{"text": "The role of glutathione (GSH) and GSH-S-transferase (GST) activity in modulating the cytotoxicity of four platinum drugs and melphalan was evaluated in eight human ovarian carcinoma cell lines. The cell lines were established from solid and ascitic tumours from pretreated and untreated patients, and showed a wide spectrum of sensitivity to several platinum II and platinum IV drugs; cisplatin, carboplatin, CHIP and tetraplatin. Intracellular glutathione concentration measured in the cell lines showed a significant (P = 0.05) correlation with IC50 values for cisplatin (r = 0.91), carboplatin (r = 0.87) and CHIP (r = 0.88). The correlation between GSH levels and IC50 values for melphalan (r = 0.76) or tetraplatin (r = 0.60) was not as significant. GST activity showed no correlation with IC50 values, for the four platinum drugs. To determine the significance of the elevated GSH concentration in the refractory cell lines, the effect of D,L-buthionine-S, R-sulfoximine (BSO) mediated GSH depletion on platinum drug cytotoxicity was examined in one of the most sensitive (CH1) and two of the least sensitive  cell lines. Comparison was made with the effect of GSH depletion on melphalan cytotoxicity in these three lines. These lines were differentially sensitive to BSO, with the two most platinum drug resistant lines being more tolerant to BSO than the sensitive CH1 line. Depletion of cellular GSH, ranging between 61 and 88%, had a differential effect on the sensitivity to PtII vs PtIV drugs in the three cell lines: cytotoxicity of the PtIV drugs, tetraplatin and CHIP, was substantially enhanced in both the resistant and sensitive cell lines; in contrast, the cytotoxicity of the PtII drugs, cisplatin and carboplatin, was only significantly increased in one of the two relatively resistant lines (SKOV-3) and in the sensitive (CH1) line after GSH depletion. Moreover the dose modification factor (DMF) for the PtII agents were lower than those for PtIV agents in the three cell lines. The dose modification factor for tetraplatin after BSO treatment was similar to that observed for melphalan in all three cell lines. In the SKOV-3 cell line extending the BSO pretreatment period to 48 h from 24 h marginally reduced the cytotoxicity of cisplatin, whereas the cytotoxicity of the other three drugs remained similar to that observed after 24 h BSO pretreatment. In contrast, extending the BSO treatment to 24 h after drug exposure potentiated the cytotoxicity of cisplatin, CHIP and tetraplatin.(ABSTRACT TRUNCATED AT 400 WORDS)"}
{"text": "Cell lines selected (CHRC5) and transfected (LR-73-1A) for multi-drug resistance have total lipid compositions which are indistinguishable between resistant and parental cells. Lipid composition was evaluated by 1H NMR and the total fatty acid content by GLC. No change in surface hydrophobicity, as measured with the fluorescent probe dansyl-PE, was observed as a result of transfection of CHO cells with the mdr1 gene. However, the selected cell line, CHRC5, showed a decreased surface hydrophobicity. This decreased surface hydrophobicity was indicated by an 8 nm increase in the fluorescence emission of dansyl-PE in the CHRC5 cell line compared with the AB1. Both resistant cell lines showed an increase in the polarisation of the fluorescent probe, TMA-DPH in the plasma membranes corresponding to a 14% and a 24% change in fluorescence polarisation for the selected and transfected cell lines, respectively. This is indicative of reduced mobility of the acyl chains in the resistant cell lines. Both the CHRC5 and the transfected cell lines showed almost a 2-fold increase in the initial rate of membrane cycling. The membrane cycling could be inhibited by a known bilayer stabiliser, the N-carbobenzoxy-D-Phe-L-Phe-Gly. These results indicate that the properties of the plasma membrane from resistant cells are altered compared with their parental cell line."}
{"text": "Congenital arhinia is an extremely rare anomaly consisting of an absence of external nasal structures and nasal passages. Fewer than 30 cases have been reported. Patients with a familial absence of the nose have been reported, but the effects of genetic and maternal factors are unknown. Midface hypoplasia may accompany arhinia. Accompanying malformations are thought to be caused by an absent or rudimentary nose. A patient with partial congenital arhinia is presented and the embryology and literature review are discussed. Arhinia may be accompanied by midline defects, or ear, palatal, ocular, or facial abnormalities. Severe airway and feeding problems may accompany arhinia in newborns.Congenital absence of the nose, Our case is a female patient, born to a 28-year-old mother. This was the mother's first pregnancy. The mother denied any antenatal problems, and the baby was born vaginally at full term. Family history revealed no interrelative marriage or history of congenital malformation. Results of a chromosomal analysis showed a normal female. The baby was born with severe nasal deformation. On gross inspection the external nose was absent. The nasal dorsum structures and remnants of the alar cartilages were barely palpable. The columella and right nostril were absent; the left nostril was severely stenotic Figure . ProbingDuring facial development, cranial neural crest cells migrate from the trigeminal nerve region to the face . DevelopThe pathogenesis of arhinia is not clearly understood. The proposed mechanism may be a developmental defect in the medial and lateral nasal processes or overdevelopment and early fusion in the medial nasal processes . Arrest Olsen and associates reviewed the literature through 2001 and collected 22 additional cases . McGloneCongenital arhinia is a very rare defect during embryogenesis. Most cases are sporadic but familial cases have been reported. Mostly the karyotype is normal. In 2 cases, anomalies in chromosome 9-and in a single case, chromosome 3\u201312 translocation \u2013 have been detected . A varieWe considered our patient as having partial arhinia because there were some remnants of the external nose and a cul-de-sac was present. This was accompanied by central nervous system and ear anomalies, a submucosal cleft palate, and hypotelorism.Congenital arhinia is an extremely rare condition of unknown etiology. Facial anomalies and other concomitant distant anomalies may be present. These patients experience serious problems with regard to an open airway and feeding. Any accompanying anomalies should be detected and surgical correction should be planned.Written informed consent was obtained from the patient's parents for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The author(s) declare that they have no competing interests.GA made substantial contributions with regard to the manuscript's conception and design and was involved in drafting the manuscript; the author will give final approval of the version to be published. BA made substantial contributions with regard to the manuscript's conception and design and was involved in drafting the manuscript; the author will give final approval of the version to be published. EA made substantial contributions with regard to the manuscript's conception and design and was involved in drafting the manuscript; the author will give final approval of the version to be published. MD made substantial contributions with regard to the manuscript's conception and design and was involved in drafting the embryologic part of the manuscript. LO was involved in revising the manuscript critically; the author will give final approval of the version to be published."}
{"text": "In vitro recordings of medial entorhinal layer II stellate cells have revealed subthreshold membrane potential oscillations (MPOs) whose temporal periods, and time constants of excitatory postsynaptic potentials (EPSPs), both increase along this axis. Slower (faster) subthreshold MPOs and slower (faster) EPSPs correlate with larger  grid spacings and field widths. A self-organizing map neural model explains how the anatomical gradient of grid spatial scales can be learned by cells that respond more slowly along the gradient to their inputs from stripe cells of multiple scales, which perform linear velocity path integration. The model cells also exhibit MPO frequencies that covary with their response rates. The gradient in intrinsic rhythmicity is thus not compelling evidence for oscillatory interference as a mechanism of grid cell firing. A response rate gradient combined with input stripe cells that have normalized receptive fields can reproduce all known spatial and temporal properties of grid cells along the MEC dorsoventral axis. This spatial gradient mechanism is homologous to a gradient mechanism for temporal learning in the lateral entorhinal cortex and its hippocampal projections. Spatial and temporal representations may hereby arise from homologous mechanisms, thereby embodying a mechanistic \u201cneural relativity\u201d that may clarify how episodic memories are learned.Place cells in the hippocampus of higher mammals are critical for spatial navigation. Recent modeling clarifies how this may be achieved by how grid cells in the medial entorhinal cortex (MEC) input to place cells. Grid cells exhibit hexagonal grid firing patterns across space in multiple spatial scales along the MEC dorsoventral axis. Signals from grid cells of multiple scales combine adaptively to activate place cells that represent much larger spaces than grid cells. But how do grid cells learn to fire at multiple positions that form a hexagonal grid, and with spatial scales that increase along the dorsoventral axis?  Spatial navigation is a critical competence of all higher mammals, and place cells in the hippocampus represent the large spaces in which they navigate. Recent modeling clarifies how this may occur via interactions between grid cells in the medial entorhinal cortex (MEC) and place cells. Grid cells exhibit hexagonal grid firing patterns across space and come in multiple spatial scales that increase along the dorsoventral axis of MEC. Signals from multiple scales of grid cells combine to activate place cells that represent much larger spaces than grid cells. This article shows how a gradient of cell response rates along the dorsoventral axis enables the learning of grid cells with the observed gradient of spatial scales as an animal navigates realistic trajectories. The observed gradient of grid cell membrane potential oscillation frequencies is shown to be a direct result of the gradient of response rates. This gradient mechanism for spatial learning is homologous to a gradient mechanism for temporal learning in the lateral entorhinal cortex and its hippocampal projections, thereby clarifying why both spatial and temporal representations are found in the entorhinal-hippocampal system. Navigating the world requires the brain to learn and maintain memory of spatial positions within various environments. Place cells in the hippocampal areas CA1 and CA3 demonstrate a neural code for position in large spaces that higher mammals inhabit In particular, Brun and colleagues The presence of multiple spatial scales on the dorsoventral axis of MEC has important implications for the development of the hippocampal place cells In vitro whole-cell patch clamp recordings Excitatory projections to the hippocampal formation from layer II of MEC are primarily from stellate cells This article develops a SOM neural model, called Spectral Spacing for reasons summarized below, to explain the above data. This model shows how a gradient of cell response rates along the dorsoventral axis of MEC can control the development of grid cells whose hexagonal grid firing fields exhibit a gradient of spatial scales and whose MPOs exhibit a gradient of frequencies. These results combine several conceptual and technical advances.First, these results are part of an emerging general entorhinal-hippocampal model architecture see also , which sSecond, these SOM laws have been proposed to control development and learning in many other parts of the brain, notably the visual cortex. Thus, both grid and place cells may develop using general SOM principles of brain map organization.Third, the linear velocity and angular velocity path integration inputs that drive model learning are derived from realistic trajectories of rats in spatial learning and memory experiments.Fourth, these linear velocity and angular velocity estimates can both be transformed into position codes by ring attractors.Fifth, the rate gradient mechanism for spatial learning in the MEC pathway and its hippocampal projections is homologous to a rate gradient mechanism that has been used to model temporal learning in the lateral entorhinal cortex (LEC) pathway and its hippocampal projections. Spatial and temporal representations in the medial and lateral processing streams may hereby arise from homologous mechanisms, thereby embodying a mechanistic \u201cneural relativity\u201d in the entorhinal-hippocampal system. This homology may clarify why spatial and temporal representations both occur in hippocampus, and provides new clues about how episodic memories may be learned.In summary, this model system exhibits parsimony and unity, both in its use of similar ring attractor mechanisms to code the linear and angular velocity path integration inputs that drive learning, and in its use of a rate gradient mechanism that can support the learning of both spatial and temporal codes.Even more striking is the fact that both grid cell and place cell receptive fields emerge by detecting, learning, and remembering the most frequent and energetic coactivations of their inputs. This co-occurrence property is different from the property of oscillatory interference that some other models have proposed e.g., . OscillaSimulation Settings). These simulations demonstrate that, at least among these variations, only a response rate gradient, combined with input cells that have normalized receptive fields, can explain all the data mentioned above.In order to better understand what aspects of the Spectral Spacing model are needed to explain how spatial and temporal properties of grid cell firing change along the dorsoventral extent of MEC, several model and input variations were simulated  and weight decreases (long-term depression). It hereby enables the weights to adapt to the Simultaneously active stripe cells are more likely to strongly activate map cells whose bottom-up weight patterns closely match their activity pattern. Adaptation of the weights to a map cell occurs only when its activity is above a threshold see  in . This poThe GRIDSmap model Until recently, SOM models of place cell learning used idealized or hand-crafted grid cells e.g., . Pilly aBoth the GRIDSmap and the GridPlaceMap models learn hexagonal grid firing fields whose spatial scale is derived from that of the input stripe cells. In particular, stripe cells with the same period were used to learn grid fields of a given spatial scale. Stripe cells of different spatial scales were assumed to activate different locations along the dorsoventral axis in layer II of MEC, thereby giving rise to grid cells with different spatial scales. But how is the selection of just one spatial scale of stripe cells realized for each grid cell scale? What would happen if stripe cells of multiple scales initially projected to the map layer before grid cell learning began, as in in vitro to a steady current input. Whereas a dorsoventral gradient in either response rate or habituation rate can explain the corresponding gradient in learned spatial scale and MPO frequency of grid cells, only a gradient in response rate was found to be consistent with data regarding the associated dorsoventral gradient in peak and mean firing rates of grid cells This article shows that the rates at which the category cells and their corresponding habituative transmitters respond, called the response rate . For a cell to respond with contrast-enhanced, or above-threshold, activity at any moment with the help of its self-excitatory feedback signal, its habituative transmitter needs to be at a sufficient high level. But each time the cell responds intensely, there is a collapse of the transmitter , which tThis property directly explains learned scale selectivity for the case of a rat running forward at a constant speed on a linear track. Then dorsal MEC cells in the model respond better to inputs at periodic positions with relatively smaller spacings, while ventral MEC cells respond better to those with relatively larger spacings. However, the situation is more complicated when the rat navigates along the type of two-dimensional real trajectory used in our simulations, for which the running speed of the rat through time varies between 0 cm/s and 146.6 cm/s with a mean of 14.03 cm/s, a standard deviation of 9.8 cm/s, and a mean length of piecewise linear segments of only 0.9 cm. How different response rates selectively learn different spatial scales in response to such realistic trajectories is discussed in the next subsection.Emergent properties of model simulations  emulate As noted earlier, for each input stripe scale considered separately, the most frequent and energetic activations of grid cells occur when sets of three stripe cells are coactivated whose preferred directions differ by 60\u00b0 peak firing rate of MEC cells decreases from the dorsal to the ventral ends  exhibit lower gridness scores  and inte problem \u20137 is not problem  that wasells see . This reIn Case 8, unlike the data, dorsal cells learned hexagonal grid fields derived from large-scale stripe cells. This case imposes the same peak activity across both small-scale and large-scale stripe cells. Thus, the stripe cell receptive fields are not normalized across scales, and the large-scale stripe cells have a competitive advantage since they are sampled by map cells for a longer time. This advantage seems to be sufficient for them to win over small-scale stripe cells with the same peak activity, despite the lower frequency of their favored coactivations across space , in driving the learning of large-scale grid cells even for faster response rates. Thus, if stripe field widths increase with stripe spacing, similar to grid cells Normalized receptive fields occur in many other examples of multi-scale processing in the brain, and may be a general principle of brain design. The general design theme is how to achieve selective processing across multiple scales, so that the largest scales do not always win the competition to represent incoming data. Normalization ensures that the degree of brain commitment covaries with the amount of evidence for that choice gradient , and 7. gradient , in contin vitro studies have showed that layer II MEC stellate cells exhibit subthreshold MPOs, in response to steady current injection, whose temporal period increases from the dorsal to the ventral end of MEC  to convert a stripe cell population code that implicitly represents an animal's position using multiple small spatial scales into a place cell code in which a single place cell can explicitly represent spatial position in large environments. The intermediate stage of grid cells converts input stripe cell signals into a form conducive to the learning of such unimodal place cell spatial fields, which thereby significantly increase the scale of spatial representation compared to the inducing grid cells. Simulations in Can place cells be learned directly from stripe cells, without the intervention of hexagonal grid fields? The presence of the animal at a given spatial position strongly activates just one or few stripe cells in each directional ring attractor. So, a unimodal spatial field at that position could be learned, in principle, if a map cell could become tuned to the combination of all these coactive stripe cells across directions and scales. However, such input combinations are not favored by the self-organization process because they occur only at single positions in the environment, as opposed to the multiple positions at which the stripe cell combinations suitable for hexagonal grid fields are activated. As we mentioned above, map cell learning at both the grid cell and place cell levels is naturally sensitive to both the energy and frequency of input coactivations. How, then, are place cells learned, given they are activated only at single positions in the environment? If inputs to a SOM come from comprise grid cells of multiple spatial scales, then sets of co-active grid cells involving a greater number of scales are more likely to gain control of hippocampal map cells in vivo may be modulated by running speed, because of which the slope and intercept of the dorsal-ventral gradient in grid spacing may not be significantly altered in response to very fast or slow running speeds. It would be instructive to explicitly test this prediction.The spacings of grid fields in our model are adaptively selected based on cell response rate, which is inversely correlated with the minimum temporal duration between two episodes of intense activity. Therefore, it is important to discuss how the learned grid cells may respond if an adult animal were to run around an environment with a mean speed that is higher or lower than when the grid cells are learned during development. However, these extreme speed cases may be relevant only for theoretical purposes because of two reasons; namely, the distribution of running speeds in the realistic trajectory, used for our simulations, is relatively broadly tuned with a standard deviation of 9.8 cm/s, and existing relevant data indicates that the average running speed of rats increases by just \u223c2 cm/s from P16 to adulthood . The nature of our model's incompleteness clarifies data about how and when deformations in grid cell receptive fields do occur In summary, our model here and in More work needs to be done to study how the response rate gradient and the habituative gating mechanism in our model relate to the HCN and leak potassium channels, which control the varied temporal integrative properties of MEC layer II stellate cells The model suggests several predictions regarding the development of grid cells at different anatomical locations along the dorsoventral axis of MEC as young animals begin to navigate for the first time. These predictions are tempered by the awareness that the model does not yet incorporate various known mechanisms, such as top-down matching and attentional mechanisms from hippocampus, that may influence model properties, notably their malleability after the predicted dynamical stabilization of grid field structures sets in due to attentive matching.Existing empirical studies on the development of grid cells The Spectral Spacing model illustrates how control by a single rate parameter can determine a gradient of grid cell spatial scales in response to inputs from multiple stripe cell spatial scales. Multiple small grid cell scales can then be adaptively combined in the hippocampus to generate place cell scales that are large enough to support spatial navigation In support of this prediction, MacDonald and coworkers Where cortical stream go through postrhinal cortex and medial entorhinal cortex on their way to hippocampal cortex, and object representations in the What cortical stream go through perirhinal cortex and lateral entorhinal cortex on their way to hippocampal cortex Thus, dorsoventral gradients in single rate parameters within the entorhinal-hippocampal system may create multiple small spatial and temporal scales that can be fused into larger spatial and temporal scales in the hippocampal cortex that are large enough to control adaptive behaviors. The mechanistic homology between these spatial and temporal mechanisms suggests why they may occur side-by-side in the medial and lateral streams through entorhinal cortex into the hippocampus. In particular, spatial representations in the This subsection highlights and justifies differences between the GRIDSmap model First, we introduced a threshold in the signal function that transforms membrane potentials of map cells into their output activities, which both govern the recurrent inhibitory interactions and gate the competitive adaptation of corresponding bottom-up weights .Increased stability of learned spatial fields as the bottom-up weights do not adapt in response to noisy or non-optimally activated map cells.Second, we initialized the pre-development synaptic weights of the connections from stripe cells to grid cells  in  using a Third, the inactivation and recovery dynamics of the habituative transmitter depend only on the self-excitatory feedback signal see  in the eFourth, the adaptive weights from stripe cells to category cells use a different version of the instar learning law (Equation 1.6) that more robustly enabled category cells to become tuned to coactivations of stripe cells In the GRIDSmap model, the stripe cells of different spacings were assumed to have the same maximal firing rate but different field widths (i.e., Several models exist to explain the generation of grid cells, but the Spectral Spacing model differentiates itself by providing for the first time a principled explanation of how grid cells learn not only their characteristic hexagonal grid firing patterns band cellsPrior grid cell models can be generally classified into two categories based on whether the linear velocity path integration happens before or at the level of grid cells. In addition to the SOM type of model, the former possibility has been modeled using mechanisms of oscillatory interference in vivo, result if this constraint is not met Oscillatory interference models The 2-D attractor models"}
{"text": "We introduce a grid cell microcircuit hypothesis. We propose the \u2018grid in the world\u2019 (evident in grid cell discharges) is generated by a \u2018grid in the cortex\u2019. This cortical grid is formed by patches of calbindin-positive pyramidal neurons in layer 2 of medial entorhinal cortex (MEC). Our isomorphic mapping hypothesis assumes three types of isomorphism: (i) metric correspondence of neural space  and the external two-dimensional space within patches; (ii) isomorphism between cellular connectivity matrix and firing field; (iii) isomorphism between single cell and population activity. Each patch is a grid cell lattice arranged in a two-dimensional map of space with a neural : external scale of approximately 1 : 2000 in the dorsal part of rat MEC. The lattice behaves like an excitable medium with neighbouring grid cells exciting each other. Spatial scale is implemented as an intrinsic scaling factor for neural propagation speed. This factor varies along the dorsoventral cortical axis. A connectivity scheme of the grid system is described. Head direction input specifies the direction of activity propagation. We extend the theory to neurons between grid patches and predict a rare discharge pattern (inverted grid cells) and the relative location and proportion of grid cells and spatial band cells. These 2.(a)We first provide a minimal sketch of the operation of the grid cell system in layer 2 of the rat MEC. The isomorphic mapping hypothesis is inspired by the similarity between the pattern of patches in the MEC and grid cell discharge patterns in space . It is s(b)a shows the grid-like arrangement of these patches in layer 2 of the MEC. Calbindin patches have a diameter of about 150 \u00b5m and are arranged in a regular array According to our hypothesis, we assume three types of isomorphism: (i) a metric correspondence of neural space  and the external two-dimensional Cartesian space within calbindin grid cell patches; (ii) an isomorphism between the connectivity matrix of a grid cell and its firing field ; (iii) an isomorphism between single grid cell and population grid activity within a patch. All these assumptions are simplifications, and only assumption (i) is very strong and essential to the theory. Assumptions (ii) and (iii) greatly simplify the interpretation of entorhinal circuits, but they are less critical to the theory and can easily be relaxed.b). A layer 2 grid cell patch, seen in a top view, is a metric representation of repeating segments of real space (figure 1c). We show a patch in the shape of a hexagon (figure 1b), which makes it easy to illustrate how real space (figure 1c) is represented and tiled up by the grid patch. The hexagonal shape of a grid patch is not essential to our theory, however, and anatomically calbindin-patches look typically circular. While the hexagonal shape of a grid patch is not essential to the model, the arrangement of multiple grid patches in a hexagonal grid is of significance for the connectivity scheme proposed later and our ideas how spatial band cells might be generated from grid cell inputs (see below). Spatial direction applies in both the real and neural space. This is our first isomorphism assumption. Figure 1b illustrates, in a colour-coded population, firing rate in the patch at a set of positions a in real space, whereas figure 1c illustrates, in a colour-coded single neuron, firing rate of a grid cell, which has its peak firing rate at position A. The correspondence of single grid cell and population grid activity illustrated here is our third isomorphism assumption.figure 1a. It can be seen that different from conventional attractor models there is not one but many \u2018bumps of activity\u2019 [figure 2a, left), these bumps of activity move from left to right in the entorhinal cortex . Although these bumps of activity are at analogous positions and move at identical speed in laterally neighbouring patches, the bumps of activity move at different speeds in patches at different dorsoventral positions.figure 2ctivity\u2019 \u201320. Spec(d)b) and in real space (figure 1c). A calbindin patch has a diameter of 150 \u00b5m [a,b). Specifically, we assume that there is an intrinsic difference in propagation speed of neural activity between dorsal and ventral patches (b). Note that the actual speed, with which activity travels through the patch, depends also on the animal's velocity in space. Spatial scale is therefore not interpreted as an absolute propagation speed of neural activity but rather as an intrinsic speed propagation factor to be multiplied with the animal's velocity. The idea that neural propagation of activity differs between dorsal and ventral entorhinal cortex is in line with experimental evidence on dorsal/ventral differences in intrinsic properties [figure 1f 150 \u00b5m  and in d patches b. Note toperties , synaptioperties \u201329.\u22121, a rat will cross a dorsal patch in 3 s and will cover 1.14 cm in one theta cycle. In external space, 1.14 cm corresponds to 5.7 \u00b5m in neural space. If one imagines the about 120 calbindin-positive pyramidal cells in one plane (the patch has an area of about 15 000 \u00b5m2), then they have a spacing of approximately 11 \u00b5m [These assumptions predict fast neural propagation in dorsal patches  and slow neural propagation in ventral patches . What do these numbers mean in neural terms? At a standard running speed of 10 cm sly 11 \u00b5m . Thus, w(e)c illustrates how head direction information drives the propagation of activity through the patch. We assume that the grid cell activity rests statically at a low level that is exactly self-sustaining in the absence of head direction input. While it seems fairly clear that head direction inputs control the direction of propagation activity, how this exactly occurs is not predicted by the theory. To give the reader a better idea how this may occur, we specify one possible implementation of head direction input to grid patches in figure 2c,d, but we emphasize that this scheme\u00a0is not essential to our theory. As shown in figure 2c, directionally delayed inhibition activated by head direction inputs can be used to push the population activity forward (figure 2d). The local and long-range wrap-around connectivity between grid cell patches (see \u00a72f) assures that the grid activity peak wraps around in time . The velocity signal could involve either the head direction cells or could be implemented through a higher grid cells activity with increasing velocity, which will also lead to faster propagation. Most likely, velocity is implemented in multiple ways in the grid/head direction circuit.The propagation of activity through a grid patch is what leads to grid cell firing, but several aspects of this process are not yet understood. Figure\u00a02(f)The core of our hypothesis is to devise a connectivity scheme that generates grid cell discharges and relates spatial discharge in an unambiguous manner across spatial scales and grid cell patches. We assume that all patches have the same grid orientation. Patches at the same dorsoventral height of MEC are thought to have the same grid map. A set of six connectivity rules that connect grid cells within and across patches is illustrated in Is this connectivity scheme compatible with the experimental evidence? The mutual excitatory connectivity between layer 2 pyramidal cells postulated here differs sharply from the connectivity observed in paired recording studies of layer 2 stellate neurons, which show almost no mutual excitatory connections and which are coupled by strong disynaptic inhibition ,30. MorpThe fact that even same-sized grids can have deviations, such as slightly different tilts in their main axes , poses cGrids of different scales shift relative to each other in situations that cause place cell remapping . We thin(g)a. As we have seen in the description of the patchy anatomy, this is not the case, however. Instead, putative grid patches are displaced from each other as shown in figure 4b.This brief account of the isomorphic mapping hypothesis explains many features of grid cell activity, such as iterative firing and spatially coherent activity across scales. The most simple connectivity scheme to implement the entire theory outlined so far would be to arrange grid patches directly adjacent to each other, as depicted in figure 4b).We now extend our hypothesis to include this space between the grid patches in our theory. With two assumptions, one can also predict spatial firing patterns in this region. These assumptions are (i) cells in this region receive the same grid cell input as grid cell neighbours, but they do not return connections, and (ii) there is no recurrent connectivity in this region. These assumptions imply that the area outside of the patches is downstream from the grid system and that there is no metric spatial firing in between grid patches, because it is the symmetric connectivity of cells in grid patches which guarantees such a code b.c, where we show the connections of two grid cells in neighbouring patches in one dimension. The entire connectivity matrix would involve all grid cells, which make radial connections in two dimensions. Figure\u00a04d,e shows schematically the resulting spatial discharge profiles at various regions in layer 2 of the MEC. Figure\u00a04d also shows that the model predicts a gradual shift from grid-like to band-like firing patterns as one moves out from grid patches. In cells in the transition zone from grid-like to band-like firing, varying inputs from neighbouring grid and band cells might cause a flipping back and forth between such discharge patterns as observed recently [What then, are the firing fields of cells between the grid patches? We approach this issue in figure 4recently .(i)e, grid cells are observed in grid patches.As discussed before and shown in figure 4(ii)c), and the neural activity will be maximal and the same along a line connecting this position and the two neighbouring patches. Along the room axis orthogonal to this connecting line, activity falls off, because the distance of grid cells to this position increases along this axis.We predict that exactly between two neighbouring patches, one will observe cells that discharge in spatial bands. This band-like firing pattern comes about because the input from the two neighbouring patches will be the same at this point c, and tha. Because cells in the grey area between grid patches receive their inputs from grid patches and input strength from each grid cell falls off with distance, their activity will increase the closer they are to the peak of the activity bumps in grid patches. As the animal runs from left to right (figure 2a), the cells between two grid patches in the top row of figure 2a will see a constant input level. This constant input level comes about because inputs from the left-hand neighbouring grid patch increase as the activity bump approaches, but at the same time inputs from the right-hand neighbouring grid patch decrease as the activity bump moves away. If the animal runs from top to bottom, however, then input levels of the cells between two grid patches in the top row of figure 2a will vary. The cells will see maximum input when the peak of the activity bump in the neighbouring grid patches is exactly lateral from them, and a drop of inputs when the animal moves further up or down. Thus, these cells between two grid patches in the top row of figure 2a will fire in a spatial band extending from left to right.One can also understand how spatial band cells come about in our model, if one considers the dynamical scenario outlined in figure 2(iii)c, a novel discharge pattern\u2014inverted grid cells . Again, one can also understand how inverted cells come about in our model if one considers the dynamical scenario outlined in figure 2a.Based on the connectivity considerations shown in figure 4see also )\u2014can be Finally, we would like to add that the spatial firing profiles observed between grid patches depend fairly strongly on the connection parameters, such as radial spread of connections. This parameter dependence differs from the grid connectivity. Parameter dependence arises because we are dealing here with unilateral, asymmetric connections. For example, inverted grid cells come about only if not too long a radial connectivity is chosen; otherwise, one observes very little spatial modulation in this region of the entorhinal cortex. This is an important consideration because there is evidence for a large number of spatially non-modulated cells in the MEC , whereasAre the fractions of grid and band cells predicted by this model compatible with published results? There are discrepancies between the reported percentages of grid cells in superficial layer MEC in the literature. Older studies report large fractions of grid cells ,2, which(h)f may be modified by learning, such that they represent space more accurately , but we do not think that grid cells represent environment-specific landmark information. We find it difficult to imagine how individual grid cells could learn landmark information when grid scale is changing with experience, and hence the firing nodes of grid cell activity drift relative to landmark inputs [So far, we have not considered here how the grid is anchored to the world. According to our theory, the grid system is a purely metric representation of space, which needs to be aligned with the world, but which does not contain specific spatial memories. The synaptic connections specified in \u00a72k inputs . The typk inputs . As we ak inputs  and why k inputs .In line with evidence from cognitive psychology  and the We have also neglected fine temporal dynamics. Such dynamics play a major role in dorsal MEC. We expect that phase precession of grid cells occurs, because the directed propagation of excitation through the grid patch accelerates grid cells relative to theta in the running direction. This issue needs further investigation, however.3.We have described a circuit model for grid cell activity. Based on three isomorphism assumptions , a connectivity scheme for layer 2 grid cells is derived. In this scheme, intrinsic propagation speed in the patch is the neural analogue to spatial scale and is thought to differ roughly 10-fold between dorsal and ventral entorhinal cortex. Head direction input is thought to specify the direction of propagation of activity, but the details of this process are not worked out. We extend the theory to neurons between grid patches and predict the location and discharge properties in these neurons.(a)We try to explain the anatomy and the spatial discharge properties in layer 2 of MEC. We did not consider temporal dynamics, the anchoring of the grids to the environment and spatial memory. This account is an explicitly neural/microcircuit hypothesis. The neural nature of the theory implies that it predicts in great detail experimental results. We predict that there is an isomorphism between neural and external two-dimensional space in grid patches. This prediction is in conflict with experimental observations on grid cells in extracellular recordings, which did not show a systematic progression of grid phase in recording tracks . We do n(b)The core idea of this paper is that the \u2018grid in the world\u2019 (evident in grid cell discharges) is generated by a \u2018grid in the head\u2019 . Isomorphic neural representations are evident in the retinotopy of the mammalian visual cortex and the eye-catching representations of body parts such as whiskers  or nose (c)In recordings from the MEC, grid spacing does not appear to increase continuously from dorsal to ventral entorhinal cortex, but it seems to increase in discrete steps ,5. We su(d)Grid cells are also found in other layers  of MEC  as well Third, we see multiple possibilities of how grids could be propagated to the pre- and parasubiculum. It may be the case that these structures also simply inherit grid responses from layer 2 of MEC, but again it is unclear whether the connectivity to accomplish such a transfer of grid properties is present  and the (e)The proposed isoposition connectivity between patches is very similar in phenomenology to the patchy iso-orientation connectivity in visual cortex . The micRecent intracellular recording ,48 studiThe fact that we approach grid cell discharge from an anatomical perspective differentiates our hypothesis from previous models . Thus, b"}
