It has been demonstrated that motor coordination of interacting people plays a crucial role in the success of social exchanges. Abnormal movements have been reported during interpersonal interactions of patients suffering from schizophrenia and a motor coordination breakdown could explain this social interaction deficit, which is one of the main and earliest features of the illness. Using the dynamical systems framework, the goal of the current study was (i) to investigate whether social motor coordination is impaired in schizophrenia and (ii) to determine the underlying perceptual or cognitive processes that may be affected. We examined intentional and unintentional social motor coordination in participants oscillating hand-held pendulums from the wrist. The control group consisted of twenty healthy participant pairs while the experimental group consisted of twenty participant pairs that included one participant suffering from schizophrenia. The results showed that unintentional social motor coordination was preserved while intentional social motor coordination was impaired. In intentional coordination, the schizophrenia group displayed coordination patterns that had lower stability and in which the patient never led the coordination. A coupled oscillator model suggests that the schizophrenia group coordination pattern was due to a decrease in the amount of available information together with a delay in information transmission. Our study thus identified relational motor signatures of schizophrenia and opens new perspectives for detecting the illness and improving social interactions of patients. It has been demonstrated that the interaction at the level of body movements plays a crucial role in the success of social exchanges. Motor coordination of interacting people influences social cognitive functioning, for example, it impacts feelings of connectedness or interpersonal rapport Previous research has shown the relevance of theories and tools of dynamical systems to examine social motor coordination Using the dynamical systems methodology, the goal of the current study was to evaluate the dynamics of social motor coordination in people with schizophrenia in order to further characterize their abnormal interpersonal movements and understand the processes affected by the pathology. More specifically, we assumed that the stability of the coordination could be decreased in schizophrenia because of either (i) a modification of their preferred frequency of movement due to their motor disorder To investigate unintentional social coordination, each pair of participants performed three 90 s trials each containing three 30 s segments completed continuously F = 329.71, p<.05, η2 = 0.95) indicating that in accordance with previous research the preferred frequency of participants was higher when the limbs inertial loadings was less  F = 4.25, p>.05, η2 = 0.18) or interaction between Group and Pendulum  = 1.60, p>.05, η2 = 0.08).To test first whether the preferred frequencies of movements in schizophrenia could be affected and could destabilize social motor coordination, we compared the preferred frequencies of patients and control participants without and before visual interaction (first segment). A repeated-measures 2 (Group)×2 (Pendulum) ANOVA revealed a significant main effect of Pendulum  = 7.81, p<.05, η2 = 0.29), of Pendulum Combination  = 8.58, p<.05, η2 = 0.31) but no significant effect of Group  = 0.36, p>.05, η2 = 0.02) (see p<.05) and when participants oscillated the same pendulums (P1_P1) (p<.05). Although previously reported behavioral deficits in the schizophrenia To analyze the stability of unintentional social coordination, we computed the circular variance of the relative phase angles between the two times series of participants giving an index of synchronization between 0 and 1 with 1 reflecting a perfect synchronization and 0 reflecting an absence of synchronization .02) see . FurtherF = 74.84, p<.05, η2 = 0.80) and Pendulum Combination  = 5.82, p<.05, η2 = 0.23). These effects support previous research p<.05). Moreover, this analysis also revealed a significant effect of Group  = 4.61, p<.05, η2 = 0.20) and a significant interaction between Pattern, Pendulum Combination and Group  = 4.27, p<.05, η2 = 0.18). These effects are due to lower stabilities for the schizophrenia group for both in-phase (P1_P1 and P1_P2) (p<.05) and anti-phase coordination (P2_P1 and P1_P2) (p<.05)  see . These rF = 63.46, p<.05, η2 = 0.77) and a significant interaction between Pattern and Pendulum Combination  = 4.38, p<.05, η2 = 0.19) indicating, respectively, lower and higher phase shifts than P1_P1 for P2_P1 and P1_P2 (p<.05), and a larger negative phase shift in anti-phase than in-phase for P2_P1 (p<.05). Interestingly, the analysis showed also a significant effect of Group  = 8.75, p<.05, η2 = 0.32) and a significant interaction between Group and Pendulum Combination  = 3.61, p<.05, η2 = 0.16) indicating larger phase shift for P2_P1 for the schizophrenia group (p<.05) and no significant differences between the two groups for P1_P1 (p>.05) and P1_P2 (p>.05) (see t(39) = −4.07, p<.05) and P1_P2 (t(39) = 3.54, p<.05) but not significantly different for P1_P1 (t(39) = −1.01, p>.05) t(39) = 1.76, p>.05) but significantly lower than 0° for both P2_P1 (t(39) = −8.11, p<.05) and P1_P1(t(39) = −2.98, p<.05), indicating that patients suffering from schizophrenia never led in the swinging of the pendulums irrespective of the pendulum combination—that is, they did not lead even in those conditions which past research suggests that they should have.We computed then the phase shift or the deviation of the relative phasing from the intended pattern of 0° or 180° to further explore the stability of the intentional coordination and to determine which participant of the pair led within a cycle .05) see . No otheIn the current study using the dynamical systems methodology, we investigated social motor coordination in patients suffering from schizophrenia to better characterize and understand the motoric nature of their interpersonal interactions. We examined their unintentional and intentional social motor coordination using a task in which they coordinated the swinging of a pendulum with someone else.Our results demonstrate that social motor coordination is impaired in schizophrenia. These results extend previous research that indicates impairments of nonverbal behavior in schizophrenia and show that the movements of patients are abnormally coordinated while interacting with another individual ps>.05). These results suggest that examining intentional social motor coordination of patients might objectively and quantitatively assess abnormal social features that are not captured by the PANSS. Furthermore, because social motor disorders may be the earliest sign of schizophrenia, such marker may allow earlier diagnostics and thus better patient management In the intentional coordination task, our analysis of the relative phasing of the pendulum oscillations showed that the dyads in which a patient was present had a lower stability and that the patient never led in the coordination. These results demonstrate that it is possible to distinguish patients with schizophrenia from control participants by looking at their intentional social motor coordination. Such objective and quantitative differences could possibly be used as a marker of the illness and nicely complement the more subjective evaluations usually performed by clinicians. In line with this expectation is the absence of correlations between the coordination impairments of patients and the severity of their symptoms evaluated by the Positive and Negative Syndrome Scale (PANSS) We have demonstrated impairments of social motor coordination in schizophrenia but our study also aimed to understand the impaired underlying processes. We initially assumed that impairments could be the consequence of attention and visual perception deficits that may affect the visuo-motor processes underlying the maintenance of coordination. More specifically, in terms of coupled oscillators system accounting for social rhythmic coordination, we predicted a decrease of the coupling strength, which corresponds to a lower sensitivity to the movements of the other. We found a decrease in the stability of intentional coordination in the schizophrenia group that is compatible with this hypothesis Slower preferred movement frequencies in schizophrenia could explain that patients suffering from schizophrenia never led the coordination x1 and x2 represent the positions of the two oscillators and the dot notation represents derivative with respect to time. The left side of the equations represents the limit cycle dynamics of each oscillator determined by linear stiffness parameter (ω) and damping parameters  and the right side represents the coupling function determined by parameters a and b. To simulate impairments due to schizophrenia, we added the parameters K1 and K2 corresponding to the coupling strengths of the oscillators 1 and 2, To further explore these hypotheses and determine how a decrease of the strength or/and a delay in the information transmission of patients could explain their impairments of intentional social motor coordination, we compared our phase shift results with those obtained in simulations of a coupled oscillator model that has been used in the past for understanding social motor coordination K = 1.6 and τ = 0 to simulate the behavior of control participants and the parameters K = 0.6 and τ = 3 to simulate impairments of individuals with schizophrenia. Such time delay (τ = 3) represented 1% of the simulated period of P1 and 1% of the experimental period of P1 represented an increase of 75 ms compared to control participants corresponding to plausible physiological delays 1 = τ2 = 0, K1 = K2 = 1.6) and in black of the schizophrenia group with a decrease of the coupling strength     , with an2 = 1.6)  and with2 = 0.6) .As depicted in Although we have shown that increasing the delay and decreasing the strength in visuo-motor control capture the intentional coordination pattern of the schizophrenic group, further explorations may improve the understanding of the processes involved. Investigating whether such disorders are specific to social visual coordination or to visual coordination in general is an important research direction. In fact, modifications of intentional coordination in schizophrenia could be due to impairments of mechanisms mediating both visual and social visual coordination or may be specific to mechanisms mediating just social visual coordination. It has been demonstrated that patients suffering from schizophrenia have impairments of visual perception of biological motion that can be distinguished from their general visual perception deficits We have modeled the impairments of schizophrenia patients here by changes at the level of the coupling function and not changes in the natural dynamics of the individual oscillators because our experimental results did not show differences between the preferred frequencies of patients and control participants. Although we did not find differences, previous research has reported slowness in movements of patients suffering from schizophrenia More generally, by examining the coordination dynamics of swinging of handheld pendulums, this experiment demonstrates that interpersonal coordination is impaired in schizophrenia without explicitly showing the consequences of such modifications on everyday social interactions of patients. It is however important to note that the coordination dynamics observed in this laboratory task reflect the ones observed for example between hand, arm or postural movements in more ecological situations, which have been demonstrated as directly influencing the success of our social interactions Therefore, in addition to future research that aims at a further understanding of how schizophrenia affects the processes underlying social motor coordination, it will be important to develop rehabilitation protocols that can help improve the social motor coordination of patients. Although no investigations to our knowledge have yet explored the learning and rehabilitation of social motor coordination, previous research that has examined motor learning may provide interesting research directions In conclusion, our study provided