulation and greater chance of bacterial deposits originating from the environment and from the surface of the eyelashes, which makes the product more susceptible to infections.6,13 About 19.2% (95% CI=10.8%-31.9%) of in-use cosmetics were contaminated by fungus and yeast. Fungus contamination ratio in cosmetics was low compared with bacteria. It can be attributed to the more ability of cosmetics preservatives in prevention of fungus growth.17 In this study, isolation of gram-negative bacteria was more than gram-positive bacteria, while gram-positive bacteria is more predominant in the skin flora.17,18 It can be concluded by the more resistance of gram-negative bacteria to severe condition which could cause growth of them in cosmetics. Streptococci species were the most dominant isolated bacteria, which also have been reported in personal cosmetics.19 Streptococcus species can cause skin infections like Erythematous rash.20 Pseudomonas spp. was the most dominant species isolated from the eyeliners. Pseudomonas aeruginosa have been mostly reported in personal cosmetics.2,5,21 Because Pseudomonas aeruginosa is one of the natural skin microflora , it can be transferred to the cosmetics from consumer’s skin.18 Pseudomonas aeruginosa can cause skin infections.22 Also, Acinetobacter was isolated from the examined cosmetic and as skin microflora play an important role in skin infections.18,23 Bacillus spp., Staphylococcus spp. and Escherichia coli were the other isolated bacteria from the cosmetic. Staphylococcus spp. causes skin infections such as acne and desquamate.24 Bacillus species are transient skin microflora. Bacillus anthracis causes focal necrotizing cellulitis in the skin. Use of eye cosmetics contaminated with Bacillus cereus causes severe eye infections.24 Candida and Rhodotorula also were isolated from the cosmetics. Candida has been reported in other personal toiletries studies.5,25 Candida plays an important role in the establishment of skin lesions, rash and dermatitis.26 Also in this study Salmonella, Citrobacter, Klebsiella and Alcanigenes have been isolated. Higher density and diversity of bacteria isolated from shared cosmetics that obtained from beauty salons in comparison with personal cosmetics reported in the literatures.3,5,21,27 When several people share the same cosmetic an instance contamination may take place and because each individual has unique skin microflora that could be harmful to another person. The number of colony forming units of aerobic mesophilic microorganisms for eye and other cosmetics must not exceed 102 and 103CFU g-1 of products, respectively.28 In all the examined cosmetics available for public make-up in women beauty salons, the number of microbial counts was more than the maximum favorable level. Conclusion Finally, our findings showed that microbial contamination rate in cosmetics which are shared in beauty salons is higher than the rates reported for personal cosmetics in the literatures. However, the hygienic condition of the salons, socioeconomic level of the area that the beauty salons are located and even individual health behaviors of the costumers could be impressive factors on the contamination rate of cosmetics which are not assessed in this study. The skin microflora of anyone is distinctive and when several people share the same product, the rate of contamination could be increased. Therefore, it is suggested to avoid long-term use, share or use public cosmetics in toilet saloons and keep the used cosmetics in dry, cool, and fastened packets. Also, it is necessary to promote or make compulsory the use of individual cosmetic kits in the beauty salons, intensify the hygiene inspections from the beauty salons, monitor behavior of the barbers and implement continuous health education programs by the hygiene inspectors for the beauty salon workers. Acknowledgments This study is part of an MSc thesis in Environmental Health Engineering, which was submitted to Tabriz University of Medical Sciences (5/53/4643-1393.08.14). We appreciate Research Vice-Chancellor of Tabriz University of Medical Sciences for their financial support. We thank all the beauty salon lords who participated in sampling of their cosmetic kits. Ethical approval Procedures were approved by the Research Vice-Chancellor of Tabriz University of Medical Sciences review board. Competing interests No conflict of financial and non-financial interest declared between the authors. Authors’ contributions LD was involved in the conception of the study, performed data collection and the analyses and drafted the manuscript. RD was involved in the conception of the study, interpreted the results from the analyses, performed significant revisions, assisted in the revision of the manuscript and approved the final version of the manuscript. ==== Refs References 1 Enemuor S Ojih M Isah S Oguntibeju O Evaluation of bacterial and fungal contamination in hairdressing and beauty salons Afr J Microbiol Res 2013 7 14 1222 5 10.5897/AJMR12.917 2 Lundov M, Moesby L, Zachariae C, Johansen J. Contamination versus preservation of cosmetics: a review on legislation, usage, infections, and contact allergy. Contact Dermatitis. 2009(60):70-8. doi:10.1111/j.1600-0536.2008.01501.x . 3 Behravan J Bazzaz F Malaekeh P Survey of bacteriological contamination of cosmetic creams in Iran (2000) Int J Dermatol 2005 44 6 482 5 10.1111/j.1365-4632.2005.01963.x 15941436 4 Noah N. A guide to hygienic skin piercing. In: Gerson J, ed. Milady’s Standard Textbook for Professional Estheticians. New York: Milady; 1995. pp. 1-11. 5 Anelich L Korsten L Survey of micro‐organisms associated with spoilage of cosmetic creams manufactured in South Africa Int J Cosmetic Sci 1996 18 1 25 40 10.1111/j.1467-2494.1996.tb00133.x 6 Draelos ZD Special considerations in eye cosmetics Clin Dermatol 2001 19 4 424 30 10.1016/S0738-081X(01)00204-8 11535383 7 Charnock C The microbial content of nonsterile pharmaceuticals distributed in Norway J Hosp Infect 2004 3 57 233 40 10.1016/j.jhin.2004.03.016 15236853 8 Okeke I Lamikanra A Bacteriological quality of skin-moisturizing creams and lotions distributed in a tropical developing country J Appl Microbiol 2001 91 5 922 8 10.1046/j.1365-2672.2001.01456.x 11722671 9 Microbiology of cosmetics: Detection of specified and non-specified microorganisms. Tehran: Institute of Standards and Industrial Research of Iran; 1992. 10 Vos P, Garrity G, Jones D, Krieg NR, Ludwig W, Rainey FA, et al. Springer Science & Business Media; 2011. Bergey’s Manual of Systematic Bacteriology. The Firmicutes, vol 3. 11 Williams DW Lewis MA Oral Microbiology: Isolation and identification of candida from the oral cavity Oral Dis 2000 6 1 3 11 10.1111/j.1601-0825.2000.tb00314.x 10673781 12 Holland KT Bojar RA Cosmetics Am J Clin Dermatol 2002 3 7 445 9 10.2165/00128071-200203070-00001 12180892 13 Giacomel C, Dartora G, Dienfethaeler H, Haas S. Investigation on the use of expired make-up and microbiological contamination of mascaras. Int J Cosmetic Sci. 2013:375-80. doi:10.1111/ics.12053 . 14 Tran TT Hitchins AD Microbial survey of shared-use cosmetic test kits available to the public J Ind Microbiol 1994 13 6 389 91 10.1007/BF01577224 15 Kulkarni SB Bajpai ND Meghre VS Evaluation of dome marketed facepacks and cakes for microbial load Asian J Microbiol Biotechnol Environ Sci 2011 13 1 213 6 16 Kabara JJ. Preservative-Free and Self-preserving Cosmetics and Drugs: Principles and Practices. Boca Raton, FL: CRC Press; 1997. 17 Mislivec P Bandler R Allen G Incidence of fungi in shared-use cosmetics available to the public J AOAC Int 1992 76 2 430 6 18 Bojar R Holland K Review: the human cutaneous microflora and factors controlling colonisation World J Microb Biot 2002 18 9 889 903 10.1023/A:1021271028979 19 Pack LD Wickham MG Enloe RA Hill DN Microbial contamination associated with mascara use J Am Optom Assoc 2008 79 10 587 93 10.1016/j.optm.2008.02.011 20 Kolmos H Svendsen R Nielsen S The surgical team as a source of postoperative wound infections caused by Streptococcus pyogenes J Hosp Infect 1997 35 3 207 14 10.1016/S0195-6701(97)90208-5 9093919 21 Wong S Street D Delgado SI Recalls of foods and cosmetics due to microbial contamination reported to the US Food and Drug Administration J Food Protect 2000 63 8 1113 6 22 Murthy R Sengupta S Maya N Shivananda P Incidence of post operative wound infection and their antibiogram in a teaching and referral hospital Indian J Med Sci 1998 52 12 553 5 10327794 23 Jones ME Karlowsky JA Draghi DC Thornsberry C Sahm DF Nathwani D Epidemiology and antibiotic susceptibility of bacteria causing skin and soft tissue infections in the USA and Europe: a guide to appropriate antimicrobial therapy Int J Antimicrob Agents 2003 22 4 406 19 10.1016/S0924-8579(03)00154-7 14522104 24 Leyden JJ Kligman AM Acne vulgaris: new concepts in pathogenesis and treatment Drugs 1976 12 4 292 300 10.2165/00003495-197612040-00004 134884 25 Lundov MD Zachariae C Recalls of microbiologically contaminated cosmetics in EU from 2005 to May 2008 Int J Cosmetic Sci 2008 30 6 471 4 10.1111/j.1468-2494.2008.00475.x 26 Rebora A Leyden J Napkin (diaper) dermatitis and gastrointestinal carriage of Candida albicans Brit J Dermatol 1981 105 5 551 5 10.1111/j.1365-2133.1981.tb00798.x 7028077 27 Baird R Bacteriological contamination of products used for the skin care in babies Int J Cosmet Sci 1984 6 2 85 90 10.1111/j.1467-2494.1984.tb00362.x 19470003 28 Scientific Committee on Consumer Products. The SCCP’s Notes of Guidance for the Testing of Cosmetic Ingredients and Their Safety Evaluation. 2006. Available from: http://ec.europa.eu/health/scientific_committees/consumer_safety/docs/sccs_s_006.pdf.

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==== Front Health Promot PerspectHealth Promot PerspectHealth Promot PerspectTBZMEDHealth Promotion Perspectives2228-6497Tabriz University of Medical Sciences 10.15171/hpp.2016.26Original ArticleOral health education program among pre-school children: an application of health-promoting schools approach Shirzad Mahboube 1 Taghdisi Mohammad Hossein 1 *Dehdari Tahereh 1 Abolghasemi Jamileh 2 1Department of Health Education and Health Promotion, Iran University of Medical Sciences, Tehran, Iran2Department of Biostatistics, Iran University of Medical Sciences, Tehran, Iran* Corresponding Author: Mohammad Hossein Taghdisi; Tel: 00982186704756; taghdisi.mh@gmail.com2016 10 8 2016 6 3 164 170 11 6 2016 13 6 2016 © 2016 The Author(s).2016This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.http://journals.tbzmed.ac.ir/HPP Background: Preschool children have a limit ability to take care of their teeth. The aim of this study was to determine the effect of an intervention based on Albanian’s Health Promoting Schools Model (Albanian’s HPSM) on the oral health behaviors among a group of Iranian female preschool (5-6 years old) children. Methods: In this quasi-experimental study, 120 children in seventh district of Tehran, Iran were randomly recruited and assigned to either the intervention or the control groups. A scale was designed and validated to assess the oral health behaviors among the children and knowledge,attitude, self-efficacy beliefs, perceived barriers and oral health behaviors among the parents and the schoolteachers. An expert panel approved the content validity of the scale (CVR = 0.89,CVI = 0.90). The reliability was also approved applying intraclass correlation coefficient (range,0.83–0.92) and Cronbach alpha (range, 0.83–0.96). Based on the preliminary data, a 6-week intervention was designed and conducted to the intervention group. One month following the intervention, both groups were followed-up. The data were analyzed using covariance and paired t tests. Results: Following the intervention, significant differences were found in the oral health behaviors of the children in the intervention group (P < 0.05) and knowledge, attitude, oral health behaviors, self-efficacy, and perceived barriers of their parents and the schoolteachers (P < 0.05). Conclusion: Using Albanian’s health-promoting schools (HPSs) approach was useful in improving the oral hygiene behaviors among the preschool children. Oral healthPre-school childrenAlbanian’s Health Promoting Schools ModelCitation: Shirzad M, Taghdisi MH, Dehdari T, Abolghasemi J. Oral health education program among pre-school children: an application of health-promoting schools approach. Health Promot Perspect. 2016;6(3):164-170. doi: 10.15171/hpp.2016.26. ==== Body Introduction Pre-school children are at high risk for dental caries.1 About 51.7% of Iranian children, aged 3 to 5 years, have tooth decay and further efforts are essential to achieve 90% caries-free teeth among 5-year-old children.2 Various factors have been identified to affect children’s teeth decay including poor oral hygiene and nutritional status among the children as well as the level of oral health-related knowledge, habits, attitude and self-efficacy among the schoolteachers and parents.3-9 Such variables should be considered when developing oral health education programs targeting preschool children. Oral health education can be reinforced throughout the school years, an influential period in children’s lives. During school years lifelong beliefs, positive attitudes and personal skills among the children are being developed.10 As noted by Kwan et al, oral health education should form part of all subjects in the school curriculum and involve students, school staffs and parents in health promotion activities at school.10 Oral health education should be regularly reinforced at home by health-promoting school (HPS) programs, and should be also developed at key educational stages throughout the children’s school career.11 The effectiveness of interventions adopting a HPSs approach are likely to be increased.12 The schools that constantly strengthen their capacity as a healthy setting for living, learning and working are considered as HPSs.13 HPSs enable students to take control over their health and become the future active and responsible citizens in their society.14 Such schools help to involve the school and community members in planning the programs addressing their health needs and can be maintained and sustained with available resources and commitments.15 Previous literature have shown that adopting HPS approaches to develop nutrition promotion programmes increased the intake of high-fibre foods, healthier snacks, water, milk, fruit and vegetables and performing the oral health practices among students.16-18 It can also reduce ‘breakfast skipping,’ consumption of red food, low-nutrient dense foods, fatty and cream foods, sweet drinks consumption, eating disorders and smoking among students.17-19 Albanian’s Health Promoting Schools Model (Albanian’s HPSM), as one of the HPSs approaches, was developed in the field of health education for primary schools. This model consists three basic branches including pupils, teachers and parents and emphasizes good relationships and proper collaboration between these branches.20 In Albanian’s HPSM (Figure 1), the educational methods for training pupils (e.g. videos, health competitions and theatre), parents (e.g. meetings and small groups) and schoolteachers (e.g. formative courses and seminars) have been described.20 Although Albanian’s HPSM is recommended for developing educational interventions in primary schools,20 few researches have studied this model to promote healthy behaviour among preschool children.21,22 Considering the high prevalence of tooth decay among Iranian children23,24 and the effectiveness of adopting a HPSs approach to develop educational intervention on the oral health of children16,19 and, also, the lack of intervention studies in this field, this study was conducted to determine the effect of an educational intervention based on Albanian’s HPSM on oral health behaviors among a sample of pre-school (5-6 years old) children in Tehran, Iran. Materials and Methods Participants and setting This quasi-experimental study was conducted from April to September 2015. Among the 7 middle-income areas of Tehran, the area number 7 was randomly selected, from which four preschool centres were, also, randomly selected. Preschool children in two schools were assigned into the intervention group and those in the other two schools were considered as the control group. Then, according to the estimated sample size, 30 preschool (5-6 years old) children were randomly recruited from each school. Inclusion criteria in the study were the student’s agreement to participate in the study, ability to read and write Persian, residency in the city of Tehran and being in the preschool grade. The schoolteachers and parents of the students were participated in the study. None of the students and their parents and schoolteachers refused to take part in the study. Finally, 60 students, 60 parents (1 parent of every student) and 11 schoolteachers were included in each group. Demographic characteristics of the children in the two groups are presented in Table 1. At baseline, no significant differences were observed between the two groups in the demographic characteristics. Sample size calculation In this study, M (the number of clusters) = 18, V2 (the estimated variance on oral health behavior among school children in a study by Okada et al6) = 0.123, ε (the margin of errors) = 0.01, α = 0.05 and β = 0.20. To calculate the sample size, the formula (n= [(z1-α/2 +z1-β)2MV1y2/[(z1-α/2 +z1-β)2(M-1) ε2) was used. The final sample size was 120 participants with 60 in each group (control and intervention). Study instruments and measures The students’ information on oral health behaviors, as well as their parents’ and schoolteachers’ self-efficacy beliefs, perceived barriers, knowledge, attitude and oral health behaviors were collected using a self-administered questionnaire developed by the researchers. In order to develop the instruments, a literature review was done and 20 female pre-school children and their schoolteachers and parents were interviewed to collect their opinions concerning oral health. Initial instruments were generated and consequently qualitative face and quantitative content validity of the items were evaluated. Thirty female students and their parents and schoolteachers were asked to comment on the simplicity, readability and clarity of the items. According to their opinions, several questions were deleted. For calculating the content validity, an expert panel including ten specialists in the areas of health education and dentistry reviewed the necessity and the relevance of items. The necessity of the items was assessed using a 3-point rating scale: E indicated essential; U, useful but not essential; and N, not necessary. The relevance of the items was also assessed using a 4-point rating scale: (N) not relevant, (S) slightly relevant, (R) relevant, and (V) completely relevant. Based on the experts’ opinions, the content validity index (CVI) and content validity ratio (CVR) of each item were assessed. Items having CVR less than 0.62 and CVI less than 0.78 were deleted.25,26 In the present study, the CVI and CVR of the scales, as a whole, was 0.90 and 0.89, respectively. To estimate the reliability of the scales, intraclass correlation coefficient (ICC) and Cronbach alpha procedures were used with 20 female children and their parent and schoolteachers (with a 2-week interval between each test). The satisfactory value for the ICC and Cronbach alpha was considered ≥ 0.40 and ≥0.70, respectively.27,28 Knowledge of parents and teachers about oral health One question (‘in your opinion, which of the following factors affect oral health?’) with 14 items on a 3-point scale (0=No, 1=I don’t know, 2=Yes) were used to measure the knowledge of parents and teachers regarding oral health. Cronbach alpha for the knowledge scale was 0.83. The ICC for this scale was 0.78. Attitude of parents and teachers toward oral health A nine items scale was used to measure the attitude (e.g. ‘Brushing makes me feel good’). The items in this scale were measured based on a Likert-type scale ranging from 1=‘strongly disagree’ to 5=‘strongly agree’. Cronbach alpha for this scale was 0.87 and the ICC was 0.79. Oral health behaviors in teachers and parents Fourteen items constituted the oral health behaviors scale (e.g. ‘Do you brush your teeth every night before going to bed?’). The items of this scale were measured based on a Likert-type scale ranging from 1=“never” to 4=“always”’. Cronbach alpha estimated for this scale was 0.92 and the ICC was 0.93. Perceived self-efficacy of parents and teachers regarding oral health behaviors A 10 items scale was designed to measure the parents and teachers ‘s self-efficacy beliefs to adopt oral health behaviors (e.g. ‘At nights, although I am too tired, I brush my teeth before going to bed’). The items in this scale were measured on a Likert-type scale, ranging from 1=‘completely unconfident’ to 5=‘completely confident’. Cronbach alpha of this sub-scale was 0.75 and the ICC was 0.95. Perceived barriers for adopting oral health behaviors in parents and teachers Ten items were designed to measure the perceived barriers (e.g. ‘I feel nausea after using mouthwash’). These items were measured on a Likert-type scale ranging from 1=‘strongly disagree’ to 5=‘strongly agree’. Cronbach alpha of this scale was 0.84 and the ICC was 0.81. Oral health behaviors in preschool children Eight items on a 2-point scale (0=No, 1=Yes) were designed to measure the oral health behaviors among children (e.g. ‘Do you brush your teeth every night before going to bed?’). Cronbach alpha estimated for this scale was 0.96 and the ICC was 0.85. Intervention program Based on the primary diagnostic assessment, an educational intervention was designed and performed for the children their parents and schoolteachers in the intervention group. Manipulation program on children Six 45-minutes training sessions for the children were held. In the first session, after presenting a story regarding oral health, the children were encouraged to discuss their positive and negative beliefs about the oral health. In the second session, children drew paintings regarding the oral health and, by posing some open-ended questions, the students were asked to share their experiences and feelings with other participants upon their paintings. In the third session, some oral health games and entertainments such as solving puzzles, connecting points and so on were presented. In the fourth session, all children read a poem together about the oral health. In the fifth session, three films and animations about the essential behaviors to maintain good oral health were shown. Finally, in the sixth session, the correct methods of tooth brushing and flossing were demonstrated to the children. Manipulation program on parents and schoolteachers Four 45-minutes sessions for the parents and schoolteachers were held. In the first educational session for the parents and schoolteachers, the importance of preschool-aged children’s oral health and the necessity of taking care of the milk teeth among children were discussed. In this meeting, they were encouraged to pay more attention to the status of oral health and to persuade children towards good performance in oral health behaviors. In the second session, a lecture was presented on the prevention strategies of tooth decay and their role in maintaining good oral health in children. In the third and fourth sessions, by posing some open-ended questions, the parents and schoolteachers were asked to discuss about the positive and negative beliefs and experiences regarding the oral and dental health. In these sessions, through verbal persuasions, they were assured to be able to reduce the barriers in performing oral health behavior by the children. The parents in the intervention group were also given a booklet about the importance of oral health in preschool children, the benefits of tooth decay preventive behaviors, and the ways to overcome the barriers to adopt oral health behaviors among children. One month after the intervention, the questionnaires were delivered to the two groups and all completed the questionnaires again. Statistical analyses The data were analysed using the SPSS statistical software (version 16). The normality of the data was examined by Kolmogorov–Smirnov test. The homogeneity of demographic characteristics of the two groups at baseline was analysed by chi-square and Fisher exact tests. Also, paired t tests were used to test the within-group changes. Differences in the outcomes between the two groups before and after the intervention were tested using the independent-samples t test and analysis of covariance (ANCOVA), respectively. The data were expressed as mean (standard deviation [SD]). Significance of all the results was considered as P<0.05 level, at baseline. Results Outcomes for the schoolteachers and parents of children Mean scores of attitude, knowledge, perceived self-efficacy, and adopting oral health behaviors for both groups before and after intervention are shown in Table 2. The results of the paired samples t test showed a significant increase in self-efficacy beliefs, knowledge, attitude and adopting oral health behaviors scores of the schoolteachers and parents of children in the intervention group after the intervention compared to the baseline. In addition, a significant reduction was observed in perceived barriers score of this group compared to the primary data. Results also indicated that after the intervention the parents and schoolteachers of the children reported a significant increase in perceived self-efficacy, knowledge, attitude and adopting oral health behaviors compared to the parents and schoolteachers of children in the control group (Table 2). In addition, there were significant reductions in perceived barriers of parents and schoolteachers of children in the intervention group compared with the parents and schoolteachers of children in the control group (Table 2). Behavioral outcome for the children Findings showed that the educational intervention had significant effect on the mean score of oral health behaviors of children in the intervention group compared to the control group (Table 2). Discussion Results of the study showed that the Albanian’s HPSM-based intervention considerably increased adopting oral health behaviors among the preschool children in the intervention group compared to those in the control group. This finding is similar to those found in the previous studies which concluded that conducting health promoting school programs can reduce various health problems such as smoking, low consumption of fruits and water and inadequate oral health behaviors among the students.16-18 In consistent with previous studies it can be claimed that trained teachers and parents play an important role in encouraging students to adopt a sustainable healthy lifestyle for good oral health.15,29-32 Although the teachers and parents’ oral health behaviour, as role models, may influence the children’s gingival health and dental caries,6 it has been shown that many of these role models have limited knowledge and awareness about the oral health.15 Therefore, these groups need training and school is an ideal setting that can provide a participatory environment to work with them to promote the children’s oral health. In this study, after the intervention, the teachers and parents of the intervention group had higher self-efficacy belief scores towards oral health behaviors than their counterparts in the control group. Some previous studies emphasized the role of maternal oral health self-efficacy in children’s oral hygiene.5,32 As perceived self-efficacy has been recognized as one of the important predictors for adopting oral hygiene behaviors, developing programs aimed at fostering mothers’ self-efficacy may promote the healthy dental habits among children. The negative association between self-efficacy beliefs and perceived barriers has been shown in literature.33,34 The higher self-efficacy belief results in fewer perceived barriers in performing a target behavior.33-35 In line with these findings, in present study it was found that perceived barriers of parents and teachers for adopting oral health practice significantly decreased after the intervention compared to the parents and teachers of children in the control group. In the present study, the perceived barriers had a negative correlation with perceived self-efficacy for oral health behaviors. Several barriers for adopting oral health behaviors in children and their parents have been noted in a previous study.36 Focus on addressing the anticipated barriers for adopting oral health behaviors through intervention efforts may be considered as a good strategy to increase self-efficacy beliefs among parents and teachers of preschool children, as those conducted in the present study. Similar with those found by Rong et al,32 following the intervention, significant differences were found in the mean scores of knowledge, attitude and adopting oral health behaviors among the parents and teachers of children in the intervention group compared to their counterparts in the control group. Several studies have confirmed the need to enhance the knowledge and modify the attitude of teachers and parents regarding the oral hygiene.6,37-40 As these cognitive factors are potentially modifiable,5 providing educational programs aimed at addressing these factors in schools could be effective in increasing the oral health literacy among teachers, parents and establishing dental hygiene habits among their children. Strengths and limitations Although the study highlights the application of Albanian’s HPSM framework to develop an oral health education intervention, there are some limitations; firstly, the data were collected from a small sample of Iranian female preschool children in middle-income areas of Tehran, Iran which posed a constraint on the generalizability of the findings. Secondly, male children were not included in the study. Thirdly, the homogeneity of the sample may limit the generalizability of the findings to the other preschool children residing in other areas of Tehran. As a final limitation for the present study, the short duration of the follow-up sessions can be noted. This was due to the time limitations of the researchers. Implications for policy and practice Health policymakers should consider such studies applying HPSs approach to provide more evidence based policies and to build and extend the capacity of the schools in promoting the oral health of the preschool children. Practitioners, school nurses and school health workers should pay a more specific attention to the use of health-promoting approaches for developing oral health promotion interventions in the schools. Conclusion In conclusion, several benefits may be obtained from adopting a HPS approach to develop oral health education interventions. Schools can provide a participatory and supportive environment in order to involve teachers and parents in the process of oral health promotion of the preschool children. Acknowledgements This MSc thesis was funded by Iran University of Medical Sciences grant number 94-01-27-25825. Ethical approval The study was approved by the ethics committee in Iran University of Medical Sciences (code# 94-01-27-25825). Also, the present study protocol was registered in the Iranian registry of Clinical Trials Center (code# IRCT201504207352N10). Children, their parents and schoolteachers were informed about the objectives of the study and a written consent was obtained from them. Competing interests None of the authors have any conflict of interest. Authors’ contributions MS and MHT initiated and designed the study. With the help of TD, GA and MS designed and validated the scale. MS gathered the data. Data analysis of pre- and post-tests was done by GA and MS. TD, MS and MT designed and conducted the educational sessions. All co-authors made substantial contributions to data analysis and interpretation and writing of the manuscript. Table 1 Demographic characteristics of the children in the two groups Control group Intervention group P N % Mean SD N % Mean SD Age of the parents 36.9 6.54 34.9 5.09 0.135 Age of the schoolteachers 37.2 1.69 36.7 1.92 0.455 Mother’s education level <12th grade 21 36 25 44 0.560 >12th grade 37 64 32 56 Father’s education level <12th grade 29 50 31 54 0.457 >12th grade 28 40 26 46 Family income per month < US$300 10 19 9 16 US$300-600 32 60 34 62 0.120 > US$600 11 21 12 22 Occupation of mother Employee 13 22 13 23 0.134 Housewife 40 70 38 67 Other 5 6 6 10 Occupation of father Employee 20 36 25 45 0.337 Self-Employment 29 52 28 50 Other 7 12 3 5 Table 2 Comparison of the mean scores of the Albanian’s HPSM constructs and oral health behaviors scale in the student and their parents and schoolteachers before and after the educational intervention Control group Intervention group Before intervention Mean (SD) After intervention Mean (SD) P a Before intervention Mean (SD) After intervention Mean (SD) P b P c P a Parents Knowledge 44.17 ± 3.29 44.19 ± 3.29 0.931 44.23 ± 5.65 46.49 ± 3.88 0.001 0.940 0.006 Attitude 42.81 ± 29.69 43.20 ± 3.11 0.374 42.66 ± 3.34 44.13 ± 1.26 0.011 0.968 0.001 Oral health behaviors 53.34 ± 6.99 53.33 ± 6.63 0.883 54.27 ± 8.20 57.52 ± 6.62 0.001 0.513 0.001 Perceived self-efficacy 43.33 ± 5.95 42.98 ± 5.98 0.705 43.01 ± 6.08 44.85 ± 4.51 0.051 0.770 0.011 Perceived barriers 26.36 ± 9.12 26.24 ± 8.05 0.997 27.13 ± 8.60 30.57 ± 8.52 0.018 0.638 0.007 Teachers Knowledge 40.54 ± 4.46 40.63 ± 4.36 0.969 40.10 ± 3.11 44.50 ± 4.30 0.002 0.824 0.021 Attitude 33.91 ± 7.54 33.91 ± 7.55 1.000 32.27 ± 7.29 39.91 ± 4.11 0.038 0.611 0.003 Oral health behaviors 54.82 ± 5.58 54.80 ± 5.55 1.000 56.63 ± 6.83 69.36 ± 15.27 0.012 0.502 0.015 Perceived self-efficacy 39.81 ± 7.90 39.80 ± 7.91 1.000 39.72 ± 8.14 42.36 ± 5.74 0.039 0.979 0.034 Perceived barriers 34.63 ± 6.10 34.58 ± 6.14 1.000 35.63 ± 7.43 39.90 ± 8.50 0.012 0.734 0.035 Students Oral health behaviors 15.59 ± 0.27 15.64 ± 0.28 0.671 14.86 ± 0.27 20.06 ± 0.35 0.040 0.941 0.001 Abbreviation: HSPM, Health Promoting Schools Model. aMean values were significantly different from those before the intervention (paired-samples t tests): P<0.05. bMean values were significantly different from those of the control group after the intervention (ANCOVA): P<0.05. cMean values were significantly different from those of the control group before the intervention (independent-samples t test): P<0.05. Figure 1 Albanian’s Health Promoting Schools Model.20 ==== Refs References 1 Dawani N Nisar N Khan N Syed S Tanweer N Prevalence and factors related to dental caries among pre-school children of Saddar town, Karachi, Pakistan: a cross-sectional study BMC Oral Health 2012 12 49 10.1186/1472-6831-12-59 23148740 2 Ghandahari-Motlagh M Zeraati H Dental health status in 3-5 year old kindergarten children in Tehran-Iran in 2003 J Dent (Tehran) 2005 2 1 18 20 3 Kamil MA El-Ameen NM Madkhaly SH Alshamarry TH Hakami RU Nassir EM Knowledge and attitude of Saudi mothers towards health of primary teeth J Dent Oral Hyg 2015 7 7 107 112 10.5897/JDOH2015.0156 4 Oliveira LB Sheiham A Bönecker M Exploring the association of dental caries with social factors and nutritional status in Brazilian preschool children Eur J Oral Sci 2008 116 1 37 43 10.1111/j.1600-0722.2007.00507.x 18186730 5 Silva-Sanigorski A Ashbolt R Green J Calache H Keith B Riggs E Parental self-efficacy and oral health-related knowledge are associated with parent and child oral health behaviors and self-reported oral health status. Community Dent Oral Epidemiol Community Dent Oral Epidemiol 2013 41 4 345 52 10.1111/cdoe.12019 23157162 6 Okada M Kawamura M Kaihara Y Matsuzaki Y Kuwahara S Ishidori H Influence of parents’ oral health behaviour on oral health status of their school children: an exploratory study employing a causal modelling technique Int J Paediatr Dent 2002 12 2 101 8 10.1046/j.1365-263X.2002.00338.x 11966888 7 Adair PM Pine CM Burnside G Nicoll AD Gillett A Anwar S Familial and cultural perceptions and beliefs of oral hygiene and dietary practices among ethnically and socio-economicall diverse groups Community Dental Health 2004 21 1 Suppl 102 11 15072479 8 Aljanakh M Siddiqui AA Mirza AJ Teachers’ knowledge about oral health and their interest in oral health education in Hail, Saudi Arabia Int J Health Sci (Qassim) 2016 10 1 87 93 27004061 9 Mota A Oswal KC Sajnani DA Sajnani AK Oral health knowledge, attitude, and approaches of pre-primary and primary school teachers in Mumbai, India Scientifica 2016 2016 5967427 10.1155/2016/5967427 27034901 10 Kwan SY Petersen PE Pine CM Borutta A Health-promoting schools: an opportunity for oral health promotion Bull World Health Organ 2005 83 677 85 16211159 11 Peterson P, Christensen L. Oral Health Promotion: Health Promoting Schools Project. Copenhagen: World Health Organization Regional Office for Europe; 1994. 12 WHO. Research to improve implementation and effectiveness of school health programmes. Geneva: World Health Organization; 1996. 13 WHO’s Global School Health Initiative. Health-promoting schools. A healthy setting for living, learning and working. Geneva: World Health Organization; 1998. 14 Croucher R Rodgers A Humpherson W Crush L The ‘spread of effect’of a school based dental health education project Community Dent Oral Epidemiol 1985 13 4 205 7 10.1111/j.1600-0528.1985.tb01903.x 3862501 15 World Health Organization. Oral health promotion: an essential element of a health-promoting school. Geneva: WHO; 2003. 16 Moysés ST Moysés SJ Watt RG Sheiham A Associations between health promoting schools’ policies and indicators of oral health in Brazil Health Promot Int 2003 18 3 209 18 10.1093/heapro/dag016 12920141 17 Schofield MJ Lynagh M Mishra G Evaluation of a health promoting schools program to reduce smoking in Australian secondary schools Health Educ Res 2003 18 6 678 92 10.1093/her/cyf048 14654501 18 Laurence S Peterken R Burns C Fresh kids: the efficacy of a health promoting schools approach to increasing consumption of fruit and water in Australia Health Promot Int 2007 22 3 218 26 10.1093/heapro/dam016 17584783 19 Wang D Stewart D The implementation and effectiveness of school-based nutrition promotion programmes using a health-promoting schools approach: a systematic review Public Health Nutr 2013 16 6 1082 100 10.1017/S1368980012003497 22850118 20 Cheshlarov M, Havlínová M, Inchley J, Jakonen S, Jankulovska S, Bruun Jensen B, et al. Models of health promoting schools in Europe. Copenhagen: European Network of Health Promoting Schools, International Planning Committee (IPC); 2002. 21 Motamedi M. Promoting physical activity among mentally retarded male and female in primary school students using Albanian,s health-promoting schools model [Master of thesis]. Tehran, Iran: School of Health, Iran University of Medical Sciences; 2012. [In Persian]. 22 Noori Sistani M. The effect of educational methods introduced by Albanian,s health-promoting schools model on puberty health among female middle-school students in district 6 of Tehran, Iran [Master of thesis]. Tehran, Iran: School of Health, Iran University of Medical Sciences; 2008. [In persian]. 23 WHO. Report on the intercountry meeting of oral health focal points, Isfahan, Islamic Republic of Iran, 31 May–2 June 2011. http://apps.who.int/iris/handle/10665/116070. 24 Saied-Moallemi Z Virtanen J Tehranchi A Murtomaa H Disparities in oral health of children in Tehran, Iran Eur Arch Paediatr Dent 2006 7 4 262 4 10.1007/BF03262563 17164073 25 Lawshe CH A quantitative approach to content validity Pers Psychol 1975 28 4 563 75 10.1111/j.1744-6570.1975.tb01393.x 26 Polit DF, Beck CT. Nursing Research: Principles and Methods. 46th ed. Philadelphia, PA: Lippincott; 2004. p. 416-45. 27 Baumgartner TA Chung H Confidence limits for intraclass reliability coefficients Meas Phys Educ Exerc Sci 2001 5 179 88 10.1207/S15327841MPEE0503_4 28 Cronbach L Coefficient alpha and the internal structure of tests Psychometrika 1951 16 297 334 29 Wennhall I Matsson L Schröder U Twetman S Outcome of an oral health outreach programme for preschool children in a low socioeconomic multicultural area Int J Paediatr Dent 2008 18 2 84 90 10.1111/j.1365-263X.2007.00903.x 18237290 30 Kowash M Pinfield A Smith J Curzon M Dental health education: effectiveness on oral health of a long-term health education programme for mothers with young children Br Dent J 2000 188 4 201 5 10740903 31 Ramroop V Wright D Naidu R Dental health knowledge and attitudes of primary school teachers toward developing dental health education West Indian Med J 2011 60 5 576 80 22519237 32 Rong WS Bian JY Wang WJ De Wang J Effectiveness of an oral health education and caries prevention program in kindergartens in China Community Dent Oral Epidemiol 2003 31 6 412 6 10.1046/j.1600-0528.2003.00040.x 14986908 33 Finlayson TL Siefert K Ismail AI Sohn W Maternal self‐efficacy and 1–5‐year‐old children’s brushing habits Community Dent Oral Epidemiol 2007 35 4 272 81 10.1111/j.1600-0528.2007.00313.x 17615014 34 Pender NJ, Murdaugh CL, Parsons MA. Health promotion in nursing practice. Upper Saddle River, NJ: Pearson; 2006. 35 Dehdari T Rahimi T Aryaeian N Gohari MR Effect of nutrition education intervention based on Pender’s Health Promotion Model in improving the frequency and nutrient intake of breakfast consumption among female Iranian students Public Health Nutr 2014 17 3 657 66 10.1017/S1368980013000049 23360695 36 Gussy MG Waters E Kilpatrick N A qualitative study exploring barriers to a model of shared care for pre-school children’s oral health Br Dent J 2006 201 3 165 70 10.1038/sj.bdj.4813849 16902551 37 Kelly SE Binkley CJ Neace WP Gale BS Barriers to care-seeking for children’s oral health among low-income caregivers Am J Public Health 2005 95 8 1345 51 10.2105/AJPH.2004.045286 16043666 38 Almas K Al-Malik TM Al-Shehri MA Skaug N The knowledge and practices of oral hygiene methods and attendance pattern among school teachers in Riyadh, Saudi Arabia Saudi Med J 2003 24 10 1087 91 14578974 39 Gussy MG Waters E Riggs E Lo SK Kilpatrick N Parental knowledge, beliefs and behaviours for oral health of toddlers residing in rural Victoria Aust Dent J 2008 53 1 52 60 10.1111/j.1834-7819.2007.00010.x 18304242 40 Rajab L Petersen P Bakaeen G Hamdan M Oral health behaviour of schoolchildren and parents in Jordan Int J Clin Pediatr Dent 2002 12 3 168 76 10.1046/j.1365-263X.2002.00359.x 41 Tangade PS Jain M Mathur A Prasad S Natashekara M Knowledge, attitude and practice of dental caries and periodontal disease prevention among primary school teachers in Belgaum city, India Pesqui Bras Odontopediatria Clin Integr 2011 11 1 77 83

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==== Front Health Promot PerspectHealth Promot PerspectHealth Promot PerspectTBZMEDHealth Promotion Perspectives2228-6497Tabriz University of Medical Sciences 10.15171/hpp.2016.27Short CommunicationEffect of physical activity on mortality risk among Americans with retinopathy Loprinzi Paul D. * Jackson Heart Study Vanguard Center of Oxford, Physical Activity Epidemiology Laboratory, Center for Health Behavior Research, Department of Health, Exercise Science and Recreation Management, The University of Mississippi, University, MS 38677, USA* Corresponding Author: Paul D. Loprinzi, PhD; Phone: 662-915-5521; Fax: 662-915-5525; pdloprin@olemiss.edu2016 10 8 2016 6 3 171 173 26 4 2016 26 6 2016 © 2016 The Author(s).2016This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.http://journals.tbzmed.ac.ir/HPP Background: Previous work demonstrates that retinopathy is associated with increased mortality risk, with physical activity inversely associated with retinopathy and all-cause mortality. However, no study has evaluated the effects of physical activity on mortality among those with existing retinopathy, which was this study’s purpose. Methods: Data from the 2005-2006 National Health and Nutrition Examination Survey were utilized, with follow-up through 2011. Retinopathy was objectively-measured using the Canon Non-Mydriatic Retinal Camera CR6-45NM. Physical activity was objectively-measured via up to 7 days of accelerometry assessment. Results: Six-hundred and seventy one adults (40-85 years) with complete data on the study variables constituted the analytic sample. During the follow-up period, 91 deaths occurred. In the sample, 35 886 person-months occurred with a mortality incidence rate of 2.5 deaths per1000 person-months. Among participants with mild retinopathy, those who met physical activity guidelines at baseline had a 63% reduced risk of all-cause mortality (HR adjusted = 0.37; 95% CI:0.18-0.75; P = 0.007). Notably, physical activity was not associated with mortality risk among those with moderate/severe retinopathy (HR adjusted = 0.371.72; 95% CI: 0.62-4.76; P = 0.27). Conclusion: Physical activity is associated with reduced mortality risk among those with mild retinopathy, but not among those with moderate/severe retinopathy. AccelerometryEpidemiologyPhysical activityVisionCitation: Loprinzi PD. Effect of physical activity on mortality risk among Americans with retinopathy. Health Promot Perspect. 2016;6(3):171-173. doi: 10.15171/hpp.2016.27. ==== Body Introduction Retinopathy is associated with increased mortality risk.1 The mechanisms through which retinopathy may increase mortality risk is not fully elucidated. It is possible that retinopathy increases mortality risk through shared risk factors. For example, cardiovascular and mortality risk factors, such as hypertension, dyslipidemia and elevated glycated hemoglobin, are known to increase the risk of retinopathy.2,3 Our recent work demonstrates that physical activity is associated with reduced retinopathy prevalence,2 reduced mortality risk among those with visual impairment,4 and reduced mortality risk among those at high risk for a future atherosclerotic cardiovascular disease event.5 Yet to be investigated in the literature, however, is whether physical activity plays a protective role against mortality risk among those with existing retinopathy, which was this study’s purpose, written here as a short communication. The hypothesis is that physical activity will be inversely associated with mortality risk even among those with existing retinopathy. This hypothesis is plausible as previous research demonstrates that physical activity is favorably associated with both retinopathy and mortality risk.2,5 If indeed physical activity plays a protective role in mortality risk among those with existing retinopathy, then this will have important health promotion implications for those with retinopathy. Materials and Methods Design & Participants Data from the 2005-2006 National Health and Nutrition Examination Survey were used. In this sample, 671 adults (40-85 years) with complete data on the study variables constituted the analytic sample. Only those with some degree of retinopathy were evaluated herein (N=566, mild retinopathy; N=105, moderate/severe retinopathy). Retinopathy As we have described elsewhere,2 retinal imaging was performed using the Canon Non-Mydriatic Retinal Camera CR6-45NM (Canon, Tokyo, Japan). The presence of non-proliferative retinopathy (mild or moderate/severe retinopathy) was determined using the Early Treatment Diabetic Retinopathy Study grading criteria.6 Physical activity As described and validated elsewhere,7 self-reported physical activity was assessed. Consistent with government physical activity guidelines (30 min/day of moderate-to-vigorous physical activity [MVPA]), participants were classified as above or below 2000 MVPA MET-min-month. Statistical analyses All analyses were performed using survey data procedures to adjust for the complex survey design employed in NHANES. Multivariable Cox proportion hazard analysis was employed to examine the association of meeting physical activity guidelines and mortality, with analyses stratified by those with mild retinopathy and moderate/severe retinopathy. Hazard ratios (and their corresponding 95% confidence interval) were calculated as an estimate of an effect size. Schoenfeld’s residuals were used to verify the proportional hazards assumption. Analyses were adjusted for age, gender, race-ethnicity, diabetes (physician diagnosis, A1C≥6.5%, or fasting glucose ≥126 mg/dL), objectively-measured visual impairment (normal, URE, or VI),2 and comorbid illness (summed number of the following conditions: coronary artery disease, stroke, heart attack, body mass index (BMI) ≥ 25 kg/m2, hypertension diagnosis, and cancer diagnosis).Statistical significance was established as P<0.05, with all analyses evaluated in the Stata software package (v. 12, College Station, TX). Results Characteristics of the analyzed sample are shown in Table 1. Participants, on average, were 59.8 (95% CI: 58.2-61.5) years, and the mean number of comorbidities was just over 1 for the entire sample. Among those with mild and moderate/severe retinopathy, respectively, approximately 56% and 50% were men. A larger percentage of individuals with mild retinopathy were non-Hispanic white when compared to those with moderate/severe retinopathy (70.5% vs. 52.9%). As expected, diabetes was more prevalent among those with moderate/severe vs. mild retinopathy (88.8% vs. 33.7%). Further, visual impairment was also more prevalent among those with moderate/severe (7.5%; 95% CI: 0.9-14.1) vs. mild retinopathy (1.7%; 95% CI: 0.4-2.9). The median follow-up period was 55 months (IQR=43-66). During the follow-up period, 91 deaths occurred. In the sample, 35886 person-months occurred with a mortality incidence rate of 2.5 deaths per 1000 person-months. Among participants with mild retinopathy, those who met physical activity guidelines at baseline had a 63% reduced risk of all-cause mortality (HRadjusted = 0.37; 95% CI: 0.18-0.75; P=0.007); proportional hazard assumption was not violated (P=0.85). Notably, physical activity was not associated with mortality risk among those with moderate/severe retinopathy (HRadjusted = 1.72; 95% CI: 0.62-4.76; P=0.27); proportional hazard assumption was not violated (P=0.40) (Table 2). Table 1 Characteristics of the analyzed sample (N = 671) Mild retinopathy Moderate/severe retinopathy N 566 105 Age, mean (95% CI) years 59.8 (58.1-61.6) 59.8 (57.3-62.3) Comorbidities, mean (95% CI) 1.2 (1.1-1.4) 1.6 (1.4-1.9) Men, % 55.8 49.9 White, % 70.5 52.9 Diabetes, % 33.7 88.8 Visual impairment, % 1.7 7.5 Died, % 10.2 17.5 Meeting PA guidelines,a % 16.4 20.0 Abbreviations: PA, physical activity; MVPA, moderate-to-vigorous physical activity. a Meeting PA guidelines defined as ≥2000 MVPA MET-min-month. Table 2 Weighted multivariable Cox proportional hazard model evaluating the association between physical activity and mortality risk, stratified by retinopathy status Mild Retinopathy Moderate/Severe Retinopathy HR 95% CI HR 95% CI Meeting MVPA guidelines vs. not 0.37 0.18-0.75 1.72 0.62-4.76 Abbreviations: MVPA, Moderate-to-vigorous physical activity; HR, hazard ratio. Meeting PA guidelines defined as ≥ 2000 MVPA MET-min-month. Models adjusted for age, gender, race-ethnicity, diabetes, visual impairment and comorbid illness. Discussion Adults with retinopathy have an increased risk of early mortality.1,8-13 For example, in a meta-analysis of 20 studies, providing data from 19234 patients, Kramer et al1 demonstrated that, among those with type 2 diabetes, the presence of any degree of diabetic retinopathy increased the chance for all-cause mortality and/or cardiovascular disease events by 2.34. Results were similar for adults with type 1 diabetes. Consequently, these findings highlight that diabetic retinopathy, in particular, is a serious microvascular complication. As such, individuals with retinopathy should be carefully screened and monitored, along with the provision of necessary resources to mitigate cardiovascular disease and mortality risk. Encouragingly, the present findings demonstrate that physically activity adults with mild retinopathy have a reduced risk of all-cause mortality. This finding is in alignment with other studies showing that physical activity is protective of early mortality among various vulnerable populations, such as those with a high risk for cardiovascular disease,5 coronary artery disease patients,14,15 congestive heart failure patients,16,17 diabetics,18 hypertensive adults,19 chronic obstructive pulmonary disease patients,20 liver disease patients,21 and those with visual4 or hearing impairment.22,23 Physical activity, however, was not protective of early mortality among those with moderate/severe retinopathy, which may be a result of the greater degree of visual impairment (which is linked with reduced activity and increased mortality risk) among this group. As such, these findings underscore the importance of physical activity promotion among those with varying degrees of retinopathy, but particularly before the progression from mild to moderate retinopathy. Major strengths of this study include the novel investigation, national sample and prospective study design. Future replicative work is needed, which should overcome the limitations of this study, including the subjective assessment of physical activity and relatively short follow-up period. In conclusion, the results of the present study suggest that physical activity is inversely associated with mortality risk among those with mild retinopathy, but not among those with moderate/severe retinopathy. Future confirmatory work is needed, and such work would benefit by evaluating candidate mechanisms to explain the present study’s observed associations. Funding No funding was used to prepare this manuscript. Ethical approval Procedures were approved by the National Center for Health Statistics review board; written consent was obtained prior to data collection. Competing interests The authors declare no conflicts of interest. Author’s contribution PDL was involved in the conceptualization of the study, data analyses, interpretation of the results, and drafting and revising the manuscript. ==== Refs References 1 Kramer CK Rodrigues TC Canani LH Gross JL Azevedo MJ Diabetic retinopathy predicts all-cause mortality and cardiovascular events in both type 1 and 2 diabetes: meta-analysis of observational studies Diabetes Care 2011 34 5 1238 44 10.2337/dc11-0079 21525504 2 Loprinzi PD Brodowicz GR Sengupta S Solomon SD Ramulu PY Accelerometer-assessed physical activity and diabetic retinopathy in the United States JAMA Ophthalmol 2014 132 8 1017 9 10.1001/jamaophthalmol.2014.402 25124951 3 Loprinzi PD. Association of accelerometer-assessed sedentary behavior with diabetic retinopathy in the United States. JAMA Ophthalmol 2016; forthcoming. 4 Loprinzi PD Joyner C Accelerometer-determined physical activity and mortality in a national prospective cohort study: Considerations by visual acuity Prev Med 2016 87 18 21 10.1016/j.ypmed.2016.02.005 26861750 5 Loprinzi PD Accelerometer-determined physical activity and mortality in a national prospective cohort study of adults at high risk of a first atherosclerotic cardiovascular disease event Int J Cardiol 2016 202 417 8 10.1016/j.ijcard.2015.09.061 26432494 6 Grading diabetic retinopathy from stereoscopic color fundus photographs--an extension of the modified Airlie House classification. ETDRS report number 10. Early Treatment Diabetic Retinopathy Study Research Group. 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The Beijing Eye Study Graefes Arch Clin Exp Ophthalmol 2008 246 6 923 5 10.1007/s00417-008-0773-z 18299875 14 Loprinzi PD The fat-but-fit paradigm and all-cause mortality among coronary artery disease patients Int J Clin Pract 2016 70 5 406 8 10.1111/ijcp.12799 27040330 15 Loprinzi PD Addoh O The effects of free-living physical activity on mortality after coronary artery disease diagnosis Clin Cardiol 2016 39 3 165 9 10.1002/clc.22508 26748944 16 Loprinzi PD The effects of free-living physical activity on mortality after congestive heart failure diagnosis Int J Cardiol 2015 203 598 9 10.1016/j.ijcard.2015.11.017 26574935 17 Loprinzi PD Physical activity, weight status, and mortality among congestive heart failure patients Int J Cardiol 2016 214 92 94 10.1016/j.ijcard.2016.03.180 27060265 18 Loprinzi PD The effects of objectively-measured, free-living daily ambulatory movement on mortality in a national sample of adults with diabetes Physiol Behav 2016 154 126 8 10.1016/j.physbeh.2015.11.022 26626815 19 Loprinzi PD Accelerometer-determined physical activity and all-cause mortality in a national prospective cohort study of hypertensive adults J Hypertens 2016 34 5 848 52 10.1097/HJH.0000000000000869 26828782 20 Loprinzi PD Walker JF Increased daily movement associates with reduced mortality among COPD patients having systemic inflammation Int J Clin Pract 2016 70 3 286 91 10.1111/ijcp.12778 26916560 21 Loprinzi PD VanWagner LB Survival effects of physical activity on mortality among persons with liver disease Prev Med Rep 2016 3 132 4 10.1016/j.pmedr.2015.12.011 26844199 22 Loprinzi PD Accelerometer-determined physical activity and mortality in a national prospective cohort study: considerations by hearing sensitivity Am J Audiol 2015 24 4 569 72 10.1044/2015_AJA-15-0044 26650870 23 Loprinzi PD Muscle strengthening activities and mortality with considerations by hearing sensitivity Int J Audiol 2016 55 5 320 2 10.3109/14992027.2016.1140233 26886144

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==== Front Front MicrobiolFront MicrobiolFront. Microbiol.Frontiers in Microbiology1664-302XFrontiers Media S.A. 10.3389/fmicb.2016.01340MicrobiologyOriginal ResearchDiversity, Biogeography, and Biodegradation Potential of Actinobacteria in the Deep-Sea Sediments along the Southwest Indian Ridge Chen Ping 12Zhang Limin 1Guo Xiaoxuan 12Dai Xin 12Liu Li 3Xi Lijun 1†Wang Jian 1Song Lei 4Wang Yuezhu 5Zhu Yaxin 1Huang Li 12Huang Ying 1*1State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of SciencesBeijing, China2College of Life Sciences, University of Chinese Academy of SciencesBeijing, China3Information Network Center, Institute of Microbiology, Chinese Academy of SciencesBeijing, China4China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of SciencesBeijing, China5Shanghai-MOST Key Laboratory of Disease and Health Genomics, Chinese National Human Genome Center at ShanghaiShanghai, ChinaEdited by: Mark Alexander Lever, ETH Zurich, Switzerland Reviewed by: William D. Orsi, Lüdwig-Maximilians University of Munich, Germany; Sean Patrick Jungbluth, University of Southern California, USA *Correspondence: Ying Huang huangy@im.ac.cnThis article was submitted to Extreme Microbiology, a section of the journal Frontiers in Microbiology †Present Address: Lijun Xi, State Key Laboratory of Heavy Oil Processing and Centre for Bioengineering and Biotechnology, China University of Petroleum, Qingdao, China 29 8 2016 2016 7 134022 5 2016 15 8 2016 Copyright © 2016 Chen, Zhang, Guo, Dai, Liu, Xi, Wang, Song, Wang, Zhu, Huang and Huang.2016Chen, Zhang, Guo, Dai, Liu, Xi, Wang, Song, Wang, Zhu, Huang and HuangThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.The phylum Actinobacteria has been reported to be common or even abundant in deep marine sediments, however, knowledge about the diversity, distribution, and function of actinobacteria is limited. In this study, actinobacterial diversity in the deep sea along the Southwest Indian Ridge (SWIR) was investigated using both 16S rRNA gene pyrosequencing and culture-based methods. The samples were collected at depths of 1662–4000 m below water surface. Actinobacterial sequences represented 1.2–9.1% of all microbial 16S rRNA gene amplicon sequences in each sample. A total of 5 actinobacterial classes, 17 orders, 28 families, and 52 genera were detected by pyrosequencing, dominated by the classes Acidimicrobiia and Actinobacteria. Differences in actinobacterial community compositions were found among the samples. The community structure showed significant correlations to geochemical factors, notably pH, calcium, total organic carbon, total phosphorus, and total nitrogen, rather than to spatial distance at the scale of the investigation. In addition, 176 strains of the Actinobacteria class, belonging to 9 known orders, 18 families, and 29 genera, were isolated. Among these cultivated taxa, 8 orders, 13 families, and 15 genera were also recovered by pyrosequencing. At a 97% 16S rRNA gene sequence similarity, the pyrosequencing data encompassed 77.3% of the isolates but the isolates represented only 10.3% of the actinobacterial reads. Phylogenetic analysis of all the representative actinobacterial sequences and isolates indicated that at least four new orders within the phylum Actinobacteria were detected by pyrosequencing. More than half of the isolates spanning 23 genera and all samples demonstrated activity in the degradation of refractory organics, including polycyclic aromatic hydrocarbons and polysaccharides, suggesting their potential ecological functions and biotechnological applications for carbon recycling. community structurecultivationbiodegradationmarine actinobacteriamicrobial diversitypyrosequencingSouthwest Indian Ridge ==== Body Introduction The phylum Actinobacteria is composed of a large group of morphologically and physiologically diverse Gram-positive bacteria with high genomic G+C contents, which are ubiquitous in nature (Ensign, 1992; Goodfellow et al., 2012). Members of this phylum are also successful colonizers of different extreme environments, often occurring as abundant populations (Bull, 2011). This phylum has been found to be one of the top five most abundant bacterial phyla in the deep ocean (Zinger et al., 2011; Yilmaz et al., 2016). The marine actinobacterial diversity has been mainly investigated by culture-dependent methods, and at least 66 actinobacterial genera have been cultured from different marine environments (Goodfellow and Fiedler, 2010; http://www.bacterio.net/). However, limited efforts have been devoted to exploring actinobacterial diversity and community compositions in the deep sea, particularly by using high-throughput sequencing approaches. A recent investigation using clone sequencing detected 9 actinobacterial genera from deep Arctic marine surface sediments (Zhang et al., 2014); and a targeted search for the marine actinomycetes Salinispora spp. using specific 16S rRNA gene primers revealed the presence of this genus in deep marine sediments (3814–5699 m below water surface and 0–7 m below see floor [mbsf]) from the Canary Basin and South Pacific Gyre (Prieto-Davó et al., 2013). Data from previous studies on deep-sea microbial diversity showed that the relative abundance of actinobacteria in the sediments were 0.1−3.0% in the Arctic Ocean (0.38–2.32 mbsf; Jorgensen et al., 2012), 0.7−2.4% in the Atlantic Ocean (0–0.02 mbsf; Schauer et al., 2010), and about 10.0% in the Pacific Ocean (0–350 mbsf; Inagaki et al., 2006). In the hydrothermal surface sediments, the relative abundance of actinobacteria was found to be about 5.0% at the Mid-Atlantic Ridge (López-García et al., 2003) and about 3.8% in the Guaymas Basin of Pacific Ocean (Teske et al., 2002). The metatranscriptomes of anaerobic Peru Margin sediment from 5 to 159 mbsf also indicated relatively abundant gene expression from Actinobacteria (1.2–6.1%; Orsi et al., 2013). As for planktonic actinobacteria in the ocean, it was shown that their global distribution changed along latitudinal gradients (Pommier et al., 2007); however, their composition in the brackish northern Baltic Sea was related to environmental gradients including total phosphorus (TP), dissolved organic carbon, chlorophyll a, and bacterial production (Holmfeldt et al., 2009). These findings suggest a biogeographic distribution of actinobacteria that is shaped by biogeochemical parameters such as dissolved organic carbon, TP, etc. However, little is known about the actinobacterial diversity and biogeography in deep-sea habitats. The Southwest Indian Ridge (SWIR) is one of the slowest spreading ocean ridges and has diverse geographic environments including an unexpected high frequency of hydrothermal vents and plumes (German et al., 1998; Tao et al., 2012, 2014). Hydrothermal systems are enriched with a variety of chemical substances (such as manganese [Mn], iron [Fe], methane [CH4], hydrogen gas [H2], reduced sulfur compounds, and polycyclic aromatic hydrocarbons [PAHs]) and form dynamic habitats with steep thermal and chemical gradients that may influence the microbial structure (Jannasch and Mottl, 1985; Baker et al., 1995; Simoneit and Fetzer, 1996). Due to its remote location, however, the SWIR is not readily accessible to investigate (Tao et al., 2012). Recently, a clone library study revealed that Thaumarchaeota, Acidobacteria, Actinobacteria, Bacteroidetes, and Proteobacteria dominated the archaeal and bacterial communities in a semi-consolidated carbonate sample of the SWIR and also suggested that Alphaproteobacteria and Thaumarchaeota potentially participated in sulfur (S) and nitrogen cycles in this environment (Li et al., 2014). Supporting this, another study demonstrated that Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Firmicutes, Nitrospirae, and archaea might participate in the S cycle in two low-temperature hydrothermal deposits at the SWIR (Cao et al., 2014). Nevertheless, it remains unclear about the abundance and composition of actinobacteria at the SWIR and the biogeochemical processes that they are involved in. Besides the well-known ability of actinobacteria to produce diverse bioactive compounds, they also produce various extracellular hydrolytic enzymes, which have important ecological roles and potential biotechnological applications (Peczynska-Czoch and Mordarski, 1988). These enzymes are widely reported among soil actinobacteria and have been shown to participate in the decomposition of recalcitrant organic carbons such as polysaccharides (Manucharova, 2009). Moreover, members of the actinobacteria have also been reported as highly efficient PAH degraders in PAH-contaminated soils (Uyttebroek et al., 2006; García-Díaz et al., 2013). Cellulose, chitin, and pectin are the main components of eukaryotic cell walls, and thus are abundant and widely distributed in marine environments, serving as important sources of organic matter. Many of the actinobacteria isolated from coastal and shallow marine sediments are capable of producing cellulase, chitinase, and pectinase (Veiga et al., 1983; Augustine et al., 2013). However, there are few reports corresponding to such capabilities of deep-sea actinobacteria. PAHs are the main components of petroleum and are introduced into the marine environment by oil spill, river import, natural seepage, hydrothermal activity, and even air current transfer (Latimer and Zheng, 2003). PAHs are persistent organic pollutants that tend to accumulate in marine sediments and have even been reported to be widespread in the deep Arctic Ocean and Pacific Ocean (Simoneit and Fetzer, 1996; Dong et al., 2015). A recent study indicated that hydrocarbon-degrading actinobacteria, notably Dietzia, were abundant in the Arctic deep-sea sediments (0–0.5 mbsf) and were likely to play an important role in PAH degradation in situ (Dong et al., 2015). In this study, we explored the diversity of actinobacteria at the SWIR using 16S rRNA gene pyrosequencing and culture-dependent methods. We determined how the actinobacterial community from different sampling sites varied in composition and identified the environmental factors that contributed to the distribution of actinobacteria. The degradation capacity of diverse actinobacterial isolates toward recalcitrant organic substances was also evaluated, which might help to uncover their ecological functions and biotechnological applications. Our results provide new insights into the actinobacterial community structure at the SWIR and contribute to the understanding of the underlying mechanisms behind actinobacterial distribution in the deep ocean. Materials and methods Sample collection and physicochemical analysis Eight surface sediment samples (M1–M8) and a bottom water sample (M9) were collected along the SWIR at depths ranging from 1662 to 4000 m during the DY115-21 and DY115-22 cruises of the DaYang YiHao research vessel in January 2010, March 2010, and January 2011 (Figure 1, Table 1). Samples M8 and M9 were collected from the first discovered active hydrothermal field at the SWIR (Tao et al., 2012). A television multi-core sampler (Φ10 × 60 cm) was deployed to collect surface sediment samples and a rosette water sampler equipped with 10-l Niskin bottles and a CTD unit was used to collect the water sample. The sediment samples (0–0.2 mbsf) were transferred from the cores into sterile 200-ml plastic boxes using sterile scoops. Water for physicochemical analysis was collected in a sterile 1-l glass bottle. Microbial cells were collected from 50 l of water by a tangential flow filtration device (Millipore, Bedford, MA, USA) and further concentrated by subsequent filtration onto two polycarbonate filters (diameter 47 mm, pore size 0.1 μm, Millipore) in the onboard laboratory. All samples were frozen at −20°C until further processing in the laboratory. Figure 1 The map of sampling sites along the Southwest Indian Ridge (SWIR). The hydrothermal field information was collected from previous studies (Tao et al., 2012, 2014). (A) Location of the SWIR, near the Southeast Indian Ridge (SEIR) and the Central Indian Ridge (CIR). (B) Magnified view of the sampling area marked by a red box in A using a bathymetric map. Table 1 Environmental parameters of the samples*. Parameter† M1 M2 M3 M4 M5 M6 M7 M8 M9 Sample property Sediment Sediment Sediment Sediment Sediment Sediment Sediment Sediment Seawater Sampling date 16 March 2010 24 March 2010 25 March 2010 08 January 2011 08 January 2011 09 January 2011 13 January 2010 15 January 2010 11 January 2010 Longitude 47.4069°E 55.5838°E 54.6835°E 48.6123°E 48.1095°E 47.9646°E 49.5167°E 49.6167°E 49.6500°E Latitude 38.9533°S 34.2985°S 34.8192°S 38.1668°S 38.5650°S 38.3187°S 37.7667°S 37.8333°S 37.7833°S Depth (m) 1880 3178 3523 2187 2734 1662 4000 2800 2800 EC (us/cm) 6500 12040 9920 10160 10740 7780 4170 11520 26200 pH 9.06 8.83 8.62 9.11 8.96 8.9 8.34 7.77 7.47 Ca (g/kg) 290.30 340.10 353.30 299.50 337.90 290.60 20.01 0.15 3.19 Fe (mg/kg) 2610.02 4229.17 2982.88 2159.43 3902.49 8666.48 364.74 1553.71 0.02 Mg (mg/kg) 8300.00 2900.00 1800.00 1200.00 2000.00 13300.00 158.00 62.58 9312.00 Mn (mg/kg) 1428.70 596.23 394.15 277.52 551.21 12915.74 19.19 105.02 0.01 S (mg/kg) 2067.02 2036.25 1800.20 1218.69 966.65 1133.50 300.37 44.46 1254.50 TOC (mg/kg) 1300.00 1600.00 1400.00 1700.00 1400.00 1300.00 4920.00 68255.00 18.01 TN (mg/kg) 740.00 390.00 530.00 870.00 550.00 790.00 389.00 239.00 47.00 NO-N (mg/kg) 57.00 19.00 19.00 23.00 20.00 27.00 13.91 5.12 8.84 TP (mg/kg) 170.00 370.00 230.00 150.00 180.00 210.00 1590.00 2590.00 679.00 * The temperature at all sampling locations was 2–4°C. † EC, electric conductance; TN, total nitrogen; TP, total phosphorus; TOC, total organic carbon; NO-N, nitrate nitrogen. Sample geochemical analyses were conducted at the Analysis Center of Tsinghua University. The samples were mixed with distilled water without CO2 (Millipore) at a 1:2.5 ratio, and the electric conductance and pH of the mixtures were determined using a pH/conductivity meter (HACH Sension 156). The concentrations of total organic carbon (TOC) and total nitrogen (TN) were measured using the combustion method with the Elemental Analyzer (Euroea, Eurouector, Italy). Major elements Mg, S, Ca, Mn, and Fe were analyzed using inductively coupled plasma atomic emission spectroscopy (ICP-AES) (VISTA-MPX, VARIN, USA). The concentration of TP was determined using the modified Murphy Riley method (Carter and Gregorich, 2008). Nitrate nitrogen (NO-N) was quantified using the automated spectrophotometric method (Carter and Gregorich, 2008). The longitude, latitude, depth, and temperature were recorded by the research vessel. Detailed information of the samples is provided in Table 1. The map of sampling sites was drawn using the Generic Mapping Tools (Wessel and Smith, 1998) (http://gmt.soest.hawaii.edu/). Mercator projection and NUVEL-1A plate boundaries (http://jules.unavco.org/GMT/) were used for plotting. DNA extraction, PCR amplification and pyrosequencing of the 16S rRNA gene Filters used to collect cells from the water samples were smashed in 20 ml phosphate buffered solution in an ice bath using the Tissue Tearor (Model: 985370-395, Biospec Products, INC). A sample of sediment (5 g [wet weight]) or filter homogenate (5 ml) was subjected to DNA extraction using the high-salt-SDS-heat method (Zhou et al., 1996), with a modification that the mixture of sample and extraction buffer was frozen in liquid nitrogen and thawed in a 65°C water bath for three cycles and then cooled down to 37°C before proteinase K was added. For each sample, three replicates of DNA extracts were obtained and pooled together for the subsequent PCR amplication and pyrosequencing. Due to the low coverage and relatively high false positive rate of the reported Actinobacteria-specific 16S rRNA gene primers (Stach et al., 2003b; Schäfer et al., 2010) and in order to investigate the actinobacterial relative abundance among the prokaryotic community, a pair of universal 16S rRNA gene primers was used in the present study. The total DNA was amplified in triplicate by PCR for pyrosequencing, using the primers U789F (5′-TAGATACCCSSGTAGTCC-3′) and U1068R (5′-CTGACGRCRGCCATGC-3′; Baker et al., 2003; Lee et al., 2010) with 8-nucleotide barcodes. These primers target the V5–V6 region on the 16S rRNA gene and cover about 93.0–96.0% of bacteria and archaea (Klindworth et al., 2012). PCR was performed in triplicate in 50-μl reaction system containing 5U of Pfu DNA polymerase, 1 × Pfu reaction buffer, 0.2 mM of dNTPs (TaKaRa, Japan), 0.4 μM of each barcoded primers, and 100 ng of genomic DNA template. The amplification was conducted under the following conditions: initial denaturation at 94°C for 5 min; 26 cycles of denaturation at 94°C for 30 s, annealing at 53°C for 30 s and extension at 72°C for 45 s; and a final extension at 72°C for 6 min (Lee et al., 2010). The triplicate PCR products were pooled and purified using the TaKaRa Agarose Gel DNA Purification Kit (Takara, Japan). The amplicons from nine samples were quantified by Qubit dsDNA HS Assay Kit (Life Technologies, Eugene, Oregon, USA) on the Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and then mixed in equal amounts. The prepared DNAs were transformed into single-stranded template DNA (sstDNA) libraries by using the GS DNA Library Preparation Kit (Roche Applied Science). The sstDNA libraries were clonally amplified in a bead-immobilized form by using the GS emPCR Kit (Roche Applied Science) and then sequenced on the 454 Genome Sequencer GS FLX+ Titanium Platform (Roche Applied Science) at the Chinese National Human Genome Center, Shanghai, China. Pyrosequencing data processing and statistical analyses Pyrosequencing data were processed using Quantitative Insights Into Microbial Ecology (QIIME 1.7; Caporaso et al., 2010b). Sequences that contained ambiguous bases or were shorter than 150 bp in length were discarded; only those with complete barcodes that were 100% identical to the expected barcodes, no more than three mismatches in the primer sequences (Huse et al., 2007), and an average quality score >20 were included in further analyses. Prior to the analysis, chimeric sequences were detected and excluded from the de-noised sequences using the QIIME implementation of the ChimeraSlayer algorithm (DeSantis et al., 2006; Haas et al., 2011). The high quality sequences were deposited in the NCBI database under accession number SRP046759. Uclust was used to identify phylotypes and assign sequences to operational taxonomic units (OTUs) at a distance cutoff of 0.03 (Edgar, 2010). The cluster seeds of OTUs were chosen as the representative sequences and aligned using PyNAST (Caporaso et al., 2010a) with the greengenes core set (DeSantis et al., 2006) as the template. The phylogenetic tree of the representative sequences was constructed using FastTree (Price et al., 2010). The software RDP classifier (Wang et al., 2007) was used to assign sequences to phylogenetic taxa based on the Ribosomal Database Project (Cole et al., 2013) under the condition of a bootstrap cutoff of 50%. Actinobacterial reads unclassified at the family level were further annotated by online SINA 1.2.11 (Pruesse et al., 2012) based on the SILVA SSU Ref NR 99 123.1 database. The alpha_diversity.py QIIME script was also used to calculate Shannon diversity estimators and Simpson's diversity index after rarefying reads to an even depth (n = 108 reads) across samples. The dissimilarity of phylogenetic diversity among the actinobacterial communities was measured by a weighted UniFrac distance matrix (Hamady et al., 2010). To correct the sampling error, the samples were rarefied to the minimal number of sequences for actinobacterial community. Dissimilarity matrices for geochemical characteristics were calculated using Euclidean distances, after standardizing the data to Z-score to make them comparable. A geographic distance matrix was calculated using the Vincenty formula and ln transformed for the correlation analysis as suggested by Martiny et al. (2011; Table S1). The dissimilarities of actinobacterial community compositions and environmental variables among samples were evaluated using the principal coordinate analysis (PCoA) method in QIIME, with weighted UniFrac and Euclidean distances, respectively. The Mantel test was used to assess the correlations between the actinobacterial community dissimilarity and environmental variables by 99,999 permutations in QIIME. Detrended correspondence analysis (Hill and Gauch, 1980), canonical correspondence analysis (Ter Braak, 1986), and partial canonical correspondence analysis (Borcard et al., 1992) were also performed to compute the correlations between the community structure and environmental variables in R version 3.1.0 (R Core Team, 2014) using the functions in the Vegan package (Oksanen et al., 2013). Environmental variables with variance inflation factors over 20, such as Ca and Mn, were removed from the canonical correspondence analysis in order to exclude multicollinearity in the model (Yang et al., 2014). Selective isolation of actinobacteria The samples were processed using the following three methods. Ultrasonication/dilution (Qiu et al., 2008). One gram of sediment or 1 ml of filter homogenate were added to 4 ml of sterile artificial seawater (distilled water containing 3.3% sea salts), followed by shaking for 2 h at 28°C and 180 rpm and processing in a water bath sonicator (model KQ-100DB, 40 kHz, 100 W; Kunshan Ultrasonic instruments Co., Ltd., Kunshan, China) for 2 min at 30°C. The resulting samples were further diluted (1:1) with sterile artificial seawater. Dispersion and differential centrifugation technique (Hopkins et al., 1991). One gram of sediment or 1 ml of filter homogenate were blended with 5 ml 0.1% (w/v) sodium cholate, 5 ml chelating resin (Na+, Sigma), and approximately 10 glass beads, shaken for 2 h at 10°C, and centrifuged at 500 g for 2 min to get the supernatant. The residue was resuspended in 5 ml Tris buffer (pH 7.4), shaken for 1 h at 10°C, and centrifuged at 500 g for 1 min to get the supernatant. The two supernatants were pooled together as supernatant 1. The above residue was resuspended in 5 ml sodium cholate and processed in an ultrasonic bath for 1 min, mixed with 5 ml sodium cholate and shaken for 1 h at 10°C, and then centrifuged at 500 g for 1 min to get supernatant 2. The resulting residue was resuspended in 10 ml sterile distilled water, shaken for 1 h at 10°C, and centrifuged at 500 g for 2 min to get supernatant 3. The three supernatants of each sample were combined and centrifuged at 5000 g for 20 min, and then the residue was resuspended in 1 ml sterile artificial seawater for subsequent inoculation. Enrichment/dilution. The ultrasonication/dilution sample was inoculated (1:10) in duplicate into SMP (Jensen et al., 2005) and M5 (Zhang et al., 2006) media without agar in 15-ml centrifuge tubes, followed by incubation for 8 weeks at 16°C with the addition of fresh antibiotics (50 mg l−1 each of nalidixic acid and cycloheximide) every 2 weeks. The pretreated samples were then diluted by 10 and 100 in sterile artificial seawater. Amounts of 100 μl of each of the resulting dilutions were inoculated in quadruplicate onto the surface of the following selective agar media: AMM, SMP, SNC, and SRC (Jensen et al., 2005), 1/10 AMM, ISP 2 (modified with 0.2% CaCO3), ISP 3 (Shirling and Gottlieb, 1966), and M5. All media were prepared with either natural seawater or artificial seawater and supplemented with 0.1% (v/v) vitamin solution (Janssen et al., 1997), 0.1% (v/v) trace salt solution (Shirling and Gottlieb, 1966), and 50 mg l−1 each of nalidixic acid and the anti-fungal agent cycloheximide or nystatin. The initial pH of each medium was adjusted to 7.2. The quadruplicate plates were divided into two sets and incubated respectively at 28 and 10°C for 2–15 weeks. The plates were monitored every week and almost all growing colonies were transferred onto new AMM plates until pure cultures were obtained. The pure isolates were cryopreserved in 20% glycerol at −80°C. 16S rRNA gene sequencing of the isolates and phylogenetic analysis Genomic DNA extraction from the isolates was performed as previously described (Chun and Goodfellow, 1995). Nearly complete 16S rRNA genes were PCR amplified using the universal primers 27f and 1492r (Lane, 1991). Reaction products were checked, purified, and directly sequenced using the previously described method (Guo et al., 2008). Nearly full-length 16S rRNA gene sequences were obtained and edited with MEGA 5.20 (Tamura et al., 2011). The nearest related taxa were retrieved from the GenBank non-redundant database using a BLASTN search (http://www.ncbi.nlm.nih.gov/). Phylogenetic analysis of the isolates was conducted with MEGA 5.20. The phylogenetic tree was constructed using the maximum likelihood (ML) algorithm (Felsenstein, 1981) and the tree topology was evaluated by bootstrap analysis (Felsenstein, 1985) based on 1000 resamplings. The phylogenetic relationships of the isolates and actinobacterial sequences from the pyrosequencing data set were also analyzed. The alignment of all the isolate sequences and representative sequences of actinobacterial OTUs was performed using SINA (SILVA Incremental Aligner) web-based tool (Pruesse et al., 2012). ML phylogenetic tree reconstruction was performed with RAxML version 7.3.0 (Felsenstein, 1981; Stamatakis, 2006) with 100 rapid bootstrap inferences. The jModeltest 2.1 (Darriba et al., 2012) was used to select the best-fit substitution model in the above phylogenetic analyses, which was General Time Reversible model with Gamma distribution and Invariable sites (GTR+G+I). Phylogenetic trees were edited with MEGA 5.20 (Tamura et al., 2011). The 16S rRNA gene sequences of the actinobacterial isolates were deposited in the GenBank database with accession numbers KM507587–KM507721, HQ622494–HQ622502, HQ622504–HQ622509, HQ622514–HQ622528, HQ622530–HQ622532, JF346424–JF346428, JF346418, JF346420, and JF346422. Biodegradation of recalcitrant organic matter All actinobacterial isolates were qualitatively screened for their activity in the degradation of cellulose, chitin, pectin, fluoranthene, and phenanthrene using the previously described media and methods with modifications (Kiyohara et al., 1982; Veiga et al., 1983; Augustine et al., 2013). Briefly, each actinobacterial strain was spot inoculated in triplicate on nutrient agar containing sodium carboxymethyl cellulose (1%), colloidal chitin (0.2%), pectin (0.5%), fluoranthene (10 mg l−1), and phenanthrene (10 mg l−1), respectively. Plates were incubated for 3–15 days at 28°C, and then colonies with transparent zones were considered as recalcitrant organic matter-degrading isolates. The cellulose and pectin plates were stained for 10 min with 0.5% congo red and then bleached for 10–20 min with 1 M NaCl before the observation of transparent zones. Results Environmental characteristics of the study sites Eleven physicochemical and three geographic parameters of the samples were obtained (Table 1). PCoA analysis of the Z-scored physicochemical factors showed that samples collected from different sites varied from one another, and samples M8 and M9, which were collected from a hydrothermal field, were distinct from the others (Figure 2). The pairwise geographic distance between the sampling sites ranged from 5.2 (between M8 and M9) to 964.9 km (between M1 and M2; Figure 1; Table S1). Figure 2 PCoA representing the similarity of 11 physicochemical factors of the samples. Community compositions of Actinobacteria A total of 72,889 quality-filtered 16S rRNA gene sequences with an average read length of approximately 250 bp were obtained for further analysis, with 49,717 sequences belonging to bacteria and 23,057 to archaea. Altogether, 28 bacterial and two archaeal phyla were recovered. Among them, Proteobacteria, Thaumarchaeota, Actinobacteria, Planctomycetes, and Firmicutes were the most abundant (relative abundance >1% in each sample). The phylum Actinobacteria represented 4.5% (3245 sequences) of the total reads and 1.2–9.1% of the reads among the samples. The actinobacterial sequences could be divided into 764 OTUs using Uclust at a distance of 0.03 and each sample contained 58–219 OTUs. None of the actinobacterial OTUs were distributed in all samples. The actinobacterial α-diversity, based on the Shannon and Simpson diversity indexes, was not significantly different between the samples (Table 2). Table 2 The number of OTUs and the estimators of richness and diversity at 0.03 dissimilarity. Sample ID Total reads Total OTUs Total coverage Actinobacterial reads (%) Actinobacterial OTUs (%) Shannon* Simpson* M1 7436 3548 0.65 293 (3.9) 130 (3.7) 5.34 0.97 M2 8079 3955 0.64 108 (1.3) 64 (1.6) 5.17 0.96 M3 7500 3559 0.65 136 (1.8) 65 (1.8) 5.13 0.95 M4 6442 3295 0.62 351 (5.4) 118 (3.6) 4.30 0.87 M5 11060 4794 0.68 774 (7.0) 193 (4.0) 4.51 0.93 M6 9807 3897 0.71 119 (1.2) 58 (1.5) 5.09 0.96 M7 6770 2848 0.68 617 (9.1) 171 (6.0) 4.90 0.95 M8 8826 3535 0.73 274 (3.1) 72 (2.0) 3.98 0.89 M9 6969 2493 0.74 573 (8.2) 219 (8.8) 5.05 0.94 Total 72889 23836 0.73 3245 (4.5) 764 (3.2) * Index for actinobacterial community. Five known actinobacterial classes (Acidimicrobiia, Actinobacteria, Coriobacteriia, Rubrobacteria, and Thermoleophilia) were detected, of which Actinobacteria and Acidimicrobiia were the most abundant (Figure S1). A total of 300 OTUs spanning nearly half (47.7%) of the actinobacterial reads could not be assigned to any known classes within the taxonomic framework of the phylum Actinobacteria when using the RDP classifier. These OTUs accounted for 42.0–74.1% of the actinobacterial reads in samples M1–M7 while only 5.8% in M8 and 6.6% in M9. At the rank of order, 63.2% (8.4–93.2% among the samples) of the actinobacterial reads could not be classified, however, 17 known actinobacterial orders were detected (Figure 3). Acidimicrobiales (2.1–27.0%) and Corynebacteriales (0.3–40.9%) were the most widely distributed, present in all samples, followed by Micrococcales (0.6–11.5%) and Propionibacteriales (0.7–7.0%), present in eight samples except for M6. Streptomycetales occurred in samples M7–M9, accounting for almost half (49.0%) of the reads in M9 but only 4.4 and 3.3% in M7 and M8, respectively. In contrast to the non-hydrothermal field sediment samples M1–M7, where unclassified actinobacteria at the order rank accounted for more than 68.5%, the hydrothermal field sediment sample M8 contained Corynebacteriales (40.9%) as the most abundant order and had a much higher relative abundance of Acidimicrobiales, Micrococcales, Micromonosporales, Streptomycetales, and Pseudonocardiales. The hydrothermal field bottom water sample M9 contained the highest number (13) of known orders. Figure 3 Actinobacterial community compositions at the order rank in the samples. The relative abundance represents the proportional frequencies of actinobacterial reads that were classified or unclassified at the order rank, using the RDP classifier, based on the hierarchical classification in the second edition of Bergey's Manual of Systematic Bacteriology (Goodfellow et al., 2012). Only 28.5% of the reads could be further classified into the known families (28 in total), and the majority of unclassified reads at the family rank belonged to the order Acidimicrobiales. None of the known families were discovered in all samples and seven were present in only one of the samples (Figure S2). The families Acidimicrobiaceae, Iamiaceae, and Propionibacteriaceae were obtained from eight samples, followed by Micrococcaceae and Nocardiaceae from seven samples, and Microbacteriaceae and Tsukamurellaceae from six samples. These families were relatively abundant in the actinobacterial community (>1% of the total actinobacterial reads; Figure S2). All the Streptomycetales reads could be further classified into Streptomycetaceae, which was also abundant. When looking at the genus level, at least 52 known genera were detected. Among them, 35 genera represented non-filamentous actinobacteria according to the previous descriptions of the genera (Goodfellow et al., 2012), accounting for nearly half of the total known actinobacterial reads. Six genera were found to be abundant (Figure 4), of which Iamia, Microbacterium, Propionibacterium, and Tsukamurella were reported to be non-filamentous, while Gordonia and Streptomyces were filamentous (Goodfellow et al., 2012). Figure 4 Actinobacterial genera detected by culture-dependent and -independent methods. The most abundant genera are shown in bold, and the black dots, star, and squares indicate those that were abundant in both methods, pyrosequencing only, and cultivation only, respectively. The actinobacterial sequences unclassified at the family level using the RDP classifier, belonging to 513 OTUs and accounting for 71.5% of actinobacterial reads, were further classified using SINA Alignment Service based on the SILVA database. Nearly half of these OTUs (241 OTUs) were classified as OM1 group of Acidimicrobiales, which is also an uncultured group (Yilmaz et al., 2016), accounting for 81.3% of the unclassified reads. The identities to the reference OM1 sequences were 85.5–100.0%. Most of the other OTUs (173 OTUs accounting for 10.3% of the unclassified reads) remained unclassified and showed 70.8–94.6% identities to the unclassified sequences in the SILVA database. Only 99 OTUs could be further classified. Among them, 54 OTUs were classified to known or candidatus genera or families including Acidothermus, Arthrobacter, Brevibacterium, Candidatus Microthrix, Corynebacterium, Crossiella, Frankiaceae, Geodermatophilus, Jatrophihabitans, Mycobacterium, Pseudonocardiaceae, Rothia, Solirubrobacterales 480-2, Solirubrobacterales Elev-16S-1332, Streptomyces, and Sva0996 marine group; 38 OTUs were classified to known or candidatus orders including Acidimicrobiales, Frankiales, Gaiellales, Micrococcales, PAUC43f marine benthic group, and Solirubrobacterales; and 7 OTUs were classified to known or candidatus classes of Actinobacteria, OPB41, TakashiAC-B11, and Thermoleophilia. Correlating actinobacterial community compositions with environmental factors The diversity analysis showed that the relative abundance of actinobacterial taxa varied in different samples (Figure 3, Figures S1, S2). PCoA analysis at the 0.03 OTU level further confirmed obvious variations in the actinobacterial community among the samples (Figure S3), a result in line with that of physicochemical factors (Figure 2). Simple Mantel tests between the distance matrix of actinobacterial communities and distance matrices of environmental variables showed that the community structure of the samples was significantly correlated to pH, TOC, TP, Ca and TN (P < 0.05; Table 3), where pH showed the highest correlation (r = 0.813, P = 0.020). When considering only the non-hydrothermal field samples, the actinobacterial community structure was merely correlated with Ca and pH (P < 0.05). Canonical correspondence analysis, which was chosen as the appropriate mathematical model in consideration of the axis lengths in Detrended correspondence analysis (Lepš and Šmilauer, 2003), was also performed to identify the major environmental factors shaping the actinobacterial community structure. The result demonstrated a significant model (P = 0.042) with seven environmental variables (pH, TOC, Fe, Mg, S, TN, and TP), which explained 91.2% variance of the actinobacterial community compositions with 22.6 and 20.2% explained by the first and second axes, respectively (Figure 5). Among these seven variables, pH and TOC were the most important environmental factors. Geographic distance was not a significant factor because the P-values were > 0.05 in both the Mantel test and ordination analysis. Table 3 Correlations between the actinobacterial community structure (0.03 OTU level) and environmental variables by Mantel tests. Factor r P pH 0.813 0.020 TOC 0.640 0.027 TP 0.526 0.041 Ca 0.523 0.040 TN 0.487 0.022 Ln_Distance −0.167 0.726 EC 0.390 0.170 Fe 0.016 0.286 Mg −0.005 0.315 Mn −0.122 0.388 S 0.121 0.291 NO-N 0.140 0.240 The significant correlations are shown in bold. Figure 5 Canonical correspondence analysis of actinobacterial OTUs (cutoff = 0.03) detected by pyrosequencing. Arrows indicate the environmental variables and the communities in the samples are represented by squares. The distance between the squares represents the dissimilarity of the communities. The length of the arrows and the included angle of the axes indicate the influence of the specified environmental factors on the community structure. TN, total nitrogen; TP, total phosphorus; TOC, total organic carbon. The relative abundance of the phylum Actinobactreia showed a significantly negative correlation with Fe (r = −0.676, P = 0.045). The relationship between the relative abundance of the major groups of actinobacteria and environmental factors was further analyzed. The class Actinobactreia showed significant correlations with Mn (r = −0.849, P = 0.004), pH (r = −0.715, P = 0.004), NO-N (r = 0.754, P = 0.019), Fe (r = −0.724, P = 0.027), and Mg (r = 0.676, P = 0.046). At the order rank, significant correlations were found between Corynebacteriales and TP (r = 0.838, P = 0.005), Micrococcales and Ca (r = −0.920, P = 0.000), pH (r = −0859, P = 0.003), TN (r = −0.749, P = 0.020), Fe (r = −0.715, P = 0.030), and TP (r = 0.713, P = 0.031), and between Propionibacteriales and electric conductance (r = 0.904, P = 0.001), TN (r = −0.862, P = 0.003), and pH (r = −0.817, P = 0.007). For the known actinobacterial families, only Micrococcaceae and Propionibacteriaceae showed negative correlations to TN and pH and a positive correlation to electric conductance (Figure S4). The results also indicated that unclassified Actinobacteria (phylum) positively correlated with pH and TN, unclassified Actinobacteria (class) correlated with TN, NO-N, and pH, and unclassified Acidimicrobiales highly correlated with Fe (Figure S4). Some of the OTUs within these unclassified/uncultured groups were more influenced than others, including the OTUs detected in most of the samples. For instance, OTU00513 and OTU00620 showed significant correlations with pH (r = 0.782, P = 0.013; r = 0.692, P = 0.039) and TN (r = 0.689, P = 0.040; r = 0.764, P = 0.017), OTU00305 and OTU00066 significantly correlated to TN (r = 0.791, P = 0.011; r = 0.712, P = 0.032), OTU00085 and OTU00624 correlated to NO-N (r = 0.771, P = 0.015; r = 0.721, P = 0.028), and OTU00183 had a highly significant correlation with Fe (r = 0.854, P = 0.003). All the above mentioned OTUs were classified to Acidimicrobiales OM1 clade using the SILVA database. Besides the geochemical parameters, correlations between the relative abundance of the phylum Actinobactreia and other bacterial and archaeal phyla present in the samples were also evaluated. The result showed a significant correlation between Actinobactreia and Chloroflexi (r = 0.678, P = 0.045). Diversity of culturable actinobacteria To further investigate the diversity and potential functions of actinobacteria at the SWIR, the samples were subjected to isolation of actinobacterial strains using three pretreatments and eight selective media. A total of 176 actinobacterial strains were purified from the samples. Most of the strains were isolated from ISP 3 (62 strains) and AMM (46 strains) media. The 16S rRNA gene sequences of the isolates were affiliated with 29 genera, 18 families, and 9 orders within the Actinobacteria class, of which 15 genera, 13 families and 8 orders were also found in the pyrosequencing data set (Figures 4, 6). The isolates showed 97.3–100% 16S rRNA gene sequence similarities to the closest known actinobacterial species, with the most divergent isolate belonging to Nocardioides. Figure 6 Phylogenetic diversity, taxonomic affiliation and degradation capacity of culturable actinobacterial isolates. The maximum-likelihood tree was based on nearly full-length 16S rRNA gene sequences of the isolates and closely related type strains. The GTR+G+I evolutionary model was selected to construct the tree. Coriobacterium glomerans DSM 20642T and Thermoleophilum album ATCC 35263T were set as outgroups. Numbers at branch nodes are percentages of bootstrap replicates of 1000 resamplings (only values above 50% are shown). The bar represents 0.1 substitutions per nucleotide position. Genera that were also recovered in the pyrosequencing data set are in bold. Numbers following each genus indicate the number of isolates or degrading isolates. I, number of isolates; DI, number of degrading isolates; A, number of cellulose-degrading isolates; B, number of chitin-degrading isolates; C, number of pectin-degrading isolates; D, number of fluoranthene-degrading isolates; E, number of phenanthrene-degrading isolates. A total of 77.3% (136/176) of the isolates showed ≥97% 16S rRNA sequence similarities to the pyrosequencing reads, whereas only 10.3% (335/3245) of the in situ actinobacterial community were represented by the isolates when mapping the actinobacterial reads against the sequences of the isolates at the 97% similarity cutoff. The phylogenetic analysis on all the isolates and representative actinobacterial sequences from the pyrosequencing data set also supported the above results (Figure 7). Most of the isolates clustered closely with the environmental sequences, formed 8 of the 18 known orders detected in total. Nine known orders contained sequences from only the pyrosequencing but not the isolates, while the order Kineosporiales contained only the isolate sequences. On the other hand, most of the unclassified/uncultured pyrosequencing sequences formed independent clades that were distinguished from the isolates, notably the Acidimicrobiales OM1 clade which was also distinct from the clade of the order Acidimicrobiales and should probably be named as a new order. The phylogenetic tree also showed that there were 14 Acidimicrobiia clades and a number of unclassified actinobacterial sequences clustered with the OM1 clade, and there were at least three distinguished Actinobacteria clades (Actinobacteria clades 6–8) that might respectively represent new orders within the phylum Actinobacteria (Figure 7, Figures S5A–S5F). Figure 7 Maximum-likelihood phylogenetic tree based on partial 16S rRNA gene sequences illustrating the relationships among all the isolates and representative actinobacterial sequences from the pyrosequencing data set. The tree was constructed using the GRT+G+I model. Firmicutes was set as the outgroup. Taxonomic assignments of the sequences were based on both the RDP and SILVA databases. The tree is displayed at order level. Numbers at branch nodes are percentages of bootstrap replicates of 100 resamplings (only values above 50% are shown). Numbers in the parentheses following each order or clade indicate the relative abundance of pyrosequencing reads and isolates, respectively. The bar represents 0.1 substitutions per nucleotide position. Among the 15 genera that were present in both the pyrosequencing and cultural data sets, only three (Streptomyces, Tsukamurella, and Microbacterium) were abundant in pyrosequencing (8.8, 2.1, and 1.5%, respectively); and most of the cultured genera represented rare groups or were absent in pyrosequencing. None of the genera were recovered from all samples. The non-filamentous genera Kocuria (35 isolates) and Microbacterium (31 isolates), both belonging to the order Micrococcales, were the most abundant of the culturable genera. Microbacterium isolates were obtained from all samples except M1, while Kocuria isolates were from M1, M4, M6, and M9. The highest diversity of isolates was observed for the hydrothermal field bottom water sample M9 (13 genera), followed by sample M4 (12 genera); and the lowest diversity was observed for samples M3 and M7, each affiliated with only four genera (Table 4). Table 4 Numbers of isolates (and degrading isolates) at the genus level from different samples. Orders Family Genus M1 M2 M3 M4 M5 M6 M7 M8 M9 Total Corynebacteriales Dietziaceae Dietzia 0 0 1 (1) 0 0 0 1 (0) 0 2 (2) 4 (3) Mycobacteriaceae Mycobacterium 0 0 0 0 3 (2) 1 (0) 0 1 (1) 1 (0) 6 (3) Nocardiaceae Rhodococcus 0 1 (1) 0 8 (6) 0 0 5 (2) 1 (1) 2 (2) 17 (12) Tsukamurellaceae Tsukamurella 0 0 0 0 0 0 0 1 (1) 1 (0) 2 (1) Nocardiaceae Williamsia 0 0 0 0 1 (1) 0 0 0 0 1 (1) Frankiales Geodermatophilaceae Blastococcus 0 0 0 2 (1) 1 (1) 1 (1) 0 0 0 4 (3) Modestobacter 0 0 0 0 1 (0) 0 0 0 0 1 (0) Kineosporiales Kineosporiaceae Pseudokineococcus 0 0 0 1 (1) 0 0 0 0 0 1 (1) Micrococcales Brevibacteriaceae Brevibacterium 2 (2) 1 (1) 0 0 0 0 0 4 (2) 6 (0) 13 (5) Bogoriellaceae Georgenia 0 1 (0) 0 0 0 0 0 0 0 1 (0) Cellulomonadaceae Cellulomonas 0 0 0 0 1 (0) 0 0 0 1 (1) 2 (1) Microbacteriaceae Agrococcus 0 0 0 1 (1) 0 0 2 (0) 0 0 3 (1) Amnibacterium 0 0 0 1 (1) 0 0 0 0 0 1 (1) Curtobacterium 1 (1) 1 (1) 0 1 (1) 1 (1) 0 0 0 0 4 (4) Herbiconiux 0 1 (1) 0 0 1 (1) 0 0 0 0 2 (2) Microbacterium 0 6 (1) 1 (0) 3 (2) 10 (6) 6 (1) 1 (0) 1 (1) 3 (2) 31 (13) Micrococcaceae Arthrobacter 1 (1) 1 (1) 0 2 (2) 4 (2) 0 0 0 0 8 (6) Micrococcus 3 (1) 1 (1) 0 1 (0) 0 0 0 0 3 (2) 8 (4) Kocuria 26 (9) 0 0 3 (1) 0 3 (3) 0 0 3 (1) 35 (14) Dermabacteraceae Brachybacterium 0 2 (0) 0 0 0 0 0 0 0 2 (0) Dermacoccaceae Kytococcus 0 0 0 0 1 (0) 0 0 0 0 1 (0) Micromonosporales Micromonosporaceae Micromonospora 2 (2) 0 0 1 (1) 0 0 0 0 0 3 (3) Verrucosispora 0 0 0 0 0 0 0 0 2 (2) 2 (2) Propionibacteriales Nocardioidaceae Marmoricola 0 1 (1) 0 0 0 0 0 0 0 1 (1) Nocardioides 0 0 0 1 (0) 4 (0) 0 0 0 0 5 (0) Streptosporangiales Nocardiopsaceae Nocardiopsis 0 0 0 0 0 1 (1) 0 2 (2) 3 (3) 6 (6) Pseudonocardiales Pseudonocardiaceae Actinomycetospora 0 1 (0) 0 0 0 0 0 0 0 1 (0) Saccharopolyspora 0 0 1 (1) 0 0 0 0 0 1 (1) 2 (2) Streptomycetales Streptomycetaceae Streptomyces 4 (4) 0 2 (2) 0 0 0 0 1 (1) 2 (2) 9 (9) No. of isolates (degrading isolates) 39 (20) 17 (8) 5 (4) 25 (17) 28 (14) 12 (6) 9 (2) 11 (9) 30 (18) 176 (98) No. of genera (degrading genera) 7 (7) 11 (8) 4 (3) 12 (10) 11 (7) 5 (4) 4 (1) 7 (7) 13 (10) 29 (23) Biodegradation activity of actinobacterial isolates The majority of isolates (98 of 176) demonstrated activity in the degradation of refractory organics, i.e., cellulose (40 isolates, 22.7%), chitin (24 isolates, 13.6%), pectin (60 isolates, 34.1%), fluoranthene (14 isolates, 8.0%), and phenanthrene (14 isolates, 8.0%; Figure 6). The degrading isolates spread across 23 genera, with the pectin-degrading strains the most diverse (18 genera), followed by the cellulose degraders (15 genera), chitin degraders (10 genera), phenanthrene degraders (10 genera), and fluoranthene degraders (7 genera; Figure 6). Among them, 41 isolates belonging to 15 genera showed activity toward more than one organic substance. Nearly half of the degrading isolates were distributed in four genera, which were Kocuria (14 strains), Microbacterium (13 strains), Rhodococcus (12 strains), and Streptomyces (9 strains); and isolates classified within each of the genera Brevibacterium, Kocuria, and Rhodococcus showed degradation activity toward all of the organics tested. Almost all of the Streptomyces isolates showed activities toward cellulose (8 of 9) and chitin (9 of 9). Pectin degraders were mainly from the genera Kocuria, Microbacterium, Rhodococcus, and Streptomyces, of which Rhodococcus also accounted for >1/3 of the fluoranthene degraders (Figure 6). Cellulolytic and pectinolytic strains could be found in all nine samples. Furthermore, 9 of the 11 isolates and all 7 genera from M8 could degrade the organics, showing the highest activity rate among the samples (Table 4). Discussion The results showed a high diversity and a relatively comprehensive community structure of actinobacteria in the deep-sea surface sediments and water along the SWIR, which significantly correlated with environmental factors. The biodegradation activity toward refractory organics demonstrated by most of the diverse actinobacterial isolates suggests their potential ecological functions and biotechnological uses. Actinobacteria in the deep sea along the SWIR are highly diverse The 16S rRNA gene pyrosequencing data showed that the relative abundance of actinobacteria in the sediments collected from the SWIR was higher than that found in the sediments of the deep Arctic and Atlantic Oceans (0.1–3.0%, 0–2.32 mbsf), but less than that reported in the deep Pacific Ocean (about 10%, 0–350 mbsf) (Inagaki et al., 2006; Schauer et al., 2010; Jorgensen et al., 2012). Our results also indicate that many unclassified actinobacterial taxa may exist in the deep sea along the SWIR, particularly in the surface sediments in non-hydrothermal fields (Figures 3, 7, Figures S1, S2). While >80% of the actinobacterial unclassified reads were assigned to Acidimicrobiales OM1 clade using the SILVA database, most of these sequences, according to the recent data of Yilmaz et al. (2016), should belong to the uncultured Actinobacteria.Order3 which is distinct from the OM1 clade (Actinobacteria.Order2 in Yilmaz et al., 2016). Most of the remaining sequences were assigned into the classes Actinobacteria, similar to that reported for the surface sediments of the deep Arctic Ocean (Zhang et al., 2014). Members of the two classes have been reported to be widely distributed in various habitats; however, only members of Actinobacteria have been frequently isolated from the marine environment (Maldonado et al., 2005; Duncan et al., 2014; Zhang et al., 2014), which is also the case in our study. Most members of the class Acidimicrobiia are uncultured, and Acidimicrobiaceae, and Iamiaceae are the only two described families in this class (Jensen and Lauro, 2008; Goodfellow et al., 2012). Therefore, it is not strange to find in our study that a large number of the sequences of Acidimicrobiia could not be classified into a known family and Iamiaceae, a neutrophilic family, presented in eight samples including both the sediments and the bottom water. Interestingly, Acidimicrobiaceae, members of which prefer to grow at around pH 2 and have been isolated only from geothermal sites and acidic mine waters (Goodfellow et al., 2012), also occurred in all the SWIR sediment samples with non-negligible percentages (0.7−4.6%, Figure S2), although the pH of the samples was 7.77−9.11 (Table 1). Compared to the results of previous studies on marine actinobacterial diversity, which reported only 4−12 genera or 14 families, much more genera and families were detected in the actinobacterial community at the SWIR using either pyrosequencing or cultivation method (Figure 4, Table S2), with at least 66 known genera and 33 families detected in total (Figure 4). Most of the marine actinobacterial genera and families detected in previous studies were recovered in our results (Figure 4, Table S2), but the most abundant genera detected in deep Arctic marine surface sediments were largely different from those identified here (Table S2). Moreover, most of the known genera (46 of 66) detected at the SWIR were likely to be non-filamentous based on the previous descriptions (Goodfellow et al., 2012), similar to the finding in deep Arctic marine surface sediments (Zhang et al., 2014). In agreement with this result, most of our isolates (74.4%) were non-filamentous actinobacteria, an observation in contrast to previous reports on shallow sediments, where filamentous actinobacteria, especially Micromonospora, Pseudonocardia, and Streptomyces, were most frequently cultivated (Maldonado et al., 2005; Bredholdt et al., 2007; Zhang et al., 2014). This difference might primarily result from the change in hydrostatic pressure, which is an important parameter in shaping the microbial community structure in the ocean and leads to small cocci being the most abundant morphotype of high-pressure-surviving microbes (Marietou and Bartlett, 2014). In addition, 53 isolates showed < 99% 16S rRNA gene similarity to their closest type strains and therefore likely represented novel species according to the recommended cutoff value for actinobacterial species delineation (Stach et al., 2003a). The actinobacterial community structure at the SWIR significantly correlates to environmental factors Researchers have gradually formed a consensus on the biogeography of marine actinobacteria (Ward and Bora, 2006). However, the related factors that contribute to shape the actinobacterial community structure, especially in the deep ocean, are still not clear. This study showed that pH and Ca were significant factors along the SWIR, whether the two hydrothermal field samples, M8 and M9, were considered or not. It has been demonstrated that pH was an important contributor to actinobacterial/prokaryotic community variations in soil (pH 3.5–8.8), salt lakes (pH 7.5–10.5), and hot springs (pH 5.0–10.0; Lauber et al., 2009; Pagaling et al., 2009; Valverde et al., 2012). In our study, a less-massive change in pH from 7.47 to 9.11 demonstrated a significantly high influence on the actinobacterial community structure (Table 3), indicating that the deep-sea actinobacterial composition is sensitive to pH. This result was further supported by the evidence that pH significantly affected the relative abundance of the class Actinobactreia, the orders Corynebacteriales, Micrococcales, and Propionibacteriales, the families Micrococcaceae and Propionibacteriaceae, and unclassified Actinobacteria (both class and phylum) at the SWIR. It was not unexpected that Ca, which was an essential nutrient and involved in pH buffering, was an environmental determinant of the community structure as well. Our study also showed for the first time that TOC, TP, and TN, which represented nutrients, were significantly correlated to actinobacterial community structure in the deep sea. Dissolved organic carbon and TP were previously reported to be related to the freshwater actinobacterial community structure in the brackish northern Baltic Sea (Holmfeldt et al., 2009). Total carbon, TN, and P were also reported to be significantly correlated to the relative abundance of actinobacteria in soils (Liu et al., 2014; Zhao et al., 2014). Taken together, pH and the above mentioned nutrients are likely to be the common factors that shape actinobacterial community structures in different habitats. Interestingly, some environmental variables significantly influenced the relative abundance of a few unclassified actinobacterial groups (Figure S4). This finding suggests that the responses of these unknown taxa, particularly the specific OTUs that were more influenced, to local environments may relate to their critical requirements for pH and nutrients. The observation that the actinobacterial community structure was not affected by the geographic distance might be attributed to the fact that many actinobacteria can produce stress-resistant spores and possess various metabolic capabilities, characteristics that promoted their long-distance dispersal and survival in the resultant conditions encountered. Nevertheless, this observation may be also due to that our samples were not collected at a large spatial scale (< 1000 km in geographic distance), because the scale of sampling was suggested to affect the relative contributions of spatial distance and environmental factors to microbial distribution (Martiny et al., 2006, 2011). Hence, extensive sampling at a larger geographic scale with more hydrothermal fields is still needed to determine whether actinobacterial distribution along the Indian Ridge and even the global deep ocean is controlled by historical or contemporary environmental factors or both. Actinobacteria from the SWIR show potential ecological functions and biotechnological applications Although, the concentrations of PAHs and polysaccharides of the samples were not determined in this study due to the difficulty of sampling and the limited amount of samples we obtained, these organics have been reported to be common in the deep ocean (Simoneit and Fetzer, 1996; Dong et al., 2015). More than half of the actinobacterial isolates obtained from this research showed the capacity to degrade organic matter, suggesting that actinobacteria may contribute to the substance and energy flows in biogeochemical cycles in the deep ocean. We found a high diversity of the marine actinobacterial degraders, with 20 polysaccharide-degrading genera and 12 PAH-degrading genera (Figure 6). Most of these genera, with the exception of Streptomyces, Nocardiopsis, and Dietzia, have not been reported to contain members inhabiting deep-sea environments whilst possessing organic degradation capacity. The genera that could degrade different refractory organics may have a better chance to thrive in a range of deep-sea habitats, as exemplified by Kocuria, Microbacterium, Mycobacterium, Rhodococcus, and Streptomyces, which spread in most of the samples as indicated by the combined results of culture-dependent and -independent analyses. Hydrolytic enzyme profiles have been analyzed for marine actinobacteria from shallow sediments, and the results indicated that Streptomyces had a better ability than the other genera to degrade cellulose, chitin, and pectin (Veiga et al., 1983; Augustine et al., 2013). Among the 23 degrading genera we obtained, Streptomyces was again the most important contributor to the degradation of chitin and cellulose. The result that all of the Streptomyces isolates could degrade chitin is consistent with the report that a Streptomyces strain has at least one and usually multiple chitinase genes and genes of the chitin-binding proteins are also widely distributed in Streptomyces (Williamson et al., 2000). The observation that seven and ten actinobacterial genera in this study could degrade fluoranthene and phenanthrene, respectively, suggested a potential for discovering highly-efficient PAH degraders, which may fuel further research into the biodiversity of PAH-degrading actinobacteria in the deep marine environments. The high abundance of the order Corynebacteriales in the hydrothermal field sediment M8 may be explained by the fact that many members of Corynebacteriales are degraders of xenobiotica (e.g., PAHs) and excellent survivors of unfavorable conditions (Bock et al., 1996; Willumsen et al., 2001). PAHs and heptaheptacontane are common in hydrothermal sediments (Simoneit and Fetzer, 1996), and the genera Gordonia, Mycobacterium, Rhodococcus, and Tsukamurella within Corynebacteriales have been reported to be highly efficient hydrocarbon degraders (Peczynska-Czoch and Mordarski, 1988; Daane et al., 2001; García-Díaz et al., 2013). This is also supported by the cultivation results of our study, where all five genera (Dietzia, Mycobacterium, Rhodococcus, Tsukamurella, and Williamsia) of Corynebacteriales demonstrated PAH degradation activity (Figure 6). As most of the secondary metabolite-producing actinobacteria were filamentous (so-called actinomycetes; Tiwari and Gupta, 2012), while most of our actinobacterial isolates were non-filamentous, we did not focus on bioactive compounds of the isolates in this study. Actually we had conducted a primary screening of antimicrobial activity on 37 isolates of nine genera and found that only two isolates belonged to Micromonospora and Streptomyces, respectively, showed inhibitory activity against Micrococcus luteus only. Given the high biodegradation activity of the diverse isolates, it is likely that the ability to utilize refractory organics is important for the survival of actinobacteria in the deep sea. Recent PICRUSt predictions for the uncultured actinobacterial orders in deep pelagic zones also suggested their potential for refractory organic degradation in terms of carbon metabolism (Yilmaz et al., 2016). This characteristic implied the potential roles of actinobacteria in the recycling of organic matter in the deep-sea environments, which might enhance their competitive ability in situ. Author contributions YH, PC, XD, and LH designed the study. LS collected the samples. PC, LZ, XG, LX, and YZ conducted the lab work. PC, YH, LL, YW, and JW analyzed the data. YH, PC, XD, and LH wrote the paper. Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This research was supported by the China Ocean Mineral Resources R & D Association (DY125-15-R-02) and the National Science Foundation of China (31470142 and 41306164). We thank Dr. Xiao-Yang Zhi at the Yunnan University for assistance with pyrosequencing data processing using QIIME. Supplementary material The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb.2016.01340 Figure S1 Actinobacterial community compositions at the class rank in the samples. The relative abundance represents the proportional frequencies of actinobacterial reads that were classified or unclassified at the class rank, using the RDP classifier, based on the hierarchical classification in the second edition of Bergey's Manual of Systematic Bacteriology (Goodfellow et al., 2012). Click here for additional data file. Figure S2 Actinobacterial community compositions at the family rank in the samples. The relative abundance represents the proportional frequencies of actinobacterial reads that were classified or unclassified at the family rank, using the RDP classifier, based on the hierarchical classification in the second edition of Bergey's Manual of Systematic Bacteriology (Goodfellow et al., 2012). The most abundant families are shown in bold. Click here for additional data file. Figure S3 PCoA representing the similarity of the actinobacterial community compositions in the samples detected by pyrosequencing at the 0.03 OTU level. Click here for additional data file. Figure S4 The relationships between the relative abundance of some of the major groups of actinobacteria and environmental factors. EC, electric conductance; TN, total nitrogen; NO-N, nitrate nitrogen. Click here for additional data file. Figure S5 Parts of the zoom-in phylogenetic tree of Figure 7 showing the relationships between the 14 Acidimicrobiia clades and the OM1 clade. Taxonomic assignments of the sequences were based on both the RDP and SILVA databases. Numbers at branch nodes are percentages of bootstrap replicates of 100 resamplings (only values above 50% are shown). Numbers in the parentheses following each clade indicate the relative abundance of pyrosequencing reads and isolates, respectively. Bar = nucleotide changes per site. Click here for additional data file. Table S1 The geographical distance matrix. Click here for additional data file. 