{"text": "Grid cells in the medial entorhinal cortex (mEC) and place cells in the hippocampus are paradigms for population coding of spatial information . Both thIn order to understand the relative contributions of grid cells and non-spatial inputs in determining place field size and stability, we propose a computational model of the hippocampal-entorhinal network that includes a modular organization of grid cell inputs arranged in order of increasing spatial scale, as is seen experimentally in the mEC. Our underlying place cell model is a firing-rate based model inspired by previous work , in whicOur main findings suggest that:1.) For a wide range of parameters, the relative amounts of spatial and non-spatial inputs to place cells plays a more important role in determining place field size and stability than the spatial scale of grid cell inputs. This implies that the dorso-ventral gradient in place field size may reflect a dorso-ventral gradient in non-spatial inputs, rather than grid scale, and is agreement with prior suggestions of a functional distinction between the dorsal and ventral regions of the hippocampus .2.) In our model, place fields are sensitive to changes in the firing rates of the grid vertices of individual grid cells, emphasizing the potential implications of this grid field heterogeneity for place field formation and stability."}
{"text": "Medial entorhinal grid cells and hippocampal place cells provide neural correlates of spatial representation in the brain. A place cell typically fires whenever an animal is present in one or more spatial regions, or places, of an environment. A grid cell typically fires in multiple spatial regions that form a regular hexagonal grid structure extending throughout the environment. Different grid and place cells prefer spatially offset regions, with their firing fields increasing in size along the dorsoventral axes of the medial entorhinal cortex and hippocampus. The spacing between neighboring fields for a grid cell also increases along the dorsoventral axis. This article presents a neural model whose spiking neurons operate in a hierarchy of self-organizing maps, each obeying the same laws. This spiking GridPlaceMap model simulates how grid cells and place cells may develop. It responds to realistic rat navigational trajectories by learning grid cells with hexagonal grid firing fields of multiple spatial scales and place cells with one or more firing fields that match neurophysiological data about these cells and their development in juvenile rats. The place cells represent much larger spaces than the grid cells, which enable them to support navigational behaviors. Both self-organizing maps amplify and learn to categorize the most frequent and energetic co-occurrences of their inputs. The current results build upon a previous rate-based model of grid and place cell learning, and thus illustrate a general method for converting rate-based adaptive neural models, without the loss of any of their analog properties, into models whose cells obey spiking dynamics. New properties of the spiking GridPlaceMap model include the appearance of theta band modulation. The spiking model also opens a path for implementation in brain-emulating nanochips comprised of networks of noisy spiking neurons with multiple-level adaptive weights for controlling autonomous adaptive robots capable of spatial navigation. How our brains acquire stable cognitive maps of the spatial environments that we explore is not only an outstanding scientific question, but also one with immense potential for technological applications. For example, this knowledge can be applied in designing autonomous agents that are capable of spatial cognition and navigation in a GPS signal-impoverished environment without the need for human teleoperation.Lesion and pharmacological studies have revealed that hippocampus (HC) and medial entorhinal cortex (MEC) are critical brain areas for spatial learning, memory, and behavior Since the time of the proposal of Sensory context includes properties of the following kind: The primary determinants of grid cell firing are, however, path integration-based inputs Along similar lines, Discussion section reviews how hexagonal grid structures can be learned in the brain even when stripe cells of multiple spacings converge initially on entorhinal cells Why do grid cells learn to fire at hexagonally-located positions as an animal navigates in an open field? Most computational models focused on learning of either hippocampal place cells The original GridPlaceMap model uses neurons that interact using rate coding; that is, they interact via signals based on spiking frequency, rather than in terms of their individual spike trains. The goals of the current model are threefold; namely, to test whether the insights gained from the rate-based GridPlaceMap model can be applied and extended to simulating and explaining the development of spiking grid and place cells, as an instantiation of a general method for converting rate-based adaptive neural models, without the loss of any of their analog properties, into models whose cells obey spiking dynamics; to develop a neural system that makes it possible to address, for the first time, known temporal coding properties of hippocampal place cells and medial entorhinal layer II grid cells, such as theta band modulation Additional extensions of the GridPlaceMap and sGridPlaceMap models will be needed to achieve a general-purpose neural architecture for spatial navigation. It has, for example, been proposed how top-down attentive matching processes from hippocampal to entorhinal cortex may facilitate fast learning and dynamic self-stabilization of learned spatial memories, provide a pathway whereby environmental cues may modulate properties of grid and place cells that arise through path integration, and may help to explain a wide range of additional data about modular grid orientations, grid realignment, place remapping, and gamma and beta oscillations e.g., .A receptors, and whose synaptic plasticity is governed by a spike timing-dependent variant of the competitive instar learning law The spiking GridPlaceMap model, called sGridPlaceMap see , employs+ channels and delayed rectifier K+ channels that underlie the generation of stereotypical spike waveforms in membrane potential dynamics, synaptic transmission delays, axonal conduction latencies, and refractory periods are not considered. GABAA-gated channel conductances are approximated by single exponentially decaying traces because their rise times are typically negligible  via voltage-dependent Ca2+ channels, and an EPSP mediated by NMDA receptors, respectively The adaptive weights, These variables are initialized to zero at the start of each trial. The weights are only initialized once, at the start of the first trial, by sampling from a uniform distribution between 0 and 0.1. The learning law in A channel conductance gate that is opened by the spiking of the The membrane potential A channel conductance gate For this stage too, all gates are initialized to zero, and all membrane potentials are initialized to The adaptive weights, These variables are also initialized to zero at the start of each trial. The pre-learning weights are sampled from a uniform distribution between 0 and 0.03. The initial weights of projections from stripe cells to MEC cells have a higher individual mean to compensate for the relatively lower number of input cells; see below.The parameter values used in the simulations were The model was run with 100 HC map cells receiving adaptive inputs from three distinct populations of 100 MEC map cells, each of which was driven by adaptive inputs from stripe cells of one of three spacings. Stripe cells were activated in response to linear velocity estimates derived from realistic rat trajectories of \u223c10 min in a 100 cm\u00d7100 cm environment  from its adaptively smoothed firing rate map. Distinct fields were indentified from circular templates around local peaks based on the criteria that the maximal rate within a field is at least more than 50% of the overall peak rate 16 points) whose amplitude response is squared, and normalized to the maximal value, to yield the power spectrum between 0 Hz and 250 Hz.Temporal modulation in the spiking responses of cells was assessed by computing the power spectra of the corresponding spike trains, with a temporal resolution of 2 ms, using a standard procedure This section shows that all the results of the rate-based GridPlaceMap model ly Data: . Both arer Data: . While tsquare box of 100 cm\u00d7100 cm , each human brain consumes just about 20 W of power. This power budget contrasts dramatically with that required  to run the most advanced supercomputer in the world; namely, the Blue Gene/Q at the Lawrence Livermore National Laboratory in Livermore, CA. Moreover, such advanced supercomputers occupy a lot of physical space, and need to be explicitly programmed for each specific task that they are supposed to perform. Aggressive efforts are currently underway across the world to develop a fundamentally new class of computer chips that closely mirror biological brains to herald the arrival of a transformative new technological field of natural intelligence. With respect to sGridPlaceMap model computations, the spiking competitive instar learning law described in This form reveals a single inhibitory term  currents mentioned above, and the m-current IhIh is known to generate subthreshold MPOs The spiking grid and place model developed in this article makes it feasible to ascertain more directly the relative contributions of and interactions among different synaptic currents in setting up the spatial scale topography of grid cells, and to probe them further computationally; namely, the AHP, leak K"}
{"text": "Drinking or local application of human or animal urine for medicinal purposes has been practiced all over the world for millennia. Documented prescriptions in Europe originate from ancient Egypt, Greece and Rome. While many of the advances of antique medicine were forgotten after the fall of the Roman Empire, the use of urine and other excrements enjoyed continued popularity in mediaeval times. Ancient Indian yogic texts and ancient Chinese documents describe benefits of drinking one\u2019s own urine, and it can be assumed that people in Africa, the Americas and other parts of the world have traditionally used urine for various medical indications for a very long time, too.The almost 100,000 hits of the search for \u201curine therapy\u201d on Google, and the over 150 videos on the subject on YouTube are an indicator that drinking \u201cwaters out of thine own cistern\u201d is still, or again, rather popular today.th century application of cow\u2019s urine in the Apoz\u00e8me Suisse for oedema or ascites. Urine is sterile where it is produced in the kidney, but once it has left the body, it is usually contaminated. It is not toxic per se. There may be rare situations where urine is the cleanest liquid at hand to pour over a dirty wound, or the only liquid to drink when buried under a collapsed building or lost at sea for days, but most of the time there are better or tastier ways to improve one\u2019s health.The supposed indications for urine therapy, ancient or contemporary, are too numerous to recite. There is, it seems, virtually nothing urine won\u2019t cure. Modern proponents use pseudoscience to explain the benefits of the various, mostly exaggerated, components of urine. Some hint at a conspiracy by the medical establishment and the pharmaceutical industry to keep the knowledge of the many fantastic healing properties of cheaply available urine a secret. There is no money to be made from urine, well, unless one was to write a book about its many virtues. But, seriously, what do we really know? Urine is mostly water, lots of urea (25g/d) and uric acid (1g/d), creatinine (1.5g), various electrolytes , phosphate and organic acids (3g/d), only trace amounts of proteins , varying traces of (not necessarily active) hormones, glucose and water-soluble vitamins. Urea has a known potent diuretic effect which is at the base of the 19This said, the situation described in the paper by Ogunshe, Fawole and Ajayi in this journal , is quit"}
{"text": "This paper proposes an interactive method of model clipping for computer-assisted surgical planning. The model is separated by a data filter that is defined by the implicit function of the clipping path. Being interactive to surgeons, the clipping path that is composed of the plane widgets can be manually repositioned along the desirable presurgical path, which means that surgeons can produce any accurate shape of the clipped model. The implicit function is acquired through a recursive algorithm based on the Boolean combinations (including Boolean union and Boolean intersection) of a series of plane widgets\u2019 implicit functions. The algorithm is evaluated as highly efficient because the best time performance of the algorithm is linear, which applies to most of the cases in the computer-assisted surgical planning. Based on the above stated algorithm, a user-friendly module named SmartModelClip is developed on the basis of Slicer platform and VTK. A number of arbitrary clipping paths have been tested. Experimental results of presurgical planning for three types of Le Fort fractures and for tumor removal demonstrate the high reliability and efficiency of our recursive algorithm and robustness of the module. Computer-assisted surgical planning, a technique integrating the computer technology in presurgical planning and navigation, has been used to improve the safety and accuracy of the surgery , 2]. Or. Or2]. OMany softwares have been developed for computer-assisted surgical planning. For example, Magic Rapid Prototyping(or Magic RP), used for 3D imaging reconstruction , 6], is, is6], iOur research is related to the work of both mesh segmentation and polygon clipping algorithms in computer graphics. Firstly, a number of algorithms for mesh segmentation have been proposed to separate the semantically meaningful regions, such as the method of K-mean clustering , spectraSecondly, several polygon clipping algorithms are developed to visualize the model inside the clipping window. Such efforts include Sutherland-Hodgman algorithm, Weiler-Atherton clipping algorithm, Vatti clipping algorithm and so on. Sutherland-Hodgman algorithm obtains the intersection of the subject polygon and clip polygon  19] by  by 19] bIn this paper, an interactive method is presented for surgeons to make computer-assisted surgical planning. A module named SmartModelClip is developed on the basis of 3D-slicer , leveragThis paper is organized as follows: In section 2,we describe the recursive algorithm for resolving the implicit function and provide three different types of examples to implement the algorithm. In section 3, we develop a module named SmartModelClip and analyze the robustness of the algorithm. In section 4, we discuss the time performance of the clipping algorithm and compare it with some other polygon clipping algorithms. Our mesh segmentation method is then compared with other methods in terms of segmentation time. And finally conclusions are made in the last section.Our approach to the acquirement of the clipping path\u2019s implicit function is on the basis of a recursive algorithm. The recursive algorithm decomposes the problem into three subproblems according to the relative poses of plane widgets. The subproblems are combined through Boolean operations. The kind of Boolean operations is determined by the pose of each line segment of polyline on the transection plane of clipping path.F = 0, which separates the 3D space into three regions: on the plane = 0), the region inside the plane<0) and the region outside the plane>0). A plane widget has the handle of normal axis fixed on the center of the plane widget, such as the handle AB in Some notions about implicit function is first introduced. A plane widget is on the plane defined by the implicit equation of AB and vector OMi:Seeing from Comparing with \u03b11 \u222a \u03b12 means the Boolean union of the implicit function of plane widget \u03b11 and that of \u03b12, while \u03b11\u2229\u03b12 means the Boolean intersection of the implicit function of plane widget \u03b11 and that of \u03b12. \u03b11\u222a\u03b12 and \u03b11\u2229\u03b12. In \u03b11, while region \u2461 and region \u2462 are inside \u03b12. After the Boolean operation of \u03b11\u222a\u03b12, all the colored regions are inside \u03b11\u03b12. In \u03b11, while region \u2460 and region \u2461 are inside \u03b12. The Boolean operation of \u03b11\u2229\u03b12 makes the region \u2462 inside the \u03b11\u03b12.Two kinds of Boolean operations are used in the recursive algorithm to combine the implicit functions: Boolean union and Boolean intersection. Boolean union takes the minimum value of all implicit functions while Boolean intersection takes the maximum value of all implicit functions. For the concision of expression, Boolean union and Boolean intersection are denoted respectively by the commonly used notation of \u222a and \u2229 in the set theory. To be specific, It is noticed that the result of the Boolean operations of two implicit function is still an implicit function that the Boolean operations can still be applied to.The problem of splitting 3D space by clipping path can be de degraded into the equivalent problem of splitting 2D space by a polyline on the transection plane(perpendicular to the first and second plane widget) of the clipping path. Thus the algorithm of acquiring the clipping path\u2019s implicit function is implemented by judging the relative pose of the line segments. These line segments determine what kind of Boolean operations applies to the implicit function of each plane widget.\u03b2, forming a polyline PiPi+1Pi+2Pi+3 that\u2019s on the edge of \u03b1i\u03b1i+1\u03b1i+2. The transection plane is specified to be perpendicular to the plane widget \u03b11 and \u03b121: ifi = = jthen2: \u2003\u03b1i3: \u2003\u2003return else if (j \u2212 i) == 1 then4: \u2003ifPj+1 inside \u03b1ithen5: \u2003\u2003\u03b1i\u22c2\u03b1j6: \u2003\u2003\u2003return else7: \u2003\u2003\u03b1i\u22c3\u03b1j8: \u2003\u2003\u2003return end if9: \u2003\u2003else10: \u2003k \u2190 j11: \u2003\u2003whileLoop Conditionline26th)16: \u2003\u2003\u2003\u03b1k, \u03b1j)17: \u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u22c2 CombImpFuns19: \u2003\u2003\u2003\u03b1k, \u03b1j)20: \u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u22c3 CombImpFuns(end if21: \u2003\u2003CombinedFun22: \u2003\u2003return end if23: \u2003end function24: 25:Loop Condition:26: linePkPk+1 and polyline Pk\u2212127: i + 2 \u2a7d k \u2a7d j)28: intersect(for orrayPk\u22121Pk and polyline 29: i + 2 \u2a7d k \u2a7d j \u2212 2)30: intersect  is extended to infinitive on the both directions. k is initialized with the value of j. For the while-loop at line 12th, we check respectively whether line PkPk+1(i + 2 \u2a7d k \u2a7d j) and polyline Pk\u22121 intersect or the ray Pk\u22121Pk(i + 2 \u2a7d k \u2a7d j \u2212 2) and polyline k is declined by 1. The while-loop is broken if any one of the conditions is satisfied: (1)k < i + 2, (2)line PkPk+1 and polyline Pk\u22121Pk and polyline \u03b1k\u22121 and \u03b1k. If point Pk+1 on the plane \u03b1k is inside the plane widget \u03b1k\u22121, we apply the Boolean operation of \u03b1i\u03b1i+1 \u22ef \u03b1k\u22121 \u2229 \u03b1k\u03b1k+1 \u22ef \u03b1j. Or else we apply the Boolean operation of \u03b1i\u03b1i+1 \u22ef \u03b1k\u22121 \u222a \u03b1k\u03b1k+1 \u22ef \u03b1j. The same algorithm applies to the implicit function of \u03b1i\u03b1i+1 \u22ef \u03b1k\u22121 and that of \u03b1k\u03b1k+1 \u22ef \u03b1j. In this way, the problem has been decomposed and will finally be simplified into the subproblem1 and subproblem2.Subproblem3 first extends respectively line segment of Three different types of examples that implement the algorithm are given below to have a better understanding of the whole algorithm. Specifically, the while loop condition is explained in detail. Although all the examples contain 4 plane widgets, the poses of the plane widgets are slightly distinct and the implementation process is totally different.\u03b11 and plane widget \u03b12, leaving the polyline P1P2P3P4P5 on the edge. The shaded space in \u03b11\u03b12\u03b13\u03b14.For the first example in \u03b11\u03b12\u03b13\u03b14, line segment P2P1 in P2P1 and it becomes the ray P4P5 is extended to infinite on both directions. Since line P4P5 and polyline Q1, the value of k(3 = i + 2 \u2a7d k \u2a7d j = 4) should be decreased to 3. Then we check whether line P3P4 and polyline Q2. Therefore, the value of k should be decreased again to be 2. As the required minimum value of k is 3, seeing from line 28th of the algorithm, the loop condition is not satisfied after this decrement of k. P3 is outside plane widget \u03b11, so it\u2019s easy to get the implicit function of \u03b11\u03b12\u03b13\u03b14 on condition that the implicit function of \u03b11 and that of \u03b12\u03b13\u03b14 are known respectively because the implicit function of \u03b11\u03b12\u03b13\u03b14 is the result of \u03b11 \u222a \u03b12\u03b13\u03b14. Region \u2460 and region \u2461 are inside the implicit function of \u03b11, while region \u2461 and region \u2462 are inside the implicit function of \u03b12\u03b13\u03b14 in \u03b11 \u222a \u03b12\u03b13\u03b14 makes the regions of \u2460, \u2461 and \u2462 inside the implicit function of \u03b11\u03b12\u03b13\u03b14.To get the implicit function of \u03b11 is the solution of subprolem1, the task is to get the implicit function of \u03b12\u03b13\u03b14. As P4P5 and extended polyline Q3 in k(4 = i + 2 \u2a7d k \u2a7d j = 4) is decreased to 3. So it comes out of the loop for k = 3. As P4 is outside plane widget \u03b12, the implicit function of \u03b12\u03b13\u03b14 is available through the Boolean operation of \u03b12 \u222a \u03b13\u03b14. Region \u2460 and region \u2461 are inside the implicit function of \u03b12, while region \u2461 and region \u2462 are inside the implicit function of \u03b13\u03b14 in \u03b12 \u222a \u03b13\u03b14 makes the regions of \u2460,\u2461 and \u2462 inside the implicit function of \u03b12\u03b13\u03b14.Now that the implicit function of \u03b12 is the solution of subproblem1 and the implicit function of \u03b13\u03b14 is the solution of subproblem2, so the whole problem is solved by the Boolean combinations of implicit functions of all the plane widgets. In conclusion, the implicit function of \u03b11\u03b12\u03b13\u03b14 is available through the following equation:The implicit function of \u03b14 to that of \u03b13, from which we\u2019ll see that different poses of plane widgets lead to different expressions of the implicit function yet using the same recursive algorithm.For the second example in P4P5 and polyline Q1; line P3P4 and polyline Q2 in \u03b11\u03b12\u03b13\u03b14 is to apply the Boolean operation of \u03b11 \u222a \u03b12\u03b13\u03b14 as is illustrated in Line P4P5 and polyline \u03b12\u03b13\u03b14 is to apply the Boolean operation of \u03b12\u03b13 \u2229 \u03b14 because point P5 is inside \u03b13. Region \u2460 and region \u2461 are inside the implicit function of \u03b12\u03b13, while region \u2461 and region \u2462 are inside the implicit function of \u03b14. Boolean operation of \u03b12\u03b13 \u2229 \u03b14 makes the region of \u2461 inside the implicit function of \u03b12\u03b13\u03b14. The implicit function of \u03b12\u03b13 is the solution of subproblem2, while the implicit function of \u03b14 is the solution of subproblem1, making it possible to solve the whole problem.Seeing that line In conclusion, the solution to the implicit function of this series of plane widgets can be expressed as follows:P4P5 and polyline Q1 intersect. Then we decrease the value of k and find that line P3P4 and ray P2P3 and polyline Q2, which satisfies the loop condition at line 29th in our algorithm. Thus, the value of k should still be decreased again and the implicit function of \u03b11\u03b12\u03b13\u03b14 equals to \u03b11 \u222a \u03b12\u03b13\u03b14. The implicit function of \u03b12\u03b13\u03b14 is the same with that in example 2. So the implicit function of \u03b11\u03b12\u03b13\u03b14 is expressed as:For the last example in These three simple examples illustrate the implementation of algorithm. In fact, the correctness of this algorithm has been widely tested, not merely the clipping path with four connected plane widgets, which will be discussed in the next section.A module named SmartModelClip, served as computer-assisted surgical planning, is developed to clip the model on the basis of 3D Slicer and VTK using our recursive algorithm. The results of model clipping demonstrate the robustness of the algorithm.3D Slicer(or Slicer) is a open-source and cross-platform software that processes the analysis and visualization of biomedical images . The funOur module is developed on the platform of 3D-Slicer, making use of VTK and Qt as visualization and GUI toolkits. Le Fort fractures are common in facial trauma. The clipping path can be more complex than the previous situation. In The arrows in The clipping processes fit the reality in computer-assisted surgical planning because the yellow, green and red part of the model are clipped in sequence on a clipping path. What\u2019s more, the boundary of the clipping path in The thickness plane allows surgeons to clip model with certain thickness. Limitation in constructing the clipping path. Our recursive algorithm proves to be pretty reliable and robust for most experimental cases except for Moebius clipping path, however, the limitation may still exist in the construction of the clipping path. It\u2019s noticed that our clipping algorithm is restrained to a series of plane widgets connected from the beginning to the end because the polyline referred in the section \u2018Conversion of 3D Problem to 2D Problem\u2019 is the judgment of the type of Boolean operations applied to each plane widget. The algorithm is able to obtain the implicit function of three connected plane widgets in Another limitation in the construction of the clipping path, is the case of complex clipping path in For another case that our clipping algorithm may not work is the clipping path tangent to Moebius strip because Moebius strip is a surface with only one side. However, this kind of case may not appear in the computer-assisted surgical planning. So our algorithm still meets the need of the most clinical applications.Limitation of thickness plane. As the back side of the tumor is unobservable, we only consider using one thickness planewidget to cut the tumor. But the curved surface of the back of the tumor to be clipped sometimes could be very complex, for example, it can be seen in the The time performance of the clipping algorithm for each case is first discussed and then the algorithm is evaluated based on the model clipping experiments, which demonstrates the time performance analysis of the clipping algorithm.The time performance of the recursive algorithm depends on the number of plane widgets and their relative poses. The best case runs as fast as the linear algorithm, while the worst case runs as slowly as the sub-quadratic algorithm. But the average case has the logarithmic time performance. For most cases of the computer-assisted surgical planning to remove the tumor, our clipping algorithm has the best time performance or the average-case performance.\u03b1j \u222a \u03b1i\u03b1i+1\u22ef\u03b1j\u22121 or \u03b1j \u2229 \u03b1i\u03b1i+1\u22ef\u03b1j\u22121 in each recursion. Any case of the convex clipping path will have the best-case performance. \u03b11\u03b12\u03b13\u03b14 can be expressed respectively by\u03b11\u03b12\u03b13\u03b14.The best case happens when the condition of the while-loop (from line 27th to line 30th) in our algorithm is not satisfied all through the stage of recursion. In that case, the recursive function will only apply Boolean operation of T(N) is the time of acquiring the implicit function of N plane series, T(N \u2212 1) is the time of solving the subproblem3. Since the recursion just returns when N equals to 1 or 2, T(2)=T(1) = 1. So the above recurrence has the solution ofAs the problem is only decomposed into the subproblem1 and subproblem3 each time and the loop condition is never satisfied, the time complexity can be calculated by the sum of the time of solving subproblem1, subproblem3 and the time of the Boolean operation of the result of subproblem1 and subproblem3, which can be expressed in the following equation:j \u2212 i \u2212 1 times in each recursive call. The worst case behavior occurs in the situation that the condition inside the loop (at line 15) of the pseudo code is satisfied and the loop is executed at N plane widgets, the time complexity can be calculated by the sum of three parts. The first part is the time to go through the loop for N \u2212 1 \u2212 1 times, the second part is the time to solve the subproblem3 and the last part is to take Boolean operations of the implicit functions. Thus the time complexity can be expressed in the equations ofT(1) = T(2) = 1.For the implicit function of This recurrence has the solutionN plane widgets is the average time to execute the two recursive functions plus the time to do the loop and the time to apply the Boolean operations of the two recursive functions. The implicit function of \u03b11\u03b12\u22ef\u03b1N is decomposed into the Boolean operation of the implicit function of \u03b11\u03b12 \u22ef \u03b1i and that of \u03b1i+1\u03b1i+2\u22ef\u03b1N. And the times to do the loop is N \u2212 (i + 1). So the time complexity of the average-case performance can be expressed as:T(1) = T(2) = 1.As to the case of average time performance, we can consider the every possible looping times in the subproblem3 and take their average. If the possibility of the times that the loop will be executed is equal, then the average time to split the space with The solution of this recurrence isO(N log N).Consequently, the average time complexity of this algorithm is O(M*N), in which M stands for the number of edges in the clipping polygon and N stands for the number of vertex pairs in the subject polygon. And Vatti clipping algorithm is twice as fast as Sutherland-Hodgman Algorithm and substantially faster if the algorithm is used for both clipping and filling [O(N2), the average time complexity of the algorithm is O(N log N) and the best time complexity of O(N). In fact, as the clipping path is defined manually according to the boundary of something like tumor, the number of the clipping path widgets would not be very large(let\u2019s say 15 or 30). In that case, the time complexity of our algorithm is approximately linear, making it possible to separate the 3D space along the clipping path in a very short time.In comparison with other polygon clipping algorithms, such as Sutherland-Hodgman Algorithm and Vatti clipping algorithm, our clipping algorithm has better time complexity in the computer-assisted surgical planning. For the general case, Sutherland-Hodgman Algorithm has the time complexity of  filling . AlthougThe time to clip the model is actually composed of two parts: the first part is the time of acquiring the implicit function of the clipping path by our algorithm and the second part is the time to clip the triangles of the model by the class of \u2018vtkClipPolydata\u2019 in VTK. These two parts of time are tested respectively. The execution time of the recursive algorithm is tested using different clipping paths that have different number of plane widgets, while the second part of time performance is tested by clipping the models that have different number of triangles using the same clipping path. The experiments are tested on the laptop with the Intel CPU T1400 @1.73GHz and 2GB DDR2 RAM.We evaluate the time performance by clipping the tumor in In terms of the segmentation time, our method has an advantage over Random Walks Algorithm and fitting primitives Algorithm. Since the time to separate the model relates both to the computation speed of CPU and the complexity of the model, we only make the rough comparison of each method\u2019s average segmentation speed, in which the CPU speed is almost the same with each other.The timing performance of various models mentioned in TABLE 1 of  shows thIn this paper, a recursive algorithm is developed for clipping models based on the Boolean combination of the implicit functions of the series of the connected plane widgets. The problem of splitting 3D space by clipping path is degraded into the equivalent problem of splitting 2D space by a polyline. The recursive algorithm then decomposes the problem of splitting 2D space into three subproblems that makes the whole problem easy to solve. Three examples of four connected plane widgets illustrates the implementation process of the algorithm.A module named SmartModelClip is developed for computer-assisted surgical planning using our recursive algorithm. Three experiments of model clipping for presurgical planning has successfully demonstrated the robustness of the module: (1)presurgical planning with simple clipping path for clipping Le Forts fractures, (2)presurgical planning with complex clipping path that can be twisted from one view to another for clipping the model into several parts, (3)presurgical planning with thickness plane for tumor removal. The limitations in the construction of the clipping path and thickness plane widget are discussed in details.O(N) and the time complexity of the worst-case performance is O(N2), while the time complexity of the average-case performance is O(N log N). The algorithm tends to have linear time performance when the removed part is enclosed by the clipping path. Rough comparison with other polygon clipping algorithms shows the efficiency of our algorithm in the presurgical planning.After that, the time performance of the algorithm is analyzed. The time performance is relevant to the poses of the series of plane widgets. The time complexity of the best-case performance is The time performance of model clipping and the execution time of our algorithm to acquire the implicit function of clipping path are both evaluated highly efficient in two tests: (1)the test of time to clip different models that have different number of triangles with the same clipping path, (2)the test of time to clip the same model with the clipping paths that have different number of plane widgets. Compared with other segmentation methods, our method has an advantage over these methods in terms of the average segmentation time.From O(N log N).We want to prove that the N \u2212 1) on both sides of the N with N \u2212 1, we obtain the recurrence ofN(N \u2212 1), we obtainMultiply (1N-2T(i).Subtract1N-1T(i).Substitu"}