clear evidence of social motor coordination impairments in schizophrenia that may help us understand their social deficits observed in everyday interpersonal interactions. Our results demonstrate differences between schizophrenic and control dyads in an intentional coordination task—a task that may be used by clinicians as a marker of schizophrenia for better diagnostics. In addition, our dynamical modeling of the results revealed that such pathological intentional coordination may be due to a decrease in the amount of information as well as a delay in the information transmitted about movements of other people. Finally, such findings may lead to the development of rehabilitation protocols improving social motor coordination and successful social exchanges of patients.All participants provided written informed consent prior the experiment approved by the local Ethics Committee  conforming to the Declaration of Helsinki. A clinician who was experienced in the evaluation of mental illness assessed by a direct examination of participants, their understanding of all the procedures and capacity to consent. As proposed by Palmer et al. matched control participants for age, sex, education and premorbid IQ, as estimated by the French adaptation of the National Adult Reading Test (fNART) ps>.05). During the experiment, the patients were randomly paired with control participants 1 to compose the schizophrenia group and matched control participants were paired with control participants 2 to compose the control group. Control participants 1 and control participants 2 were matched for age, sex, education and premorbid IQ . All participants had normal or corrected-to-normal vision.Twenty outpatients who fulfilled the DSM-IV-TR criteria for schizophrenia participated in the study Participants sat on chairs approximately 1 m from one another facing in the same direction and swung wrist pendulums attached to a structure that allowed only movements from front to back. The swinging of the pendulums made no noise that could be used as a cue for coordination. The length of the two pendulums was 60 cm and a mass of 150 g was attached either at the bottom or at the middle to constitute the pendulums P1 and P2, respectively. The preferred movement frequency of participants was 0.80 Hz for pendulum P1 and 0.95 Hz for pendulum P2 . Two potentiometers measured the angular displacements of pendulums during the trials at a sampling rate of 50 Hz.matched control participants oscillated pendulums with their right hand and control participants 1 and 2 oscillated pendulums with their left hand. After the practice session with the pendulums P1 and P2, participants were tested together for unintentional social coordination.Upon arrival, participants were informed that the experiment was investigating rhythmic movements with wrist pendulums. Participants then performed practice trials individually, in which they were instructed to hold the pendulum firmly in their hand and to swing it at their own self-selected tempo, a tempo they found comfortable and could maintain for an extended period of time. Patients and control participants 1 and patients for the schizophrenia group and control participants 2 and matched control participants for the control group. Each trial contained three 30 s segments that were delimited by an auditory stimulus. Participants were instructed to swing pendulum at the same self-selected tempo whether they were looking (second segment) or not looking (first and third segments) at the movements of their partner Participants were first tested for unintentional social motor coordination. They completed three 90 s trials one for each pendulum combination P2_P1, P1_P1 and P1_P2, corresponding respectively to the pendulums used by Participant pairs were then tested for intentional social motor coordination. They were informed that they would be required to coordinate their movements together in an in-phase or anti-phase mode for 60 s The first five seconds of each intentional trial and segment of unintentional trials were discarded to avoid transient behavior. The times series of participants were low-pass filtered using a 10 Hz Butterworth filter. The frequency of oscillation during the first segment of unintentional coordination was computed as the inverse of the average time between the points of maximum angular extension as defined by the maxima of time series. The continuous relative phase between the two angular positions of pendulums was computed using the Hilbert Transform δ = −0.7, λ = γ = 1, a = −2, b = 2, ω = 6.25 for the pendulum P1 and ω = 6.30 for the pendulum P2. We used the coupling strength K = 1.6 and delay τ = 0 to simulate the behavior of control participants and K = 0.6 and τ = 3 to simulate impairments of patients suffering from schizophrenia. Only in-phase coordination was simulated because our results did not show an effect of the pattern of coordination for the phase shift modifications. Before every simulation, one cycle of each oscillator without coupling  was computed and the last samples of each were then aligned to constitute the first sample of the simulation. Such procedure was necessary to obtain initial conditions We simulated Eq. (1) to test whether a decrease of the strength or/and an increase of the time delay in the coupling function captured patients' impairments of intentional social motor coordination


We have also rigorously proved its correctness for continuous geometric domains and full robustness for sensor networks over 3D terrains.This paper proposes a high precision Gaussian Mixture Model-based novel Boundary Detection 3D (BD3D) scheme with reasonable implementation cost for 3D cases by selecting a minimum number of Boundary sensor Nodes (BNs) in continuous moving objects. It shows apparent advantages in that two classes of boundary and non-boundary sensor nodes can be efficiently classified using the model selection techniques for finite mixture models; furthermore, the set of sensor readings within each sensor node’s spatial neighbors is formulated using a Gaussian Mixture Model; different from DECOMO  and COBO One of the critical problems in sensor applications is detecting boundary sensors in a complex sensor network environment where sensed data is often required to be associated with spatial coordinates. In  a COBOM In fact, the boundary detection problem has been mostly considered for 2D sensor networks and the case of 3D sensor networks has gone practically unnoticed. Despite the fact that difference between the normal 2D and the more realistic 3D scenario is only one extra dimension, network topology could be much more complex and the location scheme has to be more robust towards network irregularities. Taking a step further to expand from 2D to 3D sensor applications, several neighborhood-measurement  based 3DMotivated by all above observations, instead of introducing miraculous assisting entities, our range-free Gaussian Mixture Model (GMM)-based approach performs a connectivity information-based segmentation algorithm  that parThe remainder of this paper is organized as follows: the next section details enhancement to the BN Array concept; Section 3 simply describes general problems in boundary detection; Section 4 presents the proposed robust Boundary Detection scheme for 3D (BD3D) sensor networks in detail; Section 5 proves BD3D by simulation results; Finally, Section 6 concludes the paper with future work.2.v) and its neighbors In  three dik = 6 in . Let us v) only indicate the relative locations of its neighbors only. Correspondingly, there is no own sensor reading, as a result, Nv judges itself by inquiring ξ(Nv) in a time and energy consuming way. In our model, we applied a head with 1 byte more space for the BN Array to store its own sensor reading as well , to receive the message, the radio expands (Eelec × δ) [This section involves the main objective of achieving a flexible and energy-efficient 3D continuous boundary detection with a clear knowledge of EBN and non-EBN. Assume that sensor nodes are randomly deployed over 3-dimensional terrain. Each sensor node has limited resources , and is equipped with an omni-directional antenna. For the radio model, Elec × δ) . This eni = {χ1, χ2, ...., χn}, n ≤ N  with each χi representing a D-dimensional random vector. Assume that ψi follows a k-component finite mixture distribution [j=1Our major contribution could be creating a statistical property of the finite mixture model, especially the Gaussian mixture model (GMM) and adopting it to distributed sensing scenarios. Suppose that we have a set of data observations ψribution  as folloj is the mixing weight or sometimes called the prior weight and θj is the set of parameters of the jth mixture component 𝒫(χi|θ). Denote θ = {α1, θ1, α2, θ2,...., αk, θk}. The objective function of estimating θ from ψi is to maximize the log-likelihood criterion as follows:where αTherefore, the maximum likelihood estimator of θ is:ML cannot be computed analytically from the above equation. Instead, GMM is applied as its general solver to iteratively find the maximum likelihood solution of θ̂ML. GMM is the most important class of finite mixture densities. GMM is formulated by using a Gaussian density 𝒢 with its mean vector μi and covariance matrix Σj to replace the general probability density function 𝒢(χi|θj) in the finite mixture model:Obviously, θ̂The Bayesian Information Criterion (BIC)  is one oi}, we assume that sensor nodes are deployed with moderate density in the spatial terrain. From a mathematical perspective, sensor readings provide a dense, but discrete sampling of the underlying continuous distribution. To check whether or not Nv is a sensor node lying on the boundary of an event, we put the data {χn} from readings of the sensor nodes in ξ(Nv) and then build our best GMM based on {χn}.In this paper, we provide an algorithm for classifying EBNs. Given a sensor network {Sn}is fed into (5) (6) for estimation of θ(J). Let BM denote the number of mixture components of the best model. We select BM where j, Σj, αj}j=1,2,....,BM.In more detail, we first set the upper bound of the mixture component number to be K. Then for each j = 1,2,…,K, the data set {χv is a EBN, the conditional probability for χi given model θ′(BM) is computed byi|θ′(BM))< γ, Nv is classified as a EBN. Where γ is used as a threshold to measure EBN which has significantly low probability density values given the final model θ′(BM). The threshold is set as γ = 0.25, the upper bound of the component number is set as K = 5. These parameters are used as the default in Sections 4 and 5, unless otherwise stated.To classify if Nf(·) is the linear or nonlinear state evolvement function and g(·) is highly nonlinear observation function. To dynamically update the estimates of observations by conducting , we have4.1.n total sensor nodes are randomly distributed in a 2D terrain, with network density μ enough to perform a boundary detection application. BD3D provides simultaneous selection of EBN and non-EBN during BN selection process by tactfully using the proposed BD3D BN Array } see  from theci)} see .Suppose n is see .In a 3D sensing space, sensor nodes are randomly distributed to form a network. To simplify the complicated operations in dealing with sensor node localization in 3D model, we apply a new concept of 2D plane that each 3D space can be divided into n → ∞see . The metn → ∞) to reconstruct. Due to the impossibility of computing n in programming, we introduce a virtual Thick Section Model (TSM).Strictly speaking, a 2D plane is definitely as a 2D section see . Therefod) with a set of representative points {3 cube (ζ is pre-determined in programming) and d ≠ r. Therefore the selected 2D section could be those in Suppose the 3D network area is ζ2*d) area [see d) is determined a priori to programming).Thus, the simulator only need to perform TSM  and continually expand it by increasing its radius by 10 meters every 10 time slots. see .Design an irregular variation object: the initial ENs that adequately covers a circle area {(x − 50)2+(y−50)2 = Rcircle2} to initiate the event. At every time slot, EN propagates by picking up a random number of neighbors to join the event (non-EN→EN). In this way, the network is guaranteed to be fully connected. see .