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==== Front Front PsychiatryFront PsychiatryFront. PsychiatryFrontiers in Psychiatry1664-0640Frontiers Media S.A. 10.3389/fpsyt.2016.00141PsychiatryCorrectionCorrigendum: Inefficient Preparatory fMRI-BOLD Network Activations Predict Working Memory Dysfunctions in Patients with Schizophrenia Baenninger Anja 12*Diaz Hernandez Laura 13Rieger Kathryn 3Ford Judith M. 24Kottlow Mara 13Koenig Thomas 131Translational Research Center, University Hospital of Psychiatry and Psychotherapy, University of Bern, Bern, Switzerland2San Francisco VA Medical Center, San Francisco, CA, USA3Center for Cognition, Learning and Memory, University of Bern, Bern, Switzerland4Department of Psychiatry, University of California San Francisco, San Francisco, CA, USAEdited and Reviewed by: Thomas W. Weickert, University of New South Wales, Australia *Correspondence: Anja Baenninger, anja.baenninger@puk.unibe.chSpecialty section: This article was submitted to Schizophrenia, a section of the journal Frontiers in Psychiatry 29 8 2016 2016 7 14114 7 2016 02 8 2016 Copyright © 2016 Baenninger, Diaz Hernandez, Rieger, Ford, Kottlow and Koenig.2016Baenninger, Diaz Hernandez, Rieger, Ford, Kottlow and KoenigThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.A corrigendum on Inefficient Preparatory fMRI-BOLD Network Activations Predict Working Memory Dysfunctions in Patients with Schizophrenia by Baenninger A, Diaz Hernandez L, Rieger K, Ford JM, Kottlow M, Koenig T. Front Psychiatry (2016) 7:29. doi: 10.3389/fpsyt.2016.00029schizophreniaworking memorytemporally coherent networksstate-dependent information processingsimultaneous EEG-fMRIcovariance mapping ==== Body Reason for Corrigendum: There is a mistake in the labeling of the two networks plotted in Figure 4: the default mode network (DMN) of controls should be indicated with circles instead of triangles such as the dorsal attention network (dAN). The authors apologize for the mistake. The error does not change the content of the article in any way. In Figure 5, there was mistakenly a red correction line below the label of the right working memory network (rWMN) that was unfortunately not spotted upon publication. This change has no impact neither on the scientific work nor the conclusions of the article in any way. We apologize for the oversight. The corrected Figures are below: Figure 4 Mean DMN and dAN from pre- to poststimulus in the WM task. Mean DMN and dAN dynamics at prestimulus and retention intervals for each load and group. X-axis: time points (prestimulus and retention period), Y-axis: mean percent signal change of variance normalized, and z-transformed TCNs. Figure 5 Mean evolution of TCNs over average trials. Upper plot shows the templates for each TCN from the study of Kottlow et al. with the three orthogonal slices through areas of maximum activation (only positive values; for detailed information about included regions, see Table S1 in Supplementary Material). Lower plots display the mean network evolutions over average trials for each load (thin line = load 2; thick line = load 5) and each group (black line = controls; red line = patients). Dashed lines indicate significance of the t-tests (two-sided, p = 0.05): (1) mean evolutions, (2) mean evolutions against 0, (3) load effect, (4) group effect (blue thin line = load 2; blue thick line = load 5), and (5) interaction of group by load. X-axis: time over trial [prestimulus: −4 to −2.5 s; stimulus (light gray block): −2.5 to 0 s; retention (dark gray block): 0–3.5 s; probe: 3.5–5.5 s], Y-axis: percent signal change of variance normalized, and z-transformed TCNs’ time courses. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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==== Front Front ImmunolFront ImmunolFront. Immunol.Frontiers in Immunology1664-3224Frontiers Media S.A. 10.3389/fimmu.2016.00315ImmunologyMini ReviewCyclic AMP Represents a Crucial Component of Treg Cell-Mediated Immune Regulation Klein Matthias 1Bopp Tobias 1*1University Medical Center, Institute for Immunology, Johannes Gutenberg-University, Mainz, GermanyEdited by: Josef Bodor, BIOCEV, Czech Republic Reviewed by: Viktor Umansky, German Cancer Research Center (HZ), Germany; Jacques A. Nunes, Centre de Recherche en Cancerologie de Marseille, France *Correspondence: Tobias Bopp, boppt@uni-mainz.deSpecialty section: This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology 29 8 2016 2016 7 31515 6 2016 02 8 2016 Copyright © 2016 Klein and Bopp.2016Klein and BoppThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.T regulatory (Treg) cells are one of the key players in the immune tolerance network, and a plethora of manuscripts have described their development and function in the course of the last two decades. Nevertheless, it is still a matter of debate as to which mechanisms and agents are employed by Treg cells, providing the basis of their suppressive potency. One of the important candidates is cyclic AMP (cAMP), which is long known as a potent suppressor at least of T cell activation and function. While this suppressive function by itself is widely accepted, the source and the mechanism of action of cAMP are less clear, and a multitude of seemingly contradictory data allow for, in principle, two different scenarios of cAMP-mediated suppression. In one scenario, Treg cells contain high amounts of cAMP and convey this small molecule via gap junction intercellular communication directly to the effector T cells (Teff) leading to their suppression. Alternatively, it was shown that Treg cells represent the origin of considerable amounts of adenosine, which trigger the adenylate cyclases in Teff cells via A2A and A2B receptors, thus strongly increasing intracellular cAMP. This review will present and discuss initial findings and recent developments concerning the function of cAMP for Treg cells and its impact on immune regulation. cyclic AMPregulatory T cellsFOXP3adenosineimmune regulationimmune tolerance networksuppressionDeutsche Forschungsgemeinschaft10.13039/501100001659BO 3306/1-1, SCHM 1014/7-1 ==== Body Introduction The immune system commands an impressive armament to repel invading microorganisms. The overwhelming part of such an antimicrobial immune response is coordinated and initiated by CD4+ T helper (Th) cells, which recognize foreign antigens through their T cell receptor after presentation by antigen-presenting cells. A huge Th cell receptor repertoire ensures that almost all pathogens can be recognized and subsequently be eliminated. However, this virtually exhaustless T cell receptor repertoire obviously bears the risk of containing autoaggressive T cells (1). This was demonstrated by the transfer of naive Th cells, which represent approximately 90% of all peripheral CD4+ Th cells into T cell-deficient mice that led to a multitude of autoimmune diseases indicating that a considerable part of this Th cell population is potentially autoreactive (2–4). These potentially autoreactive naive Th cells are usually kept in check when CD25+ peripheral Th cells are cotransferred together with CD25− T cells, thus indicating that this residual T cell compartment that was separated from naive T cells before transfer is capable to prevent autoimmune symptoms (5, 6). Today, we know that this suppressive Th cell population mainly consists of T regulatory (Treg) cells which were found to control T effector (Teff) cells in vitro in a contact-dependent manner. The Treg/Teff cell interaction was shown to suppress preferentially IL-2 production and proliferation of the Teff cells – a hallmark of clonal T cell expansion (7). Concerning the suppressive mechanism(s), the usage of cytokine-deficient and cytokine receptor-deficient mice could exclude that IL-10 and TGF-β – at least in vitro – mediated the suppressive properties of Treg cells (7, 8). Subsequently, the characterization of the transcription factor forkhead box protein 3 (FOXP3), as a lineage-specific marker for Treg cells, and the generation of FOXP3 reporter mice strongly boosted Treg cell research. Continuative analyses revealed that FOXP3 is crucial for Treg cell development and function (9–11). These findings provided the opportunity to screen the FOXP3-regulated Treg cell transcriptome which revealed that the expression of a cyclic AMP (cAMP)-degrading phosphodiesterase (PDE3b) is strongly repressed in Treg cells, whereas the expression of ectonucleotidases (CD39 and CD73) as well as expression of adenylyl cyclase 9 (AC9), an enzyme promoting de novo generation of intracellular cAMP, was upregulated (12, 13). Therefore, reduced expression of phosphodiesterase (PDE3b) implied a decreased degradation of intracellular cAMP accompanied by a strong production of cAMP due to strong expression of AC9, while elevated expression of CD39/CD73 should lead to an increased generation of extracellular adenosine in the proximity of Treg cells. Hence, FOXP3-dependent transcriptional profiling suggested that the suppressive properties of Treg cells is based at least partially on comparatively high amounts of intracellular cAMP concomitantly with an enhanced ability to generate extracellular adenosine from adenosine triphosphate (ATP) [reviewed in Ref. (14, 15)]. Intracellular cAMP Enables Treg Cells to Maintain the Balance of the Immune Tolerance Network During Immune Homeostasis Intracellular cAMP has long been recognized as a potent inhibitor of T cell activation. Especially, agents that elevated cAMP in T cells like cholera toxin, prostaglandin E2, and forskolin were found to strongly impair IL-2 production and T cell proliferation (16–19). Comparative analyses of intracellular cAMP revealed that Treg cells contained high intracellular amounts of cAMP, while it was hardly detectable in Teff cells (20). In addition, co-activation of cocultured Treg and Teff cells resulted in a considerable intracellular increase of cAMP in Teff cells, suggesting a cell contact-dependent transfer of cAMP. One possibility for cell contact-dependent transfer was gap junction intercellular communication (GJIC). GJIC was demonstrated by employing the fluorescent dye calcein which can only be transferred between T cells by gap junctions (21, 22). The functional consequence of such a GJIC-mediated transfer of cAMP between Treg and Teff cells was a strong reduction of IL-2 expression, and as a consequence, inhibition of proliferation, which was both reversed in the presence of the GJIC inhibitor GAP27. In addition, it was shown that the coculture of murine Treg cells and dendritic cells (DC) led to a strong elevation of cAMP in DC concomitantly with an immediate downregulation of CD80/CD86 costimulators (23). This Treg cell-mediated suppression of DC activation via transfer of cAMP was suggested to be decisively involved in the control of a Graft-versus-host disease (GvHD) by Treg cells. Accordingly, the potency of Treg cells to ameliorate a GvHD was found to be strongly increased in the presence of PDE-inhibitors like rolipram (24). In agreement with these findings, it was shown that neonatal human Treg cells suppress DC activation by CTLA-4 and cAMP (25). The importance of GJIC for Treg-mediated suppression was recently emphasized by the finding that diabetic NOD mice showed an impaired expression of connexin 43 (Cx43), an important component of gap junctions, leading in consequence to a strongly reduced suppressive potency of their Treg cells (26). Overexpression of Cx43 in such Treg cells increased their suppressive properties roughly to the level of non-diabetic young NOD mice. The same outcome could be observed when Cx43-mediated GJIC was strengthened via the treatment with alpha-connexin carboxyl-terminal peptide 1 (αCT-1). αCT-1 is a unique synthetic peptide that mimics a cytoplasmic regulatory domain of Cx43 and that specifically disrupts the interaction between Cx43 und its binding partner zonula occludens (ZO)-1, ultimately leading to an enhanced gap junction aggregation (27, 28). However, treatment with αCT-1 failed when Cx43-deficient Treg cells were used, indicating the specificity of action of this agent (26). The comparatively high content of cAMP in Treg cells was described to result from a strong FOXP3-mediated reduction of PDE3b expression concomitantly with a considerable suppressive activity of this T cell type (29). Further analyses of the underlying molecular mechanisms revealed that FOXP3 not only binds and regulates the Pde3b locus but also additionally downregulates expression of the miRNA miR-142-3p, a potent inhibitor of AC9, which catalyzes the conversion of ATP to 3′,5′-cAMP. As a consequence of the impaired targeting of AC9 mRNA due to reduced miR-142-3p expression in Treg cells, these cells contain high levels of AC9, driving elevated intracellular cAMP level. Accordingly, transfection of Treg cells with miR-142-3p not only reduced AC9 expression and intracellular cAMP levels but also considerably impaired the suppressive properties of these Treg cells. The same effect was observed when Treg cells were transfected with AC9 siRNA, again preventing de novo generation of intracellular cAMP by AC9. Thus, FOXP3 strongly promotes AC9-dependent intracellular cAMP production by suppressing expression of miR-142-3p and PDE3b as well. Concerning human Treg cells, it was shown that polyclonal activation as well as activation via CD4, known to induce tolerance, strongly increased the concentration of intracellular cAMP. The irreversible blockade of adenylate cyclases (AC) by MDL12 inhibited this upregulation of cAMP concomitantly with a strong impairment of their suppressive potency – in vitro as well as in vivo – and led to an increased proliferation of these AC-blocked Treg cells (30). Nevertheless, such Treg cells regained their suppressive properties after several days of expansion probably as a consequence of the dilution of the AC inhibitor MDL12. Similar to MDL12, the ectopic expression of cAMP-degrading phosphodiesterase, PDE4c, also prevented the increase of intracellular cAMP in human Treg cells and strongly impaired their suppressive capacity. By contrast, it could be shown that the inhibition of PDE4 by rolipram strongly enhanced the suppressive potency of Treg cells in vitro and in vivo. Transfer of rolipram-treated Treg cells strongly inhibited asthma symptoms in a prophylactic as well as a therapeutic preclinical animal model of experimental asthma (31). As a consequence of Treg–Th2 interaction in vivo, a strong increase of intracellular cAMP could be observed in lung-resident Th2 cells. Additional studies revealed that asthma-promoting Th1 and Th2 cells exhibited a similar increase of intracellular cAMP after contact with Treg cells in vitro and in vivo, but that Th1 cells were far more sensitive to Treg-mediated suppression (32). This was in agreement with the finding that comparatively high cAMP contents are needed to strongly inhibit Th2-derived IL-4 production, while comparatively low cAMP concentrations are sufficient in suppressing IL-2 production and subsequent proliferation of Th1 cells (18). Extracellular Adenosine is Required to Limit a Potentially Self-Destructive Inflammatory Immune Response Inflammatory immune reactions are accompanied by the release of high amounts of ATP in the extracellular space where it is converted into AMP by CD39 and dephosphorylated to adenosine by CD73 (33). Adenosine has strong anti-inflammatory influence on immune cells preferentially by triggering A2A and A2B receptors leading to the generation of intracellular cAMP. Interestingly, the anti-inflammatory action of adenosine is exploited by tumors where adenosine was shown to accumulate, thereby preventing immunological tumor regression (34, 35). The tumor-protective role of adenosine was underlined using mice deficient for the genes Adora2a, encoding for A2A or Adora2b, encoding for A2B receptors which show enhanced elimination of tumors compared to A2A and A2B receptor sufficient wild-type mice (35–37). Among mouse T cells, CD39 and CD73 were described to be preferentially co-expressed in Treg cells (38). This was in agreement with the finding that the expression of Entpd1 (CD39) and Nt5e (CD73) was shown to be upregulated by FOXP3 (12). In line with this, inhibitors of CD39 and CD73 decreased the suppressive capacity of Tregs (39–41). Conclusively, Entpd1-deficient (CD39-deficient) Treg cells exhibited impaired suppressive properties in vitro in a coculture assay as well as in vivo using an allogeneic skin graft model upon cotransfer of Treg/Teff cells indicating the importance of this ectonucleotidase for the suppressive function of Treg cells. Similarly, in a model of acute lung injury (ALI), adoptive transfer of Nt5e-deficient (CD73-deficient) Treg cells failed to resolve ALI adequately, whereas transfer of wild-type Treg cells led to normal resolution (42). Furthermore, it was shown in a model of acute kidney injury that DC treated with an A2A receptor agonist could protect the kidney by suppressing DC-dependent NKT cell activation, suggesting a strong immunosuppressive influence of adenosine on the accessory functions of such DC (43). Interestingly, Teff cells that represent a major target of Treg cell suppressive activity were found to express the T cell-typical adenosine receptor A2A not before day 4 after activation, thus excluding immediate early suppressive influence by this mechanism (38). Accordingly, the authors stated that the adenosine-driven suppression was most probably functional during the late phases of Teff cell activation. Nonetheless, the importance of A2A receptors on Teff cells for Treg cell-mediated suppression in the late phases of an adaptive immune response in vivo was demonstrated in an adoptive T cell transfer model of chronic colitis. Herein, wild-type colitogenic CD4+ Teff cells were considerably suppressed after cotransfer of Treg cells in SCID mice, whereas their Adora2a (A2A receptor)-deficient counterparts could not be inhibited by the cotransferred Treg cells so that the transfer of such Teff cells led to the development of colitis (44). In addition, and in line with the aforementioned results, A2A receptor-deficient Treg cells could not prevent colitis caused by pathogenic wild-type Teff cells, suggesting that adenosine is also of central importance for the suppressive function of Treg cells in this model of inflammatory bowel disease. Hence, it can be reasonably assumed that in addition to reduced PDE3b expression and enhanced AC9 expression, high intracellular levels of cAMP in Treg cells also depend on adenosine-driven elevation of cAMP. This assumption was corroborated by a recent publication of Sitkovsky’s group demonstrating that adenosine stimulated the expansion of Treg cells and concomitantly raised their suppressive capacity (45). Thus, adenosine can directly inhibit Teff cell activation and simultaneously improve the suppressive function of the Treg cell compartment most possibly by affecting cAMP production. In conclusion, high intracellular cAMP levels in Treg cells provide a major contribution to a balanced immune tolerance network (ITN) in the course of immune homeostasis when Treg cells are preferentially stimulated via the T cell receptor complex in combination with rather low costimulatory influences (Figure 1). However, in the course of a local inflammation which is additionally boosted by a myriad of potent microorganism-derived costimulators (bacterial, viral, fungal), an immune response can easily get out of control leading to collateral damage and with that to detrimental consequences. To prevent such a fatal scenario, the suppressive capacity of Treg cells can obviously be strongly improved by adenosine which originates from the CD39/CD73-mediated degradation of extracellular ATP that is characteristically released in huge amounts during such collateral damage of inflamed tissue. This ATP-derived adenosine profoundly increased the overall cAMP content of the involved cells as well as the suppressive arsenal of the local Treg cell compartment which is automatically reduced when inflammation is resolved enabling the ITN to return to immune homeostasis. Thus, the ITN follows an escalating suppressive strategy that allows for the initiation of an effective immune response by breaking a relatively weak suppression that is based on intracellular cAMP which subsequently exploits the locally originating ATP leading to the generation of immune suppressive adenosine to further strengthen intracellular cAMP levels, and by that, curbing a potentially life threatening immune reaction. Figure 1 Adenosine strongly improves the suppressive influence of Treg cell-derived cAMP in the course of inflammation. During immune homeostasis, Treg cells stabilize the ITN with the aid of endogenous cAMP that is driven by FOXP3, which indirectly upregulates adenylate cylase 9 (AC9) through the inhibition of miR142-3p and which concomitantly downregulates cAMP-degrading phosphodiesterase 3b (PDE3b). As a result, Treg cells contain comparatively high amounts of cAMP leading to the suppression of Teff cells and DC via gap junctional intercellular communication (GJIC). Inflammation in combination with metabolic stress and tissue injury results in a massive release of ATP, which represents a powerful danger signal that serves as an additional local inflammatory booster that bears the risk of collateral damage by uncontrollable immune reactions. Therefore, metabolization of ATP by ectonucleotidases CD39 and CD73 that leads to increased local amounts of adenosine prevents such a fatal development especially through triggering the adenosine receptor A2A (A2AR) on Treg and Teff cells and DC as well. The A2AR-mediated elevation of intracellular cAMP inhibits the activation of Teff cells and impairs the accessory function of DC and simultaneously strongly improves the suppressive activity of Treg cells. Author Contributions All authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We thank Dr. Karen Lingnau for critical reading of our manuscript. Funding This work was supported by the Deutsche Forschungsgemeinschaft (DFG BO 3306/1-1 and SCHM1014/7-1). ==== Refs References 1 Mason D A very high level of crossreactivity is an essential feature of the T-cell receptor . Immunol Today (1998 ) 19 :395 –404 .10.1016/S0167-5699(98)01299-7 9745202 2 Sakaguchi S Fukuma K Kuribayashi K Masuda T . Organ-specific autoimmune diseases induced in mice by elimination of T cell subset. I. Evidence for the active participation of T cells in natural self-tolerance; deficit of a T cell subset as a possible cause of autoimmune disease . 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==== Front Front Plant SciFront Plant SciFront. Plant Sci.Frontiers in Plant Science1664-462XFrontiers Media S.A. 10.3389/fpls.2016.01305Plant ScienceOriginal ResearchAn ATL78-Like RING-H2 Finger Protein Confers Abiotic Stress Tolerance through Interacting with RAV2 and CSN5B in Tomato Song Jianwen 12Xing Yali 12Munir Shoaib 12Yu Chuying 12Song Lulu 12Li Hanxia 12Wang Taotao 2*Ye Zhibiao 12*1Key Laboratory of Horticultural Plant Biology, Ministry of Education, Huazhong Agricultural UniversityWuhan, China2Key Laboratory of Horticultural Crop Biology and Genetic improvement (Central Region), Ministry of Agriculture, Huazhong Agricultural UniversityWuhan, ChinaEdited by: Hinanit Koltai, Agricultural Research Organization, Israel Reviewed by: Zhangjun Fei, Cornell University, USA; V. Mohan Murali Achary, International Centre for Genetic Engineering and Biotechnology, India *Correspondence: Taotao Wang, ttwang@mail.hzau.edu.cn Zhibiao Ye, zbye@mail.hzau.edu.cnThis article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science 29 8 2016 2016 7 130504 5 2016 15 8 2016 Copyright © 2016 Song, Xing, Munir, Yu, Song, Li, Wang and Ye.2016Song, Xing, Munir, Yu, Song, Li, Wang and YeThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.RING finger proteins play an important role in plant adaptation to abiotic stresses. In the present study, a wild tomato (Solanum habrochaites) cold-induced RING-H2 finger gene, ShATL78L, was isolated, which has been identified as an abiotic stress responsive gene in tomato. The results showed that ShATL78L was constitutively expressed in various tissues such as root, leaf, petiole, stem, flower, and fruit. Cold stress up-regulated ShATL78L in the cold-tolerant S. habrochaites compared to the susceptible cultivated tomato (S. lycopersicum). Furthermore, ShATL78L expression was also regulated under different stresses such as drought, salt, heat, wound, osmotic stress, and exogenous hormones. Functional characterization showed that cultivated tomato overexpressing ShATL78L had improved tolerance to cold, drought and oxidative stresses compared to the wild-type and the knockdown lines. To understand the underlying molecular mechanism of ShATL78L regulating abiotic stress responses, we performed yeast one-hybrid and two-hybrid assays and found that RAV2 could bind to the promoter of ShATL78L and activates/alters its transcription, and CSN5B could interact with ShATL78L to regulate abiotic stress responses. Taken together, these results show that ShATL78L plays an important role in regulating plant adaptation to abiotic stresses through bound by RAV2 and interacting with CSN5B. Highlight: RAV2 binds to the promoter of ShATL78L to activates/alters its transcription to adapt the environmental conditions; furthermore, ShATL78L interacts with CSN5B to regulate the stress tolerance. abiotic stressATL78LCOP9CSN5BRAV2tomatoNational Natural Science Foundation of China10.13039/501100001809315721253137208131171960 ==== Body Introduction Plants are often exposed to a variety of abiotic stresses such as drought, temperature extreme, salinity, and hypoxia. These unfavorable environmental conditions negatively affect the plant growth, development, and productivity. Nevertheless, plants have evolved a series of intricate mechanisms and regulatory networks to respond and adapt to unfavorable environmental conditions. In recent years, more and more stress responsive genes have been identified using forward or reverse genetic approaches (Liu et al., 2012; Catala et al., 2014; Ding et al., 2015; Ma et al., 2015). It is noteworthy that transcription factors play an important role in stress tolerance (Chinnusamy et al., 2007). For instance, the R2R3-MYB transcription factor MYB15 negatively regulates the expression of CBFs and accelerates the freezing tolerance in Arabidopsis (Agarwal et al., 2006). The cotton WRKY transcription factor GhWRKY17 responds to drought and salt stresses through ABA signaling and regulates the cellular ROS production in plants (Yan et al., 2014). Arabidopsis transcription factor NAC016 triggers drought stress responses by repressing AREB1 transcription through a trifurcate feed-forward regulatory loop involving NAP (Sakuraba et al., 2015). In addition, RING finger proteins also play important role in plant stress responses. The RING finger ubiquitin E3 ligase SDIR1 targets SDIR1-INTERACTING PROTEIN1 to modulate the salt stress response and ABA signaling in Arabidopsis (Zhang et al., 2015). Another RING finger E3 ligase, STRF1, is a membrane trafficking-related ubiquitin ligase, which helps the plants to respond to salt stress by monitoring intracellular membrane trafficking and ROS production (Tian et al., 2015). RING finger proteins are a special type of zinc finger proteins, which consist of 40–60 residues that binds two zinc atoms (Freemont, 1993). The RING finger domain appears to be a convenient scaffold that can be altered to provide functional specificity and the RING finger motif is defined as Cys-X2-Cys-X(9–39)-Cys-X(1–3)-His-X(2–3)-Cys/His-X2-Cys-X(4–48)-Cys-X2-Cys, where X is any amino acid (Borden and Freemont, 1996). Functional studies of the RING finger proteins revealed their wide range of roles in various biological processes including viral replication, signal transduction, and development (Tian et al., 2015). In contrast to two canonical RING types (C3H2C3 or C3HC4), additional types of modified RING domains including RING-V, RING-D, RING-S/T, RING-G, and RING-C2, were identified in Arabidopsis (Stone et al., 2005). In plants, different histidine/cysteine patterns correspond differently to C3H2C3 (RING-H2) and C3HC4 (RING-HC) RING-finger types. The consensus sequence of RING-H2 finger can be described as Cys-X2-Cys-X(9–39)-Cys-X(1–3)-His-X(2–3)-His-X2-Cys-X(4–48)-Cys-X2-Cys, whereas the C3HC4-type finger is defined as Cys-X2-Cys-X(9–39)-Cys-X(1–3)-His-X(2–3)-Cys-X2-Cys-X(4–48)-Cys-X2-Cys (Borden and Freemont, 1996). In recent years, a large number of RING finger proteins have been identified in different plant species. For example, 688 RING domains in 663 predicted proteins and nine RING types were identified in apple (Li Y. et al., 2011). 187 ANK C3HC4-type RING finger proteins were identified from 29 plant species (Yuan et al., 2013). Meanwhile, more and more RING finger proteins have been reported to be involved in plant responses to biotic and abiotic stresses. A C3HC4 RING finger E3 ligase OsDIS1 negatively regulates the drought response through transcriptional regulation of different stress-related genes and possibly through posttranslational regulation of OsNek6 in rice (Ning et al., 2011). A C3HC4-type RING finger protein, EIRP1 E3 ligase positively regulates disease resistance in plants by mediating proteolysis of the negative regulator VpWRKY11 through degradation by 26S proteasome (Yu et al., 2013). The RING-type E3 ligase CaAIR1 plays a role in regulating ABA signaling and drought stress response in pepper (Park et al., 2015). Furthermore, RING-H2 finger proteins also play an important role in stress tolerance. Upregulation of an Arabidopsis RING-H2 gene, XERICO, confers drought tolerance through increased ABA biosynthesis (Ko et al., 2006). The RING-H2 finger E3 ubiquitin ligase, OsRFPH2-10, is involved in antiviral defense at early stages of rice dwarf virus infection (Liu et al., 2014). In our previous microarray analysis of transcriptome differences between the cold tolerant (Solanum habrochaites LA1777 and its introgression line LA3969 under the genetic background of LA4024) and the sensitive (S. lycopersicum LA4024) genotypes under cold stress, a RING-H2 finger gene, ATL78L, was found to be more strongly induced in the two tolerant genotypes than in the sensitive one (Liu et al., 2012). However, little is known about the role of this gene in response to abiotic stresses in tomato. Moreover, the mechanism of drought and cold tolerance in tomato needs to be further investigated. In this study, we isolated the S. habrochaites RING-H2 finger gene, ShATL78L, and overexpressed this gene in S. lycopersicum cv. Ailsa Craig. The overexpression transgenic plants significantly improved drought and cold tolerance. Furthermore, we found that RAV2 can directly bind the promoter of ShATL78L and regulate its expression, and ShATL78L can interact with CSN5B to regulate abiotic stress responses in tomato. Our study provides new insights into the regulatory pathways involving ShATL78L in plant responses to various abiotic stresses. Materials and Methods Plant Materials and Growth Conditions Seedlings of tomato wild relative (S. habrochaites) LA1777 were grown under a natural light in a greenhouse with temperature regimes of 24–28°C and relative humidity of approximately 70–80%. Roots, stems, leaves, flowers and fruits were collected from these plants for RNA isolation. T2 generation of overexpression lines (OE7, OE8, OE9) and RNA interference (RNAi) line (Ri3), as well as the wild-type (S. lycopersicum cv. Ailsa Craig, AC), were tested for their tolerance to abiotic stresses. The abiotic stress treatments were imposed in the greenhouse. Seedlings were grown at 24–28°C and relative humidity of 70–80% of natural light. Four-week-old seedlings were used for stress tolerance assays. Abiotic Stress and Hormone Treatments The cold tolerant S. habrochaites LA1777 was used for the analysis of ATL78L expression. Four-week-old seedlings were transferred to a growth chamber at 25 ± 2°C and relative humidity of 70% with a photoperiod of 14 h light/10 h dark and 200 μmol m-2s-1light intensity. After 3 days, seedlings were used for abiotic stress and hormonal treatments, viz., drought, cold, salt, heat, wound, polyethylene glycol6000 (PEG6000), mannitol, MV, ETH, SA and IAA. For drought stress, seedlings of LA1777 were washed with tap water thoroughly to obliterate substrates, and then dehydrated on filter papers. Cold stress treatment was performed by transferring seedlings of LA1777 to a growth chamber at 4°C under the same light condition as described above. For salinity treatment, seedlings of LA1777 were irrigated once with 200 mM NaCl solution (200 mL per pot). For heat treatment, seedlings were subjected to a high temperature of 40°C. For wound treatment, the leaves of the seedlings were pricked once with a needle. For hormone treatments, the seedling leaves were sprayed once with either 30% PEG6000, 200 mM mannitol, 0.2 mM ETH, 0.1 mM SA, or 0.1 mM IAA. One hundred milliliters of hormone solution was used for three plants. The third fully expanded leaves from the growing point were collected at indicated time points after treatments. Samples were collected for RNA isolation. Sequence Alignment and Phylogenetic Analysis The multiple sequence alignments were conducted using MUSCLE1. The evolutionary history was inferred using the Neighbor-Joining method. The evolutionary distances were computed using the Poisson correction method with the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated. Phylogenetic analyses were conducted in MEGA6 (Tamura et al., 2013). The sequences of the proteins were downloaded from EnsemblPlants2. Cloning and Analysis of ShATL78L The full-length cDNA, gDNA and promoter sequences of ATL78L were obtained from The Sol Genomics Network3, and the gene identifier of ATL78L is Solyc11g005280. The primer sequences used in this study were listed in Supplementary Table S1. The homologous protein sequences of ShATL78-like were found from the NCBI4 and aligned using the DNAman software 5.2.2 (LynnonBiosoft). The PLACE database (Higo et al., 1999) was used to identify potential motifs in the ShATL78L promoter sequence. RNA Extraction and qRT-PCR Total RNA was isolated using the TRIzol reagent (Invitrogen, USA). To remove genomic DNA contamination, the RNA was treated with DNase I at 37°C for 30 min. First strand cDNA was synthesized using the HiScript II 1st cDNA Synthesis Kit (Vazyme, China) according to the manufacturer’s protocol. The expression of ATL78L under various treatments and in transgenic lines was analyzed using qRT-PCR, which was performed using SYBR® Premix Ex TaqTM (TaKaRa, China) and the LightCycler480 System (Roche, Switzerland) according to the supplier’s protocol. The PCR program was as follows: 95°C for 30 s; 40 cycles of 95°C for 5 s and 60°C for 25 s. Tomato actin was used as the internal control. All qPCR primers were designed by Primer5 and the primer sequences are listed in Supplementary Table S1. The qRT-PCR data of each gene were calculated using the 2-ΔΔCT method (Livak and Schmittgen, 2001). qRT-PCR analysis was carried out with three technical replicates. Generation and Molecular Analysis of Transgenic Tomato Plants The full-length open reading frame (ORF) of ShATL78L was amplified from the cDNA of LA1777 using the primers of ATL78L (Supplementary Table S1) designed according to the S. lycopersicum full-length cDNA. For ATL78L overexpression construct, the ORF of ATL78L was incorporated into the pDONR221 vector using the Clonase BP reaction (Invitrogen), and then the recombinant plasmid was incorporated into the pMV3 vector (modified from pHELLSGATE2) by the Clonase LR reaction (Invitrogen). For RNAi construct, a 135-bp fragment of ATL78L was amplified by ATL78L-Ri primers (Supplementary Table S1), and then incorporated into the pHGRV vector using the Clonase BP reaction (Invitrogen). All the expression vector constructs were transformed into Agrobacterium strain C58 by electroporation. Cotyledons of S. lycopersicum were used for transformation, as described previously (Ouyang et al., 2005). Tomato T0 generation was checked by PCR using CaMV 35S promoter primer and ATL78L specific reverse primer (Supplementary Table S1). The expression of ATL78L in transgenic lines was analyzed by qRT-PCR. Three independent homozygous overexpression lines (OE7, OE8, and OE9) and one RNAi line Ri3 of ShATL78L were selected for further analyses. Abiotic Stress Tolerance Assays For cold stress tolerance experiments, 4-week-old uniform-sized seedlings of T2 transgenic lines OE7, OE8, OE9, Ri3 and the wild-type (AC) were randomly chosen and grown in growth chamber with conditions of 4°C, 70% relative humidity and a 14 hr light/8 hr dark photoperiod (200 μmol m-2s-1 light intensity). The same conditions except at 25°C were used for control seedlings. After 7 days of cold treatments and 1 day of recovery, the third fully expanded leaves from the top were sampled. For drought stress tolerance experiments, seedlings from the same stage of T2 transgenic lines (OE7, OE8, OE9, Ri3) and wild-type (AC) were placed in a greenhouse at 25°C under natural light conditions. Seedlings with drought treatment were withheld from water for 8 days, while the control seedlings were watered every 2 days. After 8 days of treatments, the third fully expanded leaves from the top of the plants were collected. For salt and oxidative stress treatments, 4-week-old seedling of T2 transgenic lines OE7, OE8, OE9, Ri3 and wild-type AC were placed in a greenhouse at 25°C under natural light conditions, and the third fully expanded leaves from the top of the plants were collected. Fifteen leaf disks (0.8 cm) were detached from the third fully expanded leaves of each line and incubated in different concentrations of 15 ml solution with 0 mM NaCl, 100 mM NaCl and 5 μM MV for 72 h at 25°C, 70% relative humidity and a 16 h light/8 h dark photoperiod (200 μmol m-2s-1 light intensity) conditions before the Chl contents were measured. Measurements of Water Loss For the water loss assay, the third fully expanded leaves from the top of the control and stressed plants were detached and used for relative dehydration rate determination. The leaves were placed on a filter paper with conditions of 25°C and 60% relative humidity and weighed after 0, 1, 2, 3, 6, 9, and 18 h. Ten compound leaves of each line were weighed. The relative drainage was calculated according to the formula: the relative drainage (%) = 100 × (W0 - Wt)/W0, where W0 and Wt are weights after 0 h and t h, respectively. Protein Interaction Assay Using the Yeast Two-Hybrid System The ORF of ShATL78L was amplified using specific primers ATL78L-BD (Supplementary Table S1) containing NdeI/PstI restriction sites, thus corresponding to the yeast expression vector pGBKT7 (Clontech, USA), to construct pGBKT7-ShATL78L. The pGADT7-CSN5B was combined by the homologous recombination method (ClonExpressTM II One Step Cloning Kit, Vazyme, China). According to the manufacturer’s protocol, the plasmids of pGBKT7-ShATL78L+pGADT7-CSN5B, pGBKT7-Lam+pGADT7-RecT (negative control), and pGBKT7-53+pGADT7-RecT (positive control) were co-transformed into yeast Saccharomyces cerevisiae strain AH109. The positive and negative controls were provided with the BD Matchmaker library construction and screening kits (Clontech, USA). The transformants were tested on the SD/-Leu/-Trp and the SD/-Ade/-His/-Leu/-Trp media. CSN5B accession numbers in the SGN database are Solyc06g073150 and Solyc11g017300. Binding Assay Using the Yeast One-Hybrid System For the yeast one-hybrid assay, the promoter segments of ShATL78L were PCR amplified using the primers ATL78L-P (Supplementary Table S1) containing EcoRI/MluI restriction sites, so that the segments could be inserted into pHIS2 (Clontech, USA). Same as pGADT7-CSN5B, pGADT7-RAV2 was also combined by homologous recombination. Plasmids pHIS2-ATL78LP+pGADT7-RAV2, p53HIS2+pGAD-Rec2-53 (positive control) and pHIS2-ATL78LP+pGADT7 (negative control) were co-transferred into yeast strain Y187, and the binding was detected on SD/-Leu/-Trp/-His selective plates containing 90 mM 3-AT (3-amino-1,2,4,-triazole) according to the manufacturer’s instruction. SlRAV2 accession number in the SGN database is Solyc05g009790. Statistical Analyses Statistical analysis was carried out using SigmaPlot 10, Excel, and SPSS. A significant difference between pairs of groups was analyzed by Student’s t-test. Results Sequence Analysis of ShATL78L Isolated from S. habrochaites In our previous study, transcriptome profiles of cold tolerant (S. habrochaites LA1777 and its introgression line LA3969 under the genetic background of LA4024) and sensitive (S. lycopersicum LA4024) tomato genotypes were compared using microarray analysis, and one RING finger gene, ATL78L, was found to be more strongly induced by cold stress in the two tolerant genotypes than in the sensitive one (Liu et al., 2012). The complete ORFs of ATL78L were isolated from tomato Ailsa Craig (S. lycopersicum) and wild species LA1777 (S. habrochaites), as well as the wild species LA0716 (S. pennellii) that displays extremely high stress tolerance (Rick and Tanksley, 1981). The ORFs of ShATL78L and SpATL78L had the same size, consisting of 624 nucleotides and encoding 207 amino acid residues. Multiple alignments of amino acid sequences revealed that these three sequences showed a high degree of similarity to each other. Three amino acids at positions 20, 111, and 185 were different and one amino acid indel was found among the three genotypes (Figure 1). These four amino acids may functionally contribute to tomato stress tolerance. FIGURE 1 Alignment of ATL78L amino acid sequences from Solanum lycopersicum cv. Ailsa Craig, S. habrochaites LA1777 and S. pennellii LA0716. The RING-H2 finger domain is shown in gray background. ATL78L contains a RING finger domain (Cys-X2-Cys-X(9–39)-Cys-X (1–3)-His-X (2–3)-His-X2-Cys-X(4–48)-Cys-X2-Cys) and is classified as a RING-H2 finger protein (Figure 1). Phylogenetic analysis indicated that SlATL78L had nine paralogs in the cultivated tomato (S. lycopersicum) and showed the highest homology with PGSC0003DMT400015477 from potato (S. tuberosum). The closest homologue of SlATL78L from Arabidopsis is AT1G49230.1 (ATL78) therefore, Solyc11g005280 was named as ATL78L (Supplementary Figure S1). Differential Expression Pattern of ATL78L in Different Species of Tomato To confirm the microarray results, expression levels of ATL78L in LA1777 and AC were examined under cold stress treatment. cDNAs of ShATL78L and SlATL78L were isolated to perform the expression analysis. As shown in Figure 2A, ShATL78L transcripts exhibited a significant increase after 3 h of cold stress treatment. However, no significant difference was observed in the relative expression level of SlATL78L between cold-treated and control plants. Therefore, there is a positive correlation between ShATL78L transcripts and cold tolerance in tomato. To further understand the role of the ShATL78L in different abiotic stresses, we investigated the expression pattern of ShATL78L in LA1777 under different abiotic stresses and hormonal treatments. As shown in Figure 2C, the transcript levels of ShATL78L were increased in response to drought, salinity, heat, wound, mannitol, PEG, ETH, SA, and IAA treatments. ShATL78L was strongly induced by drought, mannitol, salinity, heat, wound, and IAA treatments. A statistically similar but lower induction level was detected in response to SA, ETH, and PEG. To investigate whether the expression of ShATL78L has any tissue/organ specificity, the expression level of ShATL78L in different tissues of LA1777 was determined by qRT-PCR. As shown in Figure 2B, ShATL78L was constitutively expressed in all examined tissues with a little lower expression levels in young leaves and stem epidermis compared with other tissues. FIGURE 2 Expression patterns of tomato ATL78L in different tissues or under various abiotic stresses and hormonal treatments. (A) Expression levels of ATL78L in Solanum habrochaites LA1777 and Solanum lycopersicum cv. Ailsa Craig under cold stress. Asterisks indicate significant differences between LA1777 and AC (∗P < 0.05; ∗∗P < 0.01, Student’s t-test). (B) Expression pattern of ShATL78L in different tissues of LA1777 plants. (C) Expression patterns of ShATL78L in LA1777 seedlings under drought, salt, heat, wound, mannitol, PEG, ETH, SA, and IAA treatments. Asterisks indicate a significant difference (∗P < 0.05; ∗∗P < 0.01, Student’s t-test) compared with the corresponding controls. Four-week-old seedlings were treated by 4°C, 200 mM NaCl, 40°C, 200 mM mannitol, 30% PEG6000, 0.2 mM ETH, 0.1 mM SA or 0.1 mM IAA, respectively, for indicated time points. Values are means ± SE of three biological replicates. Each replicated sample consists of leaves from three seedlings. Molecular Characterization of ShATL78L Transgenic Plants In order to characterize the function of ShATL78L, the full-length ORF of ShATL78L was cloned into the plant expression vector pMV3 under the control of CaMV35S promoter. This construct was introduced into AC (S. lycopersicum). A total of 28 independent transformants (T0) were generated, and 23 of them were positive as confirmed by PCR analysis using CaMV35S forward and gene-specific reverse primer sets. At the same time, the RNAi lines were also generated through knocking down the ATL78L transcripts. Transgene expression level was examined by qRT-PCR. As shown in Figure 3B, four independent lines, OE-7, OE-8, OE-9, and Ri-3 displayed expected expression levels of ATL78L. FIGURE 3 Overexpression of ShATL78L enhanced the tolerance to multiple abiotic stresses in tomato. (A) Phenotypes of seedlings from transgenic and wild-type tomato plants under cold stress conditions. Four-week-old seedlings of transgenic lines (T2 generation) and wild-type were treated at 4°C for 7 days and then recovered at 25°C for 1 day. (B) Relative expression level of ATL78L in the wild-type and transgenic lines. (C) Phenotypes of seedlings from transgenic lines and wild-type under drought stress conditions. Four-week-old seedlings of transgenic lines (T2 generation) and wild-type were withheld from water for 8 days. (D) Water loss of the detached leaves from wild-type and transgenic plants. (E) Leaf disks obtained from 4-week-old wild-type and transgenic seedlings were incubated in different solution of H2O, 100 mM NaCl and 5 μM MV for 72 h. (F) Relative chlorophyll content after the 100 mM NaCl and 5 μM MV treatment of the leaf disks. Data in (B,D,F) are means ± SE of three biologically replicated samples. AC represents the wild-type. OE-7, OE-8, OE-9, and Ri-3 represent three independent ShATL78L overexpression lines and the RNAi line, respectively. Asterisks indicate significant differences between transgenic lines and wild-type (∗P < 0.05; ∗∗P < 0.01, Student’s t-test). Overexpression of ShATL78L Enhances Tomato Plant Tolerance to Cold and Drought Stresses To characterize the functional role of ShATL78L in cold stress tolerance, 4-week-old seedlings of the four transgenic lines (T2) and the wild-type AC were examined under cold stress treatment at 4°C for 7 days, and then allowed to recover at 25°C for 1 day. After the stress treatment, seedlings of the AC line exhibited severe wilting, whereas the overexpression lines (OE-7, OE-8, OE-9) displayed much less leaf wilting symptoms, and the RNAi line Ri-3 showed higher leaf wilting symptoms compared to AC (Figure 3A). These findings demonstrated that ShATL78L plays a positive role in cold stress tolerance. To investigate whether ShATL78L has an effect on drought stress tolerance, 4-week-old seedlings of the three overexpression lines (OE7, OE8, and OE9), the RNAi line (Ri-3) and the wild-type AC were withheld from water for eight consecutive days. After the drought stress treatment, obvious dehydration symptoms (leaf wilting) were found both in the RNAi line and wild-type plants compared to overexpression lines, which exhibited inconspicuous wilting. The stem of wild-type and RNAi seedlings bent due to loss of turgor pressure while the overexpression plants remained turgid and erect (Figure 3C). Water loss assay was conducted under the dehydration stress. The detached leaf water loss was significantly lower in the overexpression lines compared to wild-type plants while RNAi line showed the maximum water loss among all the treated lines (Figure 3D). These findings demonstrated that ShATL78L plays a positive role in drought stress tolerance. Overexpression of ShATL78L Improves Tomato Oxidative Stress Tolerance The potential role of ShATL78L under salt and oxidative stress was further evaluated by incubating the leaf disks of 4-week-old seedlings in 100 mM NaCl and 5 μM MV solutions, respectively. After 72 h treatment, the leaf disks of the transgenic plants were similar to that of the wild-type AC in H2O and NaCl solutions. In the MV solutions, much more severe bleaching was observed in the RNAi line than overexpression plants, and wild-type plants showed an intermediate phenotype (Figure 3E). The Chl contents further confirmed the difference of salt and oxidative damages between transgenic and wild-type AC plants (Figure 3F). Taken together, these results supported that ShATL78L plays a positive role in tolerance to oxidative stress. Transcription Factor SlRAV2 (ABI3/VP1-Like) Specifically Binds to the Promoter of ShATL78L The induction of ShATL78L gene under various abiotic stresses prompted us to analyze the promoter of this gene (approximately 1000 bp upstream the transcription start site). The promoter sequence was isolated from LA1777 and fused with pHIS2 to construct the bait vector (pHIS2-ATL78LP). As shown in Figure 4B, 90 mM inhibitory 3-amino-1,2,4-triazole (3-AT; Sigma) was screened from 0 to 90 mM concentrations of 3-AT for the yeast one-hybrid system. Bait constructs and tomato cDNA library fusion plasmid were co-transformed into yeast strain Y187 and a positive clone of SlRAV2 was selected from 152 clones for further confirmation (Supplementary Table S2). As shown in Figure 4D, all yeast cells harboring different constructs could grow on SD/-Trp/-His/-Leu without 3-AT, which indicated that all the recombinant plasmids were introduced successfully into yeast. However, co-transformed cells with pGADT7-SlRAV2 and pHIS2-ATL78LP could grow in the presence of the 90 mM 3-AT. Moreover, the growth of transformants containing constructs but lacking SlRAV2 was completely inhibited (Figure 4E), which suggested that SlRAV2 can bind to the promoter of ATL78L in yeast. FIGURE 4 CSN5B interacts with ATL78L whose promoter is bound by RAV2. (A) Self-activation assay of ATL78L. The yeast cells containing pGBKT7-ShATL78L transformants were tested on SD/-Trp, SD/-His/-Trp, and SD/-Ade/-Trp media. (B) Screen the concentration of 3-AT for the yeast one-hybrid system. The yeast cells containing pHIS2-ATL78LP were examined on the SD/–Trp/-His/-Leu media with the 0 to 90 mM 3-AT, respectively. (C) ATL78L interacts with two CSN5B proteins. The yeast cell transformants containing pGBKT7-53+pGADT7-RecT (positive control), pGBKT7-Lam+pGADT7-RecT (negative control) and two pGBKT7-ShATL78L+pGADT7-CSN5B were tested on the SD/-Ade/-His/-Leu/-Trp media. (D,E) RAV2 binds to the promoter of ATL78L. Plasmids p53HIS2+pGAD-Rec2-53 (positive control), pHIS2-ATL78LP+pGADT7 (negative control) and pHIS2-ATL78LP+pGADT7-SlRAV2 (the right column) were co-transferred into yeast strain Y187, the interactions were detected on SD/-Leu/-Trp/-His with 0 mM 3-AT (D) and 90 mM 3-AT (E). ShATL78L Interacts with CSN5B In vivo To further investigate the molecular mechanism of the ATL78L-mediated pathway in tomato stress tolerance, a yeast two-hybrid screening was performed to identify the interacting proteins with ATL78L. As shown in Figure 4A, ATL78L does not contain self-activation due to that the yeast cells pGBKT7-ShATL78L could not grow in the SD/-His/-Trp or SD/-Ade/-Trp media while could grow in the SD/-Trp medium. In the Y2H assay, the full-length ORF of ShATL78L was used as the bait to screen a tomato cDNA Y2H library, which was constructed with the prey vector pGADT7-Rec. Two positive clones of COP9 signalosome (CSN) complex subunit CSN5B were selected from 138 clones for further analysis (Supplementary Table S3). The amino acid sequences of these two genes showed high homology (Supplementary Figure S2). As shown in Figure 4C, the yeast cells co-transformed with pGBKT7-ShATL78L and pGADT7-CSN5B could grow in the SD/-Ade/-His/-Leu/-Trp media, same as the positive control (pGBKT7-53+pGADT7-RecT). In addition, negative control transformants (pGBKT7-Lam+pGADT7-RecT) did not grow in the same media. These results indicated that ShATL78L interacts with SlCSN5B in yeast. Discussion RING-H2 Finger Gene ShATL78L is Regulated by ETH, SA, and IAA RING finger family proteins have been widely reported to play an important role in abiotic and biotic stresses (Lee et al., 2001; Cheung et al., 2007; Hong et al., 2007; Qin et al., 2008; Zhang et al., 2015). According to the motifs, ATL78L can be classified into RING-H2, a subgroup of RING finger proteins (Borden and Freemont, 1996). RING-H2 finger proteins are involved in different abiotic stress signaling pathways in plants (Ko et al., 2006; Zhang et al., 2007). However, the functions of RING-H2 proteins in tomato remain largely unknown (Hondo et al., 2007; Qi et al., 2016). Particularly, the molecular mechanism of RING-H2 finger proteins in response to stress tolerance is still unclear. ShATL78L has not been functionally characterized, though it was screened in our previous microarray experiment under cold stress (Liu et al., 2012). In this study, we showed that ShATL78L exhibited a strong response to cold, drought, salt, heat and wound stresses (Figure 2C). It has been well reported that RING finger proteins can modulate various plant hormone responses, such as ABA (Ko et al., 2006; Zhang et al., 2007; Li H. et al., 2011), IAA (Gray et al., 2002; Xie et al., 2002), GA (Park et al., 2013), ETH (He et al., 2005), JA (Hondo et al., 2007), and SA (Cheong et al., 2009). Phytohormones play critical roles in plant adaptation to adverse environmental conditions. Cross-talk between different plant hormones and the environment results in synergistic or antagonist interactions that play a significant role in plant responses to abiotic stresses (Peleg and Blumwald, 2011). Our results indicated that ShATL78L was induced by different plant hormone treatments such as ETH, IAA, and SA (Figure 2C), which indicates that ShATL78L possibly functions as a balance regulator through mediating different signaling pathways between ETH, IAA, and SA in response to abiotic stresses. ShATL78L Enhances Multiple Abiotic Stress Tolerance in Tomato In this study, we demonstrated that ShATL78L overexpression plants positively contributed to enhanced cold, drought, and oxidative stress tolerance (Figures 3A,C,E). These results are consistent with previous studies in other plant species (Hirayama and Shinozaki, 2010). On the other hand, suppression of the RING E3 ubiquitin ligase AtATL78 increased the cold stress tolerance, while decreased drought stress tolerance (Kim and Kim, 2013). Arabidopsis RING-H2 gene XERICO confers drought stress tolerance through increased ABA biosynthesis (Ko et al., 2006). In S. pimpinellifolium, the RING Finger E3 Ligase SpRing is a positive regulator of salt stress signaling (Qi et al., 2016). However, ShATL78L transgenic plants did not show significantly different response to salt stress compared to the wild type (Figure 3E). ShATL78L is Directly Regulated by RAV2 In this study, we demonstrate that RAV2 binds to the promoter of ShATL78L and activates/alters ShATL78L transcription (Figures 4D,E). The RAV transcription factor contains AP2-like and B3-like domains, which specifically bind to DNA with bipartite sequence motifs of RAV1-A (CAACA) and RAV1-B (CACCTG; Kagaya et al., 1999). The motif of RAV1-A and RAV1-B can be detected in the promoter of ShATL78L. It has been well reported that RAV2 responds to different abiotic stresses (Li C.W. et al., 2011; Fu et al., 2014; Matias-Hernandez et al., 2014; Mittal et al., 2014). The promoter of RAV2, which contains the CRT/DRE element, can be bound by the DREB transcription factors (Yamaguchi-Shinozaki and Shinozaki, 1994; Dubouzet et al., 2003; Li C.W. et al., 2011). DREB proteins specifically bind to DRE/CRT element and activate target gene transcription in Arabidopsis (Liu et al., 1998) and tomato (Li C.W. et al., 2011; Li et al., 2012). It has been well studied that DREB family play an important role in abiotic stresses in Arabidopsis, such as DREB (Liu et al., 1998) and CBF1 (Stockinger et al., 1997). In recent years, several studies have supported that DREB family genes exhibit similar role in abiotic stresses in tomato. For example, SlDREB gene acts as a positive regulator of drought stress responses by restricting leaf expansion and internode elongation (Li et al., 2012); The dehydration-responsive transcription factor SlDREB2 mediates salt stress tolerance in tomato and Arabidopsis (Hichri et al., 2016). Tomato has a complete CBF cold responsive pathway, however, its CBF regulon differs from Arabidopsis and shows much smaller and less diverse in function (Zhang et al., 2004). Our results and these previous findings clearly indicate the responsiveness of ShATL78L to multiple abiotic stresses (Figure 5). FIGURE 5 Proposed role of ShATL78L in the plant stress signaling pathway. The model shows the genetic interactions between RAV2, ATL78L, and CSN5B genes in regulating the responses to abiotic stress in tomato. ShATL78L Interacts with COP9 Signalosome Complex Subunit CSN5B in Response to Abiotic Stresses COP9 (constitutive photomorphogenesis) is a component of the signaling complex mediating light control of development in Arabidopsis (Wei et al., 1994). CSN consists of eight subunits (CSN1-8), and plays a critical role in the ubiquitin/26S proteasome proteolytic regulatory pathway (Rubio et al., 2005). We show that ShATL78L interacts with CSN5B (Figure 4C), which plays a crucial role in abiotic stress tolerance (Wang et al., 2013). In rat, CSN5 inhibits cardiac L-type Ca2+ channel activity through protein–protein interactions (Kameda et al., 2006). Taken together, we hypothesize that ShATL78L responds to multiple abiotic stresses possibly through interacting with CSN5B, which alters the Ca2+ channel activity. On the other hand, CSN regulates plant growth and development through mediating protein degradation (Serino and Deng, 2003). In addition, CSN plays a vital role in mediating E3 ubiquitin ligase-mediated responses (Schwechheimer et al., 2001; Cavadini et al., 2016). Altogether, our results indicate that ShATL78L enhances multiple abiotic stresses tolerance possibly through altering the ubiquitin-mediated protein degradation (Figure 5). Conclusion In conclusion, this study provides a possible molecular mechanism of ShATL78L in enhancing multiple abiotic stress tolerance in tomato (Figure 5). Our results clearly indicate that RAV2 binds to the promoter of ShATL78L and activates/alters transcription levels of ShATL78L in helping plants to adapt unfavorable environmental conditions. Meanwhile, ShATL78L interacts with CSN5B to regulate stress tolerances. However, further studies are still required to explore the detailed mechanism of COP9-mediated stress signaling in tomato. Author Contributions JS designed and performed experiments, analyzed the data and drafted the manuscript. YX designed and conducted the qRTPCR experiments, and performed expression analysis. SM, CY, and LS designed and carried out the stress experiments. ZY, TW, and HL designed all the experiments and revised the manuscript. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This work was supported by grants from the National Natural Science Foundation of China (31572125, 31372081 and 31171960) and the China Agricultural Research System (CARS-25-A-02) 1 http://www.ebi.ac.uk/Tools/msa/muscle/ 2 http://plants.ensembl.org/index.html 3 http://solgenomics.net/ 4 http://blast.ncbi.nlm.nih.gov/Blast.cgi Supplementary material The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2016.01305 Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. ABBREVIATIONS 3-AT3-amino-1,2,4-triazole ABAabscisic acid Chlchlorophyll DREBdehydration-responsive element binding ETHethephon IAAindole-3-acetic acid MVmethyl viologen PEGpolyethylene glycol qRT-PCRquantitative real-time PCR SAsalicylic acid ==== Refs References Agarwal M. Hao Y. Kapoor A. Dong C. H. Fujii H. Zheng X. (2006 ). A R2R3 type MYB transcription factor is involved in the cold regulation of CBF genes and in acquired freezing tolerance. J. Biol. Chem. 281 37636 –37645 . 10.1074/jbc.M605895200 17015446 Borden K. L. Freemont P. S. (1996 ). The RING finger domain: a recent example of a sequence-structure family. Curr. Opin. Struct. Biol 6 395 –401 . 10.1016/S0959-440X(96)80060-1 8804826 Catala R. Lopez-Cobollo R. Mar Castellano M. Angosto T. Alonso J. M. Ecker J. R. (2014 ). 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==== Front Front Vet SciFront Vet SciFront. Vet. Sci.Frontiers in Veterinary Science2297-1769Frontiers Media S.A. 10.3389/fvets.2016.00070Veterinary ScienceOriginal ResearchHealth and Behavioral Survey of over 8000 Finnish Cats Vapalahti Katariina 1Virtala Anna-Maija 1Joensuu Tara A. 1Tiira Katriina 123Tähtinen Jaana 4Lohi Hannes 123*1Department of Veterinary Biosciences, University of Helsinki, Helsinki, Finland2Research Programs Unit, Molecular Neurology, University of Helsinki, Helsinki, Finland3The Folkhälsan Institute of Genetics, Helsinki, Finland4Health and Education Committee of Finnish Cat Association, Helsinki, FinlandEdited by: Emily Patterson-Kane, American Veterinary Medical Association, USA Reviewed by: Paolo Buracco, University of Turin, Italy; Joseph Taboada, Louisiana State University, USA *Correspondence: Hannes Lohi, hannes.lohi@helsinki.fiSpecialty section: This article was submitted to Animal Behavior and Welfare, a section of the journal Frontiers in Veterinary Science 29 8 2016 2016 3 7004 7 2016 17 8 2016 Copyright © 2016 Vapalahti, Virtala, Joensuu, Tiira, Tähtinen and Lohi.2016Vapalahti, Virtala, Joensuu, Tiira, Tähtinen and LohiThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.A comprehensive feline health survey was conducted to reveal breed-specific inheritable diseases in Finnish pedigree cats for genetic research. Prevalence of 19 disease categories and 227 feline diseases were defined in a study population of 8175 cats belonging to 30 breeds. Dental and oral diseases, with a prevalence of 28%, and dental calculus and gingivitis (21 and 8%, respectively) were the most prevalent disease category and diseases among all cats and in most of the breeds. An exception was Korats, which were more often affected by the diseases of the respiratory tract (23%) and asthma (19%). Other prevalent disease categories affected various organ systems, such as the skin (12%), the urinary system (12%), the digestive tract (11%), eyes (10%), the musculoskeletal system (10%), and genitals of female cats (17%). Prevalent health or developmental issues included repetitive vomiting (4%), tail kink (4%), feline odontoclastic resorption lesion (4%), urinary tract infections (4%), as well as cesarean section (6%) and stillborn kittens (6%) among female cats. We found 57 breed-specific conditions by Fisher’s exact tests and logistic regression analyses, including 32 previously described and 19 new breed-specific diseases. The genetic defect has already been found in six of them: polycystic kidney disease, progressive retinal atrophy, hypertrophic cardiomyopathy, and three types of tail malformations. Behavioral profiling revealed breed-specific traits, such as an increased human avoidance in British Short and Longhairs and a higher level of aggression in Turkish vans. Our epidemiological study reveals the overall health profile in Finnish pure and mixed breed cats and identifies many breed-specific conditions without molecular identity for genetic research. health surveyfelineepidemiologydiseasebehaviorquestionnaire ==== Body Introduction The annotation of the cat genome, in 2007, has facilitated genetic research with novel genomic resources (1, 2). Pedigree cats form appropriate populations for genetic studies, since each breed represent a group of genetically similar animals that descended from few ancestors (3). It has been estimated that 85% of all current breeds have arisen only in the past 75 years, largely due to intentional breeding to influence esthetic qualities (4). There are over 40 officially approved cat breeds according to The Cat Fanciers’ Association1 (CFA), whereas The International Cat Association2 (TICA) accepts about 60, and the Finnish Cat Association3 (Suomen Kissaliittory) – 44 registered breeds. The cat is the most common pet in Europe, with an estimated 100 million domesticated cats (5). In Finland, there is a cat in every seventh household, comprising almost 600,000 cats in 360,000 households (6). The non-pedigree domestic cat forms the most common group, while Ragdolls lead the pedigree group, according to the Finnish Cat Association registry. Over 300 genetic diseases have been described in cats, of which ~70% are potential models for human diseases (7). Currently, over 55 genes have been found in various traits (7). A commercial DNA test is available for >10 inherited feline diseases (2). Examples of these are the test for polycystic kidney disease (PKD1) (8), which is common in Persian and Exotic breeds, and the tests for hypertrophic cardiomyopathy (HCM) (9, 10). In addition, over 20 mutations associated with uncommon feline diseases have been localized (2). However, the molecular background of many conditions remains unsolved. A growing biobank of cats with over 4000 samples has been established in our laboratory at the University of Helsinki to facilitate feline genetic research and to identify genetic causes of breed-specific inherited diseases and behavioral traits. Toward this aim, a comprehensive owner-completed health survey was developed to explore the overall health profiles in pedigree and non-pedigree cats. Besides general information of the cat and their living environments, the health survey comprises questionnaire data, covering altogether 227 feline diseases as well as data on behavioral traits, vaccination, clinical examinations, and possible gene tests. The present study analyzes the survey-based health profiles in 8175 Finnish cats to identify common and breed-specific conditions. Traits that are enriched in a particular breed or phylogenetic group suggest genetic susceptibility and could be prioritized for sample recruitment. The comprehensive health profiling provides useful data not only for genetic research but also for feline breeding programs and veterinary epidemiology. The survey data inform key health issues in different breeds to guide future research focus and feline biobanking activities. Materials and Methods Study Design and Questionnaire A cross-sectional online feline health survey (Presentation S1 in Supplementary Material) was established to collect information about Finnish cats. Besides data on various disorders, breed-specific behavior, living habits (including diet, outdoor activity, contacts with other animals, and hunting), and reproductive fitness were collected in the questionnaire. The survey was targeted at all Finnish cats, including pedigree and non-pedigree domestic cats. Owners were allowed to also report deceased cats. The cat breeders and owners were informed about the survey in several ways, including advertisements by the Finnish Cat breed Association, social media such as Facebook, and by sharing information bulletins in different cat shows and meetings of the breed clubs. Numbers with the set breed target numbers of submitted responses were published regularly in the research group’s website4 and Facebook site to encourage participation. The minimum sample size required for each breed to estimate disease prevalence was calculated using Epitools calculator (11) and taking into account the annual registration numbers over the past 10 years (2001–2011) by the Finnish Cat Association. To maximize the required sample size for significance, we assumed 50% disease prevalence, which is the most difficult to detect (12). Closely related breeds were grouped together, if there were not enough cats within a breed to reach the minimum sample size requirement. The questionnaire was divided into 25 sections, which covered basic information about the cat, its owner, possible offspring, living environment, feeding manners, personality and personal habits, and possible genetic and clinical tests, such as ultrasound, vaccination records, and health status, with respect to each disease category. The sections used in this study are described in more detail. The basic information of the cat included the breed, registration number (registered in the Finnish Cat Association, TICA, or CFA), date of birth, and possible death, gender, and birth control status (castration, hormone implant, or contraceptive pill combined). There were 19 disease categories (1) behavioral traits, (2) congenital developmental disorders, (3) dermatological/glandular diseases, (4) ocular diseases, (5) otic diseases, (6) dental and oral diseases, (7) diseases of the urinary system, (8) disorders of the cardiac and circulatory system, (9) blood disorders, (10) diseases of the musculoskeletal system, (11) diseases of the digestive tract, (12) diseases of the respiratory tract, (13) diseases of the nervous system, (14) genital diseases, (15) endocrine and metabolic diseases, (16) autoimmune diseases, (17) tumors (including benign tumors and cancers), (18) parasites and protozoans, and (19) diseases not mentioned in the previous categories. In all these categories, the participant was asked to report whether a given diagnosis was (1) made by a veterinarian, (2) made by the owner, (3) no such diagnosis was made, or (4) this was unknown. There were altogether 227 different diagnoses in the disease categories, varying from 3 to 27 per category. The questions concerning the cat’s personality covered general activity, contact with people, aggressiveness toward strangers, family members, or other cats, sensitivity toward new situations or unfamiliar people, and possible compulsions, such as licking. Multiple and incomplete responses were removed from the survey data. If the cat had been reported to belong to a certain breed, but the registration number was not given, the registration number was requested from the owner, the Finnish Cat Association, or searched on the internet. If the registration number was not available, the pedigree cat was moved into a special group, “Others,” as a separation from the verified pedigree and non-pedigree cats. The same procedure was used to investigate the dates of birth, which were missing or apparently wrong (the given date of birth was after the date of response or after date of death). If the date could not be verified, it was left missing. The ages of the living cats were counted as a difference between the date of response and the date of birth converted to years. For deceased cats, the age of death was similarly counted as a difference between the date of death and the date of birth. Five age groups were created: (1) <1 year, (2) 1 to <3 years, (3) 3 to <7 years, (4) 7 to <11 years, and (5) ≥11 years. The variable “Age” was determined as either the age of the living or the age of the cat at death. Also, a new variable “Alive/dead,” representing whether the cat was alive or dead, was created. Distributions of age, sex, alive/dead cats, and neutered/non-neutered cats were computed for all cats and in each breed. Before commissioning the final health survey, a pilot study was performed on Norwegian Forest cats (N = 604) (13) to test the content, usability, and advertisement strategies of the questionnaire. Prevalence Estimates Prevalence with 95% confidence intervals were used to express: (a) the number of cats having had disease/diseases belonging to a disease category (n = 19), and (b) the number of cats having had a separate disease (n = 227). In addition to the entire study population, prevalence were also determined for each breed and for subgroups of age, sex, neutered/non-neutered cats, and alive/dead cats. Prevalence of genital diseases were counted in subgroups of female or male cats depending on the disease. Phylogenetic grouping was used in tabulating disease prevalence (4, 14). In the prevalence tables, coloring (green = low, yellow = moderate and red = high prevalence) was used to point out the differences between breeds and categories. Prevalence in disease categories were counted in three ways: (1) combining diagnoses by veterinarian and owner, (2) diagnoses by veterinarian solely and (3) diagnoses by owner solely. Prevalence in separate diseases were also tabulated by breed to find the most common diseases in the breed. Breed Specificity The breed-specific diseases were tentatively deducted from the prevalence of the diseases. The disease was considered breed-specific, if the prevalence of the disease (according to coloring mentioned above and prevalence remainder) distinctly differed (upwards) in one or a few breeds from other breeds among all cats and in subgroups of sex, age, alive/dead cats, and neutered/non-neutered cats. If the disease was found to be breed-specific, according to prevalence, it was tested using the Fisher’s exact test to verify the breed specificity compared with other pedigree cats (combined) and non-pedigree cats. The p-values < 0.05 of the Fisher’s exact test were considered to indicate statistically significant difference, and, thus, potential breed specificity of the disease. No correction for the error rate α for false significance in multiple testing (15) was performed, as breed specificity analyses were aimed for preliminary information, which were later controlled in a logistic regression analysis. In addition, for each tentative breed-specific disease, a logistic regression analysis with random intercept variable “Breed” was performed to observe the effect of the breed in acquiring the disease (Model 1). The model also included variables “Age” (reference group “3 to <7 years”), “Sex,” and “Alive/dead” as fixed (level 1) independent explanatory variables to control the effect of these variables. The effect of “Breed” was measured by the median odds ratio (MOR) (16), which was counted from variance δ2 of the random variable “Breed.” MOR expressed (in this study) the MOR between a cat of a higher prevalence and a cat with a lower prevalence, when the cats with the same covariates were randomly chosen from two different breeds. A logistic regression analysis was also performed without the random variable “Breed” (Model 2) in order to compare the goodness of fit between models 1 and 2. An evidence ratio (17) was used in the comparison. Furthermore, logistic regression analysis was performed with “Breed” included as a fixed variable (Model 3). In this model, each breed, which was suspected to be overrepresented compared with other breeds (as determined by the intercept of the breed in Model 2), formed a separate category. Non-pedigree cats formed their own category, and the rest of the breeds were combined to one category and set to a reference group. The purpose of Model 3 was to display the effects of the overrepresented breeds in acquiring the disease by odds ratios (OR). OR indicated how much higher the odds were in the breed/breeds that were suspected to be prone to disease than in the other breeds. The models were basically built from the entire study population included, but, to assess the effect of the non-pedigree cats in heterogeneity of breeds (MOR), they were also built from a population of which non-pedigree cats were excluded. In some diseases, separate age groups or non-pedigree cats had to be excluded because of too few or no cases in the group. In such cases, the exclusion was executed in all three models to retain the goodness of fit comparability with Akaike Information criterion (AIC), which requires equal data sets (17). In genital diseases, the models were built from subgroups of female or male cats, and, for female cats, non-pedigree cats were excluded. The models were tested for interactions. If an interaction was detected, the MOR, evidence ratio, and OR of overrepresented breeds yielded from Models 1–3 with the interaction added were compared with corresponding values yielded from models without interactions included, to find out whether the interaction had any effect on these measures. Median odds ratio and evidence ratios were used to evaluate the breed specificity of the disease. MOR always has values greater or equal to one. The closer to one the MOR was, the less heterogeneity there was between the breeds. The evidence ratio, in turn, indicated how many times more likely Model 1 was to be the best model as compared with Model 2, according to AIC values (18). Since the smaller value of AIC indicated better goodness of fit in terms of Kullback–Leibler discrepancy (19), the evidence ratio was counted only in cases when the AIC value of Model 1 was smaller. Otherwise, Model 2 was considered the better model, and the breed was interpreted not to improve the model. Neither MOR nor the evidence ratio has any distinct cut point (threshold), which would designate that variable “Breed” had had a significant effect in acquiring the modeled disease (16, 17). In case of low value of the evidence ratio (<10) and a high value of MOR, the deduction of breed specificity was based on the whole entity of the data concerning the disease. In this deduction, in addition to MOR and the evidence ratio, the number of diseased cats, the distribution of the disease among all breeds, and the effect of non-pedigree cats in the model were used. According to the results of the logistic regression analysis and previous studies, the suspected breed-specific disease were divided into four groups: diseases with (1) established heredity, (2) known breed-specific conditions, (3) breed specificity suspected by our study for the first time, and (4) breed specificity not verified in our study. In Turkish Vans and breed groups of Cymric–Manx and Persian–Exotic, there were several cats of which the date of death was before 2005 (indicating a low possibility of these cats having lived at the same time as the alive cats of our study). To eliminate potential bias caused by deceased cats far in the past, the main results of logistic regression analyses concerning these breeds were verified in a subgroup in which cats died before January 1st, 2005 were excluded as well. Furthermore, in Cornish Rexes, Korats, Sphynxes, Bengals, Turkish Vans, and Europeans, more than 15% of the cats were associated with the same breeder. In potential breed-specific diseases, the prevalence were also counted excluding these breeders to check the effect of the breeder. Behavioral Traits Owners were asked to rate behavior at five levels in the questionnaire (“not at all,” “a little,” “quite a bit,” “often,” and “very often”), and a binary variable was created using, in case of variables concerning aggressiveness and licking, combined levels “often” and “very often,” and in case of variables concerning sensitivity, level “often” to form the first category, while the rest of the levels together formed the reference category. In the case of variables “activity” and “takes contact with people,” combined levels “not at all” and “a little” were used as the extreme features, forming the first category. Prevalence with 95% confidence intervals were computed and tabulated in a similar way to the diseases and Fisher’s exact tests and logistic regression were used in the analysis to verify behavioral differences between breeds. Statistical Analyses All statistical analyses were performed by SAS version 9.3, SAS Institute, Cary, NC, USA. Proc Freq statement with chisq and fisher options was used in testing differences and association in categorical data. Proc Npar1way statement with Wilcoxon option (producing Kruskal–Wallis test) was used in (not normally distributed) continuous data. Proc Logistic and Proc Glimmix statements were used in logistic regression analysis in testing breed specificity of the diseases. Results Demographics of the Survey Data A total of 8873 survey responses were collected, of which 8175 responses were accepted for statistical analyses after quality control (Figure 1). The data were accumulated from 29 different breeds and non-pedigree cats. When the small phylogenetically related breeds with few cats were grouped together, the study included 13 breeds, 6 breed groups (16 breeds), and non-pedigree cats. The largest populations were non-pedigree cats (N = 1545), Abyssinian–Ocicat–Somali group (N = 539), Siamese–Balinese–Oriental–Seychellois group (N = 468), and the Cornish Rex breed (N = 437). The sample size for the Cymric–Manx breed remained incomplete with 15 missing responses from the set breed target, and the results should, therefore, be considered only directional in this breed (Table 1). Figure 1 Population flow chart: cats selected into the study from responses submitted into the feline health study. Table 1 Summary of the breed-specific sample size requirements and response numbers in the health survey. Breeds and breed groups Responses Minimum sample size Extra/missing Breeds included in the study Abyssinian–Ocicat–Somali 539a 329 210 Siamese–Balinese–Oriental–Seychellois 468b 358 110 Cornish Rex 437 330 107 Turkish Van 233 157 76 British 378 323 55 Maine Coon 405 350 55 Norwegian Forest cat 406 351 55 Ragdoll 418 365 53 Russian Blue 342 295 47 Bengal 317 272 45 Birman 396 354 42 Siberian–Neva Masquerade 344c 310 34 Sphynx 272 238 34 Persian–Exotic 378d 348 30 Burmese–Burmilla–Singapura 350e 323 27 Korat 242 223 19 European 267 260 7 Devon Rex 293 290 3 Cymric–Manx 145f 160 −15 Non-pedigree cat 1545 –i All 8175 Breeds not included in the study American shorthair 2 –i Sokoke 0 12 −12 German Rex 0 15 −15 Snowshoe 0 15 −15 Selkirk Rex 8 34 −26 Kurilian Bobtail Short- and Longhair 15g 57 −42 Egyptian Mau 16 88 −72 Don Sfinx–Peterbald 17 91 −74 Chartreux 28 108 −80 American Curl Short- and Longhair 119h 200 −81 Turkish angora 49 171 −122 Others 64 –i All 318 All cats 8493 Minimal required sample size was estimated based on the 10-year registration numbers and 50% disease prevalence. aAbyssian (N = 179), Ocicat (N = 226), Somali (N = 134). bSiamese (N = 139), Balinese (N = 57), Oriental Shorthair (N = 227), Oriental Longhair (N = 31), Seychellios Shorthair (N = 12), Seychellios Longhair (N = 2). cSiberian (N = 332), Neva Masquerade (N = 12). dPersian (N = 334), Exotic (N = 44). eBurmese (N = 303), Burmilla (N = 47). fCymric (N = 41), Manx (N = 104). gKurilian Bobtail Shorthair (N = 1), Kurilian Bobtail Longhair (N = 14). hAmerican Curl Shorthair (N = 37), American Curl Longhair (N = 82). iNo sample size defined. The overall study population included more female (53%) than male cats (47%) (chi square p < 0.0001) (Data Sheet S1 in Supplementary Material). Also, there were more neutered (72%) than non-neutered cats (27%), and more alive (86%) than dead cats (12%) (p < 0.0001 between both subgroups). The mean age of the cats was 5.4 years. The ages of alive and dead cats (mean 4.9 and 9.3 years, respectively), female and male cats (mean 5.6 and 5.2 years, respectively), and neutered and non-neutered cats (mean 6.5 and 2.4 years, respectively) differed significantly (p < 0.0001 of Kruskal–Wallis test between each group). The proportions of alive and dead cats did not differ significantly in subgroups of male and female cats, but significantly more in male cats (78%) than female cats (67%), and living cats (77%) than dead cats (71%) were neutered (p < 0.001 between both subgroups) (data not shown). There was significant variation in the age distribution in different breeds (p < 0.0001 in both Kruskal–Wallis test for continuous ages and chi square test for age groups) (Figure 2 and Data Sheet S1 in Supplementary Material). For example, 30% of the Turkish vans, but less than 1% of the Sphynxes, belonged to the oldest age group of ≥11 years. The distributions of sex, alive/dead, and neutered/non-neutered cats varied considerably between breeds (p < 0.0001 for each). However, there was no significant difference in sex distributions (p = 0.1024), when Europeans and breed group of Burmese–Burmilla–Singapura were excluded, indicating that most of the variation originated from these two breeds. The largest proportion of deceased cats was among Turkish vans (28%), and the smallest – in the Siberians–Neva Masquerades group (2%). The highest proportion of neutered cats was found in non-pedigree cats (92%), and the lowest in the Persian–Exotic group (54%). Figure 2 Age distributions of all cats in all breeds. There were altogether 227 responses, of which either the age or the sex (or both) of the cat was missing. The neutering status was missing from 51 cats. In disease categories, in all other categories, 3–9 were missing values, except in “Other diseases,” where the number was 20. When the missing values of sex, age, and disease category (variables used in logistic regression models) were summed, the sample size requirements were still accomplished for all other breeds, except Cymric–Manxes. The same was true when excluding cats that had died prior to January 1st, 2005 (n = 146). In the behavioral part, information concerning “activity” was missing from 112, “contact with people” from 167, “aggressiveness toward family” from 108, “aggressiveness toward strange people” from 119, “aggressiveness toward other cats” from 207, “sensitivity toward new things” from 115, “sensitivity toward new people” from 113, and “licking” from 154 cats. The effect of missing values on the final results was not investigated, as the behavioral characterization was only preliminary and needs a more comprehensive study in the future. Prevalence of Disease Categories and Separate Diseases Prevalence of Disease Categories Altogether, 5415 cats (66%) were reported to have had a disease/diseases belonging to at least one of the 19 disease categories, including behavioral traits. The disease categories included diagnoses made either by the veterinarians or the owners (see Questionnaire in Presentation S1 in Supplementary Material). Diagnoses in the categories of “behavioral problems” and “parasites and protozoan” had mainly been made by the owner, while the diagnoses in all other disease categories had been mainly made by veterinarians (Figure 3). In the categories of “diseases of the urinary system,” “disorders of the cardiac and circulatory system,” “blood disorders,” “autoimmune diseases,” and “tumors,” almost all of the diagnoses had been made by the veterinarian. Figure 3 Proportions of disease categories diagnosed by the veterinarian or the owner. A total of 63% of pedigree cats had been diagnosed with a disease/diseases in at least one of the categories (diagnosed either by the veterinarian or the owner), whereas the percentage among non-pedigree cats was 78%. Among male and female cats, the percentages were 67 and 66%, respectively. The difference between pedigree and non-pedigree cats was significant (chi square p < 0.0001). However, when only veterinary diagnoses were considered, and diagnoses in the category of “parasites and protozoans” were excluded, the difference was non-significant (p = 0.0745), despite a modest overrepresentation of the non-pedigree cats (55%) compared with pedigree cats (52%). The difference between male and female cats was not significant (p = 0.3706); however, when diagnoses in the category of “Genital diseases” were excluded, significantly higher prevalence was found in male (66%) than female cats (59%) (p < 0.0001). The main prevalence results in disease categories diagnosed either by veterinarian or owner are presented below, and in the Data Sheet S2 in Supplementary Material, separately for diagnoses made only by veterinarian or owner (as well as for other subgroups). The most prevalent disease category among all cats was “Dental and oral diseases” (28%, Figure 4). It was the most common category in breeds and subgroups as well (Data Sheet S2 in Supplementary Material). Other prevalent categories included skin disorders (12%), the urinary system (12%), the digestive tract (11%), eyes, (10%), and the musculoskeletal system (10%). Genital diseases were also common in female pedigree cats (19%), but not as frequent in non-pedigree cats (5%) (chi square p < 0.0001). Figure 4 Prevalence of disease categories in all breeds and breed groups. Prevalence are colored with gradient filling from the lowest value to the highest (low = green, high = red) to point out the differences between the breeds. Phylogenetic grouping has been used in ordering the breeds. Age Prevalence increased by age in many disease categories. This was particularly apparent in the category of “dental and oral diseases,” but also distinctively in the urinary tract, the digestive tract, the skin, and tumors categories. In contrast, the prevalence of “congenital developmental disorders,” “blood disorders,” and “autoimmune diseases” was not affected by age, while some decrease was observed in the category of “other diseases.” The difference in the prevalence between the age groups of <1 year and ≥11 years was significant in all disease categories, except in “congenital developmental disorders” and “male genital diseases.” Sex, Alive/Dead, and Neutering In almost all the disease categories, male cats were overrepresented, as compared to female cats, deceased as compared to living cats, and neutered as compared to non-neutered cats. Between male and female, the differences were significant in the categories of “dental and oral diseases,” “genital diseases,” “diseases of the digestive tract,” “diseases of the urinary system,” “diseases of the respiratory tract,” “tumors,” “parasites and protozoans,” and “cardiac and circulatory system.” Of these categories, female cats were overrepresented only in the categories of “genital diseases” and “tumors.” Between alive and deceased cats, the differences were significant in all other categories, except in “female and male genital diseases,” “behavioral problems,” and “parasites and protozoans,” and between neutered and non-neutered cats, in all other categories, except in “other diseases” and “blood disorders.” In categories with significant difference, deceased cats had higher prevalence than living cats, and neutered cats – higher than non-neutered cats. Pedigree and Non-Pedigree Non-pedigree cats were overrepresented as compared to pedigree cats in most of the disease categories (Figure 5). They had significantly higher prevalence in “dermatological/glandular diseases,” “diseases of the digestive tract,” “diseases of the urinary system,” “behavioral problems,” “diseases of the respiratory tract,” “parasites and protozoans,” “otic diseases,” and “dental and oral diseases” as compared to pedigree cats. Pedigree cats had significantly higher prevalence only in the “genital diseases” and “disorders of the cardiac and circulatory system” categories. Figure 5 Bar chart of prevalence of disease categories among all cats, pedigree cats, and non-pedigree cats. Individual Breeds In separate breeds, like stated above, the most prevalent disease category in most breeds was “dental and oral diseases” with a minimum prevalence of 16% in Sphynxes and maximum – 40% in the breed group of Siamese–Balinese–Oriental–Somali. Non-pedigree cats had the highest prevalence in the category of “parasites and protozoans” (36%) (Figure 4). The most prominent breed-specific exceptions included high prevalence in “ocular diseases” (24%) and “female genital diseases” (32%) in Persian–Exotics, “respiratory tract diseases” in Korats (23%), “tumors” in Turkish vans (13%), and “parasites and protozoans” in Cornish Rexes and non-pedigree cats (12 and 36%, respectively). In addition, there was a relatively high prevalence of cardiac issues in Sphynxes (8%), urinary system disease in Abyssinian–Ocicat–Somali (21%), and musculoskeletal condition in Korats (16%), as well as a low prevalence of female genital diseases in non-pedigree cats (5%) compared with all the pedigree breeds (19%) (Figures 4 and 5). Prevalence of Diseases Dental calculus (21%) was the most prevalent disease among all cats and breeds, except in the age group of <1 year and in the breed group of Cymric–Manxes and the breed of Korats (Figure 6 and Data Sheet S3 in Supplementary Material). The prevalence between breeds varied from 12% in Devon Rexes to 28% in the breed group of Siamese–Balinese–Oriental–Seychellois. Common diseases for all cats and in the majority of the breeds, such as gingivitis (all cats 8%), repetitive vomiting (4%), tail kink (4%), feline odontoclastic resorption lesion (FORL) (4%), and urinary tract infection (4%), were also included. Among pedigree female cats, cesarean section (7% female) and stillborn kittens (6% female) belonged to the most prevalent issues throughout the breeds, whereas among the non-pedigree cats, the prevalence of genital disorders was comparably low. Figure 6 The 10 most prevalent diseases among all cats, pedigree cats, and non-pedigree cats. Age Examples of increasing prevalence from the youngest to the oldest age group were found in dental calculus (<1 year: 0%; ≥11 years: 43%), gingivitis (<1 year: 0%; ≥11 years: 13%), FORL (<1 year: 0%; ≥11 years: 11%), renal/kidney failure (<1 year: 0%; ≥11 years: 16%), urinary tract infection (<1 year: 1%; ≥11 years: 9%), urinary stones (<1 year: 0%; ≥11 years: 7%), and mammary tumor (<1 year: 0%; ≥11 years: 6%). Also, the same tendency was distinct in asthma (<1 year: 1%; ≥11 years: 6%). In all these diseases, the difference between the youngest and the oldest age groups was significant (chi square p < 0.0001). Alive and Dead In most diseases, the prevalence of deceased cats was higher than in alive cats, with the most prominent differences in FIP (alive 0% and dead 11%, chi square p < 0.0001) and renal/kidney failure (alive 1% and dead 14%, chi square p < 0.0001). Tumors were also overrepresented among deceased cats, particularly in the groups of other (unidentified) tumors (alive 0% and dead 9%, chi square p < 0.0001) and the mammary tumor (alive 0% and dead 7%, chi square p < 0.0001). Some tumors were represented among deceased cats only, such as the digestive tract tumor (dead 4% and alive n = 0) and liver tumor (dead 1% and alive n = 0). As noted above, higher prevalence of FIP was found in deceased than alive cats, but it was also overrepresented in non-neutered than neutered cats (3 vs. 1%, respectively), whereas in most of the other diseases, the order of prevalence between the alive and deceased cats as well as the neutered and the non-neutered cats was reversed. FIP was also the most prevalent disease among cats aged <1 year (5%). Disease Categories When separate disease categories were considered, the most prevalent diseases were moderate seborrhea (3%) in “dermatological/glandular diseases,” dental calculus and gingivitis (21 and 8%, respectively) in “dental and oral diseases,” renal failure and urinary tract infection (3 and 4%, respectively) in “diseases of the urinary system,” tail kink (4%) in “diseases of the musculoskeletal system,” repetitive vomiting (4%) in “diseases of the digestive tract,” asthma and respiratory infection (3% each) in “diseases of the respiratory tract,” cesarean section and stillborn kittens (6 and 5%, respectively) in the “female genital diseases,” undescended testis (2%) in the “male genital diseases,” and food allergy (3%) in the “endocrine and metabolic diseases.” Individual Breeds The most prevalent disorder among all breeds, except Korats and the breed group of Cymric–Manxes, was dental calculus (prevalence 21% for all cats). In turn, among Korats, the most prevalent disease was asthma (19%), and in breed group of Cymric–Manxes, variations in the length of the tail (entirely tailless 34%, stumpy 21%). Among the exceptionally high prevalence compared with other breeds, to mention some, were gingivitis in the breed group of Siamese–Balinese–Oriental–Seychellois (18%), sequester of cornea (5%) in the breed group of Persian–Exotic, renal failure (8%) in Korats, protozoans (11%) and HCM (6%) in Sphynxes, stillborn kittens (female 16%) in the breed group Burmese–Burmilla–Singapura, cesarean section (female 15%) in Birmans, different tumors in Turkish vans, and endo- and ectoparasites (23 and 18%, respectively) in non-pedigree cats. Phylogenetic Conditions Distinct phylogenetic connections of the breeds concerning association to the same disease were detected in the anal sac problem (Birman, Korat) (Data Sheet S4 in Supplementary Material), strabismus (Birman, Siamese–Balinese–Oriental–Seychellois), cesarean section (Birman, Burmese–Burmilla–Singapura, Siamese–Balinese–Oriental–Seychellois), and asthma (Korat, Siamese–Balinese–Oriental–Seychellois) (Figure 7). The prevalence of the diseases were higher in these breeds compared with other breeds (non-pedigree cats excluded). Also, the logistic regression analysis verified these breeds associated with the diseases. Figure 7 Prevalence of asthma in all breeds. Prevalence are colored with gradient filling from the lowest value to the highest (low = green, high = red) to point out the differences between the breeds. Phylogenetic grouping has been used in ordering the breeds. Breed-Specific Diseases Altogether, 78 diseases were considered tentatively breed-specific, according to disease prevalence and the number of diseased cats (Data Sheets S4 and S5 in Supplementary Material). Fisher’s exact tests’ p-values < 0.05 (Data Sheet S6 in Supplementary Material) indicated that in all 78 diseases, there were cat breeds, which were overrepresented compared with other pedigree cats, and in most of the diseases, also compared with non-pedigree cats. Reflux nephropathy (RN) was proven to be associated with Ragdolls in grounds of Fisher’s exact test p value < 0.001, and the fact that Ragdolls were the only breed which had been reported to have had the disease (Data Sheet S4G in Supplementary Material). Of the remaining 77 diseases, altogether 57 diseases were verified to be breed-specific by logistic regression analysis with variables “Age,” “Sex,” and “Alive/dead” included (Data Sheet S7A in Supplementary Material). Also, the variable “Neutered/non-neutered” was considered in the model, but omitted because of a modifying effect with the variable “Age,” and because the exact timing of the neutering with respect to the occurrence of the diseases was not known. Genetic mutation had been already revealed in 6 of the 57 diseases (Data Sheet S7B in Supplementary Material), 32 diseases were suggested as breed-specific conditions (Data Sheet S7C in Supplementary Material), 18 diseases were newly identified as likely breed-specific conditions (Data Sheet S7D in Supplementary Material), and for 21 diseases, breed specificity could not be verified, even though some of them had been proposed to be breed-specific in previous studies (Data Sheet S7E in Supplementary Material). The diseases with known mutations included polycystic kidney disease (PKD), progressive retinal atrophy (PRA), HCM, and three types of malformations of tail (Table S7B in Supplementary Material). In all these diseases, the high values of MOR and evidence ratio indicated a distinct association of the breed to the disease. For example, in PKD, both the MOR (13.8 when non-pedigree cat excluded) and the evidence ratio (>103) were very high. From the diseases with known mutations, we found the following disease–breed associations in our study: Persian to PKD, Siamese–Balinese–Oriental–Seychellois and Abyssinian–Ocicat–Somal to PRA, Sphynx and British to HCM, and Cymric–Manx to malformations of the tail. The breeds associated with these diseases in our study were the same as in which the gene mutation was found in all other diseases except in HCM. In 18 of the 32 diseases with previously suggested breed-specific conditions, at least one of the breeds found prone in our study was also proven prone in previous studies, but in 14 diseases, breeds in our study and in previous studies differed. The strongest breed-specific associations, according to the MOR and evidence ratios, were found in entropion, sequester of the cornea (Figure 8), hip dysplasia, feline eosinophilic granuloma complex, strabismus (alignment problem of eyes), fungal skin disease, asthma, and malocclusion (teeth alignment problem). All these diseases had MOR over 2.5 and the evidence ratio of over 103. Despite the low value of six of the evidence ratios, restrictive cardiomyopathy (RCM) was considered breed-specific on the grounds of high values of MOR (5.9 non-pedigree cats excluded and 3.9 non-pedigree cats included) and prevalence (Data Sheet S4H in Supplementary Material), which inevitably associated RCM to Cornish Rexes. The low value of the evidence ratio was due to only few cases (n = 9) of the disease altogether. Figure 8 Prevalence of sequester of the cornea in all breeds. Prevalence are colored with gradient filling from the lowest value to the highest (low = green, high = red) to point out the differences between the breeds. Phylogenetic grouping has been used in ordering the breeds. Of the diseases, we found breed-specific for the first time in this study, the best evidence was found for hooked sternum, oligodontia, periodontitis, traumas to the skin, and repetitive constipation with MOR > 2 and the evidence ratio of over 103. Also twisted legs, feline herpesvirus infection (FHV), and ovarian cysts (female) were considered strongly breed-specific, according to high values of MOR (5.4, 3.6, and 2.5, respectively, when non-pedigree cats excluded) and the prevalence, which associated twisted legs to the breed group of Abyssinian–Ocicat–Somali, FHV to Sphynxes and Cornish Rexes, and the breed group of Persian–Exotic to ovarian cysts (Data Sheets S4J,T,N in Supplementary Material, respectively). We found, altogether, 20 diseases without evidence for breed specificity due to the low values of evidence ratios, indicating that the variable “Breed” did not bring any additional information into the logistic regression models. According to MOR values, some heterogeneity between breeds was observed, but the variation was not large enough to conclude breed specificity. The OR of breeds proven overrepresented in our study and the references to the previous studies are summarized in Data Sheet S7 in Supplementary Material. Models 1–3 are presented without interactions included, as the interactions did not have a prominent effect on the critical values of our study (MOR, evidence ratio, OR of the breeds) in any of the diseases. Omitting the interactions was also found reasonable because the interaction should have been added in all three models, even in those in which it was not significant, to keep the comparability in counting the evidence ratio (17). Further checking, concerning breeds containing several cats that died before January 1st, 2005 or in which the responses were associated (>15%) with the one breeder, revealed that in fungal skin diseases, most of the cats in the breed group of Cymric–Manx were both associated with the same breeder and had died before January 1st, 2005. No other notable effects caused by cats that died before January 1st, 2005 were found, but other diseases in which one breeder had had an effect to the disease prevalence were detected (see Data Sheet S7A in Supplementary Material). Behavioral Abnormalities We found considerable variation in the behavioral traits between the breeds (Data Sheet S4F in Supplementary Material). For example, 10% of British, Ragdolls, and non-pedigree cats were reported with low activity (not at all or little), whereas the corresponding percentage among all pedigree cats was 5%, and in Bengals – 1%. The British were also reported to have less contact (9% not at all or a little) with people than all cats on average (4%). Turkish vans and Bengals, in turn, were more aggressive toward other cats (15 and 9% aggressive often or very often, respectively) than cats on average (5%). Turkish vans were also reported to have more aggressiveness toward unfamiliar people (6% aggressive often or very often) than cats in all the other breeds (<3% in each). Turkish vans, Russian blues, Bengals, Siamese–Balinese–Oriental–Seychellois, and non-pedigree cats indicated high proportions in sensitivity toward new things (>3% sensitive very often in each breed) as well as toward new people (>4% sensitive very often in each breed). In all pedigree cats, the corresponding proportion was 2% in both traits. Licking was common in many breeds with the highest proportions in non-pedigree cats and in the breed group of Burmese–Burmilla–Singapura (often or very often 8% in both). In the logistic regression models, breed was an essential variable in the models in terms of evidence ratio (>103) (Data Sheet S7F in Supplementary Material) in all other behavioral traits, except in “aggressiveness toward family” and “contact with people.” In “contact with people” the value of evidence ratio (60) indicated a still relatively important role for breed in the model, but in “aggressiveness toward family,” a lower value of AIC in the model without the breed indicated that the model without a variable breed was better than with the breed included. The highest MOR was in “aggressiveness toward strangers” with the Turkish van as being the most aggressive breed. Discussion The implementation of genetic research requires knowledge of breed-specific health issues to decide the best strategies for patient recruitment. Our feline health survey in a population of over 8000 cats in Finland provides a comprehensive overview of the health profiles and not only forms a strong foundation for genetic research but also gives useful information to breeders, cat fanciers, and veterinarians. This study covered 31 breeds and a large non-pedigree cat population as an appropriate reference group to pedigree cats. The living conditions of non-pedigree cats, such as environment, outdoor activities, and treatment, closely resembled those of pedigree cats (data not shown). In addition, the high percentage of non-neutered cats suggests that the owners are equally conscious as the owners of pedigree cats. The study explored the prevalence in altogether 227 separate diseases identifying the common health issues in cats and 57 breed-specific conditions as well, that can be targeted for genetic research. Disease Prevalence and Common Diseases The most common disease categories and individual diseases found in our study have also been described in earlier studies. Dental diseases were the most prevalent category in the UK study (20), and dental calculus and gingivitis the most prevalent diseases in a US study (21). Dermatological diseases were among the most prevalent disease categories in UK, Japanese, and Swedish studies (20, 22, 23). Furthermore, diseases of the urinary system and digestive tract (22, 23) and ophthalmological diseases (22) are common categories. The list of common individual diseases include dental calculus and gingivitis, parasites (21), vomiting (20, 21, 23), urinary tract infection (20), kidney disorders (20, 21), urinary stones (23), diarrhea (20, 21), and cystitis (21, 23). Although many of these were found in our study, there are also differences that can be explained by the study design (population vs. veterinary data) and size, geographical region, disease definitions, and data source. For example, the data in the US (n = 14 270) (21) and UK (n = 3584) (20) studies were based on veterinary clinical data, while the data from Sweden (n = 301 485) (23) and Japan (n = 49 450) (22) comes from an insurance database; ours is population data. Higher morbidity of male cats to common diseases (in early age) has been found in Sweden (23). The higher morbidity of male cats was rationalized by anatomical (urinary system) and behavioral (causing traumas) differences between male and female cats. The prevalence in the category of “diseases of the urinary system” was also higher for male cats in our study. Traumas were not treated like a disease category in our study, as in Swedish study, but male cats had significantly more traumas to the skin (chi square p = 0.0066) than female cats. However, omitting the diagnoses of urinary tract diseases and skin traumas, we found a higher morbidity rate in males. This is due to the fact that male cats were also overrepresented in a majority of the other disease categories. In the Japanese and Swedish studies (22, 23), domestic and cross-bred cats had been stated to be more prone to accidents, and the assumed cause was different access to outdoors. In our study, the most overrepresented breed in skin traumas was non-pedigree cats, which roamed outside more than pedigree cats (data not shown), but also, breed groups of Siamese–Balinese–Oriental–Seychellois and Abyssinian–Ocicat–Somali and Norwegian Forest cats were overrepresented, as well. The different outdoor activities did not explain their higher prevalence of traumas. The more frequent outside roaming was, instead, the most probable cause of higher prevalence of parasites among non-pedigree cats compared to pedigree cats. Our results suggest that non-pedigree cats have more diseases than pedigree cats with a higher percentage of non-pedigree cats having diseases at least in one of the disease categories (chi square p < 0.0001). However, when only veterinary diagnoses were considered, and the disease category of “Parasites” was omitted, the difference was not significant anymore (p = 0.0745), even though non-pedigreed cats were still overrepresented compared with pedigree cats. A potential explanation could relate to the differences in the selection of the reported cats between the owners of pedigree and non-pedigree cats (see limitation below). UK and Japan studies also found equal prevalence of disease in pedigree and non-pedigree populations (20, 22). In many diseases and disease categories, disease prevalence increased with age up to ≥11 years. The increase was especially strong in disease categories of “dental and oral diseases,” “diseases of the urinary system,” and “tumors.” A similar increase in these same disease categories was reported previously in Japan and Sweden (22, 23), although our study found more age-related disease categories. This is because we asked to report the health history to cover the earlier stages of life and not only at the time of reporting. For the same reason, the peak in the youngest age group, which was found in many disease categories in the Japan and Sweden studies (22, 23), remained uncovered in our study. For example, we found that the prevalence of the disease category of “parasites and protozoans” increased cumulatively throughout the age groups, due to inclusion of cured infestations from the earlier years of age (as confirmed from the additional information of the questionnaire), whereas in Japan and Sweden (22, 23), the prevalence of the “parasites and protozoans” category was high only in the first age group. The disease prevalence in most of the disease categories and diseases were higher in deceased than living cats, and neutered than non-neutered cats. An interesting exception was the prevalence of feline infectious peritonitis (FIP), which was higher among living and non-neutered cats. FIP was also the most prevalent disease in age group of <1 year. The reason why FIP was prevalent in young and deceased cats results from the clinical picture of FIP as an infectious disease of young cats with high mortality (24). The high prevalence among non-neutered cats was due to the age distribution of non-neutered cats with 73% of them being aged <3 years. Phylogenetic association to a disease, which was detected in the anal sac problem, strabismus, HCM, cesarean section, and asthma, may imply inheritance of the genetic predisposition via a common ancestor of the phylogenetic group of cats. Previous studies suggest the same to strabismus (25) and cesarean section (26). In both diseases, the phylogenetically associated breeds in our study, i.e., Birman and Siamese–Balinese–Oriental–Seychellois group, were also found to be associated in the previous studies. Instead, the anal sac problem lacks associations to any breed. Only Anal sac gland carcinoma (ASGC) was found associated with the Siamese breed (27), whereas the breeds found associated with the anal sac problems in our study were Birman and Korat. However, Siamese, Birman, and Korat breeds belong to the same eastern-derived population (14), which might indicate an inherited common predisposition to problems in anal sacs. Asthma was found phylogenetically associated with Korats and the Siamese–Balinese–Oriental–Seychellois group in our study. It has been associated with the Siamese breed in many previous studies also (28–30). No study was found, in which asthma would have been connected to Korats, but association was found to Birmans (31). A common predisposition to asthma by the breeds belonging to the same eastern-derived population could be considered. Breed-Specific Disorders One of our key objectives was to identify potential breed-specific diseases for genetic studies. Breed specificity in diseases often implies heredity (3), and, thus, breed-specific diseases are appropriate candidates to be considered taking into genetic research. We found 58 such diseases, including RN, sequester of cornea, entropion, feline eosinophilic granuloma complex, periodontitis, oligodontia, urinary tract infection, HCM (Sphynxes and British), RCM, hip dysplasia, hooked sternum, asthma, and stillborn kittens. HCM mutation has been found in Ragdolls and Maine Coons (9, 10), but the Sphynxes and British’, which were found overrepresented in our study, have also been associated with HCM in previous studies (32–34). RN was found only in Ragdolls. Of the other diseases mentioned, at least one of the breeds found overrepresented in our study had also been found overrepresented in previous studies, including sequester of cornea (35, 36), entropion (37, 38), asthma (28–30), strabismus (39, 40), and hip dysplasia (41). RCM had been found overrepresented in non-pedigree shorthairs. As potential new breed-specific conditions, we found feline eosinophilic granuloma complex, periodontitis, oligodontia, urinary tract infection, hooked sternum, and stillborn kittens. However, further studies concerning diseases suggested to be breed-specific in our study must be conducted before proceeding in genetic research. It is essential to also map other contributing factors concerning lifestyle of the cat, such as environment, diet, and outdoor activities, as well as the differences between the breeders and possible cross effects between different diseases. For example, entropion and herpes virus in the eye have been proposed as contributing factors in sequester of cornea (42). Also, our data confirmed the association between sequester and both of these diseases (Fishers’ exact test p < 0.001 for both when cats aged <1 year excluded), but the major part of the sequester cases did not have either of these associations, also allowing other possible triggers for sequester as well as for entropion. Also, nystagmus and malocclusion, which were found to be breed-specific in our study, have been proposed to be symptoms of other diseases or physical features rather than independent disorders (35, 38, 43). Ongoing studies will investigate the possible associations of above-mentioned factors to common and breed-specific disease predisposition. In diseases in which no breed specificity was detected in our study, digestive tract tumor (44), birthing complications (26, 45), FIP (46–48), and pyometra (49) were found to be breed-specific in previous studies. The reason for this was the low variation of prevalence between the breeds, which might be due to underreporting in certain breeds, diagnostic problems, or different environmental factors compared with the other studies. Behavioral Variation In the preliminary analysis of behavioral traits, most divergent behavior was found in British, Ragdolls, Turkish Vans, Korats, and Bengals. Both British and Ragdolls (along with non-pedigree cats) were the most inactive breeds, and, in addition, British had less contact with people than cats on average (Data Sheets S4 and S7 in Supplementary Material). Ragdolls have been described as less active earlier (50). Turkish Vans were distinctively the most aggressive breed toward strangers and other cats. Korats also showed aggression toward family members and unfamiliar people, and Bengals – toward other cats. This finding in Bengals has been observed before (50). We also found variation in sensitivity and licking in few breeds. Overall, these results encourage a more comprehensive future behavioral study in cats to establish proper genetic study cohorts and to identify environmental contributors. Limitations Health surveys targeted to pet breeders and owners have limitations. One of the key concerns is related to the responder distribution in the population since the cat owners were reached by information channels which might have favored breeders and other cat fanciers over owners who own cats as pets but are not really cat fanciers. This may have resulted in non-response error by some cat owners not participating in the study and in measurement error by some responders giving inaccurate or incomplete information (51). Suspicions like these have been also expressed in meetings with the cat breeders and owners during the study to encourage reports from both healthy and affected cats. Inaccurate and incomplete information may have been caused by misunderstandings or missing some diagnoses leading to under- or overestimates of prevalence, especially if the owner had made the diagnosis. Therefore, we have reported prevalence data in various diagnostic groups to allow comparisons. The third limitation may relate to the scarcity of Finnish breeders or the large proportion of breeders not participating in some breeds, which may have resulted in a concentration of responses only from a few breeders. This might have led to bias, especially in infectious diseases, making a local epidemic seem like a high prevalence in the whole breed. These kinds of effects were sought during the study, and if signs of such concentration were detected, they were marked in the Table S7 in Supplementary Material. It is important to also keep in mind that Finnish cat breeds may differ genetically from breeds in other countries because of differences in breeding practices, which can create distinct lines within a single breed (4). This may have caused genetic concentration or dilatation in Finnish breeds, yielding deviant prevalence. Also, the design of our study, with disease occurrences including earlier states of life in addition to the time of submitting the questionnaire, gives ground to presume prevalence in our study to be higher than in prevalence studies in which only occurrences of a certain point of time are accepted. In addition, it is appropriate to point out that logistic regression yields overestimates of OR when the prevalence of the disease is greater than 5% (15). However, in our study, logistic regression was targeted to designate the effect of the whole variable “Breed” by means of MOR, and the yielded OT in case of prevalence >5% in Model 3 may be considered suggestive of the approximate magnitude of the effects of separate breeds, not the exact value of odds ratio. We also recognize that “food allergies” was suboptimally placed in the category of “endocrine and metabolic diseases” instead of the categories of digestive tract diseases. This may have caused overestimates in the prevalence of the category of endocrine and metabolic diseases. However, it is more important to pay attention to the prevalence of specific conditions rather than broader categories. Despite all suggested reasons causing possible bias, prevalence in our study match well with the results stated in earlier studies and are underestimates rather than overestimates in case of disparity giving conservative inferences (20, 21). It is more likely that some breed-specific diseases have been missed rather than breed specificity has been suspected in vain. Conclusion This unique study gives valuable prevalence information of 227 feline diseases in Finnish cats and identifies nearly 60 breed-specific disorders that can be targeted for genetic characterization. Compared to previous studies, we provide the broadest countrywide health survey available so far with designed breed-specific sample size requirements. The study established a health survey platform and database, which has value not only for genetic research programs but also provides for veterinary medicine and breeding programs. Author Contributions HL conceived and designed the study with KV and A-MV. KV designed and performed statistical analyses with the help from A-MV. HL, KV, TJ, A-MV, KT, and JT contributed to the development of the questionnaire and material collection. KV and HL drafted the manuscript with the help from others. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We are grateful to all the owners and breeders who participated in the health survey to make this study possible. Carin Sahlberg is thanked for insights about Finnish cats to develop the research strategy and Annukka Salo is thanked for the help with the development and management of the questionnaire data. This study was partially funded by the Finnish Cat Association, the Jane and Aatos Erkko Foundation, and Biocentrum Helsinki. 1http://www.cfa.org/ 2http://www.tica.org/ 3http://www.kissaliitto.fi/ 4www.kissangeenit.fi Supplementary Material The Supplementary Material for this article can be found online at http://journal.frontiersin.org/article/10.3389/fvets.2016.00070 Click here for additional data file. Click here for additional data file. Click here for additional data file. 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==== Front Front PsychiatryFront PsychiatryFront. PsychiatryFrontiers in Psychiatry1664-0640Frontiers Media S.A. 10.3389/fpsyt.2016.00148PsychiatryEditorialEditorial: The Impact of Active and Passive Smoking upon Health and Neurocognitive Function Heffernan Tom 1*1Department of Psychology, Northumbria University, Newcastle upon Tyne, UKEdited and Reviewed by: Alain Dervaux, Centre Hospitalier Sainte-Anne, France *Correspondence: Tom Heffernan, tom.heffernan@northumbria.ac.ukSpecialty section: This article was submitted to Addictive Disorders, a section of the journal Frontiers in Psychiatry 29 8 2016 2016 7 14825 7 2016 15 8 2016 Copyright © 2016 Heffernan.2016HeffernanThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.active smokingpassive smoke exposurehealthneurocognitive functionsmoking cessation ==== Body The Editorial on the Research Topic The Impact of Active and Passive Smoking upon Health and Neurocognitive Function Tobacco smoking is a major risk factor for a number of chronic diseases, including a variety of cancers, lung disease, and damage to the cardiovascular system. The World Health Organization recently calculated that there were six million smoking-attributable deaths per year and that this number is due to rise to about eight million per year by the end of 2030. Recent work has demonstrated that habitual smoking in adults is associated with a range of health conditions, including cardiovascular disease, pulmonary dysfunction, and an increased risk of a variety of cancers. In terms of neurocognitive function, although some studies have found that acute smoking can enhance cognitive functions in the short term, actually chronic smoking is deleterious in the long term. Chronic smoking has been associated with reductions in working memory (the temporary storage and manipulation of information), executive function (planning tasks, focusing ones attention, and ignoring irrelevant distractions), and prospective memory (memory for everyday things, such as keeping an appointment, or taking an important medication on time). More recently, the focus on smoking-related health problems and neurocognitive deficits has expanded to include the study of “second-hand smoking” (also known as “passive smoking” – wherein a person who does not smoke him/herself inhales tobacco smoke either via side-stream smoke or via smoke being blown directly into his/her face). Research in this area has linked exposure to second-hand smoke in those who have never smoked to a range of health problems akin to smokers, including lung and cardiovascular disease, as well as deficits in neurocognitive function. In terms of neurocognitive function, exposure to second-hand smoke has been linked with an increased risk of mild cognitive impairments in older adults, reductions in working memory, as well as deficits in executive function. Interventions aimed at reducing cigarette consumption and improving the health of both smokers and those exposed to second-hand smoke continue to be developed. The aim of this Frontiers Research Topic is to bring together a collection of papers that look at what impact active and passive smoking has upon health and neurocognitive function; as well as to consider some of the wide variety of interventions aimed at reducing cigarette use and/or improving health. Copeland examined pre-treatment characteristics among daily smokers (including smoking patterns, smoking outcome expectancies, and smoking-related health information) and how these related to success on a brief motivational enhancement intervention. Marshall et al. explored whether the combined (polydrug) effect of consuming excessive amounts of alcohol and smoking cigarettes exacerbated everyday memory problems when compared with the sum of their independent effects (excessive drinking alone, or smoking alone). Philibert et al. examined whether aryl hydrocarbon receptor repressor (AHRR) can be used to determine whether AHRR methylation status is a quantifiable biomarker for progress in smoking cessation that could have substantial impact on both smoking cessation treatment and research. Ling and Heffernan reviewed evidence in relation to the cognitive consequences of exposure to second-hand smoke in those who had no history of smoking. Payne et al. evaluated chronic obstructive pulmonary disease-related health factors in flight attendants exposed to second-hand cigarette smoke and assessed whether meditative movement was effective as a treatment in improving pulmonary function in these flight attendants. Jukosky et al. demonstrated how cigarette exposure alters the innate immune response and increases an individual’s susceptibility to pathogen infection when compared with non-exposed individuals. Jovanovic and Jakovljevic discuss regulatory control of e-cigarette composition and raises concern regarding the quality control and health outcomes surrounding e-cigarettes. The commentary by Parrott discusses concerns about the paradoxical nature of using e-cigarettes; whether they may in fact be damaging to physical/psychological health of the users, as well as raising concerns about what impact e-cigarettes have upon those who are “passively vaping.” Lasebikan and Ola assessed the efficacy of screening, brief intervention, and referral for treatment package to reduce tobacco smoking in two semi-rural community settings in South-West Nigeria. Overall, the papers presented in this Frontiers in Psychiatry special topic demonstrates the broad nature of research currently being undertaken in relation to active and passive smoking and some of the current issues surrounding the use of e-cigarettes as nicotine-replacement therapy. The research cited here should pave the way for further work in this area. Areas for future research include the concern of what impact exposure to second-hand smoke might be having upon children’s health, neurocognitive function, and educational achievement, an area of particular importance given the recent estimates from the World Health Organization that approximately 40% of children across the world are regularly exposed to second-hand smoke in the home. A further area that has received very little attention at all is whether exposure to “third-hand smoke” (the residue of nicotine and other chemicals left on indoor surfaces as a result of tobacco) smoking has a detrimental impact upon those who have never smoked, both in terms of health and neurocognitive function. Author Contributions This is the sole work of the author and editor for this Special Topic. Conflict of Interest Statement The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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==== Front Front ImmunolFront ImmunolFront. Immunol.Frontiers in Immunology1664-3224Frontiers Media S.A. 10.3389/fimmu.2016.00330ImmunologyReviewHematopoietic Stem Cell Regulation by Type I and II Interferons in the Pathogenesis of Acquired Aplastic Anemia Smith Julianne N. P. 1Kanwar Vikramjit S. 2MacNamara Katherine C. 1*1Department of Immunology and Microbial Disease, Albany Medical College, Albany, NY, USA2Department of Pediatrics, Division of Pediatric Hematology-Oncology, Albany Medical Center, Albany, NY, USAEdited by: Laura Schuettpelz, Washington University School of Medicine in St. Louis, USA Reviewed by: Martijn Nolte, Sanquin, Netherlands; Megan Tierney Baldridge, Washington University School of Medicine in St. Louis, USA *Correspondence: Katherine C. MacNamara, macnamk@mail.amc.eduSpecialty section: This article was submitted to Inflammation, a section of the journal Frontiers in Immunology 29 8 2016 2016 7 33030 6 2016 17 8 2016 Copyright © 2016 Smith, Kanwar and MacNamara.2016Smith, Kanwar and MacNamaraThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Aplastic anemia (AA) occurs when the bone marrow fails to support production of all three lineages of blood cells, which are necessary for tissue oxygenation, infection control, and hemostasis. The etiology of acquired AA is elusive in the vast majority of cases but involves exhaustion of hematopoietic stem cells (HSC), which are usually present in the bone marrow in a dormant state, and are responsible for lifelong production of all cells within the hematopoietic system. This destruction is immune mediated and the role of interferons remains incompletely characterized. Interferon gamma (IFNγ) has been associated with AA and type I IFNs (alpha and beta) are well documented to cause bone marrow aplasia during viral infection. In models of infection and inflammation, IFNγ activates HSCs to differentiate and impairs their ability to self-renew, ultimately leading to HSC exhaustion. Recent evidence demonstrating that IFNγ also impacts the HSC microenvironment or niche, raises new questions regarding how IFNγ impairs HSC function in AA. Immune activation can also elicit type I interferons, which may exert effects both distinct from and overlapping with IFNγ on HSCs. IFNα/β increase HSC proliferation in models of sterile inflammation induced by polyinosinic:polycytidylic acid and lead to BM aplasia during viral infection. Moreover, patients being treated with IFNα exhibit cytopenias, in part due to BM suppression. Herein, we review the current understanding of how interferons contribute to the pathogenesis of acquired AA, and we explore additional potential mechanisms by which interferons directly and indirectly impair HSCs. A comprehensive understanding of how interferons impact hematopoiesis is necessary in order to identify novel therapeutic approaches for treating AA patients. hematopoietic stem cellsinterferon-gammainterferon type Iaplastic anemiabone marrow microenvironmentmacrophagesT lymphocytesNational Institute of General Medical Sciences10.13039/100000057R01GM105949-03 ==== Body Introduction The concept of aplastic anemia (AA) was first introduced by Paul Ehrlich in 1888 and describes patients who fail to form blood cells from all three lineages, in association with decreased or absent bone marrow precursor cells. Although there are many known etiologies, the cause of AA is generally difficult to determine in an individual patient and in the vast majority of cases no causal etiology is found (1). The focus of the current review is on the role of interferons in the pathophysiology of this bone marrow failure (BMF) syndrome. The association of disease with expansion of autoreactive T lymphocytes (2, 3) and responsiveness of disease to immunosuppressive therapies, including antithymocyte globulin (ATG) and cyclosporine (4), demonstrate the immune-mediated nature of acquired AA. Although the precise cause of acquired AA is unknown, links to radiation, chemical exposure, and infection have been made. Gene polymorphisms that alter cytokine production or stability, particularly interferon gamma [IFNγ; Ref. (5)] provide additional evidence that dysregulated inflammatory responses are an essential driving force in the BMF seen in acquired AA. Mechanisms underlying the loss of hematopoietic stem cells (HSCs) during BMF include increased apoptosis and enhanced stem cell activity resulting in exhaustion. Here, we focus on the role(s) of interferons in the pathogenesis of BMF, and highlight new questions and avenues of research that may reveal therapies for targeted treatment of acquired BMF. Regulation of HSC Function: Intrinsic and Niche-Mediated Mechanisms Quiescence preserves the self-renewal capacity and, therefore, the long-term function of HSCs. The regulators of this dormant state include intrinsic pathways as well as soluble and contact-dependent factors present in the niche microenvironment [reviewed in Ref. (6)]. Dysregulated HSC cycling may contribute to AA by enhancing differentiation over self-renewal or by sensitizing HSCs to apoptosis (7–10). Interferons have been implicated in both driving proliferation (11) and impairing proliferation of primitive hematopoietic stem and progenitor cells (HSPCs) (12), and sensitizing cells to apoptosis (13), thus supporting the notion that IFNs directly impair hematopoiesis by compromising stem cell function. An altered microenvironment may also contribute to the pathogenesis of AA. Analysis of BM mesenchymal stromal cells (MSCs) derived from AA patients revealed reduced proliferative capacity and adherence, and a propensity to differentiate into adipocytes at the expense of osteoblasts (OBs) (14, 15). Considering the essential survival and dormancy-enforcing cues provided by niche cells, it will be important to investigate more fully the defects in stromal cells in acquired AA, and the impact of IFNs, either directly or indirectly, on such cells. Interferons in Acquired AA The observation that patients with acquired AA exhibit increased levels of circulating IFNγ was made over 30 years ago (16). The presence of T cells containing intracellular IFNγ and positive for the prototypical Th1 transcription factor Tbet is an indicator of disease (17), and reduced frequencies of IFNγ positive T cells correlates with responsiveness to immunosuppressive therapy (4), suggesting that Th1 cells contribute to disease pathogenesis. Attempts to understand how IFNγ-mediated disease pathogenesis revealed that overexpression of IFNγ in vitro impairs long-term culture initiating cells LT-CIC (18), consistent with observations that neutralizing IFNγ in cultures derived from AA patients resulted in improved colony formation (16). Moreover, a polymorphism that results in enhanced stability of IFNγ transcripts is strongly associated with the risk of developing AA (5). However, the precise mechanisms whereby IFNγ drives BMF in vivo are still unclear and may involve multiple overlapping pathways and multiple cell types. Type I IFNs (IFNα/β) are key regulators of innate and adaptive immunity. Although not directly implicated in AA pathogenesis, type I IFNs mediate host responses to most infections and contribute to autoimmunity in systemic lupus erythematosus [recently reviewed in Ref. (19)] and potentially in diabetes mellitus, Sjogren’s syndrome, autoimmune myositis, and rheumatoid arthritis (20, 21). Pegylated IFNα 2a (PEG-IFNα2a) is the standard of care in hepatitis C virus (HCV) patients, but is also a treatment option for melanoma (22), hairy cell leukemia (23), and multiple sclerosis (24–26). Type I IFN therapy is not well tolerated by all patients, however, and hematologic side effects are closely monitored. HCV patients receiving both PEG-IFNα2a and the nucleoside analog ribavirin are prone to hemolytic anemia due to ribavirin processing in erythrocytes as well as PEG-IFNα2a-mediated BM suppression (27, 28). Rarely, type I IFN therapies have also been linked to persistent BM suppression and the development of AA (24, 29, 30). BM suppression appears not to require exogenous or supraphysiologic levels of IFNα/β, as anemia and BM failure have also been associated with physiologic type I IFN responses to chronic viral infection (31). Of particular relevance to AA, the impact of type I IFNs on hematopoiesis is often not immediately suppressive, but requires secondary stress, such as exposure to subsequent IFNγ during the pathogenesis of lymphocytic choriomeningitis virus (LCMV) infection (12). Herein, we will discuss the potential for direct and niche-mediated type I IFN stimulation to impair HSCs and contribute to acquired AA. Bone Marrow Failure Induced by Infection Bone marrow suppression has been observed subsequent to a number of viral infections, including parvovirus (32, 33), human immunodeficiency virus [HIV; Ref. (34)], viral hepatitis (35), Epstein–Barr virus (36), and influenza (37), among others. The ability of viral infections to suppress the BM may be due to both the ability of viruses to actively infect cells of the hematopoietic system and the host response to the virus, likely involving production of interferons and other pro-inflammatory factors. BM suppression and severe cytopenias are also common after exposure to tick bites, and are associated with the rickettsial pathogens Ehrlichia chaffeensis and Anaplasma phagocytophilum (38). Though transient, cytopenias are often severe, and infection requires antibiotic treatment (39). Human monocytic ehrlichiosis has been associated with bone marrow hypoplasia (40) and hemophagocytic lymphohistiocytosis [HLH; (41)], and murine models implicate interferon responses in mediating bone marrow suppression in rickettsial infections (42–44). Models to Study Human AA Bone marrow failure pathogenesis was first modeled in mice using exposure to toxins, instigated by the association of benzene exposure with human disease (45). Observation that AA is a result of immune-mediated pathology prompted the development of donor lymphocyte infusion models relying on the adoptive transfer of lymph node or spleen-derived lymphocytes from histocompatibility mismatched strains of mice (46). This model recapitulates many observations in human AA patients as protection can be achieved with immunosuppressive treatment and abrogation of IFNγ (47, 48). A technical hurdle of the infusion-based model is that the use of F1 recipients precludes analysis of genetically targeted mice. Thus, it has been difficult to evaluate direct and indirect roles of specific cytokines on hematopoietic versus stromal cells. However, it has allowed a deeper understanding of T cell intrinsic mechanisms necessary for initiation of disease, including Notch signaling (49) and transcriptional regulators of Tbet (50). To model human patients carrying a mutation that renders a higher risk for developing AA, a mutation was introduced to the 3′ untranslated region of the Ifng gene, stabilizing IFNγ transcripts (51). Termed “ARE-delete,” this mouse model reproduces many features of human disease and is not associated with autoreactive T cells, suggesting that elevated IFNγ, independent of activated T cells, can drive disease by impairing progenitor cell function (51). In addition, a number of insights into bone marrow suppression have come from studying bacterial and viral pathogens. In ehrlichiosis, HSC loss requires IFNγ sensing by macrophages, demonstrating that interferon signaling reduces the HSC supportive capacity of niche cells during infection-induced BM suppression (42). In LCMV, phenotypic HSCs are reduced early in the course of infection, independent of IFNγ and likely through the actions of type I IFNs (12, 31). Together, the observations made in murine infection models and in a subset of patients undergoing PEG-IFNα2a treatment provide additional evidence that interferons impair HSCs, likely via multiple direct and niche-mediated mechanisms. Mechanisms of IFNγ-Mediated AA HSC-Intrinsic Impact of IFNγ The negative impact of IFNγ on hematopoiesis is well documented [reviewed in Ref. (52)], but what is the evidence that there is a direct impact of IFNγ on the most primitive HSCs? HSC loss can occur via impaired self-renewal, increased differentiation, or induction of cell death, which may be results of both direct and/or indirect effects of IFNγ. While some studies suggest that IFNγ has an antiproliferative effect on HSCs (12, 53), evidence also suggests that IFNγ signaling promotes proliferation, and subsequent exhaustion of HSCs (11, 54). During infection with Mycobacterium avium or LCMV, IFNγ increased HSC proliferation and led to a reduction in transplantable myeloid potential (11, 55). Moreover, HSCs from a microenvironment deficient in IFNγ have more robust long-term potential, whereas excessive IFNγ signaling reduces transplantable HSPC activity (54, 56), further suggesting that tonic IFNγ signaling limits HSC function, perhaps through inducing proliferation. The discordant results with respect to whether IFNγ induces or suppresses proliferation is further confounded by the complex interaction with other cytokines, as IFNγ can augment the expansion of myelogenous leukemia cells when it signals in concert with IL-3, but can suppress proliferation in cells lacking IL-3 stimulation (57). In addition, TNFα stimulation is necessary for maximal IFNγ-induced suppression and proliferation of leukemia cell cultures (57), further emphasizing the potential for IFNγ to elicit distinct and even opposing effects dependent on the local cytokine milieu. Stem cell proliferation can result in the generation of more stem cells (self-renewal) or more committed progenitors (differentiation) and IFNγ has also been implicated in impeding self-renewing divisions (Figure 1a) (12, 58). Notably, IFNγ was shown to directly reduce HSC self-renewal during recovery from viral infection where robust type I IFNs had ablated the HSC pool (12), suggesting that type I IFNs may potentiate the suppressive impact of IFNγ on hematopoiesis during viral infection. These data highlight the importance of the cellular and cytokine context in the impact of single cytokines. Whereas IFNγ may not impede self-renewal in the steady state, prior exposure to type I IFNs may sensitize HSCs to the effects of IFNγ; at the same time, the induction of cellular stress by type I IFN-induced HSC cycling could enhance the potential for IFNγ to provoke HSC apoptosis during immune-mediated BM failure (59, 60). Figure 1 The actions of IFNγ directly on HSCs and on cells of the microenvironment can result in HSC impairment in acquired aplastic anemia. This figure summarizes key direct (A) and indirect (B) impacts of IFNγ on HSCs. The inset on the left depicts HSC-intrinsic effects of IFNγ, including STAT1-mediated hematopoietic differentiation programs (a), restriction of thrombopoietin – c-Mpl signaling by SOCS1 (b), and promotion of Fas expression (c). The inset on the right depicts cell types in the bone marrow microenvironment that are capable of regulating HSCs in an IFNγ-dependent manner include Tbet+ T lymphocytes, macrophages (MΦs), mesenchymal stromal cells, and hematopoietic progenitors. Known molecular mechanisms by which these cell types engage in IFNγ-dependent HSC regulation include: increased demand for progenitor cell differentiation to replenish downstream hematopoietic compartments (d), expression of death receptor ligands FasL and TNFα by T lymphocytes (e), propagation of MΦ-derived inflammatory signals (f) and potential impairment to MΦ-dependent regulation of HSC quiescence (g), and the production of further myelopoiesis-promoting factors by BM stromal cells (h). Hematopoietic stem cells require a variety of inputs from growth factors, chemokines, G protein-coupled receptors, and cytokines to maintain their dormant status, location, and capacity to self-renew. An intriguing role for IFNγ in limiting responsiveness to the growth factor thrombopoietin (TPO) via the increase in suppressor of cytokine signaling (SOCS1) (12) (Figure 1b) illustrates yet another direct mechanism whereby IFNγ can impede HSC function. Support for the role of TPO in HSC function comes from the promising clinical data using a TPO receptor (c-Mpl) agonist, Eltrombopag (61). When given in combination with immunosuppressive drugs, it can provide tri-lineage recovery in patients refractory to traditional therapies (62). Though precise mechanisms of Eltrombopag function have not yet been elucidated, one possibility is that the drug works by overcoming a direct impact of IFNγ on suppressing TPO signaling in HSCs. Interferon gamma is elicited by many microbial infections and plays a critical role in host defense by sensitizing cells to undergo apoptosis, thus impeding pathogen growth (63, 64). IFNγ can induce apoptosis by increasing the expression of Fas on cells subsequently targeted by Fas ligand-expressing cells, such as T lymphocytes (60). Evidence that HSCs express Fas in response to IFNγ (Figure 1c) suggests Fas-mediated destruction of HSCs contributes to their loss in AA (60). It is important, however, to consider the question of HSC sensitivity to IFNγ. Indeed, whereas some cell types respond very rapidly to IFNγ in vitro, such as macrophages, HSCs exhibit a much more subtle response, as measured by activation of STAT1 (42). This may indicate that the ability of HSCs to respond to IFNγ in vivo may be concentration dependent, and it suggests that HSCs are likely not first responders to IFNγ during an initial exposure. Under prolonged conditions of chronic exposure, however, HSCs may become direct targets. Thus, there are temporal considerations when evaluating the direct impact of IFNγ on HSCs under different inflammatory conditions. Impact of IFNγ on Progenitors The idea that HSC activation can be achieved directly or as a result of demand implies that HSC loss may result from increased progenitor cell activity or loss. Several lines of evidence support a direct role for IFNγ in impacting murine progenitor cells in the context of infection (43, 65). IFNγ promotes the emergence of a phenotypically unique, hybrid progenitor population that expresses the IL-7 receptor and has both myeloid and lymphoid potential in vitro, but has primarily myeloid potential in vivo. Similarly, an intrinsic requirement for IFNγ was found to occur during bacterial infection, directing the production and terminal differentiation of myeloid cells (43). In a model of sterile inflammation, via adoptive transfer of activated effector T lymphocytes, IFNγ acted directly on progenitors, but not HSCs (66). The ability of IFNγ to act on downstream progenitors to drive proliferation, however, may indirectly call HSCs from a dormant state, which may explain observations suggesting that IFNγ acts directly on HSCs (Figure 1d). Indirect or Niche-Mediated Effects of IFNγ on HSC Function T Lymphocytes Although T cells are the cellular source of IFNγ that drives AA pathology, T cells also sense and respond to IFNγ. The effects of IFNγ on T cells include promotion of Th1 CD4+ T cell differentiation, enhancement of CD8+ T cell response, and subversion of IFNγ-mediated apoptosis via the downregulation of IFNγ receptor (67–69). IFNγ also primes activated T cells to secrete more abundant TNFα and RANKL (70, 71), inflammatory cytokines capable of inducing hematopoietic cell death and further inflammation. Additionally, T cells derived from AA patients show elevated Fas ligand expression (72) (Figure 1e), which is IFNγ dependent in murine lymphocyte infusion models of AA (60). Thus, it is likely that IFNγ acts to expand and preserve pathologic T cells in the BM during AA. A population of T regulatory cells (Tregs) reside in the BM at homeostasis [reviewed in Ref. (73)] and establishes HSC-protective niches during transplantation and reconstitution (74). The direct HSC supportive capacity of Tregs in AA has not yet been evaluated, but Tregs derived from AA patients are reduced in number and inhibitory capacity, and show enhanced production of cytokines, including IFNγ (75, 76), suggesting that they may further contribute to immunopathology in the BM microenvironment. In Tregs, SOCS1 signaling controls IFNγ production (77) and defects in SOCS1 activity are thought to underlie autoimmunity and susceptibility to endotoxemia [reviewed in Ref. (78)]. Therefore, ex vivo expansion and treatment of autologous Tregs with small molecule SOCS1 mimetics may prove a promising therapeutic strategy for AA patients who do not respond well to conventional immunosuppression (76, 79). Osteoclasts Osteoclasts (OCLs) are bone-resorbing myeloid cells that are both directly and indirectly sensitive to IFNγ. Osteoclastogenesis requires the sensing of M-CSF and RANKL by myeloid precursors (80). Direct IFNγ sensing by myeloid precursors attenuates RANK signaling (81), but systemic IFNγ responses are associated with enhanced bone resorption due to the OCL-promoting impact of TNFα and RANKL (70, 71). Since IFNγ and TNFα levels are elevated in AA patients (82, 83), accelerated osteoclastic differentiation of myeloid precursors may occur early in AA pathogenesis. Indeed, low bone mineral density and osteoporosis are prevalent in individuals with the inherited BM failure condition Shwachman–Diamond syndrome (84) and have been observed in Fanconi anemia patients following BM transplantation (85). Whether inflammatory bone loss contributes to hematologic impairment in AA is currently unknown. OCLs and bone resorption have been found to reduce HSPC support in murine models, however, and are associated with HSPC mobilization (86, 87). The actions of bone-forming OBs and bone-resorbing OCLs are regulated primarily by the endocrine system (88). Since the responses of BM T cells to circulating hormones stimulates bone formation and short-term HSC expansion through Wnt signaling (89), T cell-based therapies warrant further investigation for their potential not only to reduce immunopathology, as mentioned above, but also to regenerate HSPCs and BM microenvironmental function in AA. Macrophages The BM microenvironment contains a heterogeneous population of tissue-resident macrophages (MΦs) that sense and respond to IFNγ [reviewed in Ref. (90)]. IFNγ stimulates MΦ cytokine production (Figure 1f) and antigen presentation (91), therefore, it stands to reason that MΦs may contribute to IFNγ-driven AA pathogenesis. We have previously established that MΦs in general, and IFNγ-stimulated MΦs in particular, reduce the pool of HSCs in a model of human monocytic ehrlichiosis, which causes transient BM suppression (42). Intriguingly, one of the few hematopoietic cell types found to be maintained in AA BM is the CD169+ MΦ (92). Tissue-resident MΦ populations, including BM-resident MΦs, are thought to be embryonically derived and maintained via self-renewal, rather than derived from HSC differentiation (93) [and recently reviewed in Ref. (94)]. This would support the idea that the maintenance of MΦs may not require an intact HSPC pool, thus explaining their persistence in the BM of patients with AA. As antigen-presenting cells, MΦs are relatively weak (95), thus, it is unlikely that MΦs drive AA pathogenesis by activating T cells directly. Mice deficient in myeloid lineage cells are resistant to severe AA induction (96), however, suggesting that MΦs are indispensable in AA pathogenesis. While further investigation is necessary to determine if MΦ number and function correlates with AA severity, it can be envisioned that MΦs play a pathologic role in AA via several mechanisms. Since HSPCs and resident MΦs interact within the BM microenvironment (97–99), it is possible that IFNγ stimulates pathologic HSPC engulfment by MΦs in AA. In fact, IFNγ is associated with hemophagocytosis-induced anemia (100), and MΦs have been implicated in the pathogenesis of human hemophagocytic disorders, such as juvenile idiopathic rheumatoid arthritis and lymphohistiocytosis (101, 102), as well as platelet clearance in immune-mediated thrombocytopenia (103). Alternatively, MΦs may contribute to HSPC loss in AA by regulating, either directly or indirectly, HSPC proliferation or differentiation. Quiescent HSCs are called to proliferate and differentiate in response to demand for mature progeny, such as myeloid cells or platelets (104, 105), but must reenter quiescence in order to avoid replication stress and ensure lifelong maintenance. MΦs have been implicated in maintaining long-term HSC quiescence, or dormancy, through the production of PGE2 and the maintenance of the quiescence-promoting tetraspanin CD82 on the surface of HSCs via Duffy antigen receptor (DARC) on MΦs (98, 99) (Figure 1g); however, functional changes to this cell–cell interaction upon inflammation have only just begun to be investigated. In conditions of inflammation and infection, MΦs may suppress dormancy as a way to enlist HSCs in demand-adapted hematopoiesis. In murine ehrlichiosis, IFNγ is required for BM-resident MΦ maintenance, and is also essential for the infection-dependent loss of HSCs. Upon MΦ depletion, HSCs proliferate, under both steady-state (98) and infectious conditions (42). McCabe et al. found that these HSCs subsequently reenter quiescence, resulting in HSC pool expansion. Thus, under infection states, and perhaps in AA, IFNγ-stimulated MΦs drive HSC loss. This may occur via inhibition of HSC proliferation and demand-adapted hematopoiesis or alternatively, via increased differentiating proliferation, at the expense of self-renewal, culminating in HSC exhaustion. Hematopoietic stem cells are motile within the BM of infected mice (106), suggesting that HSC engagement with the niche may be important for demand-adapted hematopoiesis. Since MΦs support the expression of HSPC retention factors by endosteal cells (97), MΦs may render HSCs more susceptible to T cell-mediated killing, and less capable of migration to microenvironments that support cell cycle entry and differentiation. At homeostasis, a population of endosteal MΦs, termed osteomacs, is reported to mediate osteoblastic NF-κB signaling, maintenance of bone-lining OBs, and hematopoietic progenitor cell retention in the BM (97, 107). Whether MΦs stimulated with IFNγ or other inflammatory cytokines, as in AA, drive osteoblastic dysfunction (see below), remains an open question. Since BM MΦs persist in AA patients, in spite of reductions in nearly all other BMC populations (92), and since MΦs potently respond to IFNγ, studies focused on the impact of MΦs in AA pathogenesis are warranted. Mesenchymal Stromal Cells Mesenchymal stromal cells respond to inflammatory signals, including IFNγ, to regulate the differentiation of HSCs and the mobilization of their progeny (66, 108). Cytotoxic CD8+ T cell-derived IFNγ was recently found to stimulate IL-6 production by BM MSCs, thus identifying a niche-mediated mechanism by which IFNγ stimulates myeloid transcriptional programs in hematopoietic progenitors (Figure 1h) (66). Consistent with these observations, BM stromal cells derived from AA patients and from a murine model of AA show elevated Il6 expression (15, 109). Since there is a higher prevalence among AA patients for an Il6 gene polymorphism conferring IL-6 hypersecretion (83), it is currently unclear whether elevated IL-6 in AA is IFNγ-dependent. IL-17 is increased in the BM plasma of AA patients and more potently stimulates IL-6 secretion by MΦs derived from AA BM than from healthy controls (110), suggesting that inflammation in AA primes the responses of MSCs and other cell types to amplify local cytokine production. Since MSCs exist in close proximity to HSCs, and can greatly influence HSC fate, the impact of IFNγ on MSCs in AA is a key unanswered question in the field. Adult BM MSCs are rare but exhibit heterogeneity with respect to their developmental origin, localization in the BM, and contribution to bone formation and HSPC regulation (111–114). This heterogeneity, coupled with the need for genetic reporter strains to identify and delineate MSC populations, has hindered investigation of BM MSCs in disease models, including in lymphocyte infusion-based AA models where IFNγ is known to be pathogenic. MSC dysfunction may contribute to BM failure, as MSCs possess immunoregulatory potential [reviewed in Ref. (115–117)] and are critical HSC-support cells. With regard to HSC niche function, peri-arteriolar MSCs enforce quiescence and are required for long-term HSC function (118). When the niche is activated, such as through hormonal stimulation, MSCs increase in number and mediate expansion of the HSC pool (112). Although adherent BM stromal cells, enriched in MSCs, show normal surface marker expression in AA patients, these cells fail to expand readily in culture, undergo greater apoptosis, and are impaired in osteogenic but enhanced in adipogenic differentiation, relative to normal controls (14, 15, 119). Unlike osteolineage cells, which support HSPCs and B lymphopoiesis (120, 121), BM adipocytes are detrimental to HSCs (122). MSC differentiation into either adipogenic or osteogenic progenitors is controlled by cell intrinsic and extrinsic mechanisms (123, 124). Systemic inflammation, as induced by high-fat diet, was recently linked to PPARγ activation in MSCs and resultant adipogenesis, concomitant with a reduction in HSPC support by the microenvironment (125). Severe AA, therefore, could erode BM microenvironmental function and HSC niches by a similar mechanism. Elevated IFNγ may impact MSCs in AA via a number of distinct or overlapping mechanisms. T cell-mediated MSC killing, IFNγ-induced MSC dysfunction, or bystander effects mediated by neighboring BM cell types all potentially contribute to AA pathogenesis. Although MΦs are dispensable for the maintenance of BM MSCs at homeostasis, they regulate MSC function by promoting MSC expression of the niche-retention factors Cxcl12, Angpt1, Kitl, and Vcam1 (126). Moreover, MΦs support the presence of mature OBs along the endosteum at homeostasis (97), potentially by stimulating NF-κB-mediated osteoblastic differentiation of MSCs or immature OBs (107). These data indicate that in an otherwise unperturbed system, MΦs promote the survival and/or osteolineage differentiation of bone-lining OBs. Thus, the potential for MΦs to dysregulate MSCs resulting in HSC niche destruction in AA and other disease states warrants investigation. Mechanisms of IFNα/β-Mediated HSPC Impairment HSC-Intrinsic Impact of Type I IFNs Early observations made in LCMV-infected mice (31), and in IFNα-treated HSPC cultures (127), led to the conclusion that type I IFNs suppress progenitor cell proliferation and differentiation. Indeed, IFNα induces HSPC expression of cell cycle inhibitors in vitro (13). In vivo, however, the impact of IFNα/β differs. Acute administration of the double-stranded RNA mimetic polyinosinic:polycytidylic acid (polyI:C) causes rapid, IFNα receptor (IFNαR)-dependent HSC cycling (13, 128), and has been the model of choice for studying type I IFN-mediated HSPC activation (see Table 1 for a summary of relevant findings). The impact of polyI:C-induced sterile inflammation varies depending upon the duration of stimulation and the precise HSC subset analyzed, but acute stimulation is sufficient to decrease HSC expression of cyclin-dependent kinase inhibitors and quiescence-enforcing transcriptional programs, including FoxO3a, Notch, and TGFβ (13). Table 1 Impact of acute and chronic polyI:C-induced inflammation on HSCs and HSPCs. HSCs Hematopoietic progenitors Acute Reduced in frequency but not changed in number (13) Cell cycle entry (13, 59) Increased redox stress, accumulation of DNA double-strand breaks, and engagement of Fanconi anemia DNA repair pathway (59) Increased translation of megakaryocyte- lineage proteins (104) Enhanced death in vitro (59) Increased myeloid (13) and CD41hi stem-like megakaryocyte progenitors (104) No change in Lineage− c-Kit+ cell cycling, DNA damage, or colony formation (59) Chronic Reduced in frequency, trend toward reduction in number (13) Loss of function in response to chemotherapeutic injury, transplantation, and in vitro expansion (13, 59, 128) Transiently reduced cyclin-dependent kinase inhibitor and quiescence-enforcing gene expression (13) Activation of PI3K/mTOR signaling (129) and increased m-Myc protein levels (130) Caspase 3 activation (13) Myeloid bias in transplantation (59) Transiently increased Lineage− c-Kit+ cell pool (13) Exhaustion of stem-like megakaryocyte progenitor cell function (104) Note that polyI:C was obtained from InvivoGen (59, 128, 130), GE Life Sciences (131, 132), and Invitrogen (104). The function of type I IFNs in the context of physiologic induction, such as infection, may provide insight into pathogenic role(s) of type I IFNs in AA. Somewhat paradoxically given the BM suppressive impact of IFNα in viral infection, the type I IFN response to opportunistic Pneumocystis lung infection is protective in Rag−/− mice (133). Since these mice lack all B and T lymphocytes, immunity depends entirely upon myeloid cells, which undergo greater apoptosis in the absence of type I IFNs (134). In Pneumocystis-infected Rag competent mice, IL-10 and IL-27 production by B lymphocytes is protective, and correlates with enhanced myelopoiesis (135). Thus, in the absence of lymphocytes, and the cytokines they produce, type I IFNs provide a survival signal for myeloid cells. These findings further support the notion that the complex cytokine milieu greatly impacts the outcome of IFN signaling on HSC function. At homeostasis, HSC quiescence protects against replication stress and genomic instability. Long-term label retaining studies demonstrate that ~1% of phenotypic HSCs cycle per day (136) and that a subset of multipotent progenitors is maintained in a similarly dormant state (137). PolyI:C increases HSC cycling six- to sevenfold for up to 3 days, leading to the accumulation of reactive oxygen species and DNA damage in remaining HSCs (59). DNA damage itself induces type I IFN-mediated stem cell senescence (138), in addition to the activation of cellular checkpoints and tumor suppressor genes [recently reviewed in Ref. (139)]. Additionally, type I IFNs transcriptionally regulate p53 (140), through the interferon-stimulated signaling complex ISGF3 (141, 142). Therefore, type I IFNs have the capacity to induce both proliferation as well as DNA damage-induced p53 signaling, thus priming HSCs to undergo apoptosis upon cellular stress, such as in vitro culture (13). These findings, therefore, implicate type I IFNs in the induction of replication and oxidative stress in HSCs. In vivo, repeated IFNα/β stimulation or uncontrolled type I IFN signaling is detrimental to HSCs exposed to chemotherapeutic injury or transplantation (13, 128, 143), likely by promoting cell cycle entry and heightened sensitivity to cellular stress. While this may be detrimental in some cases, complete molecular remission has been observed in several cases of chronic myelogenous leukemia (CML) where IFNα pre-treatment was followed by imatinib mesylate (144), suggesting that IFNα may induce CML stem cell exit from dormancy and subsequent sensitization to growth factor withdrawal. The sensitizing effect of type I IFNs on stem cells persists, as HSCs transplanted from mice 2 weeks after polyI:C stimulation remained functionally impaired in their repopulating capacity (13). Therefore, it is reasonable to expect that increases in endogenous IFNα/β during viral infections or chronic administration of type I IFNs may have long-term impacts on HSC response to subsequent inflammatory stimuli. Such a mechanism is consistent with HSC impairment and BM suppression during LCMV infection, which elicits initial IFNα/β followed by subsequent IFNγ (12, 31, 145). Further studies are necessary, however, to determine if apoptosis is the predominant mechanism by which HSCs are depleted upon type I IFN sensitization, or if IFNα/β also sensitizes HSCs to senescence, or to non-apoptotic cell death. In addition to HSC proliferation and apoptotic sensitization, type I IFNs influence HSPC differentiation. IFNα/β increases the synthesis of proteins required for rapid hematopoietic progenitor cell differentiation in response to inflammation and demand. This occurs via a post-transcriptional mechanism, the targets of which include the c-Myc transcription factor (130), and megakaryocyte lineage proteins (104). Expression of the megakaryocyte lineage gene von Willebrand factor and the alphaIIb integrin protein CD41 have previously been attributed to the most primitive HSCs (146, 147), but Haas et al. identified the IFNαR-dependent emergence of highly proliferative, CD41hi megakaryocyte-restricted progenitor cells within the phenotypic HSC pool upon stimulation of mice with polyI:C, TNFα, or lipopolysaccharide (104). Since CD41 was not interrogated in previous studies, it is unclear to what extent megakaryocyte-primed progenitor cells contributed to the observed effects of type I IFNs on HSPC proliferation, apoptotic sensitization, and multilineage repopulation (13, 59, 128). Hematopoietic stem cell metabolism is exquisitely regulated to protect against metabolic stress and to regulate the nature of cell division upon entry into the cell cycle (148), such as occurs upon type I IFN stimulation. One mechanism by which HSC metabolism is regulated is through autophagy and the Foxo family of transcription factors (149). FOXO3A, in particular, has been implicated in the activation of autophagy gene expression programs in HSCs that are essential for HSC survival upon cytokine withdrawal or calorie restriction-induced stress (150). Additionally, Warr et al. found that HSCs derived from aged mice had greater autophagic flux and were more reliant on autophagy for colony formation in vitro. Sterile type I IFN stimulation reduces FOXO3A expression and signaling activity in HSPC subsets (13, 104). Moreover, infection-induced type I IFNs are linked to reduced autophagic flux in the liver (151). The impact of type I IFNs on HSC autophagy has not yet been assessed, but autophagic suppression was recently identified in CD34+ BM cells from AA patients, and persisted even upon amelioration of AA symptoms (152). Therefore autophagy inhibition could represent an additional mechanism by which interferon signaling impairs HSC stress responses and exacerbates pathology in AA. Indirect or Niche-Mediated Effects of IFNα/β on HSC Function T Lymphocytes In AA pathogenesis, oligoclonally expanded CD8+ T cells infiltrate the BM and produce damaging, pro-inflammatory cytokines, including IFNγ (153). Type I IFNs regulate T cell production of IFNγ in a highly context-dependent manner, whereby type I IFNs are associated with enhanced IFNγ during extracellular bacterial infection (154), but with reduced IFNγ in response to intracellular pathogens (155–157). Type I IFNs may also contribute to the activation and expansion of pathologic T cells in AA as IFNα/β increases the survival of antigen-specific CD8+ T cell clones, as well as the generation and cytolytic activity of memory T cells (158, 159). Indeed, the blood and BM of AA patients show increased effector memory T cells (160), which may be derived from a newly identified class of progenitors termed memory stem T cells (161, 162). Since type I IFNs drive cell cycle entry and differentiation of other HSPC subsets (13, 104), they may also impact the development of CD8+ memory T cells from memory stem T cells and contribute to the etiology of infection-induced and iatrogenic BM failure through the modulation of T cell populations. Macrophages During infection, MΦs are stimulated concurrently with IFNs and TNFα and amplify inflammation through the production of additional IFNα/β and TNFα (163). Like IFNγ, TNFα is highly pathogenic in AA (164–166) and may engage in cross-talk with type I IFN signaling. TNFα levels correlate with the extent of cytopenia (165), and TNFα neutralization improves the colony-forming activity of AA patient BM (164). In addition to elevated circulating TNFα, TNF receptor 1 and 2 (TNFR1/TNFR2) expression is increased on hematopoietic progenitors derived from AA patients relative to healthy controls (165). TNFR2 ligation initiates inflammatory signaling, whereas TNFR1 drives the assembly of cytoplasmic cell death signaling complexes [reviewed in Ref. (167)]. A number of mechanisms, including caspase activity and ubiquitination of TNF receptor interacting protein kinase 1 (RIPK1), promote immunologically silent apoptosis when TNFR2 is activated [reviewed in Ref. (168)]. If caspase activity is limited, however, RIPK1–RIPK3 interactions mediate RIPK3-dependent phosphorylation of the pseudokinase MLKL. Phosphorylated MLKL then translocates to cellular membranes where its pore-forming action leads to cell lysis and the release of intracellular contents in a process known as necroptosis. MΦs in Salmonella typhimurium-infected mice were the first cell type found to undergo IFNαR- and RIP1-dependent necroptosis in vivo (169). Subsequent studies have found an absolute requirement for IFNα/β priming in the death of MΦs by necroptosis (163, 170). Thus, the combinatorial impact of TNFα and type I IFNs has the potential to drive MΦs necroptosis in autoimmune diseases, such as severe AA. BM-resident MΦs are abundant HSPC–niche cells [recently reviewed in Ref. (90)], therefore, even a low rate of MΦ necroptosis has the potential to exacerbate immunopathology through release of damage-associated molecular patterns from the lytic cells, and/or impairment of the mononuclear phagocyte system responsible for clearing dead and dying cells, including apoptotic HSPCs. Further research is also needed to discern whether myeloid progenitors, and HSPCs themselves, have the capacity to undergo necroptosis in response to IFNα/β. Stromal Cells Although IFNα/β have not been directly implicated in AA pathogenesis, TNFα stimulates autocrine type I IFN expression in MΦs and in endothelial cells (171, 172), and could, therefore, establish local IFNα/β gradients in the inflamed BM microenvironment. Type I IFN sensing by BM stromal cells is not required for IFNα-induced HSPC proliferation in response to polyI:C, but Ifnar1−/− HSPCs are induced to proliferate in 95% WT: 5% Ifnar1−/− mixed BM chimeras (128), suggesting that IFNα/β-stimulated hematopoietic cells release additional factors that act on Ifnar1−/− HSPCs. HSPC-activating cues may derive from the HSPC pool itself, as hematopoietic progenitors produce a wide repertoire of inflammatory cytokines upon toll-like receptor stimulation (173), or may originate from stromal niche cells within the BM microenvironment. Arteriolar blood vessels and megakaryocytes comprise HSC niches in the BM (174–176), although they are reported to be spatially and functionally distinct from one another. Sterile, IFNα/β-driven inflammation relocates HSPCs away from quiescence-enforcing arteriolar niches (118), though it is unclear whether this is cause or consequence of changes in HSC cycling. IFNα/β can also stimulate endothelial chemokine expression, including that of CCL5 or RANTES (177), which can impact platelet production by megakaryocytes (178). The role of megakaryocytes in HSC regulation is dynamic, as homeostatic expression of CXCL4 and TGFβ1 promotes quiescence, while concomitant increases in FGF-1 and decreases in TGFβ1 facilitate regeneration (174, 175). To our knowledge, megakaryocyte dysfunction has not been investigated in the pathogenesis of BM failure but aberrant TGFβ1 signaling is linked to pathologic extracellular matrix deposition and derangement of hematopoiesis in myelofibrosis (179). Additionally, TGFβ slows recovery from chemotherapy-induced myelosuppression by blocking HSC proliferation (180). Since type I IFNs both impair HSCs and activate a program of enhanced megakaryocyte lineage differentiation (104), it is intriguing to consider the impact this may have on HSPC–niche cell interactions during recovery from severe IFN-driven inflammation. Conclusion In severe AA, autoreactive T cells initiate immunopathology, leading to HSC depletion, and total hematopoietic collapse. IFNγ is well-known to correlate with AA disease severity in mice and humans, but the mechanisms by which IFNγ impairs HSCs remain somewhat elusive. The potential for IFNγ to both directly exhaust and deplete HSCs, as well as to indirectly reduce HSC function through microenvironmental niche cells, particularly macrophages, and MSCs (Figure 1), adds complexity to the study of AA pathogenesis but also reveals new potential therapeutic targets. Since type I IFNs have been linked to BM aplasia and sensitize HSCs to cellular stress (Table 1), it can be envisioned that initial IFNα/β exposure, as occurs in response to viral infection, may render HSCs more vulnerable to subsequent IFNγ-mediated impairment. Current understanding of how inflammatory signals impact the HSC niche is limited; thus, we discussed several potential mechanisms by which interferons may contribute indirectly to HSC loss during severe AA. Parallels emerge when considering the impact of IFNγ and IFNα/β on HSCs, including the capacity of both cytokines to (1) drive HSC proliferation, seemingly at the expense of long-term function; (2) propagate inflammatory signaling within macrophages, a critical HSC niche cell type; and (3) potentiate cell death through the regulation of death receptor signaling, suggesting that these factors may be synergistically detrimental in inflammatory disease states. The development of additional AA mouse models, in which the independent and concerted impact of interferon signaling on specific cell types can be interrogated, would be of great utility in parsing out the mechanisms that drive AA pathogenesis. Author Contributions All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MB and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review. Funding This work was supported by R01 GM105949 to KM. ==== Refs References 1 Dolberg OJ Levy Y . Idiopathic aplastic anemia: diagnosis and classification . Autoimmun Rev (2014 ) 13 (4–5 ):569 –73 .10.1016/j.autrev.2014.01.014 24424170 2 Nakao S Takami A Takamatsu H Zeng W Sugimori N Yamazaki H Isolation of a T-cell clone showing HLA-DRB1*0405-restricted cytotoxicity for hematopoietic cells in a patient with aplastic anemia . Blood (1997 ) 89 (10 ):3691 –9 .9160674 3 Risitano AM Kook H Zeng W Chen G Young NS Maciejewski JP . 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==== Front Front PediatrFront PediatrFront. Pediatr.Frontiers in Pediatrics2296-2360Frontiers Media S.A. 10.3389/fped.2016.00091PediatricsOpinionMoral Distress in the Everyday Life of an Intensivist Garros Daniel 1*1Pediatric Intensive Care Unit, Department of Pediatrics, 3A3 WC Mackenzie Health Sciences Centre, Stollery Children’s Hospital, University of Alberta, Edmonton, AB, CanadaEdited by: Stephania Cormier, University of Tennessee Health Science Center, USA Reviewed by: Michael Aloysius Freeman, Penn State Hershey College of Medicine, USA *Correspondence: Daniel Garros, dgarros@ualberta.caSpecialty section: This article was submitted to Pediatric Critical Care, a section of the journal Frontiers in Pediatrics 29 8 2016 2016 4 9104 11 2015 15 8 2016 Copyright © 2016 Garros.2016GarrosThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.moral distressethicsmedicalend-of-life caredecision-makingpediatric critical care medicine ==== Body A regular work day for intensivists can be emotionally draining, as we witness suffering, fear, pain, tragedies, unfair treatment of children, death…. We may experience the mental stress of dealing with nursing shortages, increasing family demands, and frustration related to interpersonal conflicts (e.g., between parents and specialists) among other issues (1). For the most part, we learn to manage this type of stress. Several studies involving nearly every medical and surgical specialty indicate, however, that approximately one of every three physicians experiences burnout at any given time. Burnout is characterized by behaviors such as losing enthusiasm for work (emotional exhaustion), treating people as if they were objects (depersonalization), and having a sense that work is no longer meaningful (low personal accomplishment) (2). Physicians, like other health-care professionals, can be at risk for another phenomenon, that of moral distress (MoD). This concept emerged in nursing ethics: “a challenge that arises when one has an ethical or moral judgment about care that differs from those who are in charge” (3). Thus, institutional constraints were seen as its key source (inadequate staffing, other professionals’ influence, family or patient choices, administrative agendas, institutional policies, and legislation) (3). Unlike a moral dilemma in which one is uncertain what ethical action to take, MoD is experienced by those who feel constrained from acting on their ethical judgment. Constraints are still recognized frequently as external, institutional ones (4). Internal constraints may be related to perceived powerless, lack of knowledge, increased moral sensitivity, or even lack of full understanding of a particular situation. It could also represent a lack of “moral courage” (5). In 2006, Nathaniel extended the definition, highlighting the consequences of not acting according with ones’ moral judgment and be participating in perceived moral wrongdoing (6). The word perceived is very crucial, since we may feel strongly that an action is unethical while a colleague may feel just as strongly the opposite. It is well known that MoD in pediatric intensive care (PICU) can be linked to aggressive treatment, witnessing repeated suffering, futile care, and high levels of chronic disability post discharge and may be aggravated by work environment issues such as power imbalances, improper communication, decision-making conflicts, unrealistic expectations, lack of resources or personnel, and a high index of medical errors (2, 7) Corley and colleagues have developed a scale (MDS), containing 20 clinical situations to assess the frequency to which MoD occurs, as well as the intensity of the feeling (8) This scale, now on its second version, has been utilized in several studies (4, 5), including some in the PICU environment (9). As MoD has been more thoroughly investigated, discussion about the topic has become more prominent in the bioethics literature, with several journal issues being fully dedicated to the theme (10, 11). Why Does Moral Distress Occur in Intensive Care Units? In this high-tension, rapidly changing environment, team work is paramount – and it is the way we operate (12). Indeed, the best clinical outcomes in critical care are correlated with a “team” approach (13, 14). Intrateam discordance, however, has been identified as a significant factor related to MoD among all health-care disciplines (15). Team dynamics, such as power imbalances (e.g., the lack of inclusion of the bedside nurse in overall decision-making), silencing (e.g., pressure to refrain from raising ethical concerns or voicing doubt about desired outcomes), “professional tribalism” (16), lack of trust in existing systems for ethical dialogue and patient management (e.g., worry that the “ethics people” are there to protect the organization, not patients or staff), not only affect individual PICU team member’s behaviors but also can heighten MoD. Poor team communications and lack of provider continuity have also been found as reasons for MoD among the PICU team (17, 18). In our narrative inquiry study of the MoD of PICU teams, we have found that PICU staff participants identified lack of organizational support as an important source for MoD in situations of interdisciplinary conflict (19, 20). While research indicates that genuine dialog among the team members regarding ethically difficult situations is much desired (18, 21), the sheer number of personnel necessary to provide 24-h care for a PICU patient makes such dialog a logistical challenge (13). How can it be ensured that all staff involved in a particular patient’s care are included in key discussions or even kept informed, in a timely way, of the ethical concerns being raised? Finding answers to such logistical challenges is a worthwhile endeavor. The consequences of MoD are significant for any intensive care unit, at the personal, the team, and the institutional level. Conflicting views related to life-sustaining treatment deeply impact staff members. Physicians, for instance, can become detached, and their future medical decisions compromised by such experiences (15). An experience of MoD can haunt some individuals for years, in what has been called moral residue (22). In fact, it has been found that MoD can be a reason for health-care staff to quit their position or even their practice entirely (4). There is some evidence that the intensity of MoD can vary according to two factors: personal moral sensitivity and the moral climate in the organization (23). MoD may be an expression of sensitivity to the moral aspects of practice, an appreciation of vulnerability of patients, a simple reaffirmation of one’s values expressed in codes of ethics, or perhaps an acceptance of accountability and moral responsibility (15). Such recognition allows greater openness about the experience and does much to prevent a staff member being seen as simply unable to cope in the technologically driven, fast-paced intensive care environment. Why is Moral Distress in Vogue Now? Lantos suggests that, in many areas of medicine today, there is a lack of consensus as what should be the best treatment for particular patients (24). As Morparia and colleagues noted, this lack of consensus continues to feed the controversy. Physician, in almost all cases, are equally divided in their choices (25). Parents have become – properly so – increasingly important in the determination of treatment choices for their children; this results at times in clashing beliefs and values between parents and health-care systems (26). In our study, participants’ stories suggest that professionals can be seriously distressed by taking part in treatments and/or care that they believe to be wrong for a particular patient or family (19, 20), especially when dealing with end-of-life decisions – a finding reported by others as well (27). In a recent study within an adult ICU, Dodek and colleagues found that MoD ultimately stems from three problems: uncertainty about who is in charge, cost-cutting schemes that affect patient care, and controversies about end-of-life (4, 28). An excellent review of MoD in neonatal and pediatric ICUs, by Sauerland and colleagues, concluded that situations causing the most intense distress were related to inadequate nurse staffing and perceived incompetent coworkers. The most frequently occurring distressing items were futile care and unsafe staffing (9). Moral distress, however, becomes magnified when one feels that it is unsafe to voice one’s concern. A culture of silence can prevail to the extent that professionals know that raising any ethical concern will too easily label them as a troublemaker or as someone who is unable to “take the pressure.” The lack of authentic debriefing and/or ethical dialog that occurs within such a culture appears to be a major contributor to moral distress (15). How Can we Survive in the ICU? Moral distress, like burnout, is a reality of our times. We have to find ways to meaningfully address it. Not to do so can mean that we will lose highly ethical physicians who find no recourse but to leave the job they love, forfeiting years of training and personal sacrifice (28). To prevent MoD, a culture of frank dialogue and good team communication is fundamental. In the PICU, finding the time and space for these to occur is an ongoing challenge. How Can we Address Moral Distress in the PICU? Based on the current research and personal experience, the following are some practical ways that can help physicians and their teams to prevent or to address MoD: 1. Recognize that MoD can be an alarm signal raised by a conscientious person encountering an ethical problem and worried that something ethically wrong is going to happen (29). 2. Give voice to the silence: whenever possible, foster open and authentic discussion of ethical concerns from each person’s perspective, including health-care professionals, trainees, parents, families, and patients. All practitioners on the team, regardless of discipline or “professional ranking,” should be able to safely raise their concerns of conscience (30). A culture of ethical questioning should be endorsed by institutions, similar to the openness described in the airline industry for safety concerns (31). 3. Reach for a “rapprochement”: we need to find ways to support moving to reciprocal understandings among those involved in patient care, as proposed by Carnevale (32). Respect, trust, and honesty will be required for common ground to be found in difficult situations. 4. Enhance effective communication among team members. In the fast-paced PICU environment, “real time” team sharing of information regarding treatment options and decisions made between physicians and families is crucial (30, 33). 5. Seek further ethics education: ongoing education in health ethics is one way to evolve a shared language by which the team may address ethical concerns; it may allow a deeper understanding of what is at stake (34). 6. Promote “venting/debriefing sessions”: after difficult cases, such sessions allow for open conversations and analysis. These sessions, to be successful, need to be characterized by authenticity, compassion, regrouping, and validation (30). Such encounters are crucial for the well-being of the unit and the practitioners involved and should be supported (i.e., finances, resources, and training) by the institutions (15, 35). At the personal, individual level, a general mindset that encompasses the following may be helpful: 1) Nourish “moral courage”: an ethical health-care professional will always need moral courage, i.e., be prepared to face tough decisions and confront the uncertainties associated with the resolve to do the right thing despite the consequences faced. This need for moral courage is especially true when the perceived constraints are within ourselves or when we face opposition within our own ranks. The courageous person accepts and assumes moral responsibility for the perceived consequences of his or her action; a physician may master fear without necessarily eliminating it (36). 2) Seek peer support: this type of sharing, with someone that can understand one’s struggles, has a good listening ear and acknowledges and validates what one is experiencing seems essential for survival in the profession (15). 3) Take time off: having resting periods, time offs, vacation, and long weekend getaways: these are necessary for one’s well-being. Physical activity, exercise, enjoying nature, and managing stress through mindfulness are strategies shown to increase the capacity to take new challenges with stride (1). 4) Find another passion: having another interest outside work, even one connected with the field, such as volunteering on medical missions, has been very rewarding for me. Having a favorite sport, a pet, or pursuit (e.g., mountain biking, skiing, gardening, and traveling) have been cited by many PICU colleagues as their way to recharge and continue on the job. Cultivating a spiritual life, within or outside an organized religion, has been found to be important to sustain one’s motivation and engagement. To focus on what really matters in the work I do – the children and their families – certainly helps me keep going. There is much that can be done to make the PICU a more “morally habitable” place. Given the current levels of reported MoD, health administrators need to attend to this important workplace factor and ensure the support necessary for staff to address it. As practitioners, we ought to develop insight into our feelings and reactions, contribute to an open environment where team work is a healthy practice, and adopt a lifestyle that recharges us for the next difficult case. Author Contributions The author confirms being the sole contributor of this work and approved it for publication. Conflict of Interest Statement The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author wish to thank Dr. Wendy Austin, Ph.D, Faculty of Nursing, University of Alberta, for editing and making important suggestions for this paper. ==== Refs References 1 Wallace JE Lemaire JB Ghali WA . Physician wellness: a missing quality indicator . Lancet (2009 ) 374 :1714 –21 .10.1016/S0140-6736(09)61424-0 19914516 2 Shanafelt TD Enhancing meaning in work: a prescription for preventing physician burnout and promoting patient-centered care . JAMA (2009 ) 302 :1338 –40 .10.1001/jama.2009.1385 19773573 3 Jameton A Nursing Practice: The Ethical Issues . 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J Crit Care (2016 ) 31 :271 –2 .10.1016/j.jcrc.2015.11.005 26700610 29 Bosslet GT Pope TM Rubenfeld GD Lo B Truog RD Rushton CH An official ATS/AACN/ACCP/ESICM/SCCM policy statement: responding to requests for potentially inappropriate treatments in intensive care units . Am J Respir Crit Care Med (2015 ) 191 :1318 –30 .10.1164/rccm.201505-0924ST 25978438 30 Brandon D Ryan D Sloane R Docherty D Impact of a pediatric quality of life program on providers’ moral distress . MCN Am J Matern Child Nurs (2014 ) 39 (3 ):187 –97 .10.1097/NMC.0000000000000025 31 Valentin A . The importance of risk reduction in critically ill patients . Curr Opin Crit Care (2010 ) 16 :482 –6 .10.1097/MCC.0b013e32833cb861 20613502 32 Carnevale FA Ethical care of the critically ill child: a conception of a “thick” bioethics . Nurs Ethics (2005 ) 12 :239 –52 .10.1191/0969733005ne786oa 15921341 33 Lilly C Sonna L Haley K Massaro A . Intensive communication: four-year follow-up from a clinical practice study . 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==== Front Front OncolFront OncolFront. Oncol.Frontiers in Oncology2234-943XFrontiers Media S.A. 10.3389/fonc.2016.00194OncologyOriginal ResearchAssociation of Family History of Type 2 Diabetes with Prostate Cancer: A National Cohort Study Ji Jianguang 1*Sundquist Jan 1Sundquist Kristina 11Center for Primary Health Care Research, Lund University, Malmö, SwedenEdited by: Imtiaz Ahmad Siddiqui, University of Wisconsin-Madison, USA Reviewed by: Marie-Elise Parent, Institut national de la recherche scientifique, Canada; Matthew R. Bonner, University at Buffalo, USA *Correspondence: Jianguang Ji, jianguang.ji@med.lu.seSpecialty section: This article was submitted to Cancer Epidemiology and Prevention, a section of the journal Frontiers in Oncology 29 8 2016 2016 6 19424 5 2016 18 8 2016 Copyright © 2016 Ji, Sundquist and Sundquist.2016Ji, Sundquist and SundquistThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Background Personal history of type 2 diabetes mellitus (T2DM) is associated with a lower incidence of prostate cancer, but the underlying mechanisms are largely unknown. We hypothesized that genetic factors that are involved in the development of T2DM might protect against prostate cancer. Methods We used a few Swedish registers, including the Swedish Multigeneration Register and the Cancer Register, to examine the risk of prostate cancer among men with a family history of T2DM. Standardized incidence ratios were used to calculate the relative risk. Results The overall risk of prostate cancer among men with a familial history of T2DM was 0.87 (95% CI: 0.86–0.89) as compared to matched controls. The risk was even lower for those multiple affected relatives with T2DM, and it was 0.86 for those with two affected relatives and 0.67 for those with three and more affected relatives. Conclusion Family history of T2DM was associated with a lower incidence of prostate cancer, and the risk was even lower for those with more than one affected relative. Our study strongly suggests that genetic factors or shared familial factors, such as obesity, that contributed to T2DM may protect against prostate cancer. prostate cancerfamilial riskincidencetype 2 diabetesVetenskapsrådet10.13039/501100004359K2012-70X-15428-08-3 ==== Body Introduction The incidence of type 2 diabetes mellitus (T2DM) has continuously increased worldwide and in Sweden (1), partly due to the increasing trend of obesity, which is one of the main risk factor of T2DM (2). Personal history of T2DM has consistently reported to be associated with an increased incidence and mortality of various types of cancer (3, 4), with one exception of prostate cancer (5–7). The underlying mechanisms are still largely unknown. Lower detection rate of prostate cancer due to altered health care seeking behaviors in T2DM, such as PSA examination, has been suggested to contribute to the inverse association (5, 8). The US Multiethnic Cohort study found that the frequency of PSA testing is around 44% in diabetics, whereas the frequency is 48 in non-diabetics (8). Other factors, such as low-androgen level in T2DM as well as the protective effects of diabetes medication (9), may also contribute to the lower incidence of prostate cancer. However, it is still unknown whether genetic factors that are involved in the development of T2DM might protect against prostate cancer. Shared genetic component between T2DM and prostate cancer has been investigated in many previous studies by using multiple single-nucleotide polymorphisms. Pierce and Ahsan found that the genetic score of T2DM based on 18 SNPs showed an inverse association with prostate cancer (10). Another study using individual SNPs and aggregations of 36 T2DM susceptibility loci found an association with prostate cancer (11). In the current study, we explored the hypothesis that genetic factors may partly explain the inverse association between T2DM and prostate cancer by examining the incidence of prostate cancer among Swedish men with a family history of T2DM as compared to men without a family history. Patients and Methods This cohort study was approved by the Regional Ethical Review Board of Lund University, Sweden in 2013. This study was carried out by using several nationwide Swedish Registers. The Swedish Cancer Register, which was founded in 1958 by the National Board of Health and Welfare and has almost complete nationwide coverage (12). All physicians in Sweden must report all cases of cancer to the Swedish Cancer Registry according to instructions by the National Board of Health and Welfare based on clinical and pathological reports (13). The majority of cancer cases were notified twice from separate reports, guaranteeing a high accuracy rate at a national level. A 4-digit diagnosis code according to the 7th revision of the International Classification of Diseases (ICD-7) has been used in the Swedish Cancer Register. The Multigeneration Register (14), which was created and maintained by Statistics Sweden, includes all children born in Sweden in 1932 and later (maximally 80 years old at 2012) and their siblings and biological parents. More than 14.4 million individuals (living and deceased) in more than 3.8 million families were included in the Multigeneration Register. The Swedish Hospital Discharge Register, which was founded in 1964 by the National Board of Health and Welfare and has had complete national wide coverage since 1987 (15), and the Swedish Outpatient Register, founded in 2001 with complete coverage (16), were used to identify a cohort of patients with T2DM. Diagnoses of diabetes were reported according to the different versions of ICD codes. ICD-9 code of 250 was used to retrieve patients diagnosed with diabetes in years between 1987 and 1996; ICD-10 code of E11 was used between 1997 and 2012. The quality of the Swedish Hospital Register has been examined extensively (17). As compared to the diagnoses from medical records, the positive predictive values (PPV) are generally 85–95%. Additional linkages were made to the Swedish National Population and Housing Census (18) to obtain information on individual-level characteristics, such as year of birth, gender, socioeconomic status, and region of residence; to the Cause of Death Register to identify date of death; to the Emigration Registry to identify date of emigration. All linkages were performed using individual national identification numbers, which were replaced with serial numbers in order to preserve anonymity. Study Population The study population was men who were born between 1932 and 1957 and were still alive in 1987 (age ranging between 30 and 55 at the beginning of study). Using the hospital records, we identified all the men who had a family history of T2DM. Five men from the general population without a family history of T2DM were matched according to year of birth, socioeconomic status, and regions of residence. Individuals who were diagnosed with cancer before 1987 were excluded from the current study. Outcome Variable The Swedish Cancer Register recorded both the sites and histological types of cancer. Prostate cancer was defined by the ICD-7 code of 177. Only the first primary prostate cancer was considered in the present study. Predictive Variable Familial history of T2DM was identified if individuals had one or more first degree relatives (parents and siblings) diagnosed with T2DM between 1987 and 2012. T2DM and type 1 diabetes mellitus (T1DM) were first distinguished from the Swedish Hospital Register in 1997 by using ICD-10 codes. To guard against inclusion of T1DM patients diagnosed between 1987 and 1996, we used an age at diagnosis of diabetes over 39 years to define T2DM during 1987 and 1996, as is done in the Swedish Diabetes Registry. Individual-Level Variables Adjusted in the Model Other variables that were associated with prostate cancer included age and period at diagnosis, which was categorized into 5-year groups, socioeconomic status, and regions of residence. We classified each individual’s socioeconomic status into one of six categories: (1) farmer, (2) manual worker, (3) blue collar, (4) professional, (5) private, and (6) other. Geographic region of residence was divided into large cities (those with a population of >200,000, i.e., Stockholm, Gothenburg, and Malmö), Southern Sweden, Northern Sweden, and unknown. Statistical Analysis Person-years at risk (number of persons at risk multiplied by time at risk) were calculated from the start of follow-up on 1 January 1987 until the diagnosis of cancer or death, emigration or the end of follow-up (31 December 2012). Standardized incidence ratios (SIRs) were calculated as the ratio of observed to expected number of cases. SIRs were used to measure the relative risk of prostate cancer in men with a family history of T2DM compared with matched controls. The expected number of cases was calculated for age (5-year groups), follow-up interval (5-year groups), socioeconomic status, and region of residence-specific standard incidence rates derived from individuals lacking an affected family member (19). Ninety-five percent confidence intervals were calculated assuming a Poisson distribution. Data values were accurate to two decimal places. In our exposure definition, all participants with deceased parents before 1987 would be classified as having no family history of T2DM, which may lead to misclassification of exposure. We, thus, calculated the adjusted SIR in the Table A1 in Appendix based on the percentage of misclassification of the exposure in the control group. All analyses were performed using SAS® version 9.2 (SAS Institute, Cary, NC, USA). Results A total of 198,129 men were retrieved from the databases with a family history of T2DM (Table 1). The median age was 41 at the beginning of follow-up (year 1987). The median follow-up time was 24 years. During the study period, 4.6% of them with a family history of T2DM were diagnosed with prostate cancer, whereas the proportion was 5.3% for those without a family history. Table 1 Basic characteristics among men with a family history of type 2 diabetes and matched control. Basic characteristics With family history Matched control Number % Number % Age  30–39 82,239 41.5 411,195 41.5  40–49 88,825 44.8 444,125 44.8  ≥50 27,065 13.7 135,325 13.7  Median (year) 41 41 Socioeconomic status  Farmer 4012 2.0 20,060 2.0  Manual worker 77,703 39.2 388,515 39.2  Blue collar 48,426 24.4 242,130 24.4  Professional 24,258 12.2 121,290 12.2  Private 12,875 6.5 64,375 6.5  Others 30,855 15.6 154,275 15.6 Region  Large cities 63,063 31.8 315,315 31.8  Southern 82,851 41.8 414,255 41.8  Northern 47,943 24.2 239,715 24.2  Unknown 4272 2.2 21,360 2.2 Prostate cancer  No 189,197 95.4 937,869 94.7  Yes 8932 4.6 52,776 5.3 All 198,129 100.0 990,645 100.0 The overall risk of prostate cancer in men with a family history of T2DM was 0.87 (95% CI: 0.86–0.89) as compared to matched non-exposed group (Table 2). The risk was even lower for those with more than one affected relative with T2DM, and it was 0.86 for those with two affected relatives and 0.67 for those with three and more affected relatives. Those with both parental and sibling history of T2DM has a SIR of 0.81, as compared to those with only parental history (0.95) or those with only sibling history (0.84). In addition, we examined the risk of prostate cancer among individuals with both a family history of T2DM and a personal history of T2DM, the SIR was 0.71 (N = 561, 95% CI: 0.65–0.77); the SIR was 0.94 (95% CI: 0.92–0.96) for those without personal history of T2DM but having at least one relative with T2DM. Sensitivity analyses in Table A1 in Appendix suggest that misclassification of exposure in the control group had limited effect on our observation. Table 2 Risk of prostate cancer among individuals with a family history of type 2 diabetes as compared to matched non-exposed group. Characteristics E O SIR 95% CI Overall 10216.3 8932 0.87 0.86 0.89 Numbers of affected relatives  One 8706.6 7675 0.88 0.86 0.90  Two 1278.3 1102 0.86 0.81 0.91  Three and more 231.5 155 0.67 0.57 0.78 Type of family history  Parental history 3407.2 3238 0.95 0.92 0.98  Sibling history 5994.4 5032 0.84 0.82 0.86  Both parental and sibling history 814.7 662 0.81 0.75 0.88 E, expected number of cases; O, observed number of cases; SIR, standardized incidence ratio, and adjusted for age, period, socioeconomic status, and region of residence; Bold type, 95% CI does not include 1.00. Discussion In this population-based nationwide cohort study, we found that the overall incidence of prostate cancer was significantly lower when first degree relatives (including parents and siblings) were diagnosed with T2DM as compared to matched controls. The incidence was even lower for those with more than one affected relatives, strongly suggesting that genetic factors that contributed to T2DM may protect against the development of prostate cancer. One advantage of the present study is that all the data were retrieved from nationwide databases guaranteeing reliable estimation. All the prostate cancer patients were identified from a nationwide population database with high accuracy and high coverage. In addition, the study population could be followed completely. In Sweden, patients with T2DM were normally diagnosed by two doctors, one from primary health-care center, and one from specialists in the hospitals, which can guarantee high accuracy as compared to self-reported questionnaire. Many confounding factors, including age at diagnosis, socioeconomic status, and regions of residence, were adjusted in the analyses. However, a few limitations should be kept in mind when interpreting the observed associations. One limitation of this study is that we had no information about other individual-related factors, such as diet, smoking, and obesity. Another limitation is that some T2DM patients do not require hospitalization and the present results might be applicable only to hospitalized patients with probably a severe disease (20). However, low sensitivity should not lead to differential bias in the current study. In addition, a proportion of men classified as non-prostate cancer cases in the current study might have latent undiagnosed prostate cancer. However, such non-differential bias could lead to our results to null. The probability of having a family history of T2DM depends on the number of relatives, and on their respective attained ages at the time of study. Although the matching by year of birth might have attenuated the impact of family structure disparities, it might be important and necessary to include family structures in future studies. To our knowledge, this is the first nationwide study to assess the risk of prostate cancer in relation to a family history of T2DM. The relative risk of prostate cancer was 13% lower in men with a family history of T2DM as compared to the references, which was very similar to the subsequent risk of prostate cancer in Swedish T2DM patients (adjusted SIR of 0.88) (6). Familial negative association between T2DM and prostate cancer is necessary, but not sufficient, to infer a genetic cause. Both shared genetic and environmental factors might contribute to familial aggregation. It is known that obesity is associated with T2DM, whereas obesity has also been noted to be negatively associated with non-aggressive prostate cancer and positively associated with aggressive prostate cancer (21). Families with several obese relatives will be more likely to have several members with T2DM. In such families, prostate cancer under-detection might explain a negative association with non-aggressive prostate cancer, the most common form of the disease. In addition, we found individuals with a family history of T2DM and diagnosed with T2DM had a very low risk of prostate cancer, together with the evidence that those men with more than one affected relative with T2DM had a very lower risk of prostate cancer, suggesting that genetic factors common to T2DM and prostate cancer are indeed involved, and they could contribute greatly to the current findings. Many genome-wide association studies (GWAS) were done to explore the contribution of common genetic variation on T2DM and prostate cancer (22–27), but it is still less known whether these two diseases share genetic factors. Based on our current research findings, it is highly recommended to explore the loci for both diabetes and prostate cancer that were already identified by GWAS and to understand whether there are shared genetic factors contributed for the inverse association between these two common diseases. In summary, family history of T2DM was associated with a lower incidence of prostate cancer, and the risk was even lower for those with more than one affected relative. Our study suggests that genetic factors or shared familial factor (such as obesity) that contributed to T2DM may protect against prostate cancer, but further studies are needed to explore which genetic factors contribute to the observed familial negative association. Author Contributions JJ, JS, and KS designed the study; JS and KS obtained the data; JJ did the analyses; JJ wrote the manuscript; JJ, JS, and KS approved the final manuscript. JJ was the guarantor. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This work was supported by grants to Dr. Kristina Sundquist from the Swedish Research Council (K2012-70X-15428-08-3), as well as ALF funding from Region Skåne awarded to JS, KS, and JJ. The funding agencies had no role in the design and conduct of the study; in the collection, analysis, and interpretation of the data; or in the preparation, review, or approval of the manuscript. The funding sources had no role in study design, in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. The corresponding author has full access to all the data in the study and had final responsibility for the decision to submit for publication. 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==== Front Physiol RepPhysiol Rep10.1002/(ISSN)2051-817XPHY2physreportsPhysiological Reports2051-817XJohn Wiley and Sons Inc. Hoboken 2755899910.14814/phy2.12847PHY212847Respiratory Conditions Disorder and DiseasesAgeing and DegenerationImmunologyRegulatory PathwaysOriginal ResearchOriginal ResearchExpression of polycomb protein BMI‐1 maintains the plasticity of basal bronchial epithelial cells E. Torr et al.Torr Elizabeth 1 Heath Meg 2 Mee Maureen 3 Shaw Dominick 1 Sharp Tyson V. 4 Sayers Ian 1 1 Division of Respiratory MedicineQueens Medical CentreUniversity of NottinghamNottinghamUnited Kingdom2 Cytogenetics UnitNottingham City HospitalHucknall RoadNottinghamUnited Kingdom3 School of Life SciencesQueens Medical CentreUniversity of NottinghamNottinghamUnited Kingdom4 Centre for Molecular OncologyBarts Cancer InstituteQueen MaryUniversity of LondonLondonUnited Kingdom* Correspondence Ian Sayers, Division of Respiratory Medicine, Queen's Medical Centre, University of Nottingham, Nottingham, NG7 2UH, United Kingdom. Tel: 0115 82 31066 Fax: 0115 82 31059 E‐mail: ian.sayers@nottingham.ac.uk 24 8 2016 8 2016 4 16 10.1111/phy2.2016.4.issue-16e1284707 4 2016 07 6 2016 07 6 2016 © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.Abstract The airway epithelium is altered in respiratory disease and is thought to contribute to disease etiology. A caveat to disease research is that the technique of isolation of bronchial epithelial cells from patients is invasive and cells have a limited lifespan. The aim of this study was to extensively characterize the plasticity of primary human bronchial epithelial cells that have been engineered to delay cell senescence including the ability of these cells to differentiate. Cells were engineered to express BMI‐1 or hTERT using viral vector systems. Cells were characterized at passage (p) early (p5), mid (p10), and late (p15) stage for: BMI‐1, p16, and CK14 protein expression, viability and the ability to differentiate at air–liquid interface (ALI), using a range of techniques including immunohistochemistry (IHC), immunofluorescence (IF), transepithelial electrical resistance (TEER), scanning electron microscopy (SEM), MUC5AC and beta tubulin (BTUB) staining. BMI‐1‐expressing cells maintained elevated levels of the BMI‐1 protein and the epithelial marker CK14 and showed a suppression of p16. BMI‐1‐expressing cells had a viability advantage, differentiated at ALI, and had a normal karyotype. In contrast, hTERT‐expressing cells had a reduced viability, showed limited differentiation, and had an abnormal karyotype. We therefore provide extensive characterization of the plasticity of BMI‐1 expressing cells in the context of the ALI model. These cells retain properties of wild‐type cells and may be useful to characterize respiratory disease mechanisms in vitro over sustained periods. BMI‐1bronchial epithelial cellslifespanplasticityAsthma UK10/006 source-schema-version-number2.0component-idphy212847cover-dateAugust 2016details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_NLMPMC version:4.9.4 mode:remove_FC converted:29.08.2016 E. Torr , M. Heath , M. Mee , D. Shaw , T. V. Sharp , I. Sayers . Expression of polycomb protein BMI‐1 maintains the plasticity of basal bronchial epithelial cells . Physiol Rep , 4 (16 ), 2016 , e12847, doi: 10.14814/phy2.12847 Funding Information This work was supported by Asthma UK Grant 10/006. ==== Body Introduction The airway epithelium acts as the critical interface between the environment and organ physiology. It acts as a barrier to potential pathogens and extraneous particles and helps regulate host defense mechanisms, including the inflammation process. Under normal conditions, the bronchial epithelium is composed of ciliated columnar, mucus‐secreting goblet and Clara cells that secrete surfactant. There is accumulating evidence that the airway epithelium is intrinsically different in airway diseases such as asthma and chronic obstructive pulmonary disease (COPD) if compared with the normal type. For example, bronchial epithelial cells isolated from asthma patients and cultured in vitro have: (1) defective repair mechanisms (Kicic et al. 2010), (2) altered barrier properties including reduced tight junction protein expression and formation (Xiao et al. 2011), (3) elevated expression of proremodeling factors (Lopez‐Guisa et al. 2012), and (4) when differentiated have increased numbers of goblet and reduced numbers of ciliated cells (Parker et al. 2010; Gras et al. 2012). Similarly, genetic studies have identified a large number of asthma susceptibility genes that are differentially expressed in the airway epithelium in asthma including interleukin 33 (IL33) and thymic stromal lymphopoietin (TSLP). This led to the hypothesis that the airway epithelium contributes to disease etiology. Therefore, the airway epithelium represents an attractive therapeutic target in asthma. However, more research tools and cellular assay systems are needed to rapidly move this area of important research forward. The most common approach to study bronchial epithelial cells from healthy controls and patients with respiratory disease is to isolate cells using bronchoscopic brush technique (Kelsen et al. 1992) and then culture the cells as basal cell monolayers or using air–liquid interface (ALI) differentiation (Stewart et al. 2012a,b). ALI cells form an epithelial barrier that closely resembles the in vivo architecture of the airway epithelium and is composed of basal, goblet, and ciliated cells allowing data to be generated in a more physiological context. However, the bronchoscopic procedure is invasive with risk to the individual, especially in people with severe asthma. Moreover, adequate numbers of cells are hardly collected. Similarly, once isolated and grown in vitro, the primary airway epithelial cells have a limited lifespan (i.e., the point at which the cells stop proliferating, typically 4–5 passages). This is often too short to obtain key experimental data. Importantly, after p4 bronchial epithelial cells begin to deviate in their morphology/phenotype, lose their plasticity required for the differentiation at ALI. These factors make the identification and careful characterization of a method to delay cell senescence while maintaining the plasticity of the cells highly desirable. One approach to delay cell senescence is the suppression of cyclin‐dependent kinase inhibitor, p16(Ink4a), a tumor suppressor that induces a G1 cell cycle arrest. B‐cell‐specific Moloney murine leukemia virus integration site 1 (BMI‐1) is a polycomb protein thought to repress p16(Ink4a) expression and has previously been shown to delay cell senescence (Vonlanthen et al. 2001). In this study, we set out to engineer primary human bronchial epithelial cells to express different levels of BMI‐1 via alternative promoter constructs and extensively characterize the cells for effects on viability and plasticity such as the ability to differentiate at ALI. This was assessed using a range of techniques including immunohistochemistry (IHC), immunofluorescence (IF), transepithelial electrical resistance (TEER), and scanning electron microscopy (SEM). Importantly, we also investigated genome stability/integrity through effects on karyotype. Both wild‐type early passage cells and cells engineered to express hTERT (as a commonly used immortalization technique) were included in these analyses. We demonstrate the BMI‐1‐engineered cells assessed at early (p5), mid (p10), and late (p15) passage produce elevated levels of BMI‐1 expression with associated suppression of p16. These cells retain both viability and differentiation potential of wild‐type cells while importantly not demonstrating the changes in cell karyotype. These features were apparent across all donor cell populations engineered to express the elevated levels of BMI‐1. These data are in contrast to hTERT cells that were no longer viable and did not differentiate at mid passage and demonstrated gross chromosomal abnormalities. Therefore, BMI‐1‐engineered bronchial epithelial cells delayed cell senescence and retained their plasticity including the ability to differentiate at ALI in a comparative manner to low passage unmodified cells. Materials and Methods BMI‐1 plasmid construction Human BMI‐1 full‐length cDNA was originally obtained from Geneservice™ Mammalian Gene Collection (MGC) Clone, amplified and cloned into pLVX‐Puro (Clontech) using XhoI/BamHI restriction sites and into pFLRu‐FH using MluI/BamHI restriction sites, using standard molecular biology techniques. The retroviral vector pWZL‐BLAST‐FLAG‐HA‐hTERT and corresponding empty vector backbone were purchased from Addgene (Maida et al. 2009). All vectors were sequence verified. Packaging of lentivirus and retrovirus HEK293T cells were seeded at 2 × 106 cells per 10‐cm diameter dish and were cultured in DMEM, 10% FCS, 1% penicillin/streptomycin overnight. For lentiviral production, the cells were transfected with a mix containing an 8:1 ratio of packaging: envelope vectors, that is, 4.4 μg of pCMV delta R8.9, 0.6 μg of pCMV‐VSV‐G (McKay et al. 2011), and 5 μg of the appropriate lentiviral vector (empty or BMI‐1 insert) with 36 μL of TransIT‐LT1 (Geneflow). For retroviral production, the cells were transfected with 5 μg of pKAT (Addgene) and 5 μg of retroviral vector (empty or with hTERT insert) and 36 μL of TransIT‐LT1 (Geneflow). Cells were grown for 48–72 h and the supernatant containing packaged virus was harvested and ultracentrifuged (48,384 g for 3.5 h at 4°C) to produce a concentrated viral stock which was stored at ‐80°C. Primary bronchial epithelial cell culture and air liquid interface Normal Human Bronchial Epithelial Cells (NHBEC) were purchased from Lonza, (Wokingham, UK). Donor 1 cells were isolated from a 43‐year‐old Caucasian male with no history of smoking; Donor 2 cells were isolated from a 56‐year‐old Caucasian male smoker. NHBEC were grown in a growth factor‐supplemented medium (BEGM) (Lonza) and differentiated at ALI in bronchial epithelial differentiation medium (BEDM), according to our previously published methods (Stewart et al. 2012a,b). BEDM is composed of 50:50 Dulbecco's Modified Eagle's Medium (DMEM, Sigma‐Aldrich, Dorset, UK):BEBM with Lonza singlequots, excluding triiodo‐L‐thyronine and retinoic acid, but including GA‐1000 (Gentamicin and Amphotericin‐B). BEDM is supplemented with 50 nmol/L retinoic acid, added at time of use. All cells were cultured on 6.5‐mm polyester Transwell inserts with a pore size of 0.4 μm (Corning Life Sciences, Amsterdam, The Netherlands). Cells were plated at 30,000 cells per insert in an appropriate medium. When confluent (~3 days), cells were raised to ALI. Medium was replaced and the apical face washed with media every 48 h. Cells were fixed for immunostaining, histology sectioning, and scanning electron microscopy after 28 days at ALI. Generation of genetically modified NHBEC Passage 2 NHBEC were plated in a 6‐well plate at 5 × 104 cells per well and grown overnight. These were purchased heterogeneous NHBEC and not clonal cells. The media was replaced with 800 μL of BEGM with 2 μg/mL of polybrene (Hexadimethrine bromide H9268 Sigma) and 6.25 μL of lentivirus or 12.5 μL of retrovirus stock solutions (minimal concentrations to give >90% transfection determined empirically). The plates were incubated at 37°C for 6.5 h with gentle rocking of the plate every 30 min for the first 2 h. The viral mixture was removed and replaced with BEGM. After 72 h, selection media was added to the wells with 300 ng/mL puromycin for the lentiviral‐infected cells and 600 ng/mL blasticidin for the retroviral cells. Cells with antibiotic resistance after 6 days of selection were considered to have been stably infected with the appropriate virus expressing BMI‐1 or hTERT. Western blotting A total of 2 × 105 cells were lysed in SDS loading buffer, heat denatured, and proteins were separated by electrophoresis using 12% SDS resolving gels. The proteins were transferred to nitrocellulose and probed for BMI‐1, p16, and β actin expression using mouse anti‐p16 (Millipore MAB4133; Watford, UK) at 1 in 1000 dilution (1 μg/mL), mouse anti‐BMI‐1 (Millipore Clone F6 Cat No 05‐637) primary antibody at 1 in 1000 dilution, and rabbit polyclonal anti‐β actin (Abcam ab8227, lot 712923, 0.65 mg/mL) primary antibody at 1 in 5000 dilution. Secondary antibodies were used at 1 in 10000 dilutions and consisted of goat anti‐mouse HRP (Jackson Immuno 115‐035‐062) and goat polyclonal anti‐rabbit HRP (Sigma A0545). ECL reagent was used to visualize proteins as directed by the manufacturer (GE Healthcare RPN2209; GE Healthcare, Amersham, UK). Immunofluorescence ALI‐cultured cells were fixed in situ on inserts and transferred to the glass slides for visualization. Cells were fixed using 4% formaldehyde and blocked/permeabilized with PBS, 10% goat serum, 1% BSA, and 0.15% Triton‐X. Cells were incubated with appropriate primary antibodies at 4°C overnight, and FITC labeled secondary for 1 h at room temperature before mounting in HardSet DAPI (Vector Labs). These methods were as previously described (Stewart et al. 2012b) with the addition of BMI‐1 (Millipore Clone F6) and CK14 (Chemicon MAB3232) antibodies to the panel. Cells were visualized using the Zeiss spinning disk confocal microscope and Volocity software (PerkinElmer, Cambridge, UK). Cell viability A total of 2.5 × 103 cells were plated in quadruplicate wells of a 96‐well plate in 200 μL BEGM + GA and incubated for 96–120 h before analysis. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) at 500 μg/mL in BEGM was added to the wells for 4 h before termination of the experiment. The media was then replaced with 200 μL isopropanol and incubated for 10 min to lyse the cells. The plate was read at 570 nm (with a background subtraction of 690 nm) using a Flexstation (Molecular Devices, Wokingham, UK). Transepithelial electrical resistance Transepithelial electrical resistance (TEER) was measured in differentiating cells using an EVOM2 epithelial volt‐ohm meter (World precision Instruments UK, Stevenage) to indicate development of tight junctions as described previously (Stewart et al. 2012b). Briefly, medium was aspirated and replaced with 1 mL growth medium in the basolateral and 0.5 mL growth medium in the apical compartment. Cultures were equilibrated in the incubator for 30 min before TEER measurement. Apical medium was then aspirated to restore ALI. TEER values of insert and medium alone wells was subtracted from the measured TEER and Ωcm2 was calculated by multiplying by the insert area. For comparison across cell lines and passage, we calculated area under curve using TEER data from days 1–28 using GraphPad Prism 6.02 for Windows (GraphPad Software, San Diego, CA). Karyotyping Cells in active growth phase were treated with demecolcine solution (Sigma) at 400 ng/mL for 4 h before harvesting, swelling (hypotonic solution [0.056 mol/L KCL]), and fixing in methanol/glacial acetic acid. The cells were fixed further two times before slides were produced and aged, then banded using trypsin and Giemsa staining before imaging and analyses (completed by Cytogenetics Unit, City Hospital, Nottingham, UK). Histological analyses Fixed transwells were paraffin embedded, sectioned, and then stained with hematoxylin and eosin (H & E) or alcian blue using standard approaches. Scanning electron microscopy Transwells at day 28 ALI were washed with BEGM media to remove any mucous and then fixed with 3% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer for a minimum of 24 h. The wells were washed three times with 0.1 mol/L phosphate/cacodylate buffer and post fixed for 1 h at room temperature with 1% aqueous osmium tetroxide. The samples were washed with distilled water and then dehydrated using a graded series of ethanol washes (50, 70, 90, and 100%, respectively). Samples remained submerged in 100% ethanol, processed when they were dried, and coated with gold particles. The samples were then imaged at low and high magnification using a scanning electron microscope (model JSM‐35; JOEL). Telomerase activity assay TRAPeze XL Telomerase detection kit was used as directed by the manufacturer to determine hTERT activity (Millipore S7707). A total of 1 × 105 cells were suspended in 200 μL of CHAPS lysis buffer and incubated on ice for 30 min. The positive cells (1 × 106, provided by the kit) were treated in the same way. The samples were then centrifuged at 120,000g for 20 min at 4°C. PCR tubes containing 10 μL 5X TRAPeze XL reaction mix, 0.4 μL Taq polymerase (5units/μL), 37.6 μL dH2O, and 2 μL of cell extract, positive template or buffer for the control samples were run at 30°C for 30 mins followed by 36 cycles of 94°C for 30 sec, 59°C for 30 sec, and 72°C for 1 min. This was followed by 72°C for 3 min, 55°C for 25 min, and 4°C hold. The samples were then diluted in 150 μL of buffer (10 mmol/L Tris‐HCl pH 7.4, 0.15 mol/L NaCl, 2 mmol/L MgCl2) and fluorescent signals were read at excitation 485 nm and emission 535 nm and excitation 585 nm and emission 620 nm on the Flexstation (Molecular Devices). Statistical analyses GraphPad Prism 6.02 for Windows was used for statistical analyses. Quantitative data were analyzed using Student's t test or ANOVA with Dunnett's multiple comparison test. A P <0.05 was considered significant. Results Cell generation We specifically chose two lentiviral vectors with differing promoters: Cytomegalovirus (CMV) in pLVX‐Puro or human ubiquitin C (UbiC)) in pFLRu‐FH to express BMI‐1 in human bronchial epithelial cells with the hypothesis that the CMV promoter may lead to greater BMI‐1 overexpression than the UbiC promoter (Qin et al. 2010). Lentiviruses were used to transduce passage 2 NHBECs from two donors. This protocol therefore generated four independent cell populations expressing recombinant BMI‐1 to evaluate the effect of BMI‐1 on cell plasticity. The transduction efficiency was >95%, as indicated by limited cell death following antibiotic selection and the pWZL‐based constructs gave similar findings (data not shown). Cell populations generated were followed up for ~12 months with focused analyses presented for (1) Early passage (infection passage 3/actual passage 6), (2) Mid passage (infection passage 5–7/actual passage 8–10), and (3) Late passage (infection passage 8–12/actual passage 11–15). Finally, a subset of analyses was completed using extended passage cells (infection passage 17/actual passage 20). It is important to note that we arbitrarily designated these early, mid, and late passage definitions based on passage 20 being the maximum achieved. NHBECs transduced with lentivirus containing plasmid vector that did not contain the BMI‐1 expression cassette, that is, empty vectors, stopped growing at mid passage. Similarly, for the cells containing the pWZL constructs including hTERT insert cells were not viable at mid passage, although there were donor differences in these cell senescence times. NHBEC‐BMI‐1 cell populations maintain elevated levels of BMI‐1 expression and feature p16 suppression during prolonged culture Both the pFLRu‐BMI‐1(UbiC promoter) and pLVX‐BMI‐1 (CMV promoter)‐engineered cells demonstrated elevated BMI‐1 expression as evidenced by Western blotting in comparison with control vectors without insert at early passage (Fig. 1A). Importantly, the elevated level of BMI‐1 observed in these cell populations was accompanied by a decrease in the level of CDK inhibitor p16 (Fig. 1B). Similarly, BMI‐1 immunofluorescence for wild‐type cells (control) at passage 3 showed weak BMI‐1 protein expression in comparison with high nuclear BMI‐1 expression in engineered late passage cells for both donors demonstrating robust elevated BMI‐1 through to late passage (Fig. 1C). Early and mid passage cells showed similar elevated BMI‐1 expression as determined by immunofluorescence (data not shown). In order to confirm that genetic modification of cells to overexpress hTERT translated to increased hTERT enzymatic activity, we determined telomerase activity in wild‐type cells, cells transfected with empty vector, and those cells engineered to express hTERT. hTERT activity was elevated in the hTERT‐expressing cell populations in both donors across early‐mid‐late passages as anticipated (Fig. 1D). Figure 1 BMI‐1 epithelial cell populations express elevated levels of BMI‐1 protein and show suppression of p16 protein levels. (A) Early passage‐engineered cells express elevated levels of BMI‐1 compared to empty vector control cell population as determined by Western blot and (B) have suppressed levels of p16, (C) BMI‐1 immunofluorescence analyses for wild‐type cells donor cells (control) show moderate BMI1 levels, whereas BMI‐1‐engineered late passage cells show elevated levels in both vector‐based approaches, Donor 1 (i–iii) and Donor 2 (iv–vi)). (D) hTERT activity is elevated in pWZL‐hTERT cell population in both donors compared to wild‐type cells (control) and control vector cells (individual experiment for each donor). Donor 1 (fill) and donor 2 (no fill). Positive control is provided by the assay kit. *Note late passage data not available for pWZL‐hTERT donor 2 as this cell population stopped growing. NHBEC‐BMI‐1 cell populations have extended viability In order to investigate the effect of the genetic modifications on cell viability, we used the MTT assay as described (Portelli et al. 2014). Early passage cells containing either BMI‐1 vector or pWZL‐hTERT showed increased viability in both donors over 96 or 120 h (Fig. 2A, B and C, P < 0.05). Similarly, mid passage cells showed alterations in viability compared to wild‐type cells over 96 h (empty vector controls were no longer viable at this stage). For donor 1, both BMI‐1 cell populations increased viability compared to wild‐type cells (ANOVA, post hoc P < 0.05), whereas hTERT‐engineered cells did not show altered viability compared to wild‐type control. For donor 2, pFLRu‐BMI‐1 significantly increased cell viability and hTERT reduced viability when compared to wild‐type control (ANOVA, post hoc P < 0.05) with pLVX‐BMI‐1 cell viability showing no difference (Fig. 2D). In a similar analysis of late passage cells (assayed over 120 h), both donors showed pLVX‐BMI‐1 significantly increased viability, whereas hTERT cells had a decreased viability (Fig. 2E, ANOVA, post hoc P < 0.05). Late passage pFLRu‐BMI‐1 cells showed no differences in viability compared to wild‐type control cells. These data suggest that late passage NHBECs transduced with the pLVX‐BMI‐1 lentivirus are the only cell populations to maintain high viability. Figure 2 BMI‐1 epithelial cell populations have an extended lifespan with viability increased compared to wild‐type bronchial epithelial cells. Viability (MTT assay) was determined for early passage cells at (A) pFLRu/96 h, (B) pLVX/96 h, and (C) pWZL/120 h demonstrating that the insertion of BMI1 or hTERT leads to increased viability in Donor 1 (solid bars) and Donor 2 (open bars), *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.00005. (D) Mid passage‐engineered cells show alterations in viability compared to wild‐type cells assayed for 96 h. (E) Late passage viability (assayed for 120 h). *note donor 2 hTERT cell population is mid passage (maximum passage achieved). NHBEC‐BMI‐1 cell populations maintain epithelial cell marker expression over prolonged lifespan In order to begin to characterize the cell phenotype in the genetically modified cells, we examined the expression of the epithelial lineage marker, cytokeratin (CK) 14. For both donors, a very similar level and distribution of CK14 was observed in wild‐type cells at passage 3 and late passage cells for both BMI‐1 vectors using immunofluorescence (Fig. 3). Similarly, donor 1 hTERT‐expressing cells showed comparable expression, however, for donor 2 pWZL‐hTERT, the cells did not reach late passage and infection passage 8 (passage 10) was the maximum achieved with limited CK14 expression observed. Figure 3 BMI‐1 epithelial cell populations retain epithelial cell marker expression over extended passage. Immunofluorescence for cytokeratin 14 confirmed epithelial lineage in wild‐type cells (Control) and for late passage engineered cells for Donor 1 (i–iv) and Donor 2 (vi–ix). Note pWZL‐hTERT in donor 2, mid passage was the maximum passage achieved. ALI differentiated NHBEC‐BMI‐1 cell populations show comparative morphology to nonmodified wild‐type cells To compare the morphology of genetically modified cells to low passage (p3) wild‐type cells grown at air–liquid interface, we examined sections stained with H & E and alcian blue to detect mucins. Wild‐type NHBECs generated a pseudostratifed epithelial layer upon ALI showing similarities to that observed in situ including mucin staining at the apical side and the presence of cilia (Fig. 4). Significantly, early, mid, and late passage BMI‐1‐expressing cells from both lenti‐vector constructs maintained the ability to differentiate and showed the classical pseudostratified epithelial layer in this model, however, late passage BMI‐1 cells had more diffuse mucin staining (Fig. 4). In contrast, hTERT‐expressing cells failed to show a differentiated phenotype even at early passage and in mid and late passage, it was not possible to generate sections as the cells did not grow and adhere to the transwell. Figure 4 BMI‐1 epithelial cell populations retain the ability to differentiate at air–liquid interface and show comparative cell composition and morphology to wild‐type cells. Sections of air–liquid interface cultures stained with hematoxylin and eosin or alcian blue stains. Early, mid, and late passage BMI‐1‐expressing cells maintain morphology demonstrating a pseudostratified layer with evidence of differentiation into mucus‐producing cells similar to matched wild‐type cells (passage 3). Wild‐type cells were included in each of the early, mid, and late time point experiments, hence, three panels. hTERT cell populations were not available for all passages as these did not form differentiated layers and the cells clumps did not adhere to transwells, hence, after fixing these cells became detached. Data shown for Donor 2, Donor 1 showed similar results. NHBEC‐BMI‐1 cell populations retain the ability to differentiate into multiple cell types at air liquid interface In order to further characterize the differentiated phenotype of wild‐type and genetically modified cells, we used immunofluorescence for MUC5AC which is a marker of goblet cells and beta tubulin, which is a marker of ciliated cells. These data show that early, mid, and late passage BMI‐1‐expressing cells maintain differentiation potential and generate a differentiated layer composed of multiple cell types comparable to wild‐type control cells from the same experiments (Fig. 5). As anticipated in the z‐stack images, both the beta tubulin and MUC5AC staining is particularly apical. hTERT‐expressing cells maintain these features until cell senescence for this donor. Figure 5 BMI‐1 epithelial cell populations retain the ability to differentiate at air–liquid interface and stain positively for goblet and ciliated cell markers. Immunofluorescence including z‐stacks of markers for goblet cells (MUC5AC) and ciliated cells (beta tubulin IV) generated in early, mid, and late passage BMI‐1 or hTERT‐expressing cells with wild‐type control cells (p3). Wild‐type cells were included in each of the early, mid, and late time point experiments, hence, three panels. Data from donor 1 and representative of both donors, however, donor 2 hTERT‐expressing cells did not provide robust ALI cultures post early passage. Note some data were available for hTERT mid passage cells (in contrast to Fig. 4) due to the gentler processing of the samples for IF. ALI, air–liquid interface; IF, immunofluorescence NHBEC‐BMI‐1 cell populations retain the ability to differentiate and form an epithelial barrier One of the hallmarks of the ALI model is the development of tight junctions between the epithelial cells forming a barrier. A surrogate measure of this is the development of TEER. Therefore, we examined this outcome in both wild‐type and genetically modified cells. Both donors demonstrated the capacity to develop TEER as shown by the wild‐type cells (passage 3) time course data (Fig. 6A and B). In Figure 6A, a decrease in TEER was observed between days 14–21 which we have observed for other primary human cell donors. Similar plots were generated for early, mid, and late passage‐engineered cells and to facilitate multiple comparisons area under the curve values were generated. At early passage donor 1 cells, all developed a TEER except for the pWZL empty vector cell population (Fig. 6C). The level of TEER was variable with pLVX‐BMI‐1 showing the most comparable to wild‐type control levels (Fig. 6C). Donor 2 early passage data demonstrated that only the BMI‐1‐containing cells were capable of developing a TEER and in the case of the pLVX‐BMI‐1, this was higher than the wild‐type control. All empty vector controls and the pWZL‐hTERT did not develop a measurable TEER at passage 6 (Fig. 6D). At mid passage, the empty vector controls were no longer viable and comparison of the engineered cells showed that in both donors, the BMI‐1‐expressing cells demonstrated a measurable TEER, albeit lower than wild‐type, whereas the hTERT‐engineered cells did not (Fig. 6E and F). At late passage, again the BMI‐1‐expressing cells demonstrated a TEER but at a reduced level compared to wild‐type cells (Fig. 6G and H). Across the passage stages, there was a gradual loss of TEER development in the BMI‐1 cell lines when compared to the wild‐type cells (100%) at each stage, for example, Donor 1 pFLRu‐BMI‐1: 56.5%, 28.1%, and 29.1% (of wild‐type control, 100%) across early, mid, and late passages. Similarly, Donor 1 pLVX‐BMI‐1: 89.5%, 43.5%, 42.1% (of wild‐type control, 100%) across early, mid, and late passages. Donor 2 data were more heterogeneous but showed a similar trend. These data demonstrated that the BMI‐1‐expressing cells maintain plasticity, differentiate at ALI, and develop TEER. However, due to our low number of technical replicates, we were not able to provide more quantitative comparisons of the absolute levels of differentiation, for example, TEER achieved across cell populations and/or passages with certainty. Figure 6 BMI‐1 epithelial cell populations retain the ability to differentiate at air–liquid interface and develop transepithelial electrical resistance. Representative TEER traces for (A) donor 1 and (B) donor 2 wild‐type cells at passage 3 demonstrating increasing in TEER during the ALI differentiation. Area under the curve (AUC) analyses of TEER for early passage‐engineered cells for (C) donor 1 and (D) donor 2. Mid passage TEER (AUC) for (E) donor 1 and (F) donor 2 and late passage TEER (AUC) for (G) donor 1 and (H) donor 2. Empty vector control cells failed to grow after early passage stage and so are not present on graphs (E–H). Data shows a single experiment for both donors at each passage. ALI, air–liquid interface; TEER, transepithelial electrical resistance. NHBEC‐BMI‐1 cell populations retain the ability to differentiate forming ciliated cells at the apical surface To further characterize the differentiated epithelial layer observed in the ALI model, we used scanning electron microscopy to visualize the apical surface and examine cilia. These images clearly show cilia projections on the surface of wild‐type cells as we have observed previously (Fig. 7). For genetically engineered cells, cilia were also present including the hTERT genetically modified cells and there was a large degree of heterogeneity. For mid passage pLVX‐BMI‐1 cells, there was potentially a reduced pattern of cilia compared to wild‐type control. Importantly, there was evidence for late passage BMI‐1 cells, particularly the pLVX‐BMI‐1 cells to have ciliated surfaces (Fig. 7). Figure 7 BMI‐1 epithelial cell populations retain the ability to differentiate at air–liquid interface and demonstrate surface cilia in scanning electron microscopy. Wild‐type control cells at passage 3 show evidence of ciliated cells on the surface of air–liquid interface cultures using SEM. Wild‐type cells were included in each of the early, mid, and late time point experiments, hence, three panels. Both BMI‐1‐ and hTERT (when viable)‐expressing cells grown at ALI show some evidence of ciliated cells on the surface. Data taken from donor 1 and representative of both donors were analyzed (×1700 magnification). ALI, air–liquid interface; SEM, scanning electron microscopy. NHBEC‐BMI‐1 cell populations maintain a stable genome over extended passage As many approaches to prevent cell senescence have unwanted effects on the genetic information of the cells, we completed karyotyping of wild‐type and genetically modified cells at each passage stage to identify gross changes in chromosomes (>5 megabase rearrangements). For all BMI‐1‐expressing cells assayed at early, mid, and late passage, normal karyotypes were present. In contrast, an abnormal karyotype was observed for donor 2 hTERT‐expressing cells at mid passage suggesting gross alterations on chromosomes 6, 13, and 16 and chromosomal fragments that could not be mapped (Fig. 8). Figure 8 Karyotyping identifies abnormalities in the hTERT not BMI‐1‐engineered cell populations. (A) Representative normal karyotype showing 22 autosomes and X and Y chromosomes (donor 2 passage 3 cells), (B) Abnormal karyotype including alterations on chromosomes 6, 13, and 16 and chromosomal fragments that could not be mapped observed for donor 2 hTERT mid passage cells and (C) Normal karyotype for late passage donor 2 pFLRu‐BMI‐1 cells. Discussion In this study, we set out to evaluate methods to delay cell senescence while maintaining plasticity/differentiation potential in primary human bronchial epithelial cells. This is important as these cells have a key role to play in respiratory disease warranting further research. However, such primary cells are isolated from patients by invasive procedures and have limited lifespan ex vivo. We have developed two lentiviral systems that enabled us to elevate the expression of polycomb protein BMI‐1 and importantly suppress p16 levels in cells containing these genetic modifications. Extensive characterization over 12 months demonstrated that cells expressing higher levels of BMI‐1 have (1) increased viability, (2) an extended number of cell divisions, (3) maintain basal epithelial morphology, (4) maintain plasticity, that is, the ability to form a differentiated pseudostratified air–liquid interface model, and (5) importantly maintain a normal karyotype. These data suggest that the cell populations generated using BMI‐1 induction maintain cell plasticity over extended periods in the laboratory and may be useful for primary human bronchial epithelial cell research. To date, either viral oncogenes have been used to prevent cell senescence in human bronchial epithelial cells (Willey et al. 1991; Zeitlin et al. 1991; Viallet et al. 1994) or the introduction of human telomerase reverse transcriptase (hTERT) (Zabner et al. 2003; Piao et al. 2005). These studies have shown some success, however, a major limitation is alterations in karyotype (Piao et al. 2005). Therefore, viral oncogene independent approaches for prevention of cell senescence have been sought, the most favored approach being one with an underlying mechanism that decreases the levels of cyclin‐dependent kinase inhibitor, p16(Ink4a), a tumor suppressor that induces a G1 cell cycle arrest. BMI‐1 is a polycomb transcriptional repressor protein and is part of the polycomb repressive complex‐1 (PRC1) complex. This complex regulates transcriptional activity at several loci including the Ink4a locus which encodes the tumor suppressor proteins p16(Ink4a) and p14(Arf). BMI‐1 has been extensively characterized in the context of numerous cancers, with both protein and mRNA levels correlating with disease prognosis (Cao et al. 2011). The contribution of BMI‐1 to multiple cancers is thought to be due to its role in self‐renewal and differentiation of stem cells (Lessard and Sauvageau 2003). With these unique properties of regulating cell senescence, BMI‐1 has been the focus of attention particularly in the delay of cell senescence in many cell types, for example, stem cell feeder cells (McKay et al. 2011). Recently, transduction of HBEC with combinations of hTERT and BMI‐1 has generated some promising data (Fulcher et al. 2009). The resultant hTERT/BMI‐1 cell populations continued to divide longer than nonmodified cells, and importantly, at passage 14 and 15 had a diploid karyotype and formed differentiated pseudostratified morphology following ALI culture. Interestingly, BMI‐1 alone was also introduced to bronchial epithelial cells in the study of Fulcher and colleagues and delayed cell senescence, however, these BMI‐1 cell populations were not extensively characterized for plasticity, a hallmark of low passage basal epithelial cells. In this study, we set out to significantly extend these studies and focus away from the use of hTERT to the use of BMI‐1 alone to delay cell senescence in bronchial epithelial cells and provide more definitive characterization of the plasticity of cells particularly in the context of ALI models. To this end, we developed two new lentiviral systems engineered to express elevated BMI‐1 under the control of CMV or UbiC promoter and a control retroviral system expressing hTERT for comparison. These systems were used to transduce passage 2 normal bronchial epithelial cells from two donors. These genetically engineered cells showed sustained elevation of BMI‐1 as determined by western blot and immunofluorescence and similarly elevated telomerase activity for the hTERT‐engineered cells. As anticipated, the BMI‐1‐expressing cells showed consequent suppression of p16 as observed by several groups (Meng et al. 2010). It is important to note that BMI‐1 is a master regulator of a very large number of genes and gene families of relevance to cell senescence, for example, see a recent study using microarray analyses in BMI‐1 knockout mice cells (Zacharek et al. 2011). In this study, a large number of genes were differentially expressed (elevated) when BMI‐1 was absent which included multiple homeodomain genes and Cdkn2a (p16/p19) and Cdkn2b (p15). In a more recent study using BMI‐1 −/− mouse brain tissue in conjunction with RNA‐seq, >500 genes were shown to be up regulated due to a loss of BMI‐1 including identifying a role for BMI‐1 in TGF‐β/BMP‐ER stress pathways (Gargiulo et al. 2013). Therefore, while our p16 suppression data confirms that our genetic manipulation of BMI‐1 has downstream effects on BMI‐1 responsive networks, it is highly likely that multiple pathways underlie the effects we have observed, for example, p14Arf suppression by BMI‐1 may be highly relevant for apoptosis mechanisms through the mdm2/TP53 pathway. In the first set of experiments, we demonstrated that early passage BMI‐1 cells have a slightly higher viability as did hTERT cells, however, as the passage number increased hTERT cells lost viability (mid passage and late passage for the two donors), whereas BMI‐1 cells and in particular those containing the CMV promoter, maintained this elevated viability. In order to test the limit of our system, we also grew the cells further to infection passage 17/actual passage 20. Only the BMI‐1‐engineered cells reached this extended passage as hTERT cells failed to grow. Importantly, the extended passage BMI‐1 cells had lost their plasticity as assessed by the ability to differentiate at ALI and did not adhere or grow well on the transwells and had therefore reached the end of their lifespan (data not shown). These data demonstrating delay of cell senescence following BMI‐1 introduction are in good agreement with previous work that showed that cell senescence is delayed to passage 21–24 as we have shown but not sufficient for immortalization (Fulcher et al. 2009). These data also identified a potential advantage of the pLVX‐CMV lentivirus system. The hTERT data is in contrast to a recent study that engineered primary airway epithelial cells to express hTERT and reported that these cells grew beyond 40 passages, however, it is difficult to make conclusions as this study only used cells from one donor (Walters et al. 2013). Over extended passage the BMI‐1‐expressing cells maintained epithelial marker expression (CK14), whereas this was more variable with the hTERT‐engineered cells. To date, no other study has evaluated changes in CK14 marker expression post BMI‐1 engineering, however, both BMI‐1/hTERT‐ and hTERT‐engineered cells demonstrated maintenance of an epithelial phenotype based on additional markers including KRT5 in hTERT cell populations (Walters et al. 2013). An important finding of our current study was that BMI‐1‐expressing cells (early‐mid‐late up to passage 15) maintain cell plasticity and the ability to differentiate at air–liquid interface generating a pseudostratified layer composed of basal, goblet, and ciliated cells as observed by sectioning transwells. These data are novel and significantly extend data on BMI‐1/hTERT‐ or hTERT‐engineered cells that can also maintain this capacity (Fulcher et al. 2009; Walters et al. 2013). However, the hTERT cell populations generated in this study did not maintain this plasticity and showed limited differentiation in contrast to a recent report (Walters et al. 2013). These data question the utility of hTERT alone and suggest BMI‐1 alone to be the preferred genetic tool to maintain plasticity in our study. Of note, several groups are developing combination approaches to prevent cell senescence in primary epithelial cells using hTERT, for example, hTERT/BMI‐1 can lead to cells that reach at least 38 population doublings (Fulcher et al. 2009) and hTERT/cyclin‐dependent kinase 4 (Cdk4) epithelial cells have shown >100 population doublings (Roig et al. 2010). However, in this study, we focused to the use of BMI‐1 alone to reduce transformation of the cells away from a diploid karyotype and to maintain plasticity. In agreement with the potential utility of BMI‐1 cell populations, further measures of differentiation in the context of the air–liquid interface model, mainly goblet cell (MUC5AC), ciliated cell (beta tubulin) staining, development of TEER, and SEM showing apical cilia were all maintained in the late passage BMI‐1 cell populations but not in the hTERT cell populations. Overall, these data show that for the first time BMI‐1‐expressing cells have an extended lifespan, albeit not extensive and suggest that passage 15 cells show very similar plasticity properties to wild‐type cells for these key outcomes. Of critical importance, compared to other methods that incorporate the use of hTERT, we did not observe any alterations in karyotype in BMI‐1‐expressing cells, whereas the hTERT cells (p8) had chromosomal abnormalities. These findings are in complete agreement to the recent study of hTERT‐engineered bronchial epithelial cell populations which identified chromosomal abnormalities as early as passage 9 (Walters et al. 2013). Interestingly, analyses of passage 14/15 BMI‐1/hTERT failed to identify chromosomal abnormalities suggesting that BMI‐1 may be protective in this context (Fulcher et al. 2009). This study has several strengths over previous reports such as including the evaluation of multiple BMI‐1 and hTERT vectors in parallel in multiple primary human donors. Similarly, the extensive characterization of these cells for cell plasticity in the context of the ALI model and for karyotype is novel. These data from four genetically modified cell populations show comparable effects of BMI‐1 elevation. However, we acknowledge that greater numbers of donors and technical replicates are required to provide more quantitative characterization of cell differentiation, for example, TEER. This represents a limitation of this study. We have previously identified a large degree of heterogeneity in both intra‐ and interdonor variation in the air–liquid interface model (Stewart et al. 2012b), even in the absence of genetic modification suggesting a substantial study is required involving large numbers of donors over many years to further define quantitative changes in cell plasticity using this model. In conclusion, we provide an in‐depth characterization of the plasticity of human bronchial epithelial cells that have been engineered to express BMI‐1. These cells retain the plasticity observed in wild‐type low passage cells including the ability to differentiate at ALI that can be used to model the lining of the airways. These findings suggest these cells may show utility in many aspects of respiratory research including in defining airway epithelial abnormalities in diseases such as asthma, which has the potential to lead to therapeutic opportunities. Conflicts of Interest No conflicts of interest, financial or otherwise, are declared by the authors. Acknowledgments We thank Professor Greg Longmore (Washington University) for providing the pFLRu‐FH plasmid, the Advanced Microscopy Unit (Life Sciences, University of Nottingham) for their help with SEM, and Dr John G. Foster for critical reading of this manuscript. ==== Refs References Cao , L. , J. Bombard , K. Cintron , J. Sheedy , M. L. Weetall , and T. W. Davis . 