{"text": "This study assessed the microbial quality of clay samples sold on two of the major Ghanaian markets.The study was a cross-sectional assessing the evaluation of processed clay and effects it has on the nutrition of the consumers in the political capital town of Ghana. The items for the examination was processed clay soil samples.Staphylococcus spp and fecal coliforms including Klebsiella, Escherichia, and Shigella and Enterobacterspp were isolated from the clay samples. Samples from the Kaneshie market in Accra recorded the highest total viable counts 6.5 Log cfu/g and Staphylococcal count 5.8 Log cfu/g. For fecal coliforms, Madina market samples had the highest count 6.5 Log cfu/g and also recorded the highest levels of yeast and mould. For Koforidua, total viable count was highest in the samples from the Zongo market 6.3 Log cfu/g. Central market samples had the highest count of fecal coliforms 4.6 Log cfu/g and yeasts and moulds 6.5 Log cfu/g. \u201cSmall\u201d market recorded the highest staphylococcal count 6.2 Log cfu/g. The water activity of the clay samples were low, and ranged between 0.65\u00b10.01 and 0.66\u00b10.00 for samples collected from Koforidua and Accra respectively.The clay samples were found to contain Klebsiella spp. Escherichia, Enterobacter, Shigella spp. staphylococcus spp., yeast and mould. These have health implications when consumed. The consumption of nonfood materials is a common practice in many cultures in different parts of the world. Pica is the broad term that describes this habit and it has been defined as the pathological act of eating nonfood items . A commoTraditionally, clay is used for making drinking and cooking pots with the belief that it has the ability to bind toxins. In Ghana, edible clay is called \u201ceshire\u201d by the Akan tribes, \u201cfarankese\u201d by some northern tribes, \u201cfefe\u201d by the Ewes while the Gas call it \u201cayilo\u201d. The clay soil is dug from the earth and prepared by milling the large particles and blocks to help reduce the particle size. The fine powder is then mixed with water and stirred into a uniform paste. Afterwards, the paste is molded into a sausage- like shape and then baked to reduce the moisture content and retain the shape. In some cultures the molded paste is baked and smoked to give it an appealing aroma. Both types are found commonly on the Ghanaian market. Geophagia is very prevalent among pregnant woman who are believed to develop craving for clay as a remedy for morning sickness and vomiting . This trSample collection: This study involved laboratory analysis of clay samples. Also, a survey was done in the selected markets to identify the types of clay that are sold. Microbiological analysis was carried out on the clay samples that were purchased from the selected major markets in Accra and Koforidua. The markets selected in Accra were; Madina, Makola and Kaneshie markets. For Koforidua, Zongo market, Dwakesiem  and Dwaketoam  were selected. These represented the Greater Accra and Eastern Regions of Ghana. In each of the markets, the samples were bought from different retailers. A quick market survey was done at the selected major markets in Accra  to identify the different types of clay samples sold on the market. The various types of clay samples available on the markets were examined by looking at the differences in the shapes, colors, sizes and their purpose. The simple random sampling method was used for both the survey and laboratory analysis. The clay samples were collected with stomacher bags and sealed with cello tape and properly labeled with the market it was bought from. Samples were handled with care and treated under aseptic conditions to avoid any contamination. At each market, 20 pieces of the clay were purchased randomly from different retailers and packaged into sterile stomacher bags. The stomacher bags were then carefully sealed with cello tape and labeled appropriately by the names of the specific markets they were bought from.Microbiological analyses: The bacteria yeast and molds present in the samples were enumerated using specific nutrient media. Series of morphological examinations and biochemical tests were conducted to help characterize and identify the specific microorganisms that were growing on the clay samples. Details of these procedures are described as follows; dehydrated media  were used in the analysis. The media used were: Nutrient Agar (NA) (to help in the acquisition of a pure culture), Levine Eosin Methylene Blue (EMB) (to determine the presence of coliforms), Mannitol Salt Agar (MSA) (to detect staphylococcus species), Plate Count Agar (PCA) , Yeast Extract Agar (YEA) (for detection of yeasts and molds), and Peptone Water (used as a diluent). They were prepared according to the manufacturer's instructions. For enumeration and characterization of microorganisms, samples from each market were aseptically homogenized by crushing and about 10 grams aseptically transferred into new stomacher bags and blended with 90 ml of sterile diluent (maximum recovery diluents), in a Seward stomacher blender 80  for 1 minute. Aliquots of the homogenates were serially diluted and plated for the determination of total plate count and the detection of coliforms, staphylococcal spp. and fungi, using standard procedures. For the identification and characterization of the microorganisms in the samples, some morphological and biochemical examinations were done as described by Prescott, Harley and Klein [Water holding capacity: An amount of 5 grams of the powdered sample was weighed into a centrifuge tube. 30 ml of water was added and mixed using the vortex mixer for 30 seconds. The mixture was made to stand for 30 minutes and stirred after every 10 minutes until the 30 minutes was due. This was followed by centrifugation using a bench top centrifuge set at 3000 rpm for 15 minutes. The supernatant was decanted and the weight of the residue was recorded. The amount of water absorbed, expressed as a percentage of the dry sample is equivalent to the gram of water per gram of sample that it could hold. Triplicate measurement was taken for each sample.Percent water holding capacity = weight of hydrated sample-weight of sample X 100 weight of dry sampleWater activity: The water activity of the clay samples was determined using the Retronic hycropalm water activity meter . An amount of 2 grams of the powdered sample was weighed into clean dishes. The dish was then placed into the panel of the water activity meter. The probe was then used to cover the dish containing the sample. The start button of the meter was pressed and the water activity reading was recorded after about 2 minutes when the reading had stabilized. This was done in triplicates for each sample and the average was taken.Moisture content: The air-oven method was used for this determination. The temperature of the oven was adjusted to 105 \u00b0C0 C to precondition the moisture cans for about 15 minutes. Afterwards, the moisture cans were removed and made to cool in a desiccator. Approximately 2 g of the powdered clay samples was weighed into the cans and were transferred into the oven. The sample was left overnight to allow the moisture in it to be evaporated. After 24 hours the final weight of the sample was taken and the moisture content of the sample was calculated on dry mater basis. This was repeated thrice for each of the samples average was taken.Percent Moisture content = weight of hydrated sample-weight of sample X 100 weight of dry sampleData processing and analysis: For microbiological analysis, the data was entered into Microsoft Excel (2010). Means and standard deviations were computed and graphical representations were used where appropriate.The clay samples from both Accra and Koforidua had lower moisture content thus 1.46\u00b10.30% for the Accra samples and 1.86\u00b10.24% for the Koforidua samples. The water activity of the samples were also low thus 0.66\u00b10.00 and 0.65\u00b10.01 for the Accra and Koforidua samples respectively. In another study, the moisture content of clay samples was observed to be between 0.18-0.19% when the air oven method was used . The valAnother genus of bacteria that were detected was staphylococcus spp. The detection of Staphylococcus spp also pointed to the unhygienic handling practices. Species of Staphylococcus are known food toxicants. Their presence could therefore implicate that there was some cross- contamination of the clay samples. Most spoilage bacteria, yeasts and molds require a minimum water activity of 0.9, 0.88 and 0.8 respectively . The yeaHence, pica patients consume a less varied diet and are at risk of iron deficiency anemia. Additionally, kaolin which is a common ingredient in medications to treat diarrhea, may cause constipation in high amounts. Geophagia may cause hyperkalemia because clay can bind to potassium in the intestine and lead to increased excretion. The most dangerous of the complications of Geophagia is probably lead poisoning . Lead exThe clay samples were found to contain Klebsiella spp. Escherichia, Enterobacter, Shigella spp. staphylococcus spp. yeast and mould. These findings calls for health promotion educational campaigns to halt consumptions of ayilo and related materials as this habit is detrimental to the health of particularly pregnant women and their unborn babies.The prevalence of geophagia has been studied largely and has been found to be highest among pregnant women than the non-pregnant women and men;Studies have shown that these clay samples consumed could lead to heavy metal contamination and lead to micronutrient deficiencies;The microbiology of these processed clay soil samples have been done in some markets in Accra, Ghana.This study further validated the microbiological quality of the clay samples in Greater Accra region and extended it to the Eastern region of Ghana;The different types  of clay samples available on the market was surveyed as well as the uses of these different types;Also the weight of the clay samples were determined in the Accra market to have an idea of how much clay can be consumed."}
{"text": "Disruption of riverine connectivity by artificial structures, such as culverts, can obstruct critical fish movements. We investigated the effectiveness of replacing smooth substrates with rough, naturalistic substrates (i.e. river stones) in improving fish swimming capacity and modelled fish passage through culverts. Ucrit) of two small-bodied fish, purple-spotted gudgeon  and crimson-spotted rainbowfish  were examined. Swimming trials were conducted in a hydraulic flume fitted with either a smooth acrylic substrate (control) or a rough substrate with fixed river stones. Swimming performance was improved on the rough compared to the smooth substrate, with Mo. adspersa  and Me. duboulayi  both experiencing a 26% increase in relative Ucrit. Traversable water velocity models predicted maximum water speeds allowing successful upstream passage of both species to substantially increase following roughening remediation. Together these findings suggest culvert roughening may be a solution which allows hydraulic efficiency goals to be met, without compromising fish passage.Worldwide declines in riverine fish abundance and diversity have been linked to the fragmentation of aquatic habitats through the installation of instream structures . Restoring riverine connectivity can be achieved by remediating structures impeding fish movements by, for example, replacing smooth substrates of pipe culverts with naturalistic substrates . However, empirical evaluations of the efficacy of such remediation efforts are often lacking despite the high economic cost. We assessed the effectiveness of substrate roughening in improving fish swimming performance and linked this to estimates of upstream passage success. Critical swimming speeds ( Disruption of riverine connectivity is one of the leading threats to the persistence of riverine fishes ; whereasFish passes have been developed to facilitate fish movements around instream barriers, but a comprehensive set of conditions conducive to optimizing passage  is unavailable for many species. Research in this area has predominantly focused on enabling fish to bypass large obstructions such as dams and weirs  on fish movement is estimated to be greater than that of large dams due to their high numbers  and wereAcipenser brevirostrum , and crimson-spotted rainbowfish, Melanotaenia duboulayi . These species are sympatric and endemic to Australia, with populations spread along coastal catchments in south-east Queensland and northern New South Wales (1) swimming performance would be markedly improved over rough compared to smooth substrates, and (H2) culvert remediation models would show roughening to be an effective approach, exemplified by higher maximum water velocities allowing successful upstream passage of fish.Remediation approaches can be economically costly, with finite funds directed towards waterway restoration, deeming it imperative to ensure restoration efforts benefit target species. The aim of this study was twofold: (i) to determine if fish swimming performance is improved above rough compared to smooth substrates, and (ii) to model and evaluate the effectiveness of substrate roughening as a remediation strategy. Two small-bodied , freshwater species were used to address these aims: purple-spotted gudgeon, th Wales . Both spMelanotaenia duboulayi)  and purple-spotted gudgeon (Mogurnda adspersa)  were obtained from a commercial hatchery . Fish were housed in 45 L glass aquaria  at a stocking density of approximately 3 g (body mass) L\u22121 (Mo. adspersa) and 1 g L\u22121 (Me. duboulayi). Aquaria contained Brisbane city tap water conditioned with water primer , maintained at a constant temperature (25 \u00b1 1\u00b0C). Water chemistry  was monitored weekly to ensure water quality. Fish were fed commercially supplied food pellets  daily to satiation. The photoperiod was set to a 12-h light: 12-h dark cycle.Crimson-spotted rainbowfish . Each swimming trial incorporated one substrate treatment, either a smooth acrylic panel or a custom-made, roughened substrate with fixed river stones  and crimson-spotted rainbowfish (Me. duboulayi), respectively. The top surface area (SA) of each stone was measured using the particle analysis function in ImageJ (50) = 4.99 cm2; median diameter (D50) = 2.52 cm) (see L \u00d7 W \u00d7 H) and the swim chamber walls were made of smooth acrylic in both treatments. The substrates were detachable so treatment order could be randomized.Swimming trials were conducted in a 185 L flow-controlled hydraulic flume (Loligo cm) see . The sub\u00ae, Unanderra, AUS) and custom-built air-water manometer set to a 30\u00b0 angled incline. A 5 \u00d7 5 cross-section in the centre of the swim chamber was measured for each water velocity increment (revolutions s\u22121) on the smooth and roughened substrates. A time-averaged water velocity (m s\u22121) calibration curve was determined for each substrate. The calibration curve for the smooth and roughened substrates were described by the following equations:\u22121) for the swim chamber containing smooth and rough substrates, respectively, and \u22121). The rough substrate consistently derived lower water velocities for a given propeller speed compared to the smooth substrate between 6\u201318 revolutions s\u22121, and water velocity converged for both substrates at 21 revolutions s\u22121 , and fish were tested once to avoid training effects . Body sizes of fish were similar between rough and smooth substrate treatment groups for both species . Fish were allowed 30 min to adjust to conditions in the swimming flume with water velocity switched off (i.e. 0.00 m s\u22121), after which water velocity was increased every 5 min at increments of 0.05 m s\u22121, starting at 0.05 m s\u22121, until the fish fatigued. Fatigue was defined as the fish resting on the back wall of the swim chamber for \u2265 3 s. Total swimming time until fatigue and water velocity at fatigue were recorded to calculate critical swimming speed (Ucrit), using \u22121), \u22121), Tf is the time (s) swum in the final increment and Ucrit tests provided a measurement of the maximum velocity at which a fish can sustainably swim without fatiguing (Ucrit = 0.3 m s\u22121 \u00d7 300 s = 90 m)\u2014a distance far exceeding the length of most culverts (\u22121), and both absolute (i.e. m s\u22121) and relative critical swimming speeds (BL s\u22121) are reported. The swim chamber was constantly aerated and water temperature was maintained at 25 \u00b1 0.5\u00b0C using a submersible heater . Swimming gait was observed and classified as either direct, body-caudal fin (BCF) (\u22121), blot-dried and photographed. ImageJ  was used to measure BL for each fish. Cross-sectional body area of all fish was less than 10% of the cross-sectional area of the swimming flume chamber; therefore, corrections for the solid-blocking effect . Substrate treatment was randomly assigned to fish using a random number generator ( effects  (n = 30 atiguing  and wereculverts . Criticain (BCF)  or statiin (BCF)  where peg effect  were not\u22121) within a culvert, Ucrit), d is the length of the culvert (m) and Vs (s). The endurance was 5 min, as that was the period of time in which the fish swam for before the velocity was increased in the swimming performance trials. Equation , with either smooth or roughened substrates, using Vf=Vs\u2013 was determined using a one-way analysis of covariance (ANCOVA), with body size (BL) and holding tank number included as a covariates. Minimal adequate models were determined using stepwise simplification, and separate models were run for each species. P-values < 0.05 were considered statistically significant and all data are presented as mean \u00b1 s.e.Data analyses were performed using R Studio  of both Me. duboulayi  and Mo. adspersa   and Mo. adspersa , respectively  and tank numbers were excluded from minimal adequate models. Mo. adspersa employed a combination of both station-holding and direct, BCF gaits in trials. Me. duboulayi employed direct, BCF gait in all trials but did not station-hold.Substrate treatment had a significant effect on critical swimming speeds (01) Fig. . SwimminMe. duboulayi through \u2018small\u2019 (2 m), \u2018medium\u2019 (<20 m) and \u2018large\u2019 (20 \u2264 60 m) culverts with a smooth substrate, water velocities would need to be \u22640.46, 0.40 and 0.26 m s\u22121, respectively  if culvert interiors were roughened . This heightened performance translated into the traversable water velocity models, with maximal allowable water speeds being higher in roughened compared to smooth culverts, suggesting roughening may be an effective remediation approach to improve fish passage.Roughened culverts are often assumed to improve fish swimming performance and upstream passage (Oncorhynchus kisutch), for instance, have been observed to actively exploit reduced-velocity pathways during upstream movement through a culvert test bed  and the size and number of eddies generated . For exatest bed . The nexMo. adspersa and Me. duboulayi were similarly affected by the rough substrate, with both species experiencing a ~26% increase in swimming performance, despite different gaits employed during swim trials. Mo. adspersa employed station-holding behaviour in all trials, whereas Me. duboulayi did not station-hold and instead, employed a BCF swimming mode. Species utilizing bottom-swimming behaviours (e.g. station-holding and substratum-skimming) are expected to derive a greater net benefit from substrate roughening than fishes reliant on BCF modes, as these energy-saving behaviours are largely ineffective on smooth surfaces  limit water velocities through culverts to a maximum of 0.3 m s\u22121. At this velocity maximum culvert transit is likely to be unrestricted for Me. duboulayi for culverts up to 50 m in length, but compromised for Mo. adspersa in culverts with smooth substrates. Passage is predicted to be restricted for Mo. adspersa in culverts 2\u201315 m in length with a smooth substrate , but roughening remediation increases allowable water velocities to levels exceeding current guidelines (i.e. 0.31\u20130.35 m s\u22121). Transit through very long culverts (>20 m) is likely to be restricted for Mo. adspersa even with roughening remediation, and these structures may require additional restoration efforts, such as the installation of rest areas (\u22121).Successful passage through culverts is critically important as population declines of both barriers . In agreMugil cephalus]; Galaxias maculatus]; Hypseleotris compressa]). Similar to Mo. adspersa, many small-bodied species have been identified as weak-swimmers, requiring very low water velocities for upstream movements (ranging 0.05\u20130.20 m s\u22121). Reducing water velocity to this extent can be challenging, but culvert roughening may be a solution that allows hydraulic efficiency goals to be met without compromising fish access. Examining the effect of substrate roughening on the swimming performance of a greater number of species, with variations in morphology and swimming gaits, will allow us to gauge the potential benefit and wider application of roughening fish passes. In contrast to our findings, previous research has found roughening to provide no benefit to fish swimming performance (Cyprinus carpio); but this study differed to ours with respect to the position  and type (i.e. corrugated inserts compared to river stones) of substrate. Roughening the walls of culverts/experimental swim chambers, compared to the bottom, likely differentially affects hydraulic conditions (e.g. level of TKE). Wall roughening has been suggested to generate detrimental levels of turbulence, where the energetic expense of swimming is increased and fish become disorientated/unbalanced (Outputs from our traversable water velocity models were similar to other small-bodied species (e.g. formance . Newboldbalanced . Wall roAlthough the culvert remediation model presented here can be a powerful tool for decision making, the limitations of this model need to be considered. Estimates of swimming performance derived from non-volitional, laboratory studies can underestimate true ability, as fish often attain greater swimming speeds in open-channel, volitional trials . The swiSupplementary DataClick here for additional data file.Supplementary DataClick here for additional data file.Supplementary DataClick here for additional data file."}
{"text": "Recent studies have revealed that a combination of chemical compounds enables direct reprogramming from one somatic cell type into another without the use of transgenes by regulating cellular signaling pathways and epigenetic modifications. The generation of induced pluripotent stem (iPS) cells generally requires virus vector-mediated expression of multiple transcription factors, which might disrupt genomic integrity and proper cell functions. The direct reprogramming is a promising alternative to rapidly prepare different cell types by bypassing the pluripotent state. Because the strategy also depends on forced expression of exogenous lineage-specific transcription factors, the direct reprogramming in a chemical compound-based manner is an ideal approach to further reduce the risk for tumorigenesis. So far, a number of reported research efforts have revealed that combinations of chemical compounds and cell-type specific medium transdifferentiate somatic cells into desired cell types including neuronal cells, glial cells, neural stem cells, brown adipocytes, cardiomyocytes, somatic progenitor cells, and pluripotent stem cells. These desired cells rapidly converted from patient-derived autologous fibroblasts can be applied for their own transplantation therapy to avoid immune rejection. However, complete chemical compound-induced conversions remain challenging particularly in adult human-derived fibroblasts compared with mouse embryonic fibroblasts (MEFs). This review summarizes up-to-date progress in each specific cell type and discusses prospects for future clinical application toward cell transplantation therapy. MEFs were converted into chemically induced neurones by four compounds including ISX9, Forskolin, CHIR99021, and I-BET151 . A BET fin vitro and in vivo . Re. Re67]. Pdx1, Ngn3, and MafA [in vivo, proper differentiation from pluripotent stem cells requires many kinds of culture media, growth factors, and small molecules at each step of the differentiation [The number of patients suffering from diabetes mellitus has been rapidly expanding all over the world, but the fundamental treatment is difficult without transplantation of islet or functional \u03b2 cells. So the development of induced pancreatic \u03b2 cells from other somatic cells is one of the most attractive strategies to treat diabetes. A number of studies have reported that functional \u03b2 cells were transdifferentiated from pancreatic exocrine cells, pancreatic ductal cells, intestinal crypt cells, stomach cells, and hepatocytes by forced expression of \u03b2 cell lineage-specific transcription factors such as and MafA . These sntiation ,78.In consideration of these background factors, chemical compound-based direct conversion of human dermal fibroblasts into functional \u03b2 cells is challenging. In practice, such chemical conversion into \u03b2 cells has not yet been reported. Sheng Ding and colleagues demonstrated that transient expression of the reprogramming factors such as Oct4, Sox2, Klf4, and c-Myc or P53 shRNA converted mouse and human fibroblasts into an intermediate ,80. ThenIns2 and Pdx1 [Pdx1 gene in both primary human islets and the ductal cells. 5\u2032-Azadeoxycytidine (AZA), a DNA methyltransferase inhibitor, induced Ngn3 expression in PANC-1 cells and promoted an endocrine differentiation in the cells [The high-throughput screening of small molecules from several groups have identified candidate compounds for chemical compound-based pancreatic reprogramming. BRD7389, an inhibitor of ribosomal S6 kinase (RSK), changed the cell shape of mouse pancreatic \u03b1 cells to a more \u03b2 cell-like one and increased the expression of \u03b2 cell-specific genes, and Pdx1 . BRD7552and Pdx1 . BRD7552he cells . Harminehe cells . More rehe cells .Challenges to converting either differentiated endoderm cells or fibroblasts into the progenitor cells have been noted by several groups. Wang et al.  reportedCdh1, Sox17, FoxoA2, and HNF4\u03b1. ciEPCs could be differentiated into hepatocyte-like cells in the specialized medium including chemical compounds and growth factors, DAPT, Dexamethasone, A83-01, hepatic growth factor (HGF), and oncostatin M (OSM). The hepatocyte-like cells differentiated from ciEPCs exhibited an expression pattern similar to the one of iPS cells-derived hepatocytes. ciEPCs were also differentiated into pancreatic progenitor cells expressing Pdx1; however, insulin expression was not confirmed. It is also a promising strategy to prepare clinically applicable hepatocytes and pancreatic \u03b2 cells if human adult fibroblasts are successfully converted into the endoderm progenitor cells as well. Similar combinations of the chemicals and the protocols might be useful for performing the conversion of human fibroblasts into ciEPCs.Recently MEFs were chemically converted into endoderm progenitor cells by two sets of combinations of chemical compounds and growth factors . MEFs we. [Katsuda et al.  successf. . A diffeMSCs are multipotent somatic stem cells and can be differentiated into osteocytes, chondrocytes, and adipocytes. MSCs are generally isolated from the tissues such as bone marrow, adipose tissue, and umbilical cord blood. MSCs also have immunomodulatory effects to suppress immune response and participate in tissue repair by secreting multiple cytokines . Human dIn 2013, Hou et al.  first reSall4 and Lin28a, which makes the pluripotent state more accessible. The transition via XEN-like cells in the reprogramming process is unique for chemical-based, but not observed in transcription factor-based, reprogramming. In addition, the effects of several new compounds such as AM580, EPZ004777, SGC0946, and 5-Aza-dC on the chemical reprogramming were investigated. The retinoic acid receptor agonist and epigenetic modulators in combination with VC6TF increased the efficiency up to 1000-fold greater than that described in their previous reports. Not only MEFs but also neonatal dermal fibroblasts and adult lung fibroblasts were chemically reprogrammed, suggesting a broad availability of the chemical cocktail for the reprogramming. Further studies are required to reveal in more detail the molecular mechanism by which each chemical compound facilitates the chemical reprogramming through regulation of targetted signaling pathways and epigenetic modifications. The identification of an intermediate state is an important step to further improve the experimental protocol and the combination of chemical compounds for rapid and better reprogramming to a pluripotent state that resembles that of ES cells.Approximately 2 years later, the same group showed that extraembryonic endoderm (XEN)-like cells were an intermediate in the process of CiPSC generation . The XENMoreover, their group also successfully converted mouse neural stem cells and small intestinal epithelial cells into CiPSCs . SimilarCiPSCs might have higher developmental potentials than iPSCs induced by the reprogramming transcription factors ,93,95. CBecause human somatic cells have not been reprogrammed into CiPSCs by chemical compounds only, the generation will have a great impact on this field. Several differences of signaling pathways between mouse and human pluripotent stem cells have been reported ,98. One in vivo ones.In most cases, the combination of chemical compounds required for each direct conversion were empirically determined, and the detailed molecular mechanisms of the direct conversions remain largely unknown. However, identification of the chemical compounds provides great insights into how signaling pathways regulate the direct conversion. In addition, we need to understand how multiple signaling pathways could synergistically change one cell fate to another. A better understanding of the reprogramming process is obviously helpful to improve the efficiency and to perform complete epigenetic reprogramming into various cell types which are more similar to the A number of common chemicals are frequently used in the direct conversions from both mouse and human fibroblasts. CHIR99021 is one of the most frequently used compounds in various conversions into neurones, neural stem cells, cardiomyocytes, endoderm progenitor cells, and iPS cells, suggesting that activation of WNT signaling pathway by inhibiting GSK3 might potentially increase plasticity for cell fate changes in fibroblasts. On the other hand, our report indicated that CHIR99021 severely inhibited the brown adipogenic reprogramming, which suggests that a cell type-specific regulation of signaling pathways is required for chemical direct reprogramming . TGF\u03b2 siSox2 gene and activated the expression [A few articles have reported a possible molecular mechanism underlying chemical direct reprogramming. A study on neural stem cell reprogramming suggested that downstream effectors, Elk1 and Gli2, in MAPK and SHH signaling pathways, respectively, were both directly bound to the promoter of pression . In humapression . In addiOne of the characteristics in chemically induced cells is low tumorigenicity, although whether it is really lower than that for the cells converted by transcription factors remains unknown. More careful studies will be required to evaluate the tumorigenicity in chemically induced cells by transplantation into animal models. The treatment with chemical compounds likely changes broad gene expression via downstream transcription factors of the related signaling pathways, which might also lead to an irreversible disruption of proper epigenetic states. It might increase the risk for tumorigenesis although this should be dependent on the properties of chemicals, concentration, incubation time, and so on.Compared with gene expression regulated by specific transcription factors, chemical compounds do not always activate expression of a specific set of transcription factors required for the cell fate conversion and have an influence on a wide range of gene expression and epigenetic modifications. Therefore, to more specifically and selectively regulate signaling pathways and histone-modifying enzymes, the strategy of chemical biology is required to identify novel compounds from amongst a tremendous array of chemicals. Such novel compounds might improve on the direct reprogramming efficiency, and lead to the conversions into more kinds of cell types that have not yet been reported.This review provides an overview of recent advances in chemical compound-based direct reprogramming. This field has rapidly developed based upon numerous studies on transcription factor-based direct reprogramming. The combinations of chemical compounds and the cell type-specific medium including growth factors will likely enable more cell fate conversions of somatic cells in the future. The use of chemical compounds instead of forced expression of transcription factors resolves several barriers which have been raised in the use of iPS cells and cells directly reprogrammed by transcription factors. Such small molecule-based direct conversions in a chemically defined medium will robustly facilitate the cell-based study on transplantation therapy, disease modeling, drug screening, and personalized medicine .Further studies are required to more fully manipulate cell fates by chemical compounds only. In addition, most reported protocols for chemical compound-based direct reprogramming are performed and repeated by the same groups. To increase the availability in other laboratories as well as hospitals, the same or similar experiments need to be actively repeated by independent groups. Particularly, the chemical conversion of human fibroblasts into endoderm lineage cells such as hepatocytes and pancreatic \u03b2 cells is clinically anticipated. The chemical compound-based strategy could become one of the main sources for cell transplantation therapy in the near future."}