(2D model)Design a regular variation 3D object: the object center is  and continually expand its radius by 10 meters every 10 time slots.2+(y − 50)2+(z − 50)2 = Rsphere2. EN propagates in a similar way as that used for the irregular variation object in 2D model.Design an irregular variation 3D object: the initial ENs are within a spherical area {(x − 50)(3D model)The BD3D is flexible enough to be used in a clustered network or a non-clustered network since it does not put any constraints on cluster architecture. However, BNs are usually heavily utilized to send aggregated data associated with the object/network boundary information to cluster head (in clustered networks) or the sink (non-clustered networks), they would run out of energy more quickly. Therefore, achieving a reasonable amount of BNs (the less the better) benefits energy saving.5.2.This section discusses the performance evaluations based on BD3D 2D model. Comparison of the number of BNs for a regular variation object with COBOM and DEMOCO is shown in rebound distance which indicates the end of saturated distribution around the BL, rebound distance and boundary distance.However, due to the elusive ways proposed to expand the irregular variation object, we can hardly do comparison with COBOM and DEMOCO anymore. 5.3.d < r, d > r, d >> r }r and d by using TSM for regular variation and irregular variation objects, respectively. d with r = 10m. As d < r, the values we got are approximately the same. Moreover, we varied the value of d (d > r) for simulating the cases with the significant existence of BA  using TSM once d < r. This occurrence can be clarified by the analysis illustrated in r = 5 and d = 8, it undergoes a very slow increase first and then experiences a sharp increase in the number of BNs after 40 time slots. This was caused by a phenomenon that the objects expand highly depending on the number of existing BNs. However, at network initialization, we have relatively fewer existing BNs. As the cardinal number designating the existence of BNs is over a special value (available at around 40 time slots), the performance miraculously achieves a sudden improvement.Meanwhile, we set the same parameter environment in the BD3D 3D model for evaluating the number of BNs in the network in d < r using TSM. An in depth study about the impact of localization impact on various routing protocols and its implications on design of location-dependent system are left as future work.We hereby conclude that our BD3D for continuous boundary detection in 3D case works well especially when 6.This paper has proposed a novel Gaussian Mixture Model-based BD3D scheme for boundary detection of continuously moving object in a 3D sensor network. We adequately presented the proposed protocol, and the simulation results shown support our allegation that the BD3D 2D model surely outperforms COBOM and DEMOCO in terms of average residual energy per sensor node and the number of selected BNs, and the BD3D 3D model achieves accurate boundary detections by soundly selecting EBN and non-EBN for both regular variation and irregular variation object cases. Our future work will include additional optimization desired to improve the performance of our algorithm and verification of the precision of the expected boundaries and invention of a new protocol that considers data losses and route failures due to unpredictable errors such as sensor node failures, contention, interference and fading ,36. More


We report herein studies of Aβ stimulated effects on endothelial cells. We observe an interesting and as yet unprecedented feedback effect involving Aβ1–42 fibril-induced synthesis of APP by Western blot analysis in the endothelial cell line Hep-1. We further observe an increase in the expression of Aβ1–40 by flow cytometry and fluorescence microscopy. This phenomenon is reproducible for cultures grown both in the presence and absence of serum. In the former case, flow cytometry reveals that Aβ1–40 accumulation is less pronounced than under serum-free conditions. Immunofluorescence staining further corroborates these observations. Cellular responses to fibrillar Aβ1–42 treatment involving eNOS upregulation and increased autophagy are also reported.Amyloid accumulation in the brain of Alzheimer’s patients results from altered processing of the 39- to 43-amino acid amyloid β protein (Aβ). The mechanisms for the elevated amyloid (Aβ Alzheimer’s disease (AD) is characterized by neuronal degeneration and accumulation of senile plaques, which are composed of amyloid-β (Aβ) peptides predominantly consisting of 40 and 42 amino acids Postmortem analyses of AD subjects reveal that amyloid plaques in the brain suffuse vascular cells in addition to the parenchymal. The implications of this vascular infiltration for AD has been less well studied than the parenchymal Aβ, but has generated considerable interest with studies that β-amyloid fibrils accumulate in small arteries, arterioles and capillaries of the brain The mechanism for the onset of pathological vascular changes has yet to be elucidated in vitro investigation where the interaction with Aβ fibrils resulted in the endothelial lining of the rat aorta undergoing rapid damage leading to exposure of smooth muscle cells and connective tissue The oxidative stress mechanism was used to explain an 1–42 makes it less susceptible to proteolytic degradation The “tombstone” mechanism is consistent with biochemical and biophysical studies of synthetic Aβ peptides indicating that the more toxic Aβ peptides ending at residue 42 aggregate more rapidly than peptides of 39 or 40 amino acids 1–42 in lysosomes down regulates the catabolism of APP resulting in enhanced production of amyloidogenic fragments 1–42 fibrils and established the resultant accumulation of APP and Aβ1–40.APP synthesis and processing to Aβ normally takes place only to a limited extent in endothelial cells 1–40, Aβ1–42 and monoclonal antibody against Aβ1–40 were purchased from Biosource International . Fluorescein and phycoerythrin labeled streptavidin were purchased from PharMingen .Media for cell culture was obtained from Invitrogen . Synthetic Aβ1–42 was disaggregated by pretreatment with trifluoroacetic acid (TFA) followed by treatment with trifluoroethanol (TFE) three times to remove traces of TFA. After each step, solvents were evaporated to form a film. Aβ1–42 fibrils were formed from disaggregated Aβ1–42 by following a known procedure 1–42 (250 µM) was dissolved in phosphate buffered saline, (PBS), pH 7.5, and polymerized in Eppendorf tubes (1.5 ml), at 37°C for 2 days. Newly formed fibrils were then separated from monomeric peptides by centrifugation at 4°C for 90 min at 15,000 rpm, using a Sorvall RC-5B centrifuge. The fibril pellet was re-suspended in PBS and used for in vitro studies Synthetic Aβ2. 1×106 cells were incubated with varying concentrations of Aβ1–42 fibrils in the presence or absence of FBS for 12 hrs. Cells were then harvested, washed with PBS, fixed in 2% glutaraldehyde, and probed with biotinylated monoclonal antibodies against Aβ1–40. This antibody detects low molecular weight Aβ1–40, but not fibrils. Cells were counter-labeled with 20 µg/ml phycoerythrin-streptavidin for 15 to 30 min. Labeled cells were analyzed on a FACScan flow cytometer  equipped with a 15 mW argon laser. Phycoerythrin labeled cells were excited with 488 nm light from the argon laser and emitted fluorescence was collected using the FL-2 (585/42) bands pass filter. A total of 20,000 events were analyzed for each sample. The amount of Aβ1–40 present in the endothelial cells was determined by mean fluorescence values. Light scatter signals were also collected in the forward and side scatter detectors. Experiments were repeated three times. The mean and standard error of the mean values were calculated.Human endothelial (Hep-1) cell line is a kind gift from Dr. John Kusiak at the National Institute on Aging. This is an immortal human endothelial cell line derived from an adenocarcinoma of the liver 1–40 antibody (1∶500 dilution) at room temperature, washed with PBS and stained with FITC-streptavidin conjugate. Immunofluorescence images were captured with an Axiovert S100 microscope . Images were recorded at the same exposure time using a digital SPOT camera .Cells grown on glass cover slips were incubated at 37°C for 12 hrs, and then washed with PBS. Cells with and without Aβ treatment were subsequently fixed in 2% glutaraldehyde for 10 min and washed with 100 mM glycine in PBS containing 2% bovine serum albumin. After washing, samples were incubated for 1 hr with a biotin-conjugated anti-Aβ3VO4, and 5 mM NaF. The resulting lysates were resolved on 4–15% SDS Tris-glycine gels (30 µg/lane) and transferred onto polyvinylidene difluoride membranes . The membranes were blocked in TBST  containing 5% non-fat milk and then probed with the following primary antibodies: monoclonal mouse anti-human APP , monoclonal mouse anti-human endothelial nitric oxide synthase (eNOS) , monoclonal mouse anti-human nNOS , rabbit polyclonal anti-caveolin 1 antibody  and rabbit polyclonal anti-LC3B antibody . Proteins were detected with HRP conjugated secondary antibodies and enhanced chemiluminescence (ECL) reagents . The blot was quantitated by densitometric analysis and the difference between treated and control groups were analyzed by unpaired t test (tails = 2) using Excel software.Cells were lysed in 300 µl of 20 mM HEPES, pH 7.4, supplemented with 2 mM EGTA, 50 mM β-glycerol phosphate, 1% Triton X-100, 10% glycerol, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM Na1–42 fibrils were prepared from synthetic Aβ1–42 peptides and morphologically characterized by TEM  were incubated with Aβ1–42 fibrils for 12 hrs and then harvested in a lysis buffer for Western blotting. As shown in 1–42 in a dose dependent manner.Aβd by TEM . Western1–42 used to treat Hep-1 cells by using antibodies that are specific for Aβ1–40, which only detects low molecular weight Aβ1–40, but not fibrils. The latter sequence accounts for about 90% of newly processed amyloid but no cross reactivity between Aβ1–42 and Aβ1–40 was observed, in agreement with previous reports The association of the increased APP synthesis with an increase in Aβ was investigated in the same cell model. It was possible to discriminate between newly synthesized amyloids and the Aβ1–40, which is further accentuated by the stress associated with the absence of serum. In both cases, it is clear that Aβ1–42 fibrils increase Aβ1–40 synthesis in a concentration dependent manner as shown by the plots of the mean fluorescence in The flow cytometry histograms, of cultures grown with and without FBS  and analyzed by flow cytometry. Only a negligible increase in Aβ1–40 was observed and the fluorescence histogram was similar to that of control (data not shown).Because the antibody used only detects low molecular weight amyloid, the observed Aβ1–42 treated cells revealed no difference in the forward scattering consistent indicating no change in cell size. On the other hand, changes in the granulated structure were apparent as evidenced by an increase in the intensity of side scattering  from the incident laser beam (referred to as forward scattering) is proportional to the cell size. The side scattering at larger angles is a measure of structural changes such as granulated structure on the membrane or cytoplasm that increase the scattering. Examination of changes in scattering properties of Aβattering , which w1–40 in Aβ1–42 treated cells were further corroborated by light and confocal microscopy  which catalyze the production of NO, a known modulator of CNS vascular and neuronal function. After the cells were treated with 5 and 10 µM fibrillar Aβ1–42, Western blot of different NOS isoforms showed a mild but statistically significant increase of endothelial NOS (eNOS) and no change in neuronal NOS (nNOS) level (1–42 treatment. Increased ratio of LC3B-II/I (To further study the cellular response to AβS) level . InducibS) level . Since aC3B-II/I , a relia1–42 fibril induced APP synthesis in the human endothelial cell line Hep-1.Major attention has been devoted to the role played by neuronal cells in AD development. Most of the amyloid found in AD subjects is produced in the brain and contained in plaques with a high content of highly aggregated amyloids. However, cognitive function does not generally correlate with the level of cerebral plaques. It was only in recent years that the contribution of the cerebrovascular system has been appreciated 1–42 results in elevated levels of the APP  indicating the increase of APP could also be translational in our study. As for increased Aβ production, there is ample evidence that in neuronal cells, Aβ accumulation and oxidative stress, each accelerating the other, generate a vicious circle of more Aβ production and oxidation Altered cellular function can be triggered by the aggregation state of the amyloids in addition to stress. Our observation that only Aβ2+ dependent induction of eNOS by hydrogen peroxide in endothelial cells. This upregulation has been attributed to a compensatory mechanism for increased superoxide formation 1–40 production. The intricate relations between oxidative stress and autophagy and its implication in AD vascular pathogenesis awaits further elucidation.Once inside the cell, the amyloid can affect many other cellular responses. One of the cellular responses we observed is the upregulation of eNOS  which isThe increased cellular granulation indicated both by changes in side-scattering in the flow cytometry experiment  as well 1–42 fibrils establishes, for the first time, that the exposure of endothelial cells to Aβ1–42 fibrils results in an elevated amyloid load in endothelial cells. This process provides a new potential pathway for amyloidosis with these newly formed amyloids in the endothelial cells able to contribute to both vascular amyloidosis and/or parenchyma amyloidosis.In conclusion, our observation of increased APP and amyloid in ECs incubated with Aβ