2011 BMI1 as a novel target for drug discovery in cancer . J. Cell. Biochem. 112 :2729 –2741 .21678481 Fulcher , M. L. , S. E. Gabriel , J. C. Olsen , J. R. Tatreau , M. Gentzsch , E. Livanos , et al. 2009 Novel human bronchial epithelial cell lines for cystic fibrosis research . Am. J. Physiol. Lung Cell. Mol. Physiol. 296 :L82 –L91 .18978040 Gargiulo , G. , M. Cesaroni , M. Serresi , N. de Vries , D. Hulsman , S. W. 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==== Front Physiol RepPhysiol Rep10.1002/(ISSN)2051-817XPHY2physreportsPhysiological Reports2051-817XJohn Wiley and Sons Inc. Hoboken 10.14814/phy2.12853PHY212853Maternal, Fetal and Neonatal PhysiologyCentral Nervous SystemDevelopmental NeuroscienceRegulatory PathwaysOriginal ResearchOriginal ResearchAstrocyte‐mediated regulation of multidrug resistance p‐glycoprotein in fetal and neonatal brain endothelial cells: age‐dependent effects S. Baello et al.Baello Stephanie 1 Iqbal Majid 1 Gibb William 2 3 Matthews Stephen G. 1 4 5 1 Department of PhysiologyFaculty of MedicineUniversity of TorontoTorontoOntarioCanada2 Department of Obstetrics and GynecologyFaculty of MedicineUniversity of OttawaOttawaOntarioCanada3 Department of Cellular and Molecular MedicineFaculty of MedicineUniversity of OttawaOttawaOntarioCanada4 Department of Obstetrics and GynecologyFaculty of MedicineUniversity of TorontoTorontoOntarioCanada5 Department of MedicineFaculty of MedicineUniversity of TorontoTorontoOntarioCanada* Correspondence Stephanie Baello, Department of Physiology, Faculty of Medicine, University of Toronto, Medical Sciences Building 3302, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. Tel: 416 978 2025 Fax: 416 978 4940 E‐mail: s.baello@mail.utoronto.ca 22 8 2016 8 2016 4 16 10.1111/phy2.2016.4.issue-16e1285309 5 2016 13 6 2016 14 6 2016 © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.Abstract Brain endothelial cells (BECs) form a major component of the blood–brain barrier (BBB). In late gestation, these cells express high levels of the multidrug transporter p‐glycoprotein (P‐gp; encoded by Abcb1), which prevents the passage of an array of endogenous factors and xenobiotics into the fetal brain. P‐gp levels in the BECs increase dramatically in late gestation, coincident with astrocyte differentiation. However, the role of astrocytes in modulating P‐gp in the developing BBB is unknown. We hypothesized that factors produced by astrocytes positively regulate P‐gp in BECs. Astrocytes and BECs were isolated from fetal and postnatal guinea pigs. Levels of Abcb1 mRNA and P‐gp were increased in BECs co‐cultured with astrocytes compared to BECs in monoculture. Moreover, postnatal astrocytes enhanced P‐gp function in fetal BECs but fetal astrocytes had no effect on postnatal BECs. These effects were dependent on secreted proteins with a molecular weight in the range of 3–100 kDa. LC/MS‐MS revealed significant differences in proteins secreted by fetal and postnatal astrocytes. We propose that astrocytes are critical modulators of P‐gp at the developing BBB. As such, aberrations in astrocyte maturation, observed in neurodevelopmental disorders, will likely decrease P‐gp at the BBB. This would allow increased transfer of P‐gp endogenous and exogenous substrates into the brain, many of which have neurodevelopmental consequences. Astrocytesblood–brain barrierbrain endothelial cellsfetusglial cellsP‐glycoproteinCanadian Institutes for Health ResearchFRN‐84220 source-schema-version-number2.0component-idphy212853cover-dateAugust 2016details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_NLMPMC version:4.9.4 mode:remove_FC converted:29.08.2016 S. Baello , M. Iqbal , W. Gibb , S. G. Matthews . Astrocyte‐mediated regulation of multidrug resistance p‐glycoprotein in fetal and neonatal brain endothelial cells: age‐dependent effects . Physiol Rep , 4 (16 ), 2016 , e12853 doi: 10.14814/phy2.12853 Funding Information This study was funded by the Canadian Institutes for Health Research (FRN‐84220; to S.G.M. and W.G., and the Doctoral Research Award to S.B.) ==== Body Introduction The blood–brain barrier (BBB), present at the level of cerebral capillary endothelium, is an organ‐specific barrier that protects the brain. The BBB is formed by a specialized endothelium that displays barrier characteristics, able to regulate the movement of molecules from blood into brain compartments. Tight junctions and adheren junctions form between brain endothelial cells (BECs) and limit paracellular movement of proteins and large hydrophilic molecules across the BBB (Abbott et al. 2010). These tight junctions are present in the embryonic brain and have been shown to be functionally effective (Mollgoard and Saunders 1975; Bauer et al. 1993). Lipid‐soluble substances can pass the BBB unless actively removed by transporters located on the luminal surface of BECs. P‐glycoprotein (P‐gp) is the highest expressed transporter at the BBB (van Asperen et al. 1996; Kemper et al. 2003; von Moltke et al. 2004). P‐gp is encoded by a single gene, Abcb1, in humans and guinea pigs and two genes, Abcb1a/Abcb1b, in rats and mice (Pappas et al. 2014). Deficiency in P‐gp increases the passage of its substrates into the fetal brain, resulting in teratogenesis (Lankas et al. 1998). This is clinically relevant as approximately 60% of pregnant women take prescription drugs, many of which are substrates of P‐gp (Andrade et al. 2004, 2006; Daud et al. 2015). Additionally, P‐gp regulates the movement of endogenous compounds involved in brain development, such as cytokines and steroid hormones (Bain et al. 1997; Kim and Benet 2004). Protection of the developing brain via P‐gp at the BBB becomes essential during the transition from fetal to neonatal life as the protection afforded by placental P‐gp is lost (Kalabis et al. 2005, 2009; Lye et al. 2013). Our group has shown, in mice and guinea pigs, that in early gestation, brain microvessels express very low amounts of P‐gp (Petropoulos et al. 2010; Iqbal et al. 2011). However, levels of P‐gp dramatically increase in late gestation and postnatal life. This upregulation in P‐gp is coincident with astrocyte differentiation, a process by which immature astrocytes proliferate and begin to extensively ensheathe the abluminal surface of brain microvessels (Dobbing and Sands 1979; Risau and Wolburg 1990; Byrnes et al. 2003). BECs interact very closely with astrocytes as astrocytic‐end feet ensheathe 99% of the abluminal surface of brain capillaries (Abbott 2002; Mathiisen et al. 2010). Previous studies have shown that astrocytes are key regulators in the induction and maintenance of BBB characteristics of brain endothelium. BECs that have been co‐cultured with astrocytes show enhanced activity of BBB‐specific enzymes and transporters, and reduced paracellular transport compared to BECs cultured without astrocytes (Hayashi et al. 1997; Sobue et al. 1999; Abbott 2002; Berezowski et al. 2004; Haseloff et al. 2005). Moreover, astrocytes are capable of inducing these properties in endothelial cells derived from nonneural tissue (Janzer and Raff 1987; Hayashi et al. 1997; Kuchler‐Bopp et al. 1999). Astrocyte‐conditioned medium (ACM) can mimic these effects implicating astrocyte‐derived factors. However, many of these studies use cells derived from adult animals, or co‐culture cells from different ages and in some cases, from different species. In this study, we investigated the effect of astrocytes on P‐gp function and expression at the developing BBB. Our laboratory has developed an in vitro BBB model comprised of BECs and astrocytes derived from fetal and postnatal guinea pigs. We utilized this model to investigate and characterize the astrocyte‐derived factors responsible for upregulating P‐gp at the developing BBB. We hypothesized that astrocytes would enhance P‐gp expression and function in BECs, but that the magnitude of this effect would vary with developmental age. Materials and Methods Animals Twelve‐week‐old female Dunkin‐Hartley‐strain guinea pigs were purchased from Charles River Canada Inc. (St. Constant, Quebec, Canada) and were bred as described previously (Dean and Matthews 1999). 2‐week‐old male Dunkin‐Hartley‐strain guinea pigs were also purchased from Charles River Canada Inc. The guinea pig is an ideal animal model for investigating the interaction of astrocytes and BECs in regulating BBB characteristics. In contrast to the mouse, the guinea pig has a relatively long gestation period (~68 days) and has similar placentation to humans. Our laboratory has shown that unlike other rodents, P‐gp is encoded by a single gene, Abcb1 in the guinea pig (Pappas et al. 2014). Moreover, guinea pig brain development follows a similar pattern to human brain development, such that the fetal brain growth spurt, due to rapid gliogenesis occurs in utero (Dobbing and Sands 1979; Byrnes et al. 2003). There are also many advantages to using guinea pigs for establishing an in vitro co‐culture model of the BBB. Guinea pigs give birth to large fetuses (~100 g at term) that provide sufficient brain tissue for in vitro analysis. Fetal brain tissue derived at mid‐gestation is sufficient to yield both primary BECs and astrocyte cultures. These characteristics have allowed us to generate a co‐culture model, using primary BECs and astrocytes derived from guinea pigs at various stages of fetal and neonatal development. Animal protocols used in the following studies were approved by the Animal Care Committee at the University of Toronto and in accordance with the Canadian Council on Animal Care. Isolation and culture of guinea pig primary brain endothelial cells Isolation of BECs from gestational day (GD) 50 and postnatal day (PND) 14 guinea pigs was carried out as previously described (Iqbal et al. 2011). These time‐points in development were chosen as our group has found that P‐gp expression is low at GD50 and high at PND14 at the BBB (Iqbal et al. 2011). Briefly, guinea pigs were anesthetized with isoflurane (Baxter Corp Mississauga, Ontario, Canada). Once anesthetized, guinea pigs were decapitated and brain tissue was excised, meninges removed, and brain tissue halved. One half was used for BEC isolation, while the other half was used to isolate astrocytes as explained below. Brain tissue was minced and homogenized (Potter‐Elvehjem Tissue Grinder; Sigma, St. Louis, MO). The homogenate was suspended in dextran solution (17.5%; Sigma) and centrifuged (4000g for 15 min). Microvessel pellet was mixed with type I collagenase solution (1 mg/mL; Sigma) and digested for 30 min at 37°C. At the end of digestion, the solution was centrifuged (1000g, 10 min), the pellet was resuspended in Dulbecco's Modified Eagle Medium (DMEM; Wisent Inc., Saint‐Jean‐Baptiste, Quebec, Canada) supplemented with 20% fetal bovine serum (Wisent Inc.), plated on 0.5% gelatin‐coated 75 mm2 tissue culture flasks (Becton‐Dickinson BD Biosciences, Franklin Lakes, NJ) and grown at 37°C in 5% CO2/air. BECs were frozen in liquid nitrogen. BECs isolated and cultured in this way have been previously characterized (Iqbal et al. 2011). BECs derived from different stages of development retain relative developmental levels of P‐gp activity and levels of abcb1 mRNA (Iqbal et al. 2012). Isolation and culture of guinea pig primary astrocytes Astrocytes were extracted from GD50 and PND14 guinea pigs, using a modified protocol (Babu et al. 2011). Brain tissue was cut into small pieces and digested in papain (2.5 U/mL; Worthington Biochemical Corp., Lakewood, NJ) and DNAse (250 U/mL; Worthington Biochemical Corp.) at 37°C for 30 min. After incubation, papain/DNAse was removed by centrifugation (1000g, 10 min), the pellet was resuspended in Neurobasal A medium (Invitrogen, Carlsbad, CA) and loaded onto a column containing glass beads (5 mm; Thermo Fisher Scientific, San Jose, CA). The filtrate was plated on rat tail collagen‐coated 75 mm2 tissue culture flasks (BD Biosciences) and grown at 37°C in 5% CO2/air. Astrocytes were frozen in liquid nitrogen until use. Astrocyte characterization Astrocytes from GD50 and PND14 guinea pigs were plated on 8‐well tissue culture slides (BD Biosciences). At 80% confluence, the medium was removed and cells were washed with HBSS (Invitrogen). Cells were fixed using paraformaldehyde (4%, Sigma; 22°C, 30 min), washed in Tris‐buffered saline (0.01 mol/L, pH7.5; Sigma) containing Triton X‐100 (0.1% TBS‐Triton; Sigma) and blocked in normal goat serum (NGS; 1.5% in TBS Triton; Invitrogen) for 2 h. Astrocytes were then incubated with primary antibody (diluted in NGS/TBS‐Triton) overnight at 4°C. Primary antibodies: rabbit anti‐von Willebrand Factor (vWF; 1:200; Sigma), mouse anti‐glial fibrillary acidic protein (GFAP; 1:100; Cell Signaling Technology, Inc., Danvers, MA), rabbit anti‐melanoma‐associated chondroitin sulfate proteoglycan (Ng2; 1:100; Cell Signaling Technology, Inc.), mouse anti‐Nestin (1:200; abcam, Cambridge, United Kingdom), rabbit anti‐aquaporin 4 (AQP4; 1:200; abcam), mouse anti‐synaptophysin (1:100; Millipore, Billerica, MA). Cells were washed and subsequently incubated with Alexa Fluor 488‐ or 555‐conjugated goat or secondary antibody (1:500 in NGS/TBS‐Triton; Invitrogen) depending upon the primary antibody species. As a negative control, cells were incubated with mouse IgG or rabbit IgG following secondary antibody. After the final washes, cells were mounted using mounting media with DAPI (Vector Laboratories, Burlingame, CA) and coverslipped. Guinea pig co‐culture and measurement of tight junction function BECs and astrocytes were plated on 75 mm2 tissue culture flasks (BD Biosciences) in respective media described above and grown at 37°C in 5% CO2/air. To establish co‐cultures, using GD50 and PND14 BECs and astrocytes, astrocytes were plated in the basolateral compartment of Transwell plate (polyethylene terephthalate (PET); Corning Inc., Corning, NY) coated with rat‐tail collagen at a seeding density of 1 × 104 cells/cm2. BECs were plated on the Transwell insert containing 0.45 μm pores (PET; Corning) coated with 0.5% gelatin at a seeding density of 1 × 104 cells/cm2. The insert membrane growth surface was 0.33 cm2. In co‐culture, cells were grown in BEC media. transendothelial electrical resistance (TEER) measurements were undertaken every 24 h using Millipore Millicell ERS probe MERSSTX01 and ERS‐2 Epithelial volt‐ohm meter (Millipore, Billerica, MA). Cells were washed twice with tyrode solution (Sigma). Chopstick electrodes were inserted into the donor and receiver chambers. Final resistance was calculated by subtracting the resistance of blank filters. Cell number between treatments was constant as measured by a trypan blue. Tight junction function was also assessed, using a permeability assay with FITC‐dextran (70 kDa; Sigma), which was used to mimic serum albumin (~67 kDa). This assay was carried out after 72 h in culture to further corroborate TEER values. Cells were washed twice with tyrode solution (Sigma). FITC‐dextran was added to the apical compartment (0.01 mg/mL) and after 1 h, 100 μL of medium was removed from the basal compartment and substituted with the same amount of tyrode solution (Sigma). Samples were collected on a 24‐well tissue culture plate (BD Biosciences), and fluorescence was measured at Ex/Em: 485/530 nm. Analyses using low molecular weight dextran were not carried out due to limited amount of cells. P‐gp functional assay P‐gp activity was measured using calcein‐AM as described previously (Feng et al. 2008; Iqbal et al. 2011; Baello et al. 2014). BECs were washed twice with Tyrode solution (Sigma) incubated at 37°C. Co‐cultured BECs plated on inserts were transferred into new 24‐well tissue culture plates (BD Biosciences) and washed. Subsequently, BECs were incubated with calcein‐AM (1 μmol/L calcein‐AM; Sigma) for 1 h at 37°C in 5% CO2/air. After incubation, BECs were placed on ice, washed twice with tyrode solution (Sigma), and lysed with ice‐cold 1% Triton X‐100 (Sigma) lysis buffer. Intracellular calcein was measured, using a spectrophotometer (Ex/Em: 485/510 nm). Mean background fluorescence was subtracted from all control and treated readings. Control values were divided by treatment values and expressed as fold‐change from control. To validate that the effects of co‐culturing were specific to P‐gp, cells were incubated with verapamil. Verapamil is an L‐type calcium channel blocker that has been shown to be a competitive inhibitor of P‐gp (Weiss et al. 2003). Cells were washed with Tyrode and then subsequently incubated for 1 h with either calcein‐AM (1 μmol/L) or calcein‐AM with 10−4 m verapamil (10 μmol/L; Sigma). Cells were then washed, lysed, and calcein was measured, as described above. Quantification of mRNA expression BECs were monocultured or co‐cultured on 24‐well Transwell inserts as described above (Corning). On Day 3 of co‐culture, BECs were trypsinized (0.05% trypsin‐EDTA [Invitrogen]) and centrifuged (1000g, 5 min). RNA was extracted, using TRIzol reagent (Invitrogen) as per the manufacturer's protocol. RNA was converted into cDNA via High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Waltham, MA) as per the manufacturer's protocol using the C1000 Thermal Cycler. Thereafter the cDNA (100 ng) was mixed with primer probes (Abcb1: Forward – CAATCTGGGCAAAGATACTG, Reverse – CAAGTTCTTTGCTTTGTCCTC [Ensembl ID: ENSCPOT00000012540]; β‐actin: Forward – TTTACAATGAATTGCGTGTG, Reverse –ACATGATCTGGGTCATCTTC [Ensembl ID: ENSCPOT00000013600])) and SsoFast EvaGreen Supermix (Bio‐rad Laboratories, Inc., Hercules, CA). Samples were prepared in triplicate. Primer sets were designed, using Autoprime Primer Design (Gunnar Wrobel & Felix Kokocinski; Cambridge, United Kingdom) and synthesized by Integrated DNA Technologies (Coralville, IA). Samples were loaded onto CFX96 Real‐Time System (Bio‐Rad). Relative mRNA expression was calculated as the gene of interest expression normalized [ΔΔc(t)] to the reference gene expression, β‐actin. Expression levels of β‐actin did not change with age or co‐culturing conditions. Collection of astrocyte‐conditioned medium Astrocytes from GD50 and PND14 guinea pigs were plated on 150 mm2 petri dishes at a seeding density of 1 × 104 cells/cm2. Once confluent, astrocytes were washed twice with HBSS (Invitrogen) and media was changed to phenol‐red free Neurobasal A medium (Invitrogen). After 24 h, astrocyte conditioned media (ACM) was collected, centrifuged at 1000g for 10 min, and passed through a 0.22 μmol/L filter. ACM was frozen at −80°C until use. Heat‐inactivation, protease treatment, and exosome‐depletion of astrocyte‐conditioned medium ACM was subjected to heat inactivation at 100°C for 4 h. For protease treatment, ACM was incubated with trypsin (200 μg/mL; Sigma) at 37°C for 1 h and subsequently incubated with trypsin inhibitor (400 μg/mL; Sigma) at 37°C for 30 min. As a control, trypsin and trypsin inhibitor were mixed and incubated at 37°C for 1 h and 30 min and then added to ACM. To eliminate exosomes, ACM was centrifuged at 10,0000g for 6 h and the supernatant collected. Fractionation of astrocyte‐conditioned medium To obtain fractions of ACM‐containing soluble factors of varying molecular weights, ACM was fractionated, using Centrifugal Filter Devices with different MWCO (Molecular Weight Cut‐Offs) Ultracel membranes (Millipore). First, 15 mL of ACM was loaded onto the centrifugal filter device with Ultracel membranes with a 100 kDa cut‐off and centrifuged at 2700g for 35 min. The retentate was collected and resuspended in the same volume of Neurobasal A medium (Invitrogen) as the loaded sample. The filtrate was collected and resuspended in the same volume of Neurobasal A medium as the loaded sample and subsequently loaded onto a column with a 30 kDa membrane cut‐off and centrifuged at 2700g for 35 min. The retentate and filtrate were again collected and resuspended in the same volume of medium as the loaded sample. The filtrate was loaded onto a column with a 3 kDa membrane cut‐off and centrifuged at 2700g for 35 min. BECs were then treated with these fractions of ACM containing proteins of varying molecular weights and P‐gp function was assessed. In‐solution digestion Proteins in ACM were denatured, reduced, and alkylated prior to digestion and LC‐MS/MS. Briefly, protein (10 mg) was incubated with urea (8 mol/L, Sigma), 50 mmol/L Tris‐HCl (Sigma; pH 8), and 4 mmol/L DTT (Sigma) at 60°C for 30 min and then incubated with iodoacetamide (100 mmol/L; Sigma, 22°C, 15 min). The samples were subsequently diluted five times with 0.1 mol/L Tris–HCl pH 8.3 (Sigma) and digested, using trypsin (1:50 w/w; Sigma; 37°C, 16 h). Samples were desalted, using Pierce C18 Spin Tips (Thermo Fisher Scientific) as per the manufacturer's protocol. LC‐MS/MS analysis Tryptic digests were analyzed on an Orbitrap analyzer (Q‐Exactive, Thermo Fisher Scientific) outfitted with a nanospray source and EASY‐nLC nano‐LC system (Thermo Fisher Scientific). Lyophilized peptide mixtures were dissolved in 0.1% formic acid and loaded onto a 75 μm × 50 cm PepMax RSLC EASY‐Spray column filled with 2 μmol/L C18 beads (Thermo Fisher Scientific) at a pressure of 800 Bar. Peptides were eluted over 60 min at a rate of 250 nL/min, using a 0–35% acetonitrile gradient in 0.1% formic acid. Peptides were introduced by nano‐electrospray into the Q‐Exactive mass spectrometer (Thermo Fisher Scientific). The instrument method consisted of one MS full scan (400–1500 m/z) in the Orbitrap mass analyzer with an automatic gain control (AGC) target of 1E6, maximum ion injection time of 120 msec and a resolution of 70,000 followed by 10 data‐dependent MS/MS scans with a resolution of 17,500, an AGC target of 1E6, maximum ion time of 120 msec, and one microscan. The intensity threshold to trigger an MS/MS scan was set to 1.7E4. Fragmentation occurred in the HCD trap with normalized collision energy set to 27. The dynamic exclusion was applied, using a setting of 10 sec. Protein Identification Raw data generated by LC‐MS/MS (peaklists generated by Xcalibur 2.2) were analyzed, using Xcalibur (Thermo Fisher Scientific) for ion current analysis, and were searched against the guinea pig SwissProt UniProt protein‐database (containing 20392 entries). Database searching was undertaken using SEQUEST version 1.4.0.288 (through Proteome Discoverer, Thermo Fisher Scientific). The data was then imported into Scaffold 4.3.4 (Proteome Software, Portland, OR) and X!Tandem CYCLONE (2010.12.01.1) database searching was performed. These databases were searched with a parent ion tolerance of 10.0 PPM, and a fragment mass tolerance of 0.6 Da. Peptide identifications were accepted if they could be established at greater than 95% probability by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at <95% probability and contained at least three identified peptides. Protein probabilities were assigned by the ProteinProphet algorithm (Nesvizhskii et al. 2003). Protein Quantification Proteins were quantified based on the average MS signal response for the three most intense tryptic peptides for each protein and expressed as percentage of total protein. Briefly, the MS files were searched as listed above and the SEQUEST result files were used to generate a spectral library in Skyline 2.1.0.4936 (MacLean et al. 2010), using the guinea pig sequences from SwissProt as background proteome. Employing the same parameters as for the SEQUEST search, the three most abundant peptides for each protein were manually chosen from all peptides available in the spectral library. After data import, the chromatographic traces (extracted ion chromatograms) were manually inspected and adjusted where needed to correct wrongfully assigned peaks. Proteins identified with less than two peptides were not included in the quantification. The relative abundance of proteins quantified in three samples was calculated as the average MS intensity for the two peptides for each protein divided by the sum of the average signal for all quantified proteins in the sample. Relative abundance of protein was displayed as fold change from protein levels found in GD50 ACM. Statistics Statistical analyses were performed using Prism (GraphPad Software, Inc., San Diego, CA). Effect of co‐culture or ACM on tight junction function and Abcb1/P‐gp was analyzed, using Student's unpaired t‐test. Effect of heat‐inactivated and exosome‐depleted ACM was also analyzed via Student's unpaired t‐test. Effect of age‐match and different‐aged co‐culture, protease‐treated ACM, and fractionation of ACM data was analyzed, using one‐way ANOVA, followed by Newman–Keuls post hoc analyses. Significance was set at P < 0.05. Each treatment group consisted of cells derived from 5‐8 animals. LC‐MS/MS data was analyzed, using MSstats (3.1.4; Vitek Lab, Purdue University). Three biological replicates of ACM were collected at each gestational age. Proteomic differences between GD50 and PND14 ACM were evaluated for statistical significance (P < 0.05) by Student unpaired t‐tests, and corrected for multiple testing, using the Benjamini–Hochberg correction. Results Characterization of fetal and postnatal astrocytes Astrocytes were characterized via immunofluorescence for markers of immature (Nestin) and mature astrocytes (GFAP [glial fibrillary acidic protein] and AQP4). Astrocytes derived from GD50 guinea pigs expressed Nestin and GFAP (>95%), but stained negatively for AQP4 (Fig. 1A–C). In contrast, PND14 astrocytes were positive for GFAP and AQP4 (>95%) but did not express Nestin (Fig. 1D–F). Both GD50 and PND14 astrocytes did not express (<1%) for synaptophysin (neuron marker), vWF (BEC marker), or Ng2 (pericyte marker)(data not shown), indicating little contamination by other CNS cell types. Figure 1 Fluorescent immunocytochemical images of astrocytes derived from gestational day (GD) 50 (A–C) and postnatal day (PND) 14 (D–F) male guinea pigs. Cells were stained for Nestin (nes), glial fibrillary acidic protein, and aquaporin‐4 (AQP4). Cell nuclei were stained by 4′,6‐diamidino‐2‐phenylindole (blue). Effect of astrocytes on tight junction function in BECs Co‐culture of age‐matched cells increased TEER in GD50 (P < 0.01) and PND14 (P < 0.05) BECs compared to monocultured cells after 48 h and 72 h (Fig. 2A and B). After 72 h in culture, co‐cultured PND14 BECs attained a TEER of approximately 452 Ω cm2, while co‐cultured GD50 BECs displayed a TEER of 109 Ω cm2 (Fig. 2A and B). These results were corroborated with FITC‐dextran permeability assay. Co‐cultured GD50 and PND14 BECs also demonstrated decreased permeability to FITC‐dextran (70 kDa) after 72 h in culture compared to monocultured BECs, approximately 60% and 30%, respectively (P < 0.05, P < 0.001) (Fig. 2C and D). Figure 2 Tight junction function in monocultured (MONO) or co‐cultured (CO) BECs derived from gestational day (GD) 50 and postnatal day (PND) 14 guinea pigs (n = 8/group). Tight junction function was assessed using TEER after 24 h, 48 h, and 72 h in culture (A, B). Data from TEER experiments is displayed as mean ± SEM. After 72 h in culture, FITC‐dextran was also used to measure tight junction function (C, D). Data is expressed as % dextran accumulation from control (monocultured BECs). A significant difference from monocultured cells is indicated by *P < 0.05; **P < 0.01; ***P < 0.001. BECs, Brain endothelial cells; TEER, transendothelial electrical resistance. Effect of astrocytes on P‐gp activity and levels of Abcb1 mRNA in BECs Since tight junction function was maximal after 72 h in culture, P‐gp activity and levels of Abcb1 mRNA were measured in monocultured and co‐cultured BECs at this time‐point. P‐gp activity in GD50 and PND14 BECs co‐cultured with age‐matched astrocytes increased by approximately twofold (P < 0.01) and sevenfold (P < 0.001), respectively, compared to monocultured BECs (Fig. 3A and B). This corresponded to a threefold increase in Abcb1 mRNA (P < 0.05) in co‐cultured PND14 BECs compared to monocultured PND14 BECs (Fig. 3D). However, there was no change in Abcb1 mRNA levels in co‐cultured GD50 BECs compared to monocultured GD50 BECs (Fig. 3C). Figure 3 P‐gp activity (A, B) and levels of Abcb1 mRNA (C, D) in monocultured or co‐cultured BECs derived at gestational day (GD) 50 and postnatal day (PND) 14 (n = 8/group). P‐gp activity was also measured in co‐cultured BECs (E) that was exposed to P‐gp inhibitor, verapamil (VPL) (n = 5/group). P‐gp activity is displayed as fold change from control (monocultured BECs). Abcb1 mRNA expression was normalized to β‐Actin and shown as fold change from monocultured BECs. Data are displayed as mean ± SEM. A significant difference from monocultured cells is indicated by *P < 0.05; **P < 0.01; ***P < 0.001. In order to demonstrate that this effect of co‐culturing was indeed specific to P‐gp, co‐cultured BECs derived from PND14 guinea pigs were incubated with calcein‐AM in the presence of P‐gp inhibitor, verapamil (VPL). This treatment attenuated the effects of co‐culturing on P‐gp function (Fig. 3E). Effect of age of astrocytes on P‐gp activity and levels of Abcb1 mRNA in BECs Co‐culture induced more dramatic changes in P‐gp activity and levels of Abcb1 mRNA in PND14 cultures compared to GD50 cultures. Therefore, GD50 BECs were co‐cultured with PND14 astrocytes and vice versa. Co‐culturing GD50 BECs with PND14 astrocytes induced a greater increase in P‐gp function compared to co‐culturing with GD50 astrocytes (P < 0.01; Fig. 4A). However, despite the enhancement in P‐gp activity in GD50 BECs after co‐culturing with PND14 astrocytes, there was no increase in levels of Abcb1 mRNA compared to GD50 BECs that were monocultured or co‐cultured with GD50 astrocytes (Fig. 4C). Moreover, co‐culturing PND14 BECs with GD50 astrocytes did not increase in P‐gp function or levels of Abcb1 mRNA in PND14 BECs (Fig. 4B and D). Figure 4 P‐gp activity (A, B) and levels of Abcb1 mRNA (C, D) measured in gestational day (GD) 50 and postnatal day (PND) 14 BECs after monoculture, co‐culture with age‐matched astrocytes or co‐culture with different‐aged astrocytes (n = 8/group). P‐gp activity is displayed as fold change from control (monocultured BECs). Abcb1 mRNA expression was normalized to β‐Actin and shown as fold change from monocultured BECs. Data are displayed as mean ± SEM. A significant difference from monocultured BECs is indicated by *P < 0.05; ***P < 0.001. A significant difference between two co‐culture treatments is indicated by #P < 0.01. BECs, Brain endothelial cells. Effect of astrocyte‐secreted factors on P‐gp function in BECs To determine if the effects of co‐culture on P‐gp function were due to astrocyte‐derived factors, we exposed PND14 BECs to ACM from PND14 astrocytes. P‐gp activity was increased in PND14 BECs after 24 h of exposure compared to control BECs exposed to unconditioned medium (UNM) (P < 0.05; Fig 5A). Surprisingly, the effects of ACM on P‐gp activity were not observed at the 48 h or 72 h time‐points (Fig. 5B and C). However, when ACM was changed every 24 h, the effects were rescued at the 48 h and 72 h time‐points (P < 0.001, P < 0.001; Fig. 5D and E). Figure 5 P‐gp activity in postnatal day (PND) 14 BECs exposed to unconditioned (UCM) or ACM for 24 h (A), 48 h (B) and 72 h (C). P‐gp activity was also measured in PND14 BECs to UCM or ACM, with the media being changed every 24 h (D, E). P‐gp activity is displayed as fold change from control (monocultured BECs) (n = 8/group). Data are displayed as mean ± SEM. A significant difference from control is indicated by *P < 0.05; ***P < 0.001. ACM, Astrocyte‐conditioned medium; BECs, Brain endothelial cells. To investigate whether the astrocyte‐secreted factors responsible for increasing P‐gp activity were proteins, ACM was subjected to heat‐inactivation and trypsin (protease) treatment. Heat‐inactivated ACM did not increase P‐gp activity in PND14 BECs after 24 h of exposure compared to BECs exposed to UNM (Fig. 6A). Treatment of ACM with trypsin also prevented the increase in P‐gp activity and the stimulatory‐effect was restored by inhibiting the protease (P < 0.001; Fig. 6B). We also determined the potential role of exosome‐mediated communication between astrocytes and BECs (Fig. 6C). Exosome‐depletion of ACM did not affect its stimulatory affect on P‐gp activity (P < 0.001) indicating that exosome protein cargo is not responsible for the stimulatory effect. Figure 6 P‐gp activity in postnatal day (PND) 14 BECs exposed to heat‐inactivated (A), protease‐treated (B), or exosome‐depleted (C) ACM. ACM was subjected to heat‐inactivation at 100°C for 4 h. For protease treatment, ACM was incubated with trypsin (200 μg/mL; Sigma) at 37°C for 1 h and subsequently incubated with trypsin inhibitor (400 μg/mL; Sigma) at 37°C for 30 min. As a control, trypsin and trypsin inhibitor were mixed and incubated at 37°C for 1 h and 30 min and then added to ACM. To eliminate exosomes, ACM was centrifuged at 100,000g for 6 h and the supernatant collected. P‐gp activity is displayed as fold change from control (BECs exposed to unconditioned medium [UCM]) (n = 8/group). Data are displayed as mean ± SEM. A significant difference from control is indicated by **P < 0.01; ***P < 0.001. ACM, astrocyte‐conditioned medium; BECs, Brain endothelial cells. Effect of ACM fractions on P‐gp function in BECs To determine the approximate size of the protein(s) responsible for upregulating P‐gp function in BECs, ACM was fractionated using successive filtration (molecular weight ranges: <3 kDa, 3–30 kDa, 30–100 kDa, >100 kDa). P‐gp activity was increased in PND14 BECs that were exposed to ACM containing 3–30 kDa and 30–100 kDa proteins, by approximately 4‐fold (P < 0.001, P < 0.001; Fig. 7) but was not significantly altered by ACM containing <3 kDa or >100 kDa protein fractions. Figure 7 P‐gp activity in postnatal day (PND) 14 BECs exposed ACM containing soluble factors of varying molecular weights. ACM was fractionated using centrifugal filter devices with different MWCO (molecular weight cut‐offs) ultracel membranes. P‐gp activity is displayed as fold change from control (monocultured BECs) (n = 8/group). Data are displayed as mean ± SEM. A significant difference from control is indicated by ***P < 0.001. ACM, astrocyte‐conditioned medium; BECs, Brain endothelial cells. Differential expression of proteins in GD50 and PND14 ACM Using LC‐MS/MS, 467 proteins were identified in conditioned medium from GD50 and PND14 astrocytes. Of these proteins, 85 were found to be differentially secreted by PND14 and GD50 astrocytes, all of which were elevated in PND14 ACM compared to GD50 ACM (P < 0.05; Table 1). The five principal proteins were Y‐box‐binding protein‐1 (YB‐1), high‐density lipoprotein‐binding protein, melanoma differentiation‐associated gene 20, SERPINE1 MRNA‐binding Protein 1 and Actin, Beta‐Like 2. Table 1 List of proteins found to be upregulated in PND14 astrocyte‐conditioned medium (ACM) compared to GD50 (ACM) (n = 3/group) Protein Molecular weight (kDa) Fold change Y box‐binding protein 1 36 18.75 High‐density lipoprotein‐binding protein 141 16.92 Melanoma differentiation‐associated gene 20 18 14.09 SERPINE1 MRNA‐binding protein 1 45 8.51 Actin, beta‐like 2 42 6.26 Enolase 2 (Gamma, Neuronal) 47 6.17 KH domain containing, RNA‐binding, signal transduction associated 1 48 6.1 C‐Myc‐binding protein 40 5.79 Tropomyosin 2 33 5.78 Calsyntenin 2 107 5.5 Fructose‐bisphosphate aldolase 39 5.14 Reticulocalbin 3 37 5.04 MARCKS‐Like1 20 5.02 Profilin 15 4.75 Heat Shock 27 kDa Protein 1 23 4.56 Galectin 15 4.49 Von hippel–lindau‐binding protein 1 23 4.45 Neuropilin 2 105 4.18 Microtubule‐associated protein 1B 271 4.11 Tropomyosin alpha‐4 chain 50 4 Nestin 177 3.99 radixin 69 3.96 GTPase activating protein (SH3 Domain)‐binding protein 1 52 3.89 Transketolase 68 3.86 Small nuclear ribonucleoprotein 70 kDa (U1 52 3.86 Calponin 34 3.85 ATPase inhibitory factor 1 12 3.72 Heat shock 10 kDa protein 1 11 3.7 14‐3‐3 protein beta/alpha 28 3.65 Myosin, light chain 4 22 3.46 Lipoma‐preferred partner 66 3.46 Ependymin related 1 25 3.46 Phosphoribosylaminoimidazole carboxylase 47 3.41 Dihydropyrimidinase‐like 3 62 3.37 Actin‐related protein 2 45 3.31 Proteasome subunit alpha type 26 3.21 Eukaryotic translation elongation factor 1 Beta 2 25 3.18 Thioredoxin reductase 1 71 3.16 Septin 11 49 3.12 Septin 2 41 3.04 Ribosomal protein L30 13 3 A kinase (PRKA) anchor protein 12 191 2.98 Splicing factor proline/glutamine‐rich 76 2.91 Proteasome subunit alpha type 26 2.88 Alanyl‐TRNA synthetase 107 2.84 Peroxiredoxin 4 31 2.83 Fascin actin‐bundling protein 1 55 2.71 PDZ And LIM domain 7 (Enigma) 50 2.69 14‐3‐3 protein gamma 28 2.68 Protein disulfide‐isomerase 57 2.67 PDZ and LIM domain 5 64 2.61 Talin‐1 270 2.61 Proteasome subunit beta type 25 2.6 Endoplasmic Reticulum Protein 29 29 2.6 Triosephosphate isomerase 31 2.6 Fibromodulin 43 2.59 Dachsous cadherin‐related 1 346 2.55 Calcium‐activated nucleotidase 1 45 2.54 Filamin A, alpha 281 2.53 Binding immunoglobulin protein 72 2.52 Serine/threonine kinase receptor associated protein 38 2.5 Phospholipase B domain containing 2 65 2.5 Immunoglobulin superfamily containing leucine‐rich Repeat 46 2.49 Septin 7 51 2.45 Collagen, type I, alpha 2 129 2.44 Zyxin 61 2.43 Protein disulfide‐isomerase 57 2.4 Tropomodulin‐3 40 2.39 Myosin, heavy chain 9, nonmuscle 227 2.37 Collagen triple helix repeat containing 1 26 2.36 NAD(P)H dehydrogenase [quinone] 1 31 2.34 Adenosine deaminase 41 2.25 SH3 domain‐binding glutamic acid‐rich‐like protein 128 2.24 Proteasome subunit beta type 29 2.22 Lipopolysaccharide‐associated protein 1 71 2.2 Capping protein (Actin Filament), gelsolin‐like 38 2.18 Guanine nucleotide‐binding protein (G protein), beta polypeptide 2‐like, Proliferation‐inducing gene 21 35 2.17 Nucleoside diphosphate kinase 17 2.16 14‐3‐3 protein zeta/delta 28 2.15 Proteasome subunit alpha type 30 2.08 Microtubule‐associated protein RP/EB family member 1 30 1.95 Cartilage acidic protein 1 71 1.9 Malate dehydrogenase 36 1.87 Chaperonin‐containing tcp1, subunit 8 (Theta) 60 1.83 UDP‐glucose pyrophosphorylase 2 57 1.78 John Wiley & Sons, LtdWe further analyzed the differentially secreted proteins, using gene ontology (GO) Term Finder based on cellular component (Fig. 8A). Of these 85 proteins, 59 were identified as being extracellularly located and 54 were associated with extracellular exosomes. GO analysis also associated 74 proteins with cytoplasmic localization, with some proteins related to cytoskeletal and cell junction‐related proteins. Proteins were also analyzed based on GO function (Fig. 8B). Most proteins (~92%) were classified as ligand proteins, capable of binding to other molecules, such as small molecules (i.e., monosaccharaides), organic cyclic compounds, actin, nucleic acids, and macromolecules. Figure 8 Bioinformatic analysis of differentially secreted proteins. Proteins were clustered based on gene ontology (GO) cellular component (A) and GO Function (B), using GO Term Finder. GO terms with P < 0.01 are listed in the figure. Discussion This is the first study to establish a co‐culture model, using BECs and astrocytes from two distinct developmental ages. Using this co‐culture model, we demonstrated that astrocytes enhance tight junction and P‐gp function in BECs. Moreover, postnatal astrocytes can dramatically upregulate P‐gp function in fetal BECs, while fetal astrocytes had no effect on P‐gp activity in postnatal BECs. Thus, the BBB‐inducing properties of astrocytes are dependent on developmental stage at which astrocytes were derived. We also found that ACM could mimic these effects on P‐gp function, suggesting a mechanism that involves soluble astrocyte‐derived factors. Using heat inactivation and protease treatment, we demonstrated that these astrocyte‐derived factors are dependent upon proteins. Fractionation of ACM indicated that these factors are in the molecular weight range of 3–100 kDa. Moreover, LC/MS‐MS identified 85 proteins that were significantly upregulated in PND14 ACM compared to GD50 ACM. We have previously shown that isolated BECs retain their developmental characteristics (Iqbal et al. 2011). In the present study, we demonstrate that astrocytes also maintain properties appropriate to the age at which they were derived. Astrocytes derived at GD50 and PND14 express astrocyte markers appropriate to their developmental age. Nestin is expressed by GD50 astrocytes but not PND14 astrocytes. This is consistent with astrocyte development in vivo as Nestin is expressed by immature, developing CNS cells (Laywell et al. 2000; Lim et al. 2007). Nestin expression is downregulated and is transiently co‐expressed with GFAP during astrocyte differentiation (Sergent‐Tanguy et al. 2006). Astrocytes derived at PND14 but not GD50 express AQP4. AQP4 is the major water channel expressed in brain perivascular astrocyte processes and its expression is upregulated in vivo as differentiating astrocytic endfeet that make contact with the brain microvasculature (Nicchia et al. 2004). GFAP was expressed in both GD50 and PND14 astrocytes. GFAP may be important in astrocyte‐mediated upregulation of P‐gp expression and function at the BBB since GFAP knockout (KO) mice exhibit a structurally and functionally impaired BBB (Liedtke et al. 1996). The majority of studies that have investigated the effects of astrocytes on the BBB have utilized co‐culture models composed of BECs and astrocytes from different ages or cells from different animal species. These studies demonstrated that astrocytes enhanced many BBB characteristics in co‐cultured BECs compared to monocultured BECs (Hayashi et al. 1997; Berezowski et al. 2004; Cecchelli et al. 2014). However, our study highlights the importance of the maturational status of astrocytes in the regulation of P‐gp developing BBB. We utilized a novel co‐culture model using BECs and astrocytes derived from the same animal at two precise time‐points in development to show that postnatal and fetal astrocytes can enhance P‐gp function in age‐matched BECs. However, postnatal astrocytes increased P‐gp function in fetal BECs but fetal astrocytes had no effect on postnatal BECs. Other studies have shown that the maturational state of astrocytes also affects neuronal development. Astrocytes derived from newborn and adult rats were shown to differ in their capacity to sustain neurite outgrowth in retinal ganglion cells in co‐culture (Smith et al. 1990; Bahr et al. 1995). Thus, the maturational status of astrocytes appears to influence both neuronal and BBB development. A novel finding of our study was that PND14 astrocytes exhibit different effects on P‐gp function and levels of Abcb1 mRNA in GD50 and PND14 BECs. There was strong correlation between Abcb1 mRNA levels and P‐gp function in PND14 co‐cultures. However, in GD50 BECs, enhancement in P‐gp activity in GD50 BECs as a result of co‐culturing with age‐matched or different‐aged astrocytes, did not correspond to significant increases in Abcb1 mRNA levels. One explanation for this observation may be that high variability in the data may have prevented significant differences to be statistically detected between monocultured and co‐cultured GD50 BECs. Alternatively, this data may indicate that there may be different mechanisms involved in upregulation of P‐gp function in GD50 and PND14 BECs. It is well‐established that P‐gp function can be enhanced without changes at the level of mRNA. This includes post‐translational modifications to P‐gp protein, such as glycosylation and phosphorylation, both of which lead to an increase in P‐gp function (Chambers et al. 1994; Germann et al. 1996; Draheim et al. 2010). P‐gp function can also be increased by the recruitment of intracellular storage of P‐gp in vesicles to the cell membrane (Fu and Arias 2012). Additionally, the epigenetic status of the Abcb1 promoter may vary between GD50 and PND14 BECs. The density of methylation in the Abcb1 promoter is negatively correlated with Abcb1 expression in cancer cells (Fryxell et al. 1999; Baker and El‐Osta 2010). Our laboratory has shown that global DNA methylation in brain microvessels significantly decreases from GD50 to PND14 (M. Iqbal, J. Pappas and S.G. Matthews, unpubl. obs.). However, it has yet to be determined if these global changes in methylation include specific changes in promoter methylation of the Abcb1 gene. Early in gestation, increased methylation of the Abcb1 promoter may prevent binding of transcription factors, activated by astrocytes‐derived molecules, from increasing Abcb1 mRNA levels in GD50 BECs. Moreover, GD50 and PND14 BECs may respond differently to astrocyte‐derived signals from PND14 astrocytes simply because they express different types or levels of receptors. Utilizing ACM protease treatment and heat inactivation, we demonstrated that the astrocyte‐derived factor(s) is indeed a protein or dependent on a protein. We also showed that 3–30 kDa and 30–100 kDa PND14 ACM fractions can upregulate P‐gp function in PND14 BECs. These data indicate that more than one factor is likely responsible for this effect. Moreover, of the 85 proteins that were found to be significantly upregulated in PND14 ACM compared to GD50 ACM, 63 proteins fall into these molecular weight ranges. This suggests that astrocytes may regulate P‐gp function and Abcb1 mRNA levels via multiple signaling pathways, which are activated by various astrocyte‐secreted proteins. Consistent with this hypothesis, studies have implicated many astrocyte‐derived factors in regulation of P‐gp at the adult BBB. These pathways include those linked to sonic hedgehog (shh), canonical Wnt/β ‐catenin, retinoic acid, and the transforming growth factor‐β (TGF‐β) superfamily (Prat et al. 2001; Abbott 2002; Dohgu et al. 2004; Liebner et al. 2008; Alvarez et al. 2011; Mizee et al. 2013; Baello et al. 2014). In silico pathway analysis revealed that a number of the proteins that we identified participate in these signaling pathways. However, our studies do not conclusively rule out the role of lipid or fatty acid derivatives in regulating P‐gp. For example, the stability of retinol, from which retinoic acid is derived, is dependent on proteins (Klaassen et al. 1999; N'Soukpoe‐Kossi et al. 2007) and retinol‐binding protein was identified in conditioned media from GD50 and PND14 astrocytes. Moreover, GO analysis classified most of the significantly upregulated proteins in PND14 ACM (Table 1) as ligand proteins, capable of binding to small molecules, nucleic acids, macromolecules and organic cyclic compounds. Further studies are needed to elucidate the identity of the factor(s) involved in astrocyte‐mediated upregulation in P‐gp function and expression at the fetal and postnatal BBB. The most differentially expressed protein between GD50 and PND14 astrocytes was Y‐box‐binding protein (YB‐1), which was secreted approximately 18‐fold more by PND14 astrocytes compared to GD50 astrocytes. YB‐1 is a transcription factor that regulates genes such as Abcb1 (Shen et al. 2011 ; Dolfini and Mantovani 2013). However, recent studies indicate that many cell types release YB‐1 into the extracellular space (Frye et al. 2009; Raffetseder et al. 2011). Nanomolar concentrations were shown to stimulate proliferation and migration of rat mesangial cells and human kidney cells (Frye et al. 2009). This effect was mediated by YB‐1 interaction with EGF‐repeats of the Notch3 receptor, and activation of canonical Notch signaling (Rauen et al. 2009; D'Souza et al. 2010). Currently, very little is known about how Notch signaling regulates P‐gp function and expression. The only evidence of such regulation stems from cancer cells, in which Notch signaling is generally overactivated and is associated with increased P‐gp function (Capaccione and Pine 2013; Wu et al. 2014). For example, knockdown of Notch1 receptor leads to decreased ABCB1 and ABCC1 mRNA in cultured intrahepatic cholangiocarcinoma cells (Wu et al. 2014). Moreover, overexpression of Notch1 resulted in an increase in ABCB1 mRNA in gastric human cancer cell lines (Hang et al. 2015). Future studies are needed to investigate the role of YB‐1 and Notch signaling in regulating P‐gp in normal tissues. Our laboratory was unable to carry out these experiments as there is no commercially available recombinant YB‐1 protein. High‐density lipoprotein‐binding protein (HDLBP) was also found to be increased by approximately 16‐fold in PND14 ACM compared to GD50 ACM. HDLBP binds high‐density lipoprotein (HDL), which transports cholesterol, phospholipids, and triglycerides. A positive association between cellular cholesterol content and P‐gp function has been demonstrated in various cancer cell lines (Troost et al. 2004; Belli et al. 2009). However, the mechanism by which this occurs is unclear. HDLBP may mediate this effect as it is a secreted protein that can become anchored to the cell membrane through glycosylphosphatidylinositol‐enriched domains (Beigneux et al. 2007). Binding of cholesterol to HDLBP via HDL may elicit various signaling pathways (Fidge 1999). Consistent with this hypothesis, brain cholesterol content is maximal in late gestation and postnatal life, which is coincident with the surge in P‐gp at the BBB (Dietschy and Turley 2004). Additionally, HDLBP can bind to RNA and prevent its degradation (Fidge 1999). PND14 astrocytes may secrete more HDLBP, resulting in the stabilization of Abcb1 mRNA in BECs. The link between cholesterol and P‐gp warrants further investigation, especially in the context of the developing BBB. We also found that ACM has short‐term effects on P‐gp function in BECs, as these effects disappear at 48 h and 72 h. However, when ACM was replaced every 24 h, the effect was rescued at 48 h and 72 h, indicating that BECs are responsive to these signals and that these soluble factors may be labile or metabolized. However, the in vivo induction of P‐gp at the BBB by astrocytes is constant, as the release of many astrocyte‐secreted factors are dependent on neuronal activity (Otsu et al. 2015). Recently, astrocytes have been shown to be responsible for neurovascular coupling, a process by which blood flow is matched to neuronal activity (Devonshire et al. 2012). This coupling may regulate BBB properties through development. However, very little is known about how this process occurs in the developing brain or how it may affect BBB development. It is clear that disruptions in astrocyte–neuron interaction contribute to the pathogenesis of neurodevelopmental disorders (Jacobs et al. 2010; Williams et al. 2014). However, this study highlights the importance of astrocyte–BBB interaction. Disruptions in astrocyte development may also dysregulate P‐gp at the developing BBB, which may contribute to the CNS dysfunction in neurodevelopmental disorders. Moreover, many conditions have been shown to alter the timing of astrocyte maturation. Prenatal chronic hypoxia and inflammation can delay the maturation of astrocytes in the fetal brain – an effect that has been shown to persist later in life (Tilleux and Hermans 2008; Raymond et al. 2011). Further investigation is needed to determine the effect of compromised astrocyte maturation on P‐gp function on the developing BBB. There are a number of potential limitations to these types of in vitro study. The co‐culture model presented is a noncontact model, whereby BECs and astrocytes are not in physical contract and do not share a basement membrane. Moreover, the influence of other CNS cells, such as neurons and pericytes, are not taken into account. Previous studies have shown that these factors affect BEC and astrocyte function (Abbott 2002; Wolburg et al. 2009). Another limitation is the presence of serum in co‐culture media. Cells were grown in the presence of serum as BECs do not proliferate well in vitro without this component. However, in vivo astrocytes are not exposed to serum, future more focused studies will be designed to address these potential limitations. Notwithstanding, these studies do provide novel insight into the role of astrocytes in the regulation of P‐gp in the developing brain microvasculature. In conclusion, astrocytes are key regulators of P‐gp expression and function at the developing BBB, but the magnitude of this effect is dependent on the developmental age of astrocytes. Aberrations in astrocyte maturation may alter levels of astrocyte‐derived factors and dysregulate P‐gp at the BBB. This may lead to altered movement of endogenous compounds, such as cytokines and steroid hormones, into the brain. Moreover, it is common for women to take prescribed drugs during pregnancy, many of which are P‐gp substrates. Altered levels of P‐gp at the developing BBB may result in increased fetal brain exposure to drugs and xenobiotics. 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==== Front Physiol RepPhysiol Rep10.1002/(ISSN)2051-817XPHY2physreportsPhysiological Reports2051-817XJohn Wiley and Sons Inc. Hoboken 2756191110.14814/phy2.12878PHY212878Toxins, Pollutants and Chemical AgentsRespiratory Conditions Disorder and DiseasesCellular and Molecular PhysiologyOriginal ResearchOriginal Research ADAM17 and EGFR regulate IL‐6 receptor and amphiregulin mRNA expression and release in cigarette smoke‐exposed primary bronchial epithelial cells from patients with chronic obstructive pulmonary disease (COPD) M. Stolarczyk et al.Stolarczyk Marta 1 † Amatngalim Gimano D. 2 † Yu Xiao 1 Veltman Mieke 1 Hiemstra Pieter S. 2 Scholte Bob J. 1 1 Cell BiologyErasmus MCRotterdamThe Netherlands2 PulmonologyLeiden University Medical Center (LUMC)LeidenThe Netherlands* Correspondence Bob J Scholte, ErasmusMC, Cell Biology Ee1034, PO box 2040 3000 CA Rotterdam, The Netherlands. Tel: +31 10 7043205 E‐mail: b.scholte@erasmusmc.nl † Equal contribution. 25 8 2016 8 2016 4 16 10.1111/phy2.2016.4.issue-16e1287813 6 2016 08 7 2016 09 7 2016 © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.Abstract Aberrant activity of a disintegrin and metalloprotease 17 (ADAM17), also known as TACE, and epidermal growth factor receptor (EGFR) has been suggested to contribute to chronic obstructive pulmonary disease (COPD) development and progression. The aim of this study was to investigate the role of these proteins in activation of primary bronchial epithelial cells differentiated at the air–liquid interface (ALI‐PBEC) by whole cigarette smoke (CS), comparing cells from COPD patients with non‐COPD. CS exposure of ALI‐PBEC enhanced ADAM17‐mediated shedding of the IL‐6 receptor (IL6R) and the EGFR agonist amphiregulin (AREG) toward the basolateral compartment, which was more pronounced in cells from COPD patients than in non‐COPD controls. CS transiently increased IL6R and AREG mRNA in ALI‐PBEC to a similar extent in cultures from both groups, suggesting that posttranslational events determine differential shedding between COPD and non‐COPD cultures. We show for the first time by in situ proximity ligation (PLA) that CS strongly enhances interactions of phosphorylated ADAM17 with AREG and IL‐6R in an intracellular compartment, suggesting that CS‐induced intracellular trafficking events precede shedding to the extracellular compartment. Both EGFR and ADAM17 activity contribute to CS‐induced IL‐6R and AREG protein shedding and to mRNA expression, as demonstrated using selective inhibitors (AG1478 and TMI‐2). Our data are consistent with an autocrine‐positive feedback mechanism in which CS triggers shedding of EGFR agonists evoking EGFR activation, in ADAM17‐dependent manner, and subsequently transduce paracrine signaling toward myeloid cells and connective tissue. Reducing ADAM17 and EGFR activity could therefore be a therapeutic approach for the tissue remodeling and inflammation observed in COPD. A disintegrin and metalloprotease 17 (ADAM17)amphiregulin (AREG)Chronic Obstructive Pulmonary Disease (COPD)epidermal growth factor receptor (EGFR)IL6 receptor (IL6R)TACELung Foundation Netherlands#3.3.10.027Dutch Cystic fibrosis Foundation NCFSHIT‐CF7 source-schema-version-number2.0component-idphy212878cover-dateAugust 2016details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_NLMPMC version:4.9.4 mode:remove_FC converted:29.08.2016 M. Stolarczyk , G. D. Amatngalim , X. Yu , M. Veltman , P. S. Hiemstra , B. J. Scholte . ADAM17 and EGFR regulate IL‐6 receptor and amphiregulin mRNA expression and release in cigarette smoke‐exposed primary bronchial epithelial cells from patients with chronic obstructive pulmonary disease (COPD) . Physiol Rep , 4 (16 ), 2016 , e12878, doi: 10.14814/phy2.12878 Funding Information This study (MS) was supported by grants from the Lung Foundation Netherlands (#3.3.10.027). the Dutch Cystic fibrosis Foundation NCFS (MV HIT‐CF7) and Galapagos NV, The Netherlands. ==== Body Introduction Chronic obstructive pulmonary disease (COPD) is a progressive lung disorder characterized by irreversible airflow obstruction due to airway inflammation, infection, and tissue remodeling (Vestbo et al. 2013). Airway epithelial cells play a central role in the pathogenesis of COPD through a variety of mechanisms, including production of inflammatory mediators, antimicrobial peptides, and growth factors (Hiemstra et al. 2015). Exposure of the lung tissue to triggers like cytotoxic particles and gasses, including cigarette smoke, microbes, and innate immune mediators (Koff et al. 2008; Kim et al. 2011) (Lemjabbar‐Alaoui et al. 2011) (Shao 2004) (Zhou et al. 2012) activate various matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs) expressed by airway epithelial cells (Dijkstra et al. 2009; Paulissen et al. 2009). The activity of MMPs and ADAMs contributes not only to proteolytic degradation of lung tissue, but also to regulation and processing of numerous receptor activating proteins (Richter et al. 2002; Gomez et al. 2005; Bell et al. 2007; Baumgart et al. 2010). Through these various activities, MMPs and ADAMs are implicated in a broad spectrum of processes ranging from inflammatory responses to airway epithelial repair. It has been proposed that their aberrant activity might lead to chronic inflammation and abnormal tissue remodeling in the lungs of COPD patients (Paulissen et al. 2009). One of the ADAMs, a ubiquitously expressed Zn2+‐dependent disintegrin and metalloprotease 17 (ADAM17), formerly known as TNFα converting enzyme (TACE), is recognized as an important regulator of pulmonary inflammation, cell proliferation, and epithelial barrier function (Gooz 2010; Lemjabbar‐Alaoui et al. 2011). In bronchial epithelial cells, ADAM17 modulates these processes by cleaving membrane‐bound cytokines (TNFα), several EGF receptor (EGFR) agonists (TGF‐α, amphiregulin, epiregulin, HB‐EGF), cytokine receptors (IL6R, TNF‐R), growth factor receptors (NOTCH receptors), and adhesion proteins (L‐selectin, ICAM‐1, E‐cadherin) (Gomez et al. 2005; Bell et al. 2007; Baumgart et al. 2010; Gooz et al. 2012). Moreover, ADAM17 phosphorylation and activity is enhanced in airway epithelial cell lines and in undifferentiated primary cells upon exposure to cigarette smoke extract (Lemjabbar et al. 2003; Shao 2004; Lemjabbar‐Alaoui et al. 2011). Our studies focus on two ADAM17 substrates implicated in COPD pathogenesis: the IL‐6 cytokine receptor (IL6R) and the growth factor amphiregulin (AREG), one of the EGFR agonists produced by bronchial epithelial cells (Richter et al. 2002). Elevated levels of IL6R have been observed in peripheral blood leukocytes of COPD patients (Edmiston et al. 2010), and recently genetic variants of IL6R have been linked with COPD severity (Pérez‐Rubio et al. 2016). However, the regulation of shedding of IL6R and AREG from COPD airway epithelium has not been studied. Upon shedding from epithelial cells, IL6R and AREG activate the shared interleukin receptor gp130 and EGFR, respectively, on epithelial cells (autocrine), as well as on underlying myofibroblasts and myeloid cells (paracrine) (Burgel and Nadel 2008; Nechemia‐Arbely et al. 2008; Kasina et al. 2009; Rose‐John 2012). Both IL6/IL6R/gp130 and AREG/EGFR/ERK pathways are involved in the resolution of lung inflammation and repair of injury, but also in progression of subepithelial fibrosis and collagen deposition (Zhou et al. 2012). These signaling pathways involve the JAK kinase and/or MAP kinase pathway, which are druggable targets in COPD pathology (Barnes 2013). Excessive ligand‐mediated EGFR activation results in epithelial hyperproliferation and increased production of the inducible mucin MUC5AC, processes observed in smokers with or without COPD (Lemjabbar et al. 2003; Shao 2004; Deshmukh et al. 2005; Kasina et al. 2009; Lemjabbar‐Alaoui et al. 2011; Li et al. 2011; Zhang et al. 2014). Moreover, EGFR activation results in subsequent transcriptional regulation of inflammatory mediators such as IL‐8 (Richter et al. 2002), a chemokine that has been implicated in COPD development. So far, studies on the effect of cigarette smoke on epithelial ADAMs activity has largely relied on the use of airway epithelial cell lines or undifferentiated primary cell cultures, stimulated with an aqueous extract of cigarette smoke. However, whole cigarette smoke exposure and primary differentiated airway cell cultures represent more relevant physiological conditions. Firstly, fresh whole cigarette smoke (CS) contains unstable active components and particulate matter that are largely absent from extracts. Furthermore, immortalized epithelial cells are poor models of bronchial epithelium in situ, since they are frequently karyotypically unstable and heterogeneous, do not show characteristic features of differentiation and inherently carry multiple mutations in pathways essential for growth, differentiation, cell‐cell interaction, and polarization. Furthermore, submerged cultures of primary airway epithelial cells fail to differentiate. Finally, using cell lines does not allow a comparison of patient populations. Therefore, we examined the effect of whole CS exposure on shedding of the soluble interleukin‐6 receptor (sIL6R) and the EGFR‐ligand amphiregulin (AREG) by well‐differentiated, air–liquid interface cultured human primary bronchial epithelial cells (ALI‐PBEC). This allowed us to compare CS‐induced ADAM17‐mediated protein shedding and mRNA expression of sIL6R and AREG in well‐differentiated ALI‐PBEC from COPD and non‐COPD (ex)smokers. Moreover, we established in this model the involvement of both EGFR and ADAM17 not only in shedding of ADAM17 substrates, but also in the regulation of mRNA levels of ADAM17 substrates and IL‐8. Finally, for the first time, we observed intracellular CS‐induced phosphorylated ADAM17‐substrate interaction via an in situ proximity ligation assay. Overall, our results provide novel insights into the activation of airway epithelial cells by cigarette smoke in COPD, and highlight a possible role of ADAMs and EGFR in COPD pathology. Materials and Methods Air–liquid interface cell culture of human primary bronchial epithelial cells Human airway epithelial cells were obtained from macroscopically normal, anonymous bronchial tissue obtained from lung cancer patients undergoing resection surgery for lung cancer at LUMC. This material was be used for research according to the “Code of Conduct for Responsible Use” (FEDERA code) based on the condition that the patient has no objection against such use. Primary bronchial epithelial cells (PBEC) were isolated from tumor‐free lung resection material (Wetering et al. 2000), and passage 2 expanded cells were cultured at the air–liquid interface (ALI) to achieve mucociliary differentiation as previously described (Amatngalim et al. 2015). Briefly, 40,000 cells were seeded on 0.65 cm Transwell inserts (Corning Costar, Cambridge, MA) with a 0.4 μm pore size, which were coated with 30 μg/mL PureCol (Advanced BioMatrix, San Diego, CA), 10 μg/mL Bovine serum albumin (Sigma‐Aldrich, St. Louis, MO), and 10 μg/mL Fibronectin (isolated from plasma). Cells were cultured in Bronchial epithelial growth medium (BEGM) (Lonza, Verviers, Belgium) and Dulbecco's modified Eagle's medium (DMEM) (Gibco, Bleiswijk, The Netherlands) (1:1 mixture) containing 1 mmol/L Hepes (Lonza) and supplemented with SingleQuot supplements and growth factors according to the manufacturer's instructions (bovine pituitary extract, hydrocortisone, human epidermal growth factor, epinephrine, transferrin, insulin, T3 and retinoic acid; all from Lonza), additional 15 ng/mL retinoic acid (Sigma–Aldrich), 1 mg/mL BSA (Sigma–Aldrich), 100 U/mL penicillin, and 100 μg/mL streptomycin (Lonza). PBEC were initially cultured on inserts in submerged conditions until cell layers were confluent. Next, apical medium was removed and cells were cultured at air‐exposed conditions for at least 2 weeks to allow mucociliary differentiation. Clinical history and lung function data were obtained from anonymized patients (Table 1), and COPD disease status was based on lung function data according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) classification (Vestbo et al. 2013). Donor cells were randomly allocated to experimental groups. Table 1 COPD and non‐COPD patient characteristics COPD non‐COPD P‐value Number of donors 15 11 Gender (females/males) 4/11 2/9 Age, years 70 ± 8 66 ± 6 0.1667 FEV1, % predicted 65 ± 16 81 ± 16 <0.01 FEV1/FVC % 55 ± 9 79 ± 9 <0.0001 Characteristics of PBEC donors. Age in years, and lung function as FEV1 (% predicted) and FEV1/FVC are shown as means ± SD. The mean differences were compared using the nonparametric Mann–Whitney test. COPD, chronic obstructive pulmonary disease, FEV1, Forced expiratory volume in one‐second, FVC, forced vital capacity. John Wiley & Sons, LtdCigarette smoke exposure Air–liquid interface cultured human primary bronchial epithelial cells were exposed to whole cigarette smoke (CS) in an exposure model, adapted from (Beisswenger et al. 2004) and previously described in more detail (Amatngalim et al. 2015). In this model, ALI‐PBEC cultures were placed in either a CS‐ or air (negative control) exposure chamber located in a tissue incubator at 37°C and 5% CO2. Smoke derived from one cigarette (3R4F reference cigarettes [University of Kentucky, Lexington, KY]) was infused into the exposure chamber by a mechanical pump with a constant flow of 1 L/min, and equally distributed by a ventilator inside the chamber. After infusion (approximately 4–5 min), residual smoke was removed by infusion of air from the tissue incubator for 10 min. Directly after CS exposure, the basal medium of the cell cultures was refreshed and cells were incubated for the indicated periods of time. Untreated cells used as controls were subjected to the same procedure omitting the smoke (AIR). Inhibitors TMI‐2 (1 μmol/L; PF‐5480090), a highly selective inhibitor of ADAM17 activity (Zhang et al. 2004), was obtained from Wyeth inc. (Philadelphia, Pennsylvania) and the selective EGFR inhibitor AG1478 (1 μmol/L) was from Sigma Aldrich. Cells were preincubated for 1 h with inhibitors before CS exposure, and directly after CS exposure media were replaced and inhibitors were freshly added. ELISA ALI‐PBEC conditioned culture media were collected from the basolateral side of the inserts (1 mL), and apical washes were obtained by washing the apical surface with 100 μL PBS at different time points, dependent on the experiment. Collected samples were diluted 1:1 with BEGM media and analyzed for IL6R and AREG by human IL6R or AREG ELISA kit, R&D. Further steps were performed according to the manufacturer's protocol. Data were corrected for the dilution factor and insert size, and the amount of the shed IL6R and AREG was expressed as pg/mL per cm2. RNA isolation and quantitative real‐time PCR RNA was isolated using the miRNeasy Mini Kit (Qiagen) according to the manufacturer's instructions, and cDNA was synthesized by reverse‐transcription PCR using oligo(dT) primers (Qiagen) and Moloney murine leukemia virus (M‐MLV) polymerase (Promega, Leiden, The Netherlands). mRNA expression was determined by quantitative real‐time PCR as described previously (Amatngalim et al. 2015) with primer pairs presented in Table 2. mRNA expression was quantified using the standard curve method (Larionov et al. 2005), in which arbitrary expression levels were normalized to the housekeeping genes RPL13A and ATP5B. The housekeeping genes were selected based on stable expression using the “Genorm method” (Vandesompele et al. 2002). Table 2 Quantitative‐PCR primers Primer name [ref] IL8 FW 5′‐CAGCCTTCCTGATTTCTG‐3′ Amatngalim et al. (2015) RV 5′‐CACTTCTCCACAACCCTCTGC‐3′ AREG FW 5′GTGGTGCTGTCGCTCTTGATA 3′ Clarke et al. (2013) REV 5′ACTCACAGGGGAAATCTCACT3′ IL6R full‐length form (full‐IL6R) FW 5′GCTGTGCTCTTGGTGAGGAAGTTT3′ Rath et al. (2010) REV 5′CTGAGCTCAAACCGTAGTCTGTAGAAA3′ IL6R alternatively spliced variant (spliced IL6R) FW 5′GCGACAAGCCTCCCAGGTT3′ Rath et al. (2010) REV 5′CCGCAGCTTCCACGTCTTCTT3′ FW, Forward primer; REV, Reversed primer. John Wiley & Sons, LtdProximity ligation assay Chronic obstructive pulmonary disease ALI‐PBEC werefixed with 4% paraformaldehyde and permeabilized with 0.5% Triton‐X100 in PBS twice for 15 min, blocked with 1% BSA and 0.15% glycine. Next, they were incubated overnight in 4°C with two different first antibodies simultaneously: against ADAM17 (rabbit polyclonal, C‐terminal ADAM17, 25 μg/mL, ab78162, Abcam) or ADAM17PT735 (rabbit polyclonal, phospho‐ADAM17 in position T735, 25 μg/mL, ab60996, Abcam) and IL6R (goat anti‐human IL6R recognizing extracellular domain, 20 μg/mL, AF‐227‐NA, R&D) or AREG (polyclonal goat anti‐human Areg, 25 μg/mL, AF‐262, R&D). All washing steps were repeated three times with 0.5% Triton‐X100 in PBS (Sigma‐Aldrich). Further steps were performed according to the DuoLink manufacturer's protocol. Briefly, Proximity ligation assay (PLA) probes for anti‐goat PLUS and anti‐rabbit MINUS were incubated at 37°C for 1 h. Ligation and amplification steps were performed with Detection Red Reagent. Finally, inserts with ALI‐PBEC were cut out and mounted on the slides with DAPI (Vectashield mounting medium for fluorescence with DAPI, H‐1200). z‐stacks were acquired using confocal microscopy (Leica604). PLA image analysis The number of dots was counted in the whole z‐stack with Image J software. The threshold values were adjusted with the Intermodes algorithm (the filter size set between 10 and 437 microns to exclude the small and large dots, which were in the range of 10% of the total dot count). The objects on the edges of the culture inserts were excluded from the analysis. The number of nuclei was counted in each z‐stack by hand to express the number of dots per nucleus. Statistical analysis Data were analyzed with GraphPad Prism Software, using the appropriate statistical test, as indicated underneath each figure. Cells from various donors (number indicated by n in the legends of figures) were used for each experiment, samples were collected from two or three wells from a single donor, averaged and represented as a single dot in the figure. Statistical analysis was performed on the averaged data. Values are presented as mean with SEM values. Differences at P‐values <0.05 were considered to be statistically significant. ns > 0.05, *P < 0.05, **P < 0.01, **P < 0.001, ****P < 0.0001. Results CS induces shedding of IL6R and AREG by ALI‐PBEC into basolateral medium, but not apical We first examined the effect of cigarette smoke (CS) exposure on the release of sIL6R and AREG by ALI‐PBEC at the apical surface and in the basal medium, which contains a maintenance level of EGF, associated with a basal level of EGFR activity. This was done, using a previously described whole CS exposure model (Amatngalim et al. 2015), in which CS caused a transient disruption in the airway epithelial barrier integrity, accompanied by minor cytotoxic effects measured at the apical surface. Both sIL6R and AREG were barely detectible in the apical washes collected from ALI‐PBEC of 17 COPD donors at different stages of disease, following exposure to either CS or air (Fig. 1A and B). In contrast, sIL6R and AREG were markedly released into the basal medium in both conditions. CS significantly increased release of sIL6R into the basal medium at 12 h postexposure, while AREG levels were increased at 12 and 24 h after CS exposure. These results demonstrate that shedding of sIL6R and AREG by ALI‐PBEC occurs mainly to the basolateral compartment, and is enhanced by CS exposure. Figure 1 Cigarette smoke induces shedding of IL6R and AREG by ALI‐PBEC into basolateral medium. IL6R (A) and AREG (B) were mainly shed to the basolateral compartment in ALI‐PBEC (COPD donors at different stages, n = 12 for IL6R and n = 5 for AREG). Basolateral media (basolateral) were collected and apical PBS washes (apical) were performed 12 h and 24 h after CS or air exposure. Both IL6R and AREG were readily detectable in the basolateral compartment, and barely present in the apical washes. The response of cells from each donor was analyzed within one experiment using duplicate or triplicate inserts. Statistical analysis was performed by two‐way ANOVA (Bonferroni) on the averaged data from each donor, comparing apical versus basolateral shedding at air and CS exposure, and basolateral shedding at air versus CS exposure. CS significantly induces shedding of IL6R and AREG in COPD ALI‐PBEC but not in non‐COPD ALI‐PBEC Next, we explored whether shedding of sIL6R and AREG differs between ALI‐PBEC isolated from COPD patients and non‐COPD (ex)‐smokers upon CS and air exposure. Based on the previous result, the release was only determined in the basal medium 24 h after exposure. Shedding of sIL6R and AREG did not differ between COPD and non‐COPD ALI‐PBEC exposed to air (Fig. 2A and B), indicating no differences at baseline conditions. In contrast, shedding of sIL6R (Fig. 2A) and AREG (Fig. 2B) was significantly higher after CS exposure only in COPD ALI‐PBEC, and not in non‐COPD ALI‐PBEC. These data show that CS‐induced release of sIL6R and AREG was more pronounced in airway epithelial cells from COPD in comparison to non‐COPD donors. Figure 2 Cigarette smoke significantly induces shedding of IL6R and AREG into basolateral medium by COPD ALI‐PBEC. Soluble forms of IL6R (A) and AREG (B) shed into the basolateral compartment were detected 24 h after CS or air exposure in ALI‐PBEC derived from non‐COPD and COPD donors (Table 1). (A) IL6R levels were significantly increased 24 h after CS treatment in COPD‐ALI‐PBEC (n = 15), but this increase was not significant in the non‐COPD group (n = 11 donors). (B) Similarly, CS exposure significantly increased AREG levels in ALI‐PBEC cells derived from COPD donors (n = 10), but not in non‐COPD ALI‐PBEC (n = 8 donors). Statistical analysis: paired t‐test. n refers to the number of donors, duplicate or triplicate data were averaged per donor. Statistical analysis was performed on the averaged data from each donor. CS‐induced IL6R and AREG mRNA expression is lower in COPD ALI‐PBEC compared to non‐COPD cultures We further determined mRNA expression of IL6R and AREG in CS and air exposed ALI‐PBEC cultures from COPD and non‐COPD patients. The soluble form of IL6R can be generated either by shedding of the membrane anchored form or by de novo synthesis of the alternatively spliced isoform that differs at the C‐terminus (Rose‐John 2012). Therefore, we determined mRNA expression levels of both IL6R variants: the membrane‐anchored (full‐IL6R mRNA) and the alternatively spliced (spliced‐IL6R mRNA) variant. Time‐course analysis revealed that CS increased full‐IL6R mRNA 3 h after exposure, but not at later time points (Fig. 3A). In contrast, baseline expression of spliced‐IL6R mRNA did not differ from the expression after CS treatment (Fig. 3B), suggesting that the increase in sIL6R protein levels in culture supernatants did not result from alternative splicing. Similar to full‐IL6R mRNA, CS significantly induced AREG mRNA expression 3 h after exposure, but not at later time points (Fig. 3C). These findings suggest that the CS‐induced increase in IL6R and AREG shedding is mediated at least in part via regulation of their mRNA expression levels. Figure 3 CS exposure transiently enhances IL6R and AREG mRNA expression in COPD and non‐COPD ALI‐PBEC. mRNA levels of the IL6R full‐length variant (full‐IL6R) (A), the IL6R splice variant (spliced‐IL6R) (B) and AREG (C) were determined by qPCR 3, 12, and 24 h after CS (black bars) or air exposure (open bars) (n = 14 unspecified donors). A transient induction of full‐IL6R (A) and AREG (C), but not spliced‐IL6R (B) was observed at 3 h after CS exposure. In COPD (n = 7) and non‐COPD (n = 8) ALI‐PBEC, mRNA of full‐IL6R (D), spliced‐IL6R (E), and AREG (F) were determined 3 h after CS exposure. mRNA expression of full‐IL6R and AREG, was lower on average but not statistically significant in COPD compared to non‐COPD donors. Data were normalized for expression against two reference genes (ATP5B and RPL13A). n refers to the number of donors. The response of cells from each donor was analyzed within one experiment using duplicate inserts and data were averaged per donor. Statistical analysis was performed on the averaged data from each donor. Statistical analysis: Two‐way ANOVA With Tukey's multiple comparison test. Baseline expression of full‐IL6R and AREG mRNA did not differ between COPD and non‐COPD ALI‐PBEC (Fig. 3D and F). After CS exposure, full‐IL6R and AREG mRNA were expressed at higher levels in both non‐COPD and COPD ALI‐PBEC. Interestingly, after CS induction, COPD cells expressed full‐ILR and AREG at lower levels on average but this did not reach statistical significance (Fig. 3D and F). Spliced‐IL6R mRNA expression did not differ between investigated groups either after CS or air exposure (Fig. 3E). These findings suggest that COPD patients may have impaired transcriptional or posttranscriptional responses to inflammatory and tissue regenerative triggers. The apparent contrast with the more pronounced shedding from COPD cells after CS challenge (Fig. 2) suggests that posttranslational mechanisms determine shedding rate, rather than substrate mRNA levels. ADAM17 is required for CS‐induced release of IL6R and AREG in ALI‐PBEC To confirm the previously established involvement of ADAM17 in the shedding process of IL6R and AREG in our model, we used the selective ADAM17 inhibitor TMI‐2 (Wyeth) (Zhang et al. 2004). TMI‐2 only partially decreased baseline IL6R release at all investigated time points (Fig. 4A), plausibly because release of the product of spliced‐IL6R mRNA, which cannot be distinguished from shed IL‐6R with the available antibodies, is not sensitive to inhibitors of ADAMs (Vermes et al. 2012). In contrast, TMI‐2 significantly decreased baseline AREG shedding at all time points (Fig. 4B). Importantly, CS‐induced shedding of IL6R and AREG was significantly inhibited by TMI‐2 at all time points after CS exposure, indicating that ADAM17 activity is involved in CS‐induced ADAM17 substrate release (Fig. 4). Figure 4 ADAM17 is involved in the release of soluble IL6R and AREG in ALI‐PBEC. The selective ADAM17 inhibitor, TMI‐2 (Zhang et al. 2004) decreases basal and CS‐induced IL6R (A,B) and AREG (C,D) shedding in ALI‐PBEC cells (n = 3 COPD donors) at 3, 6 (A,C), 12, and 24 h time points (B,D). n refers to the number of donors. The response of cells from each donor was analyzed within one experiment using duplicate or triplicate inserts and data were averaged per donor. Statistical analysis was performed on the averaged data from each donor, by two‐way ANOVA (Bonferroni), confirming first the effect of CS on IL6R and AREG shedding at different time points, and second the effect of TMI‐2 on shedding during air and CS exposure. ADAM17‐ and ADAM17P‐substrate interactions are increased after CS exposure in an intracellular compartment of ALI‐PBEC Next, we explored the interactions of IL6R or AREG with ADAM17 3 h after CS treatment in ALI‐PBEC with an in situ proximity ligation assay (PLA) (Fredriksson et al. 2002), using antibodies against ADAM17 phosphorylated at Thr735 (ADAM17‐PT735) or total ADAM17. Protein IL6R/AREG‐ADAM17 and IL6R/AREG‐ADAM17‐PT735 interactions were visualized as fluorescent red dots in x‐y confocal sections (representative confocal pictures shown in Fig. 5A and B). In air‐exposed cells, PLA signals were largely confined to the basal region, as in the control incubations, and not significantly higher than background (data not shown), as indicated by red lines in Figure 5C–F (relevant control data are shown in Figure S1). Interestingly, CS exposure significantly increased the total number of PLA signals for interactions of IL6R or AREG with ADAM17 (Fig. 5C and E). We observed that CS strongly enhanced interactions of IL6R or AREG with ADAM17‐PT735 (Fig. 5D and F), which further extends previous findings showing that ADAM17 is phosphorylated after smoke extract (CSE) exposure in submerged immortalized NCI‐H292 cells (Lemjabbar‐Alaoui et al. 2011). CS‐induced PLA signals of substrate‐ADAM17 and substrate‐ADAM17‐PT735 were primarily detected in the apical region of the cells and were not confined to a lateral membrane pattern suggesting an intracellular vesicular localization of protein complexes in ALI‐PBEC. These data for the first time demonstrate that CS exposure strongly increases the interaction of ADAM17 and ADAM17‐PT735 with IL6R or AREG in an intracellular vesicular compartment of ALI‐PBEC, suggesting a CS induced effect on protein trafficking. Figure 5 CS increases substrate‐ADAM17 and substrate‐ADAM17‐PT 735 interactions in pseudostratified COPD ALI‐PBEC cells. The proximity ligation assay (PLA) signal in COPD‐ ALI‐PBEC exposed to CS or air was performed for the following interactions: IL6R‐ADAM17, IL6R‐ADAM17PT 735, AREG‐ADAM17, and AREG‐ADAM17‐PT 735. Relevant control data are shown in Figure S1. Here, we show a representative figure of IL6R‐ADAM17PT 735 3 h after air (A) and CS exposure (B). Left panels show merged signals of nuclei (blue) and PLA (red) in the x–y sections of the confocal z‐stack and right panel presents PLA signal in the apical region (red dots). The number of PLA interactions was counted for all interactions as described in the methods section and expressed per nucleus 3 h after CS or air exposure in the whole z‐stack of the ALI‐PBEC (C–F). The red lines on the graphs indicate the maximal dot count in the PLA assay controls, in which one of the antibodies for the interaction was omitted (background staining not shown). For each interaction, cells from one donor were analyzed. Different filters (n = 4) were used to show distinct interactions. Statistical analysis: unpaired t‐test. EGFR is required for basal and induced AREG shedding in ALI‐PBEC ADAM17‐dependent shedding of EGFR ligands such as AREG results in activation of EGFR through an autocrine feedback loop, which modulates basal EGFR activity (DeWitt et al. 2001). This mechanism was shown to be activated by CS extract in submerged cultured PBEC and in cell lines (Lemjabbar et al. 2003). In our experimental set‐up, we have previously shown that CS enhances basal EGFR activity by increasing its phosphorylation (Amatngalim et al. 2015). To illustrate the involvement of EGFR in CS‐induced ADAM17‐related shedding in ALI‐PBEC, we assessed sIL6R and AREG shedding after starvation for growth factors, using medium devoid of EGF and bovine pituitary extract (BPE). Removing these factors from the medium substantially reduced baseline shedding of IL6R and AREG (Fig. 6), when compared to standard culture conditions including EGF and BPE (Fig. 4A and C). Both sIL6R and AREG release were significantly increased at 3 h after CS exposure. The selective ADAM17 inhibitor TMI‐2 and the EGFR tyrosine kinase inhibitor (AG1478) added prior to CS exposure, partially inhibited sIL6R shedding, consistent with a substantial contribution of the ADAM‐insensitive splice variant sIL‐6R levels in the basal medium (Fig. 6A). AG1478 strongly impaired AREG shedding, to a similar extent as TMI‐2 (Fig. 6B). These findings together demonstrate a critical role of EGFR activation in ADAM17‐mediated basal and CS‐induced shedding activity. Figure 6 IL6R and AREG shedding depends on ADAM17 and EGFR activity in COPD‐ALI‐PBEC. COPD ALI‐PBEC (n = 3 donors) were starved for growth factors for 48 h prior to CS or air exposure. Three hours after CS exposure, IL6R (A) and AREG (B) shedding were significantly increased compared to air. The ADAM17 inhibitor (1 μmol/L TMI‐2) and the EGFR inhibitor (1 μmol/L AG1478) significantly reduced AREG, but IL6R shedding to a lesser extent. The response of cells from each donor was analyzed within one experiment using triplicate inserts and data were averaged per donor. Statistical analysis was performed on the averaged data from each donor by one way ANOVA (Tukey multiple comparison test), only relevant comparisons are shown, air versus CS‐treated cells and the effect of inhibitors in CS‐treated cells. EGFR and ADAM17 are required for CS‐induced IL6R and AREG mRNA expression We previously observed that EGFR activation is involved in CS‐induced expression of several genes in ALI‐PBEC (Amatngalim et al. 2015). The molecular mechanism by which CS activates EGFR are not known. Here, we explored the effect of ADAM17 and EGFR inhibition on CS‐induced IL6R and AREG mRNA levels in ALI‐PBEC. At 3 hours after CS exposure in the absence of EGF in the medium, both TMI‐2 and AG1478 significantly impaired CS‐induced expression of full‐L6R mRNA (Fig. 7A), but not the splice variant (Fig. 7B). Both inhibitors strongly diminished CS‐induced AREG mRNA levels (Fig. 7C) as well as IL‐8 mRNA expression (Fig. 7D). Overall, these findings for the first time demonstrate that ADAM17, next to EGFR, is essential in the CS‐induced mechanism regulating not only the mRNA of ADAM17 substrates (IL6R and AREG), but also IL‐8 in ALI‐PBEC. Figure 7 IL6R and AREG mRNA expression are regulated by ADAM17 and EGFR activity in ALI‐PBEC. ALI‐PBEC (n = 3 donors) were starved for growth factors for 48 h prior to CS exposure. At 3 h, CS‐induced mRNA levels of full‐length IL6R (A), AREG (C), and IL8 (D) were diminished upon ADAM17 (1 μmol/L TMI‐2) and EGFR (1 μmol/L AG1478) inhibition, whereas that of the alternatively spliced form of IL6R was not affected (B). n refers to the number of donors. The response of cells from each donor was analyzed within one experiment using triplicate inserts and data were averaged per donor. Statistical analysis was performed on the averaged data from each donor, by one‐way ANOVA (Tukey multiple comparison test), only relevant comparisons are shown, air versus CS‐treated cells and the effect of inhibitors in CS‐treated cells. Discussion Many studies have demonstrated that airway epithelial cells are activated by exposure to environmental triggers like cigarette smoke, which contributes to COPD pathology (Rusznak et al. 2000; Schulz et al. 2004; Heijink et al. 2012; Amatngalim et al. 2015). In contrast to most studies, we used fresh whole cigarette smoke instead of (aged) cigarette smoke extract, and ALI‐differentiated PBEC from COPD and non‐COPD donors instead of submerged cultures of nondifferentiated primary cells or cell lines. While the obvious advantage of this approach is that we can study well‐differentiated primary cells from different patient populations, a limitation is that confirmation of data obtained with experimental pharmaceutics by, for example, gene editing or RNAi technology is not feasible in this context. Aside from efficiency issues and off‐target effects in primary cells, knocking down EGFR or ADAM17 likely affects the growth and differentiation of primary bronchial epithelial cells, which essentially defeats our purpose. However, the two inhibitors that we apply here to inhibit EGFR (AG1478) and ADAM17 (TMI‐2), respectively are widely used and are known to be highly selective. Importantly, our data demonstrate for the first time that CS triggered increase of basal shedding of IL6R and AREG into the basal medium, in the presence of EGF in the growth medium providing basal EGFR activity, was more pronounced in ALI‐PBEC derived from COPD patients compared to non‐COPD controls. We further report the ability of CS to increase mRNA expression of these genes in an EGFR‐ and ADAM17‐dependent way in ALI‐PBEC cells, under these conditions, with a lower tendency to induction in the COPD group. These results extend previous studies showing dysregulated responses of COPD airway epithelial cells to cellular stress, and provide novel evidence for the mechanism of CS‐induced and COPD‐related proinflammatory and profibrotic responses (Fig. 8). Figure 8 Cigarette smoke exposure activates EGFR‐ADAM17 axis in airway epithelial cells. Under basal conditions, there is ADAM17 related AREG and IL6R shedding activity, depending on the level of EGFR activity (compare Figs 4 and 6). Cigarette smoke exposure (CS) initiates an interaction of the phosphorylated form of ADAM17 (ADAM17‐P) with the full‐length transmembrane forms of IL6R and AREG in an intracellular compartment of the airway epithelial cell (Fig. 5), resulting in proteolysis and subsequent secretion (shedding) of the soluble active domains of IL6R and AREG toward the basolateral compartment. This involves both ADAM17 and EGFR activity (Figs 4 and 6). CS exposure also affects IL6R and AREG gene expression or mRNA stabilization in airway epithelial cells through ADAM17 and EGFR activation. sAREG and sIL6R secreted towards the basolateral compartment may change the level of activity of EGFR and the interleukin receptor IL6st/gp130 on the airway epithelial cells (autocrine). This may contribute to the activity of the EGFR/ADAM17 axis (positive feedback), which is likely kept in check by inactivation of internalized EGFR. Paracrine activity of sAREG and sIL6R may further transactivate EGFR and the interleukin receptor IL6st/gp130 on the underlying myofibroblasts, and myeloid cells, activating downstream pathways, including STAT3, involved in inflammation, collagen deposition, and myofibroblast proliferation. The differential effect of CS on sIL6R and AREG release between COPD and non‐COPD ALI‐PBEC might be related to differences in epithelial barrier function as previously described (Heijink et al. 2014). Using the current CS exposure system, we have previously shown that CS causes a transient decrease in epithelial barrier function (Amatngalim et al. 2015). However, in contrast to Heijink et al., we did not observe differences between COPD and non‐COPD cultures at baseline conditions and upon CS exposure (G.D. Amatngalim et al., unpubl. data), which may be explained by the fact that Heijink et al. focused on severe (GOLD stage IV) COPD. Another explanation might be differences in epithelial cell differentiation, as it has been shown that COPD epithelial cells display a more mesenchymal phenotype due to enhanced autocrine expression of TGF‐β1 (Gohy et al. 2015). As previously shown, the EGFR‐ADAM17 pathway is essential for IL‐8 release from a bronchial epithelial cell line exposed to particulate air pollution (Ovrevik et al. 2011) and implicated in CS extract‐induced expression of the mucin MUC5AC (Shao 2004). Further, autocrine production of EGFR ligands is involved in CS‐induced IL‐8 release from airway epithelial cells (Richter et al. 2002). Our studies extend these observations by showing the involvement of the ADAM17‐EGFR pathway in the release of IL6R and AREG upon CS exposure of differentiated PBEC, both in the presence (Fig. 4), and absence (Fig. 6) of EGFR ligand (EGF) in the basal medium respectively. Notably, the basal shedding rates are considerably lower in cells preincubated in medium lacking EGF, resulting in a much larger ADAM17‐ and EGFR‐dependent stimulation effect of CS (compare Figs 4 and 6). Which of these extreme conditions of basal EGFR activation apply in normal and COPD lungs in situ, and to what extent autocrine feedback signaling through ADAM‐dependent EGFR ligand shedding determines EGFR activity (Fig. 8) remains to be established. Additionally, EGFR and ADAM17 were both essential for CS‐induced IL6R and AREG mRNA expression (Fig. 7). These results provide novel insights into the mechanisms of airway epithelial cell activation by cigarette smoke in COPD, and highlight a role of ADAMs and EGFR in this process (Fig. 8). We further found that CS increases shedding of IL6R and AREG to the basal medium, but not to the apical side (Fig. 1). This is in line with report in polarized Madin‐Darby canine kidney cells (MDCK cells) showing that newly synthetized AREG is directly delivered to the basolateral surface with >95% efficiency (Brown et al. 2001). However, this is in contrast to the secretion of the innate immune mediators IL‐8 and ribonuclease 7, which were also detected at the apical surface (Amatngalim et al. 2015). A polarized ADAM17‐mediated secretion toward underlying tissue may be relevant for lung tissue remodeling through autocrine, paracrine, extracrine (exosomal targeted receptor activation) pathways in COPD (Booth et al. 2007; Zhou et al. 2012). Further examination of this phenomenon in epithelial–mesenchymal co‐culture systems is in progress. Amphiregulin release and phosphorylation of ADAM17 after CS extract treatment in ALI‐PBEC has been previously detected by ELISA or western Blotting (Lemjabbar‐Alaoui et al. 2011). Our proximity ligation assay (PLA) data show for the first time that CS‐induced shedding involves an intracellular interaction between phosphorylated ADAM17 and its substrates (Figs 5 and 8), whereas the majority of the literature suggests that shedding occurs mainly at the plasma membrane surface. This interaction likely takes place in intracellular membranes that sequester active phosphorylated ADAM17 and its transmembrane substrates upon activation. This process may relate to the transient change in barrier function upon CS treatment in our system and the subsequent activation of EGFR (Amatngalim et al. 2015). Our observation is supported by other reports showing the presence ADAM17 or its substrates in a vesicular compartment in lysosomes (Ebsen et al. 2015), endosomes (Gephart et al. 2011; Dombernowsky et al. 2015), and exosomes negative for the ER marker calreticulin (Higginbotham et al. 2011). Moreover, Gutwein et al. demonstrated that ADAM10‐mediated L1 migration factor cleavage occurs in Golgi‐derived vesicles in tumor cells (Gutwein et al. 2002). This was further supported by a recent paper suggesting that also ADAM10/ADAM17‐mediated release of soluble FasL occurs from an intracellular vesicular pool of secretory lysosomes in stimulated T lymphocytes (Ebsen et al. 2015). Moreover, after ligand binding, EGFR traffics in endosomes from the plasma membrane to an intracellular compartment to continue its signaling (Vieira et al. 1996; Teis et al. 2006). EGF‐dependent MAPK signaling occurs from late endosomes and lysosomes (de Araujo et al. 2013). Interestingly, the MAPK/ERK pathway regulates trafficking of ADAM17 phosphorylated at Thr735 from the endoplasmic reticulum toward the plasma membrane (Soond 2005; Hilliard et al. 2011), which can be also activated through ligand binding to EGFR. Higginbotham et al. showed that AREG containing exosomes are rapidly internalized by recipient cells in an EGFR‐dependent manner (Higginbotham et al. 2011), enhancing invasion of LM2‐4175 cells through Matrigel and wound healing. In our ALI‐PBEC system, we observed a predominantly lateral localization of EGFR under basal culture conditions. After exposure to CS, we observed a more cytoplasmic localization, consistent with EGFR activation (Figure S2). Therefore, in line with these and published observations, our findings suggest that in HBEC‐ALI, CS triggers EGFR‐mediated trafficking of ADAM17 and its substrates to a common subcellular compartment to allow proteolysis and subsequent secretion of soluble products (Fig. 8). At this time, we cannot establish to what extent autocrine signaling through shed ADAM substrates determine this response, or whether alternative mechanisms such as transactivation by intracellular kinases or oxidation or the extracellular receptor domain plays a role. Additional studies of triggered trafficking of EGFR, ADAM17‐P, and its substrates in polarized airway cells are required to further establish this mechanism. In addition to CS‐enhanced release through ADAM17 enzymatic activity, we observed transiently enhanced mRNA expression of both AREG and full‐IL6R in ALI‐PBEC upon CS exposure (Figure 3). CS did not affect the level of the alternatively spliced form of IL6R, so we conclude that alternative splicing unlikely contributes to CS‐enhanced release of IL6R. Previously, we observed upregulated IL‐8 mRNA expression in ALI‐PBEC exposed to CS as a result of enhanced EGFR phosphorylation and activation of the downstream MAPK/ERK1/2 signaling pathway (Amatngalim et al. 2015). Here, we report that CS‐induced IL6R and AREG mRNA expression was also reduced upon EGFR inhibition. In addition, we show here for the first time that inhibition of ADAM17 has the same effect on these mRNA levels (Fig. 7). Therefore, our data suggest that CS enhances factors common for activation of IL6R, AREG, and IL‐8 mRNA expression likely via an autocrine ADAM17‐EGFR axis (Fig. 8). The transcriptional and posttranscriptional regulation of these genes upon inhaled toxic substances has not been fully elucidated. Induced EGFR signaling is able to activate transcription of target genes. In addition, it has been shown that CS extract enhances HuR‐mediated IL‐8 mRNA stability in airway epithelial cells (Hudy and Proud 2013). Moreover, UV‐exposure of keratinocytes enhances mRNA HuR‐mediated stability of AREG, in an EGFR‐dependent manner (Nakayama et al. 2013). These observations suggest that CS‐induced activation of EGFR enhances sIL6R, AREG, and IL8 mRNA stability in ALI‐PBEC. Furthermore, mRNA regulation may be altered in cultured airway epithelial cells from COPD patients (Steiling et al. 2013). CS‐treated COPD ALI‐PBEC expressed lower AREG and IL6R mRNA levels on average compared to non‐COPD controls, but this difference was not statistically different (Fig. 3). Further studies on mRNA stability in this system are required to establish this. Nevertheless, this observation contrasts with the shedding data (Fig. 2) and suggests that ADAM17‐dependent AREG and sIL6R output is not primarily regulated on the mRNA level, but involves posttranslational regulation. Our data support the relevance of the ADAM17/EGFR pathway in COPD development and progression. Selective inhibitors of ADAM17, EGFR, and other components of this signaling pathway such as JAK and MAPK potentially expand therapeutic possibilities. The development of ADAM inhibitors for clinical use has been studied intensively (Moss et al. 2008; Duffy et al. 2011; Dreymueller et al. 2015). In cellular and animal tumor models, positive results were recorded (Witters et al. 2008). An ADAM17 inhibitor, TAPI‐0, reduced bleomycin‐induced lung inflammation (Lee et al. 2012). The selective inhibitor TMI‐2 used in this study, reduced LPS‐induced inflammation in vivo (Zhang et al. 2004). However, due to a lack of target specificity of available compounds, and side effects associated with the various other biological functions of ADAMs, chronic and systemic application of these compounds in humans is so far prohibited (Arribas and Esselens 2009). Clearly, more advanced intervention tools are required. Our data offer new insights in the regulation of mRNA expression, secretion, and release of ADAM17 substrates in airway epithelial cells upon triggering, which in combination with state of the art molecular design and advanced organotypic cellular modeling of airways could allow development of more selective inhibitors, targeted to specific cells and subcellular domains. In summary, this study provides evidence that ADAM17‐mediated release and shedding of IL‐6R and AREG is highly enhanced in airway epithelial cells in response to CS‐induced injury. Next to ADAM17, we highlight the importance of EGFR in the regulation of IL6R and AREG release and mRNA expression. Moreover, CS‐induced ADAM17‐mediated shedding of IL6R and AREG is especially high in COPD ALI‐PBEC, suggesting that reducing ADAM17 activity in COPD might be a potential therapeutic approach. Conflict of Interest Dr. Hiemstra reports receiving research grants from Galapagos NV for the submitted work, and research grants from Boehringer Ingelheim and Grifols outside the submitted work. Dr Scholte reports a research grant from Lexicon inc not related to the submitted work. Supporting information Figure S1. The proximity ligation assay (PLA) background signal. Click here for additional data file. Figure S2. Lateral EGFR in ALI‐PBEC, internalized after CS treatment. ALI‐PBEC cultured under basal conditions, including (EGF and PBE) were treated with air or CS as described in the methods section. (A) lateral EGFR immune fluorescence signal (green) becomes more diffuse 3 h after CS treatment. (B) Lateral E‐cadherin (red) illustrates the partial cytoplasmic localisation of EGFR. (C) This is confirmed in a separate experiment, after 10 min exposure to air or CS, with three separate filters each. Click here for additional data file. Acknowledgements The ADAM inhibitors TMI‐1 and TMI‐2 were kindly provided by Wyeth Research, Cambridge, MA 02140, USA. The authors thank the Department of Thoracic Surgery at LUMC for collection of lung tissue, and Winifred Broekman and Jorn Nützinger (LUMC) for their help in collecting patient data. ==== Refs References Amatngalim , G. D. , Y. van Wijck , Y. de Mooij‐Eijk , R. M. Verhoosel , J. Harder , A. N. 