{"text": "Lactobacillus helveticus LH-2 and Lactobacillus acidophilus La-5 against Salmonella Typhimurium. Non-fermented and fermented media containing 5.6% WPI were fractionated at a 3 kDa cut-off and the filtrate was analyzed by mass spectrometry. The non-fermented WPI medium contained 109 milk derived peptides, which originated from \u03b2-casein (52), \u03b1s1-casein (22), \u03b1s2-casein (10), \u03ba-casein (8), and \u03b2-lactoglobulin (17). Most of these peptides were not found in the fermented media, except for 14 peptides from \u03b2-casein and one peptide from \u03b1s2-casein. Database searches confirmed that 39 out of the 109 peptides had established physiological functions, including angiotensin-converting-enzyme (ACE) inhibitory, antioxidant, antimicrobial, or immunomodulating activity. A total of 75 peptides were found in the LH-2 cell free spent medium (CFSM): 54 from \u03b2-casein, 14 from k-casein, 4 from \u03b2-lactoglobulin and 3 from \u03b1s2-casein. From these peptides, 19 have previously been associated with several categories of bioactivity. For La-5 CFSM, a total of 15 peptides were sequenced: 8 from \u03b2-casein, 5 from \u03b1s1-casein, 2 from \u03b2-lactoglobulin. Only 5 of these have previously been reported as having bioactivity. Many of the peptides remaining in the fermented medium would contain low-affinity residues for oligopeptide binding proteins and higher resistance to peptidase hydrolysis. These properties of the sequenced peptides could explain their accumulation after fermentation despite the active proteolytic enzymes of LH-2 and La-5 strains. Down-regulated expression of hilA and ssrB genes in S. Typhimurium was observed in the presence of La-5 and LH-2 CFSM. Downregulation was not observed for the Salmonella oppA mutant strain exposed to the same CFSM used to treat the S. Typhimurium DT104 wild-type strain. This result suggests the importance of peptide transport by S. Typhimurium for down regulation of virulence genes in Salmonella.Peptides in the 3-kDa ultrafiltrate of fermented whey protein isolate (WPI) medium could be responsible for the antivirulence activity of  Salmonella enterica subsp. enterica serovar Typhimurium is considered a major foodborne pathogen with public health and economic concerns. This foodborne pathogen has developed resistance against a broad range of antibiotics  controls the production and activity of the Type III secretion system which enables the membrane ruffling process, in which the epithelial cell cytoskeleton is rearranged to engulf the Salmonella into cytoplasmic vacuoles  is the result of the balance between proteolytic activity and peptide consumption . The propeptides .S. Typhimurium. oppA gene expression in S. Typhimurium is modulated by nitrogen compounds and regulates the expression of other genes such as Lrp (leucine-responsive regulatory protein) or CodY  is one of the most abundant periplasmic proteins in  or CodY . Mutatiopeptides .oppA gene expression and virulence gene expression in Salmonella in the presence of antivirulence peptides was investigated using a S. Typhimurium oppA mutant strain.In this study, the proteolytic and peptidolytic activities of LH-2 and La-5 strains in Whey Protein Isolate (WPI) are explored through genome sequencing and biochemical assays on substrates. Peptide profile analysis by mass spectrometry was conducted to understand the potential factors that lead to accumulation of antivirulence peptides in the growth media. Furthermore, the correlation between Salmonella Typhimurium oppA mutant, which was provided by Prof. Eric Brown, McMaster University. Lactobacillus helveticus LH-2 and Lactobacillus acidophilus La-5 probiotic strains were grown under anaerobic conditions at 37\u00b0C for 48 h on De Man, Rogosa, Sharpe broth . Salmonella Typhimurium hilA::lux and Salmonella Typhimurium ssrB::lux were grown on Luria-Bertani broth  supplemented with 50 \u03bcg/ml of ampicillin (Amp). Both constructs were grown aerobically overnight on a shaking incubator at 37\u00b0C. S. Typhimurium DT104 and S. Typhimurium oppA mutant strains were grown under the same condition as S. Typhimurium constructs, but without Amp. Solid media preparation for all strains were carried out under the same conditions but with addition of 15 g/l of agar and incubated under the same conditions.All bacterial strains used in this study were obtained from the Canadian Research Institute for Food Safety (CRIFS) except for the L. helveticus LH-2 and L. acidophilus La-5, respectively. The media were filter sterilized through 0.45 \u03bcm pore-size filters . An overnight culture of each strain in MRS broth was washed and inoculated at 5% (v/v) into WPI based media and incubated anaerobically at 37\u00b0C for 48 h. Following growth, bacterial cells were removed by centrifugation at 15,000 \u00d7 g for 30 min at 4\u00b0C. The supernatant was filtered through a 0.20 \u03bcm pore-size filter (Corning) to obtain sterile CFSM which was subsequently freeze-dried  for 72 h and stored at \u221280\u00b0C.Whey Protein Isolate (WPI)  was dissolved at 5.6% (v/v) in sterile sugar solution. Glucose or sucrose  at 0.5% (v/v) were added for S. Typhimurium hilA::lux and ssrB::lux (luxCDABE operon from Xenorhabdus luminescens was isolated and cloned with an Amp resistance gene into a plasmid (pSB377), which was further fused with the hilA and ssrB promoter regions, separately. The expression of the lux genes is controlled by these promoter regions, so that light is emitted when the gene is expressed. Each overnight culture of the constructs was diluted 1:100 with fresh LB broth with and without supplementation of 10% (v/v) of neutralized (pH 7) CFSM from L. helveticus LH-2 and L. acidophilus La-5, which had been reconstituted in sterile deionized water at 10-fold concentration compared to the initial medium. The samples were incubated aerobically at 37\u00b0C with shaking for 3 h. Luminescence was measured with the FB 12 luminometer . The results are presented as Relative Light Units (RLU)/OD600 nm.For initial screening of the antivirulence effects of CFSM, bioluminescent reporter strains srB::lux  were useS. Typhimurium DT104 and S. Typhimurium oppA mutant following 3 h growth in fresh LB broth with and without 10% neutralized (pH 7) CFSM with shaking at 37\u00b0C, 2-ml samples were centrifugated at 5,000 \u00d7 g for 4 min at RT. The supernatant was discarded and cells were mixed with 1 ml of RNA Protect reagent  and incubated for 5 min at RT. Cells were collected again by centrifugation at 5,000 \u00d7 g for 10 min at RT. Cell pellets were suspended in 200 \u03bcl of Tris-EDTA buffer, pH 8.0 , 60 \u03bcl of 20 mg/ml lysozyme (Fisher Scientific) and 20 \u03bcl of proteinase K (Qiagen). The suspensions were incubated at 37\u00b0C for 1 h with shaking at 450 rpm. RNeasy Plus Mini Kit (Qiagen) was used to extract RNA from all samples following the manufacturer's instructions. DNA was eliminated by using RNase-Free DNase Set (Qiagen). In brief, 40 \u03bcl of total RNA was incubated for 10 min at RT with 2.5 \u03bcl DNase I stock solution, 10 \u03bcl RDD buffer in a total volume of 100 \u03bcl. RNA purification and concentration were performed with RNeasy MinElute Cleanup kit (Qiagen) and solubilized in 30 \u03bcl molecular-grade water. The quantity of quality RNA was determined by measuring the absorbance at 260 and 280 nm using a NanoDrop 1000 spectrophotometer . RNA integrity was verified by gel electrophoresis.RNA was extracted from The same procedures were used for a mixture of the following synthetic peptides  (Synpeptide) at a concentration of 0.2 mg/ml for each peptide.The purified RNA was used for reverse transcription by using high-capacity cDNA reverse transcription kit . RNA (1 \u03bcg) was mixed with 2 \u03bcl of 10\u00d7 RT buffer, 2 \u03bcl of 10\u00d7 random hexamer primers, 0.8 \u03bcl of 25\u00d7 dNTP (100 mM), 1 \u03bcl of Multiscribe reverse transcriptase (50 U/ml) in a total volume of 20 \u03bcl. A no reverse transcription control was included to confirm the absence of contaminating DNA. The synthesis of cDNA was conducted using a Mastercycler Gradient Thermocycler  under the following settings: 25\u00b0C for 10 min, 37\u00b0C for 120 min, 85\u00b0C for 5 min and a holding step at 4\u00b0C. The cDNA was stored at \u221220\u00b0C until use.TM 7 Real-Time PCR System  and PowerUp\u2122 SYBR\u2122 Green Master Mix (Applied Biosystems) were used for RT-qPCR. The primers (E = 10 (\u22121/slope). The PCR was performed in a total volume of 20 \u03bcl; 10 \u03bcl of PowerUp\u2122 SYBR\u2122 Green Master Mix, 1.6 \u03bcl of forward primer (5 \u03bcM), 1.6 \u03bcl of reverse primer (5 \u03bcM), 5 \u03bcl of 1/10 diluted cDNA and 1.8 \u03bcl of molecular-grade water with the final primer concentration of 400 nM for all the genes except 16S  and rpoD (sigma factor). For these two genes, a final primer concentration of 200 nM was used with reaction mixture, 10 \u03bcl of SYBR\u00ae Select Master Mix, 0.8 \u03bcl of forward primer (5 \u03bcM), 0.8 \u03bcl of reverse primer (5 \u03bcM), 5 \u03bcl of 1/10 diluted cDNA and 3.4 \u03bcl of molecular-grade water. Each PCR was performed in triplicate in the 96 well plates . PCR conditions were as follows: UDG activation at 50\u00b0C for 2 min and Dual-Lock\u2122 DNA polymerase activation at 95\u00b0C for 2 min, followed by 40 repeated cycles of denaturation, annealing and amplification, at 95\u00b0C for 15 s, 54\u00b0C for 30 s and 72\u00b0C for 45 s. Subsequently, a default dissociation curve  was performed in the instrument and specific amplicon was verified by a single melting-temperature peak. The transcript levels were normalized to the geometric average of expression for all housekeeping genes for each sample  according to the manufacturer's instructions. DNA was eluted in 10 mM Tris-HCl (pH 8.0). The concentration and the purity of the purified DNA was measured at 260 and 280 nm using a Nanodrop 1000 spectrophotometer. The integrity of the DNA was verified by agarose (1%) gel electrophoresis. Extracted DNA samples were stored at \u221220\u00b0C. Library preparation and sequencing were performed at the Plateforme d'Analyses G\u00e9nomiques of the Institut de Biologie Int\u00e9grative et des Syst\u00e8mes . In short, the libraries were prepared using 500 ng of mechanically fragmented DNA by a Covaris M220 (Covaris) using the NEBNext Ultra II kit (New England Biolabs). TruSeq HT adapters (Illumina) were ligated instead of NEBNext adaptors. The libraries were checked for quality using Bioanalyzer and quantified with Picogreen. The libraries were sequenced on a fraction of a MiSeq run  following the manufacturer's instructions. De novo assembly of the reads was performed using CLC Bio Genomic Workbench version 10.1  at the Genomics Facility of the Advanced Analysis Centre, University of Guelph. Both the assembled reads and the de novo assembled contigs were BLAST searched (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Comparative genomic analysis of the proteolytic system was conducted using other related strains L. helveticus CNRZ 32  for L. helveticus LH-2 and L. acidophilus NCFM  for L. acidophilus La-5 using the NCBI microbial genome database and Blast alignment tools.Genomic DNA was extracted from L. helveticus LH-2 and L. acidophilus La-5 were grown anaerobically in WPI\u2014sugar based medium supplemented with 10 mM CaCl2.2H2O at 37\u00b0C for 24 h. The cells were harvested by centrifugation at 15,000 \u00d7 g for 10 min at 4\u00b0C and washed twice with 10 mM CaCl2-saline solution. The supernatant designated as extracellular extract (EE) was used for the measurement of cell lysis rate while the cells were re-suspended in Tris buffer  to an optical density at 600 nm (OD600) of approximately 10 and were used for CEP and aminopeptidase enzyme assays.\u22121cm\u22121. One unit of protease activity was defined as 1 nmol of p-nitroanilide released per minute. The specific protease activity was calculated as one unit of protease from 1 mg of cell protein. The protein content was estimated using the method of Bradford  as described previously . The reaBradford . Bovine 600 of approximately 10 were used for preparation of intracellular extracts (IE) according to Pescuma et al. (g at 4\u00b0C for 5 min) and the supernatant fluid was designated as the intracellular enzymatic extract (IE) which was maintained on ice and immediately used for enzymatic assays. Intracellular aminopeptidase activity was assayed with the chromogenic substrate Lys-\u03c1Na (Sigma) as previously described . The filtrate was freeze dried for 72 h. A control sample of filter sterilized WPI CFSM was included. The freeze-dried filtrate of 30 ml CFSM was reconstituted in 300 \u03bcl Milli-Q water and peptides were identified on an Agilent 1200 HPLC liquid chromatograph interfaced with an Agilent UHD 6530 Q-Tof mass spectrometer at the Mass Spectrometry Facility of the Advanced Analysis Centre, University of Guelph  and subjected to post hoc test with a P-value of \u2264 0.05 considered significant.All experiments were carried out three independent times with triplicates of each sample. Means and standard deviations were analyzed using ANOVA followed by Tukey's S. Typhimurium hilA::lux and ssrB::lux were used for initial screening of the antivirulence effects of CFSM obtained from WPI fermented with L. helveticus LH-2 and L. acidophilus La-5. CFSM from both strains reduced luminescence of the S. Typhimurium hilA::lux while only La-5 could decrease ssrB::lux reporter luminescence; indicating down-regulation of the hilA and ssrB genes  than LH-2 CFSM . The expression of hilA and ssrB genes in S. Typhimurium oppA mutant strain was \u22122.23 and \u22121.61, respectively, in the presence of La-5 CFSM while in the presence of LH-2 CFSM, expression of both genes was \u22120.92 and \u22120.77, respectively . The relative expression of the same genes in the S. Typhimurium oppA mutant strain in the presence of the same peptide mixture was \u22121.60 and \u22121.67, respectively  with the absence of prtH, prtH2 and prtM genes   .L. helveticus LH-2 for oligopeptide transport elements and peptidases shows the presence of these proteolytic components with high identity (97\u2013100%) (L. acidophilus La-5 genome codes for oligopeptide transport elements and peptidases of L. acidophilus NCFM with high identity (99\u2013100%), except for oppB2 (33% identity)  . The L. dentity) .L. helveticus LH-2 and L. acidophilus La-5 were measured based on the in vitro hydrolysis of the chromogenic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The optimal temperature for the proteinase activity of LH-2 strain was 45\u00b0C with enzyme activity 3.60 while the maximum CEP activity of La-5 was 0.25 \u00b1 0.01 at 40\u00b0C  analysis of unfermented WPI and WPI fermented with LH-2 and La-5 showed that peptides are almost all fragments of the main milk proteins  . Out of 2-casein . Most pe2-casein \u20139.L. helveticus LH-2 CFSM, 19 of these peptides have previously been associated with several categories of bioactivity . For L. acidophilus La-5 CFSM, a total of 15 peptides were sequenced, 5 of these peptides have previously been reported with ACE-inhibitory, antioxidant, antimicrobial and anti-caries activities. Some of these peptides have multifunctional properties that can modulate two or more physiological processes  and these signals affect the growth, metabolism and virulence of bacteria  and PrtM (maturase) in L. acidophilus La-5 confirmed that La-5 can digest large proteins extracellularly and produce small peptides. Genay et al.  is not different from other previously reported L. helveticus strains (40\u201350\u00b0C) (L. acidophilus strains (45\u201350\u00b0C) previously reported  \u2013104. Thereported .L. helveticus LH2 CEP activity in WPI was lower than the CEP activity of other L. helveticus strains previously reported in skim milk  after ultrafiltration of milk during the WPI production process. The presence of these endogenous peptides in the growth media could explain the lower CEP activities of LH-2 and La-5 in WPI compared with other related strains in milk-based growth media. The CEP activity levels of lactobacilli depend on the strain, nature and quantity of peptides present in the growth media  supplemented with different nitrogen sources  in WPI fermented by LH-2 and La-5 strains indicates some similarity in the affinity of the proteinases of these strains for certain cleavage sites.The presence of endogenous peptides in unfermented WPI indicates the activity of endogenous milk proteases which are mainly plasmin, elastase and cathepsin D, B, and G . PlasminD, and G , 113. DaD, and G  concludeAccording to peptide analysis data, most of the identified peptides are casein derived, even though whey was used. The presence of casein derived peptides indicates that these proteins were exposed to proteolytic cleavage during processing . The thrL. helveticus 1198 while Sadat-Mekmene et al. . The lack of the prtH CEP gene in the L. helveticus LH-2 genome does not explain the absence of \u03b1s1-casein derived peptides in LH-2 CFSM, as some of these are present in the control medium. Strain LHC2, which has a similar combination of CEP homologs (PrtH3/PrtH4), does produce peptides from \u03b1s1-casein within a 3-h time scale  of identified peptides in the WPI and WPI fermented with LH-2 and La-5, respectively, were derived from \u03b2-casein. Thus, it can be concluded that the endogenous and microbial proteases of LH-2 and La-5 may preferentially attack \u03b2-casein. These findings are consistent with previously studies on bovine casein and kefir . The dege et al.  concludeme scale  reported strains  while \u03ba- strains . \u03b2-lacto strains . LH-2 anL. acidophilus strains tested was 6.5 with a temperature optimum of 45\u201350\u00b0C, which might explain the lower number or diversity of peptides produced by La-5 at a lower pH and temperature (pH 4.5\u20135.2 at 37\u00b0C). The La-5 genome codes for only one CEP when compared with LH-2, which could result in a lower number or diversity of released peptides accumulating in the growth medium. Even though La-5 produces fewer types of peptides, those that are produced have higher antivirulence activity compare to those produced by LH-2, which depends on the nature and structure of the peptides. None of the peptides found in WPI fermented by La-5 are similar to those found in previous studies of the same strain grown in skim milk  of these strains, thus remaining in the spent medium if they are not transported into the cell. In Salmonella, the antivirulence activity of milk protein-derived peptides is related to the presence of the oppA gene. The undigested and fermented WPI by LH-2 and La-5 strains could be considered as a possible source of natural and functional ingredients, which may be used to increase the biological activity of food products. Further studies are required to explore the antivirulence ability of individual synthetic peptides with the same sequence at different concentrations compared to the antivirulence activity of fermented WPI.https://doi.org/10.5683/SP2/43M6GX].The datasets generated for this study can be found in the the University of Guelph Research Data Repository [EA carried out the experiments, interpreted the results, and wrote the first draft of the manuscript. SN participated in interpretation and presentation of the peptide results. SA-E, AE-L, and ES provided help for interpretation of the results. GL was involved in the design of the study, interpretation of the results, and writing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
{"text": "Previous data showed that the GTPase-interacting components, Gic1 and Gic2, control cell polarity through its binding with Cdc42 and PtdInsP2 in the plasma membrane in budding yeast. However, whether the Gic proteins regulate polarized exocytosis is unknown.Cell polarity refers to spatial difference in morphology, structure, and function within different parts of a single cell, which plays important roles in a wide range of cellular processes. In eukaryotic cells, the small GTPase Cdc42 and phosphatidylinositol 4,5-bisphosphate Pgic1\u0394gic2\u0394 are synthetically sick with sec3\u0394N. We demonstrated that Gic1 and Gic2 are required for polarized exocytosis in a yeast strain harboring the N-terminal domain deletion of Sec3, which is also known as an effector of Cdc42 GTPase. Gic proteins are required for polarized localization of exocyst, growth, and efficient secretion in sec3\u2206N mutant. In addition, we found that the N-terminal domain of both Gic2 and Sec3 share the similar binding sites of Cdc42. Surprisingly, not all the Sec3/Gic binding deficient cdc42 mutants displayed defects of growth and secretion, indicating that disruption of Cdc42 binding with Gic proteins and Sec3 does not necessarily show secretion defects in cdc42 mutants.In this study, we found that Gic2 co-immunoprecipitates with the exocyst complex, suggesting Gic proteins may be involved in exocytosis. Although we could not show the direct interaction between Gic2 and exocyst, we found 2, the regulation of Gic protein and Sec3 on polarized secretion may also be controlled by PtdInsP2. Further experiments need to be performed to test this hypothesis. Our findings provide important clues for understanding the molecular mechanism of cell polarity establishment in eukaryotic cells.We conclude that Gic1/2 and Sec3 act in parallel to regulate polarized post-Golgi secretion, but this regulation is not solely controlled by their upstream factor Cdc42. Considering that N-terminal domain of Gic2 and Sec3 can bind to both Cdc42 and PtdInsPThe online version of this article (10.1186/s13578-019-0295-x) contains supplementary material, which is available to authorized users. It is essential for many processes such as cell growth, differentiation, and morphogenesis, which rely on successful implementation of polarized secretion \u20134. Previmembrane , 15\u201321. membrane \u201325. Cdc4membrane , 8. Disrmembrane , 17, 18.2 via a polybasic region [sec3\u2206N exo70-38 double mutant can be rescued by adding the N terminus of Gic2 to sec3\u2206N [The GTPase-interacting components Gic1 and Gic2, also called Gic proteins, share 39% identity and 54% similarity in protein sequence, and play critical role for establishment of cell polarity. They are involved in initiation of budding and cellular polarization through interacting with Cdc42 via the Cdc42/Rac-interactive binding (CRIB) domain and with PIPc region , 26\u201330. o sec3\u2206N , it is ncdc42 mutants. Our data suggests that Gic proteins may regulate polarized secretion by the coordination of both Cdc42 and PtdInsP2.In this study, we find that Gic2 physically interacts with exocyst complex, and Gic proteins are required for polarized exocytosis in a mutant strain harboring the deletion of Sec3N-terminal domain. But the disruption of Cdc42 binding with Gic proteins and Sec3 does not necessarily show secretion defects and growth inhibition in 2 via a polybasic region [sec3\u2206N exo70-38 double mutant can be rescued by adding the N terminus of Gic2 to sec3\u2206N [Because both Sec3 and Gic proteins bind to Cdc42 via the Cdc42/Rac-interactive binding (CRIB) domain and with PIPc region , 29, 30,o sec3\u2206N , we spec2 binding, but is mostly independent of actin [sec3\u2206N mutant protein was not able to bind to Cdc42 and PIP2 in vitro, but the exocyst subunits were polarized to bud tip in this mutant strain [sec3\u2206N, gic1\u2206 gic2\u2206 and gic1\u2206 gic2\u2206 sec3\u2206N. As shown in Fig.\u00a0gic1\u2206 gic2\u2206 and sec3\u2206N, which is similar to that in wild type cells. In gic1\u2206 gic2\u2206 sec3\u2206N triple mutant cells, however, most exocyst proteins were either depolarized or diffused inside the cells. We also examined the actin pattern in these mutant strains, and found that actin organization is mostly polarized in these mutants at 25\u00a0\u00b0C Pof actin \u201328, 30. t strain . We examsec3\u2206N mutant, we speculate that Gic2 and its a paralog, Gic1, may function in parallel with Sec3 for post-Golgi secretion, and a strain with deletion of Gic1 and Gic2 would have synthetic growth effect with sec3\u2206N mutant. To test this hypothesis, we deleted GIC1 and GIC2 either alone or in a sec3\u2206N mutant background yeast strain. As shown in Fig.\u00a0gic1\u2206 gic2\u2206 or sec3\u2206N alone at 25\u00a0\u00b0C, 32\u00a0\u00b0C, 35\u00a0\u00b0C and 37\u00a0\u00b0C. This result suggests that Gic1 and Gic2 may be involved in regulating exocyst complex in a pathway parallel to Sec3.Because Gic proteins are required for polarized localization of exocyst in sec3\u2206N and gic1\u0394 gic2\u0394 double mutant at 25\u00a0\u00b0C, and a small amount of Bgl2 was accumulated in the sec3\u2206N gic1\u0394 gic2\u0394 triple mutant. After shifting to 37\u00a0\u00b0C for 2\u00a0h, although the sec3\u2206N and gic1\u0394 gic2\u0394 double mutants still did not accumulated Bgl2 protein inside cytosol, the sec3\u2206N gic1\u0394 gic2\u0394 triple mutant showed a pronounced amount of Bgl2 accumulation. As a control, the sec10-2 cells did not accumulate Bgl2 at 25\u00a0\u00b0C, but accumulate Bgl2 at 37\u00a0\u00b0C. These results indicate that combining sec3\u2206N with gic1\u0394 gic2\u0394 greatly aggravates the Bgl2 secretion defects.To determine whether these mutations cause secretion defects, we have analyzed the secretion of two cargoes, the cell wall endo-glucanase Bgl2 and the periplasmic enzyme invertase, which are well-characterized markers for the two major classes of post-Golgi vesicles that deliver proteins to the cell surface . As showsec3\u2206N, gic1\u0394 gic2\u0394 double mutant, and sec3\u2206N gic1\u0394 gic2\u0394 triple mutant. As shown in Fig.\u00a0sec3\u2206N or gic1\u0394 gic2\u0394 mutant cells secreted >\u200985% of the total invertase outside of cytosol. However, only about 62% of invertase in sec3\u2206N gic1\u0394gic2\u0394 triple mutant cells was secreted outside at 25\u00a0\u00b0C. After a 2-h shift to 37\u00a0\u00b0C, the triple mutant cells only secreted about 30% of the total invertase.We also examined the secretion of the periplasmic enzyme invertase in the sec3\u2206N, gic1\u0394 gic2\u0394 double mutant, and sec3\u2206N gic1\u0394 gic2\u0394 triple mutant, we performed thin-section electron microscopy on these mutants. As shown in Fig.\u00a0sec3\u2206N and gic1\u0394 gic2\u0394 double mutant, whereas the sec3\u2206N gic1\u0394 gic2\u0394 triple mutant accumulated about 62.9\u2009\u00b1\u20099 vesicles/section vesicles. These results clearly show that Sec3 and Gic1/2 function at parallel to regulate exocytosis.To examine whether exocytic vesicles are accumulated in Since Gic proteins and Sec3 are downstream effectors of Cdc42, we want to know whether the regulation of Gic proteins and Sec3 on polarized exocytosis is under the control of Cdc42.Saccharomyces cerevisiae share a high degree of sequence similarity. The crystal structure of the N-terminus of Sec3 (\u201cSec3N\u201d) alone and the Sec3N\u2013Rho1 complex were resolved [cdc42 mutants were made in this study to confirm its binding to Sec3: cdc42-301 (V36A), cdc42-302 (V36A F37A), cdc42-303 (Q61A), cdc42-304 (Y64A) and cdc42-305 (Y64A R66A). Our in vitro binding assay showed that these cdc42 mutants were defective in binding to GST-Sec3N (a.a.71\u2013241) P2, and these associations are important for polarized cell growth [cdc42 mutants was performed using GST-tagged Gic2N (amino acids 1\u2013155), which contains its Cdc42-interacting domain [cdc42-302, the binding of GST-Gic2N to the other four cdc42 mutants decreased dramatically. These results suggest that the binding sites of Cdc42 in Gic2 is very similar to Sec3.The N-terminus of Gic2 contains the classic Cdc42/Rac1 interactive binding (CRIB) domain , 27. Beco sec3\u2206N , we testl growth . The in g domain . As show PtdIns4,P2, and tSaccharomyces cerevisiae under control of the endogenous Cdc42 promoter. The growth of the mutant cells was examined on YPD plates at 25\u00a0\u00b0C or 37\u00a0\u00b0C Pgic1\u0394gic2\u0394 double mutant is synthetically sick with sec3\u0394N single mutant and regulates cell gowth and exocytic secretion. Gic proteins regulate polarized secretion by targeting the tethering complex to bud tip of yeast cells P2 binding region, and the interaction of Sec3N-terminus with both Cdc42 and PtdInsP2 are important for Sec3 function. Yamashita et al. [sec3 mutants deficient in the binding of either Rho1 or PtdInsP2 can still be localized to the bud tip even in the presence of Latrunculin B, which disrupts the actin microfilament cytoskeleton, while the sec3 mutants deficient in the binding of both Rho1 and PtdInsP2 are almost completely depolarized in the presence of Latrunculin B. These results suggest that both Rho GTPase and PtdInsP2 can act as receptors for the exocyst complex in a complementary fashion. Gic proteins are considered to be targeted to the bud tip and plays an important role in early bud formation in yeast. The GTP-bound Cdc42 interacts with Gic2 through the Cdc42/Rac interactive binding domain located at the N terminus of Gic2 and activates Gic2 during bud emergence [2 in the plasma membrane. This physical interaction is also necessary for the polarized localization of Gic2 protein to the bud tip and is important for the function of Gic2 in cell polarity. These facts indicate that PtdInsP2 and Cdc42 act in concert to regulate polarized localization and function of Gic2 during polarized cell growth in the budding yeast. Based on these previous reports and the results shown in our study, we speculate that either the Cdc42\u2013Sec3/Gic interaction or PtdInsP2\u2013Sec3/Gic interaction can targeting the exocyst complex to the polarized growth sites, which explains the result that three Sec3-binding deficient cdc42 mutants did not show growth defect in Fig.\u00a0cdc42 mutants can not bind to Sec3 and/or Gic2.Zhang et al. , 10 founa et al.  also foumergence , 27. Orlmergence  have idecdc42 mutants may also be defective in interaction with other effectors. It is possible that the interaction of Cdc42 with other effectors are also destroyed in the two cdc42 mutants, cdc42-303 and cdc42-305. But it is unlikely that the ATP loading capacity of these mutants is the main reason for the binding defect because no interaction was detected between cdc42 mutants and Sec3, and neither GDP nor GTP affected the binding of Sec3 to all five mutants . Further experiments are needed to explore the molecular mechanism of Gic proteins interacting with exocyst complex.We found that Gic2 is physically associated with exocyst complex. It is likely that its paralog, Gic1, may also interacts with this protein complex. However, it is unclear whether Gic proteins bind directly to one of the exocyst subunits, or bind to another protein which interacts with exocyst complex. Another exocyst subunit, Exo70, binds to both Cdc42 and PtdInsPecretion , 18, 21.600 units of early log phase cells were collected in a 1.5-ml centrifuge tube and washed once with 0.5\u00a0ml of distilled water. 240\u00a0\u03bcl PEG 3350 (50% wt/vol), 36\u00a0\u03bcl 1.0\u00a0M LiAc, 5\u00a0\u03bcl 10\u00a0mg/ml sonicated salmon sperm DNA (Agilent Technologies), and 0.1\u201310\u00a0\u03bcg DNA were added and the tube was vortexed until the cell pellet had been completely mixed. The cells were then shocked in a water bath at 42\u00a0\u00b0C for 20\u201325\u00a0min. 2\u2013200\u00a0\u03bcl of the transformation mix was plated onto Petri dish plates containing solid synthetic complete medium. The plates were incubated at 25\u00a0\u00b0C for 2\u20134\u00a0days to recover transformants.Standard methods were used for yeast growth and genetic manipulations . All strpRS314-CDC42 plasmid [pRS316-HA-CDC42 plasmid balancer. The strains used in this study are listed in the strain table. Standard methods were used for yeast media and genetic manipulations.All mutagenesis was performed based on the  plasmid  using th plasmid  and the SEC5, SEC8, EXO70 and EXO84 by Green Fluorescence Protein (GFP) was performed as previously described [Chromosomal tagging of escribed . Cells wescribed . F-actin2, and 1\u00a0mM CaCl2). The cells were spheroplasted and fixed with 1% glutaraldehyde  at 4\u00a0\u00b0C overnight. The spheroplasts were washed in 0.1\u00a0M cacodylate buffer and were post-fixed twice with ice-cold 0.5% OsO4 and 0.8% potassium for 10\u00a0min each. After dehydration and embedding in Spurr\u2019s epoxy resin , thin sections were cut and transferred onto 600 mesh uncoated copper grids  and were post-stained with uranyl acetate and lead citrate. Cells were observed on a transmission electron microscope  at 100,000\u00d7 magnification.Cells were grown at 25\u00a0\u00b0C in YPD media to log phase, and were then processed for thin section EM analysis using a Jeol-1010 transmission electron microscope. Cells for EM were collected by vacuum filtration using a 0.45\u00a0\u00b5m nitrocellulose membrane and were fixed for 1\u00a0h at room temperature in fixation buffer  at 25\u00a0\u00b0C. NaN3 and NaF were added directly to the culture at a final concentration of 10\u00a0mM each. 10 OD600 units of cells were collected, washed with cell wash buffer . The cells were resuspended in 300\u00a0\u00b5l of spheroplast solution buffer  and incubated at 37\u00a0\u00b0C in a water bath for 30\u00a0min. The spheroplasts were pelleted gently by centrifuge at 2000\u00a0rpm for 5\u00a0min at 4\u00a0\u00b0C. 100\u00a0\u00b5l of supernatant was carefully transferred to a new tube and mixed with 20\u00a0\u00b5l of 6\u00d7 SDS loading buffer . This is the external pool. The remaining supernatant was removed and the pellet (spheroplasts) was washed once with 1\u00a0ml of spheroplast wash buffer  to remove residue external pool. The spheroplasts were dissolved in 300\u00a0\u00b5l of lysate buffer  by leaving on ice for 10\u00a0min. The cell debris was removed after the sample was centrifuged at 13,000\u00a0rpm for 5\u00a0min at 4\u00a0\u00b0C. 100\u00a0\u00b5l of supernatant (lysates) was transferred to a new tube and mixed with 20\u00a0\u00b5l of 6\u00d7 SDS loading buffer. This is the internal pool. The internal pool and external pool samples were boiled at 95\u00a0\u00b0C for 5\u00a0min, loaded into 12% SDSPAGE gel. Bgl2 was detected by Western blotting with an anti-Bgl2 rabbit polyclonal antibody (1:2000). For temperature-sensitive mutants, the cells were grown at 25\u00a0\u00b0C or shifted to 37\u00a0\u00b0C for 90\u00a0min before being processed for the Bgl2 assay.Analyses of the secretion of Bgl2 and invertase were carried out as previously described . 20\u00a0ml o600 is 0.6\u20131.0) at 25\u00a0\u00b0C. 1 OD cells were transferred to a new tube and washed with 1\u00a0ml of ice cold 1\u00a0mM NaN3. The cells were then resuspended in 1\u00a0ml YP plus glucose medium  and incubated at 25\u00a0\u00b0C for 1\u00a0h to induce invertase expression. After 1\u00a0h of incubation, the cells were collected and washed once with 1\u00a0ml 10\u00a0mM NaN3. The cells were resuspended in 1\u00a0ml 10\u00a0mM NaN3 and kept on ice. The external invertase was measured directly on the whole intact cells, whereas the internal invertase was measured after preparation of lysates. 0.5\u00a0ml of cells were removed and mixed with 0.5\u00a0ml of 2\u00d7 spheroplast cocktail mix . The cells were incubated in water bath at 37\u00a0\u00b0C for 45\u00a0min. The spheroplasts were collected and the supernatant was removed carefully without disturbing the pellet. The spheroplasts were dissolved at 4\u00a0\u00b0C in 0.5\u00a0ml 0.5% Triton X-100. The invertase assay was performed in 13\u2009\u00d7\u2009100\u00a0mm tubes. 20\u00a0\u00b5l of sample was placed in the tube and 80\u00a0\u00b5l of 50\u00a0mM NaAc, pH 5.1, was added. Then 25\u00a0\u00b5l of 0.5\u00a0M sucrose was added and the tube was incubated at 37\u00a0\u00b0C for 30\u00a0min. 150\u00a0\u00b5l of 0.2\u00a0M K2HPO4 was added and the tube was placed on ice to stop the reaction. The sample was boiled for 3\u00a0min and put on ice. 1\u00a0ml of assay mix was added  and the sample was incubated at 37\u00a0\u00b0C for 30\u00a0min. 1\u00a0ml of 6\u00a0N HCl was added into the tube and the value of A540 was measured by the Spectrophotometer . For temperature-sensitive mutants, the cells were grown at 25\u00a0\u00b0C or shifted to 37\u00a0\u00b0C for 90\u00a0min and then were grown in low-glucose medium (0.1% glucose) at the same temperature for 1\u00a0h.Invertase secretion was examined as described previously . 20\u00a0ml ocdc42 were expressed as His6-tagged fusion protein (His6-Cdc42 or His6-cdc42). 20\u00a0\u03bcg Sec3 (a.a.71\u2013241) or Gic2 (a.a.1\u2013155) and 10\u00a0\u03bcg Cdc42 or cdc42 mutant were used for in vitro binding assay as previously described [Amino-terminus of Sec3 (a.a.71\u2013241) and Gic2 (a.a.1\u2013155) were expressed as GST fusion proteins. Cdc42 and g at 4\u00a0\u00b0C, and the supernatant was further clarified by centrifugation at 100,000g at 4\u00a0\u00b0C. The published Hot-SDS protein extraction protocol was used with slight modifications [Yeast extracts were prepared using glass beads as described previously . Yeast cications . In brieFor immunoprecipitation of GFP-tagged Sec5, total proteins were extracted by using the glass beads method . A rabbiAdditional file 1: Figure S1. Polarization of actin in gic1\u2206gic2\u2206sec3\u2206N mutant. Cells were grown to log phase at 25\u00b0C, and then fixed. Actin was stained by Alexa Fluor 488-conjugated phalloidin. Scale bar, 5\u03bcm. (B) Quantification of cells with polarized actin in each mutant. 90 cells were counted for each group (n=3). Error bars represent standard deviations. The representative results from three independent experiments are shown.Additional file 2: Figure S2. Structural integrity of the exocyst complex in the gic1\u2206gic2\u2206sec3\u2206N triple mutant. The exocyst component Sec5 in wild-type and mutant strains was GFP-tagged by chromosomal integration. The cells grown to log phase were incubated at 25\u00b0C or shifted to 37\u00b0C for 2 h. The exocyst was immuno-isolated from the wild type and mutant cells by anti-GFP antibody, and individual exocyst components were detected by Western blot. At both 25\u00b0C and 37\u00b0C, the exocyst components were coprecipitated with Sec5-GFP. Our result suggests that the assembly of the exocyst complex is largely unaffected in the gic1\u2206gic2\u2206sec3\u2206N triple mutant. The representative results from three independent experiments are shown.Additional file 3: Figure S3. The cdc42 mutants were not able to bind to Sec3N in vitro with either GDP or GTP in the binding buffer. GST fusion proteins containing (a.a.71\u2013241) of Sec3 was purified and conjugated to glutathione Sepharose. Cdc42, cdc42-301, cdc42-302, cdc42-303, cdc42-304 and cdc42-305 were expressed as a Hisx6 fusion and purified from bacteria. The in vitro binding assay was performed using GST-Sec3 and Hisx6-Cdc42 in the presence of GTP\u03b3S or GDP. The Hisx6-Cdc42 fusion protein bound to the GST-Sec3N Sepharose was detected by Western blotting with anti-Hisx6 antibody (bottom). Equal amounts of Sec3 fusion proteins on beads were used in the binding assay . Equal amounts of cdc42 wild type and mutants were used . The representative results from three different experiments are shown. The representative results from three independent experiments are shown.Additional file 4: Table S1. Yeast strains and genotypes. Table S2. Plasmids used in this study."}
{"text": "Each pancreas was perfused in situ with histidine-tryptophan-ketoglutarate (HTK) or with University of Wisconsin solution and placed into a transport container. Temperature of the grafts was maintained at 4\u2009\u00b1\u20092\u00a0\u00b0C during transport to our hospital and MR scanning. A 1.5 T clinical scanner was used for the measurements. Single-voxel PRESS spectra were acquired using transmit\u2013receiver head coil.2\u2013)n , and tissue water . Average total fat, and intracellular lipids of NAPC concentrations were 79.2\u2009\u00b1\u2009100.8 (range 2.4\u2013304.4), and 2.9\u2009\u00b1\u20091.2\u00a0mmol/kg ww, respectively.Relaxation times were measured for lipid (\u2013CH1H-MRS is a useful tool for the estimation of pancreas graft lipid concentrations. Total pancreatic fat and especially content of intracellular lipids of NAPC are valuable measures for inspection of graft quality prior to transplantation or islet of Langerhans isolation.We have shown that  The pancreas plays an important role in the synthesis and secretion of digestive enzymes and metabolism-regulating hormones. The majority of pancreas volume contains exocrine glands that are dedicated to producing enzymes which help to digest proteins, fats, and carbohydrates (sugars). Approximately 2% of the pancreas mass consists of different hormone-producing endocrine cells which clump together into small clusters called islets of Langerhans. The hormones made by alpha and beta cells in the islets produce glucagon and insulin, respectively. These two hormones regulate the sugar levels in the blood and cells.Pancreas transplantation is the treatment of choice for patients whose pancreas does not make enough, or sometimes any, insulin. These patients suffer type 1 diabetes, or insulin-dependent type 2 diabetes. A relatively new and minimally invasive therapy is transplantation of the islets of Langerhans , 4.2 .Relaxation times ies Fig.\u00a0 by a Lev2O per 1\u00a0g wet weight tissue  where A and B are constants. T1 temperature coefficient decreased from dT1/dT\u2009=\u20099.5\u2009\u00b1\u20090.16\u00a0ms/\u00b0C to 5.35\u2009\u00b1\u20090.08\u00a0ms/\u00b0C in the interval from 65 to 25\u00a0\u00b0C. Linear dependence (0.9\u2009\u00b1\u20090.03\u00a0ms/\u00b0C) was found between T2 and temperature. These T1, T2 dependences and the temperature difference\u2009~\u200933\u00a0\u00b0C between in vivo and cold storage explain the shorter relaxation times.In vivo estimation of pancreatic water relaxation times e et al. . Our shoeous fat , 29. Thieous fat  reportedIn this study, we found the pancreatic fat content (f/w) to be very similar, to those reported for healthy subjects , 12, 13.1H-MRS is unable to discriminate between intracellular lipids of acinar and the islet of Langerhans cells.Pancreatic intracellular lipid accumulation is currently of considerable interest. It was shown that an increased content of cytosolic lipid droplets in islets of Langerhans leads to decreased glucose-stimulated insulin secretion , 33. ThiThe main limitation of this study is the small number of grafts and age of donors. It should be noted that the grafts were considered unsuitable for transplantation mainly due to advanced age. A further limitation is the fact that increased pancreatic fat content hinders detection of intracellular lipids of NAPC. The usability of the measured relaxation times is limited to the grafts perfused by HTK or UW solution and to the temperature of cold storage (4\u2009\u00b1\u20092\u00a0\u00b0C).1H-MRS is a useful tool for quantification of pancreas graft lipid concentrations using water as the internal concentration reference. Total pancreatic fat and especially content of intracellular lipids of NAPC are valuable measures for inspection of graft quality prior to transplantation or islet of Langerhans isolation.The present results suggest that"}