Evolutionary transitions from PXI to RXI also generally increase mean relative female fitness at the expense of decreased male fitness. These results provide a theoretical framework for predicting and interpreting the evolution of chromosome-wide expression of X-linked genes and lead to several useful predictions that could motivate future studies of allele-specific gene expression variation.X inactivation—the transcriptional silencing of one X chromosome copy per female somatic cell—is universal among therian mammals, yet the choice of which X to silence exhibits considerable variation among species. X inactivation strategies can range from strict paternally inherited X inactivation (PXI), which renders females haploid for all maternally inherited alleles, to unbiased random X inactivation (RXI), which equalizes expression of maternally and paternally inherited alleles in each female tissue. However, the underlying evolutionary processes that might account for this observed diversity of X inactivation strategies remain unclear. We present a theoretical population genetic analysis of X inactivation evolution and specifically consider how conditions of dominance, linkage, recombination, and sex-differential selection each influence evolutionary trajectories of X inactivation. The results indicate that a single, critical interaction between allelic dominance and sex-differential selection can select for a broad and continuous range of X inactivation strategies, including unequal rates of inactivation between maternally and paternally inherited X chromosomes. RXI is favored over complete PXI as long as alleles deleterious to female fitness are sufficiently recessive, and the criteria for RXI evolution is considerably more restrictive when fitness variation is sexually antagonistic  selects for preferential or complete inactivation of the paternally inherited X. Paternally biased X inactivation patterns appear to be common in nature, which suggests that sexually antagonistic genetic variation might be an important factor underlying the evolution of X inactivation. The theory provides a conceptual framework for understanding the evolution of X inactivation strategies and generates several novel predictions that may soon be tested with modern genome sequencing technologies. RXI generates an expression pattern that is more similar to diploidy, and can potentially mask the fitness costs of carrying deleterious alleles.The selective processes that might account for this observed diversity remain unclear. A leading hypothesis for the evolution of RXI is that it might be favored if segregating deleterious mutations have recessive or partially recessive fitness effects . The loge.g., e.g., While the masking hypothesis for the evolutionary origins of RXI is plausible e.g., , its feae.g., e.g., Sex differences in selection – where the fitness effects of single mutations differ in magnitude or direction between males and females – likely influence large fractions of animal genomes i.e., quantitatively unequal expression of maternally versus paternally derived X chromosomes) and predict the magnitude of biases likely to evolve. Our results suggest that conditions for evolving biased inactivation patterns are extremely permissive.It is currently unclear how the population genetic parameters of dominance, sex-differential selection, and linkage and recombination might jointly influence the evolution of X inactivation strategies. We therefore developed a mathematical model of X inactivation evolution, and used this model to identify biological conditions that favor the evolution of different X inactivation states. We first consider the dichotomous case of RXI versus PXI, describe the conditions facilitating evolutionary transitions to RXI, and characterize the consequences of such transitions for mean fitness of males and females. Overall, selection on mammalian X inactivation strategies is primarily mediated by the interaction between dominance and sex-differential selection, and the situation differs considerably from scenarios that favor the evolution of diploidy. We also examine whether sex-differential selection might favor the evolution of biased X inactivation strategies (A (the “fitness locus”) is X-linked and carries A1 and A2 alleles, which directly influence male and female fitness. Locus B (the “modifier locus”) carries B1 and B2 alleles, which influence the X inactivation rule in females within the population. The genotype at locus B can influence female fitness through its effect on the relative expression of A1 versus A2 alleles in heterozygotes. Variation at the B locus has no other phenotypic effect in males or females and is therefore neutral in males. We consider two scenarios of linkage for the B locus. Under X-linkage, A and B are physically linked, and recombine at a rate r, per female meiosis . When B is on an autosome, alleles at A and B loci segregate independently during meiosis.We focus on the simplest and most analytically tractable model that simultaneously incorporates genetic linkage, variation for fitness, and variation in the form of X inactivation. Our model follows the evolution of two bi-allelic loci. Locus B locus initially fixed for allele B1, and the A locus at polymorphic equilibrium given B1 fixed. We then characterize selection on and evolution of a novel B2 allele that changes a female carrier's X inactivation system. The population is assumed to be sufficiently large that genetic drift can be ignored, and each generation is discrete. The life cycle during each generation follows the order of birth, selection, recombination, mutation, random mating and syngamy. Our approach bears many similarities to models for the evolution of ploidy cycles  = 1−fs≤w(A1A2), w(A2A1)≤w(A2A2) = 1, where w(iAjA) is the fitness of a female with genotype iAjA, and fs describes the fitness cost to females of being homozygous or haploid for the A1 allele: 1>fs>0]. When A1 is also deleterious to males, it will be maintained at mutation-selection balance  delB1, female somatic cell lineages are assumed to silence the paternally inherited X with probability ξ11 and silence the maternal X with probability 1−ξ11 (0≤ξ11≤1). Under unbiased RXI (where ξ11 = ½), heterozygous females have fitness w(A1A2) = w(A2A1) = 1−fhs, where h represents the degree of masking of the A1 allele (0<h<1). Thus, h is a scaling factor that is analogous to the dominance coefficient of standard population genetic models. Use of the terms “dominance” and “recessivity”, applied to species with RXI, has been questioned because individual cells lack bi-allelic expression In an ancestral population fixed for ξ11≠½), fitness is function of h (as defined above) and parent-of-origin effects generated by the X inactivation rule. Assuming that female fitness decreases monotonically with the proportion of cells expressing the A1 allele, we can describe it using a generalized power function, w(x) = 1−ksfx, where x represents the proportion of cells expressing the A1 allele, and k is a positive constant that describes the specific shape of the fitness decline associated with 1A expression  has two essential properties. First, fitness approaches unity when A2 is ubiquitously expressed /ln(2). A1 is partially dominant to A2 when h>½ (k<1) and partially recessive when h<½ (k>1). Consequently, math = ξ11k represents the effective dominance coefficient when A1 is maternally inherited, and path = (1−ξ11)k represents the effective dominance coefficient when A1 is paternally inherited  can arise when the fitness of an X-linked genotype is not cell-autonomous  = 1−ξ11fs and w(A2A1) = 1−(1−ξ11)fs, which represents a special case of the generalized power function .When X inactivation is biased  are:u is the mutation rate to A1, math = ξ11k, and path = (1−ξ11)k, and k = −ln(h)/ln(2). A1 is sexually antagonistic , and with balancing selection acting at the A locus, the equilibrium frequencies of A1 are:Given the outlined assumptions, we obtain the following equilibria with respect to the X-linked fitness-determining locus. When B2 allele that causes unbiased RXI  will invade a population that is initially fixed for the PXI strategy (ξ11 = 1).PXI is thought to represent the ancestral X inactivation state, from which RXI evolved B1 fixed in the population and A1 at mutation-selection balance. As predicted, selection favors invasion of a modifier allele (B2) causing RXI when the deleterious allele is sufficiently masked in heterozygotes . Assuming weak mutation at locus A (0<u≪1), the critical dominance coefficient under RXI is approximately:h<crith , deleterious alleles need only be partially masked (h<½) for RXI to be favored. Increasing the strength of purifying selection alters crith, with stronger selection in males than females (large ms/fs ratios) expanding the parameter space in which RXI evolves, and female-biased selection decreasing it . This effect of differing selection strengths between the sexes has two contributing causes. First, expression of the paternally inherited X  is favored because the frequency of deleterious mutations is lower on the paternal X. Second, purifying selection, primarily in males, limits the buildup of linkage disequilibrium (LD) between B2 alleles and deleterious mutations, which can also prevent the invasion of B2 alleles. This LD is generated from the epistatic interaction for female fitness between A and B locus genotypes, and there is no such epistasis in males. Limited evolution of LD expands the parameter space of dominance that permits the evolution of RXI.When selection is weak , shows that RXI will evolve when the dominance coefficient for the female-deleterious allele falls below the following threshold:mt→0), female-detrimental alleles need only be partially masked for RXI to be favored, but overall, the conditions are considerably more restrictive than the mutation-selection balance model. Strong sexually antagonistic selection severely reduces the parameter space that favors the evolution of RXI  permit deleterious mutations to accumulate within populations, and reduce long-term population fitness . For the parameter space where RXI can evolve  is positive when A1 is at least partially recessive (h<½), which we assume here and below, and the ratio fhs≪1 , (1−mt)h)−ln(2)/ln. There is some evidence for alleles in mice that affect X inactivation choice . Under these conditions, criteria for invasion of a modifier allele will be iλ is the leading eigenvalue associated with the two-locus system for the modifier locus and the ith of n X-linked fitness loci  i.e., iλ≈1), the selection coefficient associated with a rare modifier allele will be mods≈nE(iλ−1), where E(iλ−1) represents the arithmetic mean, and iλ−1 represents selection