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==== Front Physiol RepPhysiol Rep10.1002/(ISSN)2051-817XPHY2physreportsPhysiological Reports2051-817XJohn Wiley and Sons Inc. Hoboken 2755098610.14814/phy2.12883PHY212883Injury, Stress and FatigueCognitive and Behavioural NeuroscienceNeurological Conditions, Disorders and TreatmentsCardiovascular Conditions, Disorders and TreatmentsOriginal ResearchOriginal ResearchPost‐exercise syncope: Wingate syncope test and visual‐cognitive function D. C. Sieck et al.Sieck Dylan C. 1 Ely Matthew R. 1 Romero Steven A. 1 Luttrell Meredith J. 1 Abdala Pedro M. 1 Halliwill John R. 1 1 Department of Human PhysiologyUniversity of OregonEugeneOregon* Correspondence John R. Halliwill, Department of Human Physiology, 122 Esslinger Hall, 1240 University of Oregon, Eugene, OR 97403‐1240. Tel: 541‐600‐4337 Fax: 541‐346‐2841 E‐mail: halliwil@uoregon.edu 22 8 2016 8 2016 4 16 10.1111/phy2.2016.4.issue-16e1288307 7 2016 15 7 2016 © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.Abstract Adequate cerebral perfusion is necessary to maintain consciousness in upright humans. Following maximal anaerobic exercise, cerebral perfusion can become compromised and result in syncope. It is unknown whether post‐exercise reductions in cerebral perfusion can lead to visual‐cognitive deficits prior to the onset of syncope, which would be of concern for emergency workers and warfighters, where critical decision making and intense physical activity are combined. Therefore, the purpose of this experiment was to determine if reductions in cerebral blood velocity, induced by maximal anaerobic exercise and head‐up tilt, result in visual‐cognitive deficits prior to the onset of syncope. Nineteen sedentary to recreationally active volunteers completed a symptom‐limited 60° head‐up tilt for 16 min before and up to 16 min after a 60 sec Wingate test. Blood velocity of the middle cerebral artery was measured using transcranial Doppler ultrasound and a visual decision‐reaction time test was assessed, with independent analysis of peripheral and central visual field responses. Cerebral blood velocity was 12.7 ± 4.0% lower (mean ± SE; P < 0.05) after exercise compared to pre‐exercise. This was associated with a 63 ± 29% increase (P < 0.05) in error rate for responses to cues provided to the peripheral visual field, without affecting central visual field error rates (P = 0.46) or decision‐reaction times for either visual field. These data suggest that the reduction in cerebral blood velocity following maximal anaerobic exercise contributes to visual‐cognitive deficits in the peripheral visual field without an apparent affect to the central visual field. Anaerobic exercisecerebrovascular circulationhypotensionorthostaticorthostatic intolerancepost‐exercise hypotensionsyncopetilt‐table testvasovagalNational Institutes of HealthHL115027 source-schema-version-number2.0component-idphy212883cover-dateAugust 2016details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_NLMPMC version:4.9.4 mode:remove_FC converted:29.08.2016 D. C. Sieck , M. R. Ely , S. A. Romero , M. J. Luttrell , P. M. Abdala , J. R. Halliwill . Post‐exercise syncope: Wingate syncope test and visual‐cognitive function . Physiol Rep , 4 (16 ), 2016 , e12883, doi: 10.14814/phy2.12883 Funding Information This research was supported in part by a grant from the National Institutes of Health Grant: HL115027. ==== Body Introduction Post‐exercise syncope is a transient loss of consciousness following an acute bout of exercise. The causes of post‐exercise syncope are multifactorial, but ultimately result in loss of consciousness due to reductions in cerebral perfusion (Halliwill et al. 2014). Before loss of consciousness occurs there is overt symptomatology of an impending faint, known as presyncope, that presents in most situations (Van Lieshout et al. 2003). It is estimated that 50–80% of healthy individuals will develop presyncopal signs and symptoms if subjected to a 15‐min passive head‐up tilt after exercise (Halliwill et al. 2014). However, it is unknown if post‐exercise reductions in cerebral perfusion lead to visual‐cognitive deficits during presyncope. Such a scenario would be of concern to populations such as emergency workers, or warfighters, who must perform complex tasks and make quick decisions in moments after vigorous physical activity resembling exercise, when vulnerability to reductions in cerebral perfusion and syncope are greatest (Luttrell and Halliwill 2015). In general, post‐exercise syncope is “neurally mediated syncope” (also known as vasovagal or neurocardiogenic syncope), with hallmarks of paradoxical bradycardia and peripheral vasodilation, during recovery from exercise (Freeman et al. 2011). The primary trigger of neurally mediated syncope is unknown; however, a sustained reduction in cardiac preload seems a prerequisite. After exercise, several mechanisms act in parallel and result in reduced preload and cardiac output. Immediately after exercise there is augmented venous pooling in dependent regions of the body. Augmented venous pooling is the combined result of sustained arterial vasodilation of the previously active skeletal muscle, combined with a reduced skeletal muscle pump, and increased hydrostatic pressure gradients due to upright posture (Pollack and Wood 1949; Krediet et al. 2004; Halliwill et al. 2013, 2014). This transient reduction in cardiac output, if large enough in magnitude, might lead to decreased autoregulatory capacity in the cerebral circulation (Halliwill et al. 2014). Further, any impact of post‐exercise hypotension on cerebral perfusion may be exacerbated after high‐intensity exercise by the presence of a hyperventilation‐induced hypocapnic cerebral vasoconstriction (Rasmussen et al. 2006; Halliwill et al. 2014; Lacewell et al. 2014). Therefore, in the setting of upright post‐exercise recovery, we expect cerebral perfusion to be transiently decreased. Neurally mediated syncope is usually preceded by symptoms of dizziness, light‐headedness, tunnel vision, and nausea (Freeman et al. 2011). These symptoms are thought to originate from a reduction in cerebral blood flow and oxygenation (Rossen et al. 1943; Van Lieshout et al. 2003). If a reduction in cerebral blood flow and oxygenation occur with neurally mediated syncope after exercise, it may also have detrimental effects on visual‐cognitive function during presyncope. On the other hand, a positive effect on visual‐cognitive performance has been demonstrated during an exercise bout, usually when subjects are in a seated position, which is generally attributed to increases in physical arousal (Brisswalter et al. 2002; Tomporowski 2002). However, changes in visual‐cognitive function during the exercise recovery period, when cardiovascular and cerebrovascular homeostasis are severely challenged, are not well defined. Therefore, the purpose of this experiment was to determine if reductions in cerebral blood velocity induced by maximal anaerobic exercise and head‐up tilt affect visual‐cognitive function prior to the onset of syncope. It was hypothesized that a modified Wingate test followed by a head‐up tilt would reduce cerebral blood velocity and result in visual‐cognitive deficits prior to the onset of syncope. Methods This study was approved by the Institutional Review Board of the University of Oregon, and conformed to the principles of the Declaration of Helsinki. Each volunteer gave written and informed consent before participation. Prior to undergoing the experimental protocols, subjects completed a screening visit that was intended to familiarize them with all testing procedures, including the reaction time test of visual‐cognitive function. Subjects Subject characteristics are summarized in Table 1. Twenty healthy, nonsmoking, normotensive subjects (10 men and 10 women) between the ages of 19 and 34 years participated in the study. Subjects were instructed to abstain from caffeine and alcohol for 12 h, and food for 2 h prior to the study. Based on the subjects’ self‐reported exercise habits over the previous month, they were classified as sedentary to recreationally active (Baecke et al. 1982; Kohl et al. 1988). Subjects completed a near‐fainting experiences index (Schrezemaier et al. 2005), to ensure they were not prone to syncope and had normal orthostatic tolerance. Due to the nature of the visual reaction time test, subjects were instructed to wear contact lenses to correct to normal vision if needed. Subjects were taking no over‐the‐counter or prescription medication other than oral contraceptives. Female subjects were studied during the early follicular phase of their menstrual cycle to minimize any potential cardiovascular effects of sex‐specific hormones and had a negative pregnancy test before completing the study. Table 1 Subject characteristics Mean ± SD Range Age (years) 22.9 ± 3.6 19–34 Height (cm) 170.0 ± 8.8 159–190 Weight (kg) 64.0 ± 12.2 144.8–90.0 Body mass index (kg m−2) 21.9 ± 2.5 17.4–27.7 Baecke sport index (arbitrary units) 3.1 ± 0.6 2–4 Physical activity index (MET h week−1) 31.1 ± 11.2 8–50.1 Near‐fainting experiences index (arbitrary units from 0 to 20) 1.9 ± 1.8 0–5 MET, metabolic equivalents; n = 19. John Wiley & Sons, LtdExperimental protocol Studies took place in a thermoneutral environment (21.7 ± 1.1°C). At the start of the protocol, subjects sat upright and were instrumented for the measurement of arterial pressure, heart rate, end‐tidal CO2, and cerebral blood velocity. The subjects then stepped onto an electronic tilt table with footboard support (Colin Medical Instruments Corporation, Valley City, ND) that was tilted to 60° head‐up. Testing started within one minute of being positioned on the tilt table, a pre‐exercise measurement period of 8 tests (16 min) was completed where arterial pressure, heart rate, end‐tidal CO2, and cerebral blood velocity were measured continuously. Visual‐cognitive function was assessed in 2‐min test blocks during the head‐up tilt both before and after exercise. Upon completion of pre‐exercise measurements, subjects were seated on a cycle ergometer (Excalibur Sport V2; Lode BV, Groningen, The Netherlands) and performed a 5‐min warm‐up at a moderate resistance (100 W for males and 75 W for females) at a self‐selected pedaling cadence. Immediately following the warm up, subjects completed a modified (1‐min) Wingate test of anaerobic power. The torque factor was set to 0.63 Nm for male subjects and 0.60 Nm for female subjects (Wingate for Windows software version 1; Lode BV, Groningen, The Netherlands). Instrumentation was left in place throughout exercise to facilitate quick transitioning between exercise and post‐exercise measurements. Following the Wingate, subjects were immediately returned to the 60° head‐up position and re‐instrumented for visual‐cognitive function assessment and end‐tidal CO2. Post‐exercise measurements were started within one minute from the end of exercise, including continuous measurements of arterial pressure, heart rate, end‐tidal CO2, and cerebral blood velocity, which were again measured in the head‐up position until the subject was unable to continue or 16 min had passed. Head‐up tilt tests were terminated if arterial pressure fell markedly (≥10 mm Hg) or heart rate slowed suddenly (≥10 beats) within one minute. In addition, subjects were asked to rate symptoms related to hypotension and cerebral hypoperfusion such as lightheadedness, nausea, and visual disturbances. Symptom scores were obtained every 2 min, each on a scale of 0–3 with 0 being no symptoms and 3 being the worst symptoms. Head‐up tilt tests were terminated if a subject reached either a 3 on any one symptom scale or a sum of 4 on the three combined scales. Measurements Heart rate, arterial pressure, and systemic hemodynamics Heart rate and arterial pressure were monitored throughout all experimental procedures. Heart rate was monitored using a 3‐lead electrocardiogram (Cardiocap/5 Critical Care Monitor; Datex‐Ohmeda, GE Healthcare, Helsinki, Finland). Arterial pressure was measured at the right brachial artery by automated auscultation (Tango+, SunTech Medical, Raleigh, NC) and mean arterial pressure was calculated, using the equation MAP = (SBP + 2 * DBP)/3. Finger photo‐plethysmography (Finometer; Finapres Medical Systems BV, Arnhem, the Netherlands) of the middle finger of the left hand, was used to monitor changes in blood pressure to determine termination of tilt test. Cerebral blood velocity A transcranial Doppler ultrasound (Spencer Technologies model ST3 with Marc 600 Headframe, Redmond, WA) was used to assess blood velocity in the middle cerebral artery. The ultrasound probe was secured with headgear at a constant angle to insonate the right middle cerebral artery over the temporal window and was adjusted for optimal signal intensity. End‐tidal CO2 A capillary line connected to a nasal cannula was used to sample end‐tidal CO2 as a percentage (CardioCap/5 Critical Care Monitor, Datex‐Ohmeda, GE Healthcare, Helsinki, Finland) and corrected to partial pressure (mmHg) of CO2 using barometric pressure. To ensure accurate measurement, subjects were instructed to breathe primarily through their nostrils. Reaction time and visual‐cognitive function A visual reaction time test was used to assess visual‐cognitive function, similar to what has been used previously (Brisswalter et al. 2002; Tomporowski 2002). A custom concave metal frame positioned an array of computer‐controlled LED lights at the subject's eye level and at a distance of 60 cm from the subject's forehead. A single yellow light was located directly in front of the subject (midline), and to each side of the subject, pairs of red and green lights were located both centrally (10° from midline) and peripherally (50° from midline). During each visual‐cognitive function test block, which lasted 95 sec, subjects focused on the midline yellow LED which was constantly illuminated throughout the test. Subjects were instructed to respond, each time a green LED was illuminated, by releasing a micro‐switch with their right thumb as fast as possible, while disregarding illumination of red LEDs. The eight green and red LEDs were programmed to illuminate briefly (100 msec) in a balanced randomized order, with a balanced randomized duration (between 2500 and 3430 msec, in 30 msec increments) between each illumination. After each visual‐cognitive function test, blood pressure was measured using automated auscultation, and subjective symptom scores were obtained, such that a test block and these additional measures were repeated in 2‐min cycles. Thus, eight visual‐cognitive function test blocks were completed pre‐exercise during the 16‐min head‐up tilt. Up to eight visual‐cognitive function tests were completed post‐exercise, determined by the termination criteria described earlier. Data analysis Continuous signals for electrocardiogram, capnogram, and transcranial Doppler velocity were sampled at 250 Hz, using a commercial analog‐to‐digital data acquisition system (Windaq, Dataq Instruments, Akron, OH). Reaction time and visual‐cognitive function For every 95 sec, visual‐cognitive function test block, reaction time, and error rates were calculated for responses to cues provided in the central and peripheral visual field. The reaction time, measured from the onset of an LED stimulus to the release of the thumb switch, was averaged for all correct responses to green LED stimulus. Error rate was calculated as the sum of incorrect, premature (<100 msec), late (>500 msec), and missed responses to the visual stimuli. Error rate is represented as a percent of total possible responses. Statistics In order to maximize statistical power, male and female subjects were analyzed as a single group. Hemodynamic and other outcome variables were analyzed with a two‐way repeated measures ANOVA (condition vs. time) with a priori contrasts of specific condition‐time combinations (SAS v9.2; SAS Institute, Inc., Cary, NC) and expressed as mean ± SE where group means are directly compared. The hypotheses were related to changes in cerebral blood velocity and visual‐cognitive function prior to the onset of syncope. These statistical tests were conducted using only data that preceded the development of syncope. In addition, the pre‐exercise values were averaged for each subject and compared to post‐exercise values at each time point by correlational analysis (Pearson Correlation Coefficient). Throughout all statistical testing, differences were considered significant when P < 0.05. Results Of the 20 subjects that participated in this study, 19 were able to complete the eight pre‐exercise test blocks, one subject could not withstand 16‐min head‐up tilt pre‐exercise, and was excluded from further analysis. Subjects tolerated post‐exercise head‐up tilt for variable durations; however, two of the subjects were able to complete the entire 16 min after exercise. Figure 1 demonstrates post‐exercise subject tolerance over time, illustrated as a survival function. Figure 1 also shows results from Lacewell et al. (2014), which used a similar exercise model but with a 2‐min supine rest prior to tilt. The general pattern of presyncope was similar, but subjects were more susceptible to presyncope without the supine rest preceding the head‐up tilt. Figure 1 Survival function. Proportion of subjects completing each stage of the head‐up tilt shown as a survival function. Solid line, post‐exercise; dashed line, previous study using similar methodology (n = 19; Reproduced from [Lacewell et al. 2014]). Hemodynamic and physiological measurements Figure 2 shows the key hemodynamic and physiological variables across time during each of the two head‐up tilt tests (pre‐ and post‐exercise). As shown in Figure 2A, prior to exercise, heart rate increased across the period from 74.0 ± 2.6 beats/min during the first test block to 86.9 ± 6.7 beats/min during the last test block (P < 0.05). After exercise, the heart rate was increased compared to pre‐exercise (P < 0.05), but subsequently decreased from the first test block (138.8 ± 3.2 beats/min) to the last test block (108.2 ± 7.1 beats/min; P < 0.05). Figure 2 Hemodynamic and physiological variables across time during each of the two head‐up tilt tests (pre‐ and post‐exercise). Heart rate, mean arterial pressure, cerebral blood flow velocity, and end‐tidal CO 2 averaged across each test block during a head‐up tilt performed before (open circles) and after exercise (closed circles). Values are means ± SE (n = 19). † P < 0.05 versus the first test block in the same condition (either before or after exercise). *P < 0.05 before versus after exercise. As shown in Figure 2B, prior to exercise, mean arterial pressure did not differ from the first test block (93.6 ± 2.2 mmHg) to the last test block (92.3 ± 1.9 mmHg; P = 0.49). After exercise, mean arterial pressure was similar to before exercise (P = 0.34), but decreased from the first test block (95.5 ± 3.2 mmHg) to the sixth test block (83.2 ± 1.2 mmHg; P < 0.05). As shown in Figure 2C, prior to exercise, cerebral blood velocity decreased from the first test block (58.6 ± 3.1 cm sec−1) to the last test block (53.1 ± 2.8 cm sec−1; P < 0.05). Cerebral blood velocity was significantly decreased post‐exercise when compared to pre‐exercise measures (P < 0.05). During the post‐exercise period, cerebral blood velocity varied more compared to the pre‐exercise period due to increased subject drop‐out, but remained lower throughout the post‐exercise period by 12.7 ± 4.0% (P < 0.05). As shown in Figure 2D, prior to exercise, end‐tidal CO2 decreased across the period from the first test block (36.5 ± 0.7 mmHg) to the last test block (34.6 ± 0.7 mmHg; P < 0.05). After exercise, end‐tidal CO2 was reduced compared to pre‐exercise (P < 0.05), and did not differ from the first test block (27.1 ± 1.5 mmHg) to the last test block (25.2 ± 0.8 mmHg; P = 0.15). Reaction time and visual‐cognitive function measurements Figure 3 shows key visual‐cognitive function and reaction time variables across time during each of the two head‐up tilt tests (pre‐ and post‐exercise). As shown in Figure 3A, prior to exercise, the reaction time for responses to cues in the central visual fields did not differ from the first test block (362 ± 13 msec) to the last test block (354 ± 11 msec; P = 0.19). After exercise, the reaction time in the central visual fields did not differ from pre‐exercise (P = 0.35), and did not differ from the first test block (360 ± 12 msec) to the last test block (372 ± 59 msec; P = 0.45). Figure 3 Reaction time and cognitive function measurements across time during each of the two head‐up tilt tests (pre‐ and post‐exercise). Central visual field reaction time, peripheral visual field reaction time, central visual field error rate, and peripheral visual field error rate averaged across each test block during a head‐up tilt performed before (open circles) and after exercise (closed circles). Values are means ± SE (n = 19). † P < 0.05 versus the first test block in the same condition (either before or after exercise). *P < 0.05 before versus after exercise. As shown in Figure 3B, prior to exercise, the reaction time for responses to cues in the peripheral visual fields did not differ from the first test block (400 ± 11 msec) to the last test block (396 ± 10 msec; P = 0.59). We note that reaction times were slower in the peripheral visual field than in the central visual fields by 11.6 ± 1.5% (P < 0.05) when averaged across the entire testing period. After exercise, the reaction time in the peripheral visual field did not differ from pre‐exercise (P = 0.48), and did not differ from the first test block (378 ± 9 msec) to the last test block (420 ± 58 msec; P = 0.17). As shown in Figure 3C, prior to exercise, error rates in the central visual fields did not differ from the first test block (5.6 ± 1.8%) to the last test block (4.3 ± 1.2%; P = 0.41). After exercise, error rates in the central visual fields were unchanged (P = 0.15), and did not differ from the first test block (1.6 ± 0.8%) to the last test block (3.1 ± 3.1%; P = 0.86). As shown in Figure 3D, prior to exercise, error rates in the peripheral visual fields did not differ from the first test block (13.2 ± 2.7%) to the last test block (12.8 ± 2.7%; P = 0.89). We note that error rates were 3 times greater (3.0 ± 0.9; P < 0.05) in the peripheral visual field than in the central visual fields when averaged across the entire testing period. After exercise, error rates in the peripheral visual fields were higher than pre‐exercise (by 63 ± 29%; P < 0.05), and did not differ from the first test block (11.1 ± 1.9%) to the last test block (21.9 ± 21.9%; P = 0.19). Since subject drop‐out increased as time from exercise increased, several methods of analysis were employed to ensure an accurate portrayal of responses, with a focus on peripheral error rates. One such method was to compare averaged pre‐exercise data and to each subject's last post‐exercise measurement. Using this approach, we saw an increase in peripheral error rates from 10.8 ± 1.2% (average pre‐exercise) to 17.8 ± 3.8% (last post‐exercise test, P < 0.05). We also note that, compared to averaged pre‐exercise values, a weak negative correlation was observed between cerebral blood velocity and error rate in the peripheral visual field (r = −0.25; P < 0.05). Discussion Passive standing, or head‐up tilt, is a physiological challenge which can lead to cardiovascular collapse in the form of pre‐syncopal signs and symptoms, or even frank syncope, if orthostasis is maintained (Blomqvist and Stone 1983). This physiological stress can be exaggerated following exercise, as there is an increased incidence of presyncope during head‐up tilt after exercise (Eichna et al. 1947; Lacewell et al. 2014), and it is estimated that presyncope will occur in 50–80% of healthy individuals if subjected to a 15‐min passive head‐up tilt after exercise (Halliwill et al. 2014). Signs and symptoms of presyncope have been attributed to reductions in cerebral perfusion and oxygenation of the brain (Rossen et al. 1943). However, it is unknown if post‐exercise reductions in cerebral perfusion associated with presyncope lead to visual‐cognitive deficits. This is of concern to populations such as emergency workers, or warfighters who must perform coordinated tasks and make quick decisions in moments after exercise when vulnerability to syncope is the greatest (Luttrell and Halliwill 2015). The results of the present study demonstrate that while the reaction time to a visual cue is well maintained in the face of orthostatic challenge after exercise, errors made while responding to visual cues in the peripheral visual field occur at higher rates (+63%) under these circumstances. Situations that affect visual‐cognitive performance Acute exercise and its effect on visual‐cognitive performance has been extensively studied, but not in the presence of a superimposed orthostatic challenge. In contrast to the current study, which focused on post‐exercise effects, most other studies have focused on visual‐cognitive effects during exercise. These studies indicate a positive effect of acute exercise on cognition. The positive effect has been reported to be intensity dependent, represented by an “inverted U” model, where both low and high duration and intensity of exercise have less benefit on cognition compared to moderate exercise (Tomporowski 2002). The increase in cognitive function seen during acute exercise has been attributed to increased arousal (Tomporowski and Ellis 1986; Tomporowski 2002) Therefore, the effect of acute exercise on cognitive performance differs by intensity and duration of exercise as well as the type of cognitive task; and has been reviewed thoroughly (Tomporowski and Ellis 1986; Brisswalter et al. 2002; Tomporowski 2002; Chang et al. 2012). The stressors of the current study differ from prior studies looking at exercise and cognitive function, and have more in common with the visual‐cognitive performance research on acute hypoxia, altitude, and pilots subjected to high‐G maneuvers, conditions which compromise global cerebral oxygen delivery. Acute hypoxia and high altitude (Vaernes et al. 1984; Kida and Imai 2015) are both associated with impaired visual‐cognitive performance due to reduction in arterial oxygen content. Studies examining visual‐cognitive function often differ in methodological approach which can make interpreting results difficult. Lie et al. found acute bouts of mild to moderate hypoxia negatively affect the visual reaction time, however error rate remained unchanged (Li et al. 2000). The present study found the reaction time was left unchanged while error rates increased. Taken together, we can conclude that visual‐cognitive function will be impaired when oxygen delivery to the brain is decreased, regardless of the mechanism. High‐G maneuvers and orthostatic challenge generate cerebral hypoxia via reductions in perfusion, rather than arterial oxygen content (such as the present study), but with a similar effect on visual‐cognitive function (Vaernes et al. 1984; Kida and Imai 2015). In many situations, especially during hypoxic conditions, loss of vision has been shown to precede loss of consciousness (Whinnery and Forster 2015). Additionally, hypoxia has been suggested to cause a wide variety of visual decrements, including impaired color vision (Connolly et al. 2008), slowed visual processing (Fowler et al. 1993), and reduced peripheral vision (Whinnery and Forster 2015). The visual system is amongst the highest energy‐consuming systems in the brain, which is partly due to the retina being one of the highest oxygen‐consuming tissues in the body. Thus, the visual system, and especially the retina, is highly sensitive to even minor changes in tissue oxygenation (Wong‐Riley 2010). Hypoxia has been shown to increase reaction times and error rates while interactions with light intensity seem to play a role (Fowler et al. 1993). The present study was conducted in a room with no outdoor light and used a consistent lighting pattern across all studies to minimize any interaction with light intensity. Unique to the Wingate syncope test, the reduction in cerebral blood velocity after exercise is most likely due to a combination of exercise‐induced hyperventilation and post‐exercise hypotension. In the present study, hyperventilation resulted in hypocapnia, which we have noted previously in this model of exercise and orthostatic challenge (Halliwill et al. 2013). Hyperventilatory‐induced hypocapnia has been shown to reduce cerebral blood velocity, likely by vasoconstricting cerebral blood vessels (Romero and Cooke 2007; Coverdale et al. 2014; Verbree et al. 2014); however, it may increase retinal blood flow by vasodilating retinal blood vessels (Brinchmann‐Hansen and Myhre 1990). The current finding of decreased blood flow velocity in the middle cerebral artery following exercise may not equate to reduced perfusion of all areas of the brain. A limitation of the transcranial Doppler technology is the inability to quantify changes in arterial diameter, and in theory, changes in diameter could offset some of the changes in velocity. That said, cerebral conduit arteries constrict in response to hypocapnia, and a decreased blood velocity with or without a cerebral conduit artery constriction would result in a reduced brain blood flow. While we did not study motor performance in the present study, it is worth noting that hypocapnia has been shown to impair motor performance (Gibson 1978), which could further exacerbate the impact of poor visual‐cognitive function during recovery from exercise. However, reaction times remained unchanged for stimuli to both visual fields, suggesting little or no motor impairment in this model. What is the nature of the cognitive deficit during presyncope? In the present study, we found reaction times were unchanged for visual‐cognitive responses following intense exercise. As indicated above, the majority of studies indicate a positive effect of acute exercise on the reaction time during similar visual‐cognitive tests. One possibility is that the cerebral hypoperfusion, which exists in our model of orthostatic challenge following exercise, had no effect on the reaction time and central processing of information which are required to respond correctly to the test. However, we would suggest another possibility, which is that the positive effect of exercise, reported by others, is being circumvented by cerebral hypoperfusion in this model. If this is the case, then it is easy to anticipate that some variation in experimental conditions could tip the scale toward improved or impaired reaction times during the combination of intense exercise and orthostatic challenge. Although we did not observe changes in error rates in response to the visual‐cognitive test when stimuli were presented in central visual fields, there was a notable increase in error rates for stimuli presented in peripheral visual fields. The selective nature of this deficit suggests that it is visual‐sensory in nature, and not secondary to an impaired cognitive decision‐making process. Along these lines, it may be related to the primary role of the rod photoreceptor in the peripheral visual field, and the sensitivity of rod photoreceptors to hypoxia. Mild hypoxia compromises threshold sensitivity during dark adaptation (Connolly and Hosking 2006), likely due to an impairment in the regeneration of rhodopsin. It has been suggested that rod photoreceptors may be functionally hypoxic when breathing normal air at the sea level (Connolly and Hosking 2006), and thus, on the shoulder of a steep dose–response curve when perfusion is challenged by orthostasis after intense exercise. This hypoxia‐derived impaired peripheral vision may be counteracted, to some extent, by hypocapnia, which enhances visual sensitivity and contrast discrimination (Connolly and Hosking 2006). In summary, a reduction in arterial pressure (post‐exercise hypotension) paired with hyperventilation‐induced hypocapnia occurred after exercise, and lead to a reduction in cerebral blood velocity. The aforementioned reduction in cerebral blood velocity occurred concurrently with an increased peripheral visual field error rate indicating that individuals experiencing pre‐syncopal symptoms after exercise are also experiencing visual‐cognitive deficits, which are likely attributable to a loss of peripheral visual function rather than overall decrements in cognitive function. These results are important in identifying and managing situations in which visual‐cognitive deficits may occur, to attenuate possibly dangerous situations. Specifically, individuals can be expected to be less likely to respond, or respond correctly, to visual cues in the periphery of their vision, during recovery from intense exercise. Presyncope versus syncope It is worth noting that our results are based on observations prior to the onset of syncope. After exercise, a clinically significant reduction in arterial pressure termed post‐exercise hypotension occurs (Kenny and Seals 1993), which can become symptomatic and result in syncope if left unchecked (Halliwill et al. 2013). One possible outcome is a rapid collapse due to neurocardiogenic syncope, a response whose trigger remains poorly understood, but may stem from vigorous contraction of an under‐filled left ventricle. It has been suggested that the hyperdynamic cardiac contraction of an under‐filled ventricle triggers a Bezold‐Jarisch‐like reflex which causes a paradoxical bradycardia via increased parasympathetic/vagal outflow to the heart and sympathoinhibition to peripheral blood vessels, resulting in vasodilation (Krediet et al. 2004; Halliwill et al. 2014). However, others have proposed mechanisms of neurocardiogenic syncope originating in the central nervous system rather than the ventricles (Hainsworth 2003). Regardless of the specific trigger for neurocardiogenic syncope, there is a reduction in cerebral perfusion after the Wingate syncope test that was employed in the current study, which is due to the post‐exercise hypotension combined with extreme hypocapnia. The hypotension is compounded by augmented venous pooling, which is the combined result loss of the muscle pump and prolonged orthostasis (Pollack and Wood 1949; Krediet et al. 2004; Barrett‐O'Keefe et al. 2013; Halliwill et al. 2014). Of greater impact on post‐exercise hypotension is the marked vasodilation in the previously active skeletal muscle. When enough muscle mass is recruited for exercise, vasoconstriction of the inactive muscle groups is insufficient to offset the rise in vascular conductance in the previously active muscle, and the overall rise in systemic vascular conductance is greater than what can be supported by the heart when preload is also compromised. The exercise model used in this experiment, a modified Wingate test, activates large amounts of muscle mass that tips the proverbial scale to induce an increase in vascular conductance that could not be maintained during the passive stand test without experiencing presyncopal symptoms (Lacewell et al. 2014). Thus, the majority of subjects were not able to continue with head‐up tilt, and one can postulate that visual‐cognitive impairments would be become marked (in response to both central and peripheral visual cues) had we allowed subjects to continue to the point of syncope. Perspectives The present study demonstrated a reduction in the ability for individuals to accurately respond to visual stimuli in their peripheral visual field after an acute bout of high intensity short duration exercise. The attenuated visual/cognitive function can be partly attributed to reductions in brain blood velocity, which occurred when individuals could not actively recover from exercise. Athletes have reported experiencing symptoms such as tunnel vision and lightheadedness after finishing a race and being stuck in a crowded finishing chute, sometimes ending in syncope. Other populations, such as emergency workers or warfighters, can be expected to find themselves in similar situations in moments after vigorous physical activity resembling exercise. These populations must perform complex tasks and make quick decisions in situations when vulnerability to reductions in cerebral perfusion and syncope are the greatest. This situation can be dangerous and become life threatening if an emergency worker cannot clearly see if the scene is safe, or a warfighter has to identify a threat versus an ally. Future research should focus on simple physical countermeasure that can be performed to minimize the effects of post‐exercise recovery on visual/cognitive function by engaging the muscle pump and maintaining adequate cerebral perfusion. Conclusion There is a reduction in cerebral blood velocity following a modified Wingate test and head‐up tilt. The reduction in cerebral blood velocity following maximal anaerobic exercise contributes to visual‐cognitive deficits as evidenced by the concurrent increase in peripheral visual field error rate during presyncope. Thus, individuals may be less likely to respond, or respond correctly, to visual cues in the periphery of their vision, during recovery from intense physical activity or exercise. Conflict of Interest None declared. Acknowledgments This study was conducted by Dylan C. Sieck in partial fulfillment of the requirements for the degree of Masters of Science at the University of Oregon. We sincerely thank the subjects involved in this study for their participation. The authors gratefully acknowledge the assistance of Kris Johnson and Clifford Dax for their roles in developing the LED array used in this work. ==== Refs References Baecke , J. A. H. , J. Burema , and J. E. R. 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==== Front Physiol RepPhysiol Rep10.1002/(ISSN)2051-817XPHY2physreportsPhysiological Reports2051-817XJohn Wiley and Sons Inc. Hoboken 10.14814/phy2.12888PHY212888KidneyBlood PressureReproductive HormonesMaternal, Fetal and Neonatal PhysiologyAgeing and DegenerationOriginal ResearchOriginal ResearchSleep restriction during pregnancy and its effects on blood pressure and renal function among female offspring R. Argeri et al.Argeri Rogério 1 Nishi Erika E. 1 Volpini Rildo A. 2 Palma Beatriz D. 3 4 Tufik Sergio 3 Gomes Guiomar N. 1 1 Department of PhysiologyEscola Paulista de Medicina – UNIFESPSão PauloBrazil2 Department of PsychobiologyEscola Paulista de Medicina – UNIFESPSão PauloBrazil3 Basic Research Laboratory – LIM12Nephrology – Faculty of MedicineUSP, São PauloSão PauloBrazil4 Centro Universitário São CamiloSão PauloBrazil* Correspondence Guiomar Nascimento Gomes, Escola Paulista de Medicina – UNIFESP, Department of Physiology, Rua Botucatu, 862, 5th floor, Vila Clementino, São Paulo, 04023‐900 SP, Brazil. Tel: +551155764848 ‐ 2354 E‐mail: guiomar.gomes@unifesp.br 22 8 2016 8 2016 4 16 10.1111/phy2.2016.4.issue-16e1288807 7 2016 11 7 2016 © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.Abstract The influence of sleep restriction (SR) during pregnancy on blood pressure and renal function among female adult offspring was investigated. Pregnant Wistar rats were distributed into control and SR groups. The SR was performed between the 14th and 20th days of pregnancy (multiple platforms method for 20 h/day). At 2 months of age, half of the offspring from both groups were subjected to an ovariectomy (ovx), and the other half underwent sham surgery. The groups were as follows: control sham (Csham), control ovx (Covx), SR sham (SR sham), and SR ovx (SR ovx). Renal function markers and systolic blood pressure (BPi, indirect method) were evaluated at 4, 6, and 8 months. Subsequently, the rats were euthanized, kidneys were removed, and processed for morphological analyses of glomerular area (GA), number of glomeruli per mm3 (NG), and kidney mass (KM). Increased BPi was observed in the Covx, SR sham, and SR ovx groups compared to Csham at all ages. Increased plasma creatinine concentration and decreased creatinine clearance were observed in the SR sham and SR ovx groups compared to the Csham and Covx groups. The SR ovx group showed higher BPi and reduced creatinine clearance compared to all other groups. The SR ovx group showed reduced values of GA and KM, as well as increased NG, macrophage infiltration, collagen deposit, and ACE1 expression at the renal cortex. Therefore, SR during pregnancy might be an additional risk factor for developing renal dysfunction and increasing BP in female adult offspring. The absence of female hormones exacerbates the changes caused by SR. Blood pressurefemale offspringrenal functionsleep restrictionFAPESPproc2013/23622‐7 source-schema-version-number2.0component-idphy212888cover-dateAugust 2016details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_NLMPMC version:4.9.4 mode:remove_FC converted:29.08.2016 R. Argeri , E. E. Nishi , R. A. Volpini , B. D. Palma , S. Tufik , G. N. Gomes . Sleep restriction during pregnancy and its effects on blood pressure and renal function among female offspring . Physiol Rep , 4 (16 ), 2016 , e12888, doi: 10.14814/phy2.12888 Funding Information We recognize the financial support from FAPESP (proc2013/23622‐7). ==== Body Introduction During critical stages of intrauterine growth, environmental alterations may predispose the fetus to the development of diseases later in life (Frasch et al. 2007). This concept has been confirmed by several studies in which nutritional deficiency (Regina et al. 2001; Franco et al. 2009), diabetes (Rocha et al. 2005; de Almeida Chaves Rodrigues et al. 2013), and the use of cortisol (Ortiz et al. 2003; Celsi et al. 1998) during pregnancy are related to the development of cardiovascular and metabolic diseases in the offspring at adulthood (Barker 1997; Langley‐Evans et al. 1999; Alexander et al. 2015). The reduction of the sleep period has become commonplace in modern society (Spiegel et al. 1999; Aldabal and Bahammam 2011). However, sleep restriction (SR) is associated with augmented production of hormones, such as corticosterone, growth hormone (GH), ACTH, and inflammatory cytokines (Spiegel et al. 1999; Schüssler et al. 2006; Aldabal and Bahammam 2011). Consequently, the reduction of sleep time during pregnancy may alter the fetal environment and may also be a threat to maternal and fetal health (Chang et al. 2010; Pires et al. 2010; Pardo et al. 2016). During pregnancy, physical and hormonal changes affect sleep quality (Sahota et al. 2003; Lopes et al. 2004; Pien and Schwab 2004). Additionally, if these changes occur alongside long working hours (Caruso 2006; National Sleep Foundation, 2007), the probability of a pregnant woman having sleep disorders increases. In the past decade, SR has been the subject of several studies; however, the impact of this condition during pregnancy on the offspring has been evaluated in only a few studies. The effect of SR during the critical period for kidney growth (the last week of pregnancy for rats) was studied by Thomal et al. (2010). In this study, the authors observed that male offspring from SR mothers presented increased blood pressure and reduced nephron number (Thomal et al. 2010). Additionally, Lima et al. showed altered sensitivity of the cardiac baroreflex response and increased blood pressure (BP) in male offspring of rats subjected to SR throughout the entire pregnancy (Lima et al. 2014). Taking into account that few studies have focused on the repercussions of SR during pregnancy on female offspring, this study aims to evaluate the effects of SR over the last week of pregnancy on blood pressure, renal morphology, and function among female offspring at adulthood. Additionally, we evaluated the expression of ACE1 and ACE2 in the kidneys in view that altered expression of RAS components have been related to renal modifications in fetal programming models (Sahajpal and Ashton 2005; Mesquita et al. 2010; Gwathmey et al. 2011). Considering that the effects of female hormones on cardiovascular and renal function could cover up SR effect over these systems, we also evaluated the effects of SR in females submitted to ovariectomy. Methods This experimental protocol was approved by the Ethical Research Committee of the Universidade Federal de São Paulo – UNIFESP (CEUA: 7647020614) and adhered to international guidelines for the care of research animals. Female Wistar rats weighing between 200 and 250 g and male Wistar rats weighing between 300 and 350 g were used in this study. The animals had free access to food and water during the entire experimental protocol and were maintained in a room with fixed temperature (22°C) and cycle of light (light and dark cycle alternating every 12 h, starting the lighting period at 7 am). Two females were caged overnight with a male, and vaginal smears were collected the following morning. The presence of sperm was regarded as a positive result and was considered as day 0 of pregnancy. The females were then distributed in two experimental groups: control and sleep‐restricted mothers. Seventeen mothers composed the control group. Twenty‐six mothers composed the SR group. Sleep restriction protocol The sleep restriction technique was based on the muscle atony that accompanies paradoxical sleep (Jouvet et al. 1964; Thomal et al. 2010). Briefly, 10 narrow circular platforms (6.5 cm in diameter) were placed inside a tiled tank (123 × 44 × 44 cm) filled with water to within 1 cm below the upper border of the platform. For the sleep‐restricted mothers group, 2–6 rats were placed on the platforms in an arrangement that allowed them to move inside the tank and jump from one platform to the other. Two days before the beginning of the study, the animals were adapted to the water tank for a period of 1 h to avoid unnecessary drops in the water. The sleep‐restricted mothers group was placed in the tank between the 14th and 20th days of pregnancy for 20 h/24 h (from 2 pm h until next day at 10 am). Following sleep restriction period, rats were placed back in their home cages and could sleep freely. At 10 am on the 20th day of pregnancy, the sleep‐restricted mothers were placed back in their home cages for maintenance until spontaneous parturition and weaning of the offspring. The animals of the control group remained in their home cages in the same room in which the sleep restriction occurred. The sleep restriction was carried out between the 14th and 20th days of pregnancy because, in rats, the last week of pregnancy is critical for the kidney development (Nigam et al. 1996), and so is more susceptible to changes in maternal environment. Birth and weaning After birth, the animals were weighed, and the litters were reduced to six offspring, preferably in the ratio of four males to two females; these pups were left with the mother for 28 days. Following the weaning period, the offspring were separated from their mothers; female offspring were placed in collective cages containing four animals per cage. The males were used in another study. The offspring were divided into two groups: control offspring (C) and offspring from sleep‐restricted mothers (SR). Experimental groups At 2 months, one female of each litter (C and SR) was submitted to ovariectomy (OVX) and the other underwent to sham surgery. The rats were anesthetized with a combination of xylazine (10 mg/kg) and ketamine (75 mg/kg) given intraperitoneally (i.p.). The surgery was performed using the transverse incision technique as described by Saadat Parhizkar and Latiff (2008). The groups were as follows: control sham (Csham), control ovx (Covx), SR sham (SRsham), and SR ovx (SRovx). Body weight The offspring were weighed after birth and at 4, 6, and 8 months of age on an analytical balance. Evaluation of ovariectomy and adiposity Considering that in the absence of female hormones, the uterine body undergoes atrophy (Naciff et al. 2007), after euthanizing the animals, the uterus was removed and weighed to confirm ovariectomy. In addition, the retroperitoneal fat pads were bilaterally collected and weighted as an index of adiposity. Indirect determination of systolic blood pressure (BPi) At 3 months of age, the offspring began the adaptation to the tail plethysmography apparatus (IITC Life Science, Woodland Hills, CA). Effective determinations of indirect systolic blood pressure (BPi) were obtained at 4, 6, and 8 months of age. The animals were placed in acrylic cylinders with appropriate dimensions for the size of the animal while the tail remained exposed. The sphygmomanometer with a sensor connected to a register system was then adjusted to the proximal tail portion of the rat. Three measurements were performed in sequence; the mean of these three measurements was considered the BPi. Renal function Renal function evaluations were performed at 4, 6, and 8 months of age. The rats were placed in metabolic cages for 24 h. Urine and blood samples were collected to measure creatinine, sodium, and potassium concentrations. Plasma and urine creatinine levels were measured using the Jaffé method, and the glomerular filtration rate (GFR) was determined based on creatinine clearance. The concentrations of sodium and potassium were measured using flame photometry (Analyser 910, SP/SP, Brazil). The quantification of urinary protein concentration was performed using the Bradford method and urine osmolarity was measured using an osmometer Advanced 3W2 (Advanced Instruments Inc., Norwood, MA). Renal morphology Kidney samples from rats of all groups were fixed in Bouin's solution (ethanol saturated with picric acid 75%, formaldehyde 20%, and acetic acid 5%) and were processed for paraffin embedding. Five‐micron histological sections were stained with hematoxylin–eosin for morphology or with picrosirius red for interstitial collagen type I and III analyses. Glomerular area and number were evaluated using a light microscope (Nikon H550L) with a camera connected to a computer with image analysis software (Nikon, NIS‐Elements 3.2, Japan). Encircled areas were determined by computerized morphometry. Analyses of morphological procedures were performed by an investigator who was blinded to the origin of the slides. Twenty‐five fields were analyzed on each slide (magnification 200×). For the interstitial collagen type I and III analyses, five fields were analyzed on each slide (magnification 100×). Images were acquired with a digital camera (AxioCam MRc) connected to a Carl Zeiss microscope with polarized light. Collagens I and III were estimated as percentages using the image analysis software ImageJ (NIH, USA). Immunohistochemistry and immunofluorescence ED1‐positive cells (macrophages/monocytes) were identified by immunohistochemistry. Tissue sections were incubated for 12 h at 4°C with a monoclonal anti‐ED1 antibody (1:1000, Serotec, Oxford, UK). The reaction product was determined with a streptavidin peroxidase complex (Dako LSAB System HRP, DAKO Corporation, Carpinteria, CA). The material was counterstained with Carazzi's hematoxylin, dehydrated, and mounted. Macrophage infiltration was evaluated using a Nikon microscope at a magnification of 200×. Each studied field had an area of 275,000 μm2. Angiotensin‐converting enzymes type 1 and 2 (ACE1 and ACE2) expressions were evaluated by immunofluorescence. Tissue sections were incubated for 12 h at 4°C with a mouse anti‐rat ACE1 monoclonal antibody (1:500 ab11738, Abcam, Cambridge, UK) or with rabbit anti‐rat ACE2 monoclonal antibody (1:100 ab108252, Abcam, Cambridge, UK), followed by incubation at room temperature for 1 h with an Alexa Fluor® 488‐conjugated anti‐mouse secondary antibody (1:500 dilution, Molecular Probes, Thermo Fisher Scientific, Waltham, MA) or an Alexa Fluor® 594‐conjugated anti‐rabbit secondary antibody (1:500 dilution, Molecular Probes, Invitrogen). Sections were then coverslipped with mounting medium containing DAPI (Sigma‐Aldrich, St. Louis, MO). Fluorescent images (magnification 630×) of the renal cortex (5 fields/rat) were collected using a Leica SP8 confocal microscope system. For these images, the laser power was set at 20%, and the excitation/emission wavelengths were 488 nm/band pass 530–555 nm for Alexa Fluor 488 and 405 nm/band pass 440–480 nm for DAPI. ACE1 and ACE2 fluorescence intensities were measured using ImageJ software (NIH, USA). The results are presented as the means ± standard error (SEM). Statistical tests used were Student's t‐test and one‐way and two‐way analysis of variance (ANOVA) when appropriate (GraphPad Prism software 5®). The level of significance was 5% (P ≤ 0.05). After one‐way ANOVA, the Bonferroni test was used for multiple comparison. Results Effects of SR on body weight There was no significant difference in birth weight between the groups (C: 5.92 ± 0.034 [n = 34]; SR: 5.90 ± 0.04 [n = 52]). However, from 4 months on, the ovariectomized animals showed increased body weight compared to controls (Fig. 1). Figure 1 Body weight (A and B) and systolic blood pressure (C) in the offspring, submitted to ovariectomy surgery (OVX) or not (Sham), of control (C) and sleep‐restricted (SR) mothers. ● P < 0.05 versus CS ham; ɸ P < 0.05 versus SRS ham; Ɛ P < 0.05 versus Covx (Bonferroni test). At 8 months, the amounts of retroperitoneal fat were augmented in Covx and SRovx rats compared to Csham and SRsham rats, suggestive of greater adiposity in ovariectomized rats (Csham: 3.7 ± 0.3 g [n = 15]; Covx: 6.8 ± 0.6 g [n = 15]; SRsham: 3.8 ± 0.4 g [n = 13]; SRovx: 8.7 ± 0.9 g [n = 15]) (two‐way ANOVA: OVX > Sham, P = 0.0001). Ovariectomy assessment The uterine weight was measured to confirm ovariectomy efficacy. Ovariectomized animals had reduced uterine weights compared to the sham groups (Csham: 1.3 ± 0.1 g [n = 17]; Covx: 0.21 ± 0.01 g [n = 17]; SRsham: 1.0 ± 0.05 g [n = 26]; SRovx: 0.19 ± 0.01 g [n = 26]). The SRsham group had decreased uterine weight compared to Csham (two‐way ANOVA: OVX < Sham, P = 0.0001; SR < C, P = 0.0024). Effects of SR on blood pressure and renal function Higher values of BPi were observed in the Covx, SRsham, and SRovx groups compared to the Csham group (Fig. 1C). The SRovx group had higher BPi compared to all of the other experimental groups at all ages. At 8 months, the BP of the Csham group was 124.9 ± 0.74 mmHg. At the same age, BP of Covx and SRsham groups were about 6 mmHg greater and in SRovx group was about 12.7 mmHg greater than the Csham. The values for creatinine plasma concentration and creatinine clearance are shown in Table 1. Increased creatinine plasma concentration and decreased creatinine clearance were observed in the SRsham and SRovx groups compared to the Csham and Covx groups at all ages studied. Yet, at 8 months, the SRovx group had decreased clearance of creatinine compared to the SRsham group. Table 1 Creatinine plasma concentration and creatinine clearance (Clcr) in the offspring, submitted to ovariectomy surgery (OVX) or not (Sham), of control (C) and sleep‐restricted (SR) mothers Groups Creatinine (mg/dL) Clcr (mL/min/kg) Two‐way ANOVA 4 months Csham (n = 17) 0.53 ± 0.02 4.64 ± 0.21 Clcr C > SR SRsham (n = 26) 0.63 ± 0.02* 3.46 ± 0.17* P = 0.001 Covx (n = 17) 0.54 ± 0.02 4.39 ± 0.15 Creatinine C < SR SRovx (n = 26) 0.65 ± 0.02*, ‡ 3.21 ± 0.14*, ‡ P = 0.001 6 months Csham (n = 17) 0.50 ± 0.02 4.81 ± 0.23 Clcr C > SR SRsham (n = 26) 0.58 ± 0.02* 3.81 ± 0.19* P < 0.001 Covx (n = 17) 0.50 ± 0.03 4.75 ± 0.43 Creatinine C<SR SRovx (n = 26) 0.59 ± 0.02*, ‡ 3.58 ± 0.20*, ‡ P = 0.001 8 months Csham (n = 17) 0.53 ± 0.01 4.56 ± 0.30 Clcr C > SR (P = 0.001) SRsham (n = 26) 0.64 ± 0.02* 3.37 ± 0.18* OVX ≠ Sham (P = 0.005) Covx (n = 17) 0.55 ± 0.02 4.09 ± 0.32 Creatinine C < SR SRovx (n = 26) 0.68 ± 0.03*, ‡ 2.48 ± 0.17*, †, ‡ P = 0.001 Significance level: *P < 0.05 versus Csham; † P < 0.05 versus SRsham; ‡ P < 0.05 versus Covx (Bonferroni test). Values are means ± standard error. n, number of animals. John Wiley & Sons, LtdThe plasma sodium and potassium concentrations were in normal ranges in all studied groups, without significant changes. The excreted loads (EL) of sodium and potassium were similar in the studied groups at 4 and 6 months, shown in Table 2. However, at 8 months, there was a significant reduction in the EL of sodium and potassium in the SRovx group compared to the Csham and Covx groups. Table 2 Plasma (P) sodium and potassium concentrations and excreted loads (EL) of sodium and potassium in the offspring, submitted to ovariectomy surgery (OVX) or not (Sham), of control (C) and sleep‐restricted (SR) mothers Groups PNa + mEq/L ELNa + mEq/L/24 h PK + mEq/L ELK + mEq/L/24 h Two‐way ANOVA 4 months Csham (n = 17) 140.8 ± 0.5 1.19 ± 0.06 3.24 ± 0.03 1.84 ± 0.08 SRsham (n = 26) 143.5 ± 0.7 1.09 ± 0.05 3.37 ± 0.05 1.65 ± 0.08 Covx (n = 17) 141.3 ± 0.7 1.18 ± 0.07 3.32 ± 0.09 1.88 ± 0.08 SRovx (n = 26) 142.8 ± 0.7 1.05 ± 0.06 3.38 ± 0.05 1.55 ± 0.1 6 months Csham (n = 17) 142.5 ± 0.5 1.26 ± 0.1 3.32 ± 0.07 1.86 ± 0.1 SRsham (n = 26) 144.5 ± 0.9 1.21 ± 0.05 3.37 ± 0.07 1.64 ± 0.06 Covx (n = 17) 143.3 ± 0.9 1.33 ± 0.05 3.36 ± 0.07 1.90 ± 0.07 SRovx (n = 26) 144.9 ± 0.8 1.15 ± 0.06 3.46 ± 0.09 1.59 ± 0.1 8 months Csham (n = 17) 143.8 ± 0.9 1.14 ± 0.05 3.31 ± 0.08 1.80 ± 0.07 ELNa + SR < C SRsham (n = 26) 145.8 ± 1.1 1.06 ± 0.05 3.52 ± 0.06 1.54 ± 0.06 (P = 0.008) Covx (n = 17) 146.0 ± 0.8 1.13 ± 0.06 3.46 ± 0.09 1.84 ± 0.08 ELK + SR < C SRovx (n = 26) 147.4 ± 0.7 0.89 ± 0.07*, † 3.58 ± 0.08 1.34 ± 0.09*, † (P = 0.0001) Significance level: *P < 0.05 versus Csham; † P < 0.05 versus Covx (Bonferroni test). Values are expressed as mean ± standard error. n, number of animals. John Wiley & Sons, LtdThe values of urinary flow, urinary osmolarity, and proteinuria are shown in Table 3. At 4 and 8 months of age, there was a reduction in urinary flow in the SRovx group compared to the Csham and SRsham groups. No significant changes were observed in the other parameters. Table 3 Urinary flow, urinary osmolarity, and proteinuria for 24 h in 4, 6, and 8 months old offspring, submitted to ovariectomy surgery (OVX) or not (Sham), of control (C) and sleep‐restricted (SR) mothers Groups Urinary flow (mL/min/kg) Urinary Osm (mOsm/KgH2O) Proteinuria (mg/24 h) Two‐way ANOVA 4 months Csham (n = 17) 0.029 ± 0.001 2445.1 ± 80.5 4.42 ± 0.25 Urinary flow SRsham (n = 26) 0.028 ± 0.002 2257.7 ± 86.9 5.55 ± 0.32 SR < C (P = 0.048) Covx (n = 17) 0.028 ± 0.002 2416.8 ± 70.2 4.61 ± 0.31 OXV < Sham (P = 0.015) SRovx (n = 26) 0.022 ± 0.001*, † 2152.5 ± 95.2 5.08 ± 0.36 6 months Csham (n = 17) 0.025 ± 0.001 2232.4 ± 87.4 4.92 ± 0.38 SRsham (n = 26) 0.027 ± 0.001 2020.8 ± 78.4 5.49 ± 0.35 Covx (n = 17) 0.023 ± 0.002 2193.2 ± 86.6 4.72 ± 0.35 SRovx (n = 26) 0.023 ± 0.002 1968.0 ± 62.2 5.46 ± 0.43 8 months Csham (n = 17) 0.025 ± 0.002 2112.0 ± 75.5 5.02 ± 0.41 Urinary flow SRsham (n = 26) 0.027 ± 0.002 1902.8 ± 75.6 6.49 ± 0.52 OXV < Sham (P = 0.0008) Covx (n = 17) 0.023 ± 0.003 2040.0 ± 76.6 4.75 ± 0.32 SRovx (n = 26) 0.018 ± 0.001*, † 1869.9 ± 83.6 5.64 ± 0.55 Significance level: *P < 0.05 versus Csham; † P < 0.05 versus SRsham (Bonferroni test). Values are expressed as mean ± standard error. n, number of animals. John Wiley & Sons, LtdEffect of SR on kidney morphology The morphological parameters are presented in Table 4. The glomerular area values obtained for the SRovx were significantly smaller than those observed in the Csham and SRsham groups. Yet, an increased number of glomeruli per mm3 was observed in the SRovx group. The kidney cross‐sectional area was reduced in the Covx compared to the Csham group and in the SRovx group in relation to the Csham and SRsham groups. Kidney weight was also significantly decreased in the Covx and SRsham groups compared to the Csham group and in the SRovx group in relation to the Csham, Covx, and SRsham groups. Table 4 Glomerular area, number of glomeruli, kidney cross‐sectional area, kidney cortical area, and kidney weight in the offspring, submitted to ovariectomy surgery (OVX) or not (Sham), of control (C) and sleep‐restricted (SR) mothers Groups Csham SRsham Covx SRovx Two‐way ANOVA Glomerular area (μm2) 7936 ± 106 (388) 7831 ± 108 (404) 7654 ± 104 (444) 7365 ± 87*, † (492) OVX < Sham (P = 0.0002) SR < C (P = 0.05) Number of glomeruli/mm3 91 ± 2 (6) 96 ± 5 (6) 106 ± 11 (6) 119 ± 6* (6) OVX > Sham (P = 0.008) Kidney cross‐sectional area (mm2) 104.6 ± 1.7 (6) 100.4 ± 1.5 (6) 97.0 ± 2.0* (6) 90.3 ± 1.8*, ‡ (6) SR < C (P = 0.006) OVX < Sham P = 0.0001) Kidney cortical area (%) 59.2 ± 0.5 (6) 58.6 ± 0.4 (6) 58.5 ± 0.3 (6) 58.2 ± 0.5 (6) Kidney weight/100 g bw 0.72 ± 0.02 (17) 0.65 ± 0.01* (26) 0.58 ± 0.01* (17) 0.53 ± 0.01*, †, ‡ (26) SR < C (P = 0.0001) OVX < Sham P = 0.0001) Significance level: *P < 0.05 versus Csham; † P < 0.05 versus SRsham; ‡ P < 0.05 versus Covx (Bonferroni test). Values are means ± standard error. n, number of animals. In the glomerular area, the number in parentheses refers to the quantity of glomeruli analyzed. John Wiley & Sons, LtdFigure 2 shows the expression of factors related to renal impairment. The presence of macrophages in renal tissue (Fig 2A and 3A) was increased in the SRsham and SRovx groups compared with the Csham and Covx groups. The presence of collagen types I and III in the renal interstitium (Figs 2B and 3B) increased significantly in the SRsham and SRovx groups compared to the Csham and Covx groups. ACE1 expression (Figs 2C and 3C) was increased in the Covx, SRsham, and SRovx groups compared to the CSham group. OVX in the SR group produced even higher expression of ACE1 expression. There was no significant difference in ACE2 expression among the groups. Figure 2 Macrophages (ED‐1) expression (A), collagen type I and III expression (B), ACE1 expression (C), and ACE2 expression (D) in renal tissue of the offspring, submitted to ovariectomy surgery (OVX) or not (Sham), of control (C) and sleep‐restricted (SR) mothers. ● P < 0.05 versus CS ham; ɸ P < 0.05 versus SRS ham; Ɛ P < 0.05 versus Covx (Bonferroni test). The number of fields analyzed is shown inside the bars. Figure 3 Representative photomicrographs of the renal tissue in the offspring, submitted to ovariectomy surgery (OVX) or not (Sham), of control (C) and sleep‐restricted (SR) mothers. (A) Macrophages (ED‐1) marked in brown color (original magnification: 200×). (B) Collagen type I and III (Picrosirius red – polarized light – original magnification: 100×). (C) ACE1 and (D) ACE2 expression by immunofluorescence (original magnification: 630×). Discussion In this study, the consequences of SR in late pregnancy on the morphological and functional aspects of the kidneys in female offspring were evaluated. Renal changes and higher blood pressure were observed in female offspring from SR mothers. The SR groups, with or without oophorectomy, showed significant reduction in creatinine clearance at the studied ages, suggesting that prenatal changes resulting from SR predispose to renal dysfunction. Ovariectomy enhanced the elevation of blood pressure caused by SR during pregnancy and at 8 months of age, renal changes were more pronounced in SRovx group (decreased: creatinine clearance, urinary flow, and excreted sodium load). The removal of ovaries resulted in increased body weight and fat deposition in both offspring of C and SR. In addition, in SR, the ovariectomy caused significant morphological renal changes: (1) reduction in glomerular area, (2) increase in the number of glomeruli per mm3, probably related to (3) significant reductions in cross‐sectional area of kidney and renal mass. In female rat, there are interactions between the estrogen (E), thyroid hormones (TH), and growth hormone (GH) (Giustina and Veldhuis 1998), and the absence of E leads to obesity by reducing the action these hormones (Ignacio et al. 2012). Our results of body weight and fat deposition in OVX groups are in agreement with previous studies (Giustina and Veldhuis 1998; Ignacio et al. 2012; Pijacka et al. 2015). Additionally, we observed reduction of renal mass in Covx, SRsham, and SRovx groups (more pronounced in SRovx group). The reduction of renal mass after ovariectomy was also observed by Pijacka et al. (2015) in rats subjected to protein restriction during fetal development. In these animals, ovariectomy was associated with greater renal damage shown by significant reduction in creatinine clearance and renal mass, changes suggestive of accelerated renal aging. Thus, it is possible that sleep restriction during pregnancy also modifies renal morphology and function of female offspring and after ovariectomy, the alterations were more evident. Hypertension and cardiovascular disease resulting from an adverse environment during fetal development have been associated with reduced renal mass and nephron number (Kett and Denton 2011); however, these changes are not common in all studies (Bertram et al. 2011; Ortiz et al. 2003; Singh et al. 2007). In 1988, Brenner et al. postulated that reduced filtering surface area due to fewer nephrons leads to sodium retention and hypertension (Brenner et al. 1988; Luyckx et al. 2013). However, other mechanisms may also be involved in the development of hypertension in adults (Oparil et al. 2003). In this study, the SR did not reduce the nephron number in female offspring, though it did cause a reduction in renal mass, suggesting changes in kidney development. Ovariectomy accentuated the reduction in renal growth, which may have led to the relative increase in the number of glomeruli per mm3. However, even with more glomeruli, a loss of kidney function and an increase in blood pressure were apparent. Sleep restriction during pregnancy resulted in increased blood pressure in both genders; however, the male rat offspring had reduced nephron numbers (Thomal et al. 2010) which did not occur in female offspring, at the same experimental protocol. Studies in rats show that stress during pregnancy increases maternal cortisol production altering fetal development (Mairesse et al. 2007; Amugongo and Hlusko 2014). Increased maternal corticosteroids affects the offspring in a sex‐specific manner especially with regard to placental levels of the enzyme 11 beta‐hydroxysteroid dehydrogenase type 2 (11β‐HSD‐2), enzyme that protect the fetus from maternal glucocorticoid excess (Cuffe et al. 2012). Thus, it is possible that changes in 11β‐HSD‐2 expression levels in the placentas of males and females are related to the different results obtained at morphological analysis. However, further experiments are needed to confirm this hypothesis. The higher BP values observed in groups subjected to ovariectomy were expected as female hormones, especially estrogen, are considered cardiovascular protective factors in premenopausal women (Murphy 2011). The main vascular effects of estrogen are vasodilatation, reduced vascular inflammation, and improved vascular reactivity (Murphy 2011). These effects occur through the activation of estrogen receptors (ERs), which can be nuclear, non‐nuclear, and G‐protein coupled (GPER or GPR30) (Murphy 2011; Prossnitz and Barton 2011). The vasodilatory effect of estrogen is mediated mainly through the generation of nitric oxide (NO), as this effect is abolished after the use of NO synthesis inhibitors (Bucci et al. 2002). Concerning renal function, the beneficial effects of female sex hormones also seem to be associated with NO production (Dubey and Jackson 2001; Nielsen et al. 2003). Physiologically, NO interacts with the renin–angiotensin system (RAS), and estrogen changes the ratio between NO and ANG II, leading to a larger proportion of intrarenal NO/ANG II (Nielsen et al. 2003). Renal changes in the expression of RAS components have been described in the model of prenatal exposure to betamethasone, namely, augmented AT1 receptor expression and decreased AT2 receptor expression in the renal cortices of young sheep offspring (Gwathmey et al. 2011). Similar results were observed in the offspring of rats subjected to protein restriction (Sahajpal and Ashton 2005; Mesquita et al. 2010). These studies suggest that insults during pregnancy lead to changes in renal RAS, which likely contribute to cardiovascular changes in the offspring. In our model, increased ACE1 expression occurred in parallel with the increase in blood pressure and the greatest increase in ACE1 expression and BP were observed when both treatments were combined (SR and ovariectomy – ovx). Differently from the results of ACE1, the expression of ACE2 was not changed in the experimental groups. These results suggest that SR during pregnancy results in renal imbalance of RAS resulting in increased blood pressure. In the absence of female hormones the interaction between NO and RAS is weakened, aggravating the changes caused by SR. The kidneys from SR groups (SRsham and SRovx), in addition to the increase in ACE1 expression, showed increased macrophage infiltration and collagen type I and III expression. These results suggest that in these groups, aside from RAS alterations, other factors appear to contribute to the decline of renal function. Hypertension itself is an important factor in the induction of renal disease (Ruiz‐Ortega et al. 2006); in this way, it is difficult to take apart the influence of high blood pressure from the effects of sleep restriction during pregnancy on the renal changes observed in this experimental model. Sleep restriction and deprivation alter the production and secretion of several hormones (Spiegel et al. 1999; Schüssler et al. 2006; Pardo et al. 2016). According to Plagemann et al. (1999) during critical ontogenetic periods, hormones such as insulin, leptin, and others, once present in nonphysiological concentrations, can act as “endogenous functional teratogens” responsible for the onset of hypothalamic dysfunction in the offspring (Plagemann et al. 1999; Tzschentke and Plagemann 2006). In this theory, hormones play a central role, as they are subject to influences from the environment and also modulate the neuroendocrine and immune systems, which, in turn, regulate all key processes in the body (Dörner 1974, 1989). Thus, it is possible that sleep restriction during pregnancy results in hormonal changes that affects renal development and additionally may also modify the hypothalamic–pituitary–adrenal axis and, thereby, alters the hormonal balance of offspring in adulthood. 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==== Front Physiol RepPhysiol Rep10.1002/(ISSN)2051-817XPHY2physreportsPhysiological Reports2051-817XJohn Wiley and Sons Inc. Hoboken 2753548110.14814/phy2.12891PHY212891Environmental PhysiologyReproductive HormonesEndocrine GlandsOriginal ResearchOriginal ResearchProgesterone has rapid positive feedback actions on LH release but fails to reduce LH pulse frequency within 12 h in estradiol‐pretreated women E. G. Hutchens et al.Hutchens Eleanor G. 1 2 Ramsey Katherine A. 3 Howard Louisa C. 4 Abshire Michelle Y. 2 Patrie James T. 5 McCartney Christopher R. 1 2 1 Division of Endocrinology and MetabolismDepartment of MedicineUniversity of Virginia School of MedicineCharlottesvilleVirginia2 Center for Research in ReproductionUniversity of Virginia School of MedicineCharlottesvilleVirginia3 University of Virginia School of MedicineCharlottesvilleVirginia4 College and Graduate School of Arts and SciencesUniversity of VirginiaCharlottesvilleVirginia5 Department of Public Health SciencesUniversity of Virginia School of MedicineCharlottesvilleVirginia* Correspondence Christopher R. McCartney, Division of Endocrinology and Metabolism, Department of Medicine, Box 801406, University of Virginia Health System, Charlottesville, VA 22908. Tel: 434‐243‐0329 Fax: 434‐243‐9143 E‐mail: cm2hq@virginia.edu 17 8 2016 8 2016 4 16 10.1111/phy2.2016.4.issue-16e1289112 7 2016 18 7 2016 © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.Abstract In women, progesterone suppresses luteinizing hormone (LH) (gonadotropin‐releasing hormone) pulse frequency, but how rapidly this occurs is unknown. In estradiol‐pretreated women in the late follicular phase, progesterone administration at 1800 did not slow sleep‐associated LH pulse frequency. However, mechanisms controlling LH pulse frequency may differ according to sleep status; and we thus hypothesized that progesterone acutely suppresses waking LH pulse frequency. This was a randomized, double‐blind, crossover study of LH secretory responses to progesterone versus placebo administered at 0600. We studied 12 normal women in the late follicular phase (cycle days 7–11), pretreated with 3 days of transdermal estradiol (0.2 mg/day). Subjects underwent a 24‐h blood sampling protocol (starting at 2000) and received either 100 mg oral micronized progesterone or placebo at 0600. In a subsequent menstrual cycle, subjects underwent an identical protocol except that oral progesterone was exchanged for placebo or vice versa. Changes in 10‐h LH pulse frequency were similar between progesterone and placebo. However, mean LH, LH pulse amplitude, and mean follicle‐stimulating hormone exhibited significantly greater increases with progesterone. Compared to our previous study (progesterone administered at 1800), progesterone administration at 0600 was associated with a similar increase in mean LH, but a less pronounced increase in LH pulse amplitude. We conclude that, in estradiol‐pretreated women in the late follicular phase, a single dose of progesterone does not suppress waking LH pulse frequency within 12 h, but it acutely amplifies mean LH and LH pulse amplitude – an effect that may be influenced by sleep status and/or time of day. Circadian rhythmestradiolgonadotropin surgegonadotropin‐releasing hormoneluteinizing hormoneluteinizing hormone surgeprogesteroneNational Institutes of HealthR01 HD058671Eunice Kennedy Shriver National Institute of Child Health and Human Development/National Institutes of HealthU54 HD28934National Institutes of Health Ruth L. Kirschstein National Research Service Award Institutional Research TrainingT32 DK07646 source-schema-version-number2.0component-idphy212891cover-dateAugust 2016details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_NLMPMC version:4.9.4 mode:remove_FC converted:29.08.2016 E. G. Hutchens , K. A. Ramsey , L. C. Howard , M. Y. Abshire , J. T. Patrie , C. R. McCartney . Progesterone has rapid positive feedback actions on LH release but fails to reduce LH pulse frequency within 12 h in estradiol‐pretreated women . Physiol Rep 4 (16 ), 2016 , e12891, doi: 10.14814/phy2.12891 Funding Information This work was supported by National Institutes of Health Grant R01 HD058671 (CRM, MYA, JTP); the Eunice Kennedy Shriver National Institute of Child Health and Human Development/National Institutes of Health through Cooperative Agreement U54 HD28934 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research (currently known as the National Centers for Translational Research in Reproduction and Infertility and funded via a P50 mechanism) (CRM, MYA); and National Institutes of Health Ruth L. Kirschstein National Research Service Award Institutional Research Training Grant T32 DK07646 (EGH). ==== Body Introduction Gonadotropin‐releasing hormone (GnRH) is secreted in a pulsatile fashion from a functionally coordinated collection of hypothalamic neurons. Variations in GnRH pulse frequency stimulate differential synthesis and secretion of luteinizing hormone (LH) and follicle‐stimulating hormone (FSH) from the pituitary, with high and low frequencies favoring LH and FSH secretion respectively (Wildt et al. 1981; Gross et al. 1987). Progesterone is a major negative feedback regulator of GnRH pulse frequency in adult women. However, the rapidity with which progesterone slows LH (and by inference GnRH) pulse frequency in women remains unclear. Although studies in cows and sheep suggest that progesterone reduces GnRH pulse frequency within 2–6 h (Bergfeld et al. 1996; Skinner et al. 1998; Robinson et al. 2000), corresponding data in humans are mixed: some studies suggest rapid slowing (within 8–14 h) (Minakami et al. 1984; Permezel et al. 1989), while others suggest that more prolonged progesterone exposure is required (Pastor et al. 1998; McCartney et al. 2007a). The rapidity with which progesterone suppresses LH pulse frequency is of particular relevance to our previously proposed hypotheses regarding day‐night GnRH pulse frequency regulation in early pubertal girls: in particular, we previously proposed that the early morning increase in progesterone concentration rapidly (within hours) inhibits GnRH pulse frequency, contributing to daytime slowing in early puberty (Blank et al. 2006; McCartney et al. 2007b, 2009). Given challenges inherent to research in early pubertal girls (e.g., blood withdrawal limits), we initially sought to interrogate a key component of this hypothesis – namely, that progesterone can acutely reduce LH pulse frequency – via a study in which eight normal, estradiol‐pretreated women received progesterone or placebo at 1800 during the latter half of the follicular phase (McCartney et al. 2007a). Compared to placebo, progesterone did not suppress LH pulse frequency within 12 h. Although the aforementioned study in adults suggested that progesterone does not rapidly suppress LH pulse frequency in women, postprogesterone (and postplacebo) assessments were performed primarily during sleep periods. We subsequently reported a small, nonrandomized study of early pubertal girls suggesting that suppression of daytime pulses (but not nighttime pulses) occurs within 3–7 h of oral progesterone administration (Collins et al. 2012). Differential control of GnRH pulse frequency based on sleep status is also consistent with studies suggesting that dietary calorie restriction slows daytime, but not nighttime, LH pulse frequency in women studied during the late follicular phase (Loucks and Heath 1994; Loucks et al. 1998). Therefore, given that sleep could have been a confounder in our earlier adult study (McCartney et al. 2007a), the current study was designed to test the primary hypothesis that morning (0600) administration of progesterone rapidly (within 12 h) suppresses waking (1000–2000) LH pulse frequency in normal, estradiol‐pretreated adult women studied during the late follicular phase (cycle days 7–11). In our earlier study (McCartney et al. 2007a), LH pulse amplitude, mean LH, and mean FSH markedly increased within 4 h of progesterone (but not placebo) administration. These findings reflected progesterone positive feedback, a previously described phenomenon that may contribute to the midcycle gonadotropin surge (Hoff et al. 1983; Liu and Yen 1983; Terasawa et al. 1987). Of interest, some studies in women (Cahill et al. 1998; Kerdelhue et al. 2002), but not all (Hoff et al. 1983), suggest that the LH surge is most likely to be initiated in the early morning hours, implying that circadian rhythms may play a role in this regard. Accordingly, as a prespecified analysis, we tested a secondary hypothesis that early morning (0600) administration of progesterone (current study) provokes greater increases in LH pulse amplitude and mean LH compared to evening (1800) administration of progesterone (previous study McCartney et al. 2007a). Materials and Methods The Institutional Review Board at the University of Virginia approved all study procedures, which were in accordance with the ethical standards of the Helsinki Declaration of 1975, as revised in 2008. The study was registered with ClinicalTrials.gov (identifier NCT00594217). Subjects Twelve healthy, nonobese women with regular menstrual cycles and no evidence of hyperandrogenism completed the study and were included in the analysis (Table 1). Self‐identified race was Asian (4 subjects), Black (1 subject), Hispanic (1 subject), Middle‐Eastern (1 subject), and White (5 subjects). None of the subjects reported previous pregnancy, recent weight loss, or excessive exercise. Study participants had taken no medications known to affect the reproductive axis in the 90 days prior to study. Table 1 Subject characteristics. The number of subjects is 12 for all variables. To convert metric units to SI units: total testosterone (ng/dL) ×0.0347 (nmol/L); free testosterone (pg/mL) ×3.467 (pmol/L); insulin (μIU/mL) ×7.175 (pmol/L); glucose (mg/dL) ×0.0555 (mmol/L) Mean SD Median Range Age (years) 20.6 3.9 19 18–31 Body mass index (kg/m2) 22.4 3.1 21.72 18.4–28.5 Body fat percentage (%) 25.0 6.7 23.3 16.4–37.8 Waist‐to‐hip ratio 0.76 0.05 0.76 0.67–0.84 Total testosterone (ng/dL) 15.8 7.7 14.14 5.1–32.2 SHBG (nmol/L) 40.1 14.3 36.85 19.5–67.1 Free testosterone (pg/mL) 2.6 1.3 2.3 0.7–5.9 Fasting insulin (μIU/mL) 4.0 3.0 2.4 <2–12.2 Fasting glucose (mg/dL) 84 4 85.5 77–88 SHBG, sex hormone binding globulin. John Wiley & Sons, LtdStudy procedures Full written informed consent was obtained from each study participant. Each volunteer underwent detailed history, physical exam, and laboratory testing to screen for hormonal and health‐related abnormalities, as previously described (McCartney et al. 2007a). All participants had normal screening laboratory tests. The study followed a crossover design, with assessment of the acute effects of progesterone and placebo (individually) on pulsatile LH secretion for each subject. Subjects were randomized to receive either progesterone or placebo during the first overnight admission, with the second overnight study occurring during a subsequent menstrual cycle. Researchers and subjects were blinded to the order of progesterone administration. Subjects received transdermal estradiol patches (0.1 mg/day/patch, 2 patches [delivering a total of 0.2 mg/day] on the abdomen and changed every 2 days) starting between cycle days 4 and 8 (inclusive) and continued for a total of 4 days, with overnight admission occurring on day 3 of estradiol administration. Exogenous estradiol was given to standardize hypothalamic exposure to estradiol and to help ensure sufficient hypothalamic progesterone receptor density (MacLusky and McEwen 1978; Romano et al. 1989; Scott et al. 2000). Immediately prior to estradiol initiation, subjects had blood drawn for progesterone and β‐hCG to help exclude a luteal phase (progesterone <1.0 ng/dL) and pregnancy respectively. On day 3 of estradiol administration (i.e., cycle day 7–11), participants were admitted to the Clinical Research Unit (CRU) at 1800. Beginning at 2000, blood for later hormone measurement was obtained through an indwelling intravenous catheter over a 24‐h period as follows: LH every 10 min; progesterone every 30 min; FSH, estradiol, and testosterone every 2 h. Sex hormone binding globulin, fasting insulin, and glucose were measured at 0600. After 10 h of sampling (i.e., at 0600), either oral micronized progesterone (100 mg) suspension or oral placebo suspension was administered according to randomization. Standard meals were given at standard times during CRU admissions, and subjects fasted between 2200 and 0600. Subjects were encouraged to sleep from 2200 to 0600, but subjects were otherwise not allowed to sleep (i.e., before 2200 or after 0600). Sleep periods were assessed throughout the admission using wrist actigraphy (Sadeh and Acebo 2002). Estradiol patches were removed at the end of the 24‐h sampling period; and volunteers were discharged on oral iron supplementation (325 mg twice daily) to help replenish iron stores. During a subsequent cycle, subjects underwent a second CRU admission identical to the first (including pretreatment with estradiol) except that oral progesterone was exchanged for placebo or vice versa according to the crossover study design. Hormonal measurements All hormone assays were performed by the Ligand Assay and Analysis Core of the Center for Research in Reproduction (University of Virginia) as previously described (McCartney et al. 2007a). All samples from an individual woman were analyzed in duplicate in the same assay for each hormone. LH, FSH, and progesterone were measured by chemiluminescence (sensitivities 0.1, 0.1 IU/L, 0.2 ng/mL; intra‐assay coefficients of variation [CVs] 3.3%, 3.2%, 4.4%; and interassay CVs 5.8%, 4.9%, 7.8%, respectively; Siemens Healthcare Diagnostics, Los Angeles, CA). Total testosterone was measured by radioimmunoassay (sensitivity 10 ng/dL, intra‐assay CV 4.3%, interassay CV 7.4%; Calbiotech, Spring Valley, CA). Estradiol was measured by radioimmunoassay (sensitivity 10 pg/mL, intra‐assay CV 6.3%, interassay CV 8.1%; American Laboratory Products Company, Salem, NH). Samples with measured values below assay sensitivity were assigned the value of the assay's sensitivity. To convert from conventional to Systeme International (SI) units: progesterone ×3.18 (nmol/L); total testosterone ×3.47 (pmol/L); estradiol ×3.671 (pmol/L). Data analysis Given that LH measurement error is unequal across the physiological range of LH concentrations (heteroscedastic), a computerized data reduction protocol (StdCurve, developed by Michael L. Johnson, Ph.D., University of Virginia) was employed to provide a variance model for experimental measurement error for each LH concentration time series (i.e., for each subject admission). StdCurve utilizes (1) unprocessed and untruncated luminometer output data in addition to (2) corresponding LH readout data from the standard curve for all LH measurements in the assay run, including blanks (no LH) and known standards (i.e., quality controls approximating LH concentrations of 2.5, 25, and 60 IU/L). In essence, StdCurve assigns statistically accurate estimates of LH concentration precision for each time point across the LH time series, eliminating truncation errors introduced at low LH concentrations by the proprietary standard curve. Experimental error is addressed by assigning and propagating uncertainty estimates for each standard curve response (including zero dose responses) by an empirically determined discrete uncertainty profile. These discrete uncertainty profiles account for both response precision (replicability) and accuracy (deviation from the predicted calibration curve) without relying on assumed theoretical variance‐assay response relationships. For each LH concentration time series, pulsatile secretion was identified and characterized using AutoDecon, a multiparameter deconvolution program (Johnson et al. 2008). This statistically based algorithm is fully automated in that it identifies initial parameter estimates while simultaneously performing deconvolution. In particular, AutoDecon iteratively inserts and tests the significance of presumed secretion events. The automated nature of AutoDecon renders it nonsubjective, in contrast with earlier deconvolution procedures. Compared to Cluster 7 (Veldhuis and Johnson 1986), AutoDecon has greater sensitivity to detect LH pulses (96% vs. 80%), but a higher false positive rate (6% vs. 1%) (Johnson et al. 2008). To limit false positives, only the following AutoDecon‐identified pulses were included in subsequent analyses: (1) pulses with at least two peak values that were at least 10% higher than the nadir value; or (2) pulses with at least one peak value that was at least 20% higher than the nadir value. Once pulse locations were established, LH pulse frequency was estimated by calculating the average interpulse interval (IPI) over time blocks using a method described in detail previously (McCartney et al. 2007a). Briefly, the number of minutes within the time block was divided by the number of LH IPIs (including fractions thereof) residing in that time block. LH pulse amplitudes were calculated by subtracting the preceding LH nadir concentration from the peak LH concentration for each pulse. Power analysis, subject dropout, and final sample size The primary endpoint for this study was the change in LH pulse frequency (average IPI over 10 h) attributable to progesterone. We hypothesized that 10‐h IPI after progesterone administration (1000–2000) would be higher than 10‐h IPI after placebo administration (i.e., that progesterone would reduce LH pulse frequency). Based on the within‐subject variability in LH IPI differences (progesterone vs. placebo) observed in our earlier study (McCartney et al. 2007a), we estimated that the study of 12 women would provide 80% power to detect a 16.7‐min difference (progesterone vs. placebo) in average 10‐h IPI. Formal study procedures were initiated in 16 women. For various personal reasons, 3 women elected to drop out of the study after completing a single admission only; data from these subjects are not included in this analysis. Thirteen women completed the full study protocol. However, one of these women was not included in the analysis because she had an average progesterone concentration of 4.8 ng/mL from 2000 to 0600 before receiving exogenous progesterone, indicating that she was inadvertently studied during the luteal phase. Thus, we formally analyzed the data for 12 women who completed both admissions during the late follicular phase. Statistical analysis The influence of exogenous progesterone on LH IPI was analyzed using a 2‐period crossover design analysis of covariance (ANCOVA) – our primary endpoint analysis. In the ANCOVA model specification, the interblock changes (i.e., the 10‐h time block after progesterone/placebo [1000–2000] minus the 10‐h time block before progesterone/placebo [2000–0600]) in loge LH IPI represented the response data. (For this and subsequent analyses, loge transformation was necessary to ensure conformity with the normality assumptions of ANCOVA procedures.) The ANCOVA model factors were sequential order of interventions (i.e. progesterone followed by placebo vs. placebo followed by progesterone), crossover period (i.e., first vs. second), and intervention (progesterone vs. placebo). Baseline loge LH IPI (i.e., during the 2000–0600 time block) served as an ANCOVA adjustment variable. With regard to hypothesis testing, our primary hypothesis was that the change in mean loge LH IPI from the baseline state (2000–0600) to the post‐treatment state (1000–2000) was the same irrespective of whether progesterone or placebo was administered at 0600. The primary hypothesis was tested based on a linear contrast of the ANCOVA least‐squared means, and a two‐sided P ≤ 0.05 decision rule was established a priori as the null hypothesis rejection rule. Traditional residual diagnostics were used to confirm model goodness of fit. With regard to secondary endpoint variables, mean LH, LH pulse amplitude, mean FSH, and progesterone were analyzed via 2‐period crossover ANCOVA in exactly the same manner as the LH IPI data. Since estradiol and testosterone concentrations were not hypothesized to change in response to progesterone administration, they were analyzed as loge total concentrations over each 24‐h admission via mixed‐effect ANCOVA models with “subject” as a random effect (i.e., blocking factor) and the intervention (progesterone, placebo) as the fixed effect factor. As a prespecified secondary analysis, we compared the influence of progesterone given at 0600 (current study) versus 1800 (previous study McCartney et al. 2007a) on mean LH concentration and mean LH pulse amplitude. We hypothesized that, compared to progesterone administered at 1800, progesterone administered at 0600 would provoke greater increases in both mean LH and LH pulse amplitude. To maximize the validity of these comparisons, we reanalyzed the data published in 2007 (McCartney et al. 2007a) using StdCurve and AutoDecon, as described above. Since this secondary analysis involved multiple simultaneous comparisons, Bonferroni correction was employed to limit experimental‐wise type I error to ≤0.05. Although the above outcome data were loge transformed prior to analysis, we report geometric means (GM) and associated 95% confidence intervals (CI) when describing the results of statistical testing. The GM is a location parameter similar to arithmetic mean, calculated as the antilog of the mean of log‐transformed values. Thus, GM provides a faithful representation of the data as analyzed (in contrast to arithmetic mean). Another benefit is that GM ratios can be interpreted as percentage and/or fold changes. Of potential importance, one subject in the current cohort had mean LH and LH pulse amplitude responses that were markedly discordant with the other 11 subjects. We suspected, but could not confirm, that the samples for her progesterone and placebo admissions had been mislabeled prior to assay. For this reason, we repeated the aforementioned analyses after excluding this subject. We do not report the results of this secondary analysis, however, because exclusion of this subject did not alter the results of our primary analysis (LH pulse frequency), and it only strengthened apparent differences between treatment conditions for mean LH, LH pulse amplitude, and mean FSH. The statistical software package SAS version 9.4 (SAS Institute Inc., Cary, NC) was used to conduct all statistical analyses. Results Progesterone admissions occurred on cycle day 9.7 ± 1.4 (mean ± SD), and placebo admissions on cycle day 9.8 ± 1.3. As measured by wrist actigraphy, sleep periods (time from first sleep to last sleep) were 7.6 ± 0.4 and 7.1 ± 0.9 h – and sleep efficiency 92 ± 4 and 92 ± 12% – during the progesterone and placebo admissions respectively. As dictated by simple (i.e., nonblocked) randomization, progesterone was given during the first admission in three subjects and placebo in nine; for all ANCOVA analyses, sequence order was not a significant predictor of results. Sex steroids Summary statistics for progesterone, estradiol, and total testosterone concentrations are shown in Table 2, and sex steroid profiles are represented graphically in Figure 1. Ten‐hour progesterone concentrations at baseline (2000–0600) were similar between the progesterone and placebo admissions (GM ratio 1.05; 95% CI: [0.75–1.48], P = 0.746). As expected, 10‐h (1000–2000) progesterone concentrations increased markedly after progesterone administration (9.4‐fold increase in GM; 95% CI: [7.71–11.48], P < 0.001). After placebo administration, 10‐h progesterone concentration increased slightly compared to baseline (27% increase in GM; 95% CI: [4–55%], P = 0.021). The increase associated with progesterone administration was 7.64‐fold greater than the increase associated with placebo administration (95% CI: [6.21–9.41], P < 0.001). Table 2 Summary statistics, sex steroid concentrations. Summary statistics for progesterone are partitioned by admission (progesterone vs. placebo) and time block (2000–0600 [preintervention] vs. 1000–2000 [postintervention]). Summary statistics for estradiol and total testosterone are for the entire 24‐h admission and, thus, partitioned by admission only. The number of subjects is 12 for all variables. To convert metric units to SI units: progesterone ×3.18 (nmol/L); estradiol ×3.67 (pmol/L); total testosterone ×0.0347 (nmol/L) Admission Time block (h) Mean SD GM Median Range Progesterone (ng/mL) Progesterone 2000–0600 0.48 0.15 0.45 0.5 0.2–0.8 1000–2000 3.96 1.35 3.77 3.7 2.4–7.1 Placebo 2000–0600 0.43 0.09 0.42 0.4 0.3–0.6 1000–2000 0.53 0.12 0.52 0.55 0.4–0.8 Estradiol (pg/mL) Progesterone 2000–2000 98.8 60.8 83.2 76.5 28–224 Placebo 2000–2000 92.9 34.1 87.1 81.5 38–162 Testosterone (ng/dL) Progesterone 2000–2000 15.5 6.5 14.2 14.7 6.1–28.0 Placebo 2000–2000 16.1 7.4 14.6 14.2 5.1–32.3 GM, geometric mean. John Wiley & Sons, LtdFigure 1 Progesterone (A), estradiol (B), and total testosterone (C) concentrations. Each data point identifies the geometric mean (GM), and vertical lines identify 95% confidence intervals for the GM. Progesterone and placebo admissions are denoted by solid and open data points, respectively. Conversion from metric to Systeme International units: progesterone ×3.18 (nmol/L); estradiol ×3.67 (pmol/L); total testosterone ×0.0347 (nmol/L). Estradiol levels were similar during both admissions: GM 83.2 pg/mL during the progesterone admission and 87.1 pg/mL during the placebo admission (GM ratio between admissions 0.96; 95% CI: [0.71–1.29], P = 0.746). Total testosterone levels were also similar between admissions (GM ratio 0.98; 95% CI: [0.84–1.12], P = 0.751). LH pulse frequency Summary statistics for LH IPI are shown in Table 3, and results are represented graphically in Figure 2. Ten‐hour GM LH IPI decreased from 79.0 min during the first 10‐h time block (2000–0600, before progesterone administration) to 64.0 min during the second 10‐h time block (2000–0600, after progesterone administration) – a 21% decrease in GM IPI (95% CI: [7–34%], P = 0.008). Similar changes occurred during the placebo admission: GM LH IPI decreased from 80.3 min before placebo to 60.5 min after placebo, representing a 24% GM IPI reduction (95% CI: [10–36%], P = 0.003). After adjusting for GM IPI during the first 10‐h time block – values of which were similar between progesterone and placebo admissions (P = 0.913) – changes in GM IPI from first to second 10‐h time block were not different between the progesterone and placebo admissions (ratio of changes, 1.05; 95% CI: [0.87–1.27], P = 0.596). Table 3 Summary statistics, gonadotropin characteristics. Summary statistics are partitioned by admission (progesterone vs. placebo) and time block (2000–0600 [preintervention] vs. 1000–2000 [postintervention]). The number of subjects is 12 for all variables Admission Time block Mean SD GM Median Range LH IPI (min) Progesterone 2000–0600 80.4 16.1 79.0 80.3 61.4–108.7 1000–2000 64.5 8.9 64.0 65.1 51.1–80.7 Placebo 2000–0600 82.1 19.3 80.3 75.1 59.7–129.9 1000–2000 61.5 11.6 60.5 57.9 47.3–82.0 Mean LH (IU/L) Progesterone 2000–0600 6.3 4.6 5.0 5.5 1.8–17.6 1000–2000 21.3 14.3 16.4 20.7 1.8–54.9 Placebo 2000–0600 5.7 2.7 4.9 6.3 0.9–10.3 1000–2000 8.4 4.3 7.2 8.8 1.9–16.5 LH amplitude (IU/L) Progesterone 2000–0600 2.8 2.1 2.2 2.4 0.7–8.5 1000–2000 7.8 3.8 6.5 8.2 1.0–12.6 Placebo 2000–0600 2.5 0.9 2.4 2.8 0.8–3.6 1000–2000 2.7 1.2 2.4 2.8 0.6–4.4 Mean FSH (IU/L) Progesterone 2000–0600 4.1 1.6 3.8 4.7 1.5–5.9 1000–2000 8.0 3.3 7.4 8.0 3.0–14.6 Placebo 2000–0600 3.5 1.0 3.3 3.5 1.4–5.4 1000–2000 4.7 1.7 4.4 4.5 2.0–7.0 John Wiley & Sons, LtdFigure 2 Luteinizing hormone (LH) secretory characteristics during progesterone and placebo admissions. Rows illustrate data for LH interpulse interval (panels A, B, and C), mean LH (panels D, E, and F), and LH pulse amplitude (panels G, H, and I). Panels A, D, and G: The first column illustrates data for 10‐h time blocks immediately before intervention (2000–0600) versus 10‐h time blocks after intervention (1000–2000). Individual data are represented by connected circles, and arithmetic mean values are shown as larger squares. Progesterone and placebo admissions are denoted by solid and open data points, respectively. Panels B, E, and H: The second column illustrates changes pre versus post‐intervention (i.e., 2000–0600 vs. 1000–2000). The points identify the percentage change (B) or fold change (E and H) in the geometric mean (GM), and the vertical lines identify the 95% confidence interval for the percentage or fold change. P values above individual data plots relate to pre versus postintervention changes within an individual admission. P values connecting admissions relate to between‐admission comparisons of pre versus postintervention changes (i.e., pre vs. postprogesterone change vs. pre vs. postplacebo change). Panels G, H, and I: The third column illustrates data for two 4‐h time blocks before intervention and three 4‐h time blocks after intervention. Each data point identifies the GM, and vertical lines identify 95% confidence intervals for the GM. Progesterone and placebo admissions are denoted by solid and open data points, respectively. Note that statistical analyses were not performed for 4‐h time blocks. IPI, interpulse interval; P4, progesterone; PBO, placebo. Mean LH and LH pulse amplitude Ten‐hour mean LH during the first 10‐h time block was similar before progesterone and placebo (GM 5.0 and 4.9 IU/L, respectively; P = 0.985) (Table 3 and Fig. 2). After progesterone administration, mean LH increased markedly (GM 16.4 IU/L) – an estimated 3.15‐fold increase in GM versus baseline (95% CI: [2.26–4.39], P < 0.001). Mean LH also increased after placebo administration (1.52‐fold estimated increase in GM; 95% CI [1.09–2.13], P = 0.016). After adjusting for mean LH in the first 10‐h time block, pre versus postintervention changes in GM mean LH (first vs. second 10‐h time blocks) were 2.07‐fold greater with progesterone compared to placebo (95% CI: [1.15–3.72], P = 0.021). Luteinizing hormone pulse amplitudes were similar in the 10 h before progesterone and placebo administration (GM 2.2 and 2.4 IU/L, respectively; P = 0.417) (Table 3 and Fig. 2). Ten‐hour GM LH amplitude increased to 6.5 IU/L after progesterone, an estimated 2.78‐fold increase (95% CI: [1.92–4.01], P < 0.001); but GM LH amplitude was unchanged after placebo (GM 2.4 IU/L; P = 0.994). Intervention‐related changes in GM LH pulse amplitude were 2.68‐fold greater with progesterone compared to placebo (95% CI: [1.46–4.92], P = 0.005). Mean FSH Ten‐hour FSH concentrations at baseline were similar between the progesterone and placebo admissions (GM 3.8 and 3.3 IU/L, respectively; P = 0.842) (Table 3 and Fig. 3). Ten‐hour FSH increased after progesterone administration (1.93‐fold estimated increase in GM FSH; 95% CI: [1.52–2.46], P < 0.001); after placebo, 10‐h mean FSH increased to a lesser degree (an estimated 31% increase in GM FSH compared to baseline; 95% CI: [3–66%], P = 0.030). Intervention‐related changes in GM FSH were 1.49‐fold higher with progesterone compared to placebo (95% CI: [1.04–2.13], P = 0.033). Figure 3 Mean follicle‐stimulating hormone (FSH) during progesterone and placebo admissions. Panel A: Data for 10‐h time blocks immediately before intervention (2000–0600) versus 10‐h time blocks after intervention (1000–2000). Panel B: Pre versus postintervention changes (i.e., 2000–0600 vs. 1000–2000). Panel C: Data for two 4‐h time blocks before intervention and three 4‐h time blocks after intervention. All data are organized as in Figure 2. P4, progesterone; PBO, placebo. Influence of time of day on changes in mean LH and LH pulse amplitude Compared to our previous study in which progesterone was administered in the evening (1800) (McCartney et al. 2007a), progesterone administered at 0600 (current study) produced similar increases in mean LH (Fig. 4). In the previous study, 10‐h GM mean LH increased 3.43‐fold (95% CI: [2.53–4.65], P < 0.001) from the first 10‐h time block (0800–1800) to the second (2200–0800) – similar to the increase observed in the current study (difference between studies, Bonferroni‐corrected P = 1.00). Ten‐hour GM mean LH pre versus postplacebo did not change significantly in the previous study (P = 0.073), while 10‐h GM LH increased between the first and second 10‐h time blocks when placebo was given at 0600 (described above). Changes in GM mean LH associated with placebo administration was 1.73‐fold greater (95% CI: [1.16–2.58]) in the current study compared to the previous study (Bonferroni‐corrected P = 0.025). Figure 4 Mean luteinizing hormone (LH) and LH pulse amplitude during progesterone and placebo admissions for the current study and for our previous study (McCartney et al. 2007a). Panel A: LH concentrations (measured every 10 min) for the current study (left) and the previous study (right). Progesterone and placebo admissions are denoted by solid and open data points, respectively. Each data point identifies the geometric mean (GM), and vertical lines identify 95% confidence intervals for the GM. Panels B and C: Fold changes in mean LH (B) and LH pulse amplitude (C), pre versus postintervention. The specific changes shown are as follows: 2000–0600 versus 1000–2000 when progesterone or placebo was administered at 0600; and 0800–1800 versus 2200–0800 when progesterone or placebo was administered at 1800. The points identify the fold change in the GM, and the vertical lines identify the 95% confidence interval for the fold change. P values (Bonferroni‐corrected) relate to between‐study comparisons of pre versus postintervention changes, ostensibly reflecting clock hour differences (e.g., pre vs. postintervention change when progesterone was given at 0600 vs. pre vs. postintervention change when progesterone was given at 1800). P4, progesterone; PBO, placebo. When progesterone was administered in our previous study, GM LH amplitude increased 5.76‐fold (95% CI: [4.01–8.27]) between the first and second 10‐h time blocks (P < 0.001); but 10‐h GM LH amplitude did not change pre versus postplacebo administration (P = 0.749). The progesterone‐associated increase in GM LH amplitude was 52% lower (95% CI: [26–68%]) in the current study (progesterone given at 0600) compared to the previous study (progesterone given at 1800) (between‐study difference in progesterone‐associated changes, Bonferroni‐corrected P = 0.004). Between the two studies, the placebo admissions were associated with similar changes in GM LH amplitude (Bonferroni‐corrected P = 0.879). Post hoc assessments Although not part of prespecified analyses, we noted that mean LH frequently increased – sometimes markedly so – within 4 h of waking during the placebo admission of the current study, as implied in Figure 4A. In particular, subjects were awakened at 0600, and 4‐h mean LH increased from 0210–0600 to 0610–1000 in 10 of 12 placebo admissions, with an average fold increase of 1.8. Five subjects with marked (ranging from 2.4‐ to 3.8‐fold) increases in mean LH within 4 h of waking had estradiol concentrations over 100 pg/mL, while the seven subjects without obvious changes in mean LH within 4 h of waking (ranging from a 24% decrease to a 25% increase) had estradiol concentrations lower than 100 pg/mL. Accordingly, average estradiol concentrations during the placebo admission positively correlated with both absolute change in mean LH (Spearman correlation: r s = 0.84, P < 0.001) and the fold‐change in mean LH (r s = 0.95, P < 0.001) within 4 h of waking (Fig. 5). Similar relationships were observed in the progesterone admissions – absolute and fold mean LH changes from 0210–0600 to 0610–1000 correlated with estradiol (for both: r s = 0.70, P = 0.011) – similar to a previous study (Permezel et al. 1989). Figure 5 Relationship between average estradiol concentrations and sleep–wake changes in mean LH during placebo admissions. Subjects were awakened at 0600, and the sleep–wake change in mean LH (y‐axis) is shown as the fold‐change in mean LH from 0210–0600 to 0610–1000. Conversion from metric to Systeme International units: estradiol ×3.67 (pmol/L). Changes in LH pulse amplitude within 4 h of waking during placebo admissions were less consistent, with LH pulse amplitude increasing in six of 12 subjects. The overall average increase was 61%, largely driven by four subjects – all with estradiol concentrations >100 pg/mL – who exhibited 2.2‐ to 3.7‐fold increases. However, estradiol concentrations did not clearly correlate with absolute or fold‐changes in LH pulse amplitude within 4 h of waking during placebo admissions (r s 0.44–0.47, P > 0.1 for both) or during progesterone admissions (for both: r s = 0.42, P = 0.18). We noted that achieved estradiol levels were highly variable. Variable absorption (e.g., variable patch adherence to the skin) could have played a role in this regard, as suggested by within‐subject (i.e., between‐admission) differences, which were as follows (pg/mL): mean ± SD, 33 ± 33; 25th percentile, 4.5; median, 24; 75th percentile, 50.5. Cycle day did not correlate with achieved estradiol level (r s = 0.004, P = 0.985, n = 24 admissions). In addition, average subject estradiol (i.e., average of two admissions) did not correlate with age, BMI, or waist‐to‐hip ratio (P > 0.1 for all, n = 12 subjects). While average subject estradiol was negatively correlated with body fat percentage (r s = −0.64, P = 0.026 [unadjusted for multiple comparisons]), achieved estradiol concentration did not correlate with body fat percentage when evaluating (1) the progesterone admissions in isolation or (2) the placebo admissions in isolation (P > 0.2 for both). Lastly, it remains possible that other (unknown) factors might explain the strong correlation between achieved serum estradiol concentration and the morning rise in mean LH during placebo admissions; but morning increases in LH did not correlate with age, BMI, body fat percentage, or waist‐to‐hip ratio. Discussion Although our previous study failed to disclose a rapid reduction in sleep‐related LH pulse frequency when a single dose of progesterone was administered at 1800 (McCartney et al. 2007a), we considered the possibility that the regulation of GnRH pulse frequency differs according to sleep status – a possibility suggested by a study in early pubertal girls (Collins et al. 2012) and other studies in late follicular phase women (Loucks and Heath 1994; Loucks et al. 1998). We thus hypothesized that progesterone administration at 0600 slows waking LH pulse frequency within 12 h in normal adult women pretreated with estradiol in the late follicular phase. Results from our current study do not support this hypothesis; however, in keeping with earlier findings (McCartney et al. 2007a), we observed (1) sleep‐related reductions in LH pulse frequency, and (2) rapid and marked increases in gonadotropin concentrations (both LH and FSH) and LH pulse amplitude associated with progesterone administration. Our confidence in these findings is enhanced by methodological improvements employed with the current study: in particular, our larger study population (n = 12 vs. 8) provided greater statistical power; we employed a more sophisticated LH pulse analysis protocol (e.g., StdCurve plus AutoDecon vs. Cluster); and we subjected the data to more sophisticated statistical analyses. The rapidity with which progesterone slows GnRH pulse frequency in women remains unclear. Although a study in postmenopausal women suggested that high dose IM progesterone – to achieve concentrations approximating 20–30 ng/mL – at 0600 suppressed LH pulse frequency by 45% within 9–14 h (Minakami et al. 1984), this study involved a markedly different patient population (older, estrogen‐deficient subjects) and a number of methodological differences, including the absence of a control state (e.g., untreated group). A study in normally cycling women studied on either cycle day 6 or 10 (n = 4 each) suggested that 10 mg intramuscular progesterone – achieving mean progesterone concentrations of 1.6 ng/mL – reduced LH pulse frequency within 8 h (i.e., the ratio of postprogesterone IPI to preprogesterone IPI was 1.24, P = 0.05) (Permezel et al. 1989). However, in a similar group of 16 women, LH pulse frequency was not acutely altered by mifepristone administration (Permezel et al. 1989). In another study, progesterone (50 mg every 8 h by vaginal suppository) and estradiol (0.2 mg/day by transdermal patch) – both started in the late follicular phase (cycle day 8–10) and achieving progesterone concentrations circa 9–10 ng/mL – appeared to reduce LH pulse frequency by 50% within 5 days (Pastor et al. 1998); but this change was not statistically significant, perhaps a reflection of the small number of subjects (n = 5) and limited statistical power. The rapidity with which progesterone slows GnRH pulse frequency in human females has been of particular interest to our group, in large part because we have previously proposed a hypothesis of day‐night LH pulse frequency regulation in early puberty that was predicated on the notion that progesterone can have rapid effects (within hours) on LH pulse frequency – specifically, we proposed that the early morning increase in progesterone rapidly (within hours) suppresses daytime LH pulse frequency, thus contributing to day–night differences in LH pulse frequency (Blank et al. 2006; McCartney et al. 2007b, 2009). Although our current study does not support the hypothesis that progesterone rapidly inhibits LH pulse frequency in women, the dynamics of progesterone suppression may vary according to hormonal milieu. For example, androgens appear to antagonize the negative feedback effects of progesterone on GnRH neuron firing rates in rats (Pielecka et al. 2006) and on LH pulse frequency in both sheep (Robinson et al. 1999) and women (Pastor et al. 1998; Eagleson et al. 2000). Therefore, when compared to feedback dynamics in adult women, progesterone‐mediated suppression may be more rapid – in addition to being more profound – when androgen concentrations are very low (e.g., in normal early puberty). This consideration may help explain the apparent discordance between our current study and the aforementioned study in early pubertal girls, which suggested a rapid suppression (within 4–7 h) of waking LH pulse frequency after progesterone administration (Collins et al. 2012). Some published data suggest that LH surge initiation tends to be initiated in the early morning hours (Cahill et al. 1998; Kerdelhue et al. 2002). Since LH surge initiation is largely a reflection of sex steroid positive feedback on pituitary LH release, we tested a predefined secondary hypothesis: progesterone positive feedback on mean LH and LH pulse amplitude would be more pronounced when administered in the morning (0600; current study) compared to evening administration (1800; previous study McCartney et al. 2007a). Progesterone‐mediated changes in mean LH were similar regardless of the timing of progesterone administration, consistent with a previous report in monkeys (Terasawa et al. 1984). In contrast, placebo‐associated changes were different: mean LH increased after placebo administered at 0600, but decreased after placebo administered at 1800. We infer that this latter difference could reflect diurnal and/or sleep–wake changes in mean LH (discussed further below). With regard to LH pulse amplitude, progesterone‐associated increases were greater with evening (1800) dosing compared to morning (0600) dosing – in contrast with our hypothesis. We considered the possibility that this particular finding reflected interstudy differences in LH pulse frequency, since LH pulse amplitude tends to increase as a function of previous IPI (O'Dea et al. 1989). That is, since LH IPI decreased from the first to second 10‐h time blocks in the current study, one might have expected a concomitant decrease in LH pulse amplitude; and since LH IPI increased from the first to second 10‐h time blocks in the previous study, one might have expected a concomitant increase in LH pulse amplitude. However, such changes in LH pulse amplitude were not clearly observed during the placebo admissions (Fig. 4C). An alternative explanation for greater progesterone‐related increases in LH pulse amplitude in the previous study is that, for unclear reasons, those subjects had higher average estradiol levels (214 ± 46 [mean ± SD] and 169 ± 23 pg/mL during the progesterone and placebo admissions, respectively) despite identical dose and duration of transdermal estradiol use. We speculate that, compared to the current study, higher estradiol levels in the previous study augmented the positive feedback responses to progesterone. We noted that mean LH commonly increased – sometimes markedly so – within 4 h of waking (at 0600) during the placebo admission in the current study; such changes correlated with ambient E2 concentrations. Mean LH tended to be highest in the early morning hours (circa 0600–1000) in our earlier study as well (see Fig. 4). As described above, some (but not all) studies in women suggest that LH surges tend to be initiated in the morning. For example, one study of 19 ovulatory women suggested that LH surges were initiated in the early morning hours (circa 0400–0800) (Kerdelhue et al. 2002). Given that average estradiol concentrations positively correlated with changes in mean LH within 4 h of waking, we speculate that this could represent nascent surge‐like activity – specifically associated with time of day and/or the transition from sleep to wake – that that will be further amplified as preovulatory estradiol (and progesterone) concentrations are achieved. Conclusion Herein, we provide data suggesting that daytime/waking LH pulse frequency is not suppressed within 12 h by a single 100 mg oral dose of progesterone in estradiol‐pretreated women studied in the late follicular phase. However, mean LH and LH pulse amplitude are acutely amplified by progesterone in this setting. Our data suggest that progesterone‐mediated augmentation of LH pulse amplitude may be affected by time of day and/or sleep status – being more prominent in the evening/nighttime hours compared to the morning/daytime hours – although differences in achieved estradiol concentrations represent an important potential confounder in this regard. Conflicts of Interest The authors have no conflicts of interest to disclose. Acknowledgments We gratefully acknowledge Lauren Lockhart and Anne Gabel for subject recruitment, study scheduling, and assistance with data management; the nurses and staff of the Clinical Research Unit at the University of Virginia for implementation of these sampling protocols; and the Center for Research in Reproduction Ligand Assay and Analysis Core Laboratory for performance of all assays. ==== Refs References Bergfeld , E. G. , F. N. Kojima , A. S. Cupp , M. E. Wehrman , K. E. Peters , V. 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==== Front Physiol RepPhysiol Rep10.1002/(ISSN)2051-817XPHY2physreportsPhysiological Reports2051-817XJohn Wiley and Sons Inc. Hoboken 10.14814/phy2.12895PHY212895Endocrine and Metabolic Conditons, Disorders and TreatmentsSkeletal MuscleCardiovascular Conditions, Disorders and TreatmentsVasculatureSignalling PathwaysOriginal ResearchOriginal ResearchHyperinsulinemia augments endothelin‐1 protein expression and impairs vasodilation of human skeletal muscle arterioles A. M. Mahmoud et al.Mahmoud Abeer M. 1 2 3 Szczurek Mary R. 3 Blackburn Brian K. 1 2 Mey Jacob T. 1 2 Chen Zhenlong 4 Robinson Austin T. 1 2 Bian Jing‐Tan 3 Unterman Terry G. 5 Minshall Richard D. 4 Brown Michael D. 1 2 Kirwan John P. 6 Phillips Shane A. 1 2 3 Haus Jacob M. 1 2 1 Department of Kinesiology and NutritionUniversity of Illinois at ChicagoChicagoIllinois2 Integrative Physiology LaboratoryUniversity of Illinois at ChicagoChicagoIllinois3 Department of Physical TherapyUniversity of Illinois at ChicagoChicagoIllinois4 Department of Pharmacology and AnesthesiologyUniversity of Illinois at ChicagoChicagoIllinois5 Department of MedicineDivision of Endocrinology, Diabetes and MetabolismUniversity of Illinois at ChicagoChicagoIllinois6 Department of PathobiologyLerner Research InstituteCleveland ClinicClevelandOhio* Correspondence Jacob M. Haus, Department of Kinesiology and Nutrition, University of Illinois at Chicago, 1919 West Taylor Street, Room 530 (MC 517), Chicago, IL 60612. Tel: 312‐413‐1913 Fax: 312‐413‐0319 E‐mail: hausj@uic.edu 22 8 2016 8 2016 4 16 10.1111/phy2.2016.4.issue-16e1289515 7 2016 21 7 2016 © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.Abstract Hyperinsulinemia is a hallmark of insulin resistance‐associated metabolic disorders. Under physiological conditions, insulin maintains a balance between nitric oxide (NO) and, the potent vasoconstrictor, endothelin‐1 (ET‐1). We tested the hypothesis that acute hyperinsulinemia will preferentially augment ET‐1 protein expression, disrupt the equilibrium between ET‐1 expression and endothelial NO synthase (eNOS) activation, and subsequently impair flow‐induced dilation (FID) in human skeletal muscle arterioles. Skeletal muscle biopsies were performed on 18 lean, healthy controls (LHCs) and 9 older, obese, type 2 diabetics (T2DM) before and during (120 min) a 40 mU/m2/min hyperinsulinemic‐euglycemic (5 mmol/L) clamp. Skeletal muscle protein was analyzed for ET‐1, eNOS, phosphorylated eNOS (p‐eNOS), and ET‐1 receptor type A (ETAR) and B (ETBR) expression. In a subset of T2DM (n = 6) and LHCs (n = 5), FID of isolated skeletal muscle arterioles was measured. Experimental hyperinsulinemia impaired FID (% of dilation at ∆60 pressure gradient) in LHCs (basal: 74.2 ± 2.0; insulin: 57.2 ± 3.3, P = 0.003) and T2DM (basal: 62.1 ± 3.6; insulin: 48.9 ± 3.6, P = 0.01). Hyperinsulinemia increased ET‐1 protein expression in LHCs (0.63 ± 0.04) and T2DM (0.86 ± 0.06) compared to basal conditions (LHCs: 0.44 ± 0.05, P = 0.007; T2DM: 0.69 ± 0.06, P = 0.02). Insulin decreased p‐eNOS (serine 1177) only in T2DM (basal: 0.28 ± 0.07; insulin: 0.17 ± 0.04, P = 0.03). In LHCs, hyperinsulinemia disturbed the balance between ETAR and ETBR receptors known to mediate vasoconstrictor and vasodilator actions of ET‐1, respectively. Moreover, hyperinsulinemia markedly impaired plasma NO concentration in both LHCs and T2DM. These data suggest that hyperinsulinemia disturbs the vasomotor balance in human skeletal muscle favoring vasoconstrictive pathways, eventually impairing arteriolar vasodilation. Endothelin‐1hyperinsulinemiamicrovasculaturenitric oxideskeletal muscleCTSAUL1RR024989UL1RR029879Central Society for Clinical and Translational Research – Junior Faculty AwardAmerican Diabetes Association1‐14‐JF‐32NIHR01s HL095701HL095701‐01A2SHL085497DK089547P01 HL60678American Heart Association15POST24480172 source-schema-version-number2.0component-idphy212895cover-dateAugust 2016details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_NLMPMC version:4.9.4 mode:remove_FC converted:29.08.2016 A. M. Mahmoud , M. R. Szczurek , B. K. Blackburn , J. T. Mey , Z. Chen , A. T. Robinson , J.