{"text": "Alzheimer\u2019s disease (AD) is a progressive neurodegenerative disease. The study of blood-based biomarkers has lasted for a long time in AD, because it supports the concept that peripheral changes are involved in AD pathology. But it is still unclear how peripheral blood is involved in the temporal characteristic molecular mechanisms of AD from aging to mild cognitive impairment (MCI) and which cells are responsible for the molecular mechanisms. The main purpose of our study is to gain a systematic and comprehensive understanding of temporal characteristic networks of peripheral blood in AD using whole blood samples with 329 case-control samples, including 104 normal elderly control subjects (CTL), 80 MCI who are susceptible to AD, and 145 AD, by the weighted gene co-expression network analysis (WGCNA). The module-trait relationships were constructed and module preservation was validated with independent datasets GSE63061, GSE97760, GSE18309, GSE29378, GSE28146, and GSE29652. Our results indicate that the down-regulated protein modification and ubiquitin degradation systems, and the up-regulated insulin resistance both play a major role in MCI, while the up-regulated inflammatory cascade dominates in AD, which is mainly mediated by monocytes, macrophages. Although there is mixed activation of M1 and M2 macrophages in all stages of AD, the immune neutral state or M2 polarization may predominate in MCI, and M1 polarization may predominate in AD. Moreover, we found that TRPV2, NDUFV1, ATF4, HSPA8, STAT3 and LUC7L3 may mediate the pathological changes in MCI, while SIRPA, LAMP-2, NDUFB5, HSPA8, STAT3 and FPR2 may mediate the conversion from MCI-AD or the pathological changes in AD, which provide a basis for the treatment based on the peripheral blood system. In addition, we also found that the combined diagnosis based on a panel of genes from the red, blue, and brown modules have a moderate diagnostic effect on distinguishing MCI and AD from CTL, suggesting that those panels of genes may be used for detection of MCI and prediction of this conversion from MCI to AD. Our research emphasizes that pathological changes, based on temporal characteristics of peripheral blood, provide a theoretical basis for targeted peripheral treatment based on appropriate times and identified several diagnostic markers. Alzheimer\u2019s disease (AD), the most common type of dementia, affects more than 50 million people worldwide Hodson, . More imAlthough accurate diagnosis has been achieved by novel CSF biomarkers , a bioinformatics analysis method, has been proven to effectively detect the complex module-trait relationships , 80 MCI who are susceptible to AD, and 145 AD. Then, we identified seven modules associated with different stages of AD and module preservation for each of the seven modules was validated with independent datasets. Next, we tested the overlap between the module and cell signature gene lists using GeneOverlap and performed functional enrichment analysis. Finally, we obtained the hub genes of modules using cytohubba plugin in Cytoscape and performed receiver operating characteristic curve analysis to detect its diagnostic power.The human whole blood mRNA expression dataset GSE63060 provided by AddNeuroMed Cohort, which is a large cross-European prospective biomarker study, was downloaded from the Gene Expression Omnibus (GEO) database . GO terms and KEGG pathways  suggest that RNA metabolism and inflammatory signaling pathway were down-regulated in both microglia and peripheral blood monocytes and macrophages, while ubiquitin-mediated proteolysis and protein modification process were down-regulated in peripheral blood monocytes and macrophages. The black module with high preservation in peripheral whole blood or monocyte datasets indicates that ribonucleoprotein complex biogenesis and RNA processing were up-regulated in regulatory T cells and Th1 cells. It is worth emphasizing that those modules are almost simultaneously involved in M1 and M2 macrophages or regulatory T cells and Th1 cells, which suggests that mixed activation of the peripheral blood immune system may exist in MCI. In these modules, we found that a part of the hub genes may be involved in the pathology, development and diagnosis of AD. For example, Enoyl-CoA hydratase 1 (ECH1), a member of the hydratase/isomerase superfamily, was proven to be a promising marker for early diagnosis of AD  cell type specific genes are based on reference datasets, and do not completely reflect cell type proportions. Changes in monocytes can occur in AD compared to controls, and this change within the same cell type will be lost or incorrectly interpreted using the currently used approach of assigning cell specificity to particular genes; and (2) since this is a cross-sectional study rather than a longitudinal study, the above results cannot fully represent the pathological changes from CTL-MCI-AD, and further experimental verification is needed.HL and RT: study design. RT: literature search, data analysis and article writing. HL: article revision. Both authors approved the final version of the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
{"text": "N-acetylglutamate synthase deficiency  is an autosomal recessive disorder of the urea cycle that results from absent or decreased production of N-acetylglutamate (NAG) due to either decreased NAGS gene expression or defective NAGS enzyme. NAG is essential for the activity of carbamylphosphate synthetase 1 (CPS1), the first and rate-limiting enzyme of the urea cycle. NAGSD is the only urea cycle disorder that can be treated with a single drug, N-carbamylglutamate (NCG), which can activate CPS1 and completely restore ureagenesis in patients with NAGSD. We describe a novel sequence variant NM_153006.2:c.-3026C\u2009>\u2009T in the NAGS enhancer that was found in three patients from two families with NAGSD; two patients had hyperammonemia that resolved upon treatment with NCG, while the third patient increased dietary protein intake after initiation of NCG therapy. Two patients were homozygous for the variant while the third patient had the c.-3026C\u2009>\u2009T variant and a partial uniparental disomy that encompassed the NAGS gene on chromosome 17. The c.-3026C\u2009>\u2009T sequence variant affects a base pair that is highly conserved in vertebrates; the variant is predicted to be deleterious by several bioinformatics tools. Functional assays in cultured HepG2 cells demonstrated that the c.-3026C\u2009>\u2009T substitution could result in reduced expression of the NAGS gene. These findings underscore the importance of analyzing NAGS gene regulatory regions when looking for molecular causes of NAGSD. NAG is an essential allosteric activator of carbamylphosphate synthetase 1 , which catalyzes the initial step of ureagenesis \u2013 ATP-dependent synthesis of carbamylphosphate from ammonia and bicarbonate2. Primary NAGS deficiency  results from defects in NAGS gene and protein leading to decreased abundance or absence of NAG, decreased or absent CPS1 activity, reduced or absent urea synthesis and hyperammonemia4. Hyperammonemia is the principal biochemical symptom of NAGSD which manifests clinically with nausea, vomiting, cognitive changes, seizures and, in severe cases, coma and death; other biochemical symptoms include high plasma glutamine and low or absent plasma citrulline4. Additionally, NAGSD can arise secondary to defects in organic and fatty acid metabolism4.N-acetylglutamate synthase  is a urea cycle enzyme that catalyzes formation of N-acetylglutamate (NAG) from glutamate and acetyl coenzyme ANAGS gene is located on chromosome 17 within band 17q21.31; NAGS spans approximately 8.5\u2009kb and has seven exons that encode a 1605 bp open reading frame, starting at chr17:42,082,032 (GRCh37/hg19), six introns, a promoter, and an enhancer located about 3\u2009kb upstream of the transcription start sites9. The NAGS promoter directs transcription of the NAGS gene from multiple transcription start sites located between 20 and 120\u2009bp upstream of the NAGS translation initiation codon9. The NAGS promoter controls transcription of the NAGS gene through binding of specificity protein 1 (Sp1) and cAMP response element binding (CREB) transcription factors9. The NAGS enhancer binds hepatic nuclear factor 1 (HNF1) and nuclear factor Y (NF-Y) transcription factors and is responsible for liver-specific expression of the NAGS gene9.Human 10. Majority of patients with NAGSD are homozygous for the disease causing NAGS allele11. Most NAGSD causing sequence variants are within NAGS coding region or affect splicing of the NAGS mRNA15; a sequence variant in the NAGS enhancer (NM_153006.1:c.-3064\u2009C\u2009>\u2009A) caused NAGSD by reduced NAGS expression due to decreased binding of HNF1 to its binding site8. Quick and accurate diagnosis of NAGSD is essential because it is the only urea cycle disorder that can be completely treated with a single drug, i.e. N-carbamylglutamate (NCG), a chemical analog of NAG that binds to and activates CPS117 to completely restore ureagenesis in patients with NAGSD18. NAGSD can be diagnosed by sequencing of the NAGS gene and the diagnosis is supported by the markedly enhanced ureagenesis in response to treatment with NCG8.Primary NAGSD is an autosomal recessive disorder that affects approximately 1 in 2,000,000 peopleNAGS enhancer that occurred independently in two families whose members had NAGSD, totaling to three independent families with NAGS enhancer variants with apparent clinical consequences. This novel variant is pathogenic because it is associated with recurrent episodes of hyperammonemia (and epilepsy) that were promptly resolved upon treatment with NCG and caused reduced expression in a functional assay consisting of a reporter gene in cultured cells.We describe a novel sequence variant in the All methods were performed in accordance with the relevant guidelines and regulations of the participating institutions. Mutation analysis was conducted with the approval of the Institutional Review Boards of the Erasmus Medical Center of the Sophia Children\u2019s Hospital and the University Hospital of the University of Padua. Informed consent was obtained either from all study subjects or their parents/guardians.The female subject is the only child of non-consanguineous parents. Mother is from Russia while father is Dutch. Pregnancy, delivery and neonatal period were normal. She was breastfed until the age of 3 months. She had a normal development, smiling, first words at 6 months and walking at 18 months. At the age of 2\u20133 years, she refrained from milk and dairy products, fish and meat.3 18.6\u2009mmol/L, BE \u22127), elevated lactate 4.0\u2009mmol/L (ref. 1.0\u20131.8) and ammonia . Amino acids in plasma (prior to treatment) showed increased alanine 1043 \u00b5mol/L (ref. 176\u2013480), normal glutamine 482 \u00b5mol/L (ref. 400\u2013720), and decreased citrulline 10 \u00b5mol/L (ref. 17\u201350) and arginine 17 \u00b5mol/L (ref. 32\u2013128); isoleucine, leucine and valine were also decreased. Urinary orotic acid was normal as was blood acylcarnitine profile. She was started on a high caloric drip, without protein but did not improve clinically and was therefore transferred to the Pediatric Intensive Care Unit (PICU). The patient suffered a similar period with abnormal behavior a month prior to this event, but the cause of her strange behavior remained unclear.A female patient, 3 years and 9 months old, was referred because of encephalopathy . Her CT scan indicated diffuse bilateral obliteration of peripheral cerebral spinal fluid, hypodensity of white matter with loss of white-gray demarcation, and diffuse hypodense aspect of the white matter as a result of global cerebral ischemia. Four days prior, she had been restless, drowsy/lethargic, and repeating incomprehensible sentences. She seemed tired, but would get up in the night. With increasing loss of consciousness, she was admitted to the hospital. An EEG was suspicious for focal epilepsy. She was treated with diazepam and valproate. Brain MRI, taken after treatment, was normal. Laboratory investigations revealed compensated metabolic acidosis , she was started on i.v. arginine . Ammonia levels decreased from maximum 462 \u00b5mol/L to 103 \u00b5mol/L, but increased again after 3 days to 194 \u00b5mol/L. Therefore, nitrogen scavengers were increased to 400\u2009mg/kg/d and NCG started simultaneously (5\u2009\u00d7\u2009200\u2009mg/d). With this treatment and after reintroduction of protein at final dose of 1\u2009g/kg/d, ammonia levels normalized and remained normal.Based on initial results, ornithine transcarbamylase (OTC) deficiency was suspected and NCG treatment was stopped. Two weeks later, another episode of hyperammonemia occurred which resolved immediately after reintroduction of NCG as the only treatment. This fast response to NCG together with the self-imposed protein restriction suggested NAGSD.NAGS and CPS1 coding exons, including the flanking intronic regions, did not reveal any sequence variants. Liver enzyme activity analyses were all normal: NAGS 94 \u00b5IE/mg protein (ref. >34), NAGS after arginine stimulation 197 \u00b5IE/mg protein (ref. >144), CPS1 26.7 mIE/mg protein (ref. >12), and OTC 489 mIE/mg protein (ref. >160).Routine DNA analysis of NAGS enhancer region was identified  of chromosome 17  which remained elevated despite continuous i.v. glucose fluids. Due to persistent hyperammonemia, treatment with sodium benzoate was initiated. A complete metabolic work-up including the plasma amino acid profile showed high glutamine (895 \u00b5mol/L), alanine (719 \u00b5mol/L) while urinary organic acids and blood acylcarnitine profile were normal. No orotic acid was detected in the urine collected during acute hyperammonemic episode. Subsequently, an allopurinol loading test was performed showing neither orotic acid nor orotidine. Screening of the gene panel for UCD, which includes Three months after this episode, she presented again with a similar psychotic picture that required hospitalization. Metabolic investigations revealed hyperammonemia (236 \u00b5mol/L), elevated glutamine in plasma (855 \u00b5mol/L) and in the CSF . These data, consistent with a proximal urea cycle defect, including NAGSD, suggested to change therapy from sodium benzoate to NCG at an initial dose of 100\u2009mg/kg/d. Shortly after this change, ammonia normalized and the child regained her normal activity.th percentile and the neurological and developmental assessments were normal. Despite the negative results for NAGS mutation(s) in the coding region and splice sites we decided to continue the therapy with NCG. After mutation analysis of patient 1 revealed the NM_153006.2:c.-3026C\u2009>\u2009T variant in the NAGS enhancer we revisited the previous sequencing results in this patient and found that she is also homozygous for the c.-3026C\u2009>\u2009T sequence variant  and the conservation scores for each position were downloaded from the UCSC Genome Browser (https://genome.ucsc.edu/). Sequences of NAGS enhancers from 30 mammals  according to manufacturer\u2019s instructions. Presence of the enhancer mutation was confirmed by DNA sequencing of the resulting plasmid.Expression plasmid p4.23hE_Mut, which harbors 2. Cells were plated in the 24-well plates at 1.5\u2009\u00d7\u2009105 cells/well; 24\u2009hr. later cultured cells reached 90\u201395% confluency. Cells were transfected with Lipofectamine 3000 reagent (Life Technologies) mixed with 0.5\u2009\u00b5g of plasmid DNA. The ratio of experimental plasmids, which express firefly luciferase luc2, and pGL4.74, which expresses Renilla reniformis luciferase and was used as a transfection efficiency control, was 1000:1.Human HepG2 hepatoma cells (ATCC) were cultured in complete EMEM medium (ATCC) supplemented with 10% fetal bovine serum (ATCC) and 5% penicillin/streptomycin (Life Technologies); cells were cultured at 37\u2009\u00b0C in atmosphere containing 5% CORenilla luciferase to correct for differences in transfection efficiency, and to firefly luciferase activity from the p4.23hE plasmid harboring wild-type NAGS enhancer. Results are average of three independent experiments, each carried out in triplicate. Values were expressed and mean\u2009\u00b1\u2009SEM and analyzed using Student t-test.Cells were harvested 24\u2009hr. after transfection and used for reporter gene assay with the Dual Luciferase Reporter Assay System (Promega) and Berthold Centro 960 luminometer according to the manufacturer\u2019s instructions. Firefly luciferase activities were normalized to activity of NAGS gene regulatory region encompasses approximately 4\u2009kb; it harbors several regions of high conservation in mammalian genomes that cluster in the NAGS promoter, immediately upstream of NAGS exon 1, and in the NAGS enhancer, which is located about 3\u2009kb upstream of the NAGS translation initiation codon . ENCODE project revealed that human NAGS enhancer also binds transcription factor NF1C in HepG2 cells .Human don Fig.\u00a0. Human Nver Fig.\u00a0 and8,9. lls Fig.\u00a0 and22.FiNAGS exons to include the NAGS gene regulatory regions.All three patients in this study presented with hyperammonemia and a metabolic profile suggestive for a proximal urea cycle disorder. Treatment with NCG led to normalization of plasma ammonia in patients 1 and 2 while withdrawal of this treatment resulted in recurrence of hyperammonemia in both patients. The third patient in this study markedly increased dietary protein intake after initiation of treatment with NCG. Therefore, genetic investigations were expanded beyond the routine sequencing of the coding NAGS regulatory region in all three patients revealed a C to T change at position chr17: 42,079,006 (GRCh37/hg19 human genome assembly), which is 3026\u2009bp upstream of the NAGS translation initiation codon, within intron1 of PYY, and within the Nuclear Factor IC (NFIC) transcription factor binding site 25 and Phylo-P26 scores for the base pair affected by the c.-3026C\u2009>\u2009T variant are 4.51  and 2.749 , respectively, indicating evolutionary constraint at this position while the PhastCons score26 of 0.999  indicates that the c.-3026C is part of a conserved element. The CADD (Combined Annotation-Dependent Depletion) scaled C-score, which includes the previous scores in a weighted model, for the c.-3026C\u2009>\u2009T is 19.36 indicating that this variant is among 1\u20132% of the most deleterious variants in the human genome27, and MutationTaster228 classified this variant as disease causing. Additionally, c.-3026C\u2009>\u2009T variant was classified as deleterious by each of the five variant prediction tools  that are combined into PredictSNP2 platform29. Because computational prediction of the effects of sequence variants are not always accurate we carried out functional testing in cultured cells.Sequencing of the ite Fig.\u00a0. This seNAGS gene and NAGSD in three patients. The C to T change corresponding to the c.-3026C\u2009>\u2009T variant was engineered into p4.23Enh construct8 and resulting construct p4.23hE_Mut was transfected into HepG2 cells. The p4.32Enh construct, harboring wild-type NAGS enhancer, was used as a positive control and p4.23, which lacks NAGS enhancer, was a negative control. The relative firefly luciferase activity was approximately 20% lower in HepG2 cells transfected with the p4.23hE-Mut construct than in cells transfected with the construct harboring wild-type NAGS enhancer  binding site, which has been identified using chromatin immunoprecipitation followed by sequencing carried out as part of the ENCODE project31. Although NFIC transcription factor is a DNA-binding protein that interacts with transcription machinery32, the presence of its binding site within an enhancer is not unusual as many enhancers have been shown to interact with transcription machinery when regulating expression of their target genes33. The high conservation scores of the base pair affected by the c.-3026C\u2009>\u2009T variant indicate that it resides in a conserved element, and suggests evolutionary constraint in vertebrates and importance for the NAGS enhancer function. This is reflected in computational predictions that c.-3026C\u2009>\u2009T variant is deleterious (PredictSNP2) and disease causing (MutationTaster2). Because computational predictions of the functional effects of sequence variants do not always agree with the expert opinion34, we carried out functional testing with reporter gene constructs, which revealed that the c.-3026C\u2009>\u2009T variant could affect expression of the NAGS gene. Taken together clinical, biochemical and molecular evidence strongly suggest that the c.-3026C\u2009>\u2009T variant is pathogenic and caused NAGSD in three patients experiencing physiological stress. As a consequence, and including the previous single case, we recommend adding sequencing of the NAGS enhancer region both in direct NAGS gene analysis using Sanger sequencing and when next generation approaches are performed.The c.-3026C\u2009>\u2009T variant maps in the vicinity of the NF-Y transcription factor binding siteFigureS1 and TableS1Dataset 1"}
{"text": "According to multivariate logistic regression analysis, the variability of the renin concentration was associated with NTG (p = 0.006). In conclusion, the systemic concentration and variability of renin levels were elevated in NTG patients. An altered renin concentration could represent a difference in RAAS function in NTG patients.The purpose of this study was to investigate the function of the renin\u2013angiotensin\u2013aldosterone system (RAAS) in normal tension glaucoma (NTG) patients by measuring the level of renin and angiotensin II (AngII) in the plasma. Twenty-four patients with NTG and 38 control subjects were included in this study. Renin and AngII were measured in the blood samples of all subjects by enzyme-linked immunosorbent assay (ELISA). No significant differences were found in the complete blood count, fasting glucose, low-density lipoprotein (LDL), and high-sensitivity C-reactive protein (hs-CRP) levels between the control and NTG groups. The systemic concentration and variability of the renin concentration in the blood was significantly higher in the NTG group ( Glaucoma is characterized by the progressive death of retinal ganglion cells (RGCs) and the associated visual field (VF) defect. Elevated intraocular pressure (IOP) is traditionally known to be the main cause of glaucoma . HoweverGlaucoma patients with normal IOP, known as normal tension glaucoma (NTG), have distinct clinical features associated with vascular dysregulation. According to several studies, orthostatic hypotension, systemic arterial hypotension, Raynaud\u2019s phenomenon, and migraines are common in NTG patients ,5,6. AccThe renin\u2013angiotensin\u2013aldosterone system (RAAS) is important for regulating hemodynamic stability and fluid volume in the body . When hyThe purpose of this study was to investigate the systemic hemodynamic features associated with RAAS function in patients with NTG. To the best of our knowledge, no study has been conducted measuring renin and AngII levels in the blood of NTG patients. We measured the concentration of renin and AngII, as well as the complete blood count, fasting glucose, total cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels in the blood of the study subjects. The results of the plasma concentrations of renin and AngII, including the variability, were compared to those of normal controls.This study was performed according to the tenets of the Declaration of Helsinki and was approved by the Institutional Review and Ethics Board of Incheon St. Mary\u2019s Hospital, South Korea. All subjects of this single-center, case\u2013control study provided written, informed consent. Normal controls were recruited from a routine ocular examination group, and patients with NTG were recruited from the glaucoma clinic of Incheon St. Mary\u2019s Hospital. Patients were excluded if they met any of the following criteria: (1) a history of non-glaucomatous optic neuropathy; (2) a history of eye trauma or surgery, except for uncomplicated cataract extraction; (3) pathologic myopia ; (4) other retinal diseases, including diabetic retinopathy and retinal vascular diseases such as vascular occlusion or uveitis.All subjects underwent comprehensive ophthalmic examinations, including best-corrected visual acuity (BCVA), refraction, Goldmann applanation tonometry, slit-lamp examination, gonioscopy, and dilated fundus bimicroscopy. Fundus photography, stereoscopic photography of the optic disc , and perimetry with the Swedish Interactive Threshold Algorithm (SITA) 24-2 test on a Humphrey Field Analyzer were performed to diagnose NTG. The diagnostic criteria for NTG were as follows: (1) open angle by gonioscopy; (2) IOP <22 mmHg; (3) glaucomatous-appearing optic disc morphology corresponding to glaucomatous VF defects on SITA 24-2 results, as follows: a glaucomatous-appearing optic disc accompanied by increased cup-disc ratio (CDR) of more than 0.7, a difference in vertical CDR of more than 0.2 between both eyes, retinal nerve fiber layer (RNFL) defects, or neural rim thinning. We defined glaucomatous VF defects as meeting two of the following three criteria: (1) a cluster of 3 points with a probability lower than 5% on the pattern deviation map in at least one hemifield and including at least 1 point with a probability lower than 1% or a cluster of 2 points with a probability lower than 1%; (2) glaucoma hemifield test results outside normal limits; (3) a pattern standard deviation outside 95% of the normal limits. VF defects were confirmed by at least two consecutive and reliable tests . All NTG patients had anti-glaucomatic eyedrops and showed stable disease status without VF progression for three years.BP was measured after the study subjects had rested in a sitting position for 5 min. Trained clinicians took measurements of two consecutive systolic and diastolic BPs, and the average value was used. For hypertension patients, the type of hypertensive medication was identified, and subjects with angiotensin-converting enzyme (ACE) inhibitors or angiotensin II receptor blockers (ARBs) were excluded, as these medications could interfere with systemic renin and angiotensin II levels.After fasting for 12 h and resting for 30 min, blood samples for the biochemical analysis were obtained by standard venipuncture. Blood was collected into tubes containing EDTA as an anticoagulant for plasma preparation. As soon as a blood sample was collected, the tube was frozen at \u221220 \u00b0C, and repeated freeze\u2013thaw cycles were avoided. Frozen samples were thawed before conducting ELISA of renin  and EIA of AngII  using commercially available kits and according to the manufacturer\u2019s instructions. In brief, standards, samples, and controls were added to each well of a microplate pre-coated with a secondary antibody specific for renin and AngII. After incubation and washing the plate, a primary antibody was added, and the plate was again incubated and washed. Subsequently, a chromogenic substrate was added to each well for the development of color, and the intensity was measured by a microplate reader. All measurements were assayed in duplicate and the mean values were calculated. In addition, the standard deviations (SDs) of the measurements were calculated to assess the variability of renin and AngII.t-tests were used to compare the continuous variables between the control and NTG groups. Chi-square tests were used to compare the categorical variables between the two groups. To identify the factors associated with NTG, logistic regression analyses were performed. Variables with p-values <0.1 in the univariate analyses were included in the multivariate analysis. The odds ratio (OR) and 95% confidence interval (CI) were calculated for each variable. All statistical analyses were performed with SPSS version 24.0 . p < 0.05 was considered statistically significant.All data are expressed as mean \u00b1 standard deviation. Independent This study included 24 patients with NTG and 38 controls as study subjects. p = 0.027) and HDL was lower (p = 0.036) in the NTG patients.The laboratory findings were comparable between the two groups, as shown in p = 0.005; SD: 17.79 \u00b1 14.29 vs. 49.45 \u00b1 49.41, p = 0.005). The mean concentration and SD of AngII were higher in the NTG group but not statistically significant . According to The mean concentration and SD of renin and AngII from each group are reported in p = 0.033, 0.003, and 0.002, respectively). In the multivariate analysis, including the variables with p-values \u22640.10 from the univariate analyses, only the SD of renin was significantly associated with NTG (p = 0.006).The factors related to NTG from the clinical and laboratory findings were evaluated using logistic regression analyses . In the The systemic features representing hemodynamic instability include systemic arterial hypotension, orthostatic hypotension, and a nocturnal BP dip. These features have been reported as commonly found characteristics in NTG patients ,13,14. AWhen systemic hypotension occurs in the body, the RAAS starts to regulate the blood volume and systemic vascular resistance . The RAAIn the present study, we found that the systemic mean concentration and variability of renin was higher in the NTG patients. Additionally, the SD of the renin concentration was a meaningful factor associated with NTG. AngII was thought to be elevated in response to an increase in renin; however, there was no statistically significant difference between the NTG and control groups. In short, this study identified a link between systemic RAAS functioning and NTG through the measurement of the systemic concentration of renin and AngII. RAAS function showed a different pattern in the NTG patients when compared to normal controls.According to our previous study, the systemic concentration of endothelin-1 (ET-1) and macrophage chemoattractant protein-1 (MCP-1) are elevated in NTG patients . ET-1 isIn healthy eyes, retinal blood flow is mediated by endothelial cells because there is no autonomic nervous system innervation . AngII, In addition, most RAAS components and receptors were found in the retinal tissue. The local RAAS from tissue, including the retina, has been reported as a system that maintains the homeostasis of tissue . ProreniIn this study, total cholesterol and HDL were different between the two groups. The NTG group had worse lipid profiles in terms of dyslipidemia. However, lipid profiles were not independently associated with NTG in the regression analyses. Indeed, there is controversy regarding the relationship between dyslipidemia and glaucoma. Wang et al. suggested that hyperlipidemia is related to an increased risk of glaucoma and IOP elevation . It has The other limitations of this study should be mentioned. First, clinical features associated with vascular dysregulation, such as optic disc hemorrhage, migraine, and Raynaud\u2019s phenomenon, were not included in our analysis. Since normal controls were recruited from routine medical examinations, information on their hemodynamic features was not available. Several studies have already reported the correlation of vascular dysregulation with optic disc hemorrhage , migrainIn conclusion, NTG patients showed elevation and fluctuation of systemic renin concentration. The difference in RAAS function in NTG patients may be associated with diverse features of vascular dysregulation. Further studies investigating the relationship between RAAS and NTG could suggest a novel approach for disease modulation."}
{"text": "Correspondence: Paola Dama - dmapla@gmail.com, p.dama@sussex.ac.uk; Hongtao Liu - hliu2@medicine.bsd.uchicago.eduJournal of Translational Medicine 2020, 18(Supp 1):1Background: Activation of immune checkpoint pathways in Acute Myeloid Leukemia (AML) may interfere with effective T-cell anti-tumor immunity, and is associated with immune evasion in pre-clinical leukemia models as it has been demonstrated . It was previously reported that overexpression of CTLA4 and PD-1 is associated with more aggressive leukemia and progression from MDS to AML or AML relapse. While PD-1/PD-L1 blockade therapy can be effective as cancer immunotherapy, interruption of PD-1/PD-L1 interactions alone does not completely restore T cell function in some patients indicating the involvement of additional negative regulatory pathways, such as Tim-3/Gal-9, in T cell exhaustion. Immune checkpoint pathways active in Acute Myeloid Leukemia (AML) patients, especially during the course of remission induction chemotherapy, have not been well-studied. We characterized these pathways in newly diagnosed AML patients enrolled in a phase I dose escalation trial that combined Selinexor a Selective Inhibitor of Nuclear Export (SINE) with high-dose cytarabine (HiDAC) and mitoxantrone (Mito) (NCT02573363) as induction therapy.Methods and study design: Multi-parameter flow-cytometry was performed on bone marrow specimens at diagnosis and following remission induction therapy in 26 patients with AML enrolled to the study to monitor the changes in expression of immune checkpoint receptors. Expression of CD47, PD-L1, PD-L2 and Gal-9 was assessed on CD34+ AML blasts and CD34- cell populations. In parallel, expression of inhibitory  and stimulatory co-receptors  on CD4+ and CD8+ T cell subsets were evaluated. The positivity and frequency of parent in percentage of each markers was gauged by comparing with their FMO controls. Samples were analyzed using LSR Fortessa or LSRII Cytometers. The Mann\u2013Whitney Test, Spearman\u2019s rank correlation and Runs Test analysis were applied. For all analyses, P-values\u2009<\u20090.05 were considered statistically significant.Results: The percentage of CD34\u2212 Gal9+ cells was significantly higher and was positively correlated with higher numbers of TIM-3-expressing T cells at the time of diagnosis in patients who experienced treatment failure (TF) after chemotherapy, compared to those in complete remission (CR). When comparing TIM-3 expression on CD4+ and CD8+ T cells in pre-treatment (diagnosis) to post induction therapy samples, the magnitude of increase measured by median fluorescence intensity (MFI) inversely correlated to response to therapy with increase TIM-3 MFI of >\u200950% in patients with TF.Conclusions: This study provides preliminary evidence to support a rationale for incorporating antibodies against the Gal9/TIM3 pathway during and/or following remission induction therapy for AML.ReferencesZhang L, Gajewski TF, Kline J, PD-1/PD-L1 interactions inhibit antitumor immune responses in a murine acute myeloid leukemia model. Blood. 2009; 114(8):1545\u201352.Zhou Q, Munger ME, Blazar BR, Coexpression of Tim-3 and PD-1 identifies a CD8+ T-cell exhaustion phenotype in mice with disseminated acute myelogenous leukemia. Blood. 2011;117(17):4501\u201310.The study was approved by the Institutional Review Board at The University of Chicago IRB15-0412) :2Background: HLA-A*02, a common allele in the Scandinavian population, is a negative prognostic factor in epithelial ovarian cancer. It is a strong predictor of patient outcome, only inferior to clinical staging. This prognostic trait in epithelial ovarian cancer is stronger by the presence of the gene compared with the expression of its protein, MHC class I. Microsatellite instability (MSI) is used as a biomarker for prognosis and is suggested an increased tumor mutational burden which can make the tumor more susceptible for T cell mediated immunotherapy. Our aim was to analyze the prognostic markers HLA-A*02 genotype, MHC class I on tumor cells, the CD8+ lymphocyte infiltration and MSI status in colon cancer patients with randomized treatment.Methods: Clinical information and primary tumors were collected from 520 colon cancer patients and followed for overall survival for 120\u00a0months. Patients hade stage II and III colon cancer and were randomized to surgery alone or surgery and adjuvant chemotherapy. HLA-A*02 genotype was determined by conventional PCR. MHC class I, MSI status and CD8+ lymphocyte infiltration were determined by immunohistochemistry.Results: Female patients with a stage III tumor and HLA-A*02 genotype had a better outcome if they had received adjuvant chemotherapy instead of just surgery (p\u2009=\u20090.03), whereas this was not the case for patients with other HLA-A genotypes or in the male patients where HLA-type did not correlate to outcome. MHC class I expression did not act as a prognostic factor, however the presence of CD8+ lymphocytes in the invasive margin and inside the tumor was a positive prognostic factor for overall survival (p\u2009=\u20090.01), although only statistically significant in the male patients (p\u2009=\u20090.03). 21% patients had a tumor with MSI . MSI tumors had a slightly better outcome and this was irrespective of gender and HLA-type.Conclusions: The prognostic traits of HLA-A*02 appear in this colon cancer cohort to act differently in male and female patients. Also CD8+ infiltration is different between genders. These findings suggest that men and women may have two different immune responses to malignancy :3Background: Melanoma remains one of the most aggressive and heterogeneous skin cancer, which is often refractory to conventional chemotherapy. Nevertheless, it responds well to both immuno-and targeted therapy, which is focused on inhibiting the most common signaling pathway involved in melanoma transformation including the mitogen-activated protein kinase (MAPK) pathway. However, mechanisms of drug resistance have been described, some involving the release of extracellular vesicles (EVs). EVs are play an important role as intercellular communication mediators that can influence the phenotype and function of receiving cells. The aim of our study is to investigate the role of EVs in the mechanisms of drug resistance and phenotypic alteration in primary melanoma cell lines MEL50 BRAF-V600mut and M257 BRAF-Wild Type.Materials and methods: In order to define phenotypic and functional differences between the two cell types, we characterized their surfaceome with a panel of 361-PE-conjugated antibodies specific for cell surface proteins. We compared the extracellular vesicles produced by both cell line, quantitatively and qualitatively by NTA and flow cytometry.Results: We identified 49 markers expressed by more than 30% of MEL50 cells and 69 markers expressed by more than 30% of M257 cells. Among these markers, 10 are exclusively expressed by MEL50 and 36 are exclusively expressed by M257. Defining a distinctive surfaceome for both cell lines. We have also characterized the EVs produced by these cell lines and showed that MEL50 produces 3 times as much EVs than M257. These EVs are indistinguishable by Nanoparticle tracking analysis. Preliminary flow cytometric characterization of individual EVs did not show a significant difference in the expression of the classic EVs markers CD81, CD82, CD63 and CD9.Conclusions: The characterization of the cancer cell surfaceome of two primary melanoma cell lines, one BRAF-V600mut and one BRAF-Wild Type, uncovered very distinctive phenotypes. While the expression of classic EVs markers was similar for EVs produced by either cell line, the extension of EVs marker characterization to the whole surfaceome of the parental cell line, may reveal the same heterogeneity, which could be used as biomarkers to identify BRAF mutated or wild type melanomas in liquid biopsies, and opens the door to investigating the role of specific EVs in drug resistance and phenotypic transformation.Correspondence: Maria Libera Ascierto - ml.ascierto@gmail.comJournal of Translational Medicine 2020, 18(Supp 1):4Introduction: The role of CD8 cells in determining clinical outcome to programmed death ligand-1 (PD-L1) blocking treatments has been well characterized, however, the contribution of NK cells is not well understood. This is partly due to the paucity of NK cell-specific markers that can identify NK cells in the tumor microenvironment (TME). We developed an NK cell-specific transcriptional signature to estimate the NK cell abundance in the TME. This signature, together with NK-chemokines shown to modulate the priming of adaptive immunity1 were investigated in patients with advanced non-small cell lung cancer (NSCLC) treated with a PD-L1 inhibitor, durvalumab.Methods: Peripheral blood mononuclear cells (PBMCs) and Fluorescence-Activated Cell Sorted (FACS) NK/CD8 populations from three heathy donors were subjected to single cell RNA sequencing  and transcriptome analysis (Affymetrix), respectively. Fresh frozen tumor biopsies from 97 NSCLC were profiled with RNA sequencing prior to durvalumab treatment; 29 of these had paired tumors procured 29\u00a0days following treatment with durvalumab. Kaplan\u2013Meier (KM) analyses were performed to identify predictive effects of the NK cell-specific signature. Clinical trial: 1108/NCT01693562Results: Transcripts over-expressed in sorted NK relative to CD8 cells were first identified  and intersected with 28 mRNAs up-regulated in the NK cell cluster determined by scRNAseq, providing an 8 gene NK cell-specific transcriptional signature defined as MEDI-NK. MEDI-NK correlated with NK signatures recently described2, and included chemokines shown to induce an effective NK-response1. When evaluated in TCGA, higher expression of MEDI-NK was associated with good prognosis  of patients with melanoma and breast cancer .3 and was associated with Progression Free Survival  of NSCLC patients treated with durvalumab. Following treatment with durvalumab, the increased expression of MEDI-NK and of additional genes leading to NK-priming of adaptive immunity1 was observed to be associated with patients\u2019 overall survival . Similar findings were not observed prior to durvalumab treatment.At baseline, MEDI-NK was highly correlated with the previously identified IFN\u03b3 signatureConclusions: Using single cell analysis, an NK cell-specific signature was developed to better define the role of NK cells in anti-PDL1 therapy. The increased expressions of the NK cell-specific gene signature and of genes leading to NK-cell priming of adaptive immune response were associated with clinical benefit to durvalumab.ReferencesB\u00f6ttcher JP et al. Cell. 2018.Barry KC et al. Nature Medicine. 