contributed by the ith locus .For reasons of tractability, and in parallel with theory on the evolution of diploidy e.g., , we havei.e., if E(iλ−1)≠0, then mods scales approximately linearly with n, the number of contributing loci; this scaling is in agreement with multilocus models for the evolution of diploidy e.g., To the extent that X-linked loci segregate for deleterious alleles, and these have similar selection and dominance coefficients, the net effects of multiple X-linked loci on a modifier will be reinforcing, and the strength of selection on the X inactivation strategy will increase with the number of contributing loci  there is genetic variation for RXI ; (ii) X-linked fitness variation is largely caused by segregating deleterious mutations; and (iii) the fitness costs of these mutations are at least partially masked under RXI . The availability of mutations to RXI could potentially constrain the convergent evolution of RXI in marsupials e.g., Evolution of RXI from an ancestral population with PXI should be relatively unconstrained, provided three conditions are met: , selection can favor reduced expression of the maternally inherited X when fhs<ms, as implied by mu/fu>1) reduces this parameter space to (mu/fu)fhs<ms, because males transmit a higher fraction of de-novo mutations to their daughters. We predict that preferential inactivation of the paternally inherited X will be more common (or more severe) in species with strongly male-biased mutation rates.Mammalian mutation rates are often higher in males than females vely see . With une.g.: Sexual selection could similarly favor paternally biased X inactivation. Although strong purifying selection in males via sexual selection can reduce paternal transmission rates of deleterious alleles e.g.: , it willx1 = [A1B1], x2 = [A2B1], x3 = [A1B2], and x4 = [A2B2]. Haplotype frequencies in sperm are y1 = [A1B1], y2 = [A2B1], y3 = [A1B2], and y4 = [A2B2]. Following random mating, females of the next generation will carry a maternally inherited haplotype i = {1, 2, 3, 4} and paternally inherited haplotype j = {1, 2, 3, 4} with probability iyjx. Males of the next generation inherit a single haplotype i from their mothers, with probability ix. The fitness of a female carrying haplotypes i and j is ijf, and the fitness of males carrying haplotype i is im. Haplotype frequencies after selection and recombination, but prior to mutation, are described by the following set of recursion equations:F is the sum of the numerators of x1′, x2′, x3′, and x4′; M is the sum of numerators of y1′, y2′, y3′, and y4′; and Θ = x1y4f14−x2y3f23−x3y2f32+x4y1f41.In a given generation, let the haplotype frequencies in eggs be A1 is the deleterious allele. Assuming mutation rates at locus A are much smaller than the strength of selection against A1, mutations from A1 to A2 can be safely ignored. The frequency of each haplotype after a single generation, including mutation, will be:u is the mutation rate at the A locus, per gamete. When variation is maintained by sexual antagonism , recurrent mutation will marginally affect equilibrium allele frequencies at the A locus For the mutation-selection balance scenario, we consider fitness variation contributed by alleles that are deleterious to both sexes, and arbitrarily assume that B2 allele is favored when the leading eigenvalue of the Jacobian is greater than one; B2 cannot invade when the leading eigenvalue is less than one. One submatrix has leading eigenvalue less than one as a condition of the population initially being fixed for B1 and at stable equilibrium at the A locus. Stability at B is therefore determined by the leading eigenvalue of the remaining submatrix, J  were determined by hand, and leading eigenvalues were obtained numerically by Newton-Raphson iteration.Stability criteria m1+(x2+x4)m2.When the modifier locus, B2 alleles) follows the same approach as before, with initial conditions of variation at locus A remaining unchanged. Under the case of variation maintained by sexually antagonistic selection, effects of mutation are ignored (we set u to zero). The generic characteristic polynomial is:x1 = 1−x2, y1 = 1−y2, and x3 = x4 = y3 = y4 = 0. Values of Stability analysis  = delλ−1, where delλ is the leading eigenvalue at the equilibrium with B1 fixed and A1 at mutation-selection balance . A sexually antagonistic locus imposes selection on a modifier s(SA) = SAλ−1, where SAλ is the leading eigenvalue at the equilibrium with B1 fixed and A1 at deterministic balanced polymorphism . The y-axis plots the relative strength of selection imposed by the two types of fitness loci, i.e., the ratio: s(del)/s(SA).The relative strength of selection on a modifier allele. This example plots the strength of selection for preferential inactivation of the maternal X  relative to selection for a paternal X inactivation bias . In both cases, the modifier locus is linked to an autosome. A locus under purifying selection imposes selection on a modifier of strength (TIF)Click here for additional data file.Table S1Fitness for the two locus system.(PDF)Click here for additional data file.Text S1A locus.Polymorphism at the (DOC)Click here for additional data file.Text S2Two locus fitness and stability.(DOC)Click here for additional data file.


Dear Editor,We thank the author for showing interest in our case series of patients admitted to our multidisciplinary intensive care unit last year during the first wave of the 2009 H1N1 pandemic. ContraryProlonged ventilatory support is often required for respiratory failure from 2009 H1N1 infection. The median duration of ventilator support in our series was 10 days with an IQR of 4–22 days, which is similar to previous experience.4 It is clThe lung bears the brunt of the disease in 2009 H1N1 infection. The author suggests that myocardial dysfunction may be a relatively rare manifestation of 2009 H1N1 infection. However, extrapulmonary organ failure including shock is a common feature of severe 2009 H1N1 infection in the intensive care unit. A signif


After publication of our article Pérez et al Int J Equity Health 2012 11:43, we became aware of some errors in the final published version, for which the corresponding author accepts full responsibility.Page 3: third paragraph, Lines 2–4: “…, microcredit, and technical training for those ages 15 to 30” should be read “…, microcredit for those ages 15 to 65, and technical training for those ages 18 to 40”.The original Table four Table has someBecause new corrected figures in the original Table four Table the follWe regret any inconvenience that this oversight may have caused.


Using this multivariate data set, a systematic and inherently unbiased partial least squares (PLS) approach showed that for both peptides the abundance of oligomers in the tetramer to 13-mer range contributed positively to both pore formation and cytotoxicity, while monomers, dimers, trimers, and the largest oligomers (>210 kDa) were negatively correlated to both phenomena. Multivariate PLS analysis is ideally suited to handle complex data sets and interdependent variables such as relative oligomer concentrations, making it possible to elucidate structure function relationships in complex mixtures. This approach, therefore, introduces an enabling tool to the field of amyloid research, in which it is often difficult to interpret the activity of individual species within a complex mixture of bioactive species.Aggregates of amyloid-beta (Aβ) peptides are thought to be involved in the development of Alzheimer’s disease because they can change synaptic plasticity and induce neuronal cell death by inflammation, oxidative damage, and transmembrane pore formation. Exactly which oligomeric species underlie these cytotoxic effects remains unclear. The work presented here established well-controlled aggregation conditions of Aβ To make this analysis quantitative, we adopted a partial least squares (PLS) approach and found that cytotoxicity and pore formation have identical dependencies on the oligomer distribution, supporting a causative relationship between these phenomena.Here, we established a well-controlled and reproducible preparation method to form Aβ aggregates over 0, 1, 2, 3, 10 and 20 days in water. We examined the ability of these populations to form ion pores in planar lipid bilayers and separately quantified their cytotoxic effects on a human neuroblastoma cell line. For each time point during the aggregation process, we quantified the relative abundance of different Aβ oligomers and correlated individual oligomer levels with pore formation and toxicity data as well as with β-sheet content and degree of fibrillization. We carried out all assays with the same Aβ preparation at each time point in order to enable a systematic study of cross-correlation of abundance of oligomer species in two Aβ preparations  went through a maximum after approximately 2 to 3 days. We observed, as expected, that the relative abundance of high molecular weight oligomers (>18-mers) gradually increased over time.1–40 showed that both detection techniques revealed similar trends for the change of relative abundance of small oligomer sizes but quantification of abundance for aggregates larger than hexamers was difficult with silver staining, as reported previously  and increased only slowly after the third day . Data from CD spectroscopy revealed that the relative amount of β-sheet followed a similar trend as the ThT signal, i.e., a rapid increase during the first 1–2 days, followed by a slower increase as aggregation continued (We used thioflavin T (ThT) fluorescence as a surrogate to monitor fibrillization hird day . In caseontinued . TogetheIn order to develop a model that describes the effect of oligomer populations on pore formation and cytotoxicity, we adopted a multivariate regression approach. This approach is particularly well suited here since we had observations at six time points over twenty days that included the measured levels of pore formation, cytotoxicity, β-sheet content, and ThT fluorescence, as well as the relative abundance of nine different oligomer species for two Aβ peptides. A multiple linear regression approach might seem logical in this case, but was not appropriate given the relative uncertainties in the explanatory variables (oligomer levels) and response variables  as well as the significant correlations in the oligomer population. Instead, we used a partial least squares (PLS) formalism since it is suited for limited data sets and correlated predictor variables, and has been shown to work well for similar problems ranging from pharmaceutics to neuroimaging 1–40 and Aβ1–42 independently and found for each case that a model with two components or latent variables 1–40 and Aβ1–42 are nearly identical, suggesting that the dependence of pore formation, cytotoxicity, β-sheet content, and ThT signal on the underlying oligomer distributions must be similar for both peptides.We performed PLS regression using the oligomer abundance as predictor variables and simultaneously carried out regression for all four observables: pore formation, cytotoxicity, β-sheet content, and ThT fluorescence. We carried out this analysis twice by treating data from Aβ1–40 and Aβ1–42 peptides. In this plot, positive or negative coefficients indicate a positive or negative effect on the given observation, and the asterisks indicate the statistical significance of a given coefficient based on jackknife test 1–40 constitutes the majority of species binding to neuronal cells at physiological Aβconcentration and that these binding events were associated with sporadic Ca2+ leakage 1–40 forms stable annular tetramers, while Aβ1–42 forms quasiplanar, annular hexamers as well as dodecamers (from stacking two such hexamers) 2+-imaging that 5- to 40-mers of Aβ induced pores for Ca2+ in plasma membranes These results agree well with previous observations, in particular that the smallest oligomers may in fact be protective rather than pathologic In order to further examine the plausibility for the formation of these proposed Aβ pore structures, we applied a simple barrel-stave model of such an Aβ pore to estimate the numbers of monomers per pore from single channel recordings of Aβ-induced ion flux  and compThe size range from dimers to 8-mers constitutes the predominant Aβ oligomer fraction in the brain Unlike the tetramer