‐T. Bian , T. G. Unterman , R. D. Minshall , M. D. Brown , J. P. Kirwan , S. A. Phillips , J. M. Haus . Hyperinsulinemia augments endothelin‐1 protein expression and impairs vasodilation of human skeletal muscle arterioles . Physiol Rep , 4 (16 ), 2016 , e12895, doi: 10.14814/phy2.12895 Funding Information This research was supported by the following funding sources: CTSA grants UL1RR024989 and UL1RR029879 (J. M. H.), Central Society for Clinical and Translational Research – Junior Faculty Award (J. M. H.), American Diabetes Association Grant 1‐14‐JF‐32 (J. M. H.), NIH R01s HL095701, HL095701‐01A2S (S. A. P.), HL085497 (M. D. B.), DK089547 (J. P. K.), P01 HL60678 (R. D. M.), and American Heart Association Grant 15POST24480172 (A. M. M.). ==== Body Introduction Compensatory hyperinsulinemia associated with insulin resistance is considered one of the primary mechanisms that promotes vascular dysfunction and the subsequent development of cardiovascular disease (CVD) (Muniyappa et al. 2007). Understanding the mechanism by which hyperinsulinemia induces vascular dysfunction is essential for advancing treatment and prevention strategies of insulin resistance‐related vascular complications. At physiological levels, insulin regulates vascular function by maintaining the balance between endothelial‐derived nitric oxide (NO) and, the potent vasoconstrictor, endothelin‐1 (ET‐1). Insulin initiates signaling cascades through both phosphatidylinositol 3‐kinase (PI3K) and mitogen‐activated protein kinase (MAPK) pathways that subsequently activate eNOS phosphorylation and ET‐1 protein expression, respectively (Muniyappa et al. 2007). The concept of selective insulin resistance, characterized by impairment of PI3K signaling with preservation of MAPK signaling, may be the mechanism by which chronic hyperinsulinemia promotes vascular dysfunction (Kim et al. 2006). Thus, the unopposed activation of the MAPK signaling arm of insulin action, and subsequent transcriptional upregulation of ET‐1, will favor the vasoconstrictor effects of insulin. However, it remains unclear whether or not mere hyperinsulinemia, in isolation from insulin resistance, augments ET‐1 signaling and, thus, disturbing the vasomotor balance and compromising endothelial function. Numerous studies have reported enhanced limb blood flow in response to insulin stimulation (Clerk et al. 2006; Fujita et al. 2006; Eggleston et al. 2007), whereas others demonstrate either null effect or reduction in blood flow in lean, healthy individuals (Hedman et al. 2001; Arcaro et al. 2002; Morgantini et al. 2012). Insulin infusion in rats has been shown to increase plasma ET‐1 and precipitate hypertension (Juan et al. 2004; Potenza et al. 2005). Similarly, in vitro studies have consistently demonstrated increased ET‐1 gene expression in insulin‐treated endothelial cells (Formoso et al. 2006; Yang and Li 2008). Collectively, these observations support the notion that hyperinsulinemia, not confounded by insulin resistance, induces ET‐1 production and disturbs vasomotor balance. The effect of insulin infusion on ET‐1 signaling in humans has mainly been deduced from circulating concentrations of ET‐1 (Ferri et al. 1995b,c; Seljeflot et al. 1998; Surdacki et al. 1999). However, circulating ET‐1 concentrations do not correlate with vasoconstrictor activity in peripheral tissues as ET‐1 acts predominantly in a local fashion after being released from endothelial cells (Wagner et al. 1992). This emphasizes the importance of measuring ET‐1 and microvascular responses to insulin in peripheral tissues. Among the insulin sensitive tissues, skeletal muscle accounts for more than 80% of insulin‐stimulated glucose disposal (Defronzo et al. 1985) and receives more than 25% of the cardiac output at rest (Korthuis 2011). Also, skeletal muscle insulin resistance is the primary defect in the development of T2DM and future risk of CVD (Petersen et al. 2007). To our knowledge, no data exist that examine human isolated skeletal muscle microvessel function in response to experimental hyperinsulinemia. Furthermore, direct comparisons between a healthy reference group and T2DM subjects are also lacking. Thus, the primary objective of this study was to investigate the effect of systemic hyperinsulinemia on human skeletal muscle microvascular function and differentiate the potential differences in chronic hyperinsulinemia, as experienced with insulin resistance and T2DM versus acute experimental hyperinsulinemia in lean healthy adults. We hypothesized that acute hyperinsulinemia would disturb the balance of vasoactive mediators through biased augmentation of the ET‐1 pathway, with subsequent impairment of endothelial function. In healthy and T2DM subjects, we measured skeletal muscle protein expression of the major vasoactive mediators (ET‐1 and eNOS) collected under basal and insulin‐stimulated conditions of a hyperinsulinemic‐euglycemic clamp. In addition, microvessels isolated from skeletal muscle biopsy samples, obtained at basal and insulin‐stimulated conditions, were studied via in vitro flow‐induced dilation (FID). Materials and Methods Study design We designed this cross‐sectional study to obtain as much information as possible about skeletal muscle microvessel responses to experimental hyperinsulinemia. Our intent was to examine these responses in a population of obese, hypertensive, T2DM subjects and directly contrast these findings with lean healthy controls (LHCs). Comparisons to a LHC group, representing an implied state of good health, allowed for a reference of “normal” protein expression and physiological responses, thus highlighting the importance of the disease phenotype. Hyperinsulinemic‐euglycemic clamps were used to induce experimental hyperinsulinemia and characterize whole‐body insulin sensitivity. Skeletal muscle needle biopsies were obtained during basal and insulin‐stimulated conditions of the clamp procedure for each subject. Skeletal muscle tissue samples were probed for protein expression of vasoactive regulatory proteins, and fresh tissue samples (when available) were dedicated to the isolation, and ex vivo study, of resistance microvessels. To our knowledge, this is one of the first studies of isolated skeletal muscle resistance arterioles from humans due to tissue limitations and technical burden. Despite these technical challenges, we were successful in obtaining FID data from both LHCs and T2DM participants. Also, skeletal muscle tissue and plasma samples, when available, were used for histological analysis of capillary density and NO bioavailability, respectively. Subjects Participants were recruited from the Cleveland, OH, and Chicago, IL, metropolitan areas. All subjects were screened via health history, medical exam, resting EKG, and fasting blood chemistry in the Clinical Research Centers of the Cleveland Clinic and the University of Illinois at Chicago. Glucose tolerance was characterized by a 75 g oral glucose tolerance test (OGTT) following a standard fasting period for all subjects. Individuals were excluded if they used nicotine, had undergone greater than 2‐kg weight change in the last 6 months, or had evidence of hematological, renal, hepatic, or CVD. LHCs were excluded if results of OGTT indicated impaired fasting glucose or impaired glucose tolerance. T2DM subjects self‐reported diabetes duration of 4 ± 1 years. All studies were approved by the Institutional Review Boards of the Cleveland Clinic and the University of Illinois at Chicago and performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all research participants during the initial screening visit. Metabolic control All subjects were instructed to maintain their regular dietary eating habits and completed 3‐day diet records (2 weekday and 1 weekend day). Subjects were asked to refrain from physical activity outside of their normal activities of daily living, consumption of alcoholic beverages, and consumption of foods and beverages containing caffeine for 48 h prior to the metabolic testing day. On the day preceding metabolic testing, subjects were counseled to consume ~55% of calories as carbohydrate in order to meet a goal of 250 g (Solomon et al. 2011; Haus et al. 2013). The evening prior to testing, participants were provided a balanced metabolic meal (55% carbohydrate, 35% fat, and 10% protein). The meal composition was free of foods that are known to contain high amounts of nitrates. After the meal consumption, the participants fasted overnight for a period of 10–12 h. All participants were asked to withhold medications and supplements the morning of metabolic testing that were known to influence primary outcome variables. This included all medications for the treatment of allergies, asthma, hypercholesterolemia, hypertension, pain, and T2DM. A full list of medication use is provided in Table 1. Female participants (n = 13), who were premenopausal with regular menstrual cycles, were studied in the follicular phase. Two women were postmenopausal without use of hormone replacement therapy. Table 1 Medication use Drug class LHC (n) T2DM (n) Aldosterone receptor antagonist 0 1 Alpha blocker 0 2 Angiotensin converting enzyme inhibitor 0 1 Angiotensin II receptor antagonists 0 2 Antiallergy 4 3 Antimineralcorticoid 1 1 Beta‐blocker 0 2 Bronchodilator 1 1 Calcium channel blocker 0 1 Diuretic 0 2 DPP4 inhibitor 0 2 Insulin 0 3 Metformin 0 5 NSAID 2 4 Oral contraceptive 3 0 Proton pump inhibitor 1 1 Statin 0 4 Sulfonylurea 0 3 LHC, lean healthy control. John Wiley & Sons, LtdInsulin sensitivity Whole‐body insulin sensitivity was assessed using the hyperinsulinemic (40 mU/m2/min)‐euglycemic (5 mmol/L) clamp procedure. Arterialized venous blood was sampled for glucose concentrations at 5‐min intervals and continued over the course of 2 h (120 min) (YSI 2300; STAT Plus, Yellow Springs, OH). Adjustments to the glucose infusion rate were made according to the calculations of Defronzo et al. (1979). Mean space‐corrected exogenous glucose infusion rates during the final 30 min of steady‐state hyperinsulinemia are presented as peripheral tissue glucose disposal rates (GDR: mg/kg/min). We have previously described these procedures in detail (Haus et al. 2009; Solomon et al. 2010, 2013; Williamson et al. 2015). Skeletal muscle biopsy Skeletal muscle needle biopsies (vastus lateralis) were performed during the baseline (0 min) and insulin‐stimulated (120 min) periods of the hyperinsulinemic‐euglycemic clamp procedure (Haus et al. 2007; Williamson et al. 2015). Infusion of steady‐state insulin and glucose was not terminated until completion of the muscle biopsy procedure. Muscle tissue was immediately blotted, trimmed of adipose and connective tissue, and aliquoted as described below. Skeletal muscle tissue was either: (1) flash frozen in liquid nitrogen for protein expression analyses; (2) placed in ice‐cold (4°C) HEPES buffer solution (pH 7.4) for resistance vessel isolation and subsequent FID experiments; and/or (3) mounted in tragacanth gum and frozen in liquid nitrogen‐cooled isopentane for histological analysis. Protein and histology samples were stored at −80°C until analysis. FID experiments were completed immediately following biopsy. FID experiments Resistance arterioles of ~50 μm in diameter and ~2 mm in length were carefully dissected from the skeletal muscle tissue and cleaned of fat and connective tissue. In an organ perfusion chamber, single vessels were cannulated with glass micropipettes (outer tip diameter ∼40 μm) filled with cold bicarbonate buffer consisting of 123 mmol/L NaCl, 4.4 mmol/L KCl, 2.5 mmol/L CaCl2, 1.2 mmol/L MgSO4, 20 mmol/L NaHCO3, 1.2 mmol/L KH2PO4, and 11 mmol/L glucose. Both ends of the vessel were secured with 10‐0 nylon Ethilon monofilament suture, and the vessels were maintained at an intraluminal pressure of 20 mmHg for 30 min. Each preparation was transferred to the stage of an inverted microscope (magnification ×200) attached to a video camera, monitor, and video‐measuring device (model VIA‐100; Boeckeler Instruments, Tucson, AZ). The external bathing medium was continuously superfused with heated buffer solution (pH = 7.4 ± 0.05, Po2 = 140 ± 10 mmHg) aerated with a gas mixture of 21% O2–5% CO2–74% N2 and maintained at 37°C. The pressure was slowly increased to 100 mmHg and maintained for 30 min. Vessels were preconstricted 30–50% with ET‐1 (100–200 pM). Vessels that did not constrict >30% were excluded from the analysis. Flow was produced by simultaneously changing the heights of the reservoirs in equal and opposite directions to generate an intraluminal pressure gradient of ∆10–∆100 cmH2O (equivalent to ~7–70 mmHg), which covers the physiological range of arteriolar pressure in the human body (Phillips et al. 2007; Grizelj et al. 2015). In separate experiments, vasoreactivity measures were determined in response to incremental doses of acetylcholine (ACh) (10−9–10−4 mol/L). Steady‐state internal arterial diameters were measured before and during intraluminal flow or ACh in the absence or presence of the NO synthase (NOS) inhibitor Nω‐nitro‐l‐arginine methyl ester (L‐NAME; 10−4 mol/L). Maximal diameter of every vessel was determined in the presence of papaverine (10−4 mol/L), and the diameter in response to flow at a gradient of 100 cmH2O was measured in the presence of papaverine first in one direction, and then with the gradient reversed to verify pipette resistance matching (Phillips et al. 2007). Immunoblotting Basal and insulin‐stimulated protein expression of eNOS (sc‐654; Santa Cruz Biotechnology, Dallas, TX), p‐eNOS (serine 1177) (9570S; Cell Signaling Technology, Beverly, MA), endothelin‐1 (ET‐1) (ab2786; Abcam, Cambridge, MA), endothelin‐converting enzyme (ECE) (sc‐376017; Santa Cruz Biotechnology), ETAR, and ETBR (ab178454 & ab129102; Abcam) was achieved via immunoblotting for all subjects. Of technical note, the ET‐1 antibody detects the 203 amino acid pre‐proendothelin‐1 protein using an epitope corresponding to amino acids 8–16. This epitope is shared by all endothelin‐1 posttranslational products (pre‐proendothelin‐1 (24 kDa), pro‐endothelin‐1 (4 kDa) and endothelin‐1 (2 kDa)). Given the difficulties in resolving small protein products via SDS‐PAGE, it is widely accepted for the 24 kDa pre‐proendothelin‐1 to be representative of active ET‐1. Approximately 10–15 mg (wet weight) of frozen muscle tissue from each sample was homogenized (lysing matrix D beads; FastPrep®‐24 homogenizer, MP Biomedicals, Santa Ana, CA) in ice‐cold buffer consisting of 20 mmol/L Tris‐HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L Na2 EDTA, 1 mmol/L EGTA, 2.5 mmol/L Na pyrophosphate, 1 mmol/L β‐glycerophosphate, 1 mmol/L Na3VO4, 1% Triton, and 1 μg/mL leupeptin (Cell Signaling Technology) with an added protease and phosphatase inhibitor cocktail (Sigma Aldrich, St. Louis, MO). Total protein concentration was determined via BCA Protein Assay (Pierce Biotechnology, Rockford, IL), and 30 μg protein were separated via 10% SDS‐PAGE and immunoblotted using primary antibodies. Protein expression of glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (D16H1; Cell Signaling Technology) or β‐Actin (612657; BD Biosciences, San Jose, CA) was utilized as a loading control. Densitometry was performed using NIH Image J software. Plasma NO As a surrogate marker of NO bioavailability, concentrations of nitrate and nitrite, stable end products of NO metabolism, were measured in basal and insulin‐stimulated plasma samples using the Griess reaction (Cayman Chemicals, Ann Arbor, MI) (Solomon et al. 2011). Briefly, nitrate was converted into nitrite utilizing nitrate reductase; then the Griess reagents were added which convert nitrite into a dark purple azo compound. The absorbance was measured at 540 nm using a plate reader. Samples were run in triplicates, and the concentration of nitrate was calculated using a nitrate standard curve. Capillary density Serial 10‐μm cross‐sections were obtained using a cryostat (Leica CM 3050; Leica Microsystems, Bannockburn, IL) and mounted on poly‐ l‐lysine‐coated glass slides. Capillaries were identified using a periodic acid‐Schiff stain (Sigma) following digestion of glycogen by amylase and hematoxylin counterstain, as described previously (Solomon et al. 2011). Images were acquired by using a Nikon microscope and analyzed using NIS‐Elements Microscope Imaging Software (Nikon, Melville, NY). The number of capillaries per square millimeter of surface area and the average number of capillary contacts per fiber were determined. Statistical analyses Baseline subject characteristics for each group (LHCs vs. T2DM) were compared using an independent samples t‐test. A two‐way (group [LHCs vs. T2DM]) × condition [basal vs. insulin]) ANOVA, with repeated measures for “condition,” was used to compare protein expression and plasma NO data. FID data collected at basal, basal + L‐NAME, insulin, insulin + L‐NAME, ACh basal, ACh basal + L‐NAME, ACh insulin, and ACh insulin + L‐NAME were compared using a two‐way (group [LHCs vs. T2DM] × dose [10, 20, 40, 60, 100 cmH2O]) ANOVA. Bonferroni/Dunn post hoc tests were used for multiple comparisons when appropriate. Univariate analysis of variance was used to explore absolute change (Δ; insulin – basal), percentage Δ (% Δ; insulin), and capillary density data for group differences. Bivariate correlation analyses were performed with Pearson correlation coefficients. SPSS v22 (IBM, Armonk, New York) was used to perform all statistical analyses. P < 0.05 was considered significant and data are presented as mean ± SEM. Results Subject characteristics Baseline subject characteristics including anthropometric and metabolic variables are presented in Table 2 for each group. As expected, and by design, LHCs were significantly different from T2DM subjects in age, body composition, systolic blood pressure, and glucose metabolism with the exception for fasting insulin and steady‐state plasma insulin (90–120 min) during the hyperinsulinemic‐euglycemic clamp. Table 2 Subject characteristics Variable LHC T2DM n 18 (7 ♂) 9 (5 ♂) Age, year 31 ± 2 58 ± 4a Weight, kg 62.8 ± 2.8 104.7 ± 7.7a BMI, kg/m2 22.3 ± 0.6 34.1 ± 2.1a Body fat, % 25.3 ± 1.4 40.2 ± 2.8a Systolic BP, mmHg 116 ± 4 135 ± 6a Diastolic BP, mmHg 69 ± 3 79 ± 4 FPG, mg/dL 91 ± 1 127 ± 12a FPI, μU/mL 8.4 ± 1.4 8.0 ± 1.0 HbA1c, % 5.4 ± 0.1 7.2 ± 0.6a 2 h‐OGTT, mg/dL 101 ± 3 233 ± 39a GDR, mg/kg/min 6.6 ± 0.4 2.8 ± 0.3a Clamp Insulin, μU/mL 91.0 ± 10.2 89.2 ± 6.0 Total chol, mg/dL 156 ± 6 152 ± 10 HDL, mg/dL 63 ± 4 53 ± 5 LDL, mg/dL 79 ± 7 79 ± 7 VLDL, mg/dL 15 ± 1 21 ± 5 TG, mg/dL 74 ± 6 104 ± 26 Data represent mean ± SEM. a.u., arbitrary units; BMI, body mass index; chol, cholesterol; fat%, percentage of body fat; FPG, fasting plasma glucose; FPI, fasting plasma insulin; GDR; hyperinsulinemic‐euglycemic clamp‐derived glucose disposal rate; clamp insulin, steady‐state plasma insulin concentrations at end of clamp; HbA1c, hemoglobin A1c; LHC, lean healthy control; OGTT, oral glucose tolerance test; TG, triglycerides. a Significant difference between LHC and T2DM (P < 0.05). John Wiley & Sons, LtdExperimental hyperinsulinemia impairs vasodilation of human skeletal muscle arterioles Basal endothelial‐mediated vasodilation of isolated skeletal muscle arterioles was greater in LHCs versus T2DM subjects as determined by FID (P = 0.049) and Ach (P = 0.046), at all pressure gradients and doses (P < 0.001, Figs. 1A, B and 2A, B). Isolated skeletal muscle arterioles demonstrated impaired FID with 2 h of hyperinsulinemia; % of maximum dilation at ∆60 cmH2O, which reflects the mean of physiological arteriolar pressure inside the human body, was reduced by 23% in LHCs (P = 0.003) (Fig. 1A) and by 22% in subjects with T2DM (P = 0.01) (Fig. 1B) relative to basal states. Compared to baseline, FID at ∆60 was also reduced in the presence of L‐NAME (LHCs: −27%, P = 0.03; T2DM: −31%, P < 0.001). Interestingly, L‐NAME‐induced impairment of FID was greater in insulin‐stimulated arterioles compared to basal (LHCs: −39%, P = 0.03; T2DM: −32%, P < 0.01), suggesting an increased dependence of FID on NO under hyperinsulinemic conditions. These findings were recapitulated by exposing isolated arterioles to increased concentrations of ACh (Fig. 2A and B), confirming the involvement of endothelium‐dependent mechanisms in the insulin‐induced impairment of vasodilation. Figure 1 Flow‐induced dilation (FID) is reduced in isolated skeletal muscle arterioles of lean healthy controls (n = 5) (A) and T2DM (n = 6) (B) at 2 h of hyperinsulinemia. FID was measured during intraluminal flow corresponding to pressure gradients of 10–100 cmH2O in the absence or presence of L‐NAME. All values are plotted as means ± SE. *(P < 0.05) for L‐NAME and † (P < 0.05) for insulin. Figure 2 Acetylcholine‐induced dilation is attenuated in isolated skeletal muscle arterioles of lean healthy controls (n = 5) (A) and T2DM (n = 6) (B) at 2 h of hyperinsulinemia. Arteriolar dilation was measured in response to increased concentrations of acetylcholine (10‐9 to 10‐4 mole/L) in the absence or presence of L‐NAME. All values are plotted as means ± SE. *(P < 0.05) for L‐NAME and † (P < 0.05) for insulin. Hyperinsulinemia reduced circulating NO To determine if the observed impairment in FID was accompanied by changes in plasma concentrations of NO, plasma nitrite/nitrate concentrations were measured in LHC and T2DM subjects at basal and insulin‐stimulated conditions. Although not significant, the average plasma NO concentration was higher in LHCs than in T2DM in the basal state (37.4 ± 10.1 μmol/L vs. 21.6 ± 3.9 μmol/L, P = 0.08). Plasma NO concentrations during hyperinsulinemia were reduced by 65.9% in LHCs and by 37.8% in T2DM subjects (P < 0.01, Fig. 3). Circulating NO was also found to be correlated with FID in the basal state (R = 0.7; P = 0.04). Figure 3 Hyperinsulinemia reduces plasma nitric oxide (NO) (nitrate/nitrite) concentrations in lean healthy controls (n = 10) and T2DM (n = 9). (A) Plasma nitrite/nitrate concentrations, a marker of NO production, were measured in the basal and hyperinsulinemic states. Results represent the means ± SE for each group. *P < 0.05 for insulin versus basal. Hyperinsulinemia increases ET‐1 protein expression in skeletal muscle tissue Skeletal muscle ET‐1 protein expression was greater in T2DM subjects (57%, P = 0.003) (Fig. 4A) compared to LHCs. Hyperinsulinemia increased ET‐1 protein expression by 43.2% in LHCs (P = 0.007) and by 24.6% in T2DM subjects (P = 0.02) compared to basal (Fig. 4A). Similarly, ECE protein, which is responsible for cleaving inactive proendothelin to the active endothelin‐1 peptide, was upregulated in response to hyperinsulinemia in LHCs (basal: 0.80 ± 0.06; insulin: 1.08 ± 0.17, P = 0.02); however, no significant changes were detected in T2DM subjects. Basal ET‐1 protein expression was found to be inversely correlated with insulin‐stimulated GDR (R = −0.71, P = 0.002) (Fig. 4B). Figure 4 Hyperinsulinemia induces ET‐1 protein expression in skeletal muscle of lean healthy controls (LHCs) (n = 8) and T2DM (n = 8). (A) Western blot analysis of ET‐1 protein expression in skeletal muscle homogenate. Signal relative intensity was normalized to GAPDH and results represent the means ± SE for each group. *P < 0.05 for insulin versus basal and † (P < 0.05) for LHCs versus T2DM. (B) Correlation between ET‐1 protein expression and clamp‐derived glucose disposal rate in the study subjects (n = 16). Hyperinsulinemia increases p‐eNOS (serine 1177) in skeletal muscle of LHCs Figure 5 compares total and phosphorylated eNOS between LHCs and T2DM. Under basal conditions, total eNOS protein was 1.9‐fold higher in LHCs than T2DM subjects (P = 0.003), however, the phosphorylated fraction of eNOS (p‐eNOS) was 1.3‐fold greater in the T2DM than the LHCs (P = 0.01) (Fig. 5B). Hyperinsulinemia did not induce significant changes in total eNOS protein in LHCs or T2DM subjects (Fig. 5A). In contrast, p‐eNOS showed differential responses to hyperinsulinemia depending on the subject group. In LHCs, p‐eNOS was increased by 75% (P = 0.03), whereas in T2DM subjects it was reduced by 39% (P = 0.03) (Fig. 5B). Accordingly, p‐eNOS/eNOS ratio was fivefold higher in T2DM (P = 0.004) at basal and then reduced by 57.7% during hyperinsulinemia (P = 0.02) (Fig. 5C). Significant correlations were observed between GDR and basal expression of eNOS protein (R = 0.7, P = 0.001) and insulin‐induced changes in p‐eNOS (Δ p‐eNOS) (R = 0.51, P = 0.03) (Fig. 5D and E, respectively). Figure 5 Hyperinsulinemia increases phosphorylated eNOS protein expression in skeletal muscle of lean healthy controls (LHCs) (n = 10) but not in T2DM (n = 8). Western blot analysis of total eNOS (A) and p‐eNOS (serine 1177) protein expression (B) in skeletal muscle homogenate. Signal relative intensity was normalized to β‐actin and results represent the means ± SE for each group. (C) Ratio between the normalized expression of p‐eNOS and eNOS proteins in T2DM subjects and LHCs at 2 h of hyperinsulinemia. Correlation between clamp‐derived glucose disposal rate in study subjects and basal expression of eNOS protein (D) and Δ p‐eNOS (E). *P < 0.05 for insulin versus basal and † (P < 0.05) for LHCs versus T2DM. Hyperinsulinemia disturbs the ET‐1/eNOS ratio in T2DM subjects but not in LHCs Due to higher ET‐1 and lower eNOS basal protein levels, T2DM subjects displayed greater basal ET‐1/eNOS ratios relative to LHCs (P < 0.001) (Fig. 6A). The ET‐1/eNOS ratio was negatively correlated with the insulin‐stimulated GDR (R = −0.82, P = 0.0001) and positively correlated with fasting plasma glucose (FPG) (R = 0.69, P = 0.003) (Fig. 6B and C, respectively). Figure 6 Effects of hyperinsulinemia on the ET‐1/eNOS ratio in skeletal muscle tissue. The ratio between ET‐1 and eNOS (A) in skeletal muscle tissue in T2DM subjects (n = 8) and lean healthy controls (LHCs) (n = 8). Results represent the means ± SE for each group. *P < 0.05 for insulin versus basal and † (P < 0.05) for LHCs versus T2DM. The ratio between the ET‐1 and eNOS protein expression in skeletal muscle tissue of the study subjects (n = 16) correlates negatively with glucose disposal rate (B) and positively with FPG (C). Hyperinsulinemia modifies ET receptor expression in skeletal muscle of LHCs ET‐1 action is mediated via two types of receptor, ETAR and ETBR. To test the potential downstream effects of hyperinsulinemia‐induced increased ET‐1 protein expression, we measured the protein expression of ETAR and ETBR receptors in LHCs and T2DM subjects at basal and insulin‐stimulated conditions. T2DM subjects had higher basal levels of ETAR (1.8‐fold, P = 0.004) and ETBR proteins (1.6‐fold, P < 0.0001) relative to LHCs (Fig. 7A and B, respectively). Hyperinsulinemia increased ETAR protein expression in skeletal muscle tissue of LHCs (P = 0.004) without any significant effect on ETBR protein expression (Fig. 7A and B). As a result, during hyperinsulinemia, the ETAR/ETBR ratio was increased by 2.7‐fold in LHCs (P = 0.03, Fig. 7C). Hyperinsulinemia had no significant effect on either ETAR or ETBR in T2DM subjects. Figure 7 Effects of hyperinsulinemia on ET‐1 receptor protein expression in skeletal muscle of lean healthy controls (LHCs) (n = 8) and T2DM (n = 8). Western blot analysis ETAR (A) and ETBR (B) protein expression in skeletal muscle homogenate. Signal relative intensity was normalized to GAPDH and results represent the means ± SE for each group. (C) The ratio between normalized expression of ETAR and ETBR proteins in LHCs (n = 8) and T2DM (n = 8). *P < 0.05 for insulin versus basal and † (P < 0.05) for LHCs versus T2DM. *Significant difference between LHC and T2DM (P < 0.05). Skeletal muscle capillary density is lower in T2DM than LHCs As expected, the average number of capillaries per square millimeter of surface area (capillary density (CD) and mean capillary to muscle fiber (C/F) ratio were lower in skeletal muscle of T2DM subjects (CD: 3.48 ± 0.17 and C/F ratio: 1.16 ± 0.06) relative to LHCs (CD: 4.85 ± 0.15; C/F ratio: 1.62 ± 0.05, P < 0.0001). Although CD and C/F ratios were not found to be correlated with basal plasma NO concentrations (R = 0.37, P = 0.13), the ratios correlated positively with GDR (R = 0.85, P < 0.0001) and negatively with body mass index (R = −0.75, P = 0.0004), FPG (R = −0.65, P = 0.004), HbA1c (R = −0.68, P = 0.002), and ETAR protein expression (R = −0.81, P = 0.01). Discussion The concept of selective insulin resistance, in the face of chronic hyperinsulinemia, is now recognized as a major mechanistic factor in the development of endothelial dysfunction and subsequent CVD. The major findings of this work are that acute, experimental hyperinsulinemia impairs endothelial‐mediated FID, reduces plasma NO bioavailability, and disrupts the balance between the vasodilator and the vasoconstrictor pathways in isolated skeletal muscle arterioles in LHCs and T2DM subjects. Furthermore, hyperinsulinemia disturbed the balance between the two main ET‐1 receptors, ETAR and ETBR. Collectively, our data suggest that hyperinsulinemia, independent of impaired insulin action, may contribute to endothelial dysfunction by favoring the predominance of vasoconstrictor signaling. Also, our novel data highlight potential candidate mechanisms for therapuetic target, such as agents that alter the ET‐1 receptor density or ETAR/ETBR ratio. Of clinical relevance, ETAR upregulation is implicated in the pathogenesis of hypertension, endothelial dysfunction, inflammation, and fibrosis (Vignon‐Zellweger et al. 2012). Furthermore, ETAR antagonists are the first‐line treatment option for pulmonary arterial hypertension, and interestingly, are currently being studied for the treatment of diabetic nephropathy (Reichetzeder et al. 2014). We previously reported that impairments of NO bioavailability and capillary density across the glucose tolerance continuum in obese adults were associated with clamp derived GDRs (Solomon et al. 2011). Consistent with our previous data, we show here that the basal concentrations of plasma NO were considerably lower in the T2DM group than in LHCs. Previous work by Tsukahara et al. (1997) demonstrated increased plasma NO concentrations during experimental hyperinsulinemia in LHCs. In contrast, our data showed a marked decrease in plasma NO in both LHCs and T2DM groups with 2 h of 40 mU/m2/min experimental hyperinsulinemia. Interestingly, the magnitude of reduction we observed in plasma NO was greater in the LHC group than the T2DM, which could be explained by the lower basal concentrationsof plasma NO in T2DM subjects. Our data suggest that hyperinsulinemia is capable of reducing circulating NO bioavailability independent of insulin resistance. Despite our observed differences, the use of circulating nitrate and nitrite as a surrogate for NO bioavailability may be limiting due to the contribution of nitrates from the diet. While we made efforts to control dietary intake prior to metabolic testing, we cannot exclude residual dietary effects on our plasma measures. Furthermore, under certain conditions, the nonenzymatic production of NO can be significant and thus we cannot derive the exact proportion of insulin‐mediated NO production from eNOS. Despite the established vasodilatory action of insulin, human studies show an inconsistent vasomoter response to experimental hyperinsulinemia (Mahmoud et al. 2015). In this study we tried to bridge these gaps by: (1) examining the differential effects of hyperinsulinemia between T2DM subjects and LHCs in order to isolate effects of experimental hyperinsulinemia versus insulin resistance; (2) using an insulin dose (40 mU/m2/min) that produces physiological range of peripheral hyperinsulinemia which generally corresponds to peripheral insulin concentrations after a mixed meal (Roden 2007); and (3) employing an innovative ex vivo approach in skeletal muscle that allows direct measurement of arteriolar microvascular diameter under conditions of controlled flow. Using this integrated approach, we demonstrated lower baseline FID and ACh‐induced dilation of isolated skeletal muscle arterioles in subjects with T2DM compared to LHCs. This is consistent with previous studies that reported diminished muscle microvascular perfusion in obese and diabetic individuals using contrast‐enhanced ultrasound (Clerk et al. 2006). With hyperinsulinemia, FID in LHCs was reduced to levels comparable with baseline FID in T2DM subjects, which may indicate a causitive role of hyperinsulinemia in endothelial dysfunction. Interestingly, L‐NAME induced greater reduction in FID in insulin‐stimulated arterioles than arterioles from baseline conditions (Figs. 1 and 2), suggesting an increased dependence of FID on NO bioavailability in the presence of hyperinsulinemia. Previous studies have reported similar findings where larger reductions in blood flow were achieved in response to L‐NAME during hyperinsulinemia than at baseline (Scherrer et al. 1994; Steinberg et al. 1994). In this study, baseline expression of eNOS phosphorylation in T2DM subjects was unexpectedly higher than LHCs; nevertheless, plasma NO concentrations were lower in T2DM than those in LHCs. Data by Kashyap et al. (2005) reported lower basal NOS activity in T2DM than LHCs assessed by measuring the conversion of l‐arginine into l‐citrulline in skeletal muscle tissue, which is downstream of eNOS phosphorylation. When interpreted in combination with our finding, this might indicate downstream interference with the ability of p‐eNOS to catalyze the conversion of l‐arginine into l‐citrulline and, hence, diminish NO production. This might also promote the assumption that higher basal expression of p‐eNOS in T2DM is a compensatory response to the compromised NO production. However, further work is necessary to confirm this statement and explain this paradoxical finding. Our data also demonstrate a reduction in eNOS phosphorylation in skeletal muscle tissue of T2DM with insulin stimulation which together with the concomitant increase in ET‐1 protein expression may explain the impaired FID in T2DM subjects. Furthermore, in accordance with findings from previous studies (Kashyap et al. 2005; Bradley et al. 2007), LHCs have exhibited consistent increases in eNOS phosphorylation. Experimental evidence supports the role of insulin in regulating endothelial cell ET‐1 production (Ferri et al. 1995a,b; Chisaki et al. 2003). However, clinical data are contradictory and depend mainly on circulating plasma ET‐1 data that may be of limited value, as ET‐1 predominantly acts in a paracrine manner (Webb 1997). Also, the extent to which insulin‐stimulated ET‐1 production contributes to maintenance of vascular homeostasis in physiological settings versus insulin‐resistant states has not been assessed. Although we did not measure circulating ET‐1 concentrations, we show higher basal protein expression of ECE and ET‐1 in skeletal muscle tissue of T2DM subjects compared with LHCs. These findings are in agreement with previous studies that reported higher circulating concentrations of ET‐1 in obese and T2DM versus healthy individuals (Ferri et al. 1995a,c). It has also been reported that elevated ET‐1 plays a critical role in the development of insulin resistance and endothelial dysfunction in skeletal muscle (Shaw and Boden 2005). Although the insulin‐mediated induction of ET‐1 protein expression in skeletal muscle of T2DM subjects is consistent with others (Ferri et al. 1995c; Rab et al. 2004), our study is the first to demonstrate similar effects in LHCs. This paradoxical similarity in FID and ET‐1 response to insulin between LHCs and T2DM subjects suggests either absence of vascular insulin resistance in T2DM or transient development of vascular insulin resistance in LHCs under conditions of acute hyperinsulinemia. Furthermore, we cannot rule out the possibility that the active form of ET‐1 may not actually be released from skeletal muscle microvessels due to our limitations in detecting the 2 kD active form of ET‐1. However, we are not aware of defects in the processing of pre‐proendothelin‐1 to ET‐1 with insulin stimulation or metabolic disease. Despite the induction of ET‐1 protein expression, the concomitant stimulation of eNOS phosphorylation led to a preservation of the ratio between these two vasoactive mediators. Thus, investigating the pathway downstream to ET‐1 protein, i.e., ET‐1 action‐mediating receptors, ETAR and ETBR, were essential in this study to detect a deleterious response to hyperinsulinemia that might explain impaired FID in LHCs. Previous work has reported enhanced leg blood flow and skeletal muscle glucose uptake in obese subjects after blockade of ETAR (Lteif et al. 2007). It has been also shown that flow‐mediated dilation with ET‐1 receptor antagonism in obese and T2DM yielded similar data to LHCs (Lteif et al. 2007; Shemyakin et al. 2010), indicating that the basal activity of ET‐1 or the basal expression of ET‐1 receptors may be elevated in subjects with insulin resistance. Furthermore, Shemyakin et al. (2010) demonstrated that dual inhibition of ETAR and ETBR during hyperinsulinemia caused greater improvement of skeletal muscle blood flow and glucose uptake in lean, healthy people than insulin infusion alone. Ross et al. (2007) also showed that whereas ET‐1 infusion alone did not induce significant changes in skeletal muscle hemodynamics or glucose metabolism, concomitant infusion of ET‐1 with insulin reduced blood flow and glucose uptake. These data, along with our current findings, provide evidence that normal levels of insulin maintain a balance between the two opposing ET‐1 receptors and that loss of this balance might be a primary feature in the pathogenesis of hyperinsulinemia‐induced vascular dysfunction even in the absence of underlying insulin resistance. Given the inherent nature of human investigations, our study is not without limitations in establishing causality and detailed mechanistic insight. Future studies in transgenic animal models or more elaborate human clinical trials of ET‐1 antagonists are needed. Our observations were performed in a small sample size and the data are underpowered for some variables such as the FID measurements due to difficulty in isolating adequate vessels samples. Also, ex vivo measures of FID or reactivity in isolated arterioles removes biological regulation such as neurohumoral activation and metabolic‐induced vasomotor changes (Young et al. 2010). However, this model is a unique aspect of our study that allowed us to observe a microvascular functional consequence to experimental hyperinsulinemia. Furthermore, it was our intent not to match subjects for age or obesity status in an attempt to establish a disease phenotype against a health reference group. We acknowledge that the T2DM disease phenotype is complex and that additional confounding factors that influence vascular function, such as age, obesity, hypertension, hyperglycemia, and inflammation, require both independent and combined study with hyperinsulinemia. In conclusion, hyperinsulinemia is a hallmark of obesity and T2DM. Mechanistic insight into the role of hyperinsulinemia in skeletal muscle microvascular dysfunction during controlled acute hyperinsulinemia may, therefore, help to uncover treatments for patients exhibiting cardiometabolic risk factors. Our study suggests hyperinsulinemia as an independent and primary factor for the disturbed balance among vasoactive mediators and for the development and advancement of microvascular dysfunction. Moreover, this study serves as a first step toward changing the prevailing concepts about hyperinsulinemia, which may promote reevaluation of the current therapeutic approaches for T2DM such as insulin secretagogues and administration of exogenous insulin. Conflicts of Interest No conflicts of interest were disclosed. Acknowledgments The authors would like to thank the research participants and the nursing staff of the Clinical Research Centers of the Cleveland Clinic and the University of Illinois at Chicago. ==== Refs References Arcaro , G. , A. Cretti , S. Balzano , A. Lechi , M. Muggeo , E. Bonora , et al. 2002 Insulin causes endothelial dysfunction in humans: sites and mechanisms . Circulation 105 :576 –582 .11827922 Bradley , S. J. , B. A. Kingwell , B. J. Canny , and G. K. 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==== Front Physiol RepPhysiol Rep10.1002/(ISSN)2051-817XPHY2physreportsPhysiological Reports2051-817XJohn Wiley and Sons Inc. Hoboken 2753548210.14814/phy2.12900PHY212900Blood PressureRenal Conditions, Disorders and TreatmentsGastrointestinal, Hepatic and Pancreatic PhysiologyEndocrine and Metabolic Conditons, Disorders and TreatmentsEndocrine GlandsOriginal ResearchOriginal ResearchOxidative stress increases the risk of pancreatic β cell damage in chronic renal hypertensive rats S. Gao et al.Gao Shan 1 2 † Park Byung M. 1 † Cha Seung A. 1 † Bae Ui J. 3 Park Byung H. 3 Park Woo H. 1 Kim Suhn H. 1 1 Department of PhysiologyChonbuk National University Medical SchoolJeonjuKorea2 Department of PharmacologyTaishan Medical UniversityShandongChina3 Department of BiochemistryResearch Institute for Endocrine SciencesChonbuk National University Medical SchoolJeonjuKorea* Correspondence Suhn Hee Kim, 2‐20 Keum‐Am‐Dong‐San, Department of Physiology, Chonbuk National University Medical School, Jeonju 561‐180, Korea. Tel: 82 63 270 3078 Fax: 82 63 274 9892 E‐mail: shkim@chonbuk.ac.kr † These authors contributed equally to this manuscript. 17 8 2016 8 2016 4 16 10.1111/phy2.2016.4.issue-16e1290013 7 2016 21 7 2016 25 7 2016 © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.Abstract Hypertension often occurs in conjunction with insulin resistance. The purpose of this study was to evaluate whether sustained renal hypertension increases the risk of diabetes mellitus in rats, and to define the underlying mechanisms. Two‐kidney, one‐clip hypertensive (2K1C) rats received captopril (50 mg/kg/day), α‐lipoic acid (100 mg/kg/day), or vehicle treatment for 3 months after surgery. Blood pressure was measured by tail cuff plethysmography. Oral glucose tolerance test (OGTT), immunohistochemistry, and western blotting were performed. In addition, insulin secretion from islet cells was measured. OGTT yielded abnormal results, and the number of islet cells and the size of pancreatic β/α cells were decreased in 2K1C rats. Basal insulin levels were also reduced in the plasma. Insulin secretion from pancreatic islet cells in response to high glucose was also attenuated in 2K1C rats compared with sham rats. The levels of oxidative stress markers, including 8‐hydroxydeoxyguanosine and NADPH oxidase‐4, were increased in pancreatic tissue and pancreatic islets in 2K1C rats. The abnormalities observed in 2K1C rats were improved by captopril or α‐lipoic acid treatment. These findings indicate that sustained renal hypertension may lead to pancreatic dysfunction, increasing oxidative stress in pancreatic islets. 8‐hydroxydeoxyguanosineadvanced glycation end productsangiotensin IIdiabetesH2O2hypertensionNADPH oxidase‐4oxidative stressβ cellNational Research Foundation of Korea2008‐0062279 source-schema-version-number2.0component-idphy212900cover-dateAugust 2016details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_NLMPMC version:4.9.4 mode:remove_FC converted:29.08.2016 S. Gao , B. M. Park , S. A. Cha , U. J. Bae , B. H. Park , W. H. Park , S. H. Kim . Oxidative stress increases the risk of pancreatic β cell damage in chronic renal hypertensive rats . Physiol Rep , 4 (16 ), 2016 , e12900, doi: 10.14814/phy2.12900 Funding Information This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2008‐0062279). ==== Body Introduction Hypertension and cardiovascular diseases are the leading causes of morbidity and mortality (Kannel and Wilson 1995). Hypertension often occurs in conjunction with insulin resistance (Carlsson et al. 1998) and other components of the cardiometabolic syndrome (Cersosimo and DeFronzo 2006). Angiotensin II (Ang II) is the major peptide hormone of the renin–angiotensin system (RAS) and plays a pivotal role in the pathogenesis of hypertension and insulin resistance (Richey et al. 1999; Ran et al. 2006). Studies have indicated that Ang II inhibits insulin activity in vascular and skeletal muscle tissues, in part, by interfering with insulin signaling through the phosphatidylinositol 3‐kinase and protein kinase B pathway (Ogihara et al. 2002; Sowers 2004). Ang II also suppresses adiponectin production, which impairs insulin sensitivity (Ran et al. 2006). Insulin resistance promotes hypertension by up‐regulating vascular Ang II type 1 receptor (AT1R) (Nickenig et al. 1998). Most of the studies were performed in animals suffering with diabetic mellitus as a causal factor of hypertension. However, there is a few evidence that hypertension causes an impairment of insulin secretion leading to diabetes mellitus. Conen et al. (2007) reported hypertensive patients showing an approximately 3.3‐fold increased risk of new‐onset diabetes, relative to nonhypertensive patients. This study seems to be the first report showing hypertension to become a causal factor of diabetes mellitus. It has been indicated by experimental evidence that oxidative stress plays an important role in the pathophysiology of hypertension and target organ damage in hypertension, especially focusing on atherosclerosis, heart disease, renal failure, and cerebrovascular disease (Dinh et al. 2014; Rubattu et al. 2015; Sinha and Dabla 2015). Oxidative stress induces modification of DNA, protein, and lipid peroxidation, which contribute to the pathogenesis of various diseases (Nickenig and Harrison 2002). Ang II signaling promotes the production of reactive oxygen species (ROS) via NADPH oxidase (NOX) in adipose tissue, skeletal muscle, cardiovascular tissue (Nickenig and Harrison 2002; Sowers 2002, 2004), and the pancreas (Chan and Leung 2011). Tikellis et al. reported that components of RAS are expressed in the pancreas and that the RAS blockade has beneficial effects on pancreatic structure and function (Tikellis et al. 2004). Moreover, Ang II suppresses glucose‐induced insulin secretion (Fliser et al. 1997) and reduces blood flow to islets (Carlsson et al. 1998). These findings suggest that oxidative stress induced by the Ang II signaling pathway is one of the causal factors connecting hypertension to the impairment of insulin secretion and/or production leading to diabetic condition. We hypothesized that hypertension‐induced oxidative stress may be a predisposing factor for diabetes. To test this hypothesis, oral glucose tolerance test (OGTT) was performed in two‐kidney one‐clip (2K1C) rats, 5 weeks, 3 months, and 6 months after surgery (postop 6‐month). The fasting blood glucose (FBS) level in 2K1C rats tended to be high without significance and the impairment of OGTT was more prominent in postop 6‐month 2K1C rats (Table 1). Therefore, we examined the morphology, number, and insulin secretion of islets in postop 6‐month 2K1C rats. Table 1 Fasting blood sugar, area under curve during oral glucose tolerance test, plasma hormone levels in sham and two‐kidney one‐clip hypertensive rats Sham 5 weeks 3 months 6 months FBS (mg/dL) 96.0 ± 8.3 112.3 ± 5.9 102.4 ± 5.9 104.6 ± 8.0 AUC of OGTT (mg/dL/3 h) 367.2 ± 20.5 385.4 ± 25.2 415.4 ± 25.6 435.7 ± 17.2* PAng II (pg/mL) 43.9 ± 5.5 925.5 ± 65.6* 824.2 ± 219.3 56.0 ± 15.7 PAldo (pg/mL) 235.0 ± 70.5 1454.6 ± 165.6* 925.2 ± 89.3* 168.4 ± 74.9 Values are mean ± SEM of sham rats and two‐kidney one‐clip hypertensive rats (5 weeks, 3 months, and 6 months after surgery) (n = 4–8). FBS, fasting blood sugar; AUC of OGTT, area under the curve of oral glucose tolerance test; PAng II, PAldo, plasma levels of angiotensin II and aldosterone levels, respectively. *versus Sham rats, P < 0.05. John Wiley & Sons, LtdMethods Animals and treatments Male Sprague–Dawley (SD) rats aged 5–6 weeks were purchased from Orient Bio (Seoungnam, Korea) and were housed in a temperature‐controlled room with a 12:12‐h light–dark cycle. The animals had free access to standard laboratory chow (5L79 Purina rat & mouse 18% chow; Charles River Laboratories, Wilmington, MA) and water. All experimental protocols conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication No. 85–23, revised 1996) and were approved by Chonbuk National University Medical School. Following an acclimatization period of 3 days, surgery was performed to make 2K1C hypertensive rats. Animals were anesthetized with a mixture of ketamine and xylazine (9:1, 2 mL/kg), then a silver clip (0.2 mm ID) was inserted on the left renal artery (Yuan et al. 2008). Sham rats received the same treatment except for placement of silver clips. Systolic blood pressure (SBP) was measured once a week by tail cuff plethysmography (Power Lab 2/20, AD instruments, Australia), and rats with SBP >140 mmHg were considered hypertensive and used for experiments. Three months after surgery, 2K1C rats were randomly divided into three groups: group 1 rats received vehicle (2K1C‐vehicle rats, n = 13), group 2 rats received captopril (Ang II‐converting enzyme inhibitor, 50 mg/kg/day, n = 10) (2K1C‐captopril rats), and group 3 rats received α‐lipoic acid (antioxidant, 100 mg/kg/day, n = 10) (Yu et al. 2012) (2K1C‐lipoic acid rats) orally for 3 months. Sham rats were maintained for 6 months after surgery as a control group (n = 7). OGTT and plasma hormone levels A 50% glucose solution (2.0 g/kg) was orally administered to overnight‐fasted rats. Blood was sequentially collected from the tail vein before the administration of glucose and 0.5, 1, 2, and 3 h after glucose administration. Blood glucose levels were measured using a hand‐held glucometer (Accucheck, Roche Diagnostic, Basel, Switzerland). Levels of insulin, Ang II, and aldosterone were measured in the plasma using an insulin ELISA kit (Millipore, St. Charles, Missouri), Ang II ELISA kit (Enzo Life Sciences, Plymouth Meeting, PA), and aldosterone radioimmunoassay kit (DiaSorin, Northwestern Avenue Stillwater, MN), respectively. Glucose‐induced insulin secretion by islet cells in vitro Pancreatic islets were isolated from sham and 2K1C rats as described previously (Kim et al. 2007). Rats were anesthetized with a mixture of ketamine and xylazine, then the common bile duct was cannulated. After cannulation, the bile duct was infused with 50 ml of collagenase solution (0.15 mg/mL collagenase P [Roche, Indianapolis, IN] in Hanks’ balanced salt solution supplemented with 1 g/L glucose and 0.2 g/L bovine serum albumin [BSA]). After infusion, the pancreas was removed and incubated in a water bath at 37°C for 30 min with gentle shaking every 10 min. The digested pancreas was washed with Tris‐HCl with Tween (TBST), and the islets were separated by centrifugation over a discontinuous Ficoll gradient. Isolated pancreatic islets were washed three times in Krebs–Ringer bicarbonate buffer (KRB) (25 mmol/L HEPES, 115 mmol/L NaCl, 24 mmol/L NaHCO3, 5 mmol/L KCl, 1 mmol/L MgCl2, 2.5 mmol/L CaCl2, 3 mmol/L D‐glucose, and 0.1% BSA, pH 7.4). Islets were incubated fivefold in 1.5 mL Eppendorf tube (10 islets/tube) in KRB buffer with 5.5 mmol/L or 20 mmol/L glucose at 37°C for 60 min. Finally, the islet samples were centrifuged and supernatant was collected for insulin assays. Immunohistochemistry of pancreatic islet cells Pancreatic tissue was fixed in 10% formalin overnight and then embedded in paraffin. Ten 4‐μm sections were cut from each tissue block (n = 5 for each group). For immunohistochemistry, the tissue sections were blocked with peroxidase blocking agent for 5 min and washed with TBST, then blocked with protein blocking serum‐free buffer (DAKO, Carpinteria, CA) for 5 min. Sections were incubated in primary antibodies against insulin (Santa Cruz Biotechnology, Dallas, TX), glucagon (Bioworld, St. Louis Park, MN), NOX‐4 (Abcam, Cambridge, MA), or 8‐hydroxydeoxyguanosine (8‐OHdG; Abcam) for 2 h at room temperature. After washing with TBST, samples were then incubated in secondary antibodies (DAKO) at room temperature for 30 min, then the chromogen was added. All sections were H&E stained and observed at 200× magnification with a light microscope (Nikon, Tokyo, Japan). Islet areas and entire section areas were measured using iSolution DT 36 software (Carl Zeiss, Oberkochen, Germany) (Ka et al. 2015). Measurement of pancreatic oxidative stress markers Rats were killed and pancreatic tissue was removed and stored at −80°C until use. To measure oxidative stress, pancreatic tissue was cut into small pieces, homogenized, and M‐PER lysed in T‐PER mammalian protein extraction reagent (Thermo Scientific, Rockford, IL) according to the manufacturer's instructions. Hydrogen peroxide (H2O2) and Mn‐superoxide dismutase (Mn‐SOD) were measured in pancreatic tissue using ELISA assay kits (Enzo Life Sciences). Advanced glycation end products (AGE), 8‐OHdG, and oxygen radical antioxidant capacity (ORAC) were measured using ELISA assay kits (Cell Biolabs, San Diego, CA). For western blot analysis, pancreatic tissue was cut into small pieces, homogenized, and lysed using T‐PER mammalian protein extraction reagent (Thermo Scientific). After separation by gradient SDS‐polyacrylamide gel electrophoresis, proteins were transferred to an Immobilon‐polyvinylidene fluoride membrane and blocked with TBST containing 5% skim milk. The membranes were incubated with primary antibodies against AT1R, AT2R (Sigma‐Aldrich, St. Louis, MA), Mn‐SOD (Stressgen Biotechnologies, Victoria, BC), NOX‐4 (Santa Cruz Biotechnology), and GAPDH (Sigma‐Aldrich). Proteins were detected by incubation in horseradish peroxidase‐conjugated secondary antibodies (Zymed, South San Francisco, CA) at room temperature for 1 h. Immunoreactivity was detected using chemiluminescence and analyzed quantitatively using Image J (NIH, Bethesda, MD) (Shah et al. 2012). Statistical analysis Results are presented as means ± SEM. Statistical significance of the differences was assessed using analysis of variance followed by the Bonferroni multiple comparison test. Student's t test was also used. The statistical significance was set at P < 0.05. Results OGTT in 2K1C rats During the first 2 months after surgery, there was no difference in body weight between sham and 2K1C rats (data not shown). After the third month, the body weight of 2K1C rats was significantly reduced compared with sham rats (429.7 ± 32.3 g vs. 492.7 ± 12.2 g, P < 0.05). 2K1C rats with a SBP of >140 mmHg were allocated to the hypertensive group. The SBP of hypertensive rats gradually increased up to 190 mmHg and was maintained at that level throughout the experiment (190.0 ± 12.1 mmHg, n = 17 vs. 123.2 ± 0.8 mmHg, n = 9, P < 0.01). The levels of glucose and insulin in response to glucose administration in postop 6‐month rats are shown in Figure 1. The blood glucose level after 8 h fasting (FBS) was not different between 2K1C and sham rats (Fig. 1Aa). The blood glucose levels were substantially increased in 2K1C and sham rats 30 min after glucose administration (Fig. 1Aa). The elevated glucose level of 2K1C rats was slightly reduced 3 h after glucose administration, while the elevated glucose level significantly decreased to basal levels in sham rats. Similarly, the glucose area under the curve (AUC) of 2KlC rats was higher than that of sham rats. Glucose administration increased the plasma insulin level of sham rats, but the insulin level of 2K1C rats did not significantly change (Fig. 1Ab). Basal plasma insulin levels were lower in 2K1C rats than sham rats (Fig. 1Ba). There were no significant differences in plasma Ang II (Fig. 1Bb) and aldosterone levels (Fig. 1Bc) between the two groups. In postop 5‐week and 3‐month 2K1C rats, the FBS levels tended to be high without significance and the impairment of OGTT was not prominent even though plasma levels of Ang II and aldosterone were higher than sham rats (Table 1). Therefore, we examined the morphology, number, and insulin secretion of islets in postop 6‐month 2K1C rats. Figure 1 (A) Blood glucose level (left), area under the curve of glucose disposal (AUC, right) (a), and changes in plasma insulin level (b) during the glucose tolerance test (OGTT) in sham and 2K1C rats. (B) Comparison of basal plasma levels of insulin (a), angiotensin (Ang) II (b), and aldosterone (c) in both groups. (C) Comparison of the total number of islets labeled with an insulin antibody (a), ratio of the size of β/α cells (b), and glucose‐stimulated insulin secretion (c) in both groups. Pancreatic α and β cells were labeled with primary antibodies against glucagon and insulin, respectively. The number of islets was calculated as the islet number divided by the size of the pancreatic tissue. Insulin secretion was measured using isolated islets incubated with 5.5 or 20 mmol/L glucose at 37°C for 1 h. Data are presented as mean ± SEM of 7–9 rats in each group. 2K1C, two‐kidney, one‐clip hypertensive rats; sham, age‐matched sham rats. Bar indicates 100 μm. *versus sham rats, *P < 0.05, **P < 0.01. Change in pancreatic islets in 2K1C rats To evaluate the function of pancreatic β cells, the number of pancreatic islets and size of pancreatic α and β cells were determined using immunohistochemistry. As shown in Figure 1C, the number of Langerhans islets (Fig. 1Ca) and the size of β/α cells (Fig. 1Cb) were markedly decreased in 2K1C rats. In addition, glucose‐stimulated insulin secretion by β cells was also reduced in 2K1C rats (20 mmol/L) compared with sham rats (Fig. 1Cc). Effect of captopril or α‐lipoic acid treatment on SBP, glucose tolerance, and plasma hormone levels in 2K1C rats To evaluate whether pancreatic β‐cell dysfunction is caused by hypertension or oxidative stress, postop 3‐month 2K1C rats received vehicle, captopril, or α‐lipoic acid treatment for 3 months. The SBP was lower in 2K1C‐captopril rats than 2KIC‐vehicle rats (Fig. 2A). Treatment with α‐lipoic acid did not have a significant effect on SBP (Fig. 2A). The basal blood glucose level was similar after 8 h fasting and the blood glucose level increased significantly after glucose administration in all groups. The peak of glucose increase after glucose administration was not different. However, blood glucose levels and the AUC during OGTT were significantly lower 3 h after glucose administration in 2K1C‐captopril rats and 2K1C‐lipoic acid rats compared with 2K1C‐vehicle rats (Fig. 2B). Basal insulin levels in the plasma were also higher in 2K1C‐captopril and 2K1C‐lipoic acid rats compared with 2K1C‐vehicle rats (Fig. 2Ca). However, there were no significant differences in plasma Ang II (Fig. 2Cb) and aldosterone levels (Fig. 2Cc) among the four groups. Figure 2 Changes in systolic blood pressure (SBP) (A) and blood glucose level (B) during OGTT in sham and 2K1C rats treated with vehicle, captopril, or α‐lipoic acid. (C) Basal plasma levels of insulin (a), Ang II (b), and aldosterone (c) among four groups. Data are presented as mean ± SEM of 7–10 rats in each group. α‐LA, α‐lipoic acid. *versus sham rats, P < 0.05; #versus 2K1C rats administered with vehicle, # P < 0.05, ## P < 0.01. Morphological changes of islets in 2K1C rats after vehicle, captopril, or α‐lipoic acid treatment Figure 3A shows the morphological changes in pancreatic islets of 2K1C rats after water, captopril, or α‐lipoic acid administration compared with sham rats. The number of pancreatic islets and the size of pancreatic β/α cells were decreased in 2K1C rats, and this was improved by captopril or α‐lipoic acid treatment (Fig. 3A). Quantification of pancreatic islets and pancreatic β/α cell size confirmed a significant improvement after treatment with captopril or α‐lipoic acid in 2K1C rats (Fig. 3Ba and Bb). Ex vivo, glucose‐stimulated insulin secretion (20 mmol/L) by isolated islet cells was improved to control levels in 2K1C‐captopril rats and 2K1C‐lipoic acid rats (Fig. 3Bc). Figure 3 (A) Representative pancreatic islets stained with H&E and immunohistochemistry for insulin (β cell) and glucagon (α cell) from sham and 2K1C rats treated with vehicle, captopril, or α‐lipoic acid. (B) Quantification of the total number of islets (a), the ratio of β/α‐cell size (b), and high‐glucose‐induced insulin secretion (c) using iSolution DT 36 software. Values are expressed as mean ± SEM of seven rats in each group. Bar indicates 100 μm. *versus sham group, *P < 0.05, **P < 0.01; #versus 2K1C rats fed vehicle, # P < 0.05, ## P < 0.01. Effect of vehicle, captopril, or α‐lipoic acid treatment on oxidative stress in the pancreas of 2K1C rats To evaluate oxidative stress in pancreatic tissues, Ang II, H2O2, Mn‐SOD, AGEs, 8‐OHdG, and ORAC levels were measured. The level of H2O2 in pancreatic tissues increased in 2K1C‐vehicle rats but not in 2K1C‐captopril and 2K1C‐lipoic acid rats (Fig. 4B). Ang II and Mn‐SOD levels did not differ significantly among the four groups (Fig. 4A). AGE formation is increased by hyperglycemia; therefore, AGEs are implicated in diabetes and pancreatic β‐cell dysfunction (Schleicher et al. 1997). The AGE level was significantly higher in pancreatic tissue of 2K1C rats compared with sham rats (Fig. 4D) and there was a positive relationship between AGE levels and SBP (y = 0.067x − 5.92, r = 0.77, P < 0.001 for sham and 2K1C rats) (Fig. 4G). 8‐OHdG is a biomarker of oxidative DNA damage and was also higher in pancreatic tissue of 2K1C rats compared with sham rats (Fig. 4E). There was a positive correlation between 8‐OHdG levels and SBP (y = 0.05x − 1.14, r = 0.69, P < 0.05) (Fig. 4H). The ORAC was lower in pancreatic tissue of 2K1C rats (Fig. 4F), and there was no significant correlation between ORAC levels and SBP (Fig. 4I). Captopril or α‐lipoic acid treatment reduced AGE and 8‐OHdG levels (Fig. 4D and E) and enhanced ORAC levels in the pancreas (Fig. 4F). Figure 4 Changes in pancreatic levels of Ang II (A), hydrogen peroxide (B), Mn‐SOD (C), AGE (D), 8‐OHdG (E), and ORAC (F) of sham and 2K1C rats treated with water, captopril, or α‐lipoic acid. Correlation between SBP and AGE (G), 8‐OHdG (H), or ORAC level (I). Data are expressed as mean ± SEM of six rats in each group. Ang II, angiotensin II; Mn‐SOD, Mn‐superoxide dismutase; AGE, advanced glycation end product; 8‐OHdG, 8‐hydroxydeoxyguanosine; ORAC, oxygen radical antioxidant capacity. *versus sham group, P < 0.05; #versus 2K1C rats fed vehicle, P < 0.05. Figure 5 shows changes in AT1R, AT2R, NOX‐4, and SOD expression in the pancreatic tissue of sham and 2K1C rats. AT1R (Fig. 5Ba) and NOX‐4 (Fig. 5Bc) expression was increased in the pancreatic tissue of 2K1C rats, and this was reduced by captopril or α‐lipoic acid treatment. However, no significant changes in AT2R (Fig. 5Bb) and Mn‐SOD (Fig. 5Bd) expression were observed. Pancreatic islets were immunostained with antibodies against the oxidative stress markers NOX‐4 and 8‐OHdG (Fig. 6). NOX‐4 and 8‐OHdG staining was increased in pancreatic islets of 2K1C‐vehicle rats and reduced to control levels in 2K1C‐captopril and 2K1C‐lipoic acid rats. Figure 5 (A) Western blot showing AT 1R, AT 2R, NOX‐4, and Mn‐SOD protein expression in sham and 2K1C rats treated with vehicle, captopril, or α‐lipoic acid. (B) Quantitative analysis of western blotting using Image J. Values are expressed as mean ± SEM of 5–7 rats in each group. AT1R, AT2R, angiotensin II type 1 and type 2 receptor, respectively; NOX 4, NADPH oxidase 4. *versus sham group, P < 0.05; #versus 2K1C rats fed vehicle, # P < 0.05, ## P < 0.01. Figure 6 (A) Pancreatic islets immunostained with antibodies against insulin, NOX‐4, or 8‐OHdG in sham and 2K1C rats treated with vehicle, captopril, or α‐lipoic acid. (B) Quantification of insulin (a), NOX‐4 (b), and 8‐OHdG (c) staining in pancreatic islets Islet areas and entire section areas were measured using iSolution DT 36 software. Values are expressed as mean ± SEM of five rats in each group. Bar indicates 100 μm. *versus sham group, *P < 0.05, **P < 0.01; #versus 2K1C rats fed vehicle, # P < 0.05, ## P < 0.01. Discussion We have used a hypertension rat model to demonstrate that sustained hypertension induces abnormal islet morphology and insulin secretion in response to high glucose and increases oxidative stress in the pancreas. These effects were reversed by captopril or α‐lipoic acid treatment. In addition, we observed a close correlation between blood pressure and oxidative stress in pancreatic tissue. These findings suggest that sustained hypertension increases oxidative stress (ROS generation), thereby altering the structure and function of islets (e.g., β cells). Hypertension is an important underlying symptom of cardiometabolic syndrome (Cersosimo and DeFronzo 2006). Ang II causes insulin resistance by interfering with insulin signaling (Ogihara et al. 2002; Sowers 2004) and impairs insulin sensitivity by suppressing adiponectin production (Ran et al. 2006). 2K1C rats are an established model for early stage Ang II‐dependent hypertension (Morishita et al. 1991). Postop 6‐month 2K1C rats had abnormal OGTT results and insulin response after glucose loading. Moreover, the number and size of islets in pancreas of 2K1C rats and the size of β/α cells were markedly reduced. High‐glucose‐induced insulin secretion from 2K1C islets was significantly attenuated compared with sham rats. To determine whether hypertension or Ang II is responsible for islet dysfunction, Ang II levels were measured in the plasma. The plasma Ang II level was increased from 43.9 ± 5.51 to 925.5 ± 65.6 pg/mL and 824.2 ± 219.3 pg/mL on postop 5‐week and postop 3‐month 2K1C rats, respectively, and then declined to control level (Table 1). Ang II and aldosterone levels were no different between sham and postop 6‐month 2K1C rats. Morishita et al. (Morishita et al. 1991) reported that in 2K1C rats 4 weeks after clipping, the plasma renin and angiotensin II levels were significantly higher than those in sham‐operated rats. Sixteen weeks after clipping, the plasma renin and angiotensin II levels in 2K1C rats were not significantly higher than those in sham‐operated controls. It has been suggested that the roles of the RAS in maintenance of hypertension in 2K1C rats differ in the acute and chronic phases (Morishita et al. 1991). Therefore, it is possible that the early increase in Ang II levels (i.e., postop 3‐month) could induce substantial damage that was not observed until a later time point. These findings suggest that early increase in Ang II or aldosterone may be partly involved in an impairment of insulin secretion during hypertension and that the duration of hypertension is more important for pancreatic islet dysfunction. Even though RAS is not activated in essential hypertension, captopril still decreases BP. These data suggest that extrarenal RAS may be more important in the regulation of BP (Morishita et al. 1991; Romero and Reckelhoff 2000; Dinh et al. 2014; Rubattu et al. 2015; Sinha and Dabla 2015). To define whether pancreatic β‐cell dysfunction is caused by high blood pressure or oxidative stress, the rats were treated with ACE inhibitor (captopril) or antioxidant (α‐lipoic acid). Treatment with captopril or α‐lipoic acid improved OGTT results and increased the size and number of pancreatic islets. Treatment also enhanced high‐glucose‐induced insulin secretion. H2O2 was increased in the pancreas and NOX‐4 expression was higher in pancreatic β cells of 2K1C rats; both were decreased by treatment with captopril or α‐lipoic acid. Our data are partly consistent with the report showing a role of NOX‐4 in ventricular H2O2 generation during pulmonary hypertension (Tyagi et al. 2005). Ang II activates NOX‐4, increases production of superoxide, and activates MAPKs in rat pancreatic islets (Alves et al. 2012). However, Ang II levels were not different among groups on postop 6 month. Therefore, Ang II in early stage and/or other factors in late stage of 2K1C rats may induce oxidative stress in the pancreatic β‐cell dysfunction. Although blood pressure was not reduced by α‐lipoic acid treatment, α‐lipoic acid may reduce oxidative stress in the pancreas and restore pancreatic islet function. These results are consistent with previous findings that α‐lipoic acid reduces H2O2‐induced apoptosis in INS‐1 cells and isolated islets (Lee et al. 2009) and improves hyperglycemia through antioxidative properties (Midaoui et al. 2003). Therefore, improving pancreatic β‐cell dysfunction may be necessary to reduce oxidative stress and blood pressure in 2K1C rats. Oxidative stress is a physiological condition and is enhanced by an imbalance between ROS and antioxidants. Therefore, it is important to evaluate ROS levels and antioxidant capacity. AGEs (a source of ROS) and 8‐OHdG (a biomarker of oxidative DNA damage) are associated with age‐related diseases, such as diabetes mellitus (Schleicher et al. 1997) (Dandona et al. 1996) and hypertension (Negishi et al. 1999). In addition to monitor the effects of oxidative stress by the measurement of ROS products, it is also important to evaluate the antioxidant capacity (ORAC). Fruits, vegetables, and low‐fat diet have beneficial effect on BP, serum cholesterol concentrations, and insulin sensitivity by inducing ORAC and suppressing oxidative stress (Botero et al. 2009). A higher ORAC level (antioxidant capacity) is associated with lower risk of hypertension in type 2 diabetes (Rodrigo et al. 2011). In this study, the levels of AGE and 8‐OHdG were substantially increased in the pancreas of 2K1C rats. Moreover, enhanced AGE and 8‐OHdG levels correlated positively with SBP and captopril or α‐lipoic acid treatment decreased AGE or 8‐OHdG levels. ORAC was reduced in the pancreas of 2K1C rats and this was reversed by treatment with α‐lipoic acid or captopril. Our data are partly consistent with other findings that sustained hypertension stimulates AGE generation and increases 8‐OHdG levels followed by deterioration of pancreatic islet cells, especially β cells (Luciano Viviani et al. 2008; Puddu et al. 2010; Gonzalez et al. 2014). Taken together, these results indicate that an imbalance between ROS generation and antioxidant capacity in the pancreas of 2K1C rats causes damage to islet cells. In conclusion, our findings indicate that sustained hypertension increases pancreatic oxidative stress followed by pancreatic β‐cell dysfunction. The relevance of this study in clinical area is that if a person with high blood pressure does not recognize or take antihypertensive drugs during certain period, pancreatic β cells may be disrupted and diabetes mellitus will be combined. To prevent the progression of diabetes in hypertension, early treatment of hypertension with antihypertensive drugs and/or antioxidants may be necessary. 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==== Front Physiol RepPhysiol Rep10.1002/(ISSN)2051-817XPHY2physreportsPhysiological Reports2051-817XJohn Wiley and Sons Inc. Hoboken 2753548310.14814/phy2.12903PHY212903Cardiovascular Conditions, Disorders and TreatmentsEndurance and PerformanceSkeletal MuscleOriginal ResearchOriginal ResearchComparison of regional skeletal muscle tissue oxygenation in college athletes and sedentary control subjects using quantitative BOLD MR imaging M. R. Stacy et al.Stacy Mitchel R. 1 Caracciolo Christopher M. 1 Qiu Maolin 2 Pal Prasanta 2 Varga Tyler 1 Constable Robert Todd 2 3 Sinusas Albert J. 1 2 1 Department of Internal MedicineYale University School of MedicineNew HavenConnecticut2 Department of Radiology and Biomedical ImagingYale University School of MedicineNew HavenConnecticut3 Department of NeurosurgeryYale University School of MedicineNew HavenConnecticut* Correspondence Mitchel R. Stacy, Section of Cardiovascular Medicine, Yale University School of Medicine, Dana 3, P.O. Box 208017, New Haven, CT 06520‐8017. Tel: 203‐737‐5917 Fax: 203‐737‐1030 E‐mail: mitchel.stacy@yale.edu 17 8 2016 8 2016 4 16 10.1111/phy2.2016.4.issue-16e1290322 7 2016 28 7 2016 © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.Abstract Blood oxygen level‐dependent (BOLD) magnetic resonance (MR) imaging permits noninvasive assessment of tissue oxygenation. We hypothesized that BOLD imaging would allow for regional evaluation of differences in skeletal muscle oxygenation between athletes and sedentary control subjects, and dynamic BOLD responses to ischemia (i.e., proximal cuff occlusion) and reactive hyperemia (i.e., rapid cuff deflation) would relate to lower extremity function, as assessed by jumping ability. College football athletes (linemen, defensive backs/wide receivers) were compared to sedentary healthy controls. BOLD signal of the gastrocnemius, soleus, anterior tibialis, and peroneus longus was assessed for peak hyperemic value (PHV), time to peak (TTP), minimum ischemic value (MIV), and time to recovery (TTR). Significantly higher PHVs were identified in athletes versus controls for the gastrocnemius (linemen, 15.8 ± 9.1%; defensive backs/wide receivers, 17.9 ± 5.1%; controls, 7.4 ± 3.5%), soleus (linemen, 25.9 ± 11.5%; backs/receivers, 22.0 ± 9.4%; controls, 12.9 ± 5.8%), and anterior tibialis (linemen, 12.8 ± 5.3%; backs/receivers, 12.6 ± 3.9%; controls, 7.7 ± 4.0%), whereas no differences in PHV were found for the peroneus longus (linemen, 14.1 ± 6.9%; backs/receivers, 11.7 ± 4.6%; controls, 9.0 ± 4.9%). In all subject groups, the gastrocnemius and soleus muscles exhibited the lowest MIVs during cuff occlusion. No differences in TTR were found between muscles for any subject group. PHV of the gastrocnemius muscle was significantly and positively related to maximal vertical (r = 0.56, P = 0.002) and broad jump (r = 0.47, P = 0.01). These results suggest that BOLD MR imaging is a useful noninvasive tool for evaluating differences in tissue oxygenation of specific muscles between active and sedentary individuals, and peak BOLD responses may relate to functional capacity. Blood oxygen level dependentexercise trainingmagnetic resonance imagingtissue oxygenationvascular functionNational Football League (NFL) CharitiesNIHT32 HL098069 source-schema-version-number2.0component-idphy212903cover-dateAugust 2016details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_NLMPMC version:4.9.4 mode:remove_FC converted:29.08.2016 M. R. Stacy , C. M. Caracciolo , M. Qiu , P. Pal , T. Varga , R. T. Constable , A. J. Sinusas . Comparison of regional skeletal muscle tissue oxygenation in college athletes and sedentary control subjects using quantitative BOLD MR imaging . Physiol Rep , 4 (16 ), 2016 , e12903, doi: 10.14814/phy2.12903 Funding Information This research was supported in part by a grant from the National Football League (NFL) Charities (Sinusas). The views expressed in this work are those of the author/s and do not necessarily reflect the views or policies of the NFL or NFL Charities. This work was also supported in part by NIH grant T32 HL098069 (Sinusas). ==== Body Introduction The beneficial effects of exercise training on cardiovascular health have been well documented, and evidence suggests that many of the health‐related benefits associated with exercise are modulated by repetitive increases in vessel wall shear stress, which may result in improved vascular function as well as positive vascular remodeling (Green et al. 2012). Studies evaluating vascular adaptation in response to chronic aerobic exercise have demonstrated that exercise training leads to an increased size of peripheral conduit arteries (Huonker et al. 2003; Schmidt‐Trucksass et al. 2000; Zeppilli et al. 1995), increased microvascular density of skeletal muscle (Cocks et al. 2013), and improved vasodilator capacity of both peripheral (Martin et al. 1987; Sinoway et al. 1986; Snell et al. 1987) and coronary vessels (Hildick‐Smith et al. 2000; Pelliccia et al. 1990). Skeletal muscle is composed of several fiber types, which all possess different contractile and metabolic properties and can be generally classified as slow twitch (ST) and fast twitch (FT) (Engel 1962). In humans, FT fibers are further subdivided into FTa fibers, which are more aerobic (or oxidative), and FTb fibers, which are more anaerobic (or glycolytic) fibers (Brooke and Kasier 1970). A large amount of heterogeneity in muscle fiber type composition can exist between people due to the ability of skeletal muscle tissue to adapt to a multitude of variables, such as exercise training, which can lead to changes in muscle mass, strength, and composition (i.e., increased vascular density), and potentially explain some of the differences in fiber types that exist between athletes who undergo different training programs (Costill et al. 1976; Fink et al. 1977; Saltin et al. 1977). Noninvasive imaging is commonly applied to evaluate the effects of exercise training on skeletal muscle and the peripheral vasculature, with the majority of research directed at the application of ultrasound to assess skeletal muscle blood flow and vascular reactivity (Green et al. 2012). Blood oxygen level‐dependent (BOLD) magnetic resonance (MR) imaging is a noninvasive technique that can be used to evaluate dynamic changes in skeletal muscle oxygenation in response to various stimuli, such as reactive hyperemia (Ledermann et al. 2006a; Schulte et al. 2008; Utz et al. 2005) or muscle contraction (Towse et al. 2011, 2005). BOLD MR image signal intensity is based on relative changes in paramagnetic deoxyhemoglobin and diamagnetic oxyhemoglobin in the microcirculation of skeletal muscle, where an increase in blood oxygen saturation results in an increase in T2*‐weighted BOLD image signal intensity (Ledermann et al. 2006b). Due to the sensitivity of BOLD for detecting dynamic changes in intravascular hemoglobin oxygenation, the BOLD image signal intensity can also be affected by multiple factors, such as blood volume, fluid shifts, metabolic factors, vascular architecture, and magnetic field angulation; however, fluctuations in BOLD signal intensity are believed to be largely dependent on dynamic changes in blood flow within the microcirculation (Jacobi et al. 2012). Changes in BOLD image signal intensity have been shown to correlate well with laser Doppler flow and transcutaneous oxygen pressure (TcPO2) during the assessment of transient ischemia and reactive hyperemia paradigms in the lower extremities (Ledermann et al. 2006b; Partovi et al. 2013, 2014), and is in close agreement with near infrared spectroscopy (NIRS) measures of blood volume and saturation following maximal muscle contractions (Towse et al. 2011). Prior studies evaluating dynamic changes in BOLD signal intensity during reactive hyperemia have demonstrated impaired BOLD responses with increasing age (Kos et al. 2009; Schulte et al. 2008), in the setting of peripheral arterial disease (PAD) (Ledermann et al. 2006a), and in young smokers (Nishii et al. 2015). The coregistration of functional BOLD images with anatomical MR images allows for evaluation of volumetric changes in skeletal muscle oxygenation within lower extremity muscle groups (Ledermann et al. 2006a) or vascular territories of interest (Stacy et al. 2016). Specifically, functional BOLD images have been paired with anatomical images to demonstrate impaired BOLD signal intensity responses to reactive hyperemia in PAD patients within specific calf muscles (Ledermann et al. 2006a), and have also shown significantly higher and more prolonged BOLD responses to muscle contraction in the anterior tibialis muscle of physically active individuals when compared to sedentary counterparts (Towse et al. 2005). However, BOLD MR imaging has not been applied to evaluate which lower extremity muscles undergo, or are most susceptible to, changes in tissue oxygenation following chronic exercise training. Prior work in young athletes suggests that exercise training may not improve vascular function measures above those of sedentary controls due the preexistence of healthy vessels in young individuals; however, prior work in this field has primarily focused on evaluating macrovascular, and not microvascular function (Montero et al. 2014). We hypothesized that BOLD MR imaging during transient ischemia and reactive hyperemia would allow for quantitative assessment of regional volumetric differences in lower extremity skeletal muscle oxygenation at the microvascular level between sedentary healthy control subjects and college football athletes. We also hypothesized that peak hyperemic BOLD responses would relate to lower extremity functional capacity, as assessed by maximal vertical and broad jumping ability. Materials and Methods Research subjects Young healthy men (n = 33) were recruited to participate in the MR research protocol. Of the subjects recruited, 23 were college football athletes of two distinctly different playing positions, which consisted of linemen (n = 12) and defensive backs/wide receivers (n = 11). Linemen, whose main objective in American football is to block and/or push opposing linemen off the line of scrimmage, are characterized by larger body sizes and high levels of upper and lower body strength, which is typically utilized during quick bursts. By contrast, the role of backs and receivers is to advance the football by running with the ball or passing it, or cover opposing players. Therefore, these athletes are characterized by smaller body sizes and are generally much faster to allow for covering or dodging of opposing players. In addition to football athletes, 10 sedentary control subjects were recruited for evaluating differences in skeletal muscle oxygenation between exercise‐trained and ‐untrained individuals, as well as assessment of test–retest repeatability of our BOLD imaging approach. In all subjects, maximal vertical jump was measured using a vertec vertical jump tester, and maximal broad jump was evaluated via the standing long jump test. Body fat composition was also evaluated using a three‐site skin‐fold assessment (abdomen, suprailiac, and triceps), with all measurements taken by an experienced individual. A standard medical history questionnaire was administered to evaluate each subject's risk factors for cardiovascular, pulmonary, and metabolic disease, which included history of smoking, hypertension, diabetes, hypercholesterolemia, and family history of coronary artery disease. Additionally, the International Physical Activity Questionnaire (IPAQ) was administered to evaluate each subject's activity levels for the week prior to study involvement. The study protocol was approved by the Institutional Review Board for Human Subjects Research and Review Committee, and was in accordance with the guidelines set forth by the Declaration of Helsinki. Subjects reported for imaging following an 8‐h fast that also consisted of abstinence from caffeine and alcohol. All individuals provided written informed consent after receiving an explanation of the experimental procedures and potential risks associated with participating in this study. Exercise training program College football athletes recruited for the research protocol were actively involved in a structured exercise training program, which consisted of a resistance training and plyometric regimen 3 days per week, in addition to full on‐field practice sessions 5–7 days per week. Specifically, resistance training exercises that involved the lower extremities consisted of compound, dynamic full‐body barbell lifts that involved multiple joints, including the hips, knees, and ankles. Lifts consisted of the back squat (once/week, 5 sets of 2–5 repetitions), hang clean (twice/week, 3 sets of 1–2 repetitions), and clean pulls (twice/week, 5 sets of 2 repetitions). All lifts activated a large range of lower extremity muscles, including but not limited to the quadriceps, glutes, adductors, hamstrings, soleus, and gastrocnemius muscles. In addition to barbell lifts, athletes performed hamstring curls (once/week, 5 sets of 10 repetitions). Plyometric exercises consisted of two sets each of bounding exercise and the triple broad jump. MR imaging protocol Sedentary control subjects reported for MR imaging on two separate occasions for repeatability testing of our quantitative BOLD analyses, with each visit being separated by at least 1 week. All athletes reported for a single visit. Resting blood pressure and heart rate measurements were manually acquired prior to the start of each imaging session. All subjects underwent BOLD MR imaging of the dominant leg at the level of the mid‐calf on a 3 Tesla MR System (TIM Trio; Siemens Healthcare, Erlangen, Germany) using spine and body phased array coils (Siemens Healthcare, Erlangen, Germany). The leg was firmly secured to restrict movement. A blood pressure cuff was secured around the thigh for rapid inflation (ischemia) and deflation (reactive hyperemia) using a Hokanson E20 AG101 Rapid Cuff Inflation System (D.E. Hokanson, Inc; Bellevue, WA). Serial BOLD images were acquired over 15 min, with the first 5 min representing baseline assessment, followed by a 5 min occlusion phase (cuff pressure inflated to 50 mmHg above resting systolic pressure), and 5 min of reactive hyperemia following cuff deflation. BOLD imaging parameters were as follows: 10 mm effective slice thickness (i.e., six slices with a slice thickness of 5 mm and a distance factor of 100%), FOV = 360 × 180 mm2, in‐plane matrix size = 128 × 64, TR=3000 ms, TE = 40 ms, and flip angle = 90°. Following each BOLD acquisition, coregistered high‐resolution anatomical images were acquired with a T1‐FLASH (i.e., fast low‐angle shot) gradient echo sequence to guide segmentation of calf muscles of interest. T1‐FLASH imaging parameters were as follows: 1.4 mm slice thickness, FOV = 320 × 320 mm2, in‐plane matrix size = 256 × 256, TR = 9.83 ms, TE = 4.9 ms, and flip angle = 10°. Image analysis Volumes of interest (VOIs) were generated from BOLD MR images of the calf with the guidance of coregistered T1‐FLASH anatomical images, which assisted with identification and manual segmentation of calf muscles. This image segmentation approach has been previously applied in our laboratory for analysis of BOLD responses in the foot (Stacy et al. 2016), as well as lower extremity single photon emission computed tomography/computed tomography (SPECT/CT) perfusion imaging (Stacy et al. 2014). All image segmentation and analysis was performed using BioImage Suite (http://www.bioimagesuite.org), an image analysis toolkit. Time‐course data were generated to evaluate dynamic changes in BOLD signal intensity for each calf muscle VOI. Image intensity values were expressed as a percent change from resting baseline signal intensity (i.e., image intensity value – baseline SI ÷ baseline SI). Four parameters were evaluated during transient ischemia and reactive hyperemia. Parameters included: (1) minimum ischemic value (MIV); (2) peak hyperemic value (PHV), defined as the highest 3‐second average value recorded following cuff deflation; (3) time to peak (TTP), which refers to the time from cuff deflation to peak BOLD signal; and (4) time to recovery (TTR), which was the time required to return to baseline BOLD signal intensity. Test–retest repeatability was evaluated for each parameter within all calf muscle VOIs in sedentary control subjects. Statistical analyses Analysis of variance (ANOVA) was used to identify differences in BOLD signal intensity responses between each calf muscle VOI (four factors: gastrocnemius, soleus, anterior tibialis, and peroneus longus) as well as between‐subject groups (three factors: controls, linemen, and receivers/backs). Normality was assessed using the D'Agostino and Pearson omnibus normality test. Paired analysis was performed for evaluating differences in BOLD parameters between study visits in the control group. Pearson's correlation coefficient was used to assess the relation between maximal jumping ability (maximal broad and vertical jump) and peak hyperemic value. Two control subjects were excluded from correlation analysis due to missing data related to jumping ability, and three athletes were excluded from analysis as outliers. Normality was assessed using the D'Agostino and Pearson omnibus normality test. All statistical analyses were performed using commercially available software (GraphPad Prism v6.0 for Mac OS X, GraphPad Software, La Jolla, CA). Statistical significance for all analyses was set at P < 0.05. All values are expressed as means ± SD unless stated otherwise. Results Subject demographics Subjects were separated into three groups: linemen, defensive backs/wide receivers, and sedentary controls. Subject group characteristics are summarized in Table 1. Linemen presented with significantly greater body weight, height, body fat composition, and systolic blood pressure when compared to backs/receivers and control subjects. Control subjects presented with significantly higher resting heart rate values than both linemen and backs/receivers. Control subjects also possessed lower diastolic blood pressure when compared to both athlete subject groups. In the assessment of maximal jumping ability, backs/receivers demonstrated significantly greater vertical and broad jumping ability when compared to both linemen and control subjects, and linemen possessed significantly greater broad jumping ability compared to control subjects. Table 1 Subject characteristics Linemen (n = 12) Backs/Receivers (n = 11) Controls (n = 10) Age, years 20.9 ± 1.0 20.6 ± 1.2 21.8 ± 2.2 Body Weight, kg 119.2 ± 6.6b 86.9 ± 7.2 80.2 ± 16.2a Height, cm 191.5 ± 4.8b 180.1 ± 11.2 179.7 ± 5.3a Body composition, % body fat 23.5 ± 3.1b 10.7 ± 2.4 N/A Resting HR, bpm 63.2 ± 7.5 57.8 ± 8.1 76.3 ± 8.9a , b Systolic BP, mmHg 138.8 ± 12.0b 128.0 ± 9.5 119.7 ± 12.8a Diastolic BP, mmHg 82.2 ± 8.3 78.7 ± 6.7 69.9 ± 11.0a , b Maximum vertical jump, cm 56.6 ± 6.5b 72.8 ± 5.1 50.7 ± 8.1b Maximum broad jump, cm 239.3 ± 12.8b 273.2 ± 12.3 201.6 ± 32.3a , b N/A, not available. All values are means ± SD. a Significantly different from linemen. b Significantly different from backs/receivers (P < 0.05). John Wiley & Sons, LtdThe IPAQ revealed no differences in the average weekly amount of moderate and vigorous intensity exercise between athlete subject groups, as well as similar weekly totals for hours spent walking and sitting between athlete groups (Table 2). Control subjects significantly differed from both athlete groups with regard to the amount of time performing moderate and vigorous intensity exercise, as well as the average number of hours spent per week sitting. Table 2 International physical activity questionnaire responses Linemen (n = 12) Backs/Receivers (n = 11) Controls (n = 10) Vigorous exercise, h/week 27.7 ± 8.4 28.0 ± 8.3 0.3 ± 0.7a , b Moderate exercise, h/week 6.5 ± 5.8 9.0 ± 12.0 0.5 ± 1.3a , b Walking, h/week 10.3 ± 7.6 9.4 ± 8.2 8.8 ± 10.1 Sitting, h/week 25.7 ± 13.5 27.6 ± 11.9 70.4 ± 28.8a , b All values are means ± SD. a Significantly different from linemen. b Significantly different from backs/receivers (P < 0.05). John Wiley & Sons, LtdTest–retest repeatability Analysis of quantitative BOLD indices between study visits in sedentary control subjects demonstrated repeatability of PHV, TTP, MIV, and TTR measures for each muscle group of interest (Table 3). Table 3 Test–Retest repeatability in sedentary control subjects Visit 1 CV (%) Visit 2 CV (%) P‐value Peak hyperemic value (%) Gastrocnemius 7.4 ± 3.5 47.1 6.2 ±3.0 48.2 0.4 Soleus 12.9 ± 5.8 45.0 10.4 ±4.6 44.0 0.4 Anterior Tibialis 7.7 ± 4.0 52.1 8.0 ±3.9 48.6 0.9 Peroneus Longus 9.2 ± 5.1 55.2 6.7 ±1.8 26.2 0.2 Minimum ischemic value (%) Gastrocnemius −13.3 ± 4.3 32.7 −10.2 ± 3.1 30.1 0.1 Soleus −12.4 ± 6.4 51.7 −10.4 ± 3.7 36.1 0.4 Anterior Tibiatis −7.5 ± 3.4 45.5 −6.1 ± 2.1 34.9 0.4 Peroneus Longus −9.4 ± 5.5 58.8 −6.3 ± 1.8 29.5 0.2 Time‐to‐Peak (sec) Gastrocnemius 28.5 ± 5.6 19.5 29.3 ± 15.9 20.3 0.7 Soleus 21.0 ± 2.8 13.2 21.4 ± 7.9 37.1 0.9 Anterior Tibialis 26.3 ± 5.0 19.1 28.6 ± 9.5 33.2 0.4 Peroneus Longus 25.1 ± 6.6 26.3 29.3 ± 8.3 28.4 0.3 Time‐to‐Recovervy (sec) Gastrocnemius 129.4 ± 28.5 22.1 134.6± 37.8 28.1 0.6 Soleus 134.1 ± 21.5 16.0 139.5 ± 55.6 39.8 0.7 Anterior Tibialis 136.5 ± 40.2 29.4 153.4 ± 52.7 34.3 0.5 Peroneus Lougus 140.6 ± 48.4 34.4 124.9 ± 30.2 24.2 0.5 CV, coefficient of variation All values are means ± SD. N = 10 subjects. John Wiley & Sons, LtdBOLD analysis between‐subject groups Analysis of dynamic changes in BOLD signal between‐subject groups within specific muscle groups (gastrocnemius, soleus, anterior tibialis, and peroneus longus) revealed regional variability in the time‐course responses to cuff occlusion and reactive hyperemia (Fig. 1). Specifically, significantly higher PHVs were identified in both athlete groups for the gastrocnemius, soleus, and anterior tibialis muscle groups when compared to control subjects. No significant differences were found between any subject groups in evaluating the PHV of the peroneus longus (Fig. 2). Additionally, no significant differences were found between‐subject groups for all other evaluated BOLD indices (i.e., MIV, TTP, TTR; data not shown). Figure 1 Regional assessment of dynamic changes in skeletal muscle tissue oxygenation during cuff occlusion and reactive hyperemia. (A) Anatomical T1‐FLASH images were used to identify and guide segmentation of lower extremity calf muscles. (B) Muscle groups of interest were segmented on T2*‐weighted BOLD images to generate (C) averaged dynamic time‐course data in each muscle group for each of the three subject groups. Arrows indicate time of cuff inflation (initialization of ischemia) and deflation (start of reactive hyperemia phase). Plotted data represents average values for all subjects within each group. Figure 2 Evaluation of peak hyperemic value between‐subject groups. All values are means ± SD. *significantly different from linemen. †significantly different from backs/receivers (P < 0.05). BOLD analysis between skeletal muscle groups All quantitative BOLD indices were assessed and compared across each muscle group of interest (Fig. 3). In the assessment of PHV in control subjects, the soleus muscle demonstrated a significantly higher PHV than all other muscle groups (Fig. 3A). The soleus muscle also demonstrated a significantly higher PHV than all other muscle groups in the linemen. Additionally, the gastrocnemius of linemen exhibited a higher PHV than the anterior tibialis muscle group. In the receivers/backs subject group, both the gastrocnemius and soleus muscles exhibited higher PHVs than the peroneus longus and anterior tibialis muscles. Figure 3 Regional evaluation and comparison of quantitative BOLD indices between calf muscles of interest. All values are means ± SD. *significantly different from gastrocnemius. †significantly different from soleus. ‡significantly different from peroneus longus (P < 0.05). In the assessment of TTP for the hyperemic value of control subjects and receivers/backs, the soleus muscle demonstrated a significantly faster TTP when compared to all other muscle groups (Fig. 3B). In the linemen subject group, the soleus and peroneus longus muscles both exhibited a significantly faster TTP than the gastrocnemius muscle group. Evaluation of MIV demonstrated a similar trend between muscle groups for all three subject groups, where the gastrocnemius and soleus muscles on average exhibited the lowest MIVs during the cuff occlusion phase of our BOLD imaging protocol when compared to the peroneus longus and anterior tibialis muscles (Fig. 3C). In the evaluation of TTR following reactive hyperemia, no significant differences were found between any muscle groups within any of the subject groups (Fig. 3D). Relationship between BOLD response and jumping ability The PHV of the gastrocnemius muscle was significantly and positively related to both maximal vertical (r = 0.56; P = 0.002) and broad jump (r = 0.47; P = 0.01; Fig. 4). No other significant relationships were found between muscle‐specific BOLD responses and maximal jumping ability. Figure 4 Relationship between peak hyperemic value of the gastrocnemius muscle and maximal vertical and broad jump. Peak hyperemic BOLD response in the gastrocnemius was significantly and positively related to both (A) vertical and (B) broad jumping ability. N = 28 subjects. Discussion In this study, we have demonstrated for the first time the utility of BOLD MR imaging for evaluating differences in peak tissue oxygenation of lower extremity skeletal muscles between athletes and sedentary healthy control subjects by using a cuff occlusion and reactive hyperemia paradigm (Fig. 2). In addition to noninvasively identifying significant differences in regional microvascular tissue oxygenation between‐subject groups of varying physical activity levels, we have demonstrated significantly different vascular responses between muscle groups within the same‐subject groups (Fig. 3). Furthermore, we have identified a significant relationship between peak reactive hyperemia of the gastrocnemius muscle and maximal jumping ability, indicating that BOLD MR imaging may provide a noninvasive tool for tracking serial improvements in lower extremity function. Previous studies have found higher BOLD responses in physically active versus sedentary control subjects following 1‐sec maximal voluntary contractions (Towse et al. 2005), and that peak BOLD responses after maximal muscle contraction are also significantly correlated with physical activity level (active vs. inactive) (Towse et al. 2011). However, prior studies evaluating the effects of physical activity on the BOLD signal have only assessed the anterior tibialis muscle in response to a single maximal muscle contraction paradigm. Given that research subjects could display differences in muscle contraction patterns, as well as the possibility for preexisting differences in vascular density between lower extremity muscle groups, we sought to evaluate a reactive hyperemia paradigm that would be capable of generating a more diffuse hyperemic BOLD response across all muscles of the calf in an effort to assess muscle group‐specific differences in tissue oxygenation between athletes and sedentary subjects. Using our reactive hyperemia approach to assess regional BOLD responses, we found that both athlete groups (linemen and backs/receivers) possessed higher PHVs than sedentary control subjects in the gastrocnemius, soleus, and anterior tibialis muscle groups, with no significant differences in PHV between athletes and sedentary controls for the peroneus longus muscle (Fig. 2). This finding suggests that specific lower extremity muscles may be more susceptible to exercise‐induced changes in tissue oxygenation and increased vascular density. These significant differences in peak BOLD responses between muscle groups also stresses the value of assessing multiple muscle groups in future investigations evaluating skeletal muscle adaptation to exercise training. In addition to comparing BOLD responses between athletes and sedentary controls, we found no significant differences between linemen and backs/receivers for any of the BOLD indices of vascular function. This finding is not too surprising given the similarities in physical activity levels of our athlete groups, as well as prior studies suggesting that exercise training‐induced changes in vascular function may be less apparent in younger athletes (Montero et al. 2014). In the evaluation of muscle‐specific differences in our quantitative BOLD indices, the soleus muscle was found to have a higher PHV than other muscle groups, a finding that persisted in both athletes and sedentary control subjects (Fig. 3A). This finding is in agreement with prior studies (Ledermann et al. 2006a; Schulte et al. 2008) using similar reactive hyperemia paradigms and may be indicative of inherently high levels of vascular density in the soleus muscle group. In support of this argument, the soleus muscle also revealed a trend toward possessing a faster TTP than other muscle groups (Fig. 3B), which may be due to greater intravascular filling space to accommodate hyperemic blood flow, and thus suggestive of higher vascular density. In addition to demonstrating differences in PHV and TTP, the soleus, along with the gastrocnemius muscle, exhibited the lowest MIVs during the cuff occlusion phase of the imaging protocol (Fig. 3C). This finding indicates that the soleus and gastrocnemius muscles may be most susceptible to dramatic changes in tissue ischemia in the setting of acute arterial occlusion. However, in spite of the regional heterogeneity displayed across muscle groups during cuff occlusion and reactive hyperemia, no significant differences in TTR were found between muscles (Fig. 3D), suggesting that no differences in functional recovery exist between muscle groups. Future studies evaluating dynamic changes in BOLD signal during cuff occlusion and reactive hyperemia may be elucidated by incorporation of time‐of‐flight imaging or contrast‐enhanced MR angiography to provide complementary information related to tissue vascularization, and thus offer an explanation for the regional variations and adaptation in skeletal muscle oxygenation within athletes exposed to chronic exercise. In evaluating the relationship between maximal vertical and broad jumping ability and peak BOLD response during reactive hyperemia (i.e., PHV), we found a significant and positive relationship between PHV of the gastrocnemius muscle and jumping ability, which existed for both vertical (Fig. 4A) and broad jump (Fig. 4B). However, the PHV of other muscle groups did not demonstrate a similar relationship with regard to jumping ability. This finding may be related to the important role that the gastrocnemius muscle is thought to play in vertical jumping exercise (van Soest et al. 1993), and may also be associated with exercise‐induced changes in fiber‐type composition of the gastrocnemius. Future application of BOLD MR imaging in patients undergoing exercise therapy for improved lower extremity function could provide opportunities for better evaluating this relationship and elucidate the functional relationship between peak vascular responses of individual muscles and functional capacity. 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==== Front Physiol RepPhysiol Rep10.1002/(ISSN)2051-817XPHY2physreportsPhysiological Reports2051-817XJohn Wiley and Sons Inc. Hoboken 10.14814/phy2.12904PHY212904Genetic Conditions Disorders and TreatmentsEndurance and PerformanceDigestive Conditions, Disorders and TreatmentsNutritionRespiratory Conditions Disorder and DiseasesLungCase ReportCase ReportsExercise capacity following a percutaneous endoscopic gastrostomy in a young female with cystic fibrosis: a case report O. W. Tomlinson et al.Tomlinson Owen W. 1 2 Barker Alan R. 1 Oades Patrick J. 2 Williams Craig A. 1 2 1 Children's Health and Exercise Research CentreSport and Health ScienceUniversity of ExeterExeterU.K2 Royal Devon and Exeter NHS Foundation Trust HospitalExeterU.K* Correspondence Craig A. Williams, Children's Health and Exercise Research Centre, Sport and Health Sciences, College of Life and Environmental Sciences, University of Exeter, St Luke's Campus, Exeter EX1 2LU, UK. Tel: 44 (0)1392‐724890 E‐mail: c.a.williams@exeter.ac.uk 22 8 2016 8 2016 4 16 10.1111/phy2.2016.4.issue-16e1290425 5 2016 25 7 2016 28 7 2016 © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.Abstract Cystic fibrosis (CF) is a genetic condition affecting the respiratory and gastrointestinal systems, with patients experiencing problems maintaining weight, especially during rapid growth periods such as puberty. The aim of this case report was to monitor the effect of gastrostomy insertion and implementation of overnight supplemental feeding upon clinical outcomes, including body mass index (BMI), lung function (FEV 1), and exercise‐related variables (maximal oxygen uptake [VO 2max] and ventilatory efficiency [VE/VO 2]) in an 11‐year‐old female with CF. Combined incremental and supramaximal exercise testing to exhaustion was performed at four time points: 3 months prior to the procedure (T1), 2 days prior to (T2), 4 months (T3), and 1 year following the procedure (T4). Improvements following gastrostomy insertion were observed at the 1 year follow‐up with regards to BMI (+20%); whereas absolute VO 2max remained stable and lung function fluctuated throughout the period of observation. Declines in function with regards to body weight relative VO 2max (−16.3%) and oxygen uptake efficiency (+7.5%) were observed during this period. This case report is the first to consider exercise‐related clinical outcomes in assessing the effect of implementing gastrostomy feeding in CF. The varied direction and magnitude of the associations between variables shows that further investigations are required. Adolescenceexercise testingrespiratory diseaserisk factors source-schema-version-number2.0component-idphy212904cover-dateAugust 2016details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_NLMPMC version:4.9.4 mode:remove_FC converted:29.08.2016 O. W. Tomlinson , A. R. Barker , P. J. Oades , C. A. Williams . Exercise capacity following a percutaneous endoscopic gastrostomy in a young female with cystic fibrosis: a case report . Physiol Rep , 4 (16 ), 2016 , e12904, doi: 10.14814/phy2.12904 Funding Information No funding information provided. ==== Body Introduction Cystic fibrosis (CF) is a genetically inherited, life‐shortening disease characterized by respiratory and digestive problems which manifests in a decreased exercise capacity (Saynor et al. 2014) and malnutrition (Panagopoulou et al. 2014). Increased mortality risk is reported when patients exhibit decreased lung function and poor nutritional status (Liou et al. 2001). However, exercise‐related predictors of mortality, including maximal oxygen uptake (VO2max) (Pianosi et al. 2005a) and peak ventilatory equivalent ratio for oxygen (a measure of ventilatory efficiency; VE/VO2) (Hulzebos et al. 2014), are also reported in this patient group. Patients with CF are encouraged to increase their exercise levels (Swisher et al. 2015) and daily caloric intake (Stallings et al. 2008) to improve clinical outcomes, in line with clinical practice guidelines (Cystic Fibrosis Trust, 2011). However, nutritional targets are not always met despite a high level of calorie intake relative to non‐CF controls (Woestenenk et al. 2014). When patients fail to gain weight as predicted and conservative dietary interventions fail, invasive support through the insertion of a percutaneous endoscopic gastrostomy (PEG) may be required. This procedure has been shown to improve nutritional status (Williams et al. 1999) and stabilize lung function (Bradley et al. 2012). Exercise testing is a valuable tool for evaluating interventions and profiling the clinical status of patients with CF (Cystic Fibrosis Trust, 2011), but has yet to be utilized to assess the effectiveness of this procedure. Therefore, this case report is the first to describe exercise‐related changes alongside nutritional status and lung function following a PEG implant, and supplemental feeding, in a pediatric patient with CF, over a 15‐month period. Patient Information The subject of this case report, an 11‐year‐old female, presented at birth with meconium ileus requiring surgery and was subsequently confirmed to have CF (sweat chloride >60 mmol L−1 and homozygous for the ΔF508 mutation). Her clinical course through childhood was complicated by Pseudomonal and Staphylococcal chest infections as well as relapsing Allergic Broncho‐Pulmonary Aspergillosis, treated with recurrent courses of antibiotics, inhaled mucoactives (DNase and hypertonic saline), corticosteroids, antifungals, and chest physiotherapy (autogenic drainage and oscillating PEP). She developed impaired glucose tolerance at age 9, then CF‐related diabetes requiring insulin treatment at 10 years of age (as shown in Fig. 1). Figure 1 Changes in body mass index as measured by percentile in the 3‐year period preceding the procedure and 1 year following. Dashed line at 10.9 years indicates diagnosis of CFRD. Dotted lines at 11.8, 12.1, 12.4, and 13.1 years indicate T1, T2, T3, and T4, respectively. Percutaneous endoscopic gastrostomy inserted 2 days after T2. During the 15‐month period reported in this study, the patient was unstable (as shown by FEV1 in Fig. 2), and underwent 22 days of intravenous antibiotic treatment. Figure 2 Changes in predicted FEV 1 (○) (r = −0.64) and absolute VO 2max (●) (r = 0.40) over the 15‐month observation window of this case report. Four vertical lines indicate T1–T4. Percutaneous endoscopic gastrostomy inserted 2 days after T2. Timeline Changes in clinically important measures of exercise performance over a 15‐month period were assessed for this report, with anthropometric and lung function data provided for the 3 years prior to the procedure and 1 year following. Exercise testing was conducted at scheduled clinical appointments, corresponding to four time points: 3 months preprocedure (T1); 2 days preprocedure (T2); 4 months postprocedure (T3), and 1 year postprocedure (T4). A fall in body mass index (BMI) from the 64th to 5th percentile in 10 months (Fig. 1) prompted the need to investigate weight gain methods, after conservative dietary changes (visiting the patient's school, meetings with parents, and introduction of twice daily Enshake® drinks) failed. Her growth failed to respond to these noninvasive nutritional supplements, leading to consideration of overnight supplemental feeding via a PEG. Diagnostic Assessment Anthropometric measures Height was measured to 0.1 cm (Holtain wall‐mounted stadiometer, Crymych, Wales) and body weight to 0.1 kg (Seca electronic column scale, Birmingham, UK), with BMI compared to normative percentiles (de Onis et al. 2007). Lung function Lung function was assessed using a hand‐held spirometer (MicroPlus, Micro Medical Ltd, Rochester, UK), with maximal (best of three) values of forced expiratory volume in 1 sec (FEV1), forced vital capacity (FVC), and FEV1/FVC ratio being recorded and compared to normative values (Quanjer et al. 2012). Exercise parameters The patient exercised on an electronically braked cycle ergometer (Lode Excalibur Sport; Lode, Groningen, The Netherlands), completing a validated (Saynor et al. 2013a) combined ramp‐incremental and supramaximal test to exhaustion to determine VO2max and gas exchange threshold (Saynor et al. 2014). Measures of VO2max were normalized to a percentage of predicted maximum (Bongers et al. 2014). The same work rate (15 W·min−1), warm‐up, and recovery timings were used across all tests. Pulmonary gas exchange was assessed with a calibrated metabolic cart (Cortex Metalyzer 3B; Cortex Medical, Leipzig, Germany). Blood oxygen saturation (SpO2) was measured throughout the test (Nellcor N‐20; Medtronic, Minneapolis, MN) and subjective ratings of perceived effort and dyspnea were assessed on a 1–10 scale. Therapeutic Intervention A PEG tube was inserted under general anesthetic into the stomach, as described previously (Russell et al. 1984). Overnight supplemental feeding with 500 mL of Fresubin® HP Energy (630 kJ/150 kcal) was subsequently introduced. Composition of the feed (per 100 mL) was as follows: 7.5 g protein (20% total energy); 17 g carbohydrate (45%); and 5.8 g fat (35%). This volume avoided interference with morning appetite and minimized vomiting risk with physiotherapy. Follow‐Up and Outcomes Anthropometric outcomes Anthropometric and pulmonary outcomes are shown in Table 1. From T1 to T2, increases in height (+1.7 cm), but a fall in body weight (0.6 kg), resulted in a decrease in BMI by 0.63 kg·m−2 (−8.6 percentile points). Following the PEG procedure (T3), increases in height (+1.5 cm) and body weight (+3.5 kg), resulted in a gain of 11.7 BMI percentile points (+1.26 kg·m−2). At the 1‐year (T4) follow‐up, height had increased by 3.2 cm relative to T2, as had body weight (+9.2 kg) and BMI (+3.02 kg·m−2), resulting in an increase to the 38th percentile for BMI. Table 1 Changes in anthropometric and lung function measures over the 15‐month observation period Variable T1 (3M‐Pre) T2 (2D‐Pre) T3 (4M‐Post) % Change from T2 to T3 T4 (1Y‐Post) % Change from T2 to T4 Date 11 June 2014 19 September 2014 19 January 2015 21 September 2015 Age (years) 11.83 12.10 12.44 2.8 13.11 8.4 Height (cm) 146.6 148.3 149.8 1.0 153.0 3.2 Height (percentile) 29.7 30.2 28.6 −5.3 29.0 −4.0 Weight (kg) 33.9 33.3 36.8 10.5 42.5 27.6 BMI (kg·m−2) 15.77 15.14 16.40 8.3 18.16 20.0 BMI (percentile) 14.9 6.3 18.0 185.7 38.1 504.8 FVC (L) 1.67 2.06 2.17 5.3 2.23 8.3 FVC (% predicted) 64.3 76.5 78.3 2.4 75.2 −1.7 FEV1 (L) 1.61 1.84 1.76 −4.4 1.58 −14.1 FEV1 (% predicted) 69.9 77.1 71.5 −7.3 59.9 −22.3 FEV1/FVC (%) 96.41 89.32 81.11 −9.2 70.85 −20.7 Time points: 3M‐Pre (3 months prior to the procedure); 2D‐Pre (2 days prior to the procedure); 4M‐Post (4 months following the procedure); 1Y‐Post (1 year following the procedure). BMI, body mass index; FVC, forced vital capacity; FEV1, forced expiratory volume in 1 sec. John Wiley & Sons, LtdPulmonary outcomes Changes in lung function (Fig. 2) showed large variation across the 15‐month observation period, although an overall trend for a decline in function was evident (r = −0.64). There was an increase in FEV1 from T1 (69.9%) in the lead up to the procedure (T2; 77.1%), before declining at the subsequent observations, T3 (71.5%) and T4 (59.9%). Exercise outcomes Exercise‐related measures are listed in Table 2. Time to exhaustion increased by 49% across all trials (T1–T4), with an 18% increase observed at the 1‐year follow‐up (T2–T4). Absolute VO2max fluctuated over the 15‐month period, decreasing from T1 (1.15 L·min−1) to T2 (1.09 L·min−1), before increasing at T3 (1.18 L·min−1) and T4 (1.15 L·min−1). When VO2 was expressed relative to body mass a decrease was observed over the 1‐year follow‐up period (−16.3% from T2 to T4), a change associated with the observed weight gain. When normalized for body weight VO2max decreased as a percentage of predicted from 79.3% (T2) to 66.0% (T4). Table 2 Changes in exercise‐related parameters over the 15‐month observation period Variable T1 (3M‐Pre) T2 (2D‐Pre) T3 (4M‐Post) % Change from T2 to T3 T4 (1Y‐Post) % Change from T2 to T4 Peak power (W) 84 98 101 3.1 115 17.4 Exercise duration (min) 4 min 43 sec 5 min 58 sec 6 min 17 sec 5 7 min 3 sec 18 VO2max (L·min−1) 1.15 1.09 1.18 8.3 1.16 6.4 VO2max (mL·kg−1·min−1) 33.8 32.6 32.2 −1.2 27.3 −16.3 VO2max (L·min−1; % predicted) 63.8 59.0 62.0 5.1 57.6 −2.4 VO2max (mL·kg−1·min−1; % predicted) 82.4 79.3 78.1 −1.5 66.0 −16.8 VCO2 (L·min−1) 1.16 1.25 1.43 14.4 1.50 20.0 RER 1.01 1.15 1.21 5.2 1.29 12.2 VE (L·min−1) 40.06 55.78 62.70 12.4 63.80 14.4 VE/VO2 34.83 51.17 53.14 3.8 55.00 7.5 VE/VCO2 34.53 44.62 43.85 −1.7 42.53 −4.7 HRmax (beats·min−1)a 196 – 175 – GET (L·min−1) 0.77 0.71 0.73 2.8 0.65 −8.5 GET (% VO2max) 67 65 62 −4.6 56 −13.9 SpO2 96 98 94 −4.1 96 −2.0 RPE 5 6 6 0 4 −33.3 RPD 4 3 4 33.3 4 33.3 VO2max, maximal oxygen uptake; VCO2, maximal carbon dioxide production; RER, respiratory exchange ratio (VCO2/VO2); VE, minute ventilation; VE/VO2, peak ventilatory equivalent ratio for oxygen; VE/VCO2, ventilatory equivalent for carbon dioxide; HRmax, maximal heart rate; GET, gas exchange threshold; SpO2, arterial oxygen saturation; RPE, rating of perceived effort; RPD, rating of perceived dyspnea. a HRmax only available for two tests due to equipment malfunction. John Wiley & Sons, LtdChanges were observed in relation to VE/VO2, with a large increase seen between T1 (34.83) and T2 (51.17). Further, but smaller, increases were then observed at T3 (53.14, +3.8%) and T4 (55.00, +7.5%), relative to T2. VE/VCO2 decreased over the 1‐year follow‐up (T2 = 44.62, T4 = 42.53; −4.7%), although the magnitude of change was not as large as that of VE/VO2. Ventilation (VE) increased from T2 (55.78 L·min−1) to T4 (63.80 L·min−1; +14.4%). Discussion This case report shows the inclusion of exercise‐related factors among short‐term fluctuations in clinical measures, following the insertion of a gastrostomy and implementation of overnight feeding in a young CF patient. For this patient to have been considered a ‘normal’ BMI (i.e., 50th percentile), she was required to weigh 39.75 kg at T2. At T4, this requirement was 44.25 kg. The difference in required and achieved weight at T4 (1.75 kg) relative to T2 (6.45 kg) has justified the requirement of the PEG and supplemental feeding. Such gains are in accordance with prior gastrostomy feeding studies, which have shown similar increases in body weight (Levy et al. 1985) and BMI (Truby et al. 2009). Although lung function has not been shown to increase following a gastrostomy, studies have shown stabilizing of function (Williams et al. 1999; Bradley et al. 2012). However, these studies only present lung function data at distant time points following such interventions (e.g., 1 year) and do not provide serial measurements, which may bias assessment of intervention efficacy, dependent on the patients function at the time of clinical review. This case reports all clinical visits (averaging every 41 days; range 7–62 days) over the 15‐months follow‐up period and show large fluctuation and a trend for decline in lung function. As shown in Figure 2, absolute VO2max remained stable over the 15 months, despite the fluctuating FEV1, highlighting the independence between the two outcomes. As exercise‐related factors can be predictors of mortality (Pianosi et al. 2005a) and indicators of disease severity (Thin et al. 2002) when they are very low, it is therefore important to incorporate such factors in assessing progression of disease alongside FEV1 and BMI. Changes in absolute VO2max were minimal and fall within the typical error associated with the CPET over the medium term (4–6 weeks; Saynor et al. 2013b) and as such, a minimal change in predicted absolute VO2max at the 1‐year follow‐up (+0.07 L·min−1; 59.0–57.6%; Table 2) was observed. However, as VO2max is highly dependent on body size, changes are routinely expressed relative to body weight, thus resulting in a decrease in body weight relative VO2max, from T2 to T4 (−5.3 mL·kg−1·min−1; 79.3–66.0% predicted; Table 2). This change is greater than previously observed annualized declines (Pianosi et al. 2005b) and the decline in predicted relative VO2max has a greater magnitude of change than the predicted absolute VO2max value. This decline is of particular relevance given its clinical implication (i.e., risk of mortality (Pianosi et al. 2005a) and hospitalization (Pérez et al. 2014)). Although it would normally suggest a deconditioning effect, it could be proposed that the rapid increase in weight (+9.2 kg from T2 to T4, resulting in an increase of 31.8 BMI percentile points) is driving this change and deconditioning is not in fact occurring. However, to appropriately determine and interpret such changes, an accurate assessment of body composition (e.g., skin folds) is required. However, clinical constraints prevented such measures in the current report. Increases in VE without a corresponding increase in VO2max (Table 2) indicate a reduction in the efficiency of gaseous exchange. However, given the increase in VCO2 alongside the increase in VE and the stability of VE/VCO2, it can be suggested that an increase in CO2 release may be driving the change in ventilation. This is supported by the rise in RER from T1 to T4, suggesting an increased ‘non‐metabolic’ increase in CO2 at maximal exercise perhaps due to increased anaerobic metabolism, carbohydrate metabolism, and/or CO2 storage during exercise. The increase in peak power (exercise performance) indicates an increase in muscle power, but as no increase in absolute VO2max was observed, this suggests that oxidative capacity of the muscle was not enhanced and a greater contribution likely originated from anaerobic metabolism. Conclusion This case report has provided novel data combining clinical and exercise measures in a young patient with CF following the implementation of gastrostomy feeding. Of the key measures described, BMI increased, whereas relative VO2max showed a decline due to body weight changes, amid a fluctuating FEV1. Furthermore, absolute VO2max remained stable against a decreased function of VE/VO2. The direct impact of the feeding protocol upon exercise capacity cannot be directly obtained due to the patients’ clinical instability and lack of a control patient. This case report does highlight the utility of exercise and body composition testing in assessing the outcome profile of individual patients following interventions, warranting its further use in the assessment and treatment of CF. Informed Consent The patient upon whom this report is focused was previously involved in a NHS Regional Ethics Committee approved study, examining integrated approaches to exercise in CF. Observations made in this report are part of the study follow‐up, to which patient assented and parent have consented to release of data. 