2018.Higgs B et al. Clinical Cancer Res. 2018.Correspondence: Valentina Borzillo - v.borzillo@istitutotumori.na.itJournal of Translational Medicine 2020, 18(Supp 1):5Background: Ipilimumab (Ipi), an anti-cytotoxic T-lymphocyte-associated antigen4 (CTLA-4) monoclonal antibody, has been shown to improve survival in patients (pts) with advanced melanoma [1\u20133]. Several retrospective studies have shown how the combination of radiotherapy (RT) and Ipi in the treatment of melanoma brain metastases (MBMs) pts improves the outcomes, without however clarifying the exact timing of the two modalities [3\u201310]. The purpose of this study is to evaluate overall survival (OS), local control (LC) (in SRS field) of the lesion treated, and intracranial control (IC) (out SRS field) in MBMs pts receiving Ipi and Stereotactic Radiotherapy (SRT)/Radiosurgery (SRS) performed with Cyberknife\u00ae (CK) System.Materials and methods: Since December 2012 until December 2018 we treated 63 (34\u00a0M and 29 F) MBMs pts, of these 53 received RT\u2009+\u2009Ipi and 10 RT alone (NO-IPI group). Patient and treatment characteristics were in Table\u00a0Results: The median follow-up was 10.6\u00a0months (m) . 59 pts for a total of 123 lesions were valuable for the follow-up. The median OS was 10.6\u00a0m (95% CI 8.5\u201312.7) for all pts, 10.7\u00a0m for IPI\u2009+\u2009RT and 3.3\u00a0m for NO IPI (p\u2009=\u20090.96). The median OS for single group was: 7.6\u00a0m for RT POST-IPI, 10.4\u00a0m for RT CONC IPI and 11.5\u00a0m for RT PRE-IPI (p\u2009=\u20090.89). The 1-year LC (in SRS field) was 53% for all lesions, 59% in IPI\u2009+\u2009RT and 8% in NO IPI (p\u2009=\u20090.001)  for a single group was 74% for RT POST-IPI, 41% for RT CONC IPI and 48% for RT PRE-IPI groups (p\u2009=\u20090.002)  was 45% for all pts, 44% for IPI\u2009+\u2009RT and 51% for NO IPI (p\u2009=\u20090.73). The 1- and 2-year OS of patients with LC was 50% and 25% vs 30% and 4% of patients without LC respectively (p\u2009=\u20090.02).Conclusions: Our retrospective experience suggests that the combination of Ipi and SRS/SRT in MBMs pts can improve outcomes with a low toxicity profile. The optimal timing of combination Ipi and RT remains unclear, but from our experience it would seem to be a benefit on LC with SRS delivered after Ipi. The recruitment of a greater number of pts, a longer follow-up and new prospective studies are needed to demonstrate the role of Ipi in the treatment of MBMs and the better sequence with RT.ReferencesHodi FS, O\u2019Day SJ. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. 2010; 363:711\u201323.Lebbe C, McDermott DF. Ipilimumab improves survival in previously treated, advanced melanoma patients with poor prognostic factors: subgroup analyses from a phase III trial [abstract]. Ann Oncol. 2010;21(suppl 8):401.Margolin K, Ernstoff MS. Ipilimumab in patients with melanoma and brain metastases: an open-label, phase 2 trial. Lancet Oncol. 2012;13(5):459\u201365.Knisely JP, Yu JB. Radiosurgery for melanoma brain metastases in the ipilimumab era and the possibility of longer survival. J Neurosurg. 2012;117:227\u201333.Schoenfeld JD, Mahadevan A, Ipilmumab and cranial radiation in metastatic melanoma patients: a case series and review. J Immunother Cancer. 2015;3:50Patel KR1, Shoukat S. Ipilimumab and Stereotactic Radiosurgery Versus Stereotactic Radiosurgery Alone for Newly Diagnosed Melanoma Brain Metastases. Am J ClinOncol. 2015 May 16.Kiess AP1, Wolchok JD2. Stereotactic radiosurgery for melanoma brain metastases in patients receiving ipilimumab: safety profile and efficacy of combined treatment. Int J Radiat Oncol Biol Phys. 2015;92(2):368\u201375.Tazi K1, Hathaway A, Chiuzan C, Shirai K. Survival of melanoma patients with brain metastases treated with ipilimumab and stereotactic radiosurgery. CancerMed. 2015 Jan;4(1):1\u20136.Silk AW1, Bassetti MF, West BT, Tsien CI, Lao CD. Ipilimumab and radiation therapy for melanoma brain metastases. Cancer Med. 2013;2(6):899\u2013906.Mathew M1, Tam M. Ipilimumab in melanoma with limited brain metastases treated with stereotactic radiosurgery. Melanoma Res. 2013;23(3):191\u20135.Correspondence: Claire Lhuillier - cfl2002@med.cornell.eduJournal of Translational Medicine 2020, 18(Supp 1):6Background: Growing evidence suggests that mutation-associated neoantigens drive responses to immune checkpoint blockade (ICB) in tumors with high mutational burden [1]. One factor that limits the recognition of these neoantigens by T cells is the level of expression of the mutated gene product in cancer cells.\u00a0In the BALB/c-derived 4T1 mouse model of ICB-refractory metastatic breast cancer, we have previously shown that tumor-targeted radiation therapy (RT) combined with CTLA4 blockade induces CD8+ T cell-mediated regression of irradiated tumors and inhibits lung metastases [2]. Analysis of the T-cell receptor (TCR) repertoire indicated that unique clonotypes expand in treated tumors, suggesting that tumor rejection involves T cells reactive to a set of tumor antigens that are made available to the immune system by RT [3]. Therefore, we hypothesize that RT increases the expression of genes containing immunogenic mutations and hence promotes priming of neoantigen-specific T cells.Materials and methods: We performed whole-exome sequencing and RNA sequencing of untreated and irradiated (8GyX3) 4T1 cells in vitro to identify tumor-specific neoantigens and determine which ones are upregulated by RT. These mutations were also documented in vivo, in 4T1 tumors harvested before and after treatment (8GyX3\u2009+\u2009anti-CTLA4). Dedicated algorithms were used to predict MHC-I and MHC-II-binding epitopes from these mutated genes. Peptides with a predicted affinity\u2009<\u2009500\u00a0nM were synthesized and tested in vitro for binding in a MHC stabilization assay. The best candidates were used to vaccinate BALB/c mice, followed by challenge with 4T1 cells to test for the induction of protective anti-tumor immunity.Results: Out of 309 total mutations initially identified in 4T1 cancer cells, two MHC-I and one MHC-II neoepitopes were immunogenic in vaccination experiments as assessed by IFN\u03b3/TNF\u03b1 response after T cell re-stimulation. These neoepitopes were encoded by genes upregulated by RT. Vaccination with these three neoantigens induced a significant tumor growth delay in mice only when vaccination was combined with tumor-targeted RT. We observed significant changes in the intratumoral TCR repertoire in vaccinated mice. In addition, in vivo killing experiments demonstrated a potent cytolytic activity of T cells from vaccinated mice towards one of these neoepitopes. These results were confirmed in vitro after MHC-I blockade of the peptide-loaded target cells. Mass-spectrometry analyses of MHC-I-bound peptides are currently ongoing to assess the differences in presented antigens between untreated and irradiated cancer cells.Conclusions: Overall, our data demonstrate the potential of RT to modulate the expression of antigenic mutations in tumors which could enhance responses to immunotherapy.ReferencesSchumacher TN, Schreiber RD. Neoantigens in cancer immunotherapy. Science. 2015;348(6230):69\u201374.Demaria S, Kawashima N, Yang AM, Devitt ML, Babb JS, Allison JP, Formenti SC. Immune-mediated inhibition of metastases after treatment with local radiation and CTLA-4 blockade in a mouse model of breast cancer. Clinical cancer research: an official journal of the American Association for Cancer Research. 2005;11(2 Pt 1):728\u201334.Rudqvist NP, Pilones KA, Lhuillier C, Wennerberg E, Sidhom JW, Emerson RO, Robins HS, Schneck J, Formenti SC, Demaria S: Radiotherapy and CTLA-4 blockade shape the TCR repertoire of tumor-infiltrating T cells. Cancer Immunology Research. 2018; 6(2):139\u201350.Correspondence: Paolo Antonio Ascierto - p.ascierto@istitutotumori.na.itJournal of Translational Medicine 2020, 18(Supp 1):7Background: The successful deployment of immune checkpoint inhibitors (ICI) in cancer immunotherapy relies on the responsiveness of an individual\u2019s immune system for relief of that particular blockade in the cancer immunity cycle [1\u20133]. As most patients fail to respond to ICI, there is a need for biomarkers that can predict patient\u2019s clinical benefit thereby identifying the patient population most likely to respond . The goal of this study was to augment the prediction accuracy by identifying and testing novel candidate biomarkers that could envisage response to ICI in patients with metastatic melanoma. The analysis had two specific features: validation against previously published predicting biomarkers and characterization of patients\u2019 transcriptomes at individual gene and pathway levels, where network enrichment analysis (NEA) integrated disparate genes into pathway scores [6].Materials and methods: Gene expression profiles were obtained using NanoString\u00ae panels (IO 360 \u2122 beta or UIO) on formalin fixed paraffin embedded biopsies (FFPE) obtained from 30 stage IV metastatic melanoma patients treated with ipilimumab (anti-CTLA4) and 50 patients treated with Nivolumab (anti-PD1) of which 22 were first-line and 28 pretreated with ipilimumab. The samples originated from the pathological anatomy department of Istituto Nazionale Tumori IRCCS Fondazione \u201cG. Pascale\u201d of Napoli, Italy. All patients have appropriately signed informed consent. Statistical associations between treatment response and either gene or pathway score variables were estimated in linear models, which included covariates of known importance to ICI, such as mRNA expression of the checkpoint proteins and their ligands.Results: First, candidate transcription-based biomarkers were discovered in our cohorts via correlation to clinical benefit and then analyzed for significance by covariate adjustment. Secondly, the candidates performance was validated using a similar previously published NanoString-based gene dataset [7]. In the ICI-na\u00efve anti-PD1 cohort, we identified different genes which were informative on the clinical benefit regardless of the known determinants: F2RL1, ARG1 and ICAM5. In the anti-CTLA4 cohort, the individual gene analysis did not yield any significant and validated associations. However instead, we revealed a number of NEA-based correlates between \u201cprogression within 1\u00a0year\u201d and pathways e.g. \u201cCell adhesion molecules\u201d, \u201cPECAM1 interactions\u201d, as well as a number of immune-related differentially expressed gene lists.Conclusions: NanoString-based transcriptomics and the cohort designs provided high-quality data for discovery of robust biomarkers of ICI response, holding promise for development of clinically useful diagnostic panels in malignant melanoma.Cowey CL, Liu FX, Boyd M, Aguilar KM, Krepler C. Real-world treatment patterns and clinical outcomes among patients with advanced melanoma: A retrospective, community oncology-based cohort study (A STROBE-compliant article). Medicine . 2019 Jul; 98(28):e16328.Balar AV, Weber JS. PD-1 and PD-L1 antibodies in cancer: current status and future directions. Cancer Immunol Immunother. 2017; 66(5):551\u201364.2.[3] Robert C, Long GV, Brady B, Dutriaux C, Maio M, Mortier L et al. Nivolumab in previously untreated melanoma without BRAF mutation. N Engl J Med. 2015; 372(4):320\u201330.Larkin J, Minor D, D\u2019Angelo S Dutriaux C, Maio M, Mortier L et al. Overall survival in patients with advanced melanoma who received nivolumab versus investigator\u2019s choice chemotherapy in CheckMate 037: a randomized, controlled, open-label phase III trial. J Clin Oncol. 2018; 36(4): 383\u201390.Schachter J, Ribas A, Long GV, Arance A, Grob JJ, Mortier L et al. Pembrolizumab versus ipilimumab for advanced melanoma: final overall survival results of a multicentre, randomised, open-label phase 3 study (KEYNOTE-006). Lancet. 2017;390:1853\u201362.4.Franco M, Jeggari A, Peuget S, B\u00f6ttger F, Selivanova G, Alexeyenko A. Prediction of response to anti-cancer drugs becomes robust via network integration of molecular data. Sci Rep. 2019 Feb 20;9(1):2379.Chen P-L, Roh W, Reuben A, Cooper ZA, Spencer CN, Prieto PA, et al. Analysis of immune signatures in longitudinal tumor samples yields insight into biomarkers of response and mechanisms of resistance to immune checkpoint blockade. Cancer Discov. 2016 Aug 1;6(8):827\u201337.ReferencesEthics approval: The study was approved by the internal ethics board of the Istituto Nazionale Tumori IRCCS Fondazione \u201cG. Pascale\u201d of Napoli Italy, approval number of registry 17/17 OSS.Acknowledgements: The study was supported by the Institutional Project \u201cRicerca Corrente\u201d of Istituto Nazionale Tumori IRCCS Fondazione \u201cG. Pascale\u201d of Napoli, Italy.Correspondence: Ildiko Csiki - icsiki@senseibio.comJournal of Translational Medicine 2020, 18(Supp 1):8Background: SNS-301 is a first-in-class therapeutic cancer vaccine candidate targeting human aspartyl (asparaginyl) \u03b2-hydroxylase (ASPH). ASPH is a highly tumor specific antigen that is differentially overexpressed in multiple human cancers but not in healthy adult tissue and is associated with tumor cell growth, motility and invasiveness. SNS-301 is engineered to express an ASPH fusion product within an inactivated \u03bb-bacteriophage viral vector (phage display) to activate both innate and adaptive arms of the immune system. Extensive pre-clinical data demonstrated the immunotherapy\u2019s ability to overcome tumor self-tolerance and provide anti-tumor immunostimulatory effect including strong activated, functional intra-tumoral CD8+ T cell infiltration.Materials and methods: SNS-301 was tested in a phase I clinical trial via intradermal administration using a 3\u00a0M micro-needle injection system in ASPH overexpressing biochemically recurrent prostate cancer patients (pts). Twelve pts with detectable levels of ASPH received 3\u201323 doses of SNS-301.Results: The immunotherapy was well tolerated with only 3 pts. experiencing an adverse event (AE) considered at least possibly related to study drug. All AEs were\u2009\u2264\u2009grade 3 and no dose-limiting toxicity was observed. All pts. experienced NK cell activation as well as dose-dependent ASPH-specific immune responses including CD4+ and CD8+ T-cell and B cell dependent immune responses. Anti-tumor activity and disease stabilization was observed in 8/12 pts. (67%) with declines noted in both overall PSA level and increases in PSA doubling rate.Conclusions: SNS-301 is a novel immunotherapy that may overcome prior challenges of cancer vaccines and cell therapies. Based on the pre-clinical and phase I results, multiple phase II programs were initiated in ASPH positive patients across many tumor types to evaluate SNS-301 as an active product in the cancer-immunity cycle both as monotherapy and combination therapy with checkpoint inhibitors. A combination phase II study of SNS-301 with pembrolizumab in ASPH positive checkpoint resistant head and neck cancer patients is currently enrolling (NCT04034225). Additionally, ASPH is also in pre-clinical development as a cell therapy target in both heme and solid malignancies.Journal of Translational Medicine 2020, 18(Supp 1):9Background: Tumor mutational burden (TMB) has been shown to be predictive of a good response to immunotherapy in stage IV melanoma and also other tumors, and is starting to be used as an inclusion criterion in ongoing clinical trials. However, the prognostic value of this marker is jet to be validated, also in earlier stages. We analyzed data from primary melanoma of stage II patients from the TCGA database and found that TMB could be prognostic in this collective. In this study, we intended to validate the prognostic value of TMB in a stage II cohort of melanoma patients from our department.Materials and methods: We included patients with stage II melanoma diagnosed between 2000 and 2018 in the University Hospital of Tuebingen and for whom formalin-fixed and paraffine embedded normal and tumor tissue were available. Tumor and normal DNA sequencing was performed using a next generation sequencing (NGS) panel that covers 693 genes, 7 promotor regions and the intronic region of 26 genes with known fusion partners. TMB was expressed in mutations per megabase (mut/Mb) and the median TMB was used as cut-off to define high and low-TMB sub-groups. Descriptive analysis of patients\u2019 characteristics and survival analysis were performed. The follow-up time was defined as the time between diagnosis and relapse or death.Results: A total of 209 samples were included in the final analysis. More detailed information is presented in Table\u00a0The median TMB was slightly higher in the whole collective (median TMB\u2009=\u200914\u00a0mut/Mb) when compared to the subgroup of patients with BRAFV600E/K mutation (median TMB\u2009=\u200911\u00a0mut/Mb). The highest TMB was observed in patients with other BRAF mutations (median TMB\u2009=\u200955\u00a0mut/Mb).When analyzing the whole collective, we found no difference in terms of median relapse-free survival  and median overall survival  for patients with high and low-TMB. In patients harboring a BRAFV600E/K mutation the same results were observed, when the median TMB for this cohort was used as cut-off .In the multivariate Cox Hazard analysis including gender, tumor localization, histological subtype, age, tumor thickness, ulceration and TMB as a continuous variable, only age and tumor thickness were significant . The p-value for TMB was 0.2.Conclusions: Our analysis was unable to confirm the results from the TCGA database and TMB was not a prognostic marker in our cohort of stage II melanoma.Journal of Translational Medicine 2020, 18(Supp 1):10Background: Immunotherapy has become standard of care for an increasing number of tumors. Patients exposed to these drugs have a chance of developing immune-related adverse events (irAEs). In general, irAEs occur quite early, mostly within weeks to 3\u00a0months after initiation of immune checkpoint blockers. Being treatments relatively innovative, \u201clate\u201d irAEs are still unknown.Methods: This is a multicenter retrospective study of advanced cancer patients  treated with anti-PD-1/PD-L1 (mono)immunotherapy, with a minimum time to treatment failure (TTF) of 12\u00a0months. IrAEs were categorized into \u201cearly\u201d (which occurred within the first 12\u00a0months of treatment) and \u201clate\u201d. An explorative analysis of clinical outcomes  was performed. The data cut-off analysis was August 2019.Results: We evaluated 318 consecutive patients; the commencement date ranged from September 2013 to August 2018. Median age was 68.6\u00a0years (32\u201390); patients characteristics are summarized in Table\u00a0Conclusions: Late-irAEs among long responders seem to have a mild/moderate incidence. They are mostly non-serious and clinical manageable, with a low rate of treatment discontinuation. In this positive-selected population, the occurrence of any grade late-irAEs seems to be furtherly related to a prolonged OS.Keywords: immunotherapy; immune checkpoint; nivolumab; pembrolizumab; atezolizumab; immune-related adverse events.In the overall population median TTF was 28.6\u00a0months . In patients who experienced any grade late-irAEs median TTF was 30.8\u00a0months , while in patients who did not was 26.2\u00a0months  In the overall population median OS was Not Reached (274 censored). In both the patients who experienced (102 censored patients) and did not experienced any grade late-irAEs (172 censored patients) the median OS was Not Reached (p\u2009=\u20090)Journal of Translational Medicine 2020, 18(Supp 1):11Background: Recent introduction of anti-PD-1 (Nivolumab and Pembrolizumab) and anti-PD-L1  immune checkpoint inhibitors revolutionized oncological guidelines. IrAEs reported in clinical trials account to a maximum of 85%, while grade 3/4 of toxicity were reported in 10% of patients. Quality of AEs reporting in RCTs is satisfactory, but methods for data collection and analysis are unclear. The purpose of the study is to establish a cohort of cancer patients treated with immune checkpoint inhibitors (PD-1/PD-L1 inhibitors) in order to determine incidence and characteristics of irAEs in a real-world setting and improve clinical management.Materials and methods: We conducted a prospective cohort study in patients receiving anti-PD-1/PDL1 drugs for treatment of metastatic or locally advanced non-small cell lung cancer, renal cell carcinoma, squamous cell carcinoma of the head and neck, Hodgkin lymphoma starting from Jan 2019. We created a clinical pathway aimed to improve management of patients at risk for IRAEs. In particular, definite recommendations have been implemented for cases fulfilling criteria for suspected irAEs. They concern procedures for evaluation and diagnosis, specific treatments and rules for drug discontinuation. IrAEs have been defined and graded according to Common Terminology Criteria for Adverse Events vs 5.0. Management strategies have been adapted by a multidisciplinary panel, basing on the ASCO guidelines, which represent current best clinical practice.Results: Thirty-seven patients  have been enrolled. They were observed at baseline visit, and at weeks 4, 8, 12. Eleven patients had melanoma, seven had renal cell carcinoma, seventeen Non-small-cell lung carcinoma, one had Hodgkin lymphoma and one head and neck cancer. During the observation period, eight patients developed irAEs (21%) . We observed different grade of severity: G1 in two patients that developed hepatitis and hypothyroidism, G2 in three patients that developed III-V-VII cranial nerve palsy and two PMR-like. In three patients (37.5%) irAEs were severe (G3): bullous dermatitis, interstitial pneumonia and myositis. No case of G4 were observed. Median time of insurgence of irAEs was 4.5\u00a0weeks. Twenty-nine (78%) are still under treatment. Five patients stopped anti-neoplastic therapy: three due to irAEs (G2\u20133), two for radiological or clinical progression. Three patients died.Conclusions: Innovative tools are required in order to manage irAEs, prevent their potential relapse and to avoid useless interruption of therapy, with the goal to improve patients outcome.ReferencesArnaud-Coffin P. A systematic review of adverse events in randomized trials assessing immune checkpoint inhibitors. Int J Cancer. 2019 Aug 1.Champiat S, Lambotte O, Barreau E, et al. Management of immune checkpoint blockade dysimmune toxicities: a collaborative position paper. Annals of Oncology 2016;27: 559\u201374.Journal of Translational Medicine 2020, 18(Supp 1):12Background: Acute Lymphoblastic Leukemia (ALL) patients is the most common malignancy in children and represents 75\u201380% of leukemia cases. The most frequent immunophenotype is B-cell precursor ALL (B-ALL) in which, signaling via the B cell receptor (BCR) and its precursor (pre-BCR), play a crucial role in tumor promotion. It has been reported that Leukemias originate from cells with stem characteristics (LSC) well described in Acute Myeloid Leukemia (AML) but controversial in ALL. We propose to identify these cells by their dysregulated signaling pathway, using a combination of phosphoflow (SNCP) and cell surface markers in a high dimension flowcytometric approach in pediatric ALL.Methods: We enrolled a cohort of 20 B-ALL pediatric patients and adult healthy donors (HD) for a pilot study in order to set up the Single SCNP method. To evaluate the activation of ERK and STAT signal pathways, in addition to the phosphoprotein activation markers we developed a high-dimensional multicolor panel of 20 extracellular markers and applied it to 5 HD and 1 blood samples at baseline and after stimulation with Phorbol Myristate Acetate (PMA). The Spectraviewer Cytometer Aurora has been used to perform the experiments and the data have been analyzed with Cytobank using visualization tools like SPADE and viSNE algorithms.Results: In this pilot study, we show that it is possible to perform high dimension phenotypic and functional panels using fluorescently labeled antibodies, and that this constitutes a major advantage for the study of pediatric samples where sample-size is limiting. By defining SPADE trees clustered on cell surface markers, we traced multiple phosphorylation events monitored with ERK1,2 (pT202/pY204), p38MAPK (pT180/pY182), STAT1 (pY701), STAT3 (pY705) and STAT5 (pY694) in HD and B-ALL sample at basal levels and after stimulation.Conclusions: This study shows that this approach to characterize the activation pathways in different leukemia subpopulations, is feasible and potentially powerful enough to identify LSC. It can also be used as model for cancer patients were the sample size, as like pediatric samples, is very limited.Journal of Translational Medicine 2020, 18(Supp 1):13Background: Immune Checkpoint Blockade (ICB) achieves up to 45% of response in advanced non-small-cell lung cancer and melanoma. However, its use is suboptimal because the resistance mechanisms are not defined and we lack good predictive biomarkers. This study aims at identifying functional biomarkers of response to anti-PD-1 treatment.Methods: A retrospective pilot cohort of 16 patients with metastatic cutaneous melanoma treated with Nivolumab was categorized into extreme good or bad responders according to best response and treatment duration. Total RNA from FFPE tumor tissues was subjected to transcriptomics profiling by RNA-seq with ribosomal RNA depletion. Differential expression was calculated with DeSeq2, and pathway analysis with GSEA. Survival analysis was performed using Kaplan\u2013Meier method.Results: We have identified 140 genes as differentially expressed (DE) (adj p\u2009<\u20090.05) in good responders to Nivolumab. Interestingly, the genes are in their majority expressed in immune cells, in particular in the B cell lineage. GSEA shows mainly processes related to immune response, with a high B cells involvement. In addition, 22 genes are associated with improved overall survival, among which there are several genes coding for specific regions of both variable and constant domains of immunoglobulin chains, and the tumor gene LGR5, which is a cancer stem cells marker and is correlated with chemotherapy resistance in gastric cancer.Conclusion: This is the first study reporting a total-ARN profiling of patients treated with ICB. It reveals a comprehensive signature of immune-cells specific genes that delineate the response. The overrepresentation of B cell lineage genes suggests unprecedented hypotheses for the response mechanisms.Correspondence: Christian U Blank - c.blank@nki.nlJournal of Translational Medicine 2020, 18(Supp 1):14Background: Outcome of high-risk stage III melanoma patients was poor with a 5-year overall survival rate of <\u200950%. Adjuvant IPI improved 5-year RFS and OS, and adjuvant anti-PD-1 improved RFS further. Preclinical data suggested that neoadjuvant treatment might be more favorable due to a broader immune activation. The investigator-initiated OpACIN trial compared neoadjuvant with adjuvant IPI\u2009+\u2009NIVO, while the subsequent OpACIN-neo trial tested three different dosing schedules of neoadjuvant IPI\u2009+\u2009NIVO without adjuvant therapy. Concomitant neoadjuvant IPI\u2009+\u2009NIVO induced a high pathologic response rates of 77\u201380% . Here we present the 36- and 18\u00a0months RFS of the OpACIN and OpACIN-neo trial respectively.Methods: In the phase 1b feasibility OpACIN trial, 20 stage IIIB/IIIC melanoma pts with palpable nodal disease were included. Pts were randomized to receive IPI 3\u00a0mg/kg plus NIVO 1\u00a0mg/kg, either adjuvant 4 courses, or split 2 courses neoadjuvant and 2 adjuvant. In the subsequent OpACIN-neo trial 86 pts were randomized to arm A: 2\u00d7 IPI 3\u00a0mg/kg\u2009+\u2009NIVO 1\u00a0mg/kg Q3W (n\u2009=\u200930); arm B: 2\u00d7 IPI 1\u00a0mg/kg\u2009+\u2009NIVO 3\u00a0mg/kg Q3W (n\u2009=\u200930); and arm C: 2\u00d7 IPI 3\u00a0mg/kg Q3W followed immediately by 2\u00d7 NIVO 3\u00a0mg/kg Q2W (n\u2009=\u200926). Pathologic response was defined as <\u200950% viable tumor cells and was centrally reviewed by a blinded pathologist. Landmark RFS rates were estimated using Kaplan\u2013Meier method.Results: After a median FU of 36.7 and 17.7\u00a0months only one of the of the 71 pts (1.4%) with a centrally confirmed pathologic response on neoadjuvant therapy had relapsed, while 15/23 (65.2%) of pathologic non-responders had relapsed. The estimated 3-year RFS rate was 80% (95% CI 59\u2013100) for the neoadjuvant arm and 60% for the adjuvant arm (95% CI 36\u2013100) . After a median follow-up of 17.7\u00a0months, median RFS was not reached in any of the arms from OpACIN-neo. Estimated 18-months RFS was 85% for all pts (95% CI 78%\u201393%), 90% for arm A (95% CI 80%\u2013100%), 82% for arm B (95% CI 70%\u201398%) and 83% for arm C (95% CI 70%\u2013100%). Translational analyses indicate that baseline tumor mutational burden and interferon-y gene expression score are synergistic predictors of response.Conclusions: While OpACIN showed for the first time a potential benefit of neoadjuvant versus adjuvant immunotherapy, OpACIN-neo confirmed the high pathologic response rates that can be achieved by neoadjuvant IPI\u2009+\u2009NIVO. Both trials indicate that pathologic response is an excellent surrogate marker for relapse free survival.Clinical trial information: NCT02437279, NCT02977052Rozeman EA, Menzies AM, van Akkooi ACJ et al. Identification of the optimal combination dosing schedule of neoadjuvant ipilimumab plus nivolumab in macroscopic stage III melanoma (OpACIN-neo): a multicentre, phase 2, randomised, controlled trial. Lancet Oncol. 2019; 20:948\u201360.Blank CU, Rozeman EA, Fanchi LF et al. Neoadjuvant versus adjuvant ipilimumab plus nivolumab in macroscopic stage III melanoma. Nat Med. 2018; 24:1655\u201361.ReferencesCorrespondence: Pier Francesco Ferrucci - pier.ferrucci@ieo.itJournal of Translational Medicine 2020, 18(Supp 1):15Background: Unprecedented advances have been reached in the treatment of Melanoma using immune checkpoint inhibition, thanks to a better understanding of the molecular basis of tumor development and its interaction with the host.Anticipating treatment with neoadjuvant therapy has the potential to significantly improve the clinical outcome of patients with locally/regionally advanced melanoma having the advantage to allow the assessment of initial tumor response and to be probably more efficient/better tolerated, due to the lower tumor burden and the enhanced amount of neoantigens triggering the TCR in the presence of disease.In order to increase our knowledge in the field of drug resistance and/or response biomarkers, another great advantage of neoadjuvant trials is the availability of samples before and after systemic therapy for conducting novel mechanistic and biomarker studies in the circulation and the tumor microenvironment.Materials and methods: Thirty-five stage III B-D oligometastatic stage IV melanoma patients will be screened and treated with neoadjuvant therapy with Ipilimumab 1\u00a0mg/kg\u2009+\u2009Nivolumab 3\u00a0mg/kg every 3\u00a0weeks for 4 cycles, will receive surgery and then an adjuvant therapy with Nivolumab 480\u00a0mg every 4\u00a0weeks for 6 cycles.Sample collection  for diagnosis, biomarker and molecular analysis will be collected at baseline, after each cycle (except tissue) surgery and afterwards in the adjuvant setting.Results: Proteomic analysis of sera of treated patients, with particular emphasis on cytokines and chemokines, are being performed in order to identify possible markers associated with a better clinical outcome. The antitumor immune response in peripheral blood lymphocytes has been monitored, in order to evaluate whether the combination of antiCTLA4 and anti-PD1 is able to increase the number and/or the repertoire of melanoma-specific T-cells after treatments.Gene sequencing analysis and expression profiling of genes involved in immune response by different means will also be evaluated in order to detect possible variations induced by the treatment on a molecular level. Finally, data on the modification induced by the disease and treatment on the microbioma and microbiota at different time points, showed interesting influences in maintaining or creating a beneficial equilibrium.All these preliminary data will be presented and discussed together with efficacy/toxicity, based on percentages of pathological complete responses reached at surgery.Conclusion: Understanding the molecular mechanisms of metastatic spread and exploiting such knowledge in prevention will likely have a profound impact on melanoma prognosis in advanced stages.In a melanoma patient\u2019s population including stage IIIB-C, or IV with potentially resectable disease, neoadjuvant immunotherapy was feasible, while identification of biomarkers of response and prognosis is ongoing in order to allow a better patient\u2019s selection.Correspondence: Teresa Amaral - teresa.amaral@med.uni-tuebingen.deJournal of Translational Medicine 2020, 18(Supp 1):16Background: The approval of immunotherapy and targeted therapy have changed the treatment landscape of stage IV melanoma. Nevertheless, there are still patients that do not derive benefit from these therapies, particularly when primary resistance is present.Materials and methods: Here we analyzed patients diagnosed with stage IV melanoma between January 2015 and December 2018, and treated with first-line immunotherapy. Primary resistance was defined as disease progression at the time of first radiologic evaluation, after immunotherapy start. Patients with stable disease, partial response or complete response were considered to have disease control (DC). Follow-up time was defined as the time between stage IV diagnosis and dead or last contact. Descriptive analysis of patients\u2019 characteristics and prognostic factors was performed. Progression-free survival (PFS), 1, 2 and 3-y survival and overall survival (OS) were also analyzed.Results: A total of 530 patients with stage IV melanoma were analyzed; 347 patients received first-line immunotherapy and 144 patients were considered primary resistant. More information about patients\u2019 characteristics can be found in Table\u00a0p\u2009=\u20090.003), baseline level of LDH (p\u2009=\u20090.007), number of organs with metastases (p\u2009=\u20090.024) and presence of liver metastases (p\u2009=\u20090.012). Patients with primary resistance had a significantly worse prognosis compared to those that achieved DC: median PFS was 4\u00a0months (95% CI 3.62\u20134.3) for patients with primary resistance and not reached in patients DC.The prognostic factors in patients with primary resistance were baseline level of S100 (The median OS was 11\u00a0months (95% CI 8.83\u201313.17) in patients with primary resistance and was not reached in patients with disease control. The 1-y, 2-y and 3-y OS was 43.1% 17% and 10.8% in patients with primary resistance and 91.8%, 80.6% and 64.2% in the group of patients that achieved DC .There was no difference in terms of survival when the type of first-line immunotherapy (PD-1 monotherapy or CTLA-4\u2009+\u2009PD-1) was analyzed: median OS was 26\u00a0months for both sub-groups . The 1-y, 2-y and 3-y OS was 71.8%, 53.2%, 41.2% for patients receiving PD-1 monotherapy and 72.8%, 56.2%, 41% for those receiving CTLA-4\u2009+\u2009PD-1 .Conclusions: Patients with primary resistance to immunotherapy have a worse prognosis compared to those that achieve disease control. Further research is necessary to earlier identifying these patients and offering other therapeutic options.Correspondence: Michelle Krogsgaard - Michelle.Krogsgaard@nyulangone.orgJournal of Translational Medicine 2020, 18(Supp 1):17Background: T cell recognition of antigen and resulting proximal signaling are key steps in the initiation of the adaptive immune response. Identification of the specific extracellular contacts between the T cell receptor (TCR) and CD3 subunits upon recognition of peptide-major histocompatibility complexes (pMHC) gives more precise guidance for immunotherapeutic strategies that modulate T-cell immunity by targeting signaling through the TCR-CD3 complex. Previous studies that targeted the antigen binding site for enhancing T-cell responses to tumor antigens often lead to off-target effects and toxicity.Materials and methods: Recently, we used nuclear magnetic resonance (NMR) spectroscopy, mutational analysis and computational docking to derive a 3D structure of the extracellular TCR-CD3 assembly [1]. Further, biomolecular force probe (BFP) measurements allowed us to determine how 2D affinity and force-modulated TCR-pMHC kinetics depend on TCR-CD3 interaction sites and affect transduction of extracellular pMHC-TCR ligation into T cell function.Results: Based on our TCR-CD3 structural model, we mutated specific TCR-residues  to increase T cell signaling potency [2]. A TCR library for DMF5 TCR was created using site-specific mutagenesis in the C\u03b2 helix 3 and helix 4-F strand regions of the TCR :18Background: A significant proportion of melanoma patients without as well as with pre-existing immunity fail to respond to checkpoint inhibitor therapy, indicating a therapeutic potential for combining PD-(L)1 therapy with immunomodulating agents in both immunophenotypes. Domatinostat is a class I specific HDAC inhibitor in clinical development in advanced stage melanoma and gastro-intestinal cancer. In PD-(L)1 refractory mouse models without pre-existing immunity domatinostat increased inflammation and expression of genes predictive for PD-1 blockade response. In highly inflamed but exhausted tumors domatinostat beneficially affected the function of cytotoxic T cells within the tumor microenvironment. The combination of domatinostat with PD-(L)1 blockade substantially increased the anti-tumoral effects above the single agent therapies in tumors of both immunophenotypes, displaying greater benefit in highly inflamed but exhausted tumors.Here, we analyzed the baseline immunophenotypes and the impact of domatinostat mono-therapy on immune scores in melanoma patients refractory/non-responding to prior checkpoint therapy to clinically confirm our preclinical findings for domatinostat.Methods: Progressive, advanced stage melanoma patients with best response (BOR) of PD or SD to prior checkpoint inhibitor therapy were treated with domatinostat as monotherapy in a priming cycle followed by a combination treatment of domatinostat and pembrolizumab (2\u00a0mg/kg q3w) until progression or intolerability . Domatinostat was evaluated at three dose levels (100\u00a0mg once daily (OD), 200\u00a0mg OD, and 200\u00a0mg twice daily (bid) in a 14\u00a0days on\u2009+\u20097\u00a0days off schedule). Tumor biopsies for RNA-Seq analysis were taken at baseline and after the priming cycle (C1D15) to investigate the immune-modulating effect of domatinostat.Results: Analysis of immunophenotype in baseline biopsies of SENSITIZE patient revealed a very high interpatient variability comprising patients with non-inflamed to highly inflamed tumors. The majority of patients had tumors belonging to the highly inflamed, but exhausted immunophenotype. Domatinostat treatment showed a trend to increase the immune-related scores especially in patients with non- or low-inflamed tumors. In patients with high inflammation scores the effect of domatinostat was less consistent. Yet, this patient subpopulation showed the best clinical benefit confirming our pre-clinical observations. Based on murine data we postulate that domatinostat could increase the functionality of exhausted intratumoral T cells.Conclusions: In summary, these data could show that most patients refractory/non-responding to prior checkpoint therapy included into the SENSITIZE trial had an inflamed but exhausted tumor immunophenotype. For domatinostat we could confirm our preclinical hypothesis for a trend to increase the immune scores in non- or low-inflamed tumors and a greater benefit in the highly inflamed tumor patients.Acknowledgements: We thank all the patients and investigators  participating in the SENSITIZE clinical trial.Correspondence: Mark R Middleton - mark.middleton@oncology.ox.ac.ukJournal of Translational Medicine 2020, 18(Supp 1):19Background: ImmTAC molecules are unique TCR-anti-CD3 bispecifics that redirect T cells against intracellular antigens. Tebentafusp (IMCgp100), an ImmTAC targeting melanocyte-expressed gp100 antigen, has demonstrated monotherapy activity in advanced melanoma and can cause rash and cytokine-mediated AEs, hypothesized to be on-target (gp100) or effector (CD3) mediated. A preclinical MoA for T cell bispecifics suggests chemokine CXCL10 redirection of CXCR3+ T cells from blood into antigen-positive tissues; this has not been clinically validated.Methods: 84 HLA-A2+ pts with advanced melanoma  received tebentafusp. Serum (n\u2009=\u200940) and PBMC (n\u2009=\u200922) samples were taken pre- and post-infusion to analyze changes in cytokines and circulating T cells. Pre- (n\u2009=\u200916) and post-treatment (n\u2009=\u200911) tumor biopsies were analyzed by IHC for CD3, PD-L1 and gp100 expression; tumor RNA (n\u2009=\u200912) was analyzed for gene expression.Results: Tebentafusp induced a transient increase in IFN\u03b3-inducible cytokines, most prominently CXCL10. A greater increase in serum CXCL10 was associated with longer OS (p\u2009=\u20090.0002), tumor shrinkage (p\u2009=\u20090.003), and greater transient reduction in peripheral CXCR3+ CD8+ T cells (p\u2009=\u20090.001). Reduction in CXCR3+ CD8+ T cells also trended with longer OS (p\u2009=\u20090.02), and tumor shrinkage (p\u2009=\u20090.03).3/16 pre-treatment biopsies had <\u20091% gp100 expression . 