to 13-mer range of oligomers, the monomer, dimer, and trimer species were anticorrelated with both pore formation and cytotoxicity . These fThe observation that large oligomers (>210 kDa) are anticorrelated with pore formation and toxicity, rather than making no contribution, may be explained by recent findings that monomers bind to and dissociate from Aβ oligomers and fibrils in a dynamic process 1–40, both of these observables have strong, positive contributions from the 78–210 kDa and >210 kDa oligomer classes. As one might expect, the β-sheet and ThT signals from these large oligomers are largely correlated, however both observables are anticorrelated with pore formation and cytotoxicity. This result is important since it demonstrates that fibrillization, β-sheet content, pore formation, and toxicity can depend differently on various oligomer species  and largest Aβ species (>210 kDa) contributed negatively to pore formation and cytotoxicity and may therefore act as inert sinks that scavenge toxic oligomers This study introduces an unbiased, systematic approach to deduce statistical correlations between the size distribution of Aβ oligomers and pore formation, toxicity, β-sheet content and extent of fibrillization. This multivariate approach is different from previous studies on Aβ-induced toxicity and pore formation, nonetheless its results agree with many previous findings on Aβ-induced pore formation or cytotoxicity. This approach therefore supports the idea that Aβ-induced pore formation may be a significant mechanism of its toxicity. Similar multivariate PLS analyses might be valuable for other amyloidogenic diseases in which standard ensemble measurements often mask the cytotoxic contributions of individual aggregate species in complex and dynamic mixtures of amyloids.Please refer to the Supporting Information for details on chemicals, materials and methods such as formation of lipid bilayers, current recordings, preparation of AAβ samples, toxicity assays, Western blotting, silver staining, densitometry analysis, ThT fluorescence assays, CD spectrometry, transmission electron microscopy, statistical analysis, partial least squares analysis as well as for Supplementary Results, Tables, and Figures.Figure S1SDS-PAGE/Western blot of Aβ samples from different suppliers with or without treatment with HFIP followed by lyophilization. All Aβ samples were prepared freshly in deionized H2O at a conc. of 1 mg mL−1. Each well in the 18%Tris-HCl gel (Bio-rad) was loaded with 0.2 µg of Aβ. Lane 1 = Bachem (non-lyophilized); 2 = GL Biochem, Ltd (Shanghai) (non-lyophilized); 3 = Biopeptide Inc. (non- lyophilized); 4 = GL Biochem, Ltd (Shanghai) (lyophilized); and 5 = Biopeptide Inc. (lyophilized). Aggregation of Aβ varies in commercial sources. HFIP treatment followed by lyophilization for 2 d removed all aggregates of Aβ larger than ∼ 12 kDa in the case of Aβ1–40 and removed large Aβ aggregates (>225 kDa) in the case of Aβ1–42.(TIF)Click here for additional data file.Figure S219F-NMR spectroscopy of HFIP in CD3OD (left) and Aβ1–40 sample that was incubated with HFIP and then lyophilized for two days as described in the Materials and Methods section (right).19F resonance of HFIP gave a doublet at −77.8 ppm, while the peak was absent after Aβ was lyophilized in HFIP for 48 h.(TIF)Click here for additional data file.Figure S3SDS-PAGE/Western blotting of cross-linked Aβ1–40 and Aβ1–42 samples from various preparation methods.1) Method B: non-HFIP treated Aβ  in di-H2O with 0 d incubation; 2) Method C: Modified Kayed preparation; 3) Method D: Aβ proteoliposomes containing DOPS; and 4) Method E: Aβ proteoliposomes containing 30% of positively charged DOTAP lipids. The presence of high molecular weight oligomers from these four samples indicates that these preparations accelerated the aggregation of Aβcompared to preparations in diH2O (Method A) Click here for additional data file.Figure S4Silver staining after SDS-PAGE of cross-linked Aβ1–40 samples prepared by method A for 0 to 20 d. Two micrograms of sample were loaded into each well. The relative amount of intermediate aggregates (dimers to hexamers) or large aggregates of Aβ (the species in lane 10 and 20 in the stacking gel) increased with incubation time. The stacking portion of the silver stained gel appeared with a dark background even in the absence of protein (blank) as shown in the first lane on the left of the gel, making quantitative analysis of large aggregates by silver staining difficult.(TIF)Click here for additional data file.Figure S5Relative abundance of various cross-linked Aβ1–40 aggregates of different size obtained after SDS-PAGE and silver staining. The intensity on the gel was corrected by subtraction of the blank (in the absence of protein). Each point represents the average of relative abundance of aggregates from six independent gels and samples; error bars represent the standard error of the mean. Red curves are best fits of equation (S1) to the data.(TIF)Click here for additional data file.Figure S6Transmission electron micrographs of Aβ1–40 aggregates. Micrographs were taken of Aβ1–40 aggregates after the aggregates had been incubated for zero, one, two, or three days according to Method A. Two micrographs are shown for each day, each taken at different locations on the TEM grid.(TIF)Click here for additional data file.Figure S7Circular dichroism spectra of Aβ1–40 (left panel) and Aβ1–42 (right panel) samples prepared by method A as a function of incubation time. With increasing incubation time, the Aβ-sheet content in Aβ samples increased, while the random coil content decreased.(TIF)Click here for additional data file.Figure S8Loadings of the first and second components (latent variables) for both Aβ peptides as determined by PLS regression. The first components for Aβ1–40 and Aβ1–42 (solid lines) and the second components for Aβ1–40 and Aβ1–42 (dotted lines) are nearly identical and have inner products of 0.91 and 0.87, respectively. The percentages listed indicate the percent of the variance explained by each component. These results indicate that the relationship between the four observables and the oligomer ensemble must be similar for the two peptides.(TIF)Click here for additional data file.Figure S9Comparison of experimentally measured single channel conductance values of Aβ1–40 pores with theoretically predicted values. These box plots were constructed from the amplitude of single-step current jumps in current versus time traces such as those shown in d, of 1 nm (approximate diameter of a transmembrane peptide) l, of 3.5 nm (approximate thickness of a lipid bilayer). The centers of the cross-section of each cylinder are located at the corners of the regular polygon and the lengths of the polygon sides are equal to d. The theoretically predicted box plot was constructed from estimated single channel conductance values of eleven possible regular polygons ranging from the smallest one (assembly of three monomers to a triangle) to a regular polygon of 13 monomers. We estimated the single channel conductance as a function of the number of Aβ monomers, N, in these assemblies by calculating the area of the lumen LA(N) in such a pore. We obtained this area by calculating the area of a regular polygon with N sides, RPA(N) and subtracting the area of circle sectors, CSA(N), that overlap with the polygon area. The area of a regular polygon is given by: RPA(N) = d2 × N/[4 × tan(180°/N)] and the area of circle sectors depends on the angle, α, between the sides of a regular polygon – which is given by α (N) = 180° (N –2)/N – in the following way: CSA(N) = α (N) × π × d2/(4 × 360°). Therefore, the area of the lumen is given by: LA(N) = RPA(N) – N × CSA(N) = (d2/4) × [N/tan(180°/N) – (π/2) × (N –2)]. With LA(N), we calculated the area equivalent radius of a circle as a function of the number of monomers, r(N), i.e. r(N) = (LA(N)/π)0.5, which we then used to calculate the single channel conductance,γ(N), through a pore with radius r(N) and length l in an electrolyte with resistivity ρ using the relationship γ(N) = [(l + π × r(N)/2) ×ρ/[π × r2(N)]]−1et alN) values of oligomers with 3 to 13 monomers, weighed by the relative abundance of each of these oligomers as shown in th to 75th percentile, the whiskers enclose the 5th to 95th percentile, the crosses enclose the 1st to 99th percentile, the hyphens indicate the minimum and maximum single channel conductance value, while the horizontal line in the box indicates the median and the open square symbol indicates the mean single channel conductance value.(TIF)Click here for additional data file.Table S1Comparison of different Aβ aggregation procedures with regard to the propensity of the resulting Aβ preparation to form pores in planar lipid bilayers and to kill cells.(DOCX)Click here for additional data file.Table S2Deconvolution of CD spectra of Aβin different preparation methods.(DOCX)Click here for additional data file.


The coordination dynamics and coordination mechanism of Et2Cit with Ca2+ ions and the effect of pH on the complex were examined. The result was compared with that for the conventional anticoagulant sodium citrate (Na3Cit). The reaction order (n) of Et2Cit and Na3Cit with Ca2+ was 2.46 and 2.44, respectively. The reaction rate constant (k) was 120 and 289 L·mol−1·s−1. The reverse reaction rate constant (kre) was 0.52 and 0.15 L·mol−1·s−1, respectively. It is indicated that the coordination ability of Et2Cit with Ca2+ was weaker than that of Na3Cit. However, the dissociation rate of the calcium complex of Et2Cit was faster than that of Na3Cit. Increased pH accelerated the dissociation rate of the complex and improved its anticoagulant effect. The Et2Cit complex with calcium was synthesized and characterized by elemental analysis, XRD, FT-IR, 1H NMR, and ICP. These characteristics indicated that O in –COOH and C–O–C of Et2Cit was coordinated with Ca2+ in a bidentate manner with 1 : 1 coordination proportion; that is, complex CaEt2Cit was formed. Given that CaEt2Cit released Ca2+ more easily than Na3Cit, a calcium solution was not needed in intravenous infusions using Et2Cit as anticoagulant unlike using Na3Cit. Consequently, hypocalcemia and hypercalcemia were avoided.Diethyl citrate (Et Sodium citrate (Na3Cit) is an important anticoagulant used in clinical settings  at t time was also calculated. Given that CaCl2 was mixed with Na3Cit or Et2Cit (1 : 1) and that the reaction of Ca2+ with Na3Cit or Et2Cit was equal in solution  with t in reaction system of Et2Cit and Na3Cit with CaCl2 is shown in c(Ca2+) was observed with prolonged t from 0 s to 30 s. This finding indicated that Et2Cit or Na3Cit was rapidly coordinated with Ca2+. At t = 30 s, c(Ca2+) decreased from 1.0 mmol/L to 0.49 mmol/L in the Na3Cit system and from 1.0 mmol/L to 0.87 mmol/L in the Et2Cit system. c(Ca2+) slowly decreased when t > 120 s, indicating that the system was in a dynamic equilibrium of complexation dissociation.The change in concentration of free Cadx/dt) of points on the curve, that is, the reaction rate r of each point formula (r  versus  logc(Ca2+)  , the slo(formula ). The in2Cit and Na3Cit with Ca2+ were as follows:The reaction rate equations of Etk can directly reflect the reaction rate,  in serum, as well as the reduced concentration of free Ca2+ in plasma that disturbed the blood clotting process from reaching the anticoagulation effect in vitro [3Cit, particularly as an anticoagulant, can coordinate a large number of Ca2+ ions in the blood. This phenomenon can lead to the low serum concentration of calcium in patients, as well as to hypocalcemia and all kinds of complications [3Cit [2+ after entering the body in the dialysis process. Additionally, hypercalcemia easily ensued in patients with presupplementary Ca2+. Therefore, the incidence of hypocalcemia and hypercalcemia can be reduced if we can reduce the coordination ability of anticoagulant.The anticoagulant mechanism of Nain vitro –11. Howeications –15. Therns [3Cit . MeanwhiThe reaction rate was equal to the inverse reaction rate when the reaction reached equilibrium, as shown in the following:k is the reaction rate constant, kre is the inverse reaction rate constant, and Ks is the complex stability constant.The above equation can be written as follows :(7)xn, wherein Na3Cit was alkaline. Therefore, when Na3Cit was used as an anticoagulant, the blood pH decreased and metabolic alkalosis likely ensued.In the dialysis process, the pH values of different dialysates varied. The acidities of different anticoagulants also differed. Therefore, the pH of blood in the dialysis process also changed. Considering that Na2Cit molecule only had one –COO−, the possibility of causing alkalosis was significantly reduced when Et2Cit was used as anticoagulant. With increased pH from 6.0 to 8.0, free c(Ca2+) decreased faster in the system , indicating a hydrogen bond.The peak at 3430 cm2Cit split into two peaks, which were 1709 and 1624 cm−1, respectively, indicating two different coordination environments in carbonyl. The position of both peaks red-shifted by approximately more than 30 and 110 cm−1 compared with the carbonyl absorption peaks of Et2Cit at 1736 cm−1. This finding indicated that the carbonyl of Et2Cit was coordinated with the calcium ions and was consistent with the change in the carbonyl characteristic absorption peak before and after coordination, as reported in [The carbonyl absorption peak (C=O) of CaEtorted in .2Cit was at 1100 cm−1. However, the peak split into two in the complex, that is, at 1081 and 1041 cm−1, respectively. This phenomenon was ascribed to one of the three C–O–C groups of the Et2Cit molecular complex with Ca2+, in which C–O–C absorption was bimodal and red shifted.The absorption peak of the symmetric stretching vibrations of (C–O–C) in C–O–C of Et−1 was the absorption peak of the methyl hydrocarbon of CaEt2Cit. It did not significantly change compared with the absorption peak of Et2Cit methyl hydrocarbon (2986 cm−1).The peak at 2982 cmThe FT-IR spectra of Et1H NMR spectra of Et2Cit and CaEt2Cit were studied using CDCl3 as a solvent, and the results are shown in 1H NMR are shown in δ = 7.26 and 6.28 ppm disappeared, indicating that –COOH participated in the coordination reaction. Meanwhile, the hydrogen in –OH group is very active; it can be easily dissociated and be partially or entirely substituted by deuterium in CDCl3 solution.The proton peaks of the ligand at 2Cit quartets were –CH2C=O , this peak of the complex (δ = 4.12 ppm to  4.17 ppm) shifted to a high field. This finding indicated the weakening of the induction effect of attracting electrons of O in –OCH2 from H after the O atom in –OCH2 coordinated with Ca. Thus, the total electron density of H increased, and the absorption peaks moved to a high field.The peak at OOCH2CH3 . CompareThe 1H NMR revealed that Et2Cit formed a 1 : 1 complex with Ca2+, that is, CaEt2Cit.Elemental analysis, ICP, XRD, FT-IR, and 2+ was coordinated with Et2Cit. O in –COO and C–O–C of Et2Cit was coordinated with Ca2+ in bidentate ligand. Two kinds of –OCH2CH3 had different chemical environments in the crystals, that is, 1,3-CaEt2Cit and 1,5-CaEt2Cit. However, their proportions were still difficult to ascertain because of their similar physical and chemical properties. Based on the above characterization results, two kinds of coordination of Et2Cit with Ca2+ are shown in The above results showed that Ca2Cit based on the reason that the FT-IR  of coordination with CaCl2 were 0.52 and 0.15 L·mol−1·s−1 for Et2Cit and Na3Cit, respectively. The dissociation rate of Ca2+ of CaEt2Cit was faster than that of CaCit. The increased pH accelerated the dissociation of the complex. With increased pH from 6.0 to 8.0, kre of Et2Cit-CaCl2 increased from 0.04 to 19.80, which was beneficial in improving the anticoagulant effect. Et2Cit and Ca2+ were coordinated to form a 1 : 1 complex, and O atoms in –COOH and C–O–C of Et2Cit were coordinated with Ca2+ in bidentate ligand. Et2Cit was able to coordinate with Ca2+, and its release capacity of Ca2+ was stronger than that of Et2Cit. Thus, it did not require an intravenous infusion of calcium when used as an anticoagulant, thereby avoiding hypocalcemia and hypercalcemia that can be caused by Na3Cit. Overall, Et2Cit was a better anticoagulant than Na3Cit.The coordination dynamics and effect of Et


Sensors in 2010 [Herewith please find corrected structures for Figure 8 in our paper published in 17α-ethynylestradiol used for the analysis is shown below:The structure for With product number E4876-1G from sigma Aldrich.naphthalene is below with no double bond in between carbon 2 and 7.The corrected structure of


CYP2C19 have reported wide ranges in the metabolic ratios of its substrates. These discrepancies could be attributed to the variations in the promoter region and this aspect has been reported recently. The observations in the recent reports on the influence of promoter region variants on the metabolism of CYP2C19 substrates might also have been influenced by the copy number variations of CYP2C19. In this paper, we describe copy number variations of CYP2C19 using real-time polymerase chain reaction by comparative Ct method. No copy number variations were observed in the south Indian population indicating the observed discrepancies in genotype-phenotype association studies might be due to the regulatory region polymorphisms as reported earlier.CYP2C19 is a polymorphic enzyme involved in the metabolism of clinically important drugs. Genotype-phenotype association studies of  CYP2C19 gene on chromosome 10, playing a major role in metabolizing about 5% of the clinically used drugs [CYP2C19 have been reported till date . Most of the genotype-phenotype association studies of CYP2C19 in different ethnic groups were focused on exonic region variants, but the promoter region variants and copy number variations were not explored to a greater extent [CYP2C19, namely, c.636G>A  or c.681G>A ; extensive metabolizers (EMs) carrying no variant alleles; ultra-rapid metabolizers (UMs) carrying −806C>T and −3402C>T variations  in the promoter region of CYP2C19 [The CYP2C19 is a clinically important drug metabolizing enzyme encoded by polymorphiced drugs . About 4r extent –11. Base CYP2C19 –18.CYP2C19 gene and copy number variants of the gene [CYP2C19 in different populations [CYP2C19 were due to promoter region polymorphisms influencing the expression of CYP2C19. However, the existence of copy number variations and the variability in the basal levels of expression of CYP2C19 between individuals was not considered, which might have affected the observations. Thus an alternative explanation for these discrepancies could be the copy number variations of this gene. Therefore, here in this paper we describe the results of an explorative study analyzing copy number variations of CYP2C19 in the study subjects of the previous report.CYP2C19 activity differs among extensive metabolizers (EM), demonstrated by a wide range in the metabolic ratios (MR) of probe drugs, and some discrepancies were commonly noted in these reports –20. Thesthe gene . Few stuulations , 22–24. ulations , 24. In IL-2 as reference gene using genomic DNA obtained from the peripheral white blood cells [CYP2C19  and IL-2  from genomic sequence were evaluated using serial 10-fold dilutions of human genomic DNA (n = 50) ranging from 0.2 to 200 ng. The demographic details of the subjects were discussed elsewhere [CYP2C19 amplification are specific to prevent the amplification of other homologous genes such as CYP2C9 located on the same chromosome. Total reaction volume consists of 25 μL and contained the following: 12.5 μL of 2X SYBR Green Master Mix , human gDNA  and the appropriate primer pair (400 nM). The PCR thermoprofile consisted of initial activation of the polymerase at 95°C for 15 min followed by 40 PCR cycles of 95°C for 15 s, and 60°C for 1 min (40 cycles) followed by a melting curve to verify the presence of a single amplification product. Assays were performed in duplicate on an Applied Biosystems 7300 Fast Real-Time PCR system using genomic DNA of 2 μL (approximately 20 ng). The PCR was performed in a 96-well clear optical reaction plate MicroAmp (Applied Biosystems). IL-2 gene was coamplified with CYP2C19 gene and served as internal standard (reference gene). CYP2C19 copy number was calculated relative to the human gDNA sample with no genetic variations in exonic and promoter regions, showing a good genotype-phenotype correlation (standard). The following equation was used: relative copy number = 2∗2−ΔΔCt, where  −ΔΔCt = (Ct  of reference gene −  Ct  of target gene) of standard − (Ct  of reference gene −  Ct  of target gene) of sample. Mean Ct values were taken for each sample derived from duplicate reactions. Efficiency of the assay was calculated using the formula E = 10(−1/slope) − 1 × 100. The calculated relative copy numbers were confirmed by recalculating the haploid copy number from the observed  Ct  values, and then multiplied by 2 to give the diploid copy number. The alternate formula used for calculation of haploid copy number was 2−ΔΔCt = (1+E)test  gene−ΔCt/(1=E) reference  gene−ΔCt, where E = Efficiency of the PCR reaction;  ΔCt test  gene  = Difference in the threshold cycle value between the test sample and calibrator sample for the gene (test gene) under investigation and  ΔCt reference  gene  = Difference in the threshold cycle value between the test sample and calibrator sample for reference gene.The relative copy number was estimated using a real-time PCR (comparative  ΔΔCt  method of relative quantification) approach based on existing methodologies with gene-specific primers using od cells , 26. Cyclsewhere . The priGraphPad Instat version 3.06  was used for statistical analysis. Data were expressed as mean ± SD. The copy number range from 1.7–2.1 was considered as the normal diploid copy number.IL-2 and CYP2C19 was between 87–100% and 94–100%, respectively. The efficiencies were similar and the assay stability was good. The slope of the standard curve for IL-2 was in the range of −2.7 to −4.3 with R2 (mean) of 0.99. The slope of the standard curve for CYP2C19 was in the range of −3.2  to −4.5 with R2 (mean) of 0.99. The product sizes for CYP2C19 and IL-2 were 193 bp and 93 bp, respectively. Dissociation curves have shown the specific melting temperatures for both IL-2 and CYP2C19 products and/or any primer dimer formed during amplification. The mean Ct values observed for the CYP2C19 and IL-2 standards are given in CYP2C19 and IL-2 from healthy volunteers are given in CYP2C19 and IL-2 are given in the Supplementary Material available online at doi: 10.1155/2012/643856. No copy number variations of CYP2C19 were detected in south Indian population. All the samples studied were found to have two copies of the gene. The CYP2C19 copy numbers were in the range of 1.7–2.17 with slight deviation from the set value 1.7–2.1 .The efficiency of the assay for  1.7–2.1 . The valCYP2C19 in south Indian population indicates that the genetic variations such as single-nucleotide polymorphisms may predict the changes in the activity of the enzyme. This observation also indicates that the discrepancies in genotype-phenotype association studies and wide ranges in the metabolic ratios of CYP2C19 substrates in EMs are due to other variations in the regulatory region. The involvement of other CYP isoforms in the metabolism of CYP2C19 substrates may also contribute to the discrepancies observed in genotype-phenotype association studies. Further studies are needed on a larger scale to investigate the copy number variations of CYP2C19 in different disease states in comparison to the healthy volunteers.Absence of the copy number variations of CYP2C19 were found in south Indian population suggesting that the previously observed discrepancies of phenotype-genotype studies could be due to the variations in the regulatory region as reported earlier.We here by conclude that no copy number variations of CYP2C19, IL-2 gene amplification are depicted in the supplementary figures 1 and 2 . The dissociation curves specific for CYP2C19 and IL-2 amplification are given in the supplementary figures 3 and 4 (where x-axis represents temperature in °C and y-axis represents the derivative of fluorescence with respect to temperature). The standard curves for CYP2C19 and IL-2 gene quantification are given in the figures 5 and 6 (where x-axis represents the log transformed concentrations of the standards used and y-axis represents the cycle threshold (Ct) reflecting the cycle number at which the fluorescence generated within a reaction crossing the threshold level.The amplification plots for Click here for additional data file.


We propose a novel reconstruction method using a spline-based image model in both spatial and temporal dimensions that takes the advantage of the precise timing of each k-space sample to reconstruct image series at high time frames, independently from the original sampling rate of data and avoiding the temporal blurring that can affect other reconstruction methods like sliding window.While MRI techniques have undergone considerable improvements since the early days, performing real time imaging is still challenging today. Despite the various original methods proposed until very recently, the usual way to reconstruct data at a higher frame rate is the sliding window technique that brings along intrinsic temporal blurringρn1,n2,n3cn1,n2,n3β1=Σ (1x-n)1β(2y-n)αβ(3t-n) where αβ is a spline function (α the spline degree). The spline model has the main advantage of allowing image reconstruction at arbitrary time points. In order to recover the Nyquist criteria, more data samples are included in the reconstruction by increasing the spline degree but they are weighted with the temporal information . The model was validated on an analytic phantom and then applied to real time data acquired from a healthy volunteer on a 3T scanner using a spiral sequence.The proposed image model is the following: th degree splines was comparable to the one obtained with the classical sliding window. Note that the number of spline coefficients (degrees of freedom of the model) remains fixed.Results obtained on the numerical phantom demonstrated that the time resolution of the reconstructed data could be improved by a factor of 3 when using a high enough spline degree, without sacrifying the SNR  figure . The temThis novel approach is able to increase up to 3 times the temporal resolution of reconstructed images without introducing temporal blurring.


At 14 d after Ca treatment, half of these plants were harvested (H1), and the rest of the plants were exposed to 100 μM Al for additional 6 d and harvested (H2). Severe Al injury was found only in the plants with the lowest supply of Ca before Al treatment. Aluminum concentration in the apoplastic fluid was very high at 125 μM Ca probably because the plasma membrane of some of the cells was destroyed due to the attack of 100 μM Al. Aluminum content in roots decreased with increasing supply of Ca before Al treatment. Calcium content decreased drastically at harvest (H2) in the plants with 100 μM Al. Under Al stress conditions, the plant responded to Al in different ways due to not only the different Ca supply but also the variation of Ca content in the plant tissues. Actually, the plants having the largest Ca content in the roots before Al treatment can receive less Al injury during Al treatment. To substantiate this idea, a companion study was conducted to investigate the effects of 2500 μM Ca supply during, before, and after 100 μM Al treatment on root growth. The results indicated clearly that exogenous Ca supply before Al treatment is able to alleviate Al injury but less effective than Ca supply during Al treatment.Aluminum (Al) sensitive wheat cultivar kalyansona was grown for 14 d in a range of Ca solution (125, 625, and 2500  The effects of Aluminum on the uptake and accumulation of divalent cations such as Ca and Mg have been extensively studied , 2. It hμM Ca during, before, and after Al treatment on root growth. This study will also clarify the importance of accumulated Ca in the root tissues during, before, and after Al treatment in alleviating Al injury.Considerable evidence suggests that the phytotoxic effects of Al on roots can be partially or completely overcome by increasing the concentration of Ca in the culture solution , 12, 13.Triticum aestivum L.) cultivar, Kalyansona, was used in our research work. Seeds were sterilized with 1% NaClO, stirred for 10 min, washed in deionized water, and soaked in distilled water for 24 h. Then the seeds were placed on planting trays with nylon fabric screen for germination in a plastic box containing full nutrient solution (mgL−1): 50N (as NH4NO3), 20P (Na2HPO4·12H2O), 50K (K2SO4), 3Fe (FeSO4·7H2O), 0.5Mn (MnSO4·4H2O), 0.2Zn (ZnSO4·7H2O), 0.5B (H3BO3), 0.05Cu (CuSO4·5H2O), 0.05Mo {(NH4)6Mo7O24·4H2O}, 10Ca (CaCl2·2H2O) and 10Mg (MgCl2·6H2O), and kept for 7 d at pH 5. The seedlings by then had developed 5–7 roots. The seedlings were then transferred to 3.5 L pots containing 125, 625, and 2500 μM Ca along with other nutrients. The pH of these treatment solutions was adjusted to 5.0 with NaOH and HCl, and these solutions were renewed every 3 d. Treatments consisted of three replications and each of them was composed of 3 pots. Each pot had 4 plants. A control plot was set up with 625 μM Ca from the beginning. Constant aeration of the nutrient solution was provided. After two weeks, harvest one (H1) was performed with half seedlings, when growth difference appeared  due to different levels of Ca. Then the remaining plants were exposed to 100 μM Al containing only 125 μM Ca at pH 4.5 for 6 d. The solutions were renewed every 3 d. Constant aeration to the nutrient solution was also provided. Then harvest two (H2) was performed at 6 d after Al treatment. At each harvest, the roots were washed in water and root lengths were measured. Then the roots were used for chemical analyses.A Bangladeshi wheat (μM Ca was considered as low dose and Ca treatment duration before Al treatment was reduced to 7 d instead of 14 d. Three weeks treatment duration was designated by three letters either L (250 μM Ca) or H (2500 μM Ca) consecutively . C stands for the control (without Al). In the first week, plants were treated with 250 and 2500 μM Ca without Al (before Al treatment). In the second week, plants were treated with 250 and 2500 μM Ca with 100 μM Al (during Al treatment) but the control was kept without Al. In the third week, plants were treated with 250 and 2500 μM Ca without Al (recovery phase). At the end of every week, one-third plants were harvested and root lengths were measured. Here, the harvests were designated as HI, HII, and HIII.In the companion study, same plant materials and same nutrient solution were used. During Al treatment, other nutrients were not applied except Ca. In this study, 250 2SO4 and 30% H2O2. Then Al in the samples was determined by pyrocatechol violet (PCV) method using a spectrometer [After completion of root growth measurement, the harvested plants (root and shoot) were dried in an oven at 70–800°C for 24 h and dry weights were determined. The Al content in roots was determined as described [trometer . CalciumμM Al, about 2 cm (from the apex) long roots were detached from a plant of each treatment, weighed, vacuum-infiltrated with demineralized water, blotted, and reweighed. The infiltrated roots were arranged on strips (2 cm × 10 cm) of thin polyvinyl sheeting (cut-up of plastic shopping bags). The all cut ends of roots were arranged in the same direction as described [2SO4-H2O2. Aluminum in the AF was determined by the same method as described above.The method was used as described . After 6escribed . The strμM for 14 d had adistinct effect on the shoot growth as well as on root growth  produced 20% higher root dry matter than the control with 250 μM Ca (cLLL). The root dry matter in other plots was similar to the control (In the absence of Al (H1), increasing supply of Ca from 125 to 2500 t growth . The lowdy at HI . That is control .μM Ca before Al treatment. It was the highest (104%) in the control plot (data not shown). About 34, 46, and 62% increase in root dry matter was recorded at H2 in comparison with their corresponding root dry matter at H1 in the plots pretreated with 125, 625, and 2500 μM Ca, respectively. The shoot dry matter production in the presence of Al was greater than the root dry matter production and showed a similar tendency.Under Al stress conditions (H2), the plants with different Ca supply before Al treatment responded to the same level of Al in different ways . Dry matμM Al was applied during the second week. After 7 d exposure to 100 μM Al, root dry matter at HII was in the following order: LHL > HLL > LLH ≥ LLL , new root development was only observed in LHL and HLL plots (μM Ca during Al treatment (LHL) than the plants with the same level of Ca before Al treatment (HLL). Furthermore, the root tip of the former (LHL) was not strongly affected as observed in HLL. New root development at the root tips was not observed in the plants with 2500 μM Ca for 7 d after Al treatment (LLH). As a consequence at HIII, about 6% more roots dry matter than the control was observed in LHL, whereas it was 88% of the control roots dry matter in HLL (μM Ca (LLH) and 250 μM Ca (LLL) during the recovery phase, although more new roots were observed at the base of shoots in LLH than LLL . The plants with the highest Ca (3010 μgg−1 DM) content at H1 showed a remarkable reduction in Ca (197 μgg−1 DM) content after 6 d exposure to 100 μM Al. Aluminum was undetected in the plant at H1 and the control plant at H2. The plants pretreated with different levels of Ca had different Al content in the roots at H2. The highest Al content was determined in the roots with the lowest Ca supply before Al treatment. Aluminum content decreased gradually with increasing supply of Ca before Al treatment.In the absence of Al (H1), Ca content in the roots increased with increasing supply of Ca in the culture solution . The higμM Ca (HLL), whereas it was 44% in the plants that treated with 2500 μM Ca (LHL) during Al exposure. In the control plants (cLLL), Ca uptake increased by 33% during second week treatment without Al. During recovery phase (HIII), Ca content increases in all treatments and the highest was in LHL. The highest Al content at HII was observed in LLL and LLH. It was 1.5 and 1.4 times higher than LHL and HLL, respectively. The results further showed that the difference in Al content between LHL and HLL was lesser extent than the difference observed in their root dry matter yields . Therefore, other factors such as plant age may cause the difference in the short-term and long-term experiments as reported [In the absence of Al, there were differences among the Ca levels in terms of plant (root + shoot) growth Figures  and 2 anμM Ca  before Al treatmentnot only enable the Ca2+ ions to occupy most of the CBS of the roots [Severe Al injury was found in the plants that pretreated with 125 μM Ca . It is aogically , 19. Thiogically . In our 25 μM Ca . Severe 25 μM Ca  which pohe roots  but alsohe roots , boundedhe roots  in the pThe disturbance of Ca homeostasis via Ca displacement from CBS and increased cytosolic Ca level by Al is a well-established event during Al-Ca interactions in plants . In our μM Ca was supplied during Al treatment. The results also showed that 2500 μM Ca supply before Al treatment is able to alleviate Al injury but lesser extent than the same level Ca supply during Al treatment (μM Ca supply (LLH) at the recovery phase could not improve root growth inhibition caused by Al toxicity (In the companion study, the highest root growth at HII  as well reatment . The difreatment  showed treatment . Therefotoxicity , and newtoxicity . This matoxicity  indicate