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==== Front Physiol RepPhysiol Rep10.1002/(ISSN)2051-817XPHY2physreportsPhysiological Reports2051-817XJohn Wiley and Sons Inc. Hoboken 2756590310.14814/phy2.12905PHY212905NutritionReproductive Conditions, Disorders and TreatmentsMaternal, Fetal and Neonatal PhysiologyMetabolic PathwaysOriginal ResearchOriginal ResearchIncreasing fetal ovine number per gestation alters fetal plasma clinical chemistry values M. Zywicki et al.Zywicki Micaela 1 Blohowiak Sharon E. 1 Magness Ronald R. 1 2 Segar Jeffrey L. 3 Kling Pamela J. 1 1 Departments of PediatricsUniversity of Wisconsin‐Madison School of Medicine and Public HealthMadisonWIUSA2 Obstetrics and Gynecology Perinatal Research LaboratoriesUniversity of Wisconsin‐Madison School of Medicine and Public HealthMadisonWIUSA3 Department of PediatricsUniversity of Iowa Children's HospitalIowaIAUSA* Correspondence Pamela J. Kling, Department of Pediatrics, Meriter Hospital, University of Wisconsin, 202 S. Park St., Madison, WI 53715, USA. Tel: +608 417‐6236 Fax: +608 417‐6377 E‐mail: pkling@pediatrics.wisc.edu 26 8 2016 8 2016 4 16 10.1111/phy2.2016.4.issue-16e1290508 6 2016 22 7 2016 27 7 2016 © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.Abstract Intrauterine growth restriction (IUGR) is interconnected with developmental programming of lifelong pathophysiology. IUGR is seen in human multifetal pregnancies, with stepwise rises in fetal numbers interfering with placental nutrient delivery. It remains unknown whether fetal blood analyses would reflect fetal nutrition, liver, and excretory function in the last trimester of human or ovine IUGR. In an ovine model, we hypothesized that fetal plasma biochemical values would reflect progressive placental, fetal liver, and fetal kidney dysfunction as the number of fetuses per gestation rose. To determine fetal plasma biochemical values in singleton, twin, triplet, and quadruplet/quintuplet ovine gestation, we investigated morphometric measures and comprehensive metabolic panels with nutritional measures, liver enzymes, and placental and fetal kidney excretory measures at gestational day (GD) 130 (90% gestation). As anticipated, placental dysfunction was supported by a stepwise fall in fetal weight, fetal plasma glucose, and triglyceride levels as fetal number per ewe rose. Fetal glucose and triglycerides were directly related to fetal weight. Plasma creatinine, reflecting fetal renal excretory function, and plasma cholesterol, reflecting placental excretory function, were inversely correlated with fetal weight. Progressive biochemical disturbances and growth restriction accompanied the rise in fetal number. Understanding the compensatory and adaptive responses of growth‐restricted fetuses at the biochemical level may help explain how metabolic pathways in growth restriction can be predetermined at birth. This physiological understanding is important for clinical care and generating interventional strategies to prevent altered developmental programming in multifetal gestation. FetalnutritionovineplacentaHilldale Undergraduate/Faculty Research FellowshipUW Department of PediatricsNIHHL087144HL079020P01 HD38843Meriter Foundation source-schema-version-number2.0component-idphy212905cover-dateAugust 2016details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_NLMPMC version:4.9.4 mode:remove_FC converted:29.08.2016 M. Zywicki , S. E. Blohowiak , R. R. Magness , J. L. Segar , P. J. Kling . Increasing fetal ovine number per gestation alters fetal plasma clinical chemistry values . Physiol Rep , 4 (16 ), 2016 , e12905, doi: 10.14814/phy2.12905 Funding Information Financial Support was provided by Hilldale Undergraduate/Faculty Research Fellowship (MZ), UW Department of Pediatrics (PJK), NIH HL087144 Supplement (PJK), NIH HL079020 (RRM & PJK), P01 HD38843 (RRM), and Meriter Foundation (PJK). ==== Body Introduction More than 3% of human pregnancies carry multiple fetuses (Martin et al. 2010). When uterine space for implantation is limited, intrauterine growth restriction (IUGR) can develop, often resulting in perinatal morbidity and even fetal mortality. Defining the critical point of deteriorating fetal function in IUGR has been of much clinical interest (Cox et al. 1988). Additional research is needed to understand biomarkers of fetal health deterioration in IUGR. This is especially true under the common conditions of uterine space restriction (USR) from either uterine anomalies or multifetal gestation pregnancies. Ovine gestational models have been used to study placental insufficiency and other causes of IUGR, with maternal–fetal blood sampling allowing better understanding of oxygen and nutrient transfer by the placenta (Barry and Anthony 2008). The vascular structures of both human and ovine placenta share many similarities, with an additional benefit of discrete cotyledons in the ovine placenta allowing precise quantification of placental attachment sites (Meyer et al. 2010). However, limited biochemical information is available for the study of USR‐induced growth restriction (Meyer et al. 2010; Meyer‐Gesch et al. 2013). We previously reported that with restricted uterine space for placental implantation, changes in placental efficiency and deficits in placental nutrient delivery cause compensatory adaptations to promote a viable, albeit compromised fetus (Meyer et al. 2010; Meyer‐Gesch et al. 2013); however, these alterations may become maladaptive later in life, altering metabolic pathways and promoting adult diseases, including hypertension and diabetes as described in the Barker hypothesis (Anthony et al. 2003; de Boo and Harding 2006). In addition, studies found that ewes carrying multiple fetuses exhibited decreased birth weight and survival of the lambs (Gootwine et al. 2007). By examining plasma biochemical markers of ovine placental–fetal nutrition, fetal liver enzymes, and placental–fetal excretory function associated with growth restriction, we could identify fetal biomarkers of maladaptive compensation. Understanding these fetal blood biochemical biomarkers may also explain how fetal dysfunction in growth restriction impacts the developmental programming of adult disease and ultimately to aid understanding of potential therapeutic strategies. Although data were also obtained from a cohort at gestational day (GD) 120 and are for reference in the supplementary materials, relationships between fetal number and clinical chemistries at GD130 would better inform underlying physiology because growth arrest became apparent between GD120 and GD130. In an ovine model, it was hypothesized that fetal plasma biochemical values would reflect progressive placental, fetal liver, and fetal kidney dysfunction as the number of fetuses per gestation rose. The aims of this study are to provide fetal plasma biochemical reference values for future research in ovine multifetal gestation and utilize these biochemical values in examining relationships between uterine–placental space restriction and fetal growth. Methods Animals Mixed Western breed ewes previously selected for the Polypay and Booroola genes were obtained from the University of Wisconsin‐Madison farm facility, group‐housed, and fed identical diets that were a mixture of hay and corn silage that met daily NRC feed requirements of pregnant sheep (Meyer et al. 2010; Meyer‐Gesch et al. 2013). The University of Wisconsin‐Madison Research Animal Care and Use Committee of the School of Medicine and Public Health and the College of Agriculture and Life Sciences approved the protocols. To accurately determine gestational age, a synchronization protocol was performed using an intravaginal controlled internal drug release (Progesterone CIDR; 0.3 g; Pfizer, Aukland, New Zealand) for 10–14 days, followed by intramuscular prostaglandin F2 α (15 mg; Pfizer) and equine chorionic gonadotropin (500 IU; EMD Biosciences, San Diego, CA) injections before breeding (Meyer‐Gesch et al. 2013; Sun et al. 2013). Pregnancy was confirmed using ultrasound by GD60. Surgical procedures The study's intent was to investigate the impact of multifetal gestation without any previous procedural manipulation to decrease uterine space. Nonsurvival surgery was performed at GD120 or GD130 (±4 days), with term at GD145. GD120, or 80% gestation, was selected because previous data showed only subtle impact of uterine space restriction with more than one fetus per uterine horn (Meyer et al. 2010). GD130, or 90% gestation, was selected because asymmetric IUGR became apparent with more than one fetus per horn (Meyer et al. 2010; Meyer‐Gesch et al. 2013). After administering sodium pentobarbital and ketamine anesthesia to the pregnant ewe, a uterine incision was performed and simultaneous umbilical artery and vein blood sampling (12 mL) with a 20G needle was performed from each fetus and the collected blood immediately placed in heparinized tubes that were kept on ice. Fetal blood samples were collected from anesthetized ewes without supplemental oxygen, at an estimated time of 25 min from the phenobarbital administration and 15–20 min after the ewe was placed supine on the table. Mean maternal artery pO2 was 55.5 and mean fetal umbilical vein pO2 was 22.4 mmHg. After blood sampling, the ewe and fetuses were euthanized and fetal organ morphometry was obtained. Placental and body growth was assessed by placentome count, fetal body weight (kg), kidney weight (g), liver weight (g), and brain weight (g) (Meyer et al. 2010). Plasma chemistries were assessed after centrifugation of heparinized blood for 10 minutes at 3600 X g at 4°C. A comprehensive metabolic panel was measured within 5 h in the Clinical Chemistry Laboratory at Meriter Laboratories using the Cobas Integra 800 (Roche Pharmaceuticals, Basel, Switzerland). Plasma macronutrients and minerals included glucose (mg/dL), triglycerides (mg/dL), albumin (g/dL), and total protein (g/dL), as well as Alkaline (Alk) Phosphatase (U/L) representing calcium metabolism. Plasma liver enzymes measured include aspartase transaminase (AST) (U/L), gamma‐glutamyl transpeptidase (GGT) (U/L), alanine transaminase (ALT) (U/L), and lactate dehydrogenase (LDH) (U/L). Tests of placental and fetal excretory function included cholesterol (mg/dL), creatinine (mg/dL), BUN (mg/dL), and sodium (mmol/L). Additional methods included plasma iron (μg/dL) (Cobas Integra), iron binding capacity (μg/dL), and transferrin saturation (%) from Pointe Scientific, and hematocrit % (pocH‐100i, Sysmex, Mundelein, IL), see Tables S1 and S2. Normality of distribution was assessed. Nonparametric data were natural log (ln)‐converted to normalize the distribution. Analysis of Variance (ANOVA) with Fisher's post hoc analyses compared fetal organ weight or chemistry values by fetal lamb number per ewe. Simple regression analyses were performed by comparing fetal organ or chemistry values by fetal weight and the highest order relationship models are reported. Data S1 show 95% confidence intervals for parameters. Also included are qualitative directional comparisons of fetal lamb values with human fetal growth restriction at the comparable analogous time points in gestation. “Placental dysfunction” in multifetal gestation was defined as the point when inadequate space for placentomal attachment sufficiently disrupts fetal nutrient delivery resulting in fetal growth restriction, altered nutritional or metabolic parameters, or placental clearance to impact fetal biochemical indices. Although it is typically a label used for postnatal live born lambs, “small for gestational age (SGA)” was defined as <10% percentile for our population, fetal singleton carcass weight within that gestational age group. Results Placental and body growth The multiparous ewes ranged from 2.1 years to 9.79 at delivery, although ewe age was not recorded in a few instances. Mean age of the ewes delivering singletons was 4.54 years, twins 4.48 years, triplets 4.59 years, and quadruplets+ 4.92 years, with no differences in ewe age when compared by fetal number. As the onset of growth restriction fell between GD120–GD130 (Meyer et al. 2010; Meyer‐Gesch et al. 2013), we examined and found that the mean gestational age did not differ between the singleton, twin, and triplet and the quadruplet and quintuplet groups. The 10th percentile for GD130 singleton fetuses in our flock is 2.626 kg and defines SGA. It was found that 10.8% of singletons were SGA, 26.8% of twins, 34.7% of triplets, and 61.5% of quadruplet and quintuplet group were SGA. The figures display data from GD130 data because previously (Meyer et al. 2010; Meyer‐Gesch et al. 2013), in a mixed group that included both surgically reduced uterine space and naturally decreased due to multifetal gestation, growth and clinical chemistry values showed more variability based on fetal number per ewe at GD130. Reference values grouped by natural singletons, twins, triplets, or quadruplet/quintuplets at both GD120 and GD130 are located in Data S1. The number of discrete placentome attachment sites fell as number of fetuses per ewe increased (P < 0.0001), supporting a loss in maternal–fetal connections, (Fig. 1A). At GD130, fetal weight (kg) exhibited a stepwise fall as fetal number per ewe increased, P < 0.0001, (Fig. 1B). Fetal kidney weight at GD130 fell in a graded fashion as fetal number per ewe rose, P < 0.0001 (Fig. 1C). Because fetal weight was so strongly related to fetal number, we utilized fetal weight as the surrogate for space restriction caused by greater fetal number, a direct linear relationship was found between fetal weight and fetal kidney weight at GD130 (P < 0.0001), (Fig. 1D). Kidney weight (g) expressed relative to fetal weight (kg) was unchanged as fetal number per ewe rose. A stepwise fall in fetal liver weight at GD130 was observed as fetal number per ewe rose (P < 0.0001), (Fig. 1E), and was directly related to fetal weight at GD130 (P < 0.0001), (Fig. 1F). Liver weight (g) expressed relative to fetal weight (kg) did not differ as fetal number per ewe rose, until 4 when it was lower than fetal weight (P < 0.01). At GD130, brain weight (g) did not differ until fetal number reached 4 at which point brain weight fell (P < 0.02), but fetal brain weight expressed proportionate to fetal weight (g/kg) exhibited a stepwise rise as fetal number per ewe rose (P < 0.0001), (Fig. 1G), and was directly related to fetal weight (P < 0.0001), (Fig. 1H). Figure 1 Growth Parameters at GD130 on the vertical axis were examined based on fetal number per ewe with S (singletons blue), Tw (twins red), Tr (triplets green), and Q (quadruplets/quintuplets purple) on horizontal axis. Error bars represent Means ± SEM. Lower case letters indicate post hoc differences (s from singletons, tw from twins, tr from triplet, and q from quadruplets/quintuplets). Regression lines follow the same color scheme; S in squares, Tw in circles, Tr in triangles, and Q in diamonds. (A) Placentomes; as fetal number per ewe increased, a stepwise fall in placentome number was seen, P < 0.001. (B) Fetal Weight (kg); as fetal number per ewe increased, a stepwise fall in fetal weight was seen, P < 0.0001. Samples sizes were 14 singletons, 45 twins, 34 triplets, and 8 quadruplets+. (C) Fetal Kidney Weight (g); as fetal number per ewe increased, a stepwise fall in fetal kidney weight was seen, P < 0.0001. Sample sizes were 18 singletons, 58 twins, 42 triplets, and 14 quadruplets+. D. Fetal Kidney Weight (g) and Fetal Weight (kg). Kidney weight was directly related to fetal weight, P < 0.0001. (E) Fetal Liver Weight (g); as fetal number per ewe increased, a stepwise fall in fetal liver weight was seen, P < 0.0001. Sample sizes were 16 singletons, 56 twins, 42 triplets, and 14 quadruplets+. (F) Fetal Liver Weight (g) and Fetal Weight (kg). Liver weight was directly related to fetal weight, P < 0.0001. (G) Fetal Brain Weight (g) Relative to Fetal Weight (kg); Fetal brain weight was relatively spared, with stepwise increase observed when expressed relative to fetal weight, P < 0.0001. Sample sizes were 16 singletons, 52 twins, 40 triplets, and 8 quadruplets+. (H) Fetal Brain Weight (g) and Fetal Weight (kg). Brain weight was directly related with fetal weight, P < 0.0001. Male fetuses weighed more than females (kg) in each fetal number grouping, P < 0.0001, but BMI did not differ by sex in these groupings. Liver and kidneys organ weights (g) expressed relative to fetal weight (kg) did not differ by sex, although these organ weights (g) in twins and triplets were lower in females (P < 0.05). Brain weights (g) did not differ by sex, although brain weights (g) expressed relative to fetal weight (kg) in twins and triplets were lower in males (P < 0.03). Observations on sample source sex differences In clinical chemistry values with paired data, there were no measurable differences between fetal vein and fetal artery. Comparing clinical chemistry values from singletons, twins, triplets, and quads/quints, the only difference by sex was glucose values lower in males when fetal number was at least 4, compared to females in that group. Macronutrients and minerals At GD130, fetal plasma glucose levels fell in a stepwise fashion as total lambs per ewe increased (P < 0.0001), (Fig. 2A). Fetal plasma glucose levels were directly related to fetal weight (P < 0.0001), (Fig. 2B). Fetal plasma triglycerides levels were lower in the quadruplets/quintuplets than twins or triplets (P < 0.015), (Fig. 2C) and were directly related to fetal weight (P < 0.005), (Fig. 2D). Fetal plasma Alk Phosphatase levels, a biomarker of bone calcium metabolism, were lower in triplets and quadruplets/quintuplets (P < 0.002), (Fig. 2E), and were directly related to fetal weight (P < 0.01), (Fig. 2F). Additional measures of fetal iron nutrition are contained in the Data S1. Figure 2 Macronutrients and Minerals at GD130 on the vertical axis were examined based on fetal number per ewe with S (singletons blue), Tw (twins red), Tr (triplets green), and Q (quadruplets/quintuplets purple) on horizontal axis. Error bars represent Means ± SEM. Lower case letters indicate post hoc differences (s from singletons, tw from twins, tr from triplet, q from quadruplets/quintuplets). Regression lines follow the same color scheme, S in squares, Tw in circles, Tr in triangles, and Q in diamonds. (A) Fetal Plasma Glucose levels (mg/dL); as fetal number per ewe increased, a stepwise fall in glucose was seen, P < 0.0001. Sample sizes were 18 singletons, 58 twins, 42 triplets, and 8 quadruplets+. (B) Plasma Glucose Levels (mg/dL) and Fetal Weight (Kg). Glucose was directly related to fetal weight, P < 0.0001. (C) Fetal Plasma Triglyceride levels (mg/dL); triglyceride differed, with lowest values in quads/quints, P < 0.0015. Sample sizes were 18 singletons, 58 twins, 40 triplets, and 8 quadruplets+. (D) Fetal Plasma Triglyceride levels (mg/dL) and Fetal Weight (kg). Plasma triglyceride was directly proportionate to fetal weight, P < 0.005. (E) Fetal Plasma Alkaline (Alk) Phosphatase levels (U/L); Alk Phosphatase was lower in triplets and quadruplets/quintuplets than other groups, P < 0.002. Sample sizes were 18 singletons, 58 twins, 42 triplets, and 8 quadruplets+. (F) Fetal Alk Phosphatase levels (U/L) and Fetal weight (kg). Alk Phosphatase was directly proportionate to fetal weight, P < 0.01. Liver enzyme tests At GD130, fetal plasma AST levels rose in the quadruplet/quintuplet group (P < 0.0001), (Fig. 3A), but were unrelated to fetal weight, (Fig. 3B). Fetal plasma LDH levels fell as fetal number rose (P < 0.0001), (Fig. 3C), and were also directly related to fetal weight (P < 0.025), (Fig. 3D). Figure 3 Liver Enzymes at GD130 on the vertical axis were examined based on fetal number per ewe with S (singletons in blue), Tw (twins red), Tr (triplets green), and Q (quadruplets/quintuplets purple) shown on the horizontal axis. Error bars represent Means ± SEM. Letters indicate post hoc differences from other groups (s from singletons, tw from twins, tr from triplets, q from quadruplets/quintuplets). Regression lines follow the same color scheme; S in squares, Tw in circles, Tr in triangles, and Q in diamonds. (A) Fetal Plasma AST levels (U/L); AST was increased in quadruplets/quintuplets, P < 0.0001. Sample sizes were 14 singletons, 56 twins, 42 triplets, and 14 quadruplets+. (B) Fetal Plasma AST levels (U/L) and Fetal Weight (kg). AST was not related to fetal weight. (C) Fetal Plasma LDH levels (U/L); LDH was lower in triplets and quadruplets/quintuplets, P < 0.0001. Sample sizes were 14 singletons, 56 twins, 36 triplets, and 14 quadruplets+. (D) Fetal LDH levels (U/L) and Fetal weight (kg). LDH was directly related to fetal weight (kg), P < 0.025. Placental and fetal renal excretory function tests Fetal plasma cholesterol levels are known to represent placental clearance from fetal production. At GD130, a stepwise increase in fetal plasma cholesterol was observed as fetal number per ewe rose (P < 0.0001), (Fig. 4A). Fetal plasma cholesterol levels were indirectly related to fetal weight (P < 0.0001), (Fig. 4B). Levels of fetal plasma BUN, recognized to be freely diffusible across the placenta (Robinson and Sprayberry 2009), were higher in triplets and quadruplets/quintuplets, compared to other groups (P < 0.015), (Fig. 4C), and were inversely related to fetal weight (P < 0.0001), (Fig. 4D). Fetal plasma creatinine rose with increasing fetal number per ewe, with twins and triplets similar (P < 0.003), (Fig. 4E). Fetal plasma creatinine levels were inversely related to fetal weight (P < 0.0001), (Fig. 4F). Ratio of fetal/maternal levels of both BUN and creatinine examined the relationship between maternal and fetal excretory function. The ratio of fetal/maternal BUN (Fig. 4G) did not differ by fetal number (P < 0.06), but fetal/maternal plasma creatinine, although similar in singletons and twins, rose in a stepwise fashion in the triplet and quadruplet/quintuplet group (P < 0.0001), (Fig. 4H). Figure 4 Placental and Fetal Excretory Function at GD130 on the vertical axis was examined based on fetal number per ewe with S (singletons in blue), Tw (twins in red), Tr (triplets in green), and Q (quadruplets/quintuplets in purple) shown on the horizontal axis. Error bars represent Means ± SEM. Letters on bar graphs indicate post hoc differences from other groups (s = differs from singletons, tw = differs from twin, tr = differs from triplet, q = differs from quad group). Regressions of growth parameters use the same color scheme, inset with S in squares, Tw in circles, Tr in triangles, and Q in diamonds. (A) Fetal Plasma Cholesterol levels (mg/dL); as fetal number per ewe increased, cholesterol rose, P < 0.0001. Sample sizes were 18 singletons, 58 twins, 42 triplets, and 8 quadruplets+. (B) Plasma Cholesterol Levels (mg/dL) and Fetal Weight (kg). Cholesterol was indirectly related to fetal weight, P < 0.0001. (C) Fetal Plasma Blood Urea Nitrogen (BUN) levels (mg/dL); as fetal number per ewe increased to triplets, BUN rose slightly, P < 0.015, with post hoc differences shown. Sample sizes were 18 singletons, 58 twins, 30 triplets, and 8 quadruplets+. (D) Fetal Plasma BUN levels (mg/dL) and fetal weight (kg). BUN was inversely related to fetal weight, P < 0.001. (E) Fetal Plasma Creatinine levels (mg/dL); as fetal number per ewe increased, creatinine rose, P < 0.0001. Sample sizes were 18 singletons, 58 twins, 30 triplets, and 8 quadruplets+. (F) Creatinine Levels (mg/dL) and Fetal Weight (mg/dL). Creatinine was inversely related to fetal weight, P < 0.0001. (G) Ratio of Fetal:Maternal Plasma BUN; although minimal change was appreciated by fetal number, (H) Ratio of Fetal:Maternal Plasma Creatinine rose, P < 0.0001. Demarcation as SGA status We analyzed the data in dichotomous fashion (y/n) for body weight falling below 2.626 kg, SGA. All morphometric parameters were lower in the SGA group, including brain weight (g), P < 0.015 for all, although brain weight (g) relative to body weight (kg) was still higher, P < 0.005. Plasma levels of glucose, triglycerides, and LDH were lower, whereas creatinine and cholesterol were higher in the SGA group, P < 0.015 for all. However, plasma levels of Alk Phosphatase, AST, and BUN did not differ when demarcated by SGA status. Discussion This study defined normal plasma biochemical values for a group of nutritional measures, liver enzymes, and placental and fetal kidney excretory measures for late gestation fetal sheep, based on fetal number per ewe, showing stepwise space restriction inhibiting growth and disrupting placental nutrition, liver enzymes, and placental/fetal renal excretory tests. There is limited research on the effects of multifetal gestation on fetal wellbeing, but our study indicates that these fetuses exhibit short‐term metabolic consequences similar to other etiologies of IUGR. As anticipated, as fetal number rose, so did the percentage that weighed <10th percentile of our fetal singleton population. Qualitative comparisons (Table 1) from our current multifetal gestation ovine data are contrasted to findings on human fetuses with typical placental insufficiency‐induced IUGR; both have head sparing, with altered nutrition indices and increased accumulated wastes, but iron status was more protected in fetal sheep, compared to humans. Table 1 Outline of placental efficiency parameters from Chem 20 and growth measurements into the categories of placental nutrition/growth, liver nutrition and function, iron nutrition, and waste excretion. Sources are listed below the table Test Type Test Human fetuses limited space/IUGR Sheep natural IUGR (multifetal gestation) Placental nutrition/Growth Kidney weight ↓ ↓ Liver weight ↓ ↓ Heart weight ↓ ↓ Brain weight No change No change Liver nutrition & Function AST ↑ No change GGT ↑ ↓ Glucose ↓ ↓ Alk Phos Unknown ↓ Triglycerides ↓ ↓ Iron nutrition Serum Iron ↓ No change Waste excretion (Renal/Placental) BUN ↑ ↑ Creatinine ↑ ↑ GFR ↓ ↓ Sodium (Electrolytes) Unknown No change Cholesterol ↑ ↑ AST, Aspartamine transaminase; GGT, Gamma‐glutamyltranspeptidase; Alk Phos, Alkaline phosphatase; BUN, Blood urea nitrogen; GFR, Glomerular filtration rate. Source: Cox et al. (1988); Meyer et al. (2010);Meyer‐Gesch et al. (2013); Sun et al. (2013); Verspyck et al. (1999); Alvino et al. (2008); Onyesom et al. (2009), Nieto‐Diaz et al. (1996); Rao and Georgieff (2007). Arrows up or down denote values relatively to normally grown fetuses. John Wiley & Sons, LtdLoss in placentome number paralleled the observed drop in fetal and organ weights. The study found increased placentomal weight representing better placental efficiency, but similar to what we found in more extreme space restriction (Meyer et al. 2010; Meyer‐Gesch et al. 2013), after GD120, the ability to compensate in higher‐order multifetal gestation was limited. Thus, in this situation, placental efficiency does not accurately reflect functional indices including placental clearance or delivery of nutrients and metabolites. As anticipated, brain weights (g) did not fall, but rose when expressed as ratio of brain weight (g) per body weight, (kg) as fetal number per ewe rose, defining head‐sparing asymmetric growth restriction, with other organ weights proportionate to body weight. As anticipated, males weighed more than females. Brain weight fell slightly (86% of singletons) when fetal number reached four. Many of the fetal body weights fell below our flock's 10th percentile for singletons, representing growth restriction as a compensatory adaption (Barker 1992), potentially leading to developmental programming described by the Barker hypothesis (Fang 2005). As fetal number rose, the stepwise lower fetal plasma glucose levels reflected ineffective placental facilitated diffusion because fetal liver contributes little to gluconeogenesis. The adaptive response to fetal hypoglycemia is to increase efficiency of tissue glucose uptake for fuel, as set forth by the thrifty phenotype hypothesis (Limesand et al. 2007). These findings are consistent with previous studies finding hypoglycemia in growth‐restricted fetal sheep (Morrison 2008), and with altered protein or mRNA expression of proteins within the insulin and gluconeogenic signaling pathways (Lie et al. 2014). The fall in glucose and triglyceride levels likely reflected lost substrate, as both are supplied by placenta. Finding lower circulating glucose and triglycerides as nutrient sources is consistent with that previously seen in multifetal gestation lambs after birth (Moallem et al. 2012). Current data are consistent with the finding of altered energy generation pathways in hearts and livers of twin fetuses and also to singleton lamb fetuses with preconception/preimplantation under nutrition that are epigenetically programmed in the anticipation of a future poor substrate supply (Lie et al. 2013, 2014). In human fetal growth restriction, fetal plasma triglycerides, normally synthesized by placenta after the breakdown of maternal lipids, reflect the etiology, with abnormal placental implantation of preeclampsia raising triglycerides and malnutrition of placental insufficiency lowering triglycerides (Flores et al. 1974; Alvino et al. 2008). The current data support the later, with a threshold, nongraded drop in triglyceride levels in the quadruplet/quintuplet group. Low triglyceride levels might be explained by previous work that found epigenetic differences in genes involved in the adrenal IGF/growth hormone axis in late gestation twins, which may represent an adaptation to mitigate the poor placental substrate supply (Williams‐Wyss et al. 2014). Because poor placental triglyceride delivery can negatively impact cardiac growth and metabolism, increasing the risk of cardiovascular disease (Lie et al. 2013), the impact of multifetal gestation on future lipid metabolism should be further investigated. Although fetal plasma levels of calcium and phosphorus did not differ, Alk Phosphatase, a vital hormone driving the mineral deposition in bone, fell in triplets and quadruplets groups. Alk Phosphatase, present in every tissue, rises with fetal maturation and rapid postnatal growth in premature humans (Tinnion and Embleton 2012). Placental–fetal Alk Phosphatase levels were previously found to parallel plasma glucose in IUGR (Onyesom et al. 2009). Maintaining fetal plasma calcium and phosphorus levels for fetal homeostatic enzymatic and metabolic pathways is vital, and thus lower Alk Phosphatase likely represents a diversion of minerals from bone development. Liver enzymes were minimally changed, except that plasma AST levels were higher in the quadruplets/quintuplets. This finding could potentially reflect either liver ischemia or more likely intravascular hemolysis because ALT was not elevated. LDH, clinically considered a liver enzyme, was lower as fetal number per ewe increased, contrasting with a study showing total LDH was higher in human IUGR, although LDH 5 isoenzyme was reported lower in IUGR (Verspyck et al. 1999). LDH normally converts pyruvate, the final product of glycolysis, into lactate under hypoxia. Lactate accumulation is known to inhibit LDH synthesis and higher lactates were likely present because we, and others previously, found poorer oxygenation (Meyer et al. 2010; Meyer‐Gesch et al. 2013) or increased lactate levels (Morrison 2008) in growth‐restricted fetal sheep. Limited data have been published on plasma electrolytes or fetal placental and renal excretory function parameters in multifetal gestation (Gibson and Lumbers 1999; Morrison 2008). Impaired placental excretion of wastes was supported by a graded rise in fetal plasma cholesterol level as fetal number rose (Wallach 1983). Fetal cholesterol and fetal adrenal glands both provide precursors to placental production of steroid hormones (Sanderson 2009). In contrast to our findings, cholesterol was previously reported to fall somewhat as fetal number rose in newborn lambs (Moallem et al. 2012), and it remains unclear why we found the opposite. The placenta serves to clear cholesterol from fetal production, so perhaps the additional 15 days to term gestation resulted in lower fetal cholesterol synthesis or neonatal factors dominated. The near‐complete fetal growth arrest in multifetal gestation became apparent between GD120 and GD130, and the additional days in gestation may further compromise transfer of fetally derived cholesterol to the placenta. Greater levels of fetal creatinine and a higher ratio of fetal:maternal creatinine were likely due to poor excretory function. Work with fetal unilateral nephrectomy (halving fetal kidney function) reported a doubling of fetal plasma creatinine, concluding that excretory function is a joint function of placenta and fetal kidney (Anderson et al. 1999; Douglas‐Denton et al. 2002). In support, this study found 75% higher fetal creatinine with nephrons cut by 25% (Martin et al. 2010). Previously, lower plasma BUN and higher creatinine were seen in the smaller of discordant human twins (Arad et al. 2001), supporting that BUN reflected fetal protein, whereas creatinine reflected less placental–fetal renal excretion. If GFR fell by 50%, creatinine and potassium tests rose (Wallach 1983; Cheung and Lafayette 2013). In this study, the rise in plasma creatinine was greater than BUN, perhaps because BUN is more tightly regulated, or being of smaller molecular weight was more readily diffusible through the placenta (Meyer et al. 2010). Normally, initial fetal plasma creatinine levels reflect maternal levels, falling to infantile levels at 1 week, as renal blood doubles (Su and Stonestreet 2010). In human asymmetric IUGR, altered glomerular development and postnatal compensation predisposes to glomerular hyperfiltration and adult hypertension (Hinchliffe et al. 1992). Strengths to the data include the large number of samples collected and the number that were growth restricted. Demarcating data as SGA or normally grown showed a similar direction for most biochemical values and supported that growth restriction strongly influenced multifetal gestation effects. A study limitation includes the slight delay in the time from anesthetic to fetal sampling, but finding that the mean maternal pO2 did not differ, the mean singleton fetal pO2 only mildly lower than reported values, and lack of fetal arteriovenous impact on measurements supported adequate fetal placental function despite this sampling delay. This study quantitatively examines plasma biochemical parameters in multifetal gestation; providing normative data and aiding understanding of placental–fetal physiology. As fetal number rose, biochemical evidence supported increased functional adaptations in placental–fetal nutrition, placental and fetal renal excretory function, and fetal liver enzymes. Future research could further identify biomarkers and cut‐off limits to detect the most compromised fetal status (Sun et al. 2013) and to help understand mechanisms behind placental adaptation and developmental programming of long‐term effects. Understanding fetal blood biochemical indices in multifetal gestation may help understand lifelong health of multifetal gestation infants (Baschat 2004). Conflict of Interest None declared. Supporting information Table S1. Data at GD120 with 95% confidence intervals. Table S2. Data at GD130 with 95% confidence intervals. Click here for additional data file. Data S1. Iron nutritional indices. Click here for additional data file. Acknowledgments The Kling & Magness Laboratories, Bryan Ampey, Jason Austin, Natalie Dosch, Jason Habeck Sheik Omar Jobe, Jill Koch, Colin Korlesky, Rachel Kranch, Gladys Lopez, Steven Marmer, Timothy J. 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==== Front Physiol RepPhysiol Rep10.1002/(ISSN)2051-817XPHY2physreportsPhysiological Reports2051-817XJohn Wiley and Sons Inc. Hoboken 2755098710.14814/phy2.12906PHY212906Cellular and Molecular Conditions, Disorders and TreatmentsRegulatory PathwaysDigestive Conditions, Disorders and TreatmentsOriginal ResearchOriginal Research BMI1 is downregulated by the natural compound curcumin, but not by bisdemethoxycurcumin and dimethoxycurcumin T. A. Adeyeni et al.Adeyeni Temitope A. 1 2 Khatwani Natasha 1 San KayKay 1 Ezekiel Uthayashanker R. 1 1 Department of Biomedical Laboratory ScienceSaint Louis UniversitySt. LouisMissouri2 Department of Health Science and InformaticsSaint Louis UniversitySt. LouisMissouri* Correspondence Uthayashanker R. Ezekiel, Department of Biomedical Laboratory Science, Saint Louis University, 3437 Caroline Mall, St. Louis, MO 63104. Tel: (314)‐977‐8689 Fax: (314) 977‐1155 E‐mail: uezekiel@slu.edu 22 8 2016 8 2016 4 16 10.1111/phy2.2016.4.issue-16e1290624 5 2016 21 7 2016 25 7 2016 © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.Abstract The B‐cell‐specific Moloney murine leukemia virus integration site 1 (BMI1) locus encodes a 37‐kD protein that is a key regulatory component of the polycomb regulatory complex 1 (PRC1). When overexpressed in various cancer types, the BMI1 protein induces cell growth and promotes tumor growth in vitro and in vivo. Curcumin, a major phytochemical in turmeric (Curcuma longa), inhibits the proliferation and survival of many types of cancer cells, both in vitro and in vivo, and has been reported to reduce BMI1 expression in breast cancer cells. In this study, effects of curcumin and two analogs (bisdemethoxycurcumin and dimethoxycurcumin) on BMI1 expression were evaluated in DLD‐1 colorectal cancer cells. Bisdemethoxycurcumin (BDMC) is naturally occurring in turmeric, whereas dimethoxycurcumin (DMC) is a synthetic analog of curcumin. All three compounds reduced cell survival, but only the natural compound downregulated BMI1 protein expression; curcumin significantly reduced BMI1 levels more than bisdemethoxycurcumin and dimethoxycurcumin. In addition, curcumin and BDMC inhibit survival of the DLD‐1 colorectal cancer cells by inducing apoptosis, whereas DMC inhibits survival by a mechanism other than apoptosis. ApoptosisBMI1colorectal cancerphytochemicalDeNardo Education and Research FoundationAmerican Society of Clinical Laboratory Science Education and Research FundThe Saint Louis University President Research Fund source-schema-version-number2.0component-idphy212906cover-dateAugust 2016details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_NLMPMC version:4.9.4 mode:remove_FC converted:29.08.2016 T. A. Adeyeni , N. Khatwani , K. San , U. R. Ezekiel . BMI1 is downregulated by the natural compound curcumin, but not by bisdemethoxycurcumin and dimethoxycurcumin . Physiol Rep , 4 (16 ), 2016 , e12906, doi: 10.14814/phy2.12906 Funding Information TAA, NK, and KS were supported by the DeNardo Education and Research Foundation. TAA was also supported by the American Society of Clinical Laboratory Science Education and Research Fund (I. Dean Spradling Graduate Student Grant). The Saint Louis University President Research Fund supported URE. ==== Body Introduction Colorectal cancer is the third most common cancer among men and women in the United States (Siegel et al. 2013). Higher incidence rates were observed in people 40 years and older (Siegel et al. 2013). However, it was recently reported that there has been an increased incidence of colorectal cancer in the 20‐ to 34‐year‐old age group (Bailey et al. 2015). This was attributed to the western diet, which is high in processed foods and red meat but low in fruits and vegetables (Bailey et al. 2015). The anticancer properties of fruits and vegetables may be partly due to their abundant phytochemicals (Srivastava et al. 2015). Phytochemicals refer to a variety of compounds that are produced by plants (Srivastava et al. 2015). Curcumin (CUR), the major phytochemical in turmeric (Curcuma longa), has been shown to inhibit the proliferation of many types of cancers without creating adverse effects in patients taking oral doses up to 8–10 g per day (Hatcher et al. 2008; Shehzad et al. 2010; Carroll et al. 2011). Although CUR shows great potential as an anticancer drug, in vivo pharmacokinetic studies have shown it has low bioavailability (Carroll et al. 2011). However, some analogs of CUR are reported to have higher bioavailability. Two of these analogs were evaluated in this study — bisdemethoxycurcumin (BDMC) and dimethoxycurcumin (DMC). BDMC is naturally occurring in turmeric, whereas DMC is a synthetic CUR analog designed to have higher solubility and bioavailability than CUR (Anand et al. 2007; Shehzad et al. 2010; Hassan et al. 2015). B‐cell‐specific Moloney murine leukemia virus integration site 1 (BMI1) encodes a 37‐kD polycomb group protein that serves as a key regulatory component of the polycomb regulatory complex 1 (PRC1) (Cao et al. 2011). The PRC1 complex is a chromatin structure modulator that normally regulates transcription of many important genes. BMI1 protein is involved in self‐renewal of adult stem cells (Cao et al. 2011). Overexpression of BMI1 in breast, prostate, lung, and ovarian cancers promotes the stem state of tumor cells and is associated with therapy failure (Cao et al. 2011). Reduction in BMI1 expression, however, leads to programmed cell death (apoptosis), senescence in tumor cells, and increased tumor cell susceptibility to cytotoxic agents and radiation therapy (Cao et al. 2011). Chowdhury et al. reported that acute myeloid leukemia patients with lower BMI1 expression levels have significantly longer overall survival, relapse‐free survival, and remission duration than patients with higher BMI1 expression levels (Chowdhury et al. 2007). Guo et al. reported that CUR suppressed BMI1 protein expression in MDA‐MB‐231 and MDA‐MB‐435 breast cancer cells (Guo et al. 2013), but there are no studies that have reported such effects in other cancer cells. Thus, the objective of this present study was to assess the selected compounds’ (CUR, BDMC, DMC) effect on BMI1 expression in DLD‐1 colorectal cancer cells. Previously we identified that curcumin showed a similar inhibitory effect on genetically dissimilar DLD‐1, LoVo, and HCT‐116 cancer cell lines (Montgomery et al. 2016). Therefore, for this report, an exploratory study was done on the effect of the above three compounds on BMI1 expression using DLD‐1 cancer cells as a model system. We found that all three compounds inhibited survival of DLD‐1 cells. However, only curcumin did so by downregulation of BMI1 protein expression. Materials and Methods Chemical reagents and cell culture Curcumin (purity >80%), bisdemethoxycurcumin (purity ≥98%), and dimethoxycurcumin (purity ≥90%) were purchased from Cayman Chemical Company (Ann Arbor, MI). All solutions were prepared as 100 mM stocks in DMSO and stored at −20°C. DMSO alone (0.2%) served as vehicle‐only control. DLD‐1 colorectal cancer cell isolates were purchased from the American Type Culture Collection (Manassas, VA). They were isolated from an adult male with Dukes’ type C colorectal adenocarcinoma (a subtype of colorectal cancer). Cells were grown at 37°C, 5% CO2 in DMEM (Sigma‐Aldrich, St. Louis, MO) supplemented with 10% FBS, penicillin/streptomycin, glutamine, sodium pyruvate, and HEPES buffer (Hyclone, Logan, UT). Cell survival studies Five thousand DLD‐1 cells were seeded in each well of a 96‐well cluster plate (Sigma‐Aldrich, St. Louis, MO) and incubated for 12–16 h to allow adherence to the well surface. Cells were treated with varying concentrations (3.125–50 μmol/L) of CUR, BDMC, or DMC. Dilutions of each compound were prepared immediately before treatment, all treatments were performed in triplicates, and each experiment was performed at least three times. After addition of the phytochemicals, the cells were incubated for 48 h. Remaining live cells were fixed (1.1% glutaraldehyde, 15 min rotation, 1.2 g) to the well surface, washed with water, dried, and then stained with crystal violet (0.1% crystal violet in 100 mmol/L MES, pH 6.0) (Van Schaeybroeck et al. 2005; Duessel et al. 2008; Jackson et al. 2008). Stained cells were solubilized in 10% acetic acid and absorbance at 562 nm was measured. Results were reported as percent cell survival compared to control. Cell viability and caspase‐3/7 activity Five thousand cells were seeded in each well of a flat‐bottom, cell‐grade, 96‐well cluster black plate (BrandTech Scientific, Essex, CT) and incubated for 12–16 h before compound treatment. The cells were treated with three concentrations of each compound; these were half of each compound's half‐maximal inhibitory concentration (IC50), the IC50, and twice the IC50 (CUR: 6.25, 12.5, 25 μmol/L; BDMC: 5, 10, 20 μmol/L; and DMC: 1, 2, 4 μmol/L). All treatments were performed in triplicate and each experiment was performed three times. At 48 h, the ApoToxGlo™ Triplex Assay was performed per manufacturer's instructions (Promega, Madison, WI). The assay measures fluorescence emitted by live and dead cells and also measures luminescent signals indicative of caspase‐3/7 activity. In this study, only fluorescent signals from live cells and luminescent signals from caspase‐3/7 activity were measured. In the assay, cell permeant, fluorogenic, peptide substrate (glycyl‐phenylalanyl‐aminofluorocoumarin; GF‐AFC) enter live cells, and are cleaved by proteases located in the cytoplasm. Fluorescence intensities, proportional to the number of live cells, were detected at the wavelengths 400EX/505EM nm. To measure caspase‐3/7 activity, a luminogenic substrate with attached tetrapeptide (DEVD: asp‐glu‐val‐asp) was added to the cells. Caspase digestion of the peptide releases a luciferase substrate known as aminoluciferin which is acted on by luciferase for production of luminescence: luminescence is proportional to caspase‐3/7 activity. All treatments were performed in triplicate, and each experiment was performed three times. BMI1 and apoptotic protein expression Cells were treated with CUR (6.25–25 μmol/L), BDMC (6.25–25 μmol/L), or DMC (1.56–6.25 μmol/L) for 48 h. All treatments were performed in triplicate and each experiment was repeated three times. Cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer and supplemented with protease inhibitors (Thermo Scientific, Waltham, MA). Cell lysates containing 10 μg of protein were electrophoretically separated using SDS PAGE (4–12% gradient). The separated proteins were then electrophoretically transferred onto nitrocellulose membranes (Thermo Scientific, Waltham, MA). B‐cell‐specific Moloney murine leukemia virus integration site 1, cleaved poly (ADP‐ribose) polymerase (PARP), procaspase‐3, and β‐actin (loading control) proteins were probed overnight with their respective monoclonal rabbit antibodies used at a 1:1000 dilution (Cell Signaling Technologies, Danvers, MA). Antirabbit IgG, horseradish peroxidase‐linked antibody (Cell Signaling Technology, Danvers, MA) at a 1:3500 dilution was used as secondary antibody. Signals were detected by chemiluminescence using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA) and Thermo Fisher's myECL imager (Thermo Fisher Scientific, Danvers, MA). Even though the predicted molecular weight of BMI1 is 37 kDa, antibody (Cell Signaling Technology, Danvers, MA) recognizes the protein at 41 kDa in Western blots. The blots were stripped and reprobed with β‐actin. Immunoreactive band intensity of BMI1 was normalized by comparison to β‐actin signals. Fold differences were calculated with respect to vehicle control (0.2% DMSO)‐treated cells. Statistical analysis All data were analyzed using one‐way analysis of variance (ANOVA) followed by a Student–Newman–Keuls post hoc analysis. All data were reported as mean ± SEM. Significance was reported as P < 0.05 unless otherwise noted. Results CUR, BDMC, and DMC decrease survival and viability of DLD‐1 cells We studied the effects of CUR, BDMC, and DMC on the survival of DLD‐1 colorectal cancer cells using the crystal violet method (Van Schaeybroeck et al. 2005; Duessel et al. 2008; Jackson et al. 2008). Cells were treated with varying concentrations of each compound (3.125–50 μmol/L) for 48 h. CUR, BDMC, and DMC all inhibit the survival of DLD‐1 colorectal cancer cells in a dose‐dependent manner (Fig. 1). However, inhibitory effects of DMC are significantly stronger than CUR and BDMC; it has an IC50 value of 1.90 ± 0.1 μmol/L. CUR and BDMC effects are not significantly different from one another with IC50 values of 9.83 ± 0.6 μmol/L and 8.39 ± 0.6 μmol/L, respectively. Cell viability measurements (ApoTox‐Glo™ Triplex assay, Promega, Madison, WI) showed that 48 h compound treatment (CUR: 6.25–25 μmol/L. BDMC: 5–20 μmol/L. DMC: 1–4 μmol/L) decrease the number of live cells in a dose‐dependent manner (Fig. 2). Based on previous studies (Yoon et al. 2014; Xu et al. 2015; Kim et al. 2016), concentrations of the three compounds used have no effect on cell viability of normal, noncancerous human cells. CUR (up to 40 μmol/L), BDMC (up to 80 μmol/L), and DMC (up to 30 μmol/L) showed no effect on viability of noncancerous cells (Yoon et al. 2014; Xu et al. 2015; Kim et al. 2016). Figure 1 Effect of curcumin (CUR), bisdemethoxycurcumin (BDMC), and dimethoxycurcumin (DMC) on DLD‐1 colorectal cancer cell survival. DLD‐1 cells were cultured with various concentrations of CUR, BDMC, and DMC for 48 h. Survival of the cells (percent relative to control) was determined using the crystal violet method. All values represent the mean ± SEM of three independent experiments. ***P < 0.001 compared to CUR‐treated cells. Figure 2 Effect of curcumin (CUR), bisdemethoxycurcumin (BDMC), and dimethoxycurcumin (DMC) on DLD‐1 colorectal cancer cell viability. DLD‐1 cells were cultured with three concentrations of each compound for 48 h. These were half of each compound's half maximal inhibitory concentrations (IC 50), the IC 50, and twice the IC 50. Live cells were measured using ApoTox‐Glo™ Triplex assay (Promega, Madison, WI). Fluorescence (detected at 400EX/505EM nm) is proportional to the number of live cells. All values represent the mean ± SEM of three independent experiments. ***P < 0.001 compared to vehicle control (0.2% DMSO‐treated cells). *P < 0.05 compared to vehicle control (0.2% DMSO‐treated cells). CUR downregulates BMI1 protein expression B‐cell‐specific Moloney murine leukemia virus integration site 1 is an oncogenic protein reported to be overexpressed in many cancers. Knockdown of BMI1 leads to apoptosis and senescence in tumor cells (Cao et al. 2011). Thus, we investigated whether CUR and its analogs (BDMC, DMC) could reduce BMI1 protein levels in DLD‐1 cells. Cells were treated with CUR (6.25, 12.5, 25 μmol/L), BDMC (6.25, 12.5, 25 μmol/L), and DMC (1.56, 3.13, 6.25 μmol/L) for 48 h. Western blot analysis of whole‐cell lysates from control (DMSO) and treated cells was performed. Band intensities of β‐actin (loading control) and BMI1 were determined by densitometry. BMI1 band density was normalized by comparison with β‐actin. Fold differences were calculated with respect to vehicle control (0.2% DMSO‐treated cells). B‐cell‐specific Moloney murine leukemia virus integration site 1 expression analysis by Western blot indicates that only CUR decreases BMI1 protein levels compared to vehicle control (Fig. 3A). Figure 3D shows that there was a significant decrease between 25 μmol/L CUR and vehicle control (p < 0.01); there is also a significant decrease between 25 μmol/L CUR and 12.5 μmol/L CUR (P < 0.05). No significant decrease in BMI1 is shown by BDMC and DMC (Fig. 3B–D). These results indicate that only CUR suppresses BMI1 expression. Figure 3 BMI1 protein expression in curcumin (CUR)‐, bisdemethoxycurcumin (BDMC)‐, and dimethoxycurcumin (DMC)‐treated DLD‐1 colorectal cancer cells. DLD‐1 cells were cultured with three concentrations of each compound for 48 h (CUR: 6.25, 12.5, 25 μmol/L. BDMC: 6.25, 12.5, 25 μmol/L. DMC: 1.56, 3.13, 6.25 μmol/L). Western blot analysis of whole‐cell lysates from vehicle control (0.2% DMSO) and compound‐treated cells, at 48 h, is shown ([A] CUR: 6.25, 12.5, 25 μmol/L; [B] BDMC: 6.25, 12.5, 25 μmol/L; [C] DMC: 1.56, 3.13, 6.25 μmol/L). β‐actin was used as loading control. (D) The relative intensity of BMI1 was evaluated as fold change compared to vehicle control (0.2% DMSO) after normalization with β‐actin. For curcumin, only 12.5 and 25 μmol/L band intensities were evaluated. All values represent mean ± SEM of three independent experiments. **P < 0.01 compared to vehicle control (0.2% DMSO‐treated cells). *P < 0.05 compared to 25 μmol/L CUR‐treated cells. CUR and BDMC induce apoptosis Apoptosis or programmed cell death results in distinct cellular morphological changes and biochemical events including caspase‐3/7 activation, DNA fragmentation, chromatin condensation, and poly (ADP‐ribose) polymerase (PARP) cleavage (Tawa et al. 2004; Yoon et al. 2014). Cells treated with 12.5 μmol/L CUR, 10 μmol/L BDMC, or 2 μmol/L DMC show cell rounding and morphologic changes suggestive of apoptosis (Fig. 4A–D). Cells treated singly with CUR or BDMC show more pronounced rounding and blebbing than DMC‐treated cells. Additionally, we assessed apoptotic activity of the cells by measuring caspase‐3/7 activity. Figure 4 Microscopic images (20× magnification) of DLD‐1 colorectal cancer cells. Cells were cultured with half maximal inhibitory concentrations (IC 50) of each compound for 48 h, fixed, and stained with crystal violet. (A) Vehicle control (0.2% DMSO); (B) 12.5 μmol/L curcumin (CUR); (C) 10 μmol/L bisdemethoxycurcumin (BDMC); and (D) 2 μmol/L dimethoxycurcumin (DMC). We measured apoptosis by two methods: (1) an enzymatic method that measures caspase‐3/7; and (2) a Western blot method that detects active form of caspase‐3. PARP, which catalyzes poly(ADP) ribosylation of a variety of nuclear proteins, is cleaved by caspase‐3. To further confirm apoptotic progress in treated cells, we detected the presence of cleaved PARP by Western blot. Only 12.5 μmol/L CUR and 20 μmol/L BDMC significantly (P < 0.01) increased caspase‐3/7 activity (Fig. 5D). The 12.5 μmol/L CUR concentration shows 5‐fold more apoptosis and 20 μmol/L BDMC shows 4‐fold more apoptosis than control‐treated cells (Fig. 5D). DMC (1–4 μmol/L), however, does not significantly increase caspase‐3/7 activity (n = 3, P > 0.05) (Fig. 5D). Figure 5 Apoptotic protein expression in curcumin (CUR)‐, bisdemethoxycurcumin (BDMC)‐, and dimethoxycurcumin (DMC)‐treated DLD‐1 colorectal cancer cells. DLD‐1 cells were cultured with three concentrations of each compound for 48 h (CUR: 6.25, 12.5, 25 μmol/L. BDMC: 6.25, 12.5, 25 μmol/L. DMC: 1.56, 3.13, 6.25 μmol/L). Western blot analysis of whole‐cell lysates from vehicle control and compound‐treated cells, at 48 h, is shown in (A) CUR, (B) BDMC, (C) DMC. Treated samples were probed overnight with procaspase‐3 and poly (ADP‐ribose) polymerase (PARP) antibodies. β‐actin was used as the loading control. (D) DLD‐1 cells were cultured with three concentrations of each compound for 48 h. These were half of each compound's half maximal inhibitory concentrations (IC 50), the IC 50, and twice the IC 50. Luminescence emitted was proportional to caspase‐3/7 activity. **P < 0.01 compared to vehicle control. All values represent the mean ± SEM of three independent experiments. Western blot analysis was performed to detect protein markers of apoptosis, cleaved PARP, and caspase‐3. Activation of caspase‐3 leads to proteolytic cleavage of procaspase‐3 to active p17/p12 fragments (Tawa et al. 2004; Chaitanya et al. 2010). Therefore, a decrease in procaspase‐3 expression indirectly indicates that the cells underwent apoptosis (Tawa et al. 2004). Activated caspase‐3 cleaves PARP (116 kD) to produce an 89 kD cleaved product. PARP fragment that is cleaved indicates that the cells were undergoing apoptosis (Chaitanya et al. 2010). Western blot data show that CUR and BDMC increased cleaved PARP and decreased procaspase‐3 levels (Fig. 5A and B). DMC increases only cleaved PARP levels, whereas procaspase‐3 remains uncleaved (Fig. 5C). PARP is typically cleaved by caspase‐3; however, in DMC‐treated cells, cleaved PARP is evident in Western blot but there is no increase in caspase3/7 activity. This discrepancy indicates that an additional mechanism, such as necrosis, is generating cleaved PARP (Chaitanya et al. 2010). These findings indicate that CUR and BDMC inhibit the survival of DLD‐1 colorectal cancer cells by inducing apoptosis, whereas DMC may inhibit survival by alternative mechanisms. Discussion Curcumin, bisdemethoxycurcumin, and dimethoxycurcumin inhibit the survival and viability of DLD‐1 colorectal cancer cells (Figs. 1, 2) in a dose‐dependent manner. Whereas the natural compounds (CUR and BDMC) exert similar inhibitory effects, the synthetic compound (DMC) exerts a stronger effect (Fig. 1). Interestingly, only CUR effects were mediated by the downregulation of the oncogenic protein BMI1 (Fig. 3) and the subsequent induction of apoptosis (Figs. 4, 5). BDMC and DMC did not affect the BMI1 levels but did affect cell viability. Additionally, we observed that CUR and BDMC, but not DMC, reduced cell survival by apoptosis indicating that inhibitory effects are mediated by alternative molecular mechanisms (Yoon et al. 2014). Both CUR and BDMC exhibited apoptosis and the presence of active caspase‐3. Yoon et al. observed no morphological features of apoptosis in DMC‐treated breast cancer cell lines; they also found that DMC‐induced cell death is not inhibited by caspase‐8, ‐9, or ‐3 inhibitors (Yoon et al. 2014). This suggests that apoptosis is not involved in DMC‐induced cell death. Instead, they found that DMC significantly induces another form of programmed cell death, paraptosis (Yoon et al. 2014). Paraptosis is an alternative, nonapoptotic form of programmed cell death that lacks nuclear fragmentation, chromatin condensation, and formation of apoptotic bodies characteristic of apoptosis (Yoon et al. 2014). Markers of paraptosis, such as cytoplasmic vacuolation, were observed in both CUR‐ and DMC‐treated cells; however, DMC displayed stronger inhibitory and paraptotic effects than CUR (Yoon et al. 2014). The aforementioned study supports our observation that DMC does not significantly increase caspase‐3/7 activity (Fig. 5D) and does not induce the cleavage of procaspase‐3 (Fig. 5C). However, there remains no explanation for why cleaved PARP (a hallmark of apoptosis) was expressed in 6.25 μmol/L DMC‐treated cells (Fig. 5C), despite the fact that PARP is typically cleaved by caspase‐3 during apoptosis (Chaitanya et al. 2010). Chaitanya et al. reported that when human leukemia Jurkat T cells were treated with inducers of apoptosis (150 μmol/L Vp‐16 or 150 nmol/L staurosporine) or necrosis (100 μmol/L HgCl2, 10% EtOH, or 0.1% H2O2), the cells expressed the same 89 kD fragment of PARP typically expressed only in apoptotic cells (Chaitanya et al. 2010). This was despite the fact that there was no apoptotic activity in the necrotic cells (Chaitanya et al. 2010). This observation is supported by reports of 5 μmol/L DMC's induction of both apoptotic and necrotic forms of cell death in MCF‐7 breast cancer cells (Kunwar et al. 2012). At higher doses (25 and 50 μmol/L), however, DMC primarily induces necrotic cell death (Kunwar et al. 2012). These findings explain why, in our study, DMC displays lower caspase‐3/7 activity than the other compounds (CUR, BDMC) and why the 89 kD cleaved PARP fragment is expressed even though procaspase‐3 is not cleaved (Fig. 5C). These studies show that DMC's inhibitory effects are driven by paraptotic and necrotic mechanisms (Kunwar et al. 2012; Yoon et al. 2014). A low molecular weight compound, PTC‐209, induces a dose‐dependent inhibition of BMI1 expression in HCT‐116 human colorectal cancer cells and leads to cell death (Kreso et al. 2014). BMI1 targeted by PTC209 abrogates the tumorigenic capacity of colon cancer stem cells in vivo (Kreso et al. 2014). The aforementioned study highlights the importance of discovering compounds that target BMI1. Here, we found that the natural compound CUR inhibits BMI1 expression. Future studies will focus on understanding the mechanism of this inhibition. Several studies report that BMI1 gene expression can be regulated by microRNAs (miRNAs, miR). We hypothesize that CUR upregulates miRNAs that target BMI1. MiRNAs play a role in posttranscriptional gene expression and can act as tumor promoters or tumor suppressors (Teiten et al. 2010; Jansson and Lund 2012; Shah et al. 2012). In both cases, the target mRNA is inactivated/silenced (Jansson and Lund 2012). A single miRNA can have multiple mRNA targets (Teiten et al. 2010; Jansson and Lund 2012; Shah et al. 2012). There are several miRNAs that target BMI1 in various cancer types (miRNA‐ 218, 215, 200b, 200c, 128, 15a/b, 708, 16) (Bhattacharya et al. 2009; Saini et al. 2011; He et al. 2012; Liu et al. 2012; Sun et al. 2012; Guo et al. 2014; Jin et al. 2014; Yu et al. 2014; Jones et al. 2015). When HCT‐116 and HT29 colorectal cancer cells are transfected with miRNA‐218, BMI1 gene and protein expression are significantly downregulated (He et al. 2012). A significant reduction in colony counts of the HCT‐116 and HT29 cell lines is also evident (He et al. 2012). The growth inhibitory effects have been further confirmed by tumorigenicity in vivo, where subcutaneous tumor growth is lower in pre‐miR‐218 transfected HCT‐116 cells compared with the control transfected HCT‐116 cells (He et al. 2012). Additionally, there is a significant increase in apoptotic cells and significant G2 cell‐cycle arrest (He et al. 2012). Guo et al. (2013) showed that CUR inhibits MDA‐MB‐231 and MDA‐MB‐435 breast cancer cell survival and invasion. CUR's effect is caused by the upregulation of miRNA‐34a, which subsequently reduces BMI1 levels (Guo et al. 2013). This study demonstrates that CUR reduces BMI1 expression in breast cancer cells by upregulating miR‐34a (Guo et al. 2013). However, to the best of our knowledge, our study is the first to show that CUR reduces BMI1 expression in colorectal cancer cells. Therefore, our future studies will focus on understanding if CUR reduces BMI1 levels by upregulating one or more of the miRNAs shown to downregulate BMI1 expression (Bhattacharya et al. 2009; Saini et al. 2011; He et al. 2012; Liu et al. 2012; Sun et al. 2012; Guo et al. 2014; Jin et al. 2014; Yu et al. 2014; Jones et al. 2015). Conflict of Interest None declared. 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==== Front Physiol RepPhysiol Rep10.1002/(ISSN)2051-817XPHY2physreportsPhysiological Reports2051-817XJohn Wiley and Sons Inc. Hoboken 2755098810.14814/phy2.12907PHY212907Signalling PathwaysMetabolism and RegulationSkeletal MuscleEndurance and PerformanceOriginal ResearchOriginal ResearchAcute resistance exercise‐induced IGF1 expression and subsequent GLUT4 translocation K. Kido et al.Kido Kohei 1 Ato Satoru 1 Yokokawa Takumi 2 Makanae Yuhei 3 Sato Koji 4 Fujita Satoshi 1 1 Faculty of Sport and Health ScienceRitsumeikan UniversityKusatsuJapan2 Laboratory of Sports and Exercise MedicineGraduate School of Human and Environmental StudiesKyoto UniversityKyotoJapan3 Department of Physical EducationNational Defense AcademyYokosukaJapan4 Graduate School of Human Development and EnvironmentKobe UniversityKobeJapan* Correspondence Satoshi Fujita, Faculty of Sport and Health Science, Ritsumeikan University, 1‐1‐1 Nojihigashi, Kusatsu 525‐8577, Japan. Tel: +81‐77‐561‐3760; Fax: +81‐77‐561‐3761; E‐mail: safujita@fc.ritsumei.ac.jp 22 8 2016 8 2016 4 16 10.1111/phy2.2016.4.issue-16e1290718 7 2016 21 7 2016 © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.Acute aerobic exercise (AE) is a major physiological stimulus for skeletal muscle glucose uptake through activation of 5′ AMP‐activated protein kinase (AMPK). However, the regulation of glucose uptake by acute resistance exercise (RE) remains unclear. To investigate the intracellular regulation of glucose uptake after acute RE versus acute AE, male Sprague–Dawley rats were divided into three groups: RE, AE, or nonexercise control. After fasting for 12 h overnight, the right gastrocnemius muscle in the RE group was exercised at maximum isometric contraction via percutaneous electrical stimulation (3 × 10 sec, 5 sets). The AE group ran on a treadmill (25 m/min, 60 min). Muscle samples were taken 0, 1, and 3 h after completion of the exercises. AMPK, Ca2+/calmodulin‐dependent protein kinase II, and TBC1D1 phosphorylation were increased immediately after both forms of exercise and returned to baseline levels by 3 h. Muscle IGF1 expression was increased by RE but not AE, and maintained until 3 h after RE. Additionally, Akt and AS160 phosphorylation were sustained for 3 h after RE, whereas they returned to baseline levels by 3 h after AE. Similarly, GLUT4 translocation remained elevated 3 h after RE, although it returned to the baseline level by 3 h after AE. Overall, this study showed that AMPK/TBC1D1 and IGF1/Akt/AS160 signaling were enhanced by acute RE, and that GLUT4 translocation after acute RE was more prolonged than after acute AE. These results suggest that acute RE‐induced increases in intramuscular IGF1 expression might be a distinct regulator of GLUT4 translocation. Aerobic exerciseglucose transporter type 4insulin‐like growth factor 1resistance exerciseJSPS KAKENHI2528220025560379Japanese Council for Science, Technology and Innovation, SIP14533567Bio‐oriented Technology Research Advancement Institution, NARO source-schema-version-number2.0component-idphy212907cover-dateAugust 2016details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_NLMPMC version:4.9.4 mode:remove_FC converted:29.08.2016 K. Kido , S. Ato , T. Yokokawa , Y. Makanae , K. Sato , S. Fujita . Acute resistance exercise‐induced IGF1 expression and subsequent GLUT4 translocation . Physiol Rep , 4 (16 ), 2016 , e12907, doi: 10.14814/phy2.12907 Funding Information This work was supported by JSPS KAKENHI Grant nos 25282200 and 25560379 to S. Fujita. This work was also supported by the Japanese Council for Science, Technology and Innovation, SIP (Project ID 14533567), “Technologies for creating next‐generation agriculture, forestry and fisheries” (funding agency: Bio‐oriented Technology Research Advancement Institution, NARO). ==== Body Introduction Aerobic exercise (AE) is a major physiological stimulus that induces skeletal muscle glucose uptake and improves insulin sensitivity. Acute and chronic AE affect glucose metabolism differently. Specifically, acute AE induces skeletal muscle glucose uptake incrementally and transiently (Goodyear et al. 1990). A previous study suggests that the blood glucose level is decreased approximately 30% for 4 h by acute AE, as compared with nonexercised controls (Bacchi et al. 2012). Daily blood glucose control is critical for diabetes patients to avoid diabetic complications (American Diabetes Association 2015b). Chronic AE improves insulin sensitivity, fasting blood glucose, and HbA1c levels continuously (Umpierre et al. 2011; Mann et al. 2014). The improvement of these parameters can lead to a complete reversal of type II diabetes. Thus, both acute and chronic AE are significant for improving glucose metabolism. In a recent study, chronic resistance exercise (RE), which is a skeletal muscle hypertrophy model, also improved hyperglycemia and HbA1c levels in diabetic patients (Umpierre et al. 2011). Therefore, recent exercise guidelines from the American Diabetes Association recommend RE for controlling blood glucose (American Diabetes Association 2015a). However, acute RE‐induced augmentation of skeletal muscle glucose uptake and the regulation of this process are still poorly understood. AMP‐activated protein kinase (AMPK), which is activated by increases in the cellular AMP/ATP ratio, is a key intracellular signaling protein for glucose uptake (Musi et al. 2003; Jessen and Goodyear 2005; O'Neill 2013). The activation of AMPK is mediated by acute AE that induces glucose transporter type 4 (GLUT4) translocation to the cell membrane, resulting in glucose uptake (Musi et al. 2003; Jessen and Goodyear 2005; O'Neill et al. 2011; O'Neill 2013), but no study confirmed the signaling response after RE. Ca2+/calmodulin‐dependent protein kinase II (CaMKII) is another important signaling protein for glucose uptake following acute AE. Previous studies demonstrated that CaMKII activates AMPK as an upstream event in response to AE or muscle contraction (Raney and Turcotte 2008; Morales‐Alamo et al. 2013), but other studies indicated that CaMKII may also induce glucose uptake independent of AMPK signaling pathways (Raney and Turcotte 2008; Witczak et al. 2010). Although CaMKII may have important roles in skeletal muscle glucose uptake following AE, the regulation of CaMKII signaling remains unclear. Some previous studies showed that both AMPK and CaMKII were phosphorylated following acute RE, which might contribute to glucose uptake (Witczak et al. 2010; Ogasawara et al. 2014). However, no study showed the roles of AMPK and CaMKII in acute RE‐induced glucose uptake. Phosphorylation of TBC1D1 and AS160 occurs downstream of AMPK and CaMKII (Funai and Cartee 2008; Vendelbo et al. 2014). Through the phosphorylation of TBC1D1 and AS160, AMPK and CaMKII enhance GLUT4 translocation (Chavez et al. 2008; Witczak et al. 2010). However, the relationship between AMPK/CaMKII phosphorylation and TBC1D1/AS160 phosphorylation remains unknown. Moreover, acute RE‐induced TBC1D1/AS160 responses are not fully clarified. Insulin‐like growth factor 1 (IGF1) is increased in skeletal muscle by acute RE (Ogasawara et al. 2013a,b). In contrast, there is no evidence of acute AE inducing the expression of skeletal muscle IGF1. In a cell culture study, IGF1 phosphorylated Akt which then phosphorylated AS160 at Thr642 resulting in enhanced GLUT4 translocation and glucose uptake (Ciaraldi et al. 2002; Roach et al. 2007; Baus et al. 2008; Taylor et al. 2008; Morissette et al. 2009; Peck et al. 2009). Accordingly, IGF1 may play crucial roles as a stimulator of both AMPK‐ and CaMKII‐independent glucose uptake through the activation of Akt and AS160 signaling pathways. In general, it is poorly understood how different modes of exercise, that is, acute RE and AE, differentially regulate IGF1/Akt/AS160 signaling and subsequent GLUT4 translocation. The purpose of this study was to identify specific cellular signal responses to acute RE, including IGF1 signaling, as compared with those to acute AE in respect with glucose metabolism. We additionally investigated the role of IGF1 on TBC1D1/AS160 phosphorylation and glucose uptake by using an in vitro model. Materials and Methods In vitro experiments Mouse C2C12 myoblasts (American Type Culture Collection, Manassas, VA) were cultured as described previously (Yokokawa et al. 2015). Briefly, cells were grown in Dulbecco's modified Eagle's medium (DMEM; 4.5 g glucose/L, Nacalai Tesque, Kyoto, Japan) containing 10% fetal bovine serum and 1% penicillin‐streptomycin (P/S). To initiate myogenic differentiation, the culture medium was replaced by DMEM containing 2% horse serum and 1% P/S. After 4 days of differentiation, myotubes were serum‐starved overnight and then incubated for 30 min in serum‐free medium containing 5 μmol/L 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR; Wako, Osaka, Japan), 1 μmol/L insulin (Novolin; Novo Nordisk, Bagsvaerd, Denmark), or 200 ng/mL IGF1 (PeproTech, Rocky Hill, NJ). In vivo experiments The study protocol was approved by the Ethics Committee for Animal Experiments at Ritsumeikan University, and was conducted in accordance with the Declaration of Helsinki. Forty‐two male Sprague–Dawley rats, aged 10 weeks (320–360 g), were obtained from CLEA Japan (Tokyo, Japan). The rats were divided into three groups: nonexercise control, RE, or AE. All rats were housed for 1 week in an environment maintained at 22–24°C with a 12:12‐h light–dark cycle, and were allowed food (CE2; CLEA Japan) and water ad libitum. The time course of changes in signaling protein levels was evaluated following RE and AE initiated after a 12‐h overnight fast, as detailed below. Rats were sacrificed 0, 1, or 3 h after completion of the exercise routine. Control rats were sacrificed at the basal state. Dissected gastrocnemius muscles were frozen rapidly in liquid nitrogen and stored at −80°C until use. RE protocol Under isoflurane anesthesia, hair was shaved off the right lower leg of each rat; the area was then cleaned with alcohol wipes. The rats were kept in a prone position with their right foot on the footplate and the ankle joint angle positioned at 90°. The triceps surae muscle was stimulated percutaneously with electrodes (Vitrode V, Ag/AgCl; Nihon Kohden, Tokyo, Japan) that were cut to 10 × 5 mm and connected to an electric stimulator and an isolator (SS‐104J; Nihon Kohden) (Nakazato et al. 2010). The right gastrocnemius muscle was exercised isometrically by stimulation with ten 3‐sec contractions per set for 5 sets. There was a 7‐sec interval between contractions and 3‐min rest intervals between sets. Voltage (~30 V) and stimulation frequency (100 Hz) were adjusted to produce maximal isometric tension. This exercise protocol is used widely as a RE model for animals (Ogasawara et al. 2013a,b, 2014; Tsutaki et al. 2013; Kido et al. 2015) and induces significant muscle hypertrophy (Ogasawara et al. 2013a,b). AE protocol Rats in the AE group were habituated to the treadmill by running for 30 min at 15 m/min, 45 min at 20 m/min, and 60 min at 25 m/min over a week. Three to five days after the last running habituation, rats were placed on a flat treadmill and made to run for 60 min at 25 m/min (Langfort et al. 1996). Analyses In vitro glucose uptake assay Glucose uptake was determined by measuring the glucose concentration of the medium as described previously (Yokokawa et al. 2015). In brief, culture media were collected and the glucose concentrations assayed spectrophotometrically using a Glucose II test kit (Wako). Measurements of serum IGF1, insulin, and glucose, and muscle IGF1 concentrations Insulin‐like growth factor 1 levels in the serum and skeletal muscle were determined using the mouse/rat IGF1 Quantikine ELISA kit (R&D Systems, Minneapolis, MN). Serum insulin levels were detected using a rat insulin ELISA kit (Shibayagi, Gunma, Japan), according to the manufacturer's instructions. Serum glucose concentrations were measured by the YSI 2300 STAT Plus analyzer (Yellow Springs Instrument, Yellow Springs, OH). Western blotting analyses Western blotting analyses were performed as reported previously (Goodman et al. 2011). Briefly, stimulated C2C12 myotubes were washed once with cold phosphate‐buffered saline (PBS) and lysed in radioimmunoprecipitation assay buffer containing 10 mmol/L Tris HCl (pH 7.4), 1% NP‐40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 150 mmol/L NaCl, and 5 mmol/L EDTA. The extracts were centrifuged at 13,700 g for 20 min at 4°C. Protein concentrations of the supernatants were determined using a protein assay kit (Nacalai Tesque). The lysates were mixed with 6× sample buffer containing 350 mmol/L Tris·HCl (pH 6.8), 10% SDS, 30% glycerol, 9.3% dithiothreitol, and 0.03% bromophenol blue, then boiled at 95°C for 5 min. Gastrocnemius muscles were homogenized in buffer containing 100 mmol/L Tris·HCl (pH 7.8), 1% NP‐40, 0.1% SDS, 0.1% sodium deoxycholate, 1 mmol/L EDTA, 150 mmol/L NaCl, and protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA). Homogenates were centrifuged at 13,700 g for 20 min at 4°C. The supernatant was removed, and the protein concentration determined using the Protein Assay Rapid kit (Wako). Samples were diluted in 3× sample buffer (1.0% β‐mercaptoethanol, 4.0% SDS, 0.16 mol/L Tris·HCl (pH 6.8), 43% glycerol, and 0.2% bromophenol blue), and boiled at 95°C for 5 min. Using 8–12% SDS‐polyacrylamide gels, 5 μg (for cell lysates) or 20 μg of protein (for muscle lysates) was separated by electrophoresis and transferred to polyvinylidene difluoride membranes. After the transfer, membranes were washed in Tris‐buffered saline containing 0.1% Tween 20 (TBST). Membranes were then blocked with 5% powdered milk in TBST for 1 h at room temperature. After blocking, the membranes were washed and incubated overnight at 4°C with primary antibodies against p‐Akt (Thr308), p‐Akt (Ser473), total Akt, p‐AMPK (Thr172), total AMPK, p‐AS160 (Thr642), total AS160, p‐CaMKII (Thr286), α‐tubulin (Cell Signaling Technology, Danvers, MA), total CaMKII (Santa Cruz Biotechnology, Santa Cruz, CA), or p‐TBC1D1 (Ser237) (Merck Millipore, Damstadt, Germany). The membranes were then washed again in TBST and incubated for 1 h at room temperature with the appropriate secondary antibodies. Chemiluminescent reagents (Luminata Forte Western HRP Substrate; Merck Millipore) were used to facilitate the detection of protein bands. Images were scanned using a chemiluminescence detector (ImageQuant LAS 4000; GE Healthcare, Buckinghamshire, UK). Band intensities were quantified using ImageJ 1.46 software (National Institutes of Health, Bethesda, MD). To assess the plasma membrane localization of GLUT4, plasma membranes were extracted as modified methods of previous study (Sato et al. 2009). Briefly, gastrocnemius muscles were homogenized into buffer A (20 mmol/L Tris [pH 7.4], 1 mmol/L EDTA, 0.25 mmol/L EGTA, 0.25 mol/L sucrose, 1 mmol/L DTT, 50 mmol/L NaF, 25 mmol/L sodium pyrophosphate, and 40 mmol/L β‐glycerophosphate). The homogenates were centrifuged at 400 g for 15 min at 4°C. The supernatant was centrifuged again at 249,138 g (Hitachi CS100GXII, Ibaraki, Japan) for 1 h at 4°C. The fractions were resuspended in buffer A and then homogenized to add equal volume buffer B containing 20 mmol/L Tris (pH 7.4), 1 mmol/L EDTA, 0.25 mmol/L EGTA, 2% Triton X‐100, 50 mmol/L NaF, 25 μmol/L sodium pyrophosphate, and 40 mmol/L β‐glycerophosphate. The homogenates were centrifuged at 274,052 g for 1 h at 4°C (Hitachi CS100GXII) and the supernatant was used as plasma membrane fraction. Prepared plasma membrane fraction was used for the measurement of plasma membrane GLUT4 level, which was determined by Western blotting analysis using antibodies against GLUT4 (Merck Millipore) (Sato et al. 2009). Statistical analysis All results are expressed as means ± SE. A one‐way ANOVA with a least significant difference post hoc test was used to evaluate changes among multiple groups, and the unpaired Student t‐test was used for two‐group comparisons (Yang et al. 2012). Results In vitro experiments AICAR stimulation AICAR, an AMPK activator, significantly increased the phosphorylation of AMPK (Thr172), TBC1D1 (Ser237), and AS160 (Thr642). Additionally, AICAR induced significant decreases in media glucose concentrations indicating an increase in glucose uptake (Fig. 1). Figure 1 Effects of AICAR on mouse C2C12 myotubes. Phosphorylation of AMPK at Thr172 (A), TBC1D1 at Ser237 (B), and AS160 at Thr642 (C) following stimulation with AICAR. Media glucose concentration indicating glucose uptake (D) relative to control (CON) following stimulation with AICAR. Data are presented as means ± SE (n = 5). *P < 0.05 versus CON. Insulin and IGF1 stimulation Akt phosphorylation at Thr308 and Ser473, and AS160 phosphorylation at Thr642, were increased significantly by stimulation with insulin and IGF1, whereas TBC1D1 phosphorylation at Ser237 was not changed. Additionally, insulin and IGF1 induced significant decreases in glucose concentrations in the media indicating an increase in glucose uptake (Fig. 2). Figure 2 Effects of insulin and IGF1 on mouse C2C12 myotubes. Phosphorylation of Akt at Thr308 (A) and Ser473 (B), AS160 at Thr642 (C), and TBC1D1 at Ser237 (D) following stimulation by insulin or IGF1. Media glucose concentration indicating glucose uptake (E) relative to control (CON) following stimulation by insulin or IGF1. Data are expressed as means ± SE (n = 6). *P < 0.05 versus CON. In vivo experiments Blood parameters Table 1 shows the changes in serum insulin, IGF1, and glucose levels. Serum insulin was not changed by RE and AE. Serum IGF1 was decreased significantly at 3 h after AE (P < 0.05) but not changed by RE. In the RE group, serum glucose was increased significantly immediately after exercise (P < 0.05), and returned to the baseline level by 3 h after exercise. In the AE group, serum glucose was increased significantly immediately after exercise (P < 0.05). Glucose dropped to below the baseline level at 1 h (P < 0.05) and returned to the baseline level by 3 h after AE. Table 1 Blood parameters CON RE AE 0H 1H 3H 0H 1H 3H Insulin (pmol/l) 11.3 ± 3.0 9.7 ± 1.4 7.7 ± 3.2 12.7 ± 2.1 10.3 ± 1.8 10.9 ± 1.0 9.12 ± 0.9 IGF1 (ng/mL) 1281.4 ± 51.6 1203.9 ± 115.6 1262.3 ± 60.1 1200.0 ± 54.1 1491.9 ± 57.1 1178.1 ± 115.3 937.9 ± 72.7a Glucose (mmol/L) 3.7 ± 0.3 7.2 ± 0.8a 9.9 ± 1.0a 4.0 ± 1.7 7.4 ± 0.3a 2.6 ± 0.1a 3.0 ± 0.2 Data are presented as means ± SE (n = 6). CON, control; RE, resistance exercise; AE, aerobic exercise. a P < 0.05 versus CON. John Wiley & Sons, LtdSkeletal muscle parameters AMPK was phosphorylated significantly immediately after both RE and AE (P < 0.05). AMPK phosphorylation remained high at 1 h after RE (P < 0.05). This was in contrast to AE‐induced AMPK phosphorylation that returned to baseline by 1 h after exercise (Fig. 3). Figure 3 Effects of RE and AE on AMPK. Phosphorylation of AMPK at Thr172 relative to total AMPK protein content following RE (A) or AE (B). Data are expressed as means ± SE (n = 6). *P < 0.05 versus CON. CON, control; RE, resistance exercise; AE, aerobic exercise. CaMKII phosphorylation was increased significantly immediately after RE and returned to the baseline level by 1 h after exercise. In the AE group, CaMKII was phosphorylated significantly immediately after exercise and returned to the baseline level at 3 h after AE (Fig. 4). Figure 4 Effects of RE and AE on CaMKII. Phosphorylation of CaMKII at Thr286 relative to total CaMKII protein content following RE (A) or AE (B). Data are expressed as means ± SE (n = 6). *P < 0.05 versus CON. CON, control; RE, resistance exercise; AE, aerobic exercise. TBC1D1 was phosphorylated significantly at Ser237 immediately after RE. The phosphorylation level returned to baseline by 3 h after RE. In the AE group, TBC1D1 phosphorylation tended to increase immediately after exercise and reached significance at 1 h. Phosphorylation levels returned to baseline by 3 h after exercise (Fig. 5). Figure 5 Effects of RE and AE on TBC1D1. Phosphorylation of TBC1D1 at Ser237 relative to total TBC1D1 protein content following RE (A) or AE (B). Data are expressed as means ± SE (n = 6). *P < 0.05 versus CON. CON, control; RE, resistance exercise; AE, aerobic exercise. Resistance exercise induced a significant (63%) increase in IGF1 expression in skeletal muscle at 1 h after exercise. This increase was maintained until 3 h after exercise. However, AE‐induced IGF1 expression was decreased significantly at every time point (P < 0.05) (Fig. 6). Figure 6 Effects of RE and AE on IGF1. The expression of skeletal muscle IGF1 relative to CON following RE (A) or AE (B). Data are expressed as means ± SE (n = 6). *P < 0.05 versus CON. CON, control; RE, resistance exercise; AE, aerobic exercise. Both RE and AE induced significant phosphorylation of Akt at Thr308 immediately after exercise (P < 0.05). p‐Akt (Thr308) levels returned to baseline by 1 h after exercise. Akt was also phosphorylated at Ser473 immediately after RE and AE (P < 0.05). Although AE‐induced p‐Akt (Ser473) returned to the baseline level at 1 h after exercise, this phosphorylation was maintained for 3 h after RE (P < 0.05) (Fig. 7). Figure 7 Effects of RE and AE on Akt. Phosphorylation of Akt at Thr308 and Ser473 relative to total Akt protein content following RE (A and C) or AE (B and D). Data are expressed as means ± SE (n = 6). *P < 0.05 versus CON. CON, control; RE, resistance exercise; AE, aerobic exercise. Phosphorylation of AS160 at Thr642 increased significantly at 1 h after RE (P < 0.05). This increase was sustained for 3 h (P < 0.05). In contrast, AE‐induced phosphorylation of AS160 occurred immediately after exercise (P < 0.05) and returned to the baseline level by 3 h (Fig. 8). Figure 8 Effects of RE and AE on AS160. Phosphorylation of AS160 at Thr642 relative to total AS160 protein content following RE (A) or AE (B). Data are expressed as means ± SE (n = 6). *P < 0.05 versus CON. CON, control; RE, resistance exercise; AE, aerobic exercise. The expression of GLUT4 on the plasma membrane was increased significantly 1 h after RE (P < 0.05). This increase was maintained until 3 h after RE (P < 0.05). AE induced a significant increase in plasma membrane GLUT4 immediately and 1 h after exercise (P < 0.05). Plasma membrane GLUT4 expression returned to the baseline level by 3 h after AE (Fig. 9). Figure 9 Effects of RE and AE on GLUT4. Total GLUT4 expression in plasma membrane following RE (A) or AE (B) relative to CON. Data are expressed as means ± SE (n = 6). *P < 0.05 versus CON. CON, control; RE, resistance exercise; AE, aerobic exercise. Discussion In this study, we investigated intracellular signaling and GLUT4 translocation in response to an acute bout of RE or AE in rat skeletal muscle. Skeletal muscle IGF1 expression was increased after RE but not AE, and subsequent GLUT4 translocation was sustained longer after RE as compared with AE. Additionally, we used an in vitro study to show that activation of AMPK/TBC1D1 and IGF1/Akt/AS160 signaling enhanced glucose uptake independently. These data provided crucial new information to explain the regulation of glucose uptake by acute RE through specific signals, and showed differences between acute RE and AE at the molecular level. The results of this study indicated that RE‐ and AE‐induced AMPK phosphorylation were similar. In previous studies, AMPK was also found to be phosphorylated immediately after exercise by both RE and AE, and no significant difference was observed between the responses to RE and AE (Rasmussen et al. 1998; McConell et al. 2008; Vissing et al. 2013; Ogasawara et al. 2014; Ahtiainen et al. 2015). The activation of AMPK was found previously to depend upon skeletal muscle contraction tension (Ihlemann et al. 1999). Additionally, the phosphorylation of AMPK depended upon the intensity of AE (i.e., %VO2max) (Chen et al. 2003; Sriwijitkamol et al. 2007). According to these previous studies, the magnitude of AMPK phosphorylation in response to RE and AE depends on exercise intensity. Therefore, further studies are needed to identify the magnitude of AMPK phosphorylation following RE and AE with various intensities. This is the first study comparing the time course of changes in CaMKII phosphorylation between RE and AE. The results showed that CaMKII was phosphorylated immediately after both acute RE and AE. This was consistent with a previous study showing that the phosphorylation of CaMKII was increased significantly immediately after acute AE (Rose et al. 2006). Together, these results demonstrate that both acute RE and AE induce CaMKII phosphorylation immediately after exercise. The phosphorylation of CaMKII apparently depended on Ca2+ released from the sarcoplasmic reticulum. Ca2+ release is increased by higher skeletal muscle contraction tension (Chin and Allen 1997). Accordingly, RE, which induces higher contraction tension than AE, might phosphorylate CaMKII to a greater extent than AE. However, the phosphorylation of CaMKII after acute RE returned to the baseline level faster than after acute AE. Thus, contraction tension might not be the most significant regulatory factor for CaMKII phosphorylation after AE. As a downstream signal of AMPK and CaMKII, the phosphorylation of TBC1D1 (Ser237) was measured after acute RE and AE and found to match the time course of changes in AMPK phosphorylation. To better support the functional relationship between these changes, TBC1D1 phosphorylation was measured in response to AICAR‐induced AMPK phosphorylation or insulin/IGF1 stimulation in vitro. The results showed that AMPK phosphorylation induced TBC1D1 phosphorylation. In contrast, insulin/IGF1‐induced Akt signal activation did not affect TBC1D1 phosphorylation at Ser237. A previous animal study showed that p‐TBC1D1 (Ser237) increased immediately after acute AE, and was diminished in AMPK knockout mice (Fentz et al. 2015). Based on these results, acute RE‐induced TBC1D1 phosphorylation at Ser237 may be also caused by AMPK phosphorylation. Acute RE‐induced IGF1 expression was assessed previously as a regulator of skeletal muscle hypertrophy (Ogasawara et al. 2013b). As a glucose uptake regulator, IGF1 was measured after acute RE and AE. The results showed that skeletal muscle IGF1 was increased 1 h after acute RE and maintained until 3 h. In contrast, acute AE did not augment intramuscular IGF1. Following augmented IGF1 expression, the increase in Akt phosphorylation seen in the RE was more prolonged than that in the AE group. As a downstream signal of Akt, AS160 phosphorylation was also maintained longer than AE. Moreover, using an in vitro model, we also determined that IGF1 stimulated Akt and AS160 phosphorylation. According to these results, Akt and AS160 phosphorylation after RE may be modulated by RE‐induced IGF1 expression, which may have contributed to prolonged increase in Akt and AS160 phosphorylation. Taken together, the IGF1 signaling, including prolonged Akt and AS160 phosphorylation, may be a specific signal response to acute RE. The early‐phase phosphorylation of Akt by both RE and AE may occur independently of IGF1 expression because there was no change in IGF1 expression immediately after exercise. Moreover, according to a previous study, the integrin/Akt signaling response occurred immediately after exercise (Klossner et al. 2009). Thus, Akt phosphorylation immediately after acute RE and AE may be caused by an integrin/Akt signal. This is the first study which directly compared AS160 phosphorylation in response to RE and AE. Previous studies showed that AS160 phosphorylation in rat skeletal muscle after swimming increased immediately after exercise, and it maintained until 3 h after exercise (Arias et al. 2007; Funai et al. 2009; Castorena et al. 2014). This is contrary to this study which demonstrated that AS160 phosphorylation returned to baseline level by 3 h after exercise. However, these previous studies used different mode of exercise (i.e., swimming vs. running) and analyzed epitrochlearis muscle. Acute swimming exercise‐induced PGC1α expression, which is downstream of AMPK, was different with acute running exercise on several skeletal muscles including epitrochlearis and gastrocnemius muscle (Terada and Tabata 2004). Additionally, epitrochlearis and gastrocnemius muscle have different muscle fiber composition (Castorena et al. 2011). These methodological differences between previous and this studies may have caused the differences in AS160 phosphorylation level and time course of response. This is also the first report that compared the time course of changes in GLUT4 translocation and upstream signal responses after acute RE and AE. Interestingly, enhanced GLUT4 translocation after acute RE was found at later time points, but was maintained longer than after acute AE. Previously, acute AE was reported to augment GLUT4 translocation immediately after exercise, and GLUT4 translocation returned to the baseline level by 2 h after exercise (Goodyear et al. 1990). Additional important results were that the RE‐induced increase in AS160 phosphorylation was more prolonged than TBC1D1 phosphorylation, and that acute RE‐induced GLUT4 translocation was also prolonged until the same time point. Furthermore, the induction of AS160/TBC1D1 phosphorylation and GLUT4 translocation was not as prolonged with AE as with RE. These findings may underscore the significance of AS160 signal activation on RE‐induced GLUT4 translocation. In the future study, we need to directly assess the glucose uptake to confirm our findings on GLUT4 translocation. Overall, our data showed that AMPK/TBC1D1 and IGF1/Akt/AS160 signal activation enhanced glucose uptake independently, and that acute RE activated these signals. Moreover, acute RE increased the expression of skeletal muscle IGF1 as a RE‐specific GLUT4 translocation regulator. Further studies might clarify the contribution of IGF1 signals to acute RE‐induced GLUT4 translocation by using an IGF1 knockout model. 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==== Front Physiol RepPhysiol Rep10.1002/(ISSN)2051-817XPHY2physreportsPhysiological Reports2051-817XJohn Wiley and Sons Inc. Hoboken 2756590410.14814/phy2.12912PHY212912Renal Conditions, Disorders and TreatmentsRenal FiltrationSignalling PathwaysOriginal ResearchOriginal ResearchPodocyte NF‐κB is dispensable for the pathogenesis of renal ischemia‐reperfusion injury M. Yamashita et al.Yamashita Maho 1 Yoshida Tadashi 1 2 Hayashi Matsuhiko 1 2 1 Apheresis and Dialysis CenterSchool of MedicineKeio UniversityTokyoJapan2 Department of General MedicineSchool of MedicineKeio UniversityTokyoJapan* Correspondence Tadashi Yoshida, Apheresis and Dialysis Center, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku, Tokyo 160‐8582, Japan. Tel:/Fax: 81‐3‐5363‐3908 E‐mail: tayoshida-npr@umin.ac.jp 26 8 2016 8 2016 4 16 10.1111/phy2.2016.4.issue-16e1291219 4 2016 05 7 2016 01 8 2016 © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.Abstract Podocytes play a central role in the formation of the glomerular filtration barrier in the kidney, and their dysfunction has been shown to result in multiple proteinuric kidney diseases. In this study, we sought to determine whether NF‐κB, a proinflammatory signaling, within podocytes was involved in renal ischemia‐reperfusion (I/R) injury. Podocyte‐specific IκBΔN transgenic (Pod‐IκBΔN) mice, in which NF‐κB was inhibited specifically in podocytes, were generated by the Cre‐loxP technology, and their phenotype was compared with control mice after bilateral renal ischemia. The effect of systemic administration of a NF‐κB inhibitor, pyrrolidinedithiocarbamate (PDTC), on renal I/R injury was also examined. Pod‐IκBΔN mice were phenotypically normal before surgery. Following renal I/R injury, serum concentrations of urea nitrogen and creatinine were elevated in both Pod‐IκBΔN and control mice to a similar extent, whereas PDTC treatment attenuated the elevation of these parameters. Renal histological damage in I/R‐injured Pod‐IκBΔN mice was also similar to I/R‐injured control mice, although it was improved by PDTC treatment. Moreover, I/R induced accumulation of inflammatory cells, such as neutrophils and macrophages, was reduced by PDTC treatment, but not by podocyte‐specific NF‐κB inhibition. These results provide evidence that the NF‐κB activity in podocytes does not contribute to the pathogenesis of renal I/R injury. Acute kidney injuryischemia‐reperfusionNF‐κBpodocytesJapan Society for the Promotion of Science16K0965515K09271Japanese Association of Dialysis Physicians2015‐6Kidney Foundation, JapanJKFB15‐7 source-schema-version-number2.0component-idphy212912cover-dateAugust 2016details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_NLMPMC version:4.9.4 mode:remove_FC converted:29.08.2016 M. Yamashita , T. Yoshida , M. Hayashi . Podocyte NF‐κB is dispensable for the pathogenesis of renal ischemia‐reperfusion injury . Physiol Rep , 4 (16 ), 2016 , e12912, doi: 10.14814/phy2.12912 Funding Information This study was supported by Grants‐in‐Aid for Scientific Research (KAKENHI) from Japan Society for the Promotion of Science to T.Y. (16K09655) and M.H. (15K09271) and grants from the Japanese Association of Dialysis Physicians (JADP Grant 2015‐6) to T.Y. and the Kidney Foundation, Japan (JKFB15‐7) to T.Y. ==== Body Introduction Renal ischemia‐reperfusion (I/R) injury following major surgical interventions is a leading cause of acute kidney injury (AKI) in hospitalized patients (Okusa et al. 2016). It is also the major cause of delayed graft function after cadaveric kidney transplantation. For the development of novel therapeutic approaches to prevent and/or treat these disease conditions, it is critical to identify the molecular factors and mechanisms underlying renal I/R injury. The nuclear factor‐κB (NF‐κB) family of transcription factors is involved in the inflammatory process in a variety of cells (Brown et al. 1995; Traenckner et al. 1995; Sanz et al. 2010). In resting cells, NF‐κB exists in the cytoplasm as an inactive dimer by binding to an inhibitory protein, IκB. Upon stimulation with inflammatory signals, IκB is phosphorylated on specific serine residues, serines 32 and 36, leading to its ubiquitination and consecutive proteasomal degradation. Released from IκB, NF‐κB is able to translocate into the nucleus, engage DNA, and initiate transcription of many genes including cytokines, chemokines, and cell adhesion molecules. Results of previous studies showed that NF‐κB and its target molecules, such as monocyte chemoattractant protein‐1 and tumor necrosis factor‐α (TNF), were induced in rat kidneys following renal I/R injury (Donnahoo et al. 2000; Sung et al. 2002). In addition, in vivo transfection of NF‐κB decoy oligonucleotides attenuated renal I/R injury in rats (Cao et al. 2004). Intravenous injection of siRNA specific for NF‐κB also ameliorated renal I/R injury in mice (Feng et al. 2009). Moreover, adenovirus‐mediated overexpression of A20, a negative regulator of NF‐κB, significantly reduced acute tubular necrosis and NF‐κB activation in response to renal I/R injury (Lutz et al. 2008). Most recently, systemic administration of a NF‐κB inhibitor, dehydroxymethylepoxyquinomicin, has been shown to ameliorate renal I/R injury in rats (Kono et al. 2013). Another NF‐κB inhibitor, pyrrolidinedithiocarbamate (PDTC), has also been shown to improve folic acid‐induced AKI in mice (Kumar et al. 2015). Results of these studies suggest that activated NF‐κB is a potential target for treating renal I/R injury. However, the pathogenesis of renal I/R injury is very complex and involves multiple cell types, including renal tubular epithelial cells, vascular endothelial cells, neutrophils, macrophages, and possibly podocytes (Miglio et al. 2011; Zhao et al. 2015; Okusa et al. 2016). Podocytes are required for the formation of the glomerular filtration barrier in the kidney (Reiser et al. 2010). They express a repertoire of cell‐specific proteins, such as nephrin, podocin, and synaptopodin, to retain albumin and other larger proteins in the blood. Although results of previous studies by our laboratory and others have shown that NF‐κB in podocytes plays a significant role in proteinuric kidney diseases (Brähler et al. 2012; Yamashita et al. 2016), its role in renal I/R injury remains unknown. The IκBΔN mice have been developed in our laboratory (Inoue et al. 2010; Yoshida et al. 2013). They contain the human IκBΔN transgene separated from a universal CAG promoter by a floxed STOP sequence. Following the activation of Cre recombinase, they express IκBΔN, which lacks its N‐terminal of 54 amino acids including two phosphorylation sites at serines 32 and 36, thereby continuously inhibiting NF‐κB activation as a superrepressor. In this study, podocyte‐specific IκBΔN transgenic (Pod‐IκBΔN) mice were generated by crossing IκBΔN mice with Nphs1‐Cre mice, which express Cre recombinase in a podocyte‐specific manner (Asano et al. 2005). By using these mice, we determined whether podocyte‐specific inhibition of NF‐κB affected the severity of renal I/R injury. Methods Pod‐IκBΔN mice Animal protocols were approved by Keio University Animal Care and Use Committee. Mice used in this study were on the C57BL/6J background. Pod‐IκBΔN (Nphs1‐Cre+/−/IκBΔN +/−) mice and control (Nphs1‐Cre+/−/IκBΔN −/−, Nphs1‐Cre−/−/IκBΔN +/−, or Nphs1‐Cre−/−/IκBΔN −/−) mice were generated by breeding Nphs1‐Cre mice (Asano et al. 2005) and IκBΔN mice (Inoue et al. 2010; Yoshida et al. 2013). Genotyping was performed by PCR as described previously (Inoue et al. 2010; Yoshida et al. 2013). Four to eight mice per each genotype and per each treatment were analyzed. Renal ischemia‐reperfusion injury Male Pod‐IκBΔN and control mice at 12–14 weeks of age were allowed free access to water and standard mouse chow. Animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (40 mg/kg). Kidneys were exposed through flank incisions. Mice were subjected to 35 min of bilateral renal ischemia or sham surgery, as previously described (Yoshida et al. 2016). Ischemia was induced by clamping both renal pedicles with nontraumatic microvessel clamps. The incisions were temporarily closed during ischemia or sham surgery. After the clamps were removed, reperfusion in the kidneys was visually confirmed. Some mice were intraperitoneally injected with 200 mg/kg PDTC (Sigma‐Aldrich, St. Louis, MO) 3 h before surgery. A duration of 24 or 72 h after reperfusion, the mice were killed under pentobarbital anesthesia, and blood samples as well as the kidneys were harvested. Kidneys were divided into multiple pieces for histological analyses and total RNA extraction. Serum urea nitrogen and creatinine Serum concentrations of urea nitrogen were determined by the urease‐indophenol method (Wako Pure Chemical, Osaka, Japan). Serum creatinine concentrations were measured by an enzymatic method (Wako Pure Chemical). Histology and Immunostaining The kidneys were fixed in 4% paraformaldehyde and embedded into paraffin. Sections (5‐μm) were prepared and subjected to hematoxylin–eosin staining and immunohistochemistry. Histological analyses were performed in a blind manner using an arbitrary scale, as described previously (Homma et al. 2014; Yoshida et al. 2016). Proteinaceous casts and tubular necrosis were graded as follows: 0 (no damage), 1 (patchy isolated damage), 2 (damage less than 25%), 3 (damage between 25% and 50%), and 4 (more than 50% damage). Immunohistochemistry was performed with antibodies for neutrophil (7/4; Abcam, Cambridge, MA) and F4/80 (CI:A3‐1; Abcam), as described previously (Yoshida et al. 2008, 2016; Yamashita et al. 2016). Staining was visualized by diaminobenzidine, and sections were counterstained by hematoxylin. RNA extraction and real‐time RT‐PCR Total RNA was extracted, and real‐time RT‐PCR was performed as described previously (Yoshida et al. 2008). Primer sequences were as follows: NGAL‐F: 5′‐AACATTTGTTCCAAGCTCCAGGGC‐3′ and NGAL‐R: 5′‐CAAAGCGGGTGAAACGTTCCTTCA‐3′. Primary culture of podocytes Primary culture of murine podocytes was performed as described previously (Yamashita et al. 2016). Cultured podocytes derived from Pod‐IκBΔN mice and control mice, respectively, were treated with 10 ng/mL TNF for 24 h, and subjected to immunofluorescence studies with antibodies for p65 (F6; Santa Cruz Biotechnology, Santa Cruz, CA) and podocin (Abcam). Statistical analyses Data are presented as mean ± SEM. Statistical analyses were done by SigmaPlot/SigmaStat9 (Systat Software Inc, San Jose, CA). After confirming that the data passed the normality test for parametric analyses, one‐way factorial ANOVA with a post hoc Fisher protected least significant difference test was performed (Figs. 1A,B, 4, 5C,D, and 6A). Nonparametric Kruskal–Wallis test was also performed (Figs. 2B,C, and 6B). P values < 0.05 were considered significant. Figure 1 The NF‐κB inhibitor, pyrrolidinedithiocarbamate (PDTC), but not podocyte‐specific NF‐κB inhibition, attenuated renal ischemia‐reperfusion (I/R) injury. Pod‐IκBΔN and control mice were subjected to bilateral renal ischemia for 35 min (I/R) or sham‐operation. A subset of mice were treated with 200 mg/kg PDTC intraperitoneally 3 h before I/R. Serum levels of urea nitrogen (A) and creatinine (B) were measured 24 h after reperfusion. n = 5–8 per each group. *P < 0.05 compared with sham‐operated mice. #P < 0.05 compared with I/R‐injured control mice. Figure 2 Pyrrolidinedithiocarbamate (PDTC), but not podocyte‐specific NF‐κB inhibition, improved renal histological damage following ischemia‐reperfusion (I/R) injury. Pod‐IκBΔN and control mice were subjected to bilateral renal ischemia for 35 min (I/R) or sham‐operation. A subset of mice were treated with 200 mg/kg PDTC intraperitoneally 3 h before I/R. Renal histology was examined 24 h after reperfusion. (A) Representative pictures of hematoxylin–eosin staining are shown. Bar: 100 μm. Arrows indicate intratubular casts. Arrowheads indicate tubular necrosis. B, C: Levels of the formation of proteinaceous casts (B) and tubular necrosis (C) were scored semiquantitatively. n = 5–8 per each group. *P < 0.05 compared with sham‐operated mice. Results A NF‐κB inhibitor, PDTC, attenuated renal I/R injury in mice Results of the previous studies showed that systemic administration of a NF‐κB inhibitor, dehydroxymethylepoxyquinomicin, ameliorated renal I/R injury in rats (Kono et al. 2013). To confirm and extend these results, we first examined the effect of PDTC, another NF‐κB inhibitor, on renal I/R injury. Male control mice at 12–14 weeks of age were intraperitoneally injected with PDTC, and then received bilateral I/R injury for 35 min. A duration of 24 h after reperfusion, serum levels of urea nitrogen significantly increased in control mice (66 ± 16 mg/dL), compared with sham‐operated mice (28 ± 2 mg/dL) (Fig. 1A). PDTC treatment attenuated the elevation of serum urea nitrogen following I/R injury (39 ± 4 mg/dL). Serum concentrations of creatinine exhibited a similar trend (Fig. 1B). Histological analyses revealed that PDTC treatment significantly improved the formation of proteinaceous casts and tubular necrosis, two histological features of renal I/R injury (Fig. 2). These results suggest that the systemic blockade of the NF‐κB activity attenuates renal I/R injury. Podocyte‐specific NF‐κB inhibition did not improve renal I/R injury To determine the cell‐autonomous role of the NF‐κB signaling in podocytes for renal I/R injury, we utilized Pod‐IκBΔN mice, in which NF‐κB was inhibited specifically in the podocytes. Results of our previous studies showed that Pod‐IκBΔN mice were phenotypically normal at the physiological conditions, and that the amount of proteinuria was significantly lower in Pod‐IκBΔN mice than control mice in adriamycin‐induced nephropathy (Yamashita et al. 2016). In this study, we first examined the localization of p65 in cultured podocytes derived from Pod‐IκBΔN mice and control mice, respectively. Results showed that p65 was retained in the cytoplasm following TNF treatment in cultured podocytes derived from Pod‐IκBΔN mice, whereas it was translocated into the nucleus in response to TNF in cultured podocytes derived from control mice (Fig. 3). These results suggest that the NF‐κB activity is selectively inhibited in podocytes in Pod‐IκBΔN mice. Figure 3 Tumor necrosis factor (TNF) induced p65 translocation from the cytoplasm to the nucleus in cultured podocytes derived from control mice, but not from Pod‐IκBΔN mice. Primary cultures of podocytes derived from Pod‐IκBΔN and control mice, respectively, were treated with TNF for 24 h. Expression of p65 (Red) as well as podocin (Green) was examined by immunofluorescence studies. Nuclear staining was performed with 4′,6‐diamidino‐2‐phenylindole (DAPI; Blue). Bar: 100 μm. Pod‐IκBΔN and control mice were subjected to renal I/R injury. As shown in Figure 1, serum concentrations of urea nitrogen as well as creatinine did not differ between Pod‐IκBΔN and control mice following I/R injury. I/R‐induced histological damage was also similar between Pod‐IκBΔN and control mice (Fig. 2). Moreover, I/R injury‐induced increases in expression of NGAL, a marker of AKI, were similar between Pod‐IκBΔN and control mice, whereas PDTC treatment attenuated I/R injury‐induced increase in NGAL expression (Fig. 4). These results suggest that the NF‐κB signaling in podocytes does not contribute to renal I/R injury. Figure 4 Pyrrolidinedithiocarbamate (PDTC), but not podocyte‐specific NF‐κB inhibition, attenuated ischemia‐reperfusion (I/R) injury induced increases in NGAL expression. Pod‐IκBΔN and control mice were subjected to bilateral renal ischemia for 35 min (I/R) or sham‐operation. A subset of mice were treated with 200 mg/kg PDTC intraperitoneally 3 h before I/R. A duration of 24 h after reperfusion, renal NGAL expression was examined by real‐time RT‐PCR. n = 5–8 per each group. *P < 0.05 compared with sham‐operated mice. I/R induced accumulation of inflammatory cells was reduced by PDTC treatment, but not by podocyte‐specific NF‐κB inhibition I/R injury has been shown to induce the infiltration of neutrophils and macrophages in the kidneys. Accumulation of these inflammatory cells was examined by immunohistochemistry. Results showed that both neutrophils and macrophages increased following renal I/R injury (Fig. 5). Although PDTC treatment reduced the number of these inflammatory cells in the kidneys, I/R induced accumulation of these cells did not differ between Pod‐IκBΔN and control mice (Fig. 5). These results suggest that podocyte NF‐κB does not play a significant role in the infiltration of inflammatory cells during renal I/R injury. Figure 5 Ischemia‐reperfusion (I/R) induced accumulation of inflammatory cells was reduced by pyrrolidinedithiocarbamate (PDTC), but not by podocyte‐specific NF‐κB inhibition. Pod‐IκBΔN and control mice were subjected to bilateral renal ischemia for 35 min (I/R) or sham‐operation. A subset of mice were treated with 200 mg/kg PDTC intraperitoneally 3 h before I/R. Accumulation of neutrophils (A and C) and macrophages (B and D) was examined 24 h after reperfusion. n = 5–7 per each group. A and B: Representative pictures of immunohistochemical staining for neutrophils (A) and macrophages (B) are shown. Neutrophils (A) and macrophages (B) were visualized by diaminobenzidine, and sections were counterstained with hematoxylin. Bars: 100 μm. C and D: The numbers of neutrophils (C) and macrophages (D) per five random fields in the kidneys were quantified. *P < 0.05 compared with sham‐operated mice. #P < 0.05 compared with I/R‐injured control mice. Podocyte NF‐κB also did not contribute to renal damage at later stage of AKI The effect of podocyte‐specific NF‐κB inhibition on later stage of AKI was also examined. A duration of 72 h after reperfusion, I/R injury induced increases in serum concentrations of urea nitrogen and creatinine were modest in both Pod‐IκBΔN and control mice (Fig. 6). These results do not suggest that the contribution of podocyte NF‐κB is different between early and late stages of AKI. Figure 6 Renal ischemia‐reperfusion (I/R) injury was modest at later stage of AKI. Pod‐IκBΔN and control mice were subjected to bilateral renal ischemia for 35 min (I/R) or sham‐operation. A subset of mice were treated with 200 mg/kg pyrrolidinedithiocarbamate (PDTC) intraperitoneally 3 h before I/R. Serum levels of urea nitrogen (A) and creatinine (B) were measured 72 h after reperfusion. n = 4 per each group. Discussion Results of the previous studies showed that the NF‐κB signaling in various renal cells contributed to multiple renal diseases. For example, adenovirus‐mediated overexpression of IκBΔN in renal tubular cells prevented tubulointerstitial injury in protein‐overloaded rats (Takase et al. 2003). Endothelial cell‐specific overexpression of IκBΔN attenuated hypertension‐induced albuminuria and renal damage in mice (Henke et al. 2007). Fibroblast‐specific inhibition of NF‐κB by IκBΔN transgene attenuated renal fibrosis in a unilateral ureteral obstruction model (Inoue et al. 2010). Moreover, we previously showed that podocyte‐specific inhibition of NF‐κB attenuated proteinuria in adriamycin‐induced nephropathy in mice (Yamashita et al. 2016). The effect of podocyte‐specific NF‐κB inhibition on proteinuric kidney disease was also examined by the deletion of NF‐κB essential modulator (NEMO), a subunit of the IκB kinase complex required for phosphorylation and proteasomal degradation of IκB (Brähler et al. 2012). The NEMO deletion in the podocytes reduced proteinuria in nephrotoxic sheep serum‐induced glomerulonephritis in mice (Brähler et al. 2012). Although the aforementioned studies provide evidence that the NF‐κB signaling in multiple renal cells play an important role in the pathogenesis of various renal disease conditions, results of this study demonstrate that the NF‐κB activity in podocytes does not contribute to renal I/R injury. Because systemic inhibition of NF‐κB has been shown to improve renal I/R injury (Cao et al. 2004; Lutz et al. 2008; Feng et al. 2009; Kono et al. 2013), the NF‐κB activity in other renal cell types except podocytes is likely to be involved in the pathogenesis of I/R injury. In future, it is required to determine the effect of deletion of the NF‐κB activity in proximal tubular cells, distal tubular cells, endothelial cells, fibroblasts, and immune cells, serially or in combination, on renal I/R injury using genetically modified mouse models. In addition, it should be noted that systemic NF‐κB inhibitors, including PDTC, have been shown to block other intracellular signaling pathways, and therefore alter the levels of the oxidative stress and nitric oxide generation (Tapia et al. 2008; Tugcu et al. 2008). It should be careful to interpret the data using these compounds. It is hoped that specific NF‐κB inhibitors are developed in future. Although this study does not provide evidence for the involvement of NF‐κB within podocytes in renal I/R injury, podocytes per se have been shown to participate in the pathogenesis of renal I/R injury. For example, results of the previous studies showed that renal I/R injury induced structural damage to the integrity of podocytes, as assessed by the electron micrography (Zhao et al. 2015). They also showed that the structural changes in podocytes were accompanied by the upregulation of TRPC6 expression. It is possible that the induction of TRPC6 and subsequent Ca2+ influx, rather than the activation of NF‐κB, are the main signaling pathway for I/R‐induced damage in podocytes. Moreover, using cultured human podocytes, peroxisome proliferator‐activated receptor agonists were shown to prevent apoptotic cell death induced by oxygen/glucose deprivation‐reoxygenation, an in vitro model of renal I/R injury (Miglio et al. 2011). Although the results of this study showed that the NF‐κB signaling in podocytes does not play a significant role in renal I/R injury, they do not contradict these previous studies. Podocytes are still one of the possible cellular targets for the treatment of renal I/R injury. In summary, the results of this study provide evidence that the NF‐κB signaling in podocytes does not contributes to renal I/R injury. Further studies are needed to identify the renal cell types where NF‐κB plays a key role in this disease condition. Conflict of Interest None declared. ==== Refs References Asano , T. , F. Niimura , I. Pastan , A. B. Fogo , I. Ichikawa , and T. Matsusaka . 2005 Permanent genetic tagging of podocytes: fate of injured podocytes in a mouse model of glomerular sclerosis . J. Am. Soc. Nephrol. 16 :2257 –2262 .15987751 Brähler , S. , C. Ising , H. Hagmann , M. Rasmus , M. Hoehne , C. Kurschat , et al. 2012 Intrinsic proinflammatory signaling in podocytes contributes to podocyte damage and prolonged proteinuria . Am. J. Physiol. Renal. Physiol. 303 :F1473 –F1485 .22975019 Brown , K. , S. Gerstberger , L. Carlson , G. Franzoso , and U. Siebenlist . 1995 Control of IκB‐α proteolysis by site‐specific, signal‐induced phosphorylation . Science 267 :1485 –1488 .7878466 Cao , C. C. , X. Q. Ding , Z. L. Ou , C. F. Liu , P. Li , L. Wang , et al. 2004 In vivo transfection of NF‐κB decoy oligonucleotides attenuate renal ischemia/reperfusion injury in rats . Kidney Int. 65 :834 –845 .14871403 Donnahoo , K. K. , D. R. 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==== Front Physiol RepPhysiol Rep10.1002/(ISSN)2051-817XPHY2physreportsPhysiological Reports2051-817XJohn Wiley and Sons Inc. Hoboken 10.14814/phy2.12954PHY212954ErratumErratumErratum 24 8 2016 8 2016 4 16 10.1111/phy2.2016.4.issue-16e12954© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.10.14814/phy2.12871 source-schema-version-number2.0component-idphy212954cover-dateAugust 2016details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_NLMPMC version:5.0.2 mode:remove_FC converted:20.01.2017 ==== Body In 1, the surname of author Per T. Sangild was misspelled as “Sanglid.” The online version of the article has been corrected. ==== Refs Reference 1 Bergstorm , A. , S. S. Kaalund , K. Skovgaard , A. D. Andersen , B. Pakkenberg , A. Rosenorn , R. M. van Elburg , T. Thymann , G. O. Greisen , and P. T. Sangild . 2016 Limited effects of preterm birth and the first enteral nutrition on cerebellum morphology and gene expression in piglets . Physiol Rep 4 :e12871 .27462071

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==== Front Front Endocrinol (Lausanne)Front Endocrinol (Lausanne)Front. Endocrinol.Frontiers in Endocrinology1664-2392Frontiers Media S.A. 10.3389/fendo.2016.00116EndocrinologyMini ReviewThe Circulatory and Metabolic Responses to Hypoxia in Humans – With Special Reference to Adipose Tissue Physiology and Obesity Heinonen Ilkka H. A. 123*Boushel Robert 4Kalliokoski Kari K. 11Turku PET Centre, University of Turku, Turku, Finland2Department of Clinical Physiology and Nuclear Medicine, University of Turku, Turku, Finland3Division of Experimental Cardiology, Thoraxcenter, Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands4School of Kinesiology, University of British Columbia, Vancouver, BC, CanadaEdited by: Daniela Patrizia Foti, Magna Graecia University, Italy Reviewed by: Marta Letizia Hribal, Magna Graecia University, Italy; Sinan Tanyolac, Istanbul University, Turkey *Correspondence: Ilkka H. A. Heinonen, ilkka.heinonen@utu.fiSpecialty section: This article was submitted to Genomic Endocrinology, a section of the journal Frontiers in Endocrinology 29 8 2016 2016 7 11608 3 2016 10 8 2016 Copyright © 2016 Heinonen, Boushel and Kalliokoski.2016Heinonen, Boushel and KalliokoskiThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Adipose tissue metabolism and circulation play an important role in human health. It is well-known that adipose tissue mass is increased in response to excess caloric intake leading to obesity and further to local hypoxia and inflammatory signaling. Acute exercise increases blood supply to adipose tissue and mobilization of fat stores for energy. However, acute exercise during systemic hypoxia reduces subcutaneous blood flow in healthy young subjects, but the response in overweight or obese subjects remains to be investigated. Emerging evidence also indicates that exercise training during hypoxic exposure may provide additive benefits with respect to many traditional cardiovascular risk factors as compared to exercise performed in normoxia, but unfavorable effects of hypoxia have also been documented. These topics will be covered in this brief review dealing with hypoxia and adipose tissue physiology. hypoxiahumansblood flowmetabolismadipose tissue ==== Body General Cardiovascular and Metabolic Responses to Hypoxia A large body of knowledge on the physiological effects of hypoxia has been obtained over several decades from field experiments in the mountains as well as from studies in environmental chambers, where ambient air is manipulated. Hypoxia, defined as reduced or insufficient oxygen supply caused by reduced oxygen saturation of arterial blood, results in cardiovascular system adjustments to deliver more blood to tissues to compensate for reduced oxygen delivery, which is sensed by oxygen-sensing mechanisms, such as carotid bodies (1). The acute central cardiovascular response to hypoxic stress triggers an increased heart rate at an unchanged stroke volume mediated primarily by increased sympathetic neural discharge as a function of increasing hypoxic severity. At rest, lower levels of hypoxic exposure may result in some degree of systemic vasodilation, while with increasing severity of hypoxia, the peripheral vasculature constricts to redistribute oxygen delivery to the most critically dependent organs, e.g., heart (2–5), brain (6–8) needs to be ensured. This regulation is exacerbated in obstructive sleep apnea, which creates a physiological condition called chronic intermittent hypoxia, which may compromise some functions of the body. Similarly, during exercise in hypoxia, perfusion of skeletal muscle is increased to match oxygen demand, which creates circulatory competition between the locomotor skeletal muscles and other organs, and leads to decreased exercise capacity with severity of hypoxia. In addition to cardiovascular stress, hypoxia also alters energy metabolism of the body (Figure 1). Although hypoxia might theoretically even slightly decrease the oxygen requirements at the local tissue level due to reduced oxygen supply, increased sympathetic neural activation and resulting release of various stress hormones often cause whole body metabolism to increase in response to hypoxia (9–14). It has been postulated that, particularly, glucose uptake might be favorably affected by hypoxia (13–17), which has implications for the prevention and treatment of disease states, where metabolism is deranged, such as in diabetes. Not every study, however, supports that view, as decreased skeletal muscle insulin sensitivity (18) and impaired lipid metabolism (19) have also been reported after chronic hypoxic exposures. Hypoxia also alters adipose tissue circulation, which plays an integral role in its metabolism and, therefore, has implications for obesity and diabetes. Figure 1 The effects of obesity, acute exercise, hypoxic or altitude exposure, and endurance training on adipose tissue, such as subcutaneous adipose tissue, surrounding thigh musculature as illustrated by the fusion image in the middle of the figure obtained by combining magnetic resonance and positron emission tomography imaging. Obesity induces negative (red arrow) inflammatory state in adipose tissue connected with capillary rarefaction and local hypoxia. Acute low or moderate intensity exercise is on the other hand capable of increasing adipose tissue blood flow and metabolism, which, in the long run, reduces adipose tissue cell size and inflammation. This effect may be potentiated by hypoxic exposure or altitude training, but scientific evidence is still in its infancy to prove this hypothesis correct. It also remains to be investigated to what extent classical endurance training can affect adipose tissue blood flow and metabolism in humans. Hypoxia and Adipose Tissue Circulation and Metabolism Adipose tissue has an important role in regulating metabolism (20–22) – a topic of growing interest as levels of obesity have increased globally over the last several decades. Adipose tissue vasculature and oxygen supply is an important determinant of its metabolism as well as endocrine function (23, 24). Despite the fact that adipose tissue has a capillary surface area less than one-third of that in skeletal muscle (24), it has long been acknowledged that also adipocytes are surrounded by an extensive network of capillaries (23). This vascular feature importantly affects the adaptability of subcutaneous adipose tissue to excess caloric overload, which is known to be associated with a hypoxic state in adipose tissue (25–27). Thus, although opposite views have also been presented (28), it is the common consensus that due to the insufficient blood supply and capillary rarefaction connected with tissue inflammation (20, 25, 29–35), chronic low oxygen levels in expanded adipose tissue is now well appreciated to contribute to metabolic derangements of the whole body. Although there is also a noticeable extent of variability in physiological responses to hypoxia in humans (36), particularly white adipose tissue is known to respond remarkably to low levels of oxygen. This fact is well illustrated by cell culture studies, where exposure of adipocytes to low oxygen levels alters the gene expression of over 1000 genes (25). However, no change in subcutaneous adipose tissue blood flow is necessarily observed at rest in humans in response to moderate systemic hypoxia (37). Adipose tissue blood flow in humans is under the regulation of the sympathetic nervous system (38), and it is, therefore, reasonable to assume that moderate systemic hypoxia simply does not create a high enough stimulus for sympathetic neural vasoconstrictor activation to reduce blood flow in healthy human adipose tissue. On the other hand, it is also plausible that the activation of vasoconstriction by arterial chemoreceptors predominates over a local hypoxic vasodilation in adipose tissue in humans. In this regard, adipose tissue appears to be similar to bone (39). However, increased blood flow in response to systemic hypoxia has been documented in human skin (40). More studies are clearly warranted to explore whether unchanged hypoxic blood flow is also of importance to explain pathophysiological characteristics of adipose tissue under chronically low oxygen levels that is not compensated by increased blood flow (20, 25). It is known that subcutaneous adipose tissue blood flow increases in response to low intensity exercise, but levels off when exercise intensity is further increased (41). Furthermore, at rest, but not during exercise, subcutaneous adipose blood flow is under the control of nitric oxide (42). In contrast to resting conditions, subcutaneous adipose blood flow is reduced during exercise, when subjects breathe hypoxic air (37). This novel finding is likely based on the constriction of adipose tissue vasculature by hypoxia-triggered enhanced sympathetic nervous system activity, which redistributes limb blood flow to exercising muscles, which depend more critically on adequate oxygen supply in response to exercise. In this regard, we have previously reported that blood flow in subcutaneous adipose tissue is significantly lowered by local infusion of norepinephrine, which is the principal neurotransmitter released from the sympathetic nerve endings, and that the inhibition of α-adrenergic receptors by phentolamine tends to enhance adipose tissue blood flow, both at rest and during exercise (38). It has also been previously suggested by Romijn et al. (43) that the reduction of adipose tissue blood flow is likely to be one important mechanism to explain decreased free-fatty acid release in response to high intensity exercise, which then leads to preferential utilization of glucose instead of fatty acids and contributes to the increased efficiency of ATP generation for a limited O2 availability. Additionally, it has been recently documented that the inability to increase vascular resistance in adipose tissue during exercise or to maintain mean arterial pressure during orthostatic stress in aging is largely a result of reduced α-adrenergic responsiveness of adipose tissue arterioles (44, 45). Therefore, it is concluded that reduced blood flow in adipose tissue is an acute physiological response to diminished oxygen availability during exercise, while higher blood flow in adipose tissue is needed in response to prolonged exercise that also likely associates with higher lipolysis to supply more free-fatty acids into circulation to sustain muscular work for prolonged periods (46). In addition to the general hypoxic responses, the capacity of blood flow in human subcutaneous adipose tissue has remained largely unexplored, until recently. In this regard, a novel finding is that the vasodilatory capacity of human subcutaneous adipose tissue determined by infusion of exogenous dilator compounds approaches the physiological level reached during moderate intensity exercise (37). Furthermore, during this maximal vasodilation, vascular conductance can reach a level even higher than that induced by exercise. In terms of absolute values, the comparison of adipose tissue blood flow capacity to skeletal muscle is also of interest. In this regard, we have previously reported that blood flow in human skeletal muscle during a similar pharmacological vasodilation protocol increases to a level of 40 ml/min/100 g (47). As the absolute average value of pharmacologically induced adipose tissue blood flow was 10.5 ml/min/100 g, it only reaches ~26% of blood flow level in the muscle. Accordingly, the functional vascular capacity appears to be very closely followed by that of structural anatomy, as adipose tissue is known to have a capillary surface area that is slightly less than one-third than that in skeletal muscle (24). In relative terms, blood flow in adipose tissue increased 8-fold and blood flow in muscle 14-fold in response to pharmacological (adenosine) infusion, and, as such, the increase in adipose tissue flow is 57% of that of muscle. In contrast to human skeletal muscle (47), pharmacologically induced blood flow is not, however, positively and significantly related to subjects’ whole body maximal oxygen consumption determined in a separate fitness test, indicating that blood flow in adipose tissue and muscle do not simply parallel each other. Nevertheless, it can be concluded, based on these studies, that the functional blood flow capacity of adipose tissue is fairly large in healthy human subjects. It remains, however, to be measured if this capacity is lost in pathological states. Furthermore, it also remains to be determined if a loss of functional vascular capacity is linked to impaired fat storage in white adipose tissue which is known to contribute to metabolic and cardiovascular derangements in a human body (20). Hypoxia as a Treatment of Obesity and Impaired Adipose Tissue Physiology? As summarized in the beginning of the previous section, it is evident that there is a hypoxic state in adipose tissue of obese subjects, which may be caused by insufficient circulatory responses/adaptations in response to lowered oxygen supply. Despite this, chronic and/or intermittent hypoxia has also been suggested as treatment option for overweight and obesity (9, 10). This is based on findings that hypoxia alters the function of the nervous system and hormonal levels such as leptin, which lead to changes in glucose metabolism and control of appetite (9–14). These physiological responses are enhanced with increasing severity of hypoxia, such as altitude exposure. There is evidence that people living at high altitude are less likely to be overweight and/or obese, the findings which hold after adjustment for many plausible confounding factors that might also affect the association (48, 49). Protective effects of hypoxia/altitude have also been reported in regards to development of diabetes (50) and coronary heart disease, as well as stroke (51–53), meaning that hypoxia reduced the incidence of these diseases (Table 1). Furthermore, interventional trials have been conducted to test the effects of hypoxia as a treatment for weight loss and improvement of metabolic functions (Table 1). These studies demonstrated that 7 h of moderate hypoxia under resting conditions did not change postprandial glucose responses or substrate oxidation in young healthy men (54). However, when hypoxic exposure was combined with low intensity physical activity, Netzer and colleagues reported greater weight loss in obese subjects when compared to combined exercise and sham hypoxia intervention (55), although this finding could not be reproduced in their recent study (56). Beneficial effects of hypoxia regarding body weight control have also been reported in obese young adults (57). Furthermore, Haufe et al. comprehensively investigated numerous cardiovascular risk factors in response to hypoxic training and showed favorable influences on body fat content, triglycerides, fasting insulin, and insulin sensitivity, as compared to exercise training only intervention (58). These findings were confirmed in their later study in overweight and obese men with lower exercise workload, which reduces exercise burden for overweight subjects and is, thus, beneficial in terms of exercise compliance (59). Appetite regulation is not necessarily always affected, although lipid profile is improved (60). Altogether, it is concluded that training in hypoxia appears to have numerous additive and favorable effects on traditional cardiovascular risk factors, which may have important clinical implications (61, 62). Table 1 Summary of studies investigating the effects of hypoxia on cardiovascular and metabolic health in humans. Reference Type of the study Outcome(s) Voss et al. (48) Epidemiological Lower rates of new obesity diagnoses among overweight persons at high altitude Voss et al. (49) Epidemiological Obesity prevalence inversely associated with elevation and urbanization Woolcott et al. (50) Epidemiological Inverse association between diabetes and altitude Ezzati et al. (51) Epidemiological Living at higher altitude had a protective effect on ischemic heart disease and a harmful effect on chronic obstructive pulmonary disease. No net effect on life expectancy or associations with stroke and cancer after adjustments for confounders Faeh et al. (52) Epidemiological Linearly decreased ischemic heart disease mortality with increasing altitude Faeh et al. (53) Epidemiological Lower mortality from coronary heart disease and stroke at higher altitudes Morishima and Goto (54) Acute 7 h experimental trial at rest No effect of hypoxia on postprandial glucose responses or substrate oxidation in young healthy men Netzer et al. (55) Exercise training in normobaric hypoxia Significantly greater weight loss in obese persons in real hypoxia than in sham hypoxia Gatterer et al. (56) A randomized, single blind, placebo-controlled study No larger reductions in body weight due to moderate intensity exercise and rest in hypoxia compared to normoxia alone in obese subjects Kong et al. (57) Experimental trial Normobaric hypoxia training caused more weight loss than normoxia training in obese young adults Haufe et al. (58) Single blind exercise training under hypoxia or normoxia Endurance training in hypoxia resulted in a similar or even better response in terms of cardiovascular and metabolic risk factors than endurance exercise in normoxia Wiesner et al. (59) Single blind exercise training under hypoxia or normoxia Training in hypoxia elicited a similar or even better response in terms of physical fitness, metabolic risk markers, and body composition at a lower workload in obese subjects Debevec et al. (60) Hypoxic confinement at simulated altitude with and without daily moderate intensity exercise Body mass decreased in both groups, but whole body fat mass was only reduced in the exercise group. No change in hormonal appetite regulation, but improved lipid profile due to combined training and hypoxia exposure Bailey et al. (61) 4-day experimental trial An additive cardioprotective effect of normobaric hypoxia training over training in normoxia Wee and Climstein (62) A review of 25 hypoxic training trials Hypoxic training may be beneficial as an adjunct treatment to modify some cardiometabolic risk factors Despite plausible physiological mechanisms and some encouraging results that hypoxia might indeed work as a potential therapeutic tool to tackle obesity, it may also have detrimental influences that need some consideration. First, hypoxia might not be well-tolerated by all subjects, as high-altitude illness is experienced in approximately 10 to 25% of unacclimatized persons above 2500 m, and the prevalence and severity of symptoms increases with increments in altitude (63). Second, hypoxia is associated with impaired cognitive performance, which may persist even after the cessation of hypoxic exposure (64). Third, hypoxic exposure is known to impair human immune system function (65), which may be detrimental in fighting against pathogens and other triggers of communicable diseases. Fourth, hypoxia is capable of inducing fibrosis in cardiac muscle (66), which increases the stiffness of the heart. Many obese individuals already have cardiac stiffness (67, 68), which may be exaggerated by hypoxic exposure. Finally, as mentioned in the previous section, hypoxia triggers an inflammatory response in adipose tissue of obese subjects, which may be further exacerbated by hypoxia creating a vicious-cycle. Thus, hypoxia not only alters human energy metabolism, which may lead to weight loss if not compensated for by increased energy intake, but is also capable of inducing several physiologically detrimental effects on bodily functions. It is likely that the balance of all these determine the overall outcome and health effects of hypoxia in humans. Finally, as hypoxia is indeed a common feature of adipose tissue in particular, and potentially other tissues in obese subjects, it has been suggested that hyperoxia might be an option to overcome the hypoxic state. However, as oxygen is known to be toxic in high concentrations, this treatment may not be healthy in terms of circulatory and metabolic function. Hyperoxia is known to decrease adipose cell viability, increase both intra- and extracellular oxidative stress, provoke inflammation, and decrease glucose uptake of adipocytes (69). Hence, based on this information on hyperoxia and reviewed knowledge regarding hypoxia, it is concluded that there is a delicate balance of healthy oxygen supply and demand in adipose tissue that determines its overall function. While mild hypoxia over a sufficient duration of exposure may provide some additional benefits, the most feasible approach to address obesity and individual weight loss appears to reside in more traditional methods proven to be efficient in reducing adipose tissue size: physical activity and diet rich in fruits and vegetables, but low in caloric energy (Figure 1). Author Contributions IH drafted the manuscript and all authors contributed to its revision and intellectual content. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MH and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review. 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==== Front Front ImmunolFront ImmunolFront. Immunol.Frontiers in Immunology1664-3224Frontiers Media S.A. 10.3389/fimmu.2016.00322ImmunologyOriginal ResearchTransient Migration of Large Numbers of CD14++ CD16+ Monocytes to the Draining Lymph Node after Onset of Inflammation Lund Hege 1Boysen Preben 1*Åkesson Caroline Piercey 2Lewandowska-Sabat Anna Monika 2Storset Anne K. 11Department of Food Safety and Infection Biology, Norwegian University of Life Sciences, Oslo, Norway2Department of Basic Sciences and Aquatic Medicine, Norwegian University of Life Sciences, Oslo, NorwayEdited by: Fulvio D’Acquisto, Queen Mary University of London, UK Reviewed by: Lucy V. Norling, Barts and The London School of Medicine and Dentistry, UK; Paola Italiani, National Research Council, Italy *Correspondence: Preben Boysen, preben.boysen@nmbu.noSpecialty section: This article was submitted to Inflammation, a section of the journal Frontiers in Immunology 29 8 2016 2016 7 32205 7 2016 15 8 2016 Copyright © 2016 Lund, Boysen, Åkesson, Lewandowska-Sabat and Storset.2016Lund, Boysen, Åkesson, Lewandowska-Sabat and StorsetThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.The dynamics of skin-draining cells following infection or vaccination provide important insight into the initiation of immune responses. In this study, the local recruitment and activation of immune cells in draining lymph nodes (LNs) was studied in calves in an adjuvant-induced inflammation. A transient but remarkably strong recruitment of monocytes was demonstrated after onset of inflammation, constituting up to 41% of live cells in the draining LNs after 24 h. Numerous CD14+ cells were visualized in subcutaneous tissues and draining LNs, and the majority of these cells did not express dendritic cell-associated markers CD205 and CD11c. In the LNs, recruited cells were predominately of a CD14++ and CD16+ phenotype, consistent with an intermediate monocyte subset characterized to possess a high inflammatory potential. Moreover, monocytes from the draining LN showed a high expression of genes coding for pro-inflammatory cytokines, including IL-1β, IL-6, TNFa, and TGFβ. Shortly after their appearance in the LN cortical areas, the monocytes had moved into the medulla followed by an increase in peripheral blood. In conclusion, this study provides novel information on in vivo monocyte recruitment and migration after onset of inflammation. monocytesinflammationlymph nodemigrationpro-inflammatory cytokinesNorges Forskningsråd10.13039/501100005416Norges Miljø- og Biovitenskapelige Universitet10.13039/501100008119 ==== Body Introduction A protective immune response to infection or vaccination is dependent on the recruitment of immune cells to the inflamed tissue, followed by their activation and the subsequent movement of cells and antigens to the draining lymph node (LN). In this respect, the migration of antigen-loaded dendritic cells (DCs) and recirculating lymphocytes has been extensively studied [reviewed by Girard et al. (1)]. Circulating monocytes are traditionally regarded as short-lived precursors of tissue macrophages and monocyte-derived DCs (moDCs), recruited to tissues for supplementation of these cell populations during homeostasis and for expansion during inflammation (2, 3). The conventional view is that DCs rather than monocytes subsequently migrate from the inflamed tissues to LNs (1). However, monocytes display an array of pattern recognition receptors, which enables them to react to danger and pathogenic stimuli and produce cytokines, and recent studies indicate that monocytes may have distinct effector functions of their own, including the transport and presentation of antigen (4–9), functions that were previously designated to DCs only. In humans, monocytes can be classified into subsets based on their expression of the lipopolysaccharide (LPS) receptor CD14 and the FcγIIIR CD16 (10, 11). Classical monocytes are CD16-negative and form the major population in blood. The minor CD16-positive monocyte population can be further subdivided into a CD14+ CD16++ non-classical subset and a third less well-defined CD14++ CD16+ intermediate subset, suggested to represent a transitional subset between the classical and non-classical monocytes (12). Whereas the classical and intermediate subsets possess pro-inflammatory properties, the non-classical subset may serve a patrolling function (10, 13, 14). However, the precise roles of the different monocyte subsets, and in particular intermediate monocytes, are not well defined neither in the steady state nor under different inflammatory conditions. The most realistic approach to reach experimental evidence for such roles is in vivo animal studies. Circulating bovine monocytes have recently been described as phenotypically similar to humans, as the same three subsets based on CD14 and CD16 expression have been recognized in cattle (15–17). Thus, the use of the cow as an animal model may overcome some of the challenges of the large phenotypical differences between mouse and human monocytes (18). The trafficking of monocytes is mediated by a multitude of chemokine receptors, and the different subsets show different receptor expression profiles. Especially the CC-chemokine receptor 2 (CCR2) and the CX3C-chemokine receptor 1 (CX3CR1) can be applied to distinguish between different subsets in humans and mice (11, 19, 20). Human classical monocytes express high levels of CCR2 and low levels of CX3CR1 and are accordingly poised to traffic to sites of infection and inflammation, whereas non-classical monocytes have a high expression of CX3CR1 (19, 21, 22). As an intermediate subset, CD14++ CD16+ monocytes most likely express both receptors. However, since the majority of studies on this topic refer to CD14+ versus CD16+ monocytes or to mouse monocyte subsets, this is not fully resolved. Adhesion molecules, such as L-selectin (CD62L) are also important in monocyte trafficking, enabling their adhesion to endothelium and transmigration into tissue. The majority of studies describing phenotypical and functional characteristics of myeloid cells are based on in vitro differentiated blood-derived monocytes. The aim of this study was to characterize the in vivo recruitment and activation of immune cells in inflamed tissue and the draining LN, using a bovine model. For this purpose, we used a saponin-based adjuvant, which has been shown to induce both humoral and cellular immunity (23), and an efficient induction of leukocyte recruitment to the draining LN of mice (24, 25). We show here that the induction of inflammation in calves resulted in a surprisingly potent recruitment of cells to the draining LN, dominated by CD14++ CD16+ monocytes. The migrating cells retained their monocytic character rather than differentiating into moDCs, and showed a high expression of genes coding for pro-inflammatory cytokines. Altogether, these results provide novel information on the phenotype and functional capacity of monocytes after the onset of inflammation, and challenge the conventional view of monocyte trafficking in vivo. Materials and Methods Animals and Experimental Design Animals were clinically healthy Norwegian Red dairy (NRF) calves of both sexes, of 8–9 weeks of age, raised in commercial Norwegian dairy farms. The first trial included 14 animals distributed into four experimental groups and kept in separate pens: 6 calves served as non-injected controls, whereas 8 calves were injected with 500 μg Matrix-Q™ (a kind gift from Novavax AB, Uppsala, Sweden). The adjuvant was suspended in 2 ml sterile Hanks’ balanced salt solution (Gibco, Life Technologies) prior to injection and administered as a single subcutaneous dose in the left posterior flank region, in an area drained by the subiliac LN. The contralateral skin and subiliac LN were untreated. A second trial included four new calves, which received the same treatment as in the first trial in addition to an injection with a 10-fold lower dose (50 μg) of Matrix-Q™ in the left neck region, in an area drained by the superficial cervical LN. The results presented herein refer to the first trial, unless otherwise stated. Calves were given acidified milk or milk replacer, calf concentrate, water ad lib and access to straw, and the health status of the animals was examined twice daily. All experimental procedures were conducted in accordance with the laws and regulations controlling experiments using live animals in Norway: the Norwegian Animal Welfare Act of 28 December 2009 and the Norwegian Regulation on Animal Experimentation of 15 January 1996. The study was approved by the Norwegian Animal Research Authority (Norwegian Food Safety Authority). Tissue Collection and Preparation EDTA blood samples were collected prior to adjuvant injection (pre-injected samples), and from the experimental groups at 24 h (n = 3), 48 h (n = 3), and 96 h (n = 2) post-injection in the first trial, and at 24 h (n = 4) post-injection in the second trial. The 96 h group was also sampled for blood at 72 h. Hematological differential counting was performed on EDTA blood (Advia® 2120 Hematology System, Siemens AG, Erlangen, Germany). Bovine peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation (2210 × g, 30 min) on lymphoprep (Axis-Shield, Norway), and either analyzed immediately by flow cytometry (FCM), or added freezing medium (Recovery™ cell culture freezing medium, Gibco) for further storage in liquid nitrogen. Subiliac LNs from calves in the non-injected controls (n = 6) were collected at a conventional slaughterhouse. Injected calves were stunned by a captive bolt pistol and exsanguinated, and subjected to post-mortem examination at the Norwegian University of Life Sciences. Samples in the first trial were collected at 24, 48, and 96 h post-injection, and included the draining subiliac LN and the contralateral LN. In the second trial, samples were collected at 24 h and also included the draining superficial cervical LN. Skin with subcutaneous tissue and LNs on the injected side and the contralateral flank were collected and fixed for histology and immunohistochemistry (IHC). Formalin fixed samples were embedded in paraffin wax and prepared by standard procedures before staining with hematoxylin and eosin (HE) for light microscopy. Skin and LN specimens were frozen in chlorodifluoromethane (Isceon™) chilled with liquid nitrogen, and stored at −70°C until further preparation. Lymph nodes were excised vertically, and the anterior half toward the injection site was subjected to tissue preparation. LN tissue was minced mechanically by scissors in the presence of PBS/EDTA buffer. First, the LN cell suspensions were filtered through a Cell Strainer™ (BD Falcon), second, through a cotton filter pad soaked with PBS/EDTA, and finally washed in PBS/EDTA before direct analysis or freezing as described above. Immunohistochemistry Cryostat sections were cut 7 μm in thickness, mounted onto poly-lysine-coated slides and stored at −70°C before use. The sections were air dried at room temperature (RT), fixed in ice cold acetone, and finally rinsed and rehydrated in PBS pH 7.3. An indirect immunoperoxidase staining technique was performed on the sections by using an avidin–biotin complex method with the aid of a commercial kit (Vector Laboratories, Burlingame, CA, USA). To avoid non-specific binding of the biotinylated antibody, a blocking solution containing normal horse serum diluted 1:50 in 5% BSA/TBS and avidin diluted 1:6 was applied to the sections for 20 min at RT. The blocking solution was carefully tapped off the slides. Antibodies diluted in 1% BSA/TBS were added to the slides and the slides incubated overnight at 4°C. The subsequent day, the slides were washed carefully in PBS 3 × 5 min, and biotinylated horse anti-mouse IgG was diluted 1:100 in 1% BSA/TBS and added to the slides for 30 min at RT. The slides were washed carefully in PBS 3 × 5 min. Endogenous peroxidase was inhibited by treatment with 1% H2O2 in methanol for 15 min, followed by rinsing in PBS for 3 × 5 min. The avidin–biotin–horse radish peroxidase complex solution was prepared at least 30 min prior to use, according to kit instructions. The sections were incubated with the complex solution for 30 min. All incubations were done in a slowly rotating humid chamber at RT. Peroxidase activity was visualized by incubation with Imm Pact AEC peroxidase substrate. The reaction was stopped by rinsing in PBS. Slides were counterstained with Mayer’s hematoxylin for 15 s, rinsed in PBS, and mounted. To control for non-specific binding, all runs included a control section where the primary antibodies were replaced by 1% BSA/TBS. Immunofluorescence Immunofluorescent (IF) staining was performed essentially as previously described (26). Briefly, 7 μm cryostat sections were fixed in acetone and treated with 20% BSA/TBS in order to block non-specific binding. One of the following two mixtures of three primary antibodies were added to the sections: mouse anti-human CD14 (Tük4, IgG2a), mouse anti-bovine CD205 (MCA1651G, IgG2b) (both AbD Serotec), and mouse anti-bovine CD11c (BAQ153A, IgM) (VMRD), or mouse anti-human CD14, mouse anti-ovine CD21 (DU2-74-25, IgG2b) (a kind gift from W. Hein), and polyclonal rabbit anti-ki67 (Abcam) all used at previously determined optimal dilutions. Secondary antibodies were isotype-specific Alexa Fluor (350, 594, and 488) (Molecular Probes, Inc., USA). All incubations were done in a slowly rotating humid chamber for 1 h at RT. Slides were mounted in polyvinyl alcohol and stored at 4°C until examination. Control sections were included, replacing the primary antibody with 1% BSA/TBS, and replacing the secondary antibody with an irrelevant antibody. All tissue sections were examined in a Carl Zeiss Axio Imager M2 microscope equipped with a conventional camera (Axiocam HRc Rev. 3) and fluorescence camera (Axiocam HRm Rev. 3). Flow Cytometry Flow cytometry analysis was performed on fresh or previously frozen LN cell suspensions or PBMCs. Cells were first stained with LIVE/DEAD® Fixable aqua or yellow dead cell stain kit (Invitrogen), following the manufacturer’s instructions. Primary unconjugated monoclonal antibodies applied in the current study were mouse anti-bovine and against the following molecules: CD14 (CAM36A, IgG1), CD3 (MM1A, IgG1), CD62L (BAQ92A, IgG1), granulocyte marker (CH138A, IgM), CD11b (MM12A, IgG1) (all Monoclonal Antibody Center, Washington State University, USA), anti-ovine CD21 (DU2-74-25, IgG2b), and mouse anti-bovine CD205 (MCA1651G, IgG2b) (AbD Serotec/BioRad). Directly conjugated antibodies were cross-reactive anti-human CD16-FITC (KD1, IgG2a) and CD14-Pacific blue (Tük4, IgG2a) (both AbD Serotec/BioRad). Secondary isotype-specific reagents were either PE-conjugated or APC-conjugated (Southern Biotech, Birmingham, USA), or Alexa Fluor 488-or 647-conjugated (Molecular Probes/Life Technologies) polyclonal goat-anti-mouse antibodies, or PerCP- eFluor 710 conjugated rat anti-mouse monoclonal antibody (eBioscience/Affymetrix). All antibodies were used at previously determined optimal concentrations. FCM was performed with a 3-laser Gallios flowcytometer (Beckman Coulter), and gating based on staining with secondary antibodies only or isotype controls. Data were analyzed using the Kaluza software (Beckman Coulter). Isolation of CD14+ Cells and RT-qPCR Analysis Cell suspensions from the draining subiliac LNs collected at the second trial were used for isolation of CD14+ cells and RT-qPCR analysis. Cell suspensions were either snap frozen (n = 3) or used further for cell isolation (n = 4), as previously described (27). Briefly, CD14+ cells were extracted by positive selection of monocyte differentiation antigen CD14 using anti-human CD14 MACS Microbeads (coated with mAb clone Tük4) (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), according to the manufacturer’s instructions. Purity of selected cells was verified by FCM, and was found to be in the range of 95–98%. Isolated CD14+ cells were snap frozen in liquid nitrogen and transferred to −70°C for further storage. Peripheral blood mononuclear cells used for CD14+ baseline isolation were obtained from healthy NRF calves of 8–9 weeks of age (n = 3). Total RNA was isolated from LN cell suspensions (6 × 106 cells), LN CD14+ cells (16 × 106 to 2.7 × 106 cells) and blood CD14+ baseline cells (6 × 106 cells), using the MirVANA isolation kit (Ambion, Austin, TX, USA) following the manufacturer’s instructions. All RNA samples were treated with amplification grade DNase I (Invitrogen) to remove any traces of genomic DNA, and RNA concentration and quality was measured using NanoDrop 1000 (Thermo Fisher Scientific, Wilmington, USA) and 2100 BioAnalyzer (Agilent Technologies, Palo Alto, USA), respectively. All samples had a RNA integrity number (RIN) above 8.7 (except one where RIN = 6.6) and an OD A260/A280 ratio of ≥2.0. A total of 200 ng of RNA was used for cDNA synthesis reaction using Tetro cDNA synthesis kit (Nordic BioSite, Norway), and 10 ng was used in qPCR in triplicate per sample using Express SYBR GreenER SuperMix with premixed ROX (Invitrogen) according to the manufacturer’s recommendations. Transcript levels were analyzed using a 7900HT Fast Real-Time PCR System (Applied Biosystems) and the standard cycling program: 50°C for 2 min, 95°C for 2 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min, and the melting curve analyses were applied. Gene-specific primers were from either literature or designed using Primer3 ver. 0.4.0 (28). The transcript levels of the following genes were analyzed: CD14, CD16α, IL-1β, IL-6, TNFα, TGFβ, IL-12β, IL-10, CCR2, and CX3CR1. Primer sequences are presented in Table 1. The efficiencies of all primer pairs were tested by template dilution series using pooled cDNA from LN cells suspensions and CD14+ baseline cells and were 100% (±10). Negative controls with no added template were included for all primer pairs (no template control), and no RT control reactions for each sample and each primer pair were run in qPCR in order to check for genomic DNA contamination (no RT control). The peptidylprolyl isomerase A (PPIA) reference gene selected for the current study has been shown to be one of the most stable genes for gene expression studies in cattle macrophages (27) and lymphocytes (29), and in human LPS-stimulated monocytes (30). Initial analysis of the RT-qPCR data was performed using RQ Manager 1.2 (Applied Biosystems). Standard deviation of ≤0.3 per triplicate was accepted. The ΔCt method was used to calculate RT-qPCR data, i.e., ΔCt = Cttarget gene − Ctreference gene, and normalized gene expression was calculated as 2 (−ΔCt). Distribution of the data for the expression levels for each gene was tested by Shapiro–Wilk’s normality test in R (R: A Language and Environment for Statistical Computing, ver. 3.2.4, The R Core Team, 2016). The differences of normalized gene expression levels between CD14+ baseline cells and CD14+ cells from LN for each gene were tested using either unpaired t-test (for normally distributed data) or the unpaired Wilcoxon rank-sum test (for non-normally distributed data) in GraphPad (GraphPad Prism version 7.00 for Windows, GraphPad Software). Statistical significance was assigned at P ≤ 0.05. Table 1 List of primers used for reverse transcription-quantitative PCR (RT-qPCR). Gene symbol, accession no. Primers (5′ → 3′) Amp (bp) Reference CD14, NM_174008.1 CGATTTCCGTTGTGTCTGC 150 (16) TACTGCTTCGGGTTGGTGT CD16α, NM_001077402.1 Low-affinity FcγIIIR TGTCTCGTCATTCTTTCTACCTTG 138 (16) ACTTTGCCATCCCTCCATTC CX3CR1, NM_001102558.2 CX3C-chemokine receptor 1 TCACCAGAGAGAAAGAGAACGA 108 (16) GGAGCAGGAAGCCAAGAAA CCR2, NM_001194959 CC-chemokine receptor 2 GATGAAGAACCCACCACCAG 118 (16) CAAAGATGAAGACCAGCGAGTAG TGFβ1, NM_001166068.1 Transforming growth factor beta 1 CAATTCCTGGCGCTACCTCA 121 Primer 3 GCCCTCTATTTCCTCTCTGCG IL-1β, NM_174093.1 Interleukin-1 beta AAAAATCCCTGGTGCTGGCT 89 Primer 3 CATGCAGAACACCACTTCTCG IL-6, NM_173923.2 Interleukin-6 CCTGAAGCAAAAGATCGCAGA 97 Primer 3 TGCGTTCTTTACCCACTCGT IL-10, NM_174088.1 Interleukin-10 TATCCACTTGCCAACCAGCC 152 Primer 3 GGCAACCCAGGTAACCCTTA IL-12β, NM_174356.1 Interleukin-12 subunit beta GAGGTCGTGGTAGAAGCTGT 87 Primer 3 TGGGTCTGGTTTGATGATGTCC TNFα, NM_173966.3 Tumor necrosis factor alpha TCTTCTCAAGCCTCAAGTAACAAG 103 (27) CCATGAGGGCATTGGCATAC Amp, amplicon. Statistics Flow cytometry data were analyzed in the JMP Pro 12 statistical software (SAS Institute). Differences between groups consisting of different individuals were assessed by the Wilcoxon rank-sum test, and are indicated by *. Differences between groups consisting of the same individuals were assessed by the paired t-test, and are indicated by #. Statistical significance was assigned at P < 0.05. Results Adjuvant Injection Led to a Strong Recruitment of Monocytes A saponin-based adjuvant was injected subcutaneously in the flank region of calves. Calves were euthanized at different time points following injection, and blood as well as tissues at the injection and contralateral sites, including skin, draining, and contralateral LNs, were subjected to pathological and immunological analyses. On macroscopic evaluation, the injection site was characterized by a subcutaneous edema, and the draining LN was found to be two- to threefold enlarged (not shown). These changes were most pronounced at 24 h post-injection. Skin and LNs from the contralateral side did not show these changes. Histopathological evaluation of the skin on the injected side revealed a diffuse, locally extensive, and moderate-to-severe inflammation in deeper cutaneous and subcutaneous tissues (Figure 1A), consisting of an infiltration of neutrophils, lymphocytes, and large monocyte-like cells (Figure 1B). A substantial amount of the inflammatory infiltrate consisted of CD14+ cells (Figure 1C). Figure 1 Cellular recruitment to skin and subcutaneous tissues. (A) HE stained sections of skin with subcutaneous tissue from the side injected with adjuvant and the contralateral side, at 24 h post-injection. Scale bars: 200 μm. (B) Enlargement of outlined areas in A, as indicated. Scale bars: 20 μm. (C) Immunofluorescent labeling of subcutaneous tissue on the injected side with antibody against CD14 (green). Scale bar: 20 μm. Flow cytometry analysis of cells from the draining LN revealed a distinct appearance of numerous cells in the monocyte gate at 24 h post-injection, which were only scarcely present in the contralateral LN (Figures 2A,B). Also the relative percentage of cells in the granulocyte gate was increased, while the overall lymphocyte population was reduced at this time point. Detailed results are presented in Table S1 in Supplementary Material. IHC staining of the draining LN demonstrated that numerous CD14+ cells were present in the subcapsular and peri-trabecular sinuses, and in the T-cell zones of the cortex (Figure 2C). Figure 2 Cellular recruitment to lymph nodes (LN) and peripheral blood. (A) LN cells were prepared for FCM analysis and gated on forward/side scatter (FSC/SSC) characteristics. Plots from one representative animal are presented. Panels illustrate the gating of lymphocytes, monocytes, and granulocytes as indicated and in the draining LN (left) and the contralateral LN (right), at 24 h post-injection. (B) Percentages of major immune cell populations in LNs, based on the gating strategy in A. Horizontal stacked bars show mean percentages of lymphocytes (gray), monocytes (dark gray), and granulocytes (black) of the total live cell population in non-injected animals (n = 6) and at different time points after adjuvant injection (n = 2–3). (C) IHC labeling of draining and contralateral LNs at 24 h post-injection with antibody against CD14. Different regions of the LN are indicated. (D) Cellular differential counts in peripheral blood. Horizontal stacked bars show mean absolute numbers (×109) of lymphocytes (gray), monocytes (dark gray), and granulocytes (black) at pre-injection and at different time points after adjuvant injection. There was a marked and transient increase in the absolute number of granulocytes in peripheral blood, peaking at 24 h post-injection, with a mean fold increase of 2.8 from pre-injected levels (Figure 2D). The increase in the absolute number of monocytes in blood was less evident and came later, peaking to a double level at 72 h post-injection. Detailed results are presented in Table S2 in Supplementary Material. Recruited Monocytes Were CD14++ CD16+ Prior to adjuvant injection, monocytes from PBMC could be divided into three different subsets based on the expression of CD14 and CD16 (Figure 3A), coherent with previous reports (16, 17). Monocytes recruited to the draining LNs at 24 h post-injection were of an essentially homogeneous CD14++ CD16+ phenotype (Figure 3B). Moreover, the intensity of CD14 expression on these LN monocytes was increased in comparison to monocytes from PBMC (Figure 3C). CD14++ cells from draining LNs were CD11b+ (Figure 3D) and CD62L+ (Figure 3E). Monocyte-gated cells did not express the granulocyte antigen (CH138A) or the T-cell marker CD3 in FCM, both confirmed to be present on cells in the respective granulocyte and lymphocyte gates (not shown). Figure 3 Phenotype of recruited monocytes. Density plots of live cells (upper panels) from PBMC pre-injection (A) and the draining LN at 24 h post-injection (B). Plots from one representative animal are presented. Monocytes were further gated into subsets based on their expression of CD14 and CD16 (lower panels). Isotype control for CD14 on live cells from LN is shown far right. (C) CD14 expression on cells from the monocyte gate [as gated in (A,B)]. Histograms show the isotype control for CD14 (light gray line), PBMC baseline (gray line), and draining LN at 24 h (black line). (D) CD11b expression and (E) CD62L expression on CD14++ cells. Histograms show the secondary Ab control (gray line) and the draining LN at 24 h (black line). Monocytes Were Transiently Present in the LN Cortex and Migrated via the Medulla to Blood Recruited CD14++ CD16+ cells constituted 20–41% of live cells in the draining subiliac LN 24 h after adjuvant injection (Figure 4A). In PBMC, CD14+ monocytes tended to increase in percentage later; measurably already at 24 h but apparently peaking in the two consecutive days (Figure 4B). Figure 4 Percentages of monocytes in LNs and PBMC. (A) Percentages of CD14++ CD16+ monocytes of total live cells in LNs of non-injected animals, and in draining and contralateral LNs of injected animals at 24, 48, and 96 h post-injection. Symbols represent individual animals and the median value within each group is depicted as a line. Statistical significant differences between injected and non-injected groups using the Wilcoxon rank-sum test are indicated as *P < 0.05. Statistical significant differences between groups consisting of the same individuals (identical symbols) using the paired t-test are indicated as #P < 0.05. (B) Percentage of CD14+ monocytes of live cells in PBMC at pre-injection, and at 24, 48, 72, and 96 h post-injection. Symbols and statistics as in (A) (#P < 0.05). (C) Percentage of CD14++ CD16+ monocytes of live cells in the draining high dose (subiliac) LN and the contralateral LN, and in the draining low-dose (superficial cervical) LN. Symbols and statistics as in A (#P < 0.05). (D) Percentage of CD14+ monocytes of total live cells in PBMC at pre-injection and after 24 h. Symbols and statistics as in A. In the second trial, the recruitment of CD14++ CD16+ monocytes to the draining subiliac LN was again demonstrated, but in lower numbers (Figure 4C). The second trial also included an injection of a 10-fold lower dose of adjuvant in the neck region, drained by the superficial cervical LN, leading to reduced recruitment of monocytes. Like in the previous trial, an increase of CD14+ monocytes was demonstrated in blood after 24 h (Figure 4D). The transient presence of monocytes in the draining LN was also visualized by IF staining. A high number of CD14+ cells were present in the draining LN at 24 h post-injection (Figure 5A), but not in the contralateral LN (Figure 5B). Monocytes did not express Ki67, indicating that they were not in active proliferation after entry to the LNs. After 48 h, monocytes had decreased in number in the cortex of the draining LN (Figure 5C), but were present in moderate to large amounts in the medulla, including the area around efferent lymph vessels (Figure 5D). In the second trial, a similar monocyte recruitment was observed to the LN cortex, but in lower numbers, whereas more monocytes were present in the medulla already at 24 h, indicating an earlier onset or a faster migration through the lymphoid tissue than observed in the first trial (not shown). Figure 5 Distribution of monocytes in the LNs. Immunofluorescent labeling of LNs with antibodies against CD14 (green), CD21 (blue), and Ki67 (red). CD21 stains the LN follicles. (A) CD14+ cells were present in the capsule, subcapsular sinus, peri-trabecular sinus, and interfollicular areas of the draining LN at 24 h post-injection. (B) The contralateral LN was mainly devoid of CD14+ cells. Note the empty sub capsular sinus and trabecular sinus areas, as opposed to the infiltration in (A). (C) CD14+ cells were abundant in the capsule, but were decreased in numbers in the sinus and the cortex at 48 h post-injection. (D) CD14+ cells were present in the medulla of the LN at 48 h post-injection, and particularly around vessels. Follicle (f), interfollicular area (i), capsule (c), sinus area (s), vessel (v). Scale bars: 100 μm. Taken together, these findings indicated that an adjuvant injection lead to a strong and transient recruitment of monocytes to the LN cortex, followed by migration into the medullary areas before departure via efferent vessels into the blood. Monocytes in Skin and Draining LN Did Not Express DC-Associated Markers To investigate whether a differentiation of CD14+ cells toward a DC phenotype had taken place in subcutaneous tissues or in the draining LN, IF triple labeling of skin and LN tissue was performed with CD14 and the DC-associated markers CD205 and CD11c. Numerous CD14+ monocytes were present in the deep cutis and subcutis on the injected side at 24 h post-injection (Figure 6A). A limited number of CD14− CD205+ cells were present, possibly representing macrophages. Very few CD14+ cells were present in the skin on the non-injected side (Figure 6B). A moderate amount of CD11c+ CD205+ cells were observed in both the draining and the contralateral LN at 24 h post-injection, most likely representing conventional LN DCs (Figure 6C, insert, and Figure 6D). These cells did not triple label with the CD14-marker. A minor proportion of CD14+ cells in the draining LN were found to be CD11c+, while none were CD205+. CD205 also labeled cells within the lymphoid follicles, as previously assigned to B cells in cattle (31). In FCM, CD14++ cells from the draining LN did not express CD205 (not shown). Figure 6 Distribution of monocyte and DC markers in skin and LNs. Immunofluorescent labeling of subcutaneous tissues and LNs with antibodies against CD14 (green), CD205 (red) and CD11c (blue), at 24 h post-injection. (A) Large numbers of CD14+ cells were observed in the subcutis on the injected side. Note the separation of collagen bundles (appears as gray auto fluorescence, arrowheads) due to inflammatory infiltrates. A limited number of CD205+ cells were present. (B) A few CD14+ cells (arrow) were observed on the contralateral side. (C) CD14+ cells infiltrated the interfollicular areas of the cortex of the draining LN. A moderate amount of CD205+ CD11c+ DCs were observed (arrow heads in insert). (D) CD14+ cells were sparsely present in the contralateral LN. CD205+ follicles were surrounded by CD11c single labeled and CD11c+ CD205+ double labeled cells. Follicle (f), interfollicular area (i), capsule (c), sinus area (s). Scale bars: 100 μm. Monocytes in the Draining LN Expressed Genes Coding for Pro-Inflammatory Cytokines and CCR2 To investigate the functional capacity of recruited monocytes, CD14+ cells from the draining subiliac LNs of injected calves were isolated by positive selection and analyzed by RT-qPCR in the second trial. Baseline gene expression values were obtained from blood CD14+ cells isolated from calves of the same age and the same herd and were normalized to a housekeeping gene (PPIA). Expression levels of genes encoding for CD14, CD16α, IL-1β, IL-6, and TNFa were significantly higher in CD14+ cells from draining LNs compared to CD14+ cells from blood (P ≤ 0.05, Figures 7A,B). TGFβ also appeared upregulated, although the difference from baseline blood was non-significant in this limited material. No clear difference in expression of IL-12β and IL-10 was found. Of the two chemokine receptors assessed, CCR2 gene expression level was higher in most CD14+ samples from injected animals, but not to a significant degree, likely due to an outlier in the baseline samples (Figure 7C). No difference in expression of the gene coding for the chemokine receptor CX3CR1 was found. Figure 7 Gene expression of monocytes from the draining LN. Gene expression levels (normalized to the reference gene PPIA) of CD14+ cells isolated from blood (baseline non-injected) and from the draining LN at 24 h post-injection, as analyzed by RT-qPCR. Symbols represent individual animals and the median value within each group is depicted as a line. Statistical significant differences between the two groups using the unpaired t-test (for normally distributed data) are indicated as #P ≤ 0.05, and the Wilcoxon rank-sum test (for non-normally distributed data) are indicated as *P ≤ 0.05. (A) Gene expression levels of CD14, CD16α, IL-1β, and TGFβ. (B) Gene expression levels of IL-12β, TNFα, IL-6, and IL-10. Note the difference in y-axis range from (A). (C) Gene expression levels of chemokine receptors CCR2 and CX3CR1. Note the difference in y-axis range from (A,B). Discussion While most studies of cell migration from inflamed tissues to draining LNs have focused on DCs (1), recent studies have shown that also monocytes travel via this route (4, 8, 9, 32). We here demonstrate a potent in vivo recruitment of monocytes to the draining LN in a local acute inflammatory situation, and show that these cells upregulate genes for pro-inflammatory cytokines. Recruited cells were of a uniformly CD14++ CD16+ phenotype and, thus, phenotypically resembled the intermediate monocyte subset described in humans (11, 20), and recently in cattle (16, 17). However, in contrast to bovine blood monocytes, we found that monocytes recruited in vivo to LNs stained more brightly for CD14 (CD14++). This phenotype was supported by RT-qPCR findings, showing a high gene expression for both CD14 and CD16α in these cells. CD14+ cells isolated from LNs had increased expression of genes associated with induction of inflammation, including TNFα, IL-1β, IL-6, and TGFβ. This is in line with other reports of intermediate monocytes expanding under different inflammatory conditions [reviewed by Italiani and Boraschi (33) and Ziegler-Heitbrock (34)], and strongly implicates a role for these CD14++ CD16+ monocytes in inflammatory processes in vivo. Monocytes are believed to mainly differentiate into macrophages or moDCs in the inflamed tissue, after which predominantly moDCs will travel to the draining LN. These moDCs or “inflammatory” DCs should not be confused with conventional DCs that originate from an independent lineage of hematopoietic cells (35), and which have best been described in cattle in the afferent lymph (36–38). We found that the majority of CD14+ cells present in subcutaneous tissues and the draining LNs did not express the DC-associated markers CD205 and CD11c. Moreover, unlike moDCs, CD14++ cells were CD11b+ and CD62L+. The majority of studies of monocyte-derived cells in cattle are based on in vitro generated cells from blood, which have been shown to downregulate CD14 and CD62L and upregulate CD205 (39–42). In mice, moDCs have been identified based on a high surface expression of CD11c (9, 43), whereas in humans CD11c is considered specific only for those DCs found in lymphoid organs (44, 45). In cattle, both blood monocytes and in vitro derived cells express CD11c, and the latter to a lesser degree than the former (16, 41, 42, 46). However, the in vitro differentiation of blood monocytes cannot fully recapitulate the differentiation in vivo, which may be influenced by a combination of factors in the tissue environment, such as chemokines, cytokines, or administered adjuvant or antigen. Collectively, the overall phenotype indicates that the recruited cells in the present study labeling CD14++ CD16+ CD11b+ CD62L+ were monocytes rather than moDCs. Monocytes were present in the skin and subcutaneous tissue at the injection site and in high numbers in the sinus and the T-cell area of the LN cortex. This is consistent with the possibility of a monocyte migration from the skin to the draining LN via afferent lymph. Indeed, the presence of monocytes in afferent lymph of sheep (4, 47) and rat (32) has been described, and recently, adoptively transferred monocytes were shown to migrate from skin to the draining LN in mice (8, 9). We cannot exclude the possibility that monocytes also entered the LN from blood via high endothelial venules (HEVs), as CD14++ cells were strongly CD62L positive, and the recruitment of monocytes from blood across HEVs has been reported (48). However, the predominant route of monocyte trafficking to LNs is thought to be via the afferent lymph (10, 43, 49). We also did not observe any monocytes increase in blood prior to their appearance in LNs. Moreover, we found that the gene expression level of CCR2 was upregulated in CD14+ monocytes from the draining LNs, implicating this chemokine receptor in the recruitment of bovine intermediate monocytes to inflamed tissue. The recruitment of monocyte to the draining LN was transient, and after 48 h a population of monocytes remained in the medulla only, suggesting an internal migration within the LN toward an exit of these cells through efferent lymph vessels. In support of this notion, we found an increase in CD14+ monocytes in PBMC starting around 24 h post-injection. We cannot exclude the possibility that some of these circulating monocytes were recruited from the bone marrow or splenic reservoirs, as expected during an inflammation. Nevertheless, the substantial passage of monocytes through LNs represents a phenomenon that to our knowledge is not well documented in the literature, probably due to its highly transient nature. To get a gross idea of the dose–response effect of the adjuvant, a second trial included an injection of a 10-fold lower adjuvant dose, leading to reduced recruitment of monocytes. However, this was performed at a differing anatomical location, and the evaluation of the efficacy and safety of the given adjuvant in a potential vaccine context warrants additional studies, being outside the scope of the presented study. A large part of our knowledge on leukocyte recirculation derives from large animal models, and ruminants can serve as excellent in vivo models due to their size and the possibility to follow cell migration via lymphatic cannulation (4, 36, 38, 50, 51). More knowledge on the initiation of immune responses in cattle can form a basis for new vaccines in this species, but also be important for understanding processes in mammals at large, including humans. To this end, a combination of methods in experimental post-mortem analyses as presented herein can offer powerful tools for future studies of dynamics and recirculation of immune cells, in the steady state as well as under inflammatory conditions. Author Contributions HL: study design, sample collection and preparation, FCM, data interpretation, and writing of manuscript. PB: study design, FCM, data interpretation, writing, and editing of manuscript. CÅ: study design, IHC and IF stainings, data interpretation, writing, and editing of manuscript. AL-S: study design, sample collection, RT-qPCR analysis, writing, and editing of manuscript. AKS: Study design, data interpretation, writing, and editing of manuscript. All authors approved the final manuscript and are accountable for all aspects of the presented study. Conflict of Interest Statement The authors declare that no financial or commercial conflict of interest exists in relation to the content of this article. The authors have no financial involvement in Novavax AB. We are grateful to Novavax AB for supplying Matrix-Q™ adjuvant. We greatly acknowledge Grethe M. Johansen for assistance with sample preparation and flow cytometry, Mari Katharina Aas Ådland for immunohistochemical staining of tissue sections, Adam Martin and Haakon Aaen for technical assistance and care of animals, and Arild Espenes and Line Olsen for post-mortem sample collection, all at Norwegian University of Life Sciences. Funding This study was funded by the Research Council of Norway (grant 183196/S40) and the Norwegian University of Life Sciences. Supplementary Material The Supplementary Material for this article can be found online at http://journal.frontiersin.org/article/10.3389/fimmu.2016.00322 Click here for additional data file. Click here for additional data file. Abbreviations CCR2, CC-chemokine receptor 2; CX3CR1, CX3C-chemokine receptor 1; DCs, dendritic cells; FCM, flow cytometry; HE, hematoxylin and eosin staining; HEVs, high endothelial venules; IF, immunofluorescence; IHC, immunohistochemistry; LN, lymph node; moDCs, monocyte-derived dendritic cells; PBMC, peripheral blood mononuclear cells; PPIA, peptidylprolyl isomerase A; RT-qPCR, reverse transcription-quantitative polymerase chain reaction. ==== Refs References 1 Girard JP Moussion C Forster R . HEVs, lymphatics and homeostatic immune cell trafficking in lymph nodes . Nat Rev Immunol (2012 ) 12 (11 ):762 –73 .10.1038/nri3298 23018291 2 Jenkins SJ Hume DA . Homeostasis in the mononuclear phagocyte system . Trends Immunol (2014 ) 35 (8 ):358 –67 .10.1016/j.it.2014.06.006 25047416 3 van Furth R Cohn ZA Hirsch JG Humphrey JH Spector WG Langevoort HL . The mononuclear phagocyte system: a new classification of macrophages, monocytes, and their precursor cells . 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==== Front Syst BiodiversSystem. BiodiversTSABtsab20Systematics and Biodiversity1477-20001478-0933Taylor & Francis 94103810.1080/14772000.2014.941038ArticleResearch ArticlesPhylogenetic confirmation of the genus Robbea (Nematoda: Desmodoridae, Stilbonematinae) with the description of three new species Ott Jörg A. a * Gruber-Vodicka Harald R. b Leisch Nikolaus c Zimmermann Judith b a Department of Limnology and Biooceanography, University of Vienna, Althanstr. 14, A-1090Vienna, Austriab Department of Symbiosis, Max Planck Institute for Marine Microbiology, Celsiusstr. 1, D-28359Bremen, Germanyc Department of Ecogenomics and System Biology, University of Vienna, Althanstr. 14, A-1090Vienna, AustriaCorrespondence to: Jörg A. Ott. Email: joerg.ott@univie.ac.at2 10 2014 7 8 2014 12 4 434 455 13 2 2014 12 6 2014 © 2014 The Author(s). Published by Taylor & Francis.2014The Author(s)This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.ABSTRACT The Stilbonematinae are a monophyletic group of marine nematodes that are characterized by a coat of thiotrophic bacterial symbionts. Among the ten known genera of the Stilbonematinae, the genus Robbea Gerlach 1956 had a problematic taxonomic history of synonymizations and indications of polyphyletic origin. Here we describe three new species of the genus, R. hypermnestra sp. nov., R. ruetzleri sp. nov. and R. agricola sp. nov., using conventional light microscopy, interference contrast microscopy and SEM. We provide 18S rRNA gene sequences of all three species, together with new sequences for the genera Catanema and Leptonemella. Both our morphological analyses as well as our phylogenetic reconstructions corroborate the genus Robbea. In our phylogenetic analysis the three species of the genus Robbea form a distinct clade in the Stilbonematinae radiation and are clearly separated from the clade of the genus Catanema, which has previously been synonymized with Robbea. Surprisingly, in R. hypermnestra sp. nov. all females are intersexes exhibiting male sexual characters. Our extended dataset of Stilbonematinae 18S rRNA genes for the first time allows the identification of the different genera, e.g. in a barcoding approach. http://zoobank.org/urn:lsid:zoobank.org:pub:D37C3F5A-CF2B-40E6-8B09-3C72EEED60B0 Key words Belize Barrier ReefCaribbean Seachemosynthetic symbiosismarine nematodesmolecular phylogenySSU rRNAectosymbiontssystematicstaxonomyWe are grateful to the Core Facility Cell Imaging and Ultrastructure Research of the University of Vienna for technical support and the Max Planck Society for funding. This is contribution # 962 from the Carrie Bow Cay Laboratory (CCRE Program of the National Museum of Natural History, Washington, DC). N.L. was supported by an Austrian Science Fund (FWF) grant P22470-B17 (S. Bulgheresi, PI) and H.R. G.-V. by a Marie-Curie Intra-European Fellowship PIEF-GA-2011-301027 CARISYM. ==== Body Introduction Stilbonematinae are a marine subfamily within the nematode family Desmodoridae (order Desmodorida) and occur worldwide in sulphidic sediments (reviewed in Tchesunov, 2013). They are characterized by an ectosymbiosis with sulphur-oxidizing bacteria that covers their cuticle in a genus- and sometimes even species-characteristic manner (Polz et al., 1992, 1994; Ott et al., 2004). Although the family Desmodoridae is probably polyphyletic (van Megen et al., 2009), molecular data (Kampfer et al., 1998; van Megen et al., 2009) suggest a monophyly of the subfamily Stilbonematinae. A distinct morphological synapomorphy of the subfamily is the complex glandular sense organ (GSO) described by Nebelsick et al. (1992) and Bauer-Nebelsick et al. (1995), that has so far only been found in members of the Stilbonematinae. This organ produces a special set of lectins (sugar-binding proteins) that seem to be involved in sustaining the specificity of the symbiosis (Bulgheresi et al., 2006, 2011). Otherwise very few common features unite the group, such as the lack of a buccal armature and the weak development of the pharynx (Ott et al., 2004). Some characters appear to have developed independently more than once. For example, there is a tendency towards the development of an enlarged muscular portion of the anterior part of the pharynx (corpus), which is conspicuous in all species of the genera Robbea and Catanema, and has been described for the single species of Parabostrichus (Tchesunov et al., 2012), whereas in other genera (Laxus, Leptonemella, Eubostrichus) it is present only in a few species. Other examples are the reduction of the amphidial fovea or the presence of a stiff corpus gelatum protruding from the amphidial fovea (Stilbonema, Catanema, Leptonemella partim) (Tchesunov, 2013). This complicates the assessment of the relationships between the various genera described until now. In addition, the type species of several genera are either inadequately described or have features which are the exception rather than the rule in the subsequently described members of the genus. Liberal synonymization has added further confusion. The genus Robbea, introduced by Gerlach (1956), is characterized by the clear separation of a muscular anterior corpus from a mostly glandular isthmus and posterior bulbus which make up the remaining portion of the pharynx. In some species the males have peculiar papillae in the ventro-median line of the cervical and post-cervical region, which resemble suction cups and have a stout conical setae in its centre. These appear to be unique for the genus Robbea and have not been described from other stilbonematine genera. The aim of this study was to reinvestigate morphological characters that are shared by all species of the genus Robbea but clearly distinguish those from other stilbonematine nematode genera. We describe three new species of Robbea from shallow subtidal sands around the island Carrie Bow Cay on the Belize Barrier Reef (Caribbean Sea). We combine morphological and molecular analyses to clarify the status of the genus and support our findings by providing additional phylogenetic information on previously under-sampled genera. Materials and methods Sediment samples containing Robbea species were collected in the vicinity of the island of Carrie Bow Cay, Belize from shallow subtidal sand patches and from turtle grass (Thalassia testudinum) beds using buckets or cores. Samples for sequencing were obtained at the following coordinates: R. hypermnestra: 16° 48′ 47″N, 88° 04′ 58″W; R. agricola: 16° 52′ 58″N, 88° 07′ 11″W; R. ruetzleri: 16° 47′ 19″N, 88° 04′ 48″W (Fig. 1). Subtidal sediment containing Catanema sp. was collected in the bay off Capo di Sant’ Andrea, Elba, Italy (42° 48′ 26″N, 10° 08′ 28″E) from sand patches surrounding sea grass beds of Posidonia oceanica in 4–8 m water depth (Fig. 1). Leptonemella vicina was collected off the island of Sylt, Germany (55° 00′ 54″N, 08° 26′ 16″E) with sediment cores from an intertidal sand flat (Fig. 1). Worms were extracted from the sand after anaesthesia with MgCl2, shaking and decanting or simply shaking and decanting through a 32 μm mesh sieve. The live animals were sorted using a dissecting microscope and immediately fixed in 4% formaldehyde (for taxonomic preparations) or 2.5% glutaraldehyde in 0.1 sodium cacodylate buffer and postfixed in 2% OsO4 (for SEM and semi-thin sections). For light microscopy the worms were placed into glycerol:water 1:9, slowly evaporated and finally mounted in pure glycerol; for SEM specimens were critical point-dried and coated with gold. Specimens for sequencing of the 18S rRNA gene were preserved in 70% ethanol or methanol. Fig. 1. Map showing sampling locations for the nematodes used in this study. 1, Carrie Bow Cay, Belize; 2, Elba, Italy; and 3, Sylt, Germany. Insert shows detailed locations in the vicinity of Carrie Bow Cay where the three new Robbea species were found. Asterisks mark the sampling locations of the individuals that were used for sequencing. Drawings were done using a camera lucida on a Reichert Diavar or a Leitz Ortholux. Nomarsky interference contrast photos were taken on a Reichert Polyvar; SEM on a JEOL JSM-35 CF and a Phillips XL20; semi-thin sections were cut on a Reichert Ultracut and photographed in a Reichert Polyvar. DNA extraction, PCR amplification and sequencing of the 18S rRNA gene DNA for all sampled species was extracted and purified from single nematode specimens as described before (Schizas et al., 1997). The partial 18S rRNA gene (∼1800 bp) was amplified by PCR with the general eukaryotic primers 1F (5′-GGTTGATYCTGCCAGT-3′) (modified from Winnepenninckx et al., 1995) and 2023R (5′-GGTTCACCTACGRAAA-3′) (modified from Pradillon et al., 2007) using the Phusion® DNA polymerase (Finnzymes, Finland). Cycling conditions were as follows: initial denaturation at 96 °C for 5 min, followed by 35 cycles of 96 °C for 1 min, 55 °C for 1.5 min, 72 °C for 2 min and final elongation at 72 °C for 10 min. The purified PCR products (PCR Purification Kit; Qiagen, Hilden, Germany) were sequenced bidirectionally with the PCR primers and an internal reverse primer (5′-CAGACAAATCGCTCC-3′) 1272 nucleotides downstream of the 1F primer. All sequencing reactions were performed with an ABI PRISM 3100 genetic analyser (Applied Biosystems, Foster City, CA, USA). The generated 18S rRNA gene sequences and all sequences from the Stilbonematinae available in GenBank, as well as four Draconematinae sequences as outgroup were aligned using MAFFT Q-INS-I, which considers the predicted secondary structure of the RNA for the alignment (Katoh et al., 2005). Alignments were manually inspected and 5′ and 3′ end-trimmed using Geneious software version 6 (Drummond et al., 2011). The optimal substitution model was assessed using the Akaike information criterion as implemented in MEGA 5.3 (Tamura et al., 2011) and the GTR+G+I model was chosen. Phylogenetic trees were reconstructed using maximum likelihood- (RAxML) (Stamatakis, 2006) and Bayesian inference-based (MrBayes) (Ronquist & Huelsenbeck, 2003) methods. The dataset was screened for chimeric sequences by calculating maximum likelihood-based trees on three partitions of the alignments (0–600 bp, 601–1200 bp and 1201–1799 bp). The Stilbonematinae sequences Y16915 (designated Eubostrichus dianae) and Y16921 (designated Robbea hypermnestra) had statistically supported positions in 2 (Y16915) and 3 (Y16921) different genus level clades across all three partitions and were excluded from the final analysis. MrBayes was run for four Mio generations using four chains. Convergence was evaluated by plotting the generations versus logL and the burn-in was set to 1 Mio generations. Node stability was evaluated using posterior probabilities (pp, Bayesian inference) and bootstrap support (200 RAxML rapid bootstrap runs, maximum likelihood) (Stamatakis et al., 2008) with values above .80 considered significant. Nucleotide sequence accession numbers The 18S rRNA sequences from this study were submitted to GenBank under accession numbers KJ414464 (Robbea agricola), KJ414465 (Robbea ruetzleri), KJ414466-7 (Robbea hypermnestra), KJ414468 (Leptonemella vicina) and KJ414469 (Catanema sp.). Results Class Chromadorea Inglis, 1983 Subclass Chromadoria Pearse, 1942 Order Desmodorida De Coninck, 1965 Suborder Desmodorina De Coninck, 1965 Superfamily Desmodoroidea Filipjev, 1922 Family Desmodoridae Filipjev, 1922 Subfamily Stilbonematinae Chitwood, 1936 Robbea Gerlach 1956   Diagnosis (Modified from Tchesunov, 2013): Stilbonematinae. Cuticle transversely striated, except for the head region and the tip of the tail. Cephalic capsule when present with a block-layer; cephalic setae as long or longer than the subcephalic setae, usually directed straight forward. Amphidial fovea well developed, spirally coiled or loop-shaped. Pharynx distinctly tripartite, corpus muscular and clearly set off from the narrow isthmus. Pharynx bulbus largely glandular. Gubernaculum variable in shape, with or without dorso-caudal apophysis. The males of R. tenax Gerlach 1963 and the three new species have cup-shaped ventral papillae in the post-pharyngeal region. These species share also a stout body with a not exceeding 100. The species R. gerlachi Boucher 1975 from which only a female has been described may also belong to this group. Symbiotic bacteria rod shaped, corn-kernel shaped or coccoid, usually covering the body as a monolayer. Robbea hypermnestra sp. nov. http://zoobank.org/urn:lsid:zoobank.org:act:505DEA0B-614A-4086-A52F-62BA8A68F474 Synonymy Robbea sp. in: Ott & Novak (1989), Schiemer et al. (1990), Ott et al. (1991), Polz et al. (1992), Bauer-Nebelsick et al. (1995), Urbancik et al. (1996a, 1996b), Polz et al. (2000); Robbea sp. 3 in: Bayer et al. (2009), Heindl et al. (2011); Robbea hypermnestra nomen nudum in: Kampfer et al. (1998), Polz et al. (2000). Type material Holotype (male), 4 paratypes (male), 4 paratypes (female/intersex), deposited at the US National Museum, Washington, D.C. (accession numbers USNM 1231551-1231559). Measurements See Table 1. Table 1. Morphometric data for Robbea hypermnestra sp. nov. Ranges are given for the male and female paratypes. All measurements are in μm.   Holotype Paratypes (male) n = 4 Paratypes (female intersexes) n = 4 Length 3125 2765–3535 3020–3985 a 44.5 41.4–58.9 44.4–64.3 b 30.6 23.6–37.2 27.5–36.2 c 17.9 17.4–23.6 23.0–26.9 maximum width 70 55–65 65–82 pharynx length 102 92–100 90–117 tail length 174 140–162 120–150 nerve ring (% pharynx length) 50 48–54 46–58 corresponding body diameter (cbd) 45 45–52 48–55 bulbus length/width 25/32 20–28/29–30 25–38/32–45 bulbus cbd 48 47–50 48–55 V n.a. n.a. 41–45 vulva cbd n.a. n.a. 65–82 anal (cloacal) body diameter 68 55–65 50–60 tail length:anal diameter 2.6 2.3–2.9 2.3–2.5 spiculae length arc/chord 105/88 98–102/80–85 62–70/53–60 gubernaculum length/apophysis length 20/25 20–30/25–34 10–15/10–15 amphid width 15 16–20 9–12 cephalic setae number/length 4/38 4/32–40 4/30–38 subcephalic setae 1 number/length 8/37 8/30–38 8/28–32 subcephalic setae 2 number/length 8/30 8/28–32 8/28–32 Sucker-like papillae, number 17 16–18 n.a. Position of first/last papilla 85/501 65–95/440–480 n.a. n.a. = not applicable. Additional material Several specimens in the author's collection and those used for SEM. The sequences of the 18S rRNA gene are available from GenBank and have the accession numbers KJ414466-7. Type locality Subtidal sand bar in 10–50 cm depth at the north end of Carrie Bow Cay, Belize in coarse, poorly sorted calcareous sand with rubble (Fig. 1). Distribution This robust species is extremely common in the above sand bar (compare Ott & Novak, 1989) and a similar sand bar at the south end of neighbouring South Water Cay. Other localities are open sand patches between Thalassia testudinum beds and patch reefs to the west and north of Carrie Bow Cay in up to 1.5 m depth and medium, poorly sorted sand at the base of ‘sand bores’ south of Carrie Bow Cay in 5–6 m depth (Fig. 1). Etymology Named after Hypermnestra, the only one of the 50 daughters of the king of Argos, Danaos, who did not kill her husband in the wedding night as requested by her father although she was armed with a dagger. This refers to the presence of a spiculum in females (intersexes). Description Body cylindrical (Figs 2, 3), head diameter at level of cephalic setae 20–23 μm, diameter at level of posterior margin of amphidial fovea 40–54 μm in males, 33–42 μm in females, at end of pharynx 47–55 μm, maximum body diameter 55–82 μm, anal diameter 55–68 μm in males, 50–60 μm in females. Tail conical, 140–174 μm long in males and 120–150 μm in females (Figs 7, 8). Figs. 2–10. Robbea hypermnestra sp. nov. 2. Female, whole; 3. Male, whole; 4. Male, anterior body region; 5. Male, head, surface view; 6. Female, head, surface view; 7. Female, tail; 8. Male, tail (gubernaculum not drawn); 9. Female, spiculum; 10. Male, spiculum. Figs. 11–16. Robbea hypermnestra sp. nov. Female. 11. Head, in face view; 12. Amphidial fovea; 13. Vulva; 14. Annulation and bacterial coat in midbody region; 15. Tip of tail, non-striated portion; 16. Openings of the caudal glands. SEM. Cuticle finely transversely striated except for the first 28–36 μm of the head (Figs 5–8, 14) and the last 35–50 μm of the tail (Fig. 15), annules 0.45 to 0.55 μm wide (18–22 annuli/10 μm); non-striated head cuticle reinforced (cephalic capsule). The anteriormost circle of head sensillae (inner labial sensillae) is represented by 6 finger-like papillae, 2 μm long in lateral, subventral and subdorsal position surrounding the mouth opening (Fig. 19). The second circle consists of 6 short outer labial sensillae, 3–6 μm long, on the margin of the membranous buccal field; 4 cephalic setae flanking the anterior margin of the amphidial fovea, 32–38 μm long; 3 circles of 8 subcephalic setae each, 28–38 μm long, on the non-striated part of the head region, the posteriormost at the level of the posterior margin of the amphidial fovea (Figs 11, 17); 8 rows of somatic setae along the whole length of the body, 20–35 μm long somatic setae alternating with only 5–12 μm long bristles (Fig. 4). In males, there is a distinct field of about 7–10 stout, conical setae, 5 μm long on the ventral side in front of the cloacal opening (Figs 8, 25, 26). Non-striated part of the tail in females with 2 pairs of stout setae, the first pair 7 μm long, shortly behind the end of the annulation; the second pair of 3–4 μm long, close to the tip of the tail (Fig. 15); in males with a small velum ventrally (Fig. 23). Three caudal glands with separate openings at the tail tip (Fig. 16). Males have a 380–450 μm long row of 16–19 (mean 17.2, n = 16) conspicuous cuticular papillae along the mid-ventral line beginning at a distance of 65–95 μm from the anterior end at the level of the posterior bulbus of the pharynx (Figs 4, 20). Papillae cup-shaped, with a diameter of 11–15 μm, on short annulated stalks, bearing a conical seta in its centre (Figs 21, 22). Amphidial foveas situated at the anterior end bordering the buccal field, showing a distinct sexual dimorphism: small, slightly oval spirals with 1.25 turns in females, 13–18 μm long and 9–12 μm wide; in males much larger, elongated loops, 28–38 μm long and 16–20 μm wide (Figs 5, 6, 12, 18, 28, 29). Figs. 17–26. Robbea hypermnestra sp. nov. Male. 17. Head, in face view; 18. Amphidial fovea; 19. Mouth opening with fingerlike papillae; 20. Row of ventral sucker-shaped papillae; 21. Papilla withdrawn; 22. Papilla extended; 23. Tail; 24. Tips of spicule protruding from cloaca; 25. Group of precloacal setae; 26. Cloaca and precloacal setae. SEM. Figs. 27–31. Robbea hypermnestra sp. nov. 27. Pharyngeal region, optical section; 28. Female, amphidial fovea; 29. Male, amphidial fovea; 30. Female, spiculum; 31. Male, spiculum. LM Interference contrast. Pharynx (Figs 4, 27) 90–117 μm long, with a minute tubular buccal cavity, 7–12 μm long and 4 μm in diameter, leading into a conspicuous pyriform muscular corpus, 34–40 μm long and 18–25 μm wide, which is clearly set off from the following 32–40 μm long isthmus. Posterior bulbus subspherical, 20–38 μm long, 30–45 μm wide, largely glandular, containing only weak muscles. No cardia. Nerve ring 50–65 μm from anterior end; no secretory-excretory pore or ventral gland seen; 8 rows of glandular sense organs (two in each lateral and each median line) connect to the small somatic setae. Males monorchic, testis on the left side of the intestine, beginning at about 35% of body length; sperm spherical, 10 μm in diameter; spicula (Figs 8, 10, 31) strong, arcuate, slightly cephalate proximally, with blunt tips (Fig. 24), 80–88 μm (chord) or 98–105 μm (arc) long; gubernaculum massive, corpus embracing spicules, 20–30 μm long, with strong dorsocaudal apophysis, 28–34 μm long). Females didelphic, ovaries reflexed, long uteri leading to a strongly cuticularized vagina; vulva (Fig. 13) at 41–45% of body length. Large eggs (190 × 50 μm) are often present in one or both uteri, which also contain usually many sperm cells of the same shape and size as seen in males. All females are intersexes, having spicula and a gubernaculum in the anal region (Figs 7, 9, 30). Spicula and gubernaculum much smaller and of simpler construction than in males: length of the female spicula, 53–60 μm (chord) or 62–70 μm (arc); gubernaculum, 10–15 μm long with a similar sized apophysis. No traces of other parts of a male genital apparatus were found. A weakly cuticularized vulva primordium but no other female or male characteristic features could be seen in 7 of 11 large (probably 4th stage) juveniles. The bacteria covering the cuticle of the worms (Fig. 14) are distributed over the whole body, leaving only the tip of the tail and the cephalic capsule uncovered. On the ventral side the bacteria rarely reach the basis of the cephalic capsule. The bacteria are rod shaped and are usually attached with their longitudinal axis perpendicular to the cuticle surface. In a few cases, however, bacteria were seen lying parallel to the cuticular annuli of the worm (Fig. 14). The thiotrophic bacterial ectosymbionts have been previously characterized (Bayer et al., 2009) and belong to the MONTS clade of Gammaproteobacteria (Heindl et al., 2011) that also contains the symbionts of the mouthless nematode genus Astomonema and of gutless oligochaetes. The GenBank accession number for their 16S rRNA gene is EU711428. Diagnosis Species with a distinct cephalic capsule with block-layer; 16–19 cup-shaped stalked ventral papillae in the post pharyngeal region; pharynx almost equally divided into corpus, isthmus and bulbus; all females are intersexes; spicula weakly cephalate, gubernaculum with dorsocaudal apophysis, pronounced sexual dimorphism of the amphidial fovea. Symbiotic bacteria rod-shaped. Robbea ruetzleri sp. nov. http://zoobank.org/urn:lsid:zoobank.org:act:92A7E898-62D0-452D-A6A3-9A622CBD4599 Type material Holotype (male), 3 paratypes (male), 3 paratypes (female), deposited at the US National Museum, Washington, D.C. (accession numbers USNM 1231560-1231566). Measurements See Table 2. Table 2. Morphometric data for Robbea ruetzleri sp. nov. Ranges are given for the male and female paratypes. All measurements are in μm.   Holotype Paratypes (male) n = 3 Paratypes (female) n = 3 Length 2320 2280–3150 2750–3790 a 58.0 57.0–78.8 54.6–70.2 b 30.9 27.8–31.5 33.5–45.1 c 18.9 23.8–47.0 37.2–40.8 maximum width 40 40–45 50–60 pharynx length 75 72–90 72–78 tail length 72 70–88 69–75 nerve ring (% pharynx length) 53 55–60 55–58 corresponding body diameter (cbd) 38 35–40 38–42 bulbus length/width 12/17 15–17/19–20 12–18/18–20 bulbus cbd 35 34–39 33 –39 V n.a. n.a. 52–54 vulva cbd n.a. n.a. 50–60 anal (cloacal) body diameter 40 40–45 32–40 tail length:anal diameter 1.8 1.8–2.0 1.7–2.2 spiculae length arc/chord 75/60 72–85/60–66 n.a. gubernaculum length 35 36–39 n.a. amphid width 10 10–12 10–12 cephalic setae number/length 4/23 4/21–27 4/22–27 subcephalic setae 1 number/length 8/26 8/25–26 8/23–27 subcephalic setae 2 number/length 8/23 8/20–22 8/18–22 Sucker-like papillae, number 15 15–17 n.a. Position of first/last papilla 72/315 80–105/305–415 n.a. n.a. = not applicable. Additional material Several specimens in the authors’ collection and those used for SEM. The sequence of the 18S rRNA gene is available from GenBank and has the accession number KJ414465. Type locality West side of Twin Cayes, Belize; shallow fine sand among Rhizophora mangle stilt roots and Thalassia testudinum beds (Fig. 1). Distribution Rare, in fine to medium subtidal sand samples (Fig. 1). Etymology Named in honour of Klaus Ruetzler, CCRE programme director, friend and generous host on Carrie Bow Cay. Description Body slender, cylindrical (Fig. 32), head diameter at level of cephalic setae 20–25 μm, diameter at level of posterior margin of amphidial fovea 25–30 μm at end of pharynx 33–39 μm, maximum body diameter 40–60 μm, anal diameter 32–45 μm. Tail conical, 60–88 μm long (Figs 35, 36). Figs. 32–36. Robbea ruetzleri sp. nov. 32. Male, whole; 33. Female, anterior body region; 34. Male, anterior body region; 35. Female, tail; 36. Male, tail and spicular apparatus. Cuticle finely transversely striated except for the first 32–44 μm of the head and the last 30–42 μm of the tail, annules 0.5–0.65 μm wide (15–20 annuli/10 μm) (Figs 39, 42); head with a circle of 6 finger-like inner labial papillae surrounding the mouth opening (Fig. 38); 6 short outer labial sensillae, 2.5–4 μm long, at the margin of the membranous buccal field; 4 cephalic setae, flanking the anterior margin of the amphidial fovea, 21–27 μm long (Figs 33, 34, 37); a first circle of 8 subcephalic setae at the level of the amphidial fovea, a second circle approximately in the middle, a third circle at the posterior margin of the cephalic capsule; subcephalic setae, 18–27 μm long, 8 rows of somatic setae along the whole length of the body. Somatic setae of males in the region of the row of ventral papillae stout, 12–17 μm long, the following body setae thinner and shorter (8–13 μm). There is a transverse row of precloacal setae a short distance anterior to the cloacal opening (Figs 36, 42, 43). Somatic setae of females in cervical region 12–15 μm long, the following body setae 17–20 μm long. Males with a 250–320 μm long row of 15–17 conspicuous mid-ventral cuticularized papillae (Figs 34, 40), first papilla situated a short distance posterior to the end of the pharynx. Papillae with short annulated stalks, bearing central conical setae (Fig. 41). The non-striated part of the tail bears no terminal setae, in males there is a velum present (Figs 42, 44). Loop-shaped amphidial foveas (7–12 μm long, 10–12 μm wide), situated at the anterior end bordering the buccal field, with only slight sexual dimorphism. Figs. 37–44. Robbea ruetzleri sp. nov. 37. Male, head in face view; 38. Mouth opening with fingerlike papillae and circle of outer labial sensillae; 39. Annulation and bacterial coat in midbody region; 40. Male, anterior body region with row of sucker-shaped papillae; 41. Male, detail of papillae; 42. Male, tail, cloaca and precloacal setae; 43. Precloacal setae; 44. Male, Tip of tail with velum. SEM. Pharynx 72–90 μm long; minute tubular buccal cavity, 10–15 μm long and 2–4 μm in diameter, leading into a conspicuous pyriform muscular corpus, 22–30 μm long and 16–18 μm wide, clearly set off from the following 24–41 μm long isthmus. Spherical bulb, 12–18 μm long and 17–20 μm wide, mainly glandular and containing only weak muscles. No cardia (Figs 33, 34, 45). Figs. 45–47. Robbea ruetzleri sp. nov. 45. Pharyngeal region, optical section; 46. Male, sucker-shaped papillae and begin of bacterial coat; 47. Male, spiculum. LM of live animals. Nerve ring 42–60 μm from anterior end; no secretory-excretory pore or ventral gland seen; there are 8 rows of glandular sense organs (two in each lateral and each median line) connecting to the small somatic setae. Males monorchic, testis on the left side of the intestine, beginning at about 35% of body length; spicula strong, arcuate, distinctly cephalate proximally, 60–66 μm (chord) or 72–85 μm (arc) long, without velum; gubernaculum simple, embracing the distal part of the spicula laterally, with dorsally directed apophysis (35–37 μm long) (Figs 36, 47). Females didelphic, ovaries reflexed, long uteri leading to the vagina; vulva at 52–54 of body length. A dense monolayer of rod-shaped symbiotic bacteria (Fig. 39) covers almost the whole body, beginning at the level of the pharyngeal terminal bulb or at the level of the posterior region of the cup-shaped cervical papillae (Fig. 46) and terminating with the cuticle striation at the tail tip or already at the level of the cloacal opening. Rows or plaques of smaller cocci stretch forward up to the anterior bulbus of the pharynx, but not up to the non-striated part of the head. Diagnosis Species with indistinct cephalic capsule; 14–17 cup-shaped stalked ventral papillae in the post pharyngeal region; pharynx almost equally divided into corpus, isthmus and bulbus; spicula strongly cephalate, gubernaculums without apophysis; amphidial fovea in both sexes open loop-shaped. Symbiotic bacteria rod shaped. Robbea agricola sp. nov. http://zoobank.org/urn:lsid:zoobank.org:act:97995BAA-7914-4578-A4DE-64ED0DEF761B Type material Holotype (male), 3 paratypes (male), 3 paratypes (female), deposited at the US National Museum, Washington, D.C. (accession numbers USNM 1231567-1231573). Additional material Several specimens in the authors’ collection and those used for SEM. Measurements See Table 3. Table 3. Morphometric data for Robbea agricola sp. n. Ranges are given for the male and female paratypes. All measurements are in μm.   Holotype Paratypes (male) n = 3 Paratypes (female) n = 3 Length 1880 1780–2550 2280–2550 a 47.0 40.0–54.3 57.0–68.2 b 19.8 19.6–28.7 28.0–37.4 c 22.1 18.8–21.3 27.3–29.6 maximum width 40 33–38 35–42 pharynx length 95 60–75 80–90 tail length 85 78–105 70–85 nerve ring (% pharynx length) 57 53–60 53–62 corresponding body diameter (cbd) 30 28–35 30–32 bulbus length/width 15/20 12–15/17–18 12–15/18–20 bulbus cbd 30 25–32 28–33 V n.a. n.a. 52–55 vulva cbd n.a. n.a. 35–42 anal (cloacal) body diameter 30 30–35 24–30 tail length:anal diameter 2.8 2.6–3.3 2.3–3.5 spiculae length arc/chord 55/40 45–58/35–45 n.a. gubernaculum length/apophysis length 15/15 10–12/10–13 n.a. amphid width 16 12–14 8–12 cephalic setae number/length 4/21 4/20–21 4/21–22 subcephalic setae 1 number/length 8/20 8/16 –20 8/20–26 subcephalic setae 2 number/length 8/16–18 8/13–16 8/18–22 Sucker-like papillae, number 9 9 n.a. Position of first/last papilla 90/260 70–98/225–268 n.a. n.a. = not applicable. The sequence of the 18S rRNA gene is available from GenBank and has the accession number KJ414464. Type locality West side of Twin Cayes, Belize; shallow fine sand among Rhizophora mangle stilt roots and Thalassia testudinum beds (Fig. 1). Distribution Regularly in fine sand samples at several locations in the vicinity of the CBC laboratory (Fig. 1). Etymology Agricola (lat.) = farmer (German ‘Bauer’), named after Monika Bright (then Monika Bauer) who provided the first specimens. Description Body slender, cylindrical (Figs 48, 49), tapering only slightly towards anterior end, head diameter at level of cephalic setae 18–25 μm, diameter at level of posterior margin of amphidial fovea 20–29 μm, at end of pharynx 25–33 μm, maximum body diameter 33–42 μm, anal diameter 24–35 μm. Tail conical, 70–105 μm long; non-striated tail tip 17–19 μm long in males and 23–30 μm in females (Figs 52, 53, 62). Figs. 48–54. Robbea agricola sp. nov. 48. Male, total; 49. Female, total; 50. Female, head region; 51. Male, anterior body region; 52. Female, tail; 53. Male, tail; 54. Spicular apparatus. Figs. 55–62. Robbea agricola sp. nov. 55. Male, head in face view; 56. Mouth opening with fingerlike papillae; 57. Annulation and bacterial coat in midbody region; 58. Coat of corn-kernel shaped bacteria; 59. Male, anterior body region with the first part of the row of sucker-shaped papillae; 60. Papillae; 61. Papillae surrounded by bacterial growth; 62. Female, tail. SEM. Cuticle finely transversely striated except 24–28 μm of the anterior part of the head (cephalic capsule) and the tail tip, annules 0.6 to 0.7 μm wide (14–17 annuli/10 μm) (Fig. 57). Mouth opening surrounded by 6 finger-like inner labial papillae in lateral, subventral and subdorsal position (Fig. 56) followed by a circle of 6 short labial sensillae, 3–4 μm long, surrounding the membranous buccal field; a circle of 4 cephalic setae flanking the anterior margin of the amphidial fovea, 20–22 μm long; closely followed by a circle of 8 subcephalic setae, 16–26 μm long, a second circle near the end of the cephalic capsule (Figs 50, 51, 55); 8 rows of somatic setae along the whole length of the body, 5–8 μm long; a pair of 6–8 μm long setae at the begin of the non-striated part of the tail. No special precloacal setae. Mid-ventral line of males with a 150–170 μm long row of 8–9 conspicuous cup-shaped papillae (diameter 7 μm) positioned on short stalks. Papillae with central setae (1.5 μm long) (Figs 51, 59, 60). The first of these papillae is situated at a distance of 70–110 μm from the anterior end at the level of the pharyngeal terminal bulb. Amphidial foveas spiral with 2.5 turns, slight sexual dimorphism: in females oval, 12–13 μm long and 8–12 μm wide, situated directly at the anterior end bordering the buccal field; in males larger, somewhat elongated, 15–16 μm long, 12–14 μm wide, 1–4 μm from the anterior end (Figs 50, 51, 64). Figs. 63–65. Robbea agricola sp. nov. 63. Pharyngeal region, optical section; 64. Male, amphidial fovea; 65. Male, spiculum. LM Interference contrast. Figs. 66–69. Robbea agricola sp. nov. 66. Anterior body region and beginning of bacterial coat; 67. Beginning of bacterial coat, detail; 68. Large cocci on anterior body region; 69. Glandular sensory organs. LM of live animals. Fig. 70. Robbea hypermnestra sp. nov. Semi-thin longitudinal section through pharynx showing separation of corpus from isthmus and glandular bulbus. Fig. 71. Phylogenetic relationships of the genera of the Stilbonematinae based on the 18S rRNA gene. The tree shown was calculated using maximum likelihood (RAxML) and node support is given as aLRT as well as Bayesian posterior probabilities. The scale bar indicates 0.02 nucleotide substitutions per site. A minute tubular buccal cavity, 7–10 μm long and 2–3 μm in diameter, leading into a tripartite, 60–95 μm long pharynx consisting of an anterior pyriform muscular corpus (19–30 μm long, 12–18 μm wide), the following isthmus (30–50 μm long) and the terminal bulb (12–18 μm long, 17–20 μm wide). Terminal bulb largely glandular, containing only weak muscles. No cardia (Figs 50, 51, 63, 69). Nerve ring 44–67 μm from anterior end; no secretory-excretory pore or ventral gland seen; 8 rows of glandular sense organs (two in each lateral and each median line) connecting to the small somatic setae. Males monorchic, testis on the left side of the intestine, beginning at about 35% of body length; spicula strong, arcuate, slightly cephalate proximally, 35–45 μm (chord) or 55–58 μm (arc) long, with a velum; gubernaculum with a strong dorso-caudal directed apophysis (13–15μm long) ending in a spherical swelling (Figs 53, 54, 65). Females didelphic, ovaries reflexed, long uteri leading to the vagina, ventral gso enlarged in the region of the uteri; vulva at 52–55% of body length. Epigrowth of symbiotic bacteria starting at a defined line at a distance from the anterior end (Figs 66, 67) showing a sexual dimorphism: in males the bacterial coat begins at 220–340 μm (2.7–3.5 pharynx length), in females at 155–195 μm (2–2.3 pharynx length). A monolayer of corn-kernel shaped bacteria (1.5 × 0.8 μm) (Figs 57, 58) covers the remaining body except the non-striated tip of the tail. At the start of the coat the body diameter abruptly becomes smaller to accommodate the thickness of the bacterial layer without increasing the consortium's diameter (Fig. 67). In some specimens patches of bacteria occur around the cup-shaped papillae; here rods lie parallel to the cuticle surface (Fig. 61). Occasionally larger coccoid bacteria are found on the normally symbiont-free anterior body part (Fig. 68). The role of these bacteria is unknown. Suctorians are frequently attached to the cuticle in the posterior body region (Fig. 49). Diagnosis Species with indistinct cephalic capsule; 8–9 cup-shaped ventral papillae in post-pharyngeal region; isthmus occupies more than 50% of pharynx length; spicula cephalate with velum, gubernaculums with dorso-caudal apophysis ending in a spherical swelling; amphidial fovea spiral in both sexes, larger in males. Symbiotic bacteria corn-kernel shaped, bacterial coat starting at a defined line 2 to 3.5 pharynx lengths from the anterior end. Discussion Systematics Gerlach (1956) erected the genus Robbea for a male animal collected on the coast of Brazil, which he named Robbea caelestis. A second species was subsequently described from the Maldive Islands by the same author, Robbea tenax Gerlach (1963). The material consisted of one female and three males, the latter showing conspicuous cervical papillae. Both Robbea species were later placed into the genus Catanema Cobb, 1920 by Platt & Zhang (1982). Recently Tchesunov (2013) proposed diagnoses for both Robbea and Catanema in which the major distinguishing character is the shape of the amphidial fovea, which is distinct in Robbea but is reduced to a small apical opening in Catanema. Other characters such as the degree to which the pharyngeal corpus is set off from the isthmus, ‘clearly’ in Robbea, ‘distinctly’ in Catanema (see Fig. 70 for the situation in Robbea hypermnestra), and the presence (Robbea) or absence (Catanema) of a cephalic capsule are less clear and not all descriptions published so far contain explicit statements regarding these characters. According to Tchesunov's criteria, the following species belong to the genus Robbea: R. caelestis Gerlach 1956, R. tenax Gerlach 1963, R. gallica Vitiello 1974, R. (Catanema) porosum Hopper & Cefalu 1973, R. (Catanema) macintyrei Platt & Zhang 1982, R. (Catanema) smo Platt & Zhang 1982, furthermore the animal that Hopper & Cefalu (1973) described as R. tenax from Florida and the three new species described herein. R. gerlachi Boucher 1975 has only been described from a female, but according to the shape of its amphidial fovea it should be placed into the genus Robbea. One additional species of Robbea has been suggested by Tchesunov (2013), but due to the lack of male specimens the species has not been described in detail yet. In the genus Catanema only two formally described species remain, the type species C. exile Cobb 1920 and C. dambayensis Tchesunov 2013. Catanema cobbi Inglis 1967 has been placed into the genus Laxus by Ott et al. (1995), Catanema gerlachi sensu Hopper & Cefalu 1973 most probably belongs to Laxus cosmopolitus (Ott et al., 1995). To consolidate the taxonomic descriptions of the three newly described Robbea species on a molecular level we used phylogenetic 18S rRNA analyses. The tree shows a clear separation between the clade containing the three new Robbea species and the other Stilbonematinae (Fig. 71). We also provide additional 18S rRNA gene sequences for the genera Catanema and Leptonemella that only had a single 18S rRNA gene deposited prior to this study. The morphological characters of the three yet undescribed Catanema species included in the phylogenetic analyses conform to the diagnosis given by Tchesunov (2013). Our data confirm that both Robbea and Catanema, as well as all other genera of Stilbonematinae with available sequence data are represented by statistically supported genus level clades in 18S rRNA gene based phylogenetic analyses. This high resolution of the 18S rRNA gene finally allows to assign or to re-evaluate the correct taxonomic affiliation at the genus level for new or already deposited sequences. For the moment this ends the confusion around these two genera to which we have added by assigning two stilbonematine species to the genus Robbea (namely Robbea sp. 1 and 2) in Bayer et al. (2009). We have reinvestigated the nematode material used in that paper where possible. In the case of Robbea sp. 1 from Calvi (Corse), there obviously had been a mix-up during the sample sorting, and two different nematode species were present in the sample analysed: a Laxus sp. that is morphologically indistinguishable from Laxus cosmopolitus described from the Adriatic (Ott et al., 1995) and a yet undescribed Catanema species. Judging from the phylogenetic position of the deposited 18S rRNA gene, a specimen of the former species was probably sequenced. Robbea sp. 2 from the Cayman Islands is a member of the Catanema clade (Fig. 71). Robbea sp. 3 is morphologically and phylogenetically identical to the newly sequenced Robbea hypermnestra sp. nov. specimens (Fig. 71). The 18S sequences for Robbea hypermnestra sensu Kampfer 1998 (nomen nudum) that were formerly deposited under Y19621 were identified as chimeric but the last 600 bp are identical to the correct Robbea hypermnestra sp. nov. sequences from this study as well as to the Robbea sp. 3 sequence published by Bayer et al. (2009). The sequences in the Kampfer et al. (1998) paper were generated from pools of up to 50 nematodes that likely were contaminated by other nematode species. Thus, the chimeric nature of the published sequences could likely be attributed to different priming bias in the forward and reverse primers used. In contrast, all sequences generated in the present work come from single nematode specimens. This has been made possible with high-yield DNA extraction methods based on GeneReleaser (Bioventures) (Schizas et al., 1997) or the Blood and Tissue kit (Qiagen) combined with highly sensitive and efficient polymerases such as the Phusion® DNA polymerase (Finnzymes). Successful PCR based sequencing of multiple genes from the DNA of a single meiofauna individual has been performed without amplification using specimens as small as 500 μm long microturbellarians (Gruber-Vodicka et al., 2011). We thus emphasize that, wherever possible, single individuals should be used for PCR-based gene assays. While the diagnoses given by Tchesunov (2013) currently hold true for the genera Robbea and Catanema, they are based on characters such as the reduction of the amphidial foveas. However, these characters are also present in some species of other genera, such as Stilbonema and Leptonemella (e.g. in the type species L. cincta COBB 1920). Findings of new species may make emendation of diagnoses that were based on morphology alone necessary. Our results clearly indicate the necessity to provide molecular data to confirm the morphological identification and that larger taxon sampling is an important factor to be able to validate sequencing results and enable for example barcoding approaches. Intersexes Cases of intersexuality have been described from various terrestrial, parasitic or marine nematodes (e.g. Zhuo et al., 2009; Moura et al., 2014). In the latter, intersexes are most commonly reported as females with a functional reproductive system and rudimentary male sexual characters (e.g. Gourbault & Vincx, 1990; Riemann et al., 2003; Zhuo et al., 2009; Miljutina et al., 2013; Moura et al., 2014), just as observed in our newly described R. hypermnestra. However, only few intersex individuals are usually found within nematode populations (Gourbault & Vincx, 1990). As driving factor for intersexuality in nematodes, unfavourable environmental conditions (Davide & Triantaphyllou, 1968), hybridization between closely related species (Steiner, 1923; Krall, 1972) or genetic or chromosomal disorder (Roy & Gupta, 1975; Jairajpuri et al., 1977) have been hypothesized. Considering the clean and stable environment around Carrie Bow Cay and the fact that all females of R. hypermnestra are intersexes, environmental sex determination appears unlikely. We cannot exclude hybridization events with closely related species in the past. It is difficult to assign any specific function to the spicula observed in R. hypermnestra intersexes and the R. hypermnestra chromosome set has not been determined, thus our hypotheses will remain rather speculative. The spicula could be remnants from a previously hermaphroditic lifestyle, but since no hermaphroditic lifestyle is known from stilbonematine nematodes, the interpretations that R. hypermnestra females are in a transition to a hermaphroditic stage, is more likely. In fact, individuals of the well-studied terrestrial nematodes C. elegans and C. briggsae are either male or hermaphrodites where the hermaphrodites are descendants from male or female ancestors and mutations in two independent pathways were sufficient for C. elegans to develop self-fertile hermaphrodites (Baldi et al., 2009). This indicates a high plasticity and flexibility in the expression of sexual phenotypes in nematodes in general. Hermaphroditism could be favourable at low effective population densities to ensure reproduction in the absence of a mating partner (Pires-DaSilva, 2007). To test whether this is the case, the distribution patterns and gender ratios of the R. hypermnestra populations need to be monitored systematically. Ecological notes The shallow subtidal sands around Carrie Bow Cay harbour a diverse interstitial meiofauna. Among these are representatives of several taxa known to harbour bacterial symbionts. These include the ciliate genus Kentrophoros, the mouthless nematode genus Astomonema (authors’ unpublished observation), several species of the mouthless catenulid flatworm genus Paracatenula (Dirks et al., 2011; Gruber-Vodicka et al., 2011) and the gutless marine oligochaete genera Inanidrilus and Olavius (Erséus, 1990). So far, stilbonematid nematode species of several genera have been described from shallow water habitats around Carrie Bow Cay. These include Laxus oneistus, Stilbonema majum, Adelphus rolandi, Eubostrichus dianae, E.parasitiferus and E. fertilis (Ott et al., 1995, 2014; Ott, 1997; Kampfer et al., 1998; Polz et al., 1999). We add to this diversity with our description of three new Robbea species that live in vicinity of Carrie Bow Cay (Fig. 1). Available physiological and molecular evidence shows that all (Paracatenula, Stilbonematinae) or the majority of bacterial symbionts (oligochaetes) are sulphur-oxidizing chemoautotrophs (SOBs) (Dubilier et al., 2006; Bayer et al., 2009; Gruber-Vodicka et al., 2011). Associations of SOBs with motile hosts appear to be beneficial when oxygen-containing surface layers are spatially separated from sulphidic deeper layers and vertical migration by the host can alternately supply the symbiotic bacteria with sulphide and oxygen (Giere et al., 1991; Ott et al., 1991). Shallow sandbars in the immediate back reef area of Carrie Bow Cay consist of coarse to medium, unsorted carbonate sand of local origin that has been deposited under sheltered conditions and has a spacious interstitium with reduced, sulphidic conditions in layers several centimetres below the sediment surface (Ott & Novak, 1989). Here large species of Stilbonematinae (Laxus oneistus, Stilbonema majum) and also R. hypermnestra are found in high densities (Ott & Novak, 1989). In contrast, a diverse assemblage of smaller Stilbonematinae species such as E. dianae, E. fertilis, R. agricola and R. ruetzleri inhabit the fine sands in the vicinity of lagunal mangrove islands and Thalassia testudinum seagrass beds (Ott et al., 2014). Additionally to the described species, several undescribed species and possibly genera of stilbonematine nematodes can be found in the shallow water habitats in the vicinity of Carrie Bow Cay (authors’ pers. observation). Acknowledgements We thank Werner Urbancik, Veronica Novotny and Monika Bright for providing several micrographs and Sigrid Neulinger for executing the ink drawings. We gratefully acknowledge the generous help of Klaus Ruetzler and the staff of the Carrie Bow Cay Laboratory. This article was originally published with errors. This version has been corrected. Please see Corrigendum (http://dx.doi.org/10.1080/14772000.2014.960221). Associate Editor: Adrian Glover List of abbreviations in figures a amphidial fovea b pharyngeal bulbus bacbacteria cpharyngeal corpus ccscentral conical setae cgcorpus gelatum cgocaudal gland opening clcloaca cscephalic seta faamphidial fovea fovea fpfinger-like papillae ggut gsoglandular sensory organ ipharyngeal isthmus lslabial sensillae lsslong somatic setae nanon-striated tail tip psucker shaped papillae pcsprecloacal setae sssomatic setae scssubcephalic seta spspiculum sssomatic setae tsterminal seta velvelum ==== Refs References Baldi C. Cho S. 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==== Front Nucl Instrum Methods Phys Res ANucl Instrum Methods Phys Res ANuclear Instruments & Methods in Physics Research. Section A, Accelerators, Spectrometers, Detectors and Associated Equipment0168-90021872-9576Elsevier B.V S0168-9002(16)00163-710.1016/j.nima.2016.02.013ArticleA new silicon tracker for proton imaging and dosimetry Taylor J.T. jtaylor@hep.ph.liv.ac.uka⁎Waltham C. bPrice T. cAllinson N.M. bAllport P.P. cCasse G.L. aKacperek A. dManger S. eSmith N.A. aTsurin I. aa Department of Physics, University of Liverpool, Oxford Street, Liverpool L69 7ZE, UKb Laboratory of Vision Engineering, School of Computer Science, University of Lincoln, Lincoln LN6 7TS, UKc School of Physics and Astronomy, University of Birmingham, Birmingham B25 2TT, UKd Douglas Cyclotron, The Clatterbridge Cancer Centre NHS Foundation Trust, Clatterbridge Road, Bebington, Wirral CH63 4JY, UKe Department of Physics, University of Warwick, Coventry CV4 7AL, UK⁎ Corresponding author. jtaylor@hep.ph.liv.ac.uk21 9 2016 21 9 2016 831 362 366 © 2016 The Authors2016This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).For many years, silicon micro-strip detectors have been successfully used as tracking detectors for particle and nuclear physics experiments. A new application of this technology is to the field of particle therapy where radiotherapy is carried out by use of charged particles such as protons or carbon ions. Such a treatment has been shown to have advantages over standard x-ray radiotherapy and as a result of this, many new centres offering particle therapy are currently under construction around the world today. The Proton Radiotherapy, Verification and Dosimetry Applications (PRaVDA) consortium are developing instrumentation for particle therapy based upon technology from high-energy physics. The characteristics of a new silicon micro-strip tracker for particle therapy will be presented. The array uses specifically designed, large area sensors with technology choices that follow closely those taken for the ATLAS experiment at the HL-LHC. These detectors will be arranged into four units each with three layers in an x–u–v configuration to be suitable for fast proton tracking with minimal ambiguities. The sensors will form a tracker capable of tracing the path of ~200 MeV protons entering and exiting a patient allowing a new mode of imaging known as proton computed tomography (pCT). This will aid the accurate delivery of treatment doses and in addition, the tracker will also be used to monitor the beam profile and total dose delivered during the high fluences used for treatment. We present here details of the design, construction and assembly of one of the four units that will make up the complete tracker along with its characterisation using radiation tests carried out using a 90Sr source in the laboratory and a 60 MeV proton beam at the Clatterbridge Cancer Centre. Keywords Silicon tracking detectorsProton therapyDosimetryProton computed tomography ==== Body 1 Introduction Particle therapy: alongside surgery and chemotherapy, radiotherapy remains one of the three major tools used by clinicians to combat cancer today. Typically, the standard approach for radiotherapy involves the use of x-ray beams, a technique that was first developed for medicine by physicists. Radiotherapy today, still remains a field that continues to go hand in hand with developments in physics and engineering [1]. For cancers deep inside the body or close to critical structures, particle therapy has been shown to have a distinct advantage over standard x-ray radiotherapy. This is a result of the underlying physics that describes how radiation interacts with matter. For charged particles, such as protons, energy loss is described by the well established Bethe–Bloch formula [2] and results in a characteristic ‘Bragg Peak’. This is where the majority of the particles stop and where their dE/dx or Linear Energy Transfer (LET) is maximal, resulting in a concentration of the dose in this region. Taking advantage of this dose distribution for radiotherapy was first envisaged by Wilson [3]. For x-rays, energy is lost in an exponential fashion and thus much of the dose is given to healthy tissue before and after the tumour. This can be avoided by using a beam of charged particles such as protons. This sparing of healthy tissue also makes particle therapy the best choice for some childhood cancers, since growing tissues and bones are more radiosensitive than in adults and critical organs are located much closer together. In this case, the excess dose to healthy tissue can cause irreparable damage to developing cells that can cause other cancers later on in life [4]. For this reason, many of the patients that the National Health Service (NHS) in the UK currently sends abroad each year for proton therapy treatment are children [5]. Because of these advantages, approximately 50 centres offering particle therapy have been built around the world, with many new centres under construction worldwide including two new NHS proton facilities in the UK. Proton imaging: instrumental to the planning of any program of radiotherapy is a good imaging modality that can deliver accurate information on the patient׳s anatomy, and in particular the accurate location of the target volume. For proton therapy, this is carried out by an x-ray CT scan from which the proton stopping power of the tissue can be derived and the necessary range of the treatment beam calculated. During this conversion from x-ray imaging to proton stopping power, an uncertainty in the proton range is introduced, of order 1–3 mm [6]. This arises from variations in density along the proton path [7] and inaccuracies in the excitation energy or electron density value assumed for the tissue [8], as well as from the stopping power conversion method itself [9]. Uncertainties in the proton range increase the amount of dose delivered to the healthy tissue surrounding the cancer and can therefore prevent the treatment of cancers close to critical structures. If this uncertainty could be reduced, several of the limitations with proton therapy could be overcome. These uncertainties could potentially be reduced if the stopping power of the beam could be measured directly i.e. by using protons for imaging as well as for treatment [10]. In order to carry out this technique (referred to as proton computed tomography, or simply pCT), a device that can accurately measure the trajectory and energy loss of protons as they pass through an object for many different angular projections is needed [11], [12], [13], [14]. The PRaVDA Consortium [15] aims to construct a prototype of the first fully solid state pCT scanner using silicon detectors for both the tracking and range (energy) measurements of protons (see Fig. 1) [16], [17], [18], [19]. We have designed and begun construction of the silicon strip tracker, using large area (~10×10 cm) micro-strip detectors adapted from designs made for the ATLAS experiment at the high-luminosity LHC allowing the sensors to be very radiation hard [20], [21]. The sensitive area of the detectors was constrained by the available space on a standard 6-in. silicon wafer and when arranged in the proposed x–u–v configuration provides an imaging area of ~9 cm. Future designs for imaging larger, more clinically relevant areas could be done by using these detectors ganged together (with some dead regions), or by moving the imaging system itself. The current design of the tracker will demonstrate that a completely solid state system offering high-precision directional information on the path of protons can be used in conjunction with an energy-range measurement to perform a pCT scan [22]. 2 Assembly and readout of the tracking units Silicon micro-strip sensor: each silicon micro-strip detector has a nominal thickness of 150 μm and is made from n-in-p silicon. The detector contains 2048 strips in total, 1024 read out on each side of the detector by eight ASICs (see Fig. 2). Each strip has a pitch of 90.8 μm and a length of 4.8 cm and its metal layer is capacitively coupled to its implant with a measured coupling capacitance of 122 pF. Further details of the layout and electrical characteristics of the sensor can be found here [22]. Detectors are aligned to the hybrid PCB and a 12 mm thick aluminium stiffener plate that holds the detector and its associated readout electronics within the tracker unit housing (see Fig. 4). Both the PCB and the aluminium plate contain a square 10×10 cm aperture beneath the sensitive area of the detector to keep the perturbation of the proton path to a minimum. Initial mechanical alignment of the sensors is made using precision ground dowels and a custom built alignment jig with precision ground edges (see Fig. 2) and carried out as part of the gluing and assembly process for each detector used. The alignment achieved with this tooling was found to be within the strip pitch of the detector (90.8 μm) and the rotational alignment to be a few mrad. This was measured using a Smartscope metrology machine which makes optical measurements using a camera in order to estimate the height and flatness and lateral position of the silicon above the PCB surface since the detector could not be probed mechanically due to its fragile nature. Further precision in alignment will be achievable using particle tracks when multiple tracking units are available. The tracker is comprised of 12 detectors separated into four units each containing three detectors each. Two units are placed either side of the object to be imaged as shown in Fig. 1. Each tracking unit has its detectors held at an angle of 60° with respect to one another in an x–u–v co-ordinate system in order to measure precise x–y locations for particles at high fluence with minimal ambiguities. The angle is achieved by suspending the aluminium plates containing the micro-strip detectors and their associated readout electronics on six precision ground dowels (see Fig. 4). Readout electronics: the detector is read out by means of an ASIC designed specifically for this application by ISDI Ltd. [23] and known as RHEA (Rapid, High-speed Extended ASIC). RHEA is a binary chip with 128 channels and a bonding pitch of 60 μm fabricated in 0.18 μm CMOS (see Fig. 3). Each channel has two tunable thresholds (DAC1: 2000–10,000 e−, and DAC2: 20,000 – 160,000 e−) to allow for high occupancy, and the chip operates at a frequency of 104 MHz and its front-end amplifier with a shaping time of 30 ns. This corresponds to four times the average cyclotron frequency for the energy range of interest (60–200 MeV) at the facilities that will be used. There are two modes that can be used to acquire data: treatment mode and patient imaging or pCT mode. In treatment mode all strips are read out at ~100 μs intervals to allow sampling of the beam distribution for quality assurance (QA) purposes and dosimetry during the high fluences used during patient treatment. In pCT mode, it is possible to read up to four channels per ASIC with signal over threshold for the expected nominal beam spill repetition rate of 26 MHz. This allows for accurate tracking of multiple protons through the system at any one time. The hybrid PCB which can be seen in Fig. 2 is used to mount the detector and RHEA chips. It is responsible for delivering power, calibration and serial interface data to the ASICs as well as routing the data output from the ASICs to the FPGA׳s that are mounted on a data acquisition (DAQ) board which surrounds the hybrid (see Fig. 4). The DAQ boards and associated software were designed by aSpect Systems GmbH [24]. 3 Experimental work and results Tests with minimum ionising particles: preliminary tests with the assembled tracking unit were carried out in the lab using minimum ionising particles (MIPs) from a 90Sr source with a activity of 37 MBq. Although the tracker was never designed to track such particles, it served as a useful diagnostic during assembly to see, for each layer, if the signals were visible above the noise and could be read out with the DAQ. This test was repeated after construction of the tracking unit in order to verify that signals could be seen separately in each of the three layers (and six strip halves) in order to optimise the spacing of the layers and prevent crosstalk. Such tests provided valuable information for adjustments of the electronics and improvement of the DAQ software, as well as allowing sensor/ASIC characteristics to be extracted during times when tests at an accelerator were not possible. Using these methods, the number of dead or noisy strips turned off by the DAQ was found on average to be <0.5% per layer. The most probable value for the expected signal size of MIPs in the 150 μm thick detectors used is: ~12,000 e−. This signal is less than half of what will be expected in the final application when beams of protons with energies ranging from 60 to 200 MeV are used and thus, optimisation of the system for detection of MIPs allowed the system to be prepared for tests in a proton beam. In Fig. 5, the 1D hit maps for data from the 90Sr source is shown in the three layers of a tracking unit formed from the data of six strip halves. This data was collected for several thousand frames in the treatment mode readout (which reads out all available channels) and allowed the testing of all channels in both the first and second threshold. This can be seen in the blue and red histograms respectively, set at their default values of 84 and 200 mV. which correspond to the end of the first threshold (~10,000 e− and the middle of the second threshold (~90,000 e−). Tests with a proton beam: tests were carried out using a 60 MeV beam of protons from the Douglas Cyclotron at the Clatterbridge Cancer Centre. This is currently the only clinical proton beam in the UK and has been used for research as well as for the treatment of eye cancers for many years [25]. Fig. 6 shows the setup on the Clatterbridge beam line with the single tracking unit positioned at the end of the treatment nozzle. A ‘field lamp’ (shown in the inset of Fig. 6) was used to check for reasonable alignment of the active area of the sensor with the beam emerging from the nozzle. During the experiment, a beam of protons with a width of 30 mm was used and its fluence monitored by means of an ionisation chamber. The chamber (connected to an electrometer) was situated a few cm after the nozzle in front of the detector (not shown in Fig. 6) and the background current observed when the beam was off was found to be <1pA. A typical current of between 5 and 10 pA was used for all measurements which ensured that each successive frame would contain on average only one proton in order to be sure of characterising the system without any pile-up or saturation present. The reconstructed (x,y) locations of the protons for the 30 mm beam at a beam current of 7 pA can be seen for data in the first threshold in Fig. 7. The effect of the scattering foil placed upstream of the detectors is evidenced by the flat top of the distributions. Data was collected for 1 s using the CT mode where each successive event receives a timestamp depending on the time it occurs relative to the internal clock supplied to the ASIC by the FPGA on the DAQ readout board. This allowed the proton hits in the unit to be tracked across subsequent layers and reconstructed (x,y) locations calculated. A two-dimensional hit map of the beam using these locations can be seen in Fig. 8 reconstructed using two layers in the tracking unit. The addition of more layers and tracking units read out in this mode will be used in order to carry out a pCT scan. 4 Conclusion We have presented here the assembly and first results of a three layer tracking unit that will be deployed along with three other units for particle therapy applications. The results presented here show that both modes of data acquisition and both thresholds are operational and that tracking between the layers is possible. The complete tracker will be capable of fast particle tracking with minimal ambiguities and can therefore give useful measurements for dosimetry and beam QA, as well as providing the necessary directional information on the path of individual protons during a pCT scan for proton imaging. Future papers will concentrate on the reconstruction of 2D images from all layers across multiple tracking units, as well as the tracking of protons between units, in order to measure the scattering angles for a particular beam configuration. Furthermore, the ability of the tracker to track through a thick target (~75 mm phantom) in conjunction with an energy-range measurement of the protons will also be assessed. This is necessary in order to know whether it will be possible to demonstrate that a pCT scan is possible with a fully solid state system of this kind. Acknowledgements We would like to thank the members of the PRaVDA consortium, aSpect Systems GmbH, and ISDI Ltd. for their contributions and discussion of the results presented in this paper. We would also like to thank the operators and physicists at the Clatterbridge Cancer Centre for producing and maintaining the proton beams that were used to make the measurements presented here. This work was supported by a Wellcome Trust Translation Award (Grant no. 098285). Fig. 1 The PRaVDA pCT system concept. The tracker is comprised of the first four units shown here as the two which are placed in front of the object to be imaged and the two after. A range telescope (calorimeter) is placed immediately after the tracker to measure the residual energy of each proton after it has been tracked through the object to be imaged. Fig. 2 Alignment of detector to hybrid PCB and aluminium stiffener plate using precision ground tooling consisting of dowels and perspex jigs. Fig. 3 A RHEA ASIC measuring 8.2×5.2×0.85 mm with back end bonding to hybrid PCB. The staggered front-end bond pads that are empty are ready for wire-bonding to a micro-strip detector after gluing and assembly of the detector on to the hybrid PCB. Fig. 4 Setup of the three layer tracking unit assembled in the housing with a multiplexer board, HV unit and associated cables. The three layers that make up the tracking unit are secured using six dowels that provide registration and alignment at the required angle. Fig. 5 Distribution of MIPs from a 90Sr source measured in each of the three layers of the tracking unit. The upper histogram in each frame (blue data) is for the first threshold and the lower histogram in each frame (red data) for the second threshold. The top layer closest to the source is labelled as ‘U’ and is orientated at +60°. The middle layer labelled ‘X’ is orientated at 0°, and the bottom layer labelled ‘V’ is orientated at −60°. (For interpretation of the references to colour in this figure caption, the reader is referred to the web version of this paper.) Fig. 6 Experimental setup at the Clatterbridge Cancer Centre. The inset figure shows alignment of the tracking unit׳s sensitive area with the beam using the field lamp. Fig. 7 The 1D reconstructed hit maps in x and y of a 30 mm, 60 MeV proton beam at the Clatterbridge Cancer Centre. The distributions were reconstructed into 800 μm bins using two planes of the tracker unit orientated at 60° to one another. Fig. 8 The 2D reconstructed hit map of a 30 mm, 60 MeV proton beam at the Clatterbridge Cancer Centre. 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