8/11 biopsies post-tebentafusp had increased CD3+ T cells compared with matched pre-treatment samples (associated with baseline gp100 but not PD-L1 expression). Based on tumor biopsy gene expression analysis, tebentafusp increased T cell markers, IFN\u03b3-inducible and cytotoxicity-related genes.Conclusions: The association of clinical benefit with increased serum CXCL10 and decreased peripheral CXCR3+ T cells supports the MoA of tebentafusp-induced T cell redirection and activation. Tumor biopsy results support tebentafusp redirection of T cells to antigen-positive tumor. A Phase II trial in CM (NCT02535078), a Phase I/II trial in UM (NCT02570308), and a Pivotal RCT in UM (NCT03070392) are ongoing.Trial Registration: NCT01211262Correspondence: Elisa A. Rozeman - l.rozeman@nki.nlJournal of Translational Medicine 2020, 18(Supp 1):20Background: Continuous combination of MAPKi and anti-PD-(L)1 is currently tested in several trials to improve outcome of BRAFV600 mutated melanoma patients (pts). However, a major obstacle for continuous combination is the high frequency of grade 3/4 treatment-related adverse events (TRAE). In a preclinical model we showed that short\u2010time MAPKi induces T cell infiltration and is synergistic with anti-PD-1. In pts we found increased T cell infiltration upon D\u2009+\u2009T after short-term MAPKi, while this was frequently below baseline levels after >\u20092\u00a0weeks (W) MAPKi. The aim of this phase 2b study was to identify the optimal duration of D\u2009+\u2009T in combination with PEM.Methods: Treatment-na\u00efve BRAFV600E/K mutant advanced melanoma pts (n\u2009=\u200932) started PEM 200\u00a0mg Q3W and were randomized in W6 to continue PEM only (cohort 1), or to receive in addition intermittent D 150\u00a0mg BID\u2009+\u2009T 2\u00a0mg QD for 2\u00d7 1\u00a0W (cohort 2), 2\u00d7 2\u00a0W (cohort 3), or continuous for 6\u00a0W (cohort 4). All cohorts continued PEM for up to 2\u00a0years. Primary endpoints were safety and treatment-adherence. Secondary endpoints were objective response rate  at week 6, 12, 18 compared to baseline and PFS.Results: The data from the first 26 pts completed the first 18\u00a0W were presented at ESMO 2018. Grade 3/4 TRAE within the first 18\u00a0W were observed 0%, 14%, 33%, and 50% of pts in cohort 1, 2, 3, and 4, respectively. All planned D\u2009+\u2009T was given in 86%, 50%, and 33% of pts in cohort 2, 3, and 4. ORR at W6, W12, and W18 were 29%, 57%, and 57% in cohort 1, 29%, 71%, and 71% in cohort 2, 33%, 50%, and 83% in cohort 3 and 0%, 50%, and 50% in cohort 4.We will present the updated ORR and toxicity data from all 32 pts. In addition, we will present for the first time PFS and OS data from the complete four cohorts with a median FU of 18\u00a0months.Conclusion: The ESMO 2018 IMPemBra data indicated that PEM\u2009+\u2009intermittent D\u2009+\u2009T for 2\u00d7 1\u00a0W or 2\u00d7 2\u00a0W are promising combinations in terms of safety and feasibility, warranted to be tested in subsequent trials.Clinical trial information: NCT02977052Correspondence: Igor Puzanov - Igor.Puzanov@roswellpark.orgJournal of Translational Medicine 2020, 18(Supp 1):21Background: Although checkpoint inhibitor (CPI) therapy has emerged as an effective treatment option for various cancers, there is an unmet need for therapies to produce more durable and deeper responses in metastatic melanoma. Safety and clinical activity of bempegaldesleukin , a CD-122 preferential IL-2 pathway agonist, plus the anti-PD1 CPI nivolumab (NIVO), was evaluated in PIVOT-02 (NCT02983045), a multicenter phase 1/2 study in multiple solid tumor settings. At SITC 2018, PIVOT-02 reported encouraging preliminary clinical activity and safety data in metastatic melanoma  . We plan to report updated results in 1L metastatic melanoma patients, and the first report of PFS.Methods: 41 patients with previously untreated stage IV metastatic melanoma received\u2009\u2265\u20091 dose of BEMPEG (0.006\u00a0mg/kg)\u2009+\u2009NIVO (360\u00a0mg) q3w. Patients were categorized by PD-L1 status. Response was assessed every 3 cycles by RECISTv1.1. Per protocol, ORR was evaluated in the efficacy-evaluable population (\u2265\u20091 post-baseline scan) by independent central radiology review . Baseline immunohistochemistry (IHC) analysis for PD-L1 was performed (using Dako PD-L1 IHC 28-8 pharmDx) and defined as PD-L1 negative (<\u20091% tumor cell expression) and PD-L1 positive (\u2265\u20091% tumor cell expression). Safety and tolerability were assessed by CTCAEv4.03.Results: At a median follow-up of 12.7\u00a0months*, 38 patients were evaluable for efficacy. Table\u00a0Conclusions: BEMPEG plus NIVO is associated with robust clinical activity in 1\u00a0L metastatic melanoma, as demonstrated by a high rate of durable responses that deepened over time. Based on these data, the FDA granted Breakthrough Therapy Designation for this combination therapy for patients with untreated unresectable or metastatic melanoma, and a Phase 3 trial evaluating the combination of BEMPEG plus NIVO vs NIVO alone in this setting is currently enrolling (NCT03635983).ReferencesDiab A, Tykodi S, Curti B, et al. Immune monitoring after NKTR\u2010214 plus nivolumab (PIVOT\u201002) in previously untreated patients with metastatic stage IV melanoma oral presentation at SITC; November 7\u201311, 2018; Washington, DC, USA. Abstract #O4Hurwitz M, Cho DC, Balar, AV, et al. Baseline tumor-immune signatures associated with response to bempegaldesleukin (NKTR-214) and nivolumab. J Clin Oncol. 2019; 37:.Correspondence: Paolo A. Ascierto - paolo.ascierto@gmail.comJournal of Translational Medicine 2020, 18(Supp 1):22Background: Anti-PD-1 alone or in combination with anti-CTLA4 is the current standard of care for advanced unresectable or metastatic cutaneous melanoma. Despite remarkable response rates, a significant proportion of patients does not achieve any or only timely limited disease control, are refractory or do not respond to anti-PD-1-containing therapy. Epigenetic modulation, and Histone Deacetylase (HDAC) inhibition in particular is intended to enhance the immunogenicity of the tumor, alter the tumor microenvironment and thus increase the chance of clinical activity to such immunotherapy, having the potential of a new clinical approach to overcome tumor escape mechanisms.Methods: In an open label Phase Ib/II multi-center study (\u2018SENSITIZE\u2019) we investigate the combination of the selective class I HDAC inhibitor domatinostat and pembrolizumab in progressive patients with unresectable or metastatic cutaneous melanoma with best response of progressive disease (PD) or stable disease (SD) to prior checkpoint inhibitor therapy. These advanced stage melanoma patients were treated with domatinostat  with increasing dose levels in the first 3 different dose cohorts in combination with pembrolizumab in a modified \u201crolling six\u201d study design to evaluate the safety and tolerability of the combination treatment. The trial is designed to determine the optimal biological dose  and dosing schedule (dose optimization). Tumor assessments are performed every 12\u00a0weeks and evaluated per irRECIST. Sequential tumor biopsies are taken for immunohistochemical and gene expression analysis and peripheral blood for pharmacokinetic (PK) analysis.On 15-July-2019, 23 patients with progressive, unresectable or metastatic cutaneous melanoma with best response of PD or SD to prior anti-PD-1 therapy have been treated in the SENSITIZE study.Results: Domatinostat in combination with pembrolizumab is safe and well tolerated up to 200\u00a0mg twice daily (BID) in a 14\u2009+\u20097 dosing schedule). 4/23 patients experienced grade 3 treatment related AEs and no grade 4 TRAEs occurred, furthermore no increase in frequency or intensity of immune-related AEs were observed. Additionally, first signs of clinical activity have been observed showing a trend towards dose-dependency of domatinostat with a disease control in 4 out of 7 patients .Conclusion: Preliminary analyses of tumor biopsies suggest a trend towards a domatinostat-induced change in immunological tumor patterns corroborating findings from earlier preclinical work. These observations in this advanced, heavily pre-treated patient population warrant further development of domatinostat in combination with anti-PD-(L)1 antibodies to sensitize the tumor, the tumor microenvironment and the patient\u2019s immune response for synergistic anti-tumor activity :23Background: Although targeted therapies (TT) and immunotherapies (IMT) have improved survival for pts with BRAF V600 mutated stage IV MM, many pts progress and will ultimately die from this disease. Preclinical data has shown that BRAF inhibition (BRAFi) in BRAF-mutated tumors is associated with increased T cell infiltration, supporting the rationale for a clinical combinatorial approach with IMT. Although there are multicentered trials ongoing evaluating this combinatorial approach for pts with untreated MM, there are no approved therapies for pts after TT and IMT failure. Notably, pts with untreated brain metastases (BM) are often excluded from such trials. We hypothesized that N in combination with DT is safe and will demonstrate clinical activity in BRAF-mutated pts refractory to PD1 therapy and in pts with BM.Methods: We report a single arm phase II study (NCT02910700) of NDT in pts with BRAF-mutated, unresectable stage III or stage IV MM. Prior IMT is allowed, but pts who have received BRAF/MEKi are ineligible. Pts with untreated BM and asymptomatic or mildly symptomatic/requiring stable or decreasing steroids  are also allowed. Pts received 3\u00a0mg/kg Q2wks of N (later amended to 480\u00a0mg q4wks), 150\u00a0mg BID of D and 2\u00a0mg QD of T, all starting on Day 1. The primary objective of this study is to determine safety and efficacy (ORR by RECIST 1.1) of the NDT combination. This study was continuously monitored for safety and futility. Tissue and blood-based samples to assess for correlative studies are also collected.Results: Following a 6 pts safety run-in with no observed DLTs, 26 pts received NDT\u201416 pts were PD1 refractory, 10 were PD-1 naive. 9 of these 26 pts had BM. Of the 22 pts evaluable for response, 17 achieved PR and 3 CR (ORR 91%). 12 PD1 refractory were evaluable for response; 2 achieved CR and 9 PR (ORR 83%). 67% of the evaluable pts with BM achieved an intracranial response, including 2 CRs. Although the median PFS for all pts was ~\u20098\u00a0months, the median OS was not reached. 65% of pts experienced treatment related grade 3/4 AEs, but only 3 pts discontinued due to toxicities.Conclusions: NDT is well-tolerated and shows promising clinical activity in pts with IMT refractory disease and with BM. There were no significant differences in outcomes between pts with and without BM. Further translational investigation to better delineate mechanisms of response are ongoing.Journal of Translational Medicine 2020, 18(Supp 1):24Background: Bispecific antibodies have shown activity in hematologic (heme) but not solid tumors. ImmTAC molecules are unique TCR-anti-CD3 bispecifics that redirect T cells against intracellular antigens. Tebentafusp (IMCgp100), an ImmTAC targeted against melanocyte-associated lineage antigen gp100, has shown monotherapy responses in advanced melanoma with associated immune changes. Tebentafusp causes rash and cytokine-mediated AEs, hypothesized to be on-target (gp100) or effector (CD3) mediated. We explored clinical and biological characteristics of pts associated with treatment benefit.Materials and methods: 84 HLA-A2 positive advanced melanoma pts received tebentafusp on study IMCgp100-01 in 13 dose escalation cohorts. Efficacy was assessed by Kaplan\u2013Meier survival and treatment related AEs (TRAE) reported by CTCAE v4.0. Serum samples evaluated changes in cytokines. A multivariate analysis investigated the relationship between efficacy and safety variables.Results: Demographics: 73% cutaneous (CM), 23% uveal (UM) primaries; 51% LDH\u2009>\u2009ULN; 25% received prior anti-PD(L)1.83 (99%) pts had\u2009\u2265\u20091 TRAE; most commonly in skin  or cytokine-mediated (pyrexia 57%); the majority were Grade 1\u20132 and occurred and resolved within first 3 doses. The 2 most frequent Grade\u2009\u2265\u20093 TRAEs were rash (26%) and lymphopenia (13%). Tebentafusp induced transient increases in peripheral cytokines (peaking Day 1\u20132) that attenuated with subsequent doses; cytokine-mediated AE had similar kinetics.1-yr OS was 65% (95% CI 48\u201378). In multivariate analysis, longer OS was associated with: LDH\u2009\u2264\u2009ULN (p\u2009=\u20090.002) and any-grade rash occurring within 21\u00a0days (p\u2009=\u20090.003); melanoma primary site and prior anti-PD-(L)1 did not significantly affect outcome. In exploratory analyses, longer OS associated with lower baseline serum IL-6 (n\u2009=\u200943) or TNF\u03b1 (n\u2009=\u200944).Conclusions: Tebentafusp is a first-in-class, TCR-based bispecific with monotherapy efficacy in advanced melanoma. AEs were manageable and consistent with MoA. Association between tebentafusp efficacy and on-target TRAEs, previously reported for bispecifics to heme lineage antigens, is now recognized for solid tumor lineage antigens. Pivotal studies in UM are ongoing.Trial Registration: NCT01211262Journal of Translational Medicine 2020, 18(Supp 1):25Background: The relevant role played by microRNAs (miRNAs) in cancer, as in other diseases, make them possible new drugs or drug targets as well as diagnostic and prognostic disease biomarkers. MiR-193a acts as potential tumour suppressor in malignant pleural mesothelioma, gastric and non-small cell lung cancer and it regulates drug and chemoradiation resistance in bladder and oesophageal cancer, respectively [1]. As regards melanoma, actually a study evaluating the expression of miR-193a in cutaneous melanoma tissues and cell lines [2] and a pilot investigation from our laboratory on its levels in plasma of melanoma patients compared to healthy controls have been realized [3].Nevertheless, no data are reported on the role of miR-193a on the control of melanoma cell proliferation and metastasis. Here, effect of miR-193a ectopic expression was investigated in vitro and in vivo melanoma model. Parallely, its expression in plasma exosomes derived from stage IV melanoma patients was analysed in order to confirm its role as diagnostic biomarker.Materials and methods: In order to evaluate the tumour suppressor role of miR-193a in melanoma cells, we studied its influence on intracellular pathways regulating survival, proliferation, apoptosis and migration, such as MAPK/ERK, and PI3K/Akt, and on markers involved in epithelial-mesenchymal transition (EMT). The in vivo miR-193a anti-cancer effects were evaluated in the murine B16.OVA melanoma model by using a viral  platform. Exosomes were isolated from plasma samples of melanoma patients and healthy donors, and their miR-193a levels were determined via quantitative real-time PCR.Results: In vitro experiments showed a significant decrease of melanoma cell viability and migration and an increase of apoptosis in transfected cells. Furthermore, a significant decrease in B-Raf protein levels and in phosphorylation of Akt and Erk proteins was observed, suggesting the miR-193a ability to interfere with cell proliferation and survival. Vimentin and E-Cadherin transcriptional and protein levels were significantly modulated, indicating the potential of this miRNA to contrast EMT. A significant decrease of the miR-193a target PD-L1 in the in vivo murine melanoma model, suggests an efficient delivery of the functional miR by the viral platform. Finally, a statistically significant decrease in the miR-193a levels was observed in exosome-derived plasma of metastatic melanoma patients compared to healthy donors.Conclusions: Our data suggest that miR193a represents a potential therapeutic agent reducing melanoma progression and confirm its diagnostic biomarker role in this cancer type. Experiments aimed at deepened its anti-melanoma potential in the in vivo model are ongoing.ReferencesYu T, Li J, Yan M, et al. MicroRNA-193a-3p and -5p suppress the metastasis of human nonsmall- cell lung cancer by downregulating the ERBB4/PIK3R3/mTOR/S6K2 signaling pathway. Oncogene. 2015;34(4):413\u201323.Caramuta S, Egyh\u00e1zi S, Rodolfo M, et al. MicroRNA expression profiles associated with mutational status and survival in malignant melanoma. J Invest Dermatol. 2010;130(8):2062\u201370.Fogli S, Polini B, Carpi S, et al. Identification of plasma microRNAs as new potential biomarkers with high diagnostic power in human cutaneous melanoma. Tumour Biol. 2017;39(5):1010428317701646.Journal of Translational Medicine 2020, 18(Supp 1):26Introduction: Treatment with anti-PD1 induces responses in about 40% of advanced metastatic melanoma with median response induction time of 2 to 3\u00a0months. However, late responses are described, as well as atypical responses. In the real life, the decision whether to prolong or when to stop treatment in patients with slow progressing disease is still a challenge.Objectives: To evaluate anti-PD1 clinical activity in this real-life setting; to summarise the findings of CT-scans performed at 3 and 6\u00a0months after treatment starting; to correlate 3 and 6\u00a0month CT findings with BOR, Progression-Free Survival (PFS) and Overall Survival (OS).Materials and methods: Retrospective single centre study which included 112 consecutive advanced metastatic melanoma treated as first line with anti-PD1 as monotherapy since 2015. Clinical features, stage of disease, number of metastatic sites, LDH values were evaluated at baseline before treatment. CT-scan findings were evaluated at 3 and 6\u00a0months and categorised as follows: reduction/regression of lesions; stable lesions; increase of dimensions of pre-existing lesions; occurrence of new lesions; both increase of pre-existing and occurrence of new lesions.Results: The BOR was CR in 15.2% and PR in 20.5% of patients. The response rate was 35.7%, the clinical benefit was confirmed in 49.7% of patients. CT findings at 3\u00a0months were significantly correlated with BOR: 35/43 patients (81%) with reduction or stable lesions achieved a clinical benefit whilst only 8/43 (19%) developed a PD; on the other hand, among patients with new lesions, increase of pre-existing or both, only 6/53 (11%) developed a clinical benefit whilst 47 (89%) progressed (p\u2009=\u20090.0001). The same figures were obtained when analysing CT scans at 6\u00a0months. Atypical responses occurred in 6 out of 112 patients (5.3%). These patients were characterised by <\u20093 metastatic sites (6/6), good PS (6/6), normal LDH values (5/6), no brain metastases and prevalence of M1a/b score (4/6). Median OS for the entire patient cohort was 1.7\u00a0years (4\u00a0months\u20134\u00a0years) with a 3-year OS of 35%, median PFS was 10.5\u00a0months. Median OS calculated since the 3-month CT-scan showed significant benefit in patients with stable/regressing lesions with respect to the others .Conclusion: The results of this study suggest the relevant predictive value of 3-month CT-scan findings which are correlated with disease outcome in terms of BOR, clinical benefit and OS. In patients with dimension increase or new lesions at 3 or 6\u00a0months CT-scan, treatment continuation should be considered in cases with favourable PS and low tumour burden.This work was funded with the TESEO project Medicina di precisione nelle neoplasie mediante omica e big data, Progetto Strategico di Eccellenza Dipartimentale, DSM, UNITO."}
{"text": "Corynespora cassiicola, as a necrotrophic phytopathogenic ascomycetous fungus, can infect hundreds of species of plants and rarely causes human diseases. This pathogen infects cucumber species and causes cucumber target spot, which has recently caused large cucumber yield losses in China. Genome sequence and spore germination-associated transcriptome analysis will contribute to the understanding of the molecular mechanism of pathogenicity and spore germination of C. cassiicola.C. cassiicola isolate HGCC with high virulence. Although conspecific, HGCC exhibited distinct genome sequence differences from a rubber tree-sampled isolate (CCP) and a human-sampled isolate (UM591). The proportion of secreted proteins was 7.2% in HGCC. A total of 28.9% (4232) of HGCC genes, 29.5% (4298) of CCP genes and 28.6% (4214) of UM591 genes were highly homologous to experimentally proven virulence-associated genes, respectively, which were not significantly different (P\u2009=\u20090.866) from the average (29.7%) of 10 other phytopathogenic fungi. Thousands of putative virulence-associated genes in various pathways or families were identified in C. cassiicola. Second, a global view of the transcriptome of C. cassiicola spores during germination was evaluated using RNA sequencing (RNA-Seq). A total of 3288 differentially expressed genes (DEGs) were identified. The majority of KEGG-annotated DEGs were involved in metabolism, genetic information processing, cellular processes, the organismal system, human diseases and environmental information processing.First, we reported the draft genome sequences of the cucumber-sampled C. cassiicola in cucumbers and the understanding of molecular and cellular processes during spore germination.These results facilitate the exploration of the molecular pathogenic mechanism of  C. cassiicola (BerK & Curt) Wei has recently caused tremendous cucumber yield losses in China [C. cassiicola, as a necrotrophic parasitic fungus, can infect more than 500 plant species, including tomato, eggplant, tobacco, rubber, cotton, soybean and balsam pear, in addition to cucumber and cause plant spot diseases [C. cassiicola is an opportunistic pathogen in humans and causes fungal keratitis under suitable conditions [C. cassiicola against plants should be well studied to effectively control diseases caused by this pathogen.Cucumber target spot caused by in China . More imdiseases , which hnditions  and subcnditions . TherefoC. cassiicola has mainly focused on biological characteristics, pathogenicity differentiation, cloning of virulence-associated genes, etc. It was reported that C. cassiicola sampled from cucumber could grow at 10\u201335\u2009\u00b0C, and its optimum growth temperature was approximately 30\u2009\u00b0C [C. cassiicola spores germinated from one end or both ends at 25\u201330\u2009\u00b0C with >\u200990% relative humidity, and the germination rate in drops of water was the highest. C. cassiicola was able to invade cucumber leaves primarily through direct contact or via stomata [C. cassiicola showed pathogenic and genetic variation among isolates sampled in different host plants, proving that the intraspecific strains of C. cassiicola showed high host specialization [C. cassiicola isolates from perilla, cucumber, tomato, aubergine and sweet pepper in Japan were divided into seven pathogenicity groups (PG1-PG7) [C. cassiicola, and contains six different cassiicolin isoforms, Cas1, Cas2, Cas3, Cas4, Cas5 and Cas6, in different C. cassiicola isolates sampled from various hosts and geographical origins [Cas1 gene were the most aggressive to rubber trees. Additionally, some isolates with no Cas gene also generated moderate symptoms on rubber tree leaves, showing that uncharacterized effectors existed in C. cassiicola [For the past few years, research on the pathogenic mechanism of  stomata . C. casslization . Sixty-fPG1-PG7) . Cassiic origins . The aggC. cassiicola requires multiple pathogenic factors, such as cutinase, cell wall-degrading enzymes [2+ and cAMP signal pathways etc. [C. cassiicola have rarely been cloned and functionally characterized except for two MAPK genes CCk1 [CMP1 [Cas [C. cassiicola. Thus, a great number of virulence-associated genes remain to be identified, cloned and functionally characterized.Similar to other filamentous fungal pathogens,  enzymes  and cytoays etc. . To datenes CCk1  and CMP1k1 [CMP1 , and theMP1 [Cas , 17, whiC. cassiicola thus far.The fungal conidium involved in reproduction is the main type of fungal asexual spore, and is the main form of inoculation and infection . RestingC. cassiicola be globally analyzed. Although the genome sequencing of two C. cassiicola isolates, CCP from a rubber tree and UM591 from the contact lens of a patient with keratomycosis, was completed [C. cassiicola isolate (HGCC) with high virulence to cucumber, and comparatively analyzed its genome with two other C. cassiicola isolates (CCP from a rubber tree and UM591 from a human) and other phytopathogenic ascomycete fungi in multiple families or pathways of virulence-associated genes in this study. Second, we studied the relative transcriptional levels of genes during the spore germination of C. cassiicola HGCC using RNA-Seq. These research results provide a great deal of information revealing the molecular mechanism of pathogenicity and spore germination of C. cassiicola.Genome sequencing is a high throughput means to identify functional genes combined with homologous matches against functional databases or conserved domain searches , 23. Furompleted , 25, it C. cassiicola sampled from cucumber. By being subcultured on potato dextrose agar (PDA) plates at 25\u2009\u00b0C in the dark, HGCC produced a layer of fluffy aerial mycelium that was whitish gray when young and dark green when old  from the average number  of 10 other phytopathogenic fungi, including Curvularia lunata, Bipolaris maydis, Cercospora zeae-maydis, Parastagonospora nodorum, Setospaeria turcica, Pyrenophora tritici-repentis, Magnaporthe oryzae, Aspergillus flavus, Fusarium graminearum and Botrytis cinerea  using an Illumina HiSeq X 10 system. A total of 54,580,316 high-quality reads were assembled into 1032 scaffolds (N50: 500\u2009kb) with a genome size of 42.7\u2009Mb, which was slightly less than that of CCP (44.8\u2009Mb) (JGI: 1019537) and greater than that of UM591 (41.4\u2009Mb) (GenBank: JAQF00000000.1) (Table C. lunata (57.4%), B. maydis (57.1%), S. turcica (57.0%), P. nodorum (57.8%), P. tritici-repentis (56.8%), C. zeae-maydis (47.0%), A. flavus (47.5%), B. cinerea (47.2%), F. graminearum (46.0%) and M. oryzae (44.9%). More than 70% of HGCC genes had a\u2009>\u200990.0% amino acid sequence identity with CCP (75.5%) and UM591 (72.4%), which was far more than the 55% between two C. lunata isolates, CX-3 from maize and m118 from sorghum [C. cassiicola isolates had distinct differences in their genomes, although HGCC had a high amino acid sequence identity with both CCP and UM591.The HGCC genome had a 91.6 and 90.5% amino acid sequence identity with CCP and UM591, respectively, and a 44.9\u201357.8% identity with other phytopathogenic fungi, such as  sorghum . A totalnerea 47.%, F. granerea 47.%, F. granerea 47.%, F. graC. cassiicola , the major facilitator superfamily (MFS) (P\u2009=\u20090.043), cytochrome P450 enzymes (CYPs) (P\u2009=\u20090.011), G protein-coupled receptors (GPCRs) (P\u2009=\u20090.028), Pth11-like G protein-coupled receptors (P\u2009=\u20090.011), protein kinases (P\u2009=\u20090.011), proteases (P\u2009=\u20090.011), cysteine proteases (P\u2009=\u20090.017), metallo proteases (P\u2009=\u20090.022), serine proteases (P\u2009=\u20090.011), GHs (P\u2009=\u20090.011), pectate lyases (P\u2009=\u20090.011), pectinesterases (P\u2009=\u20090.044), cellulases (P\u2009=\u20090.045), glucosidases (P\u2009=\u20090.010) and secondary metabolite backbone genes (P\u2009=\u20090.042) between C. cassiicola and the 10 other phytopathogenic fungi, and the number of proteins in the each family in the former was more than that in the latter, suggesting that C. cassiicola had family expansions in these families. These protein families were expected to play important roles in fungal survival in various adverse environments.A total of 11,111 gene-encoding proteins were classified into 3892 conserved protein families in HGCC by Pfam matches with profile hidden Markov models, which was close to the 3847 families containing 11,187 proteins in CCP and the 3860 families containing 11,110 proteins in UM591. Glycoside hydrolase (GH), pectate lyase, cutinase and cellulose were important pathogenic factors that were highly abundant in C. cassiicola and other phytopathogenic fungi were conducted against the pathogen-host interaction (PHI) database at a E-value of 1e-5. A total of 28.9% (4232) of HGCC genes, 29.5% (4298) of CCP genes and 28.6% (4214) of UM591 genes were matched with PHI database, respectively. There was no significant difference (P\u2009=\u20090.866) in the percentage of PHI-associated genes between the three C. cassiicola and the 10 other phytopathogenic fungi (average: 29.7%)  from the average (113) of the 10 other phytopathogenic fungi , C  and PTH11  classes in C. cassiicola were significantly different (P\u2009<\u20090.05) from the average  of the other phytopathogenic fungi, respectively. More than 80% of Pth11-like GPCRs were identified as PHI-associated genes in HGCC (53/62), CCP (50/61), UM591 (48/57)), and the 10 other phytopathogenic fungi (average: 32/37), respectively. The G protein alpha subunit is an important component of the heterotrimeric G protein complex [Pseudocercospora fijiensis  and two G protein alpha subunits of Stemphylium lycopersici , respectively.GPCRs transduce external environmental signals by way of heterotrimeric G proteins into secondary messengers to regulate gene expression and the subsequent cellular response . These pgenicity , and its average 13 of the complex  and can  complex , 31. In 2+ signaling are part of major signaling pathways and are associated with virulence in phytopathogenic fungi, which are controlled by a series of protein kinases [Saccharomyces cerevisiae, FUS3/KSS1, Mpk1 and Hog1, have been well studied and are highly conserved in other fungi [2+ pathway-associated genes in S. cerevisiae, 48, 12 and 23 screened genes in HGCC were highly homologous with MAPK, cAMP and Ca2+ signal pathway genes of S. cerevisiae, respectively  from 116 to 140 in other phytopathogenic fungi  in the numbers of protein kinases in the Other and STE classes between C. cassiicola and the other phytopathogenic fungi, respectively. The STE class  and MAPK family  in the CMGC class were involved in the MAPK pathway. The PKA family  of the AGC class was involved in the cAMP pathway. The CLK family  and the RCK family  of the CMGC class, the CAMK class , and the PKC family  in the AGC class were related to the Ca2+ pathway. Interestingly, almost all protein kinases were PHI-associated genes in HGCC (150/154), CCP (154/156), UM591 (148/151), and the 10 other phytopathogenic fungi (average: 125/129), suggesting that protein kinases played key roles in pathogenic processes mainly mediated by the three pathogenic signal pathways.MAPK, cAMP and Ca kinases . Three Mer fungi . Based oP\u2009=\u20090.492) from the average (10) of the other phytopathogenic fungi  phosphorelay signaling is usually used by fungi, bacteria, plants and slime molds to sense and adapt to their environment . Fungal  average 0 of the P\u2009=\u20090.051) from the average (11) in the 10 other phytopathogenic fungi. There was significant difference (P\u2009=\u20090.011) in the number of pectate lyases, a type of pectinase, between C. cassiicola  and the other phytopathogenic fungi (average: 12). However, pectinesterase, another type of pectinase, was less abundant than pectate lyase in C. cassiicola  and the other phytopathogenic fungi (0\u20135). Fourteen, 17 and 16 cellulases were identified in HGCC, CCP and UM591, respectively, which were significantly different (P\u2009=\u20090.045) from the average (12) in the other phytopathogenic fungi. The hemicellulase glucanase , glucosidase  and xylanase  were abundant in C. cassiicola, of which only glucosidase showed significant difference (P\u2009=\u20090.010) in number between C. cassiicola and the other phytopathogenic fungi (average: 9). More than half of cutinases and xylanases and almost all of pectate lyases, pectinesterases, glucanases and glucosidases were PHI-associated genes in these phytopathogenic fungi.To successfully invade the host plant, phytopathogenic fungi are expected to produce and secrete multiple extracellular degrading enzymes, such as cutinase for the degradation of the cuticle , cell waC. cassiicola genomes , which were significantly different (P\u2009=\u20090.011) from the average value (521) of the other phytopathogenic fungi  from the average of the other phytopathogenic fungi   from the average (20) of the other phytopathogenic fungi  from the average (194) in the other phytopathogenic fungi. Many GH families exhibited gene expansion in C. cassiicola when compared to the other phytopathogenic fungi, including the GH1, GH2, GH3, GH7, GH12, GH20, GH27, GH35, GH43, GH53, GH61, GH72, GH88, GH106 and GH107 families; however, the GH30 and GH 65 families exhibited gene constriction. More than half of GHs were PHI-associated genes in HGCC (141/262), CCP (138/262), UM591 (141/265), and the other phytopathogenic fungi (average: 100/194). Interestingly, almost all GHs of the GH3, GH7, GH10, GH17, GH18, GH20, GH28, GH31, GH61 and GH72 families were PHI-associated genes in C. cassiicola and the 10 other phytopathogenic fungi. It was reported that the GH6, GH7, GH45 and GH61 cellulases and GH10 xlyanases were absent in insect pathogenic fungi but present in phytopathogenic fungi [GH catalyzes the hydrolysis of glycosidic bonds in complex sugars , playingic fungi . The GH C. cassiicola contained a large number of transporters , which were significantly different (P\u2009=\u20090.043) from the average (548) of the other phytopathogenic fungi  and ATP-binding cassette (ABC) superfamily  superfamily were the two largest superfamilies of transporters. The former is capable of transporting small solutes in response to chemiosmotic ion gradients, and the latter transports small molecules and macromolecules under ATP hydrolysis [P\u2009=\u20090.043) in the number of MFS transporters between C. cassiicola and the other phytopathogenic fungi (average: 160). However, there was no significant difference (P\u2009=\u20090.127) in the number of ABC transporters between C. cassiicola and the other phytopathogenic fungi (average: 43). Notably, almost all MFS transporters and all ABC transporters were PHI-associated genes in these phytopathogenic fungi, showing that MFS and ABC transporters played key roles in fungal pathogenicity.drolysis , 42. The+ antiporter (DHA) subfamilies (DHA1 and DHA2), drug transporters of the MFS superfamily, can secrete toxic compounds into the outer environment [P\u2009=\u20090.042) in the number of DHA1 transporters between C. cassiicola and the other phytopathogenic fungi (average: 32). Interestingly, all drug transporters were PHI-associated genes except for DHA2 (4/5).In phytopathogenic fungi, drug transporters of ABC and MFS superfamilies can secrete endogenous fungal virulence factors such as toxins and protect pathogens against exogenous plant defense compounds such as phytoalexins , 43. Twoironment . The mulironment . There wilies DHA and DHA2C. cassiicola HGCC (218), CCP (214) and UM591 (209) genomes and classified into 112 families; there was significant difference (P\u2009=\u20090.011) in the number of CYPs between C. cassiicola and the other phytopathogenic fungi (average: 122)  in the number of CYP65s between C. cassiicola  and the other phytopathogenic fungi (average: 11). Additionally, CYP505 was scarce in C. cassiicola  and the other phytopathogenic fungi (average: 3).CYPs are proteins of a superfamily, are ubiquitous in all biological kingdoms, and contain heme as a cofactor and are therefore hemoproteins. Fungal CYPs play key roles in various metabolic processes, such as housekeeping biochemical reactions, detoxification of chemicals and adaptation to adverse surroundings . A largela HGCC 28, CCP  in the number of backbone genes between C. cassiicola  and the other phytopathogenic fungi (average: 42)  were significantly different (P\u2009=\u20090.011) from that of the other phytopathogenic fungi (average: 17).Melanin and mycotoxin are important virulence factors in phytopathogenic fungi . To dataC. cassiicola PKSs and role-known PKSs in other pathogenic fungi  were specific to CCP when compared to HGCC. The second cluster contained nonreducing PKSs with KS and AT domains at a minimum and did not include dehydratase (DH), enoyl reductase (ER) and ketoreductase (KR) domains; this cluster contained 10 PKSs of C. cassiicola and 9 known PKSs related to melanin biosynthesis in other fungal pathogens such as Aspergillus fumigatus Alb1p, Ceratocystis resinifera PKS1, Colletotrichum lagenarium PKS1, Chaetomium globosum PKS-1, Ascochyta rabie PKS1, B. maydis PKS18, S. turcica StPKS, Bipolaris oryzae PKS1, and A. alternata ALM1. It was difficult to identify which reducing PKSs were involved in mycotoxin biosynthesis in C. cassiicola due to the complicated evolutionary relationship for the KS domain of the reducing PKSs in C. cassiicola and known mycotoxin-related PKSs in other fungi. Nevertheless, the nonreducing PKS HGCC_7666 of C. cassiicola had the closest evolutionary relationship with known melanin-associated nonreducing PKSs, suggesting that HGCC_7666 was probably the backbone gene for melanin synthesis in C. cassiicola.To analyze the relationship between the PKS domain and its function, phylogenetic analysis for the ketoacyl CoA synthase (KS) domain of PKS was performed among ngi Fig.\u00a0. These PAvr genes, trigger or suppress effector-triggered immunity (ETI) mediated by a gene-for-gene system in PHI [Avr-encoded proteins (AvrLm4\u20137) that were putative effector proteins  can be secreted directly into host plant cells to function in host recognition or colonization  and the m in PHI . Fifty-nLysin motif (LysM)-containing effectors are ubiquitous in phytopathogenic fungi and are secreted out of the cell to suppress the immune response of the host , 58. ComC. cassiicola, 6\u2009h- and 12\u2009h-germination times were selected for RNA-Seq based transcriptome analysis. After 12\u2009h at 25\u2009\u00b0C in sterile ddH2O, the majority of spores germinated at two ends, and only a few germinated at one end , suggesting that samples in the same treatment group were repeatable and that the RNA-Seq data were reliable. A total of 66\u201373% of C. cassiicola filtered reads per library mapped to the gene predictions of C. cassiicola. A total of 3288 genes were differentially expressed, of which 1552 and 1736 were up-regulated and down-regulated, respectively, during spore germination of C. cassiicola with ungerminated spores as the reference  and pathway. A total of 2600 DEGs 79.08%) were annotated by gene ontology (GO). GO term analysis for 9.08% wer2\u2009=\u20090.7434). The expression patterns of 56 DEGs were confirmed by qRT-PCR, and 10 were not , and catalase [nheC, cytK and hblC during Bacillus thuringiensis spore germination [C. cassiicola in this study. A total of 3288 genes were found to be differentially expressed, which was similar to the 3026 DEGs observed during spore germination of P. expansum [C. cassiicola and P. expansum that the majority of spore germination-associated DEGs were enriched in cellular processese, localization, metabolic processes, regulation of biological processes, single-organism processes, binding, and catalytic activity. Similar pathway classification results for DEGs were also found between C. cassiicola and P. expansum, and the majority of KEGG-annotated DEGs were classified in metabolism, genetic information processing, environmental information processing, cellular processes, and human diseases. This phenomenon showed that RNA-Seq data for spore germination were reliable and probably provided valuable information on the molecular mechanism of C. cassiicola germination.Gene expression profiles can provide insight into the molecular and cellular processes of spore germination at either the transcriptional level or at the proteome level. Zhou et al. found that a total of 3026 genes were differentially expressed during spore germination of ocessing . Liu et  RNA-Seq . Joise Hcatalase . Bassi emination . These rexpansum . It was C. cassiicola spore germination. Similar results were found in the spore germination process of Penicillium expansum [C. cassiicola spore germination, and most of these DEGs were up-regulated, which was nearly opposite to Zhou et al.\u2019s results that all DEGs involved in energy metabolism were down-regulated both in the transcriptional and protein levels [Carbohydrate metabolism involves the various biochemical processes responsible for the formation, breakdown and interconversion of carbohydrates, which serve as short-term fuel for organisms . Therefoexpansum . Energy n levels .C. cassiicola from cucumber was presented, and thousands of genome-wide virulence-associated genes were mined by homologous searches against multiple functional databases and conserved domain searches. A total of 3288 genes were differentially expressed during the spore germination of C. cassiicola. Most of the KEGG annotated DEGs were involved in metabolism, genetic information processing, cellular processes, organismal system, human diseases and environmental information processing. These results not only facilitate the understanding of the molecular pathogenic mechanism of C. cassiicola in cucumber and the molecular and cellular processes during the spore germination, but also lay the foundation for disease control.In conclusion, the genome sequence of C. cassiicola (Berk. & M.A. Curtis) strain HGCC, which we isolated from infected cucumber leaves in Shanghai in 2010, was highly virulent to cucumber; thus, it was used for genome sequencing. The isolate was cultured on PDA medium in test tubes at 25\u2009\u00b0C for 7 d and then maintained at 4\u2009\u00b0C. The HGCC isolate was subcultured on PDA plates at 25\u2009\u00b0C in the dark for 10 d, and the mycelium was then collected by peeling it off using a glass slide and ground in liquid nitrogen. Genomic DNA was extracted from finely ground material using the CTAB method [B method . DNA qua2O was added to each plate and spores were peeled off using sterile writing brushes and filtered with three layers of sterile gauze to prepare the spore suspension. Spores were collected through centrifugation at 4602\u00d7g for 5\u2009min. Spores were resuspended and adjusted to 105 spores/mL with sterile ddH2O. The spore suspension was incubated at 25\u2009\u00b0C in the dark for 6\u2009h and 12\u2009h to perform the spore germination test. Equal volumes of the germinated spore suspensions incubated for 6\u2009h or 12\u2009h were mixed. The germinated spores in the mixed suspension were collected by centrifugation at 4602\u00d7g for 5\u2009min. The germinated spores and ungerminated spores were ground for 1\u2009min using a Geno/Grinder 2010  after being frozen rapidly with liquid nitrogen for the extraction of total RNA. Total RNA was extracted using the TaKaRa MiniBEST universal RNA extraction kit following the manufacturer\u2019s recommended method and stored at \u2212\u200980\u2009\u00b0C. The test was conducted in biological triplicate.Spore production tests were conducted on PDA plates. After being activated on PDA plates at 25\u2009\u00b0C, the HGCC strain was subcultured on PDA plates at 25\u2009\u00b0C for 10 d with 12\u2009h of light and 12\u2009h of dark per day. Five milliliters of sterile ddHTotal RNA was quantified and assessed for purity using a Nanodrop instrument, and the integrity was evaluated using an Agilent 2100 instrument. RNA samples returning an RNA integrity number (RIN) greater than 6.3 were considered acceptable for sequencing. A cDNA library was made from each qualified RNA sample. The libraries were sequenced using an Illumina HiSeq X 10 instrument at the Novogene Bioinformatics Institute, Beijing.The whole genome of HGCC was de novo sequenced using an Illumina HiSeq X Ten in 2\u2009\u00d7\u2009149\u2009bp paired-end mode with a 400\u2009bp insert sizes of the library at Personalbio . High-quality data were obtained from raw data by removing adapter contamination with AdapterRemoval (version 2) , collectC. cassiicola genome sequences were predicted using Augustus software with the annotated gene information of B. cinerea [C. cassiicola was performed by sequence alignment against the Pfam database with profile hidden Markov models (http://xfam.org/) using hmmer 3.1b2 (http://www.hmmer.org/).Gene structures of  cinerea . Putativ cinerea , SignalP cinerea , TMHMM S cinerea , and Big cinerea . Putativ cinerea . ProteinC. cassiicola HGCC protein sequences against the PHI database  with a cutoff E-value of 1e-5. MAPK pathway associated genes of C. cassiicola were screened by local BLASTP against S. cerevisiae MAPK pathway-associated genes with a cutoff E-value of 1e-5 that were confirmed by experiments. GPCRs were identified by local BLASTP against the GPCRDB database (http://gpcrdb.org/) with the best hits and further confirmed by searching seven transmembrane helices with TMHMM Server v. 2.0. Kinases, proteases and transporters were identified by local BLASTP against the KinBase database with a cutoff E-value of 1e-10, the MEROPS peptidase database (https://www.ebi.ac.uk/merops/) with a cutoff E-value of 1e-20 [E-value of 1e-40, respectively [http://drnelson.uthsc.edu/CytochromeP450.html) and CAZy database (http://www.cazy.org/), respectively, with a cutoff E-value of 1e-10.Potential virulence-associated genes were identified by local BLASTP searches of of 1e-20 , and theof 1e-20  with a cectively . CYPs anhttp://smurf.jcvi.org/index.php) [C. cassiicola PKSs and other functionally known PKSs in other fungi were submitted to the SBSPKS database (http://www.nii.ac.in/~pksdb/sbspks/master.html) to modulate and analyze conserved domains. Phylogenetic analysis of PKSs was performed by aligning KS domain sequences and creating a maximum likelihood tree using MEGA 7.0 with Jones-Taylor-Thornton (JTT) model. These functionally known PKS-related toxin biosynthesis pathways included A. ochraceus AoLC35\u201312 (GenBank: AAT92023) for ochratoxin production, A. alternate ACTTS3 (GenBank: BAJ14522) for ACT-toxin production, B. maydis PKS1 (GenBank: AAB08104) and PKS2 (GenBank: AAR90257) for T-toxin production, G. zeae PKS4 (GenBank: ABB90283) for zearalenones production and G. moniliformis Fum1p (GenBank: AAD43562) for fumonisin production. Melanin-associated PKSs included A. fumigatus Alb1p (GenBank: ACJ13039), Ceratocystis resinifera PKS1 (GenBank: AAO60166), C. lagenarium PKS1 (GenBank: BAA18956), C. globosum PKS-1 (GenBank: AFP82905), A. rabie PKS1 (GenBank: ACS74449), B. maydis PKS18 (GenBank: AAR90272), S. turcica StPKS (GenBank: AEE68981), B. oryzae PKS1 (GenBank: BAD22832), and A. alternate ALM1 (GenBank: BAK64048).Secondary metabolism backbone genes such as PKS, NRPS and NRPS-PKS were identified in the SMURF system (dex.php) . C. cassC. cassiicola HGCC and the other two C. cassiicola isolates (UM591 and CCP) or other phytopathogenic fungi, the same pipeline was used for the gene prediction and annotation of the genome for all species. Accession numbers of the genomes are JAQF00000000.1 (GenBanK) for C. cassiicola UM591, GCA_003016335.1 (GenBanK) for C. cassiicola CCP, GCA_000743335.1 (GenBanK) for C. lunata, GCA_000338975.1 (GenBanK) for B. maydis, 2,761,201,826 (JGI) for C. zeae-maydis, GCA_002267025.1 (GenBanK) for P. nodorum, GCA_000359705.1 (GenBanK) for S. turcica, GCA_000149985.1 (GenBanK) for P. tritici-repentis, GCA_000002495.2 (GenBanK) for M. oryzae, GCA_009017415.1 (GenBanK) for A. flavus, GCA_000240135.3 (GenBanK) for F. graminearum, and GCA_000143535.4 (GenBanK) for B. cinerea. To construct an interspecific phylogenetic tree among these fungi, 2831 orthologous protein sequences were screened by local reciprocal Blast search with a cutoff of E-value 1e-20. These orthologous proteins were aligned using Clustal W 2.1 [To compare the gene annotation between C. cassiicola  compared to 10 other phytopathogenic fungi, including C. lunata, B. maydis, C. zeae-maydis, P. nodorum, S. turcica, P. tritici-repentis, M. oryzae, A. flavus, F. graminearum, and B. cinerea. The cutoff of significance was set at P\u2009<\u20090.05.Statistical analysis was conducted by a non-parametric test (Mann-Whitney U test) of SSPS v18.0 using the gene amount of each gene family in C. cassiicola RNA-Seq libraries were mapped on the predicted CDS of C. cassiicola using Bowtie2 with default settings [The quality of the raw reads generated from RNA-Seq was checked with FastQC . The clesettings . The numThe counts of RNA-Seq reads over transcripts were used to calculate the fold change of gene expression using DESeq2 . DEGs weGapdh gene was used as the internal control. The first strand of cDNA was synthesized from total RNA of germinated and ungerminated spores using EasyScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR  . The qRT-PCR test was conducted with a QuanStudio 6 Flex instrument . Twenty microliters of reaction mixture contained 2\u2009\u03bcL diluted cDNA (1\u2013100\u2009ng), 0.4\u2009\u03bcL of each primer (10\u2009\u03bcM), 7.2\u2009\u03bcL of nuclease-free water and 10\u2009\u03bcL of 2\u2009\u00d7\u2009TransStart\u00ae Top Green qPCR SuperMix . The reaction program was follows: one cycle at 94\u2009\u00b0C for 30\u2009s and 40\u2009cycles at 94\u2009\u00b0C for 5\u2009s, 60\u2009\u00b0C for 15\u2009s and 72\u2009\u00b0C for 10\u2009s. The specificities of the amplification products were tested through a melting-curve analysis between 60\u2009\u00b0C and 95\u2009\u00b0C after each PCR reaction. The relative gene expression data were analyzed using the 2-\u0394\u0394Ct method [Sixty-six DEGs involved in the cell growth and death of cellular processes were selected for the confirmation of the RNA-Seq data using qRT-PCR . bP value (highlighted in bold for comparisons showing significant differences) is calculated by a non-parametric test (Mann-Whitney U test) of SSPS v18.0 using the gene amount of each family in C. cassiicola  compared to the 10 other plant pathogenic fungi (grey column). The cutoff of significance is set at P\u2009<\u20090.05 (highlighted). c PHI: pathogen-host interaction. d Percentage: The percentage of PHI-associated gene in protein-coding genes. Table S2 The number of genes for selected gene families in C. cassiicola and other ascomycetes. a PHI: pathogen-host interaction. b Average is calculated using the gene/PHI-associated gene amount of each family in the 10 other plant pathogenic fungi (grey column). cP value (highlighted in bold for comparisons showing significant differences) is calculated by a non-parametric test (Mann-Whitney U test) of SSPS v18.0 using the gene amount of each family in C. cassiicola  compared to the 10 other plant pathogenic fungi (grey column). The cutoff of significance is set at P\u2009<\u20090.05 (highlighted). Fungal species: HGCC, C. cassiicola HGCC; CCP, C. cassiicola CCP; UM591, C. cassiicola UM591; CL, C. lunata; BM, B. maydis; CZM, C. zeae-maydis; PN, P. nodorum; ST, S. turcica; PTR, P. tritici-repentis; MO, M. oryzae; AF, A. flavus; FG, F. graminearum; BC, B. cinerea. Table S3 G protein-coupled receptors in different fungal genomes. a PHI: pathogen-host interaction. b Average is calculated using the gene/PHI-associated gene amount of each family in the 10 other plant pathogenic fungi (grey column). cP value (highlighted in bold for comparisons showing significant differences) is calculated by a non-parametric test (Mann-Whitney U test) of SSPS v18.0 using the gene amount of each family in C. cassiicola  compared to the 10 other plant pathogenic fungi (grey column). The cutoff of significance is set at P\u2009<\u20090.05 (highlighted). Fungal species: HGCC, C. cassiicola HGCC; CCP, C. cassiicola CCP; UM591, C. cassiicola UM591; CL, C. lunata; BM, B. maydis; CZM, C. zeae-maydis; PN, P. nodorum; ST, S. turcica; PTR, P. tritici-repentis; MO, M. oryzae; AF, A. flavus; FG, F. graminearum; BC, B. cinerea. Table S4 Gene-encoding proteins for the MAPK signaling pathway in C. cassiicola HGCC. The colour of the protein names indicates the degree of conservation between S. cerevisiae and C. cassiicola HGCC proteins based on Blastp. Blue, e-value <1e-5 and\u2009>\u20091e-20; Green, e-value <1e-100 and\u2009>\u20091e-20; Red, e-value <1e-100. Table S5 Gene-encoding proteins for the cAMP signaling pathway in C. cassiicola HGCC. Table S6 Gene-encoding proteins for the Ca2+ signaling pathway in C. cassiicola HGCC. Table S7 The number of protein kinases in different fungal genomes. a PHI: pathogen-host interaction. b Average is calculated using the gene/PHI-associated gene amount of each family in the 10 other plant pathogenic fungi (grey column). cP value (highlighted in bold for comparisons showing significant differences) is calculated by a non-parametric test (Mann-Whitney U test) of SSPS v18.0 using the gene amount of each family in C. cassiicola  compared to the 10 other plant pathogenic fungi (grey column). The cutoff of significance is set at P\u2009<\u20090.05 (highlighted). Fungal species: HGCC, C. cassiicola HGCC; CCP, C. cassiicola CCP; UM591, C. cassiicola UM591; CL, C. lunata; BM, B. maydis; CZM, C. zeae-maydis; PN, P. nodorum; ST, S. turcica; PTR, P. tritici-repentis; MO, M. oryzae; AF, A. flavus; FG, F. graminearum; BC, B. cinerea. Table S8 Protease genes classified by the MEROPS family in different fungal genomes. a PHI: pathogen-host interaction. b Average is calculated using the gene/PHI-associated gene amount of each family in the 10 other plant pathogenic fungi (grey column). cP value (highlighted in bold for comparisons showing significant differences) is calculated by a non-parametric test (Mann-Whitney U test) of SSPS v18.0 using the gene amount of each family in C. cassiicola  compared to the 10 other plant pathogenic fungi (grey column). The cutoff of significance is set at P\u2009<\u20090.05 (highlighted). Fungal species: HGCC, C. cassiicola HGCC; CCP, C. cassiicola CCP; UM591, C. cassiicola UM591; CL, C. lunata; BM, B. maydis; CZM, C. zeae-maydis; PN, P. nodorum; ST, S. turcica; PTR, P. tritici-repentis; MO, M. oryzae; AF, A. flavus; FG, F. graminearum; BC, B. cinerea. Table S9 Glycoside hydrolase in C. cassiicola and other phytopathogenic ascomycetes. a PHI: pathogen-host interaction. b Average is calculated using the gene/PHI-associated gene amount of each family in the 10 other plant pathogenic fungi (grey column). cP value (highlighted in bold for comparisons showing significant differences) is calculated by a non-parametric test (Mann-Whitney U test) of SSPS v18.0 using the gene amount of each family in C. cassiicola  compared to the 10 other plant pathogenic fungi (grey column). The cutoff of significance is set at P\u2009<\u20090.05 (highlighted). Fungal species: HGCC, C. cassiicola HGCC; CCP, C. cassiicola CCP; UM591, C. cassiicola UM591; CL, C. lunata; BM, B. maydis; CZM, C. zeae-maydis; PN, P. nodorum; ST, S. turcica; PTR, P. tritici-repentis; MO, M. oryzae; AF, A. flavus; FG, F. graminearum; BC, B. cinerea. Table S10 Transporters in C. cassiicola and other phytopathogenic ascomycetes. a PHI: pathogen-host interaction. b Average is calculated using the gene/PHI-associated gene amount of each family in the 10 other plant pathogenic fungi (grey column). cP value (highlighted in bold for comparisons showing significant differences) is calculated by a non-parametric test (Mann-Whitney U test) of SSPS v18.0 using the gene amount of each family in C. cassiicola  compared to the 10 other plant pathogenic fungi (grey column). The cutoff of significance is set at P\u2009<\u20090.05 (highlighted). Fungal species: HGCC, C. cassiicola HGCC; CCP, C. cassiicola CCP; UM591, C. cassiicola UM591; CL, C. lunata; BM, B. maydis; CZM, C. zeae-maydis; PN, P. nodorum; ST, S. turcica; PTR, P. tritici-repentis; MO, M. oryzae; AF, A. flavus; FG, F. graminearum; BC, B. cinerea. Table S11 Drug transporters in C. cassiicola and other phytopathogenic ascomycetes. a PHI: pathogen-host interaction. b Average is calculated using the gene/PHI-associated gene amount of each family in the 10 other plant pathogenic fungi (grey column). cP value (highlighted in bold for comparisons showing significant differences) is calculated by a non-parametric test (Mann-Whitney U test) of SSPS v18.0 using the gene amount of each family in C. cassiicola  compared to the 10 other plant pathogenic fungi (grey column). The cutoff of significance is set at P\u2009<\u20090.05 (highlighted). Fungal species: HGCC, C. cassiicola HGCC; CCP, C. cassiicola CCP; UM591, C. cassiicola UM591; CL, C. lunata; BM, B. maydis; CZM, C. zeae-maydis; PN, P. nodorum; ST, S. turcica; PTR, P. tritici-repentis; MO, M. oryzae; AF, A. flavus; FG, F. graminearum; BC, B. cinerea. Table S12 Cytochrome P450 genes classified by the CYP family in different fungal genomes. a PHI: pathogen-host interaction. b Average is calculated using the gene/PHI-associated gene amount of each family in the 10 other plant pathogenic fungi (grey column). cP value (highlighted in bold for comparisons showing significant differences) is calculated by a non-parametric test (Mann-Whitney U test) of SSPS v18.0 using the gene amount of each family in C. cassiicola  compared to the 10 other plant pathogenic fungi (grey column). The cutoff of significance is set at P\u2009<\u20090.05 (highlighted). Fungal species: HGCC, C. cassiicola HGCC; CCP, C. cassiicola CCP; UM591, C. cassiicola UM591; CL, C. lunata; BM, B. maydis; CZM, C. zeae-maydis; PN, P. nodorum; ST, S. turcica; PTR, P. tritici-repentis; MO, M. oryzae; AF, A. flavus; FG, F. graminearum; BC, B. cinerea. Table S13 Numbers of backbone genes for the biosynthesis of secondary metabolites in different pathogenic fungi. a PHI: pathogen-host interaction. b Average is calculated using the gene/PHI-associated gene amount of each family in the 10 other plant pathogenic fungi (grey column). cP value (highlighted in bold for comparisons showing significant differences) is calculated by a non-parametric test (Mann-Whitney U test) of SSPS v18.0 using the gene amount of each family in C. cassiicola  compared to the 10 other plant pathogenic fungi (grey column). The cutoff of significance is set at P\u2009<\u20090.05 (highlighted). Fungal species: HGCC, C. cassiicola HGCC; CCP, C. cassiicola CCP; UM591, C. cassiicola UM591; CL, C. lunata; BM, B. maydis; CZM, C. zeae-maydis; PN, P. nodorum; ST, S. turcica; PTR, P. tritici-repentis; MO, M. oryzae; AF, A. flavus; FG, F. graminearum; BC, B. cinerea. Table S14 Putative small, cysteine-rich proteins (SCRPs) in C. cassiicola HGCC.Additional file 2: Table S15 Sequencing yield from individual libraries per sample. a CC_0_1, b CC_0_2 and c CC_0_3 are triplicate ungerminated spore samples. d CC_6_12_1, e CC_6_12_2, and f CC_6_12_3 are triplicate mixed 6\u2009h- and 12\u2009h-germinated spore samples. Table S16 3288 DEGs during spore germination identified by RNA-Seq. a CC_6_12: mixed 6\u2009h- and 12\u2009h-germinated spore samples; b CC_0: ungerminated spore samples. Table S17 KEGG pathway enrichment analysis of DEGs. Table S18 qRT-PCR for selected DEGs involved in cell growth and death. * The express pattern of DEGs from RNA-Seq was not validated by qRT-PCR.Additional file 3: Fig. S1 Pearson correlation between samples in gene expression levels. CC_0_1, CC_0_2 and CC_0_3 are triplicate ungerminated spore samples. CC_6_12_1, CC_6_12_2, and CC_6_12_3 are triplicate mixed 6\u2009h- and 12\u2009h-germinated spore samples. R2 is the square of the Pearson correlation coefficient."}
{"text": "The inhibitory activities of AChE and BChE were evaluated in vitro by Ellman method. The results show that some compounds have good inhibitory activity against AChE and BChE. Among them, compound 8i showed the strongest inhibitory effect on both AChE (eeAChE IC50 = 0.39 \u03bcM) and BChE (eqBChE IC50 = 0.28 \u03bcM). Enzyme inhibition kinetics and molecular modeling studies have shown that compound 8i bind simultaneously to the peripheral anionic site (PAS) and the catalytic sites (CAS) of AChE and BChE. In addition, the cytotoxicity of compound 8i is lower than that of Tacrine, indicating its potential safety as anti-Alzheimer\u2019s disease (anti-AD) agents. In summary, these data suggest that compound 8i is a promising multipotent agent for the treatment of AD.A series of novel compounds  According to the report of the International Alzheimer\u2019s Association in the last three years, there are more than 36 million AD patients in the world. With the acceleration of the aging process of the population, the amount of AD incidence is increasing year by year, and the number of AD patients in the world will exceed 130 million by 2050 . AD is tAD was initially characterized by memory loss, cognitive dysfunction, inability to take care of themselves in daily life, and subsequent exacerbations of mental and behavioral abnormalities ,8. AD isAD is a complex neurodegenerative syndrome. Due to the complicated pathogenesis of AD, its etiology is not completely clear. The therapeutic drugs used in the clinical and research stages can only delay the course of AD, but there are no drugs that cure or delay the course of AD ,12. A laPrevious studies have identified three pathological features of AD: decreased levels of AChE in the neurotransmitter matrix, deposition of A\u03b2, and hyperphosphorylation of tau protein. Scientists have been trying to find the etiology and treatment strategy of AD through further study of the above three pathological characteristics ,20. At pThere are two types of ChEs in the central nervous system, namely AChE and BChE. AChE and BChE are important targets for the development of anti-AD drugs. The physiological function of AChE has been preliminarily understood, but the understanding of BChE is less. Studies have shown that ACh activity in some brain regions of patients with mild to severe AD decreases to 10%\u201315% of the normal value, and AChEIs have significant effects on patients with mild to moderate AD and can repair their cognitive impairment and other symptoms .When AD develops to the middle and late stage, AChE activity decreases, whereas BChE activity increases, and BChE acts as a metabolic compensation for AChE, partially compensating for the role of AChE in hydrolyzing ACh ,23,24. TWith the study of AChE crystal structure, it was found that the ligand binding pocket of AChE in general is a long and narrow channel extending from the surface to the interior . The chaG801-0274 AChE (eeAChE IC50 = 2.05 \u03bcM), BChE (eqBChE IC50 = 0.03 \u03bcM) can inhibit ChEs. In this paper, we used it as the lead compound for structural modification   triazole (11f) > 1H-Benzo[d]imidazole (6d) > 1H-Indazole (6c) > 1H-Indole (6a); its activity on BChE are 2-Oxoindoline (8i) > 1H-Benzo[d]imidazole (6d) > 1H-Indole (6a) > 1H-Benzo[d]  triazole (11f). Among them, compound 8i (50 = 0.39 \u03bcM) and BChE (eqBChE IC50 = 0.28 \u03bcM), these results indicated that compound 8i was a potent dual inhibitor against AChE and BChE. It was speculated that 2-Oxoindoline is the key structure for inhibiting two ChEs and is not selective for both ChEs. According to the molecular docking results, for eeAChE, 2-Oxoindoline of compound 8i bound with Trp286 via \u03c0-\u03c0 stacking interaction; for huAChE, 2-Oxoindoline of compound 8i bound with Trp286 and Tyr341 via \u03c0-\u03c0 stacking interaction, and for huBChE, 2-Oxoindoline of compound 8i bound with Phe329 via \u03c0-\u03c0 stacking interaction. These interactions increase the inhibitory activity by enhancing the binding affinity.First, we synthesized compounds pound 8i  showed t3, 4-CF3), compared with compound 6d, when R is 4-CF3 (6e), 4-OCH3 (11d), 2-Cl (11e), the ChEs inhibitory activity decreased. In particular, compound 11d showed little inhibitory activity on BChE at 100 \u03bcM. In general, with the same Y, when R is H, the compounds have the highest inhibitory activity against ChEs; when Y is substituted by 2-Cl or 4-CF3, the inhibitory activity of the compounds to ChEs were weakened; and when Y is substituted by 4-OCH3, the compounds have the worst inhibitory activity against AChEs. We speculate that enhancing the electron-withdrawing effect or the donor effect on the aromatic ring is not conducive to improving the performance of the analog, and the appropriate space may facilitate the analog to enter the CAS pocket of ChEs.Then, we evaluated the effect of R on ChEs activity. We modified R with different substituent  in the previous compounds was replaced by Tosyl moiety. We synthesized compounds 10s\u2013v. Except for compound 10u  has low inhibitory activity against ChEs, the compounds 10s ), 10t , 10v  all can maintain ChEs inhibitory activity at micromolar levels, indicating that the p-Toluenesulfonamide moiety is responsible for maintaining the inhibitory activity of ChEs. On one hand, we speculate that methyl occupies the pocket of the active site and interacts with amino acid residues to increase inhibitory activity. On the other hand, Sulfonamide moiety is very important for maintaining ChEs inhibitory activity, which may be related to the bond angle between Sulfonamide and molecules + 348.2070, found 348.2070.N-((1-(2-Chlorobenzyl)piperidin-4-yl)methyl)-1H-Indole-5-carboxamide (6b). 1H-Indole-5-carboxylic acid , CDI , (1-(2-Chlorobenzyl) piperidin-4-yl)methanamine , THF (15 mL) White solid, m.p.: 88\u201389 \u00b0C, yield: 66%, 1H NMR  \u03b4 8.08 , 7.58 , 7.49 , 7.41\u20137.35 , 7.30\u20137.24 , 6.51 , 3.78 , 3.05 , 2.31 , 1.77 , 1.45\u20131.33 . 13C NMR  \u03b4 170.60, 138.15, 134.64, 131.71, 129.37, 129.10, 127.70, 126.78, 125.91, 125.17, 120.18, 119.95, 110.67, 102.17, 58.57, 53.06, 44.79, 35.56, 28.93. HRMS (ESI): calcd. For C22H24ClN3O [M + H]+ 382.1681, found 382.1703.N-((1-Benzylpiperidin-4-yl) methyl)-1H-indazole-5-carboxamide (6c). 1H-Indazole-5-carboxylic acid , CDI , (1-Benzylpiperidin-4-yl)methanamine , THF (15 mL) White solid, m.p.: 111\u2013112 \u00b0C, yield: 56%, 1H NMR  \u03b4 13.30 , 8.48 , 8.35 , 8.20 , 7.87 , 7.57 , 7.33\u20137.27 , 7.24 , 3.45 , 3.18 , 2.81 , 1.92 , 1.67 , 1.59 , 1.22 . 13C NMR  \u03b4 167.07, 141.33, 138.83, 135.11, 129.24, 128.56, 127.67, 127.62, 127.30, 125.79, 122.79, 120.90, 110.16, 62.78, 53.38, 45.31, 36.17, 30.23. HRMS (ESI): calcd. For C21H24N4O [M + H]+ 349.2023, found 349.2019.N-((1-Benzylpiperidin-4-yl)methyl)-1H-benzo[d]imidazole-5-carboxamide (6d). 1H-Benzo[d]imidazole-5-carboxylic acid , CDI , (1-Benzylpiperidin-4-yl)methanamine , THF (15 mL) Yellow oil, yield: 72%, 1H NMR  \u03b4 8.53 , 8.27 , 7.78 , 7.70\u20137.61 , 7.28 , 7.21 , 7.04 , 3.39 , 3.19 , 2.76 , 1.85 , 1.65 , 1.20 . 13C NMR  \u03b4 167.40, 144.24, 139.08, 135.64, 129.16, 128.51, 127.18, 121.88, 115.66, 114.85, 62.93, 53.43, 53.35, 45.39, 36.25, 30.32. HRMS (ESI): calcd. For C21H24N4O [M + H]+ 349.2023, found 349.2023.N-((1-(4-(Trifluoromethyl)benzyl)piperidin-4-yl)methyl)-1H-benzo[d]imidazole-5-carboxamide (6e). 1H-Benzo[d]imidazole-5-carboxylic acid , CDI , (1-(4-(Trifluoromethyl)benzyl)piperidin-4-yl)methanamine , THF (15 mL) White solid, m.p.: 92\u201393 \u00b0C, yield: 77%, 1H NMR  \u03b4 8.29\u20138.27 , 8.12 , 7.87\u20137.77 , 7.62 , 7.56 , 7.39 , 3.66 , 3.30 , 3.27 , 2.90 , 2.15\u20132.06 , 1.79\u20131.63 , 1.44\u20131.24 . 13C NMR  \u03b4 169.23, 141.32, 134.81, 131.92, 130.75, 130.34, 128.00, 127.37, 127.30, 125.41, 122.55, 120.67, 114.13, 113.58, 109.69, 58.94, 57.96, 53.34, 53.01, 44.93, 44.83, 35.64, 35.52, 29.26, 29.06. HRMS (ESI): calcd. For C22H23F3N4O [M + H]+ 417.1897, found 417.1895.N-((1-(4-Methoxybenzyl)piperidin-4-yl)methyl)-1H-benzo[d]triazole-5-carboxamide (6f). 1H-Benzo[d]triazole-5-carboxylic acid , CDI , (1-(4-Methoxybenzyl)piperidin-4-yl)methanamine , THF (15 mL) Yellow oil, yield: 63%, 1H NMR  \u03b4 8.53 , 8.32 , 8.16 , 7.75 , 7.61 , 7.32 , 6.91 , 3.74 , 3.69 , 3.18 , 2.97 , 2.26 , 1.73 , 1.68 , 1.34 . 13C NMR  \u03b4 167.31, 159.33, 144.22, 131.57, 128.92, 121.94, 114.16, 60.78, 55.52, 52.34, 44.84, 35.34, 28.86. HRMS (ESI): calcd. For C21H25N5O2 [M + H]+ 380.2081, found 380.2077.N-((1-(2-Chlorobenzyl)piperidin-4-yl)methyl)-1H-benzo[d]triazole-5-carboxamide (6g). 1H-Benzo[d]triazole-5-carboxylic acid , CDI , (1-(2-Chlorobenzyl)piperidin-4-yl)methanamine , THF (15 mL) White solid, m.p.: 77\u201379 \u00b0C, yield: 57%, 1H NMR  \u03b4 8.63 , 8.44 , 7.87 , 7.47 , 7.39 , 7.30 , 7.27\u20137.25 , 7.03 , 3.52 , 3.21 , 2.82 , 2.00 , 1.68 , 1.61 , 1.23 . 13C NMR  \u03b4 166.50, 140.30, 139.38, 138.70, 131.96, 129.27, 128.56, 127.32, 125.46, 115.61, 114.32, 62.73, 53.33, 45.41, 36.08, 30.16. HRMS (ESI): calcd. For C20H22ClN5O [M + H]+ 384.1586, found 384.1584.N-((1-(4-(Trifluoromethyl)benzyl)piperidin-4-yl)methyl)-1H-benzo[d]triazole-5-carboxamide (6h). 1H-Benzo[d]triazole-5-carboxylic acid , CDI , (1-(4-(Trifluoromethyl)benzyl)piperidin-4-yl)methanamine , THF (15 mL) White solid, m.p.: 90\u201391 \u00b0C, yield: 73%, 1H NMR  \u03b4 8.38 , 7.92\u20137.90 , 7.88 , 7.81 , 7.68 , 7.61 , 7.51\u20137.42 , 3.89 , 3.33 , 3.13\u20133.01 , 2.47\u20132.31 , 1.90\u20131.68 , 1.52\u20131.32 . 13C NMR  \u03b4 166.50, 140.30, 139.38, 138.70, 131.96, 129.27, 128.56, 127.32, 125.46, 115.61, 114.32, 62.73, 53.33, 45.41, 36.08, 30.16. HRMS (ESI): calcd. For C21H22F3N5O [M + H]+ 418.1849, found 418.1850.7) , PyBOP (1.2 equiv.) and DIPEA (1.5 equiv.) were added to DMF and stirred at room temperature for 20 min. Then, intermediates (4a\u2013d) (1.0 equiv.) was added and stirred at room temperature for 4 h. After completion of the reaction, the reaction mixture was quenched with saturated NaCl solution (25 mL). The aqueous phase was extracted with DCM (25 \u00d7 3 mL). The DCM layer was combined and washed with brine solution (25 \u00d7 3 mL). The organic layer was dried over anhydrous Na2SO4 and the solvent was removed under reduced pressure. After concentration, the crude product was purified by silica gel column chromatograph using a methanol in DCM gradient (DCM: methanol= 60:1\u20135:1) yielded compounds 8i\u2013l.Intermediates (N-((1-Benzylpiperidin-4-yl)methyl)-2-Oxoindoline-5-carboxamide (8i). 2-Oxoindoline-5-carboxylic acid , (1-Benzylpiperidin-4-yl)methanamine , PyBOP , DIPEA, DMF (6 mL). White solid, m.p.:155\u2013156 \u00b0C, yield: 56%, 1H NMR  \u03b4 7.73\u20137.68 , 7.53\u20137.42 , 6.91 , 4.26 , 3.44 , 3.31 , 2.98 , 1.97 , 1.54 . 13C NMR  \u03b4 178.52, 168.90, 146.69, 130.99, 129.80, 129.40, 128.99, 127.92, 127.60, 125.95, 123.42, 123.40, 108.97, 60.21, 51.86, 43.82, 42.11, 34.01, 26.82. HRMS (ESI): calcd. For C22H25N3O2 [M + H]+ 364.2020, found 364.2032.N-((1-(2-Chlorobenzyl)piperidin-4-yl)methyl)-2-Oxoindoline-5-carboxamide (8j). 2-Oxoindoline-5-carboxylic acid , (1-(2-Chlorobenzyl)piperidin-4-yl)methanamine , PyBOP , DIPEA, DMF (6 mL). White solid, m.p.: 151\u2013152 \u00b0C, yield: 35%, 1H NMR  \u03b4 7.74\u20137.69 , 7.50 , 7.39 , 7.28 , 6.93 , 3.71 , 3.27 , 3.00 , 2.21 , 1.76 , 1.69 , 1.37 . 13C NMR  \u03b4 168.83, 146.63, 135.02, 132.40, 130.20, 129.68, 128.07, 127.58, 127.18, 125.93, 123.41, 108.96, 57.94, 52.76, 44.36, 34.83, 28.01. HRMS (ESI): calcd. For C22H24ClN3O2 [M + H]+ 398.1630, found 398.1652.N-((1-(4-Methoxybenzyl)piperidin-4-yl)methyl)-2-Oxoindoline-5-carboxamide (8k). 2-Oxoindoline-5-carboxylic acid , (1-(4-Methoxybenzyl)piperidin-4-yl)methanamine , PyBOP , DIPEA, DMF (6 mL). White solid, m.p.: 158\u2013159 \u00b0C, yield: 43%, 1H NMR  \u03b4 7.74\u20137.68 , 7.40 , 6.98 , 6.91 , 4.19 , 3.79 , 3.43 , 3.31 , 2.95 , 1.97 , 1.60\u20131.45 , 1.26 . 13C NMR  \u03b4 178.54, 168.87, 161.10, 146.66, 132.51, 127.95, 127.62, 125.93, 123.46, 121.09, 114.21, 108.98, 54.55, 51.51, 43.78, 34.03, 27.22, 26.85. HRMS (ESI): calcd. For C23H27N3O3 [M + H]+ 394.2125, found 394.2131.2-Oxo-N-((1-(4-(trifluoromethyl)benzyl)piperidin-4-yl)methyl)indoline-5-carboxamide (8l). 2-Oxoindoline-5-carboxylic acid , (1-(4-(Trifluoromethyl)benzyl)piperidin-4-yl)methanamine , PyBOP , DIPEA, DMF (6 mL). White solid, m.p.: 113\u2013115 \u00b0C, yield: 40%, 1H NMR  \u03b4 7.79 , 7.73\u20137.69 , 7.62 , 7.56 , 7.44\u20137.32 , 6.95\u20136.90 , 3.60 , 3.24 , 2.91\u20132.77 , 2.09\u20131.93 , 1.78\u20131.61 , 1.40\u20131.21 . 13C NMR ) \u03b4 168.70, 146.44, 131.73, 130.35, 130.30, 128.23, 127.47, 127.30, 126.79, 125.81, 125.26, 123.34, 108.90, 59.79, 59.79, 58.24, 53.43, 53.14, 45.07, 36.04, 36.04, 36.00, 36.00, 29.82, 29.76. HRMS (ESI): calcd. For C23H24F3N3O2 [M + H]+ 432.1893, found 432.1886.4a\u2013d)  in DCM (5 mL) was added Et3N (1.8 equiv.). The mixture was cooled to 0 \u00b0C, TsCl (1.3 equiv.) in DCM (5 mL) was added dropwise, and the reaction mixture was stirred for an additional 1 h at 0 \u00b0C. After completion of the reaction, the reaction mixture was dissolved in 15 mL saturated sodium bicarbonate (NaHCO3) and extracted with DCM (25 \u00d7 3 mL). Organic phases were combined and washed with saturated NaCl solution (25 \u00d7 3 mL). The organic layer was dried over anhydrous Na2SO4 and the solvent was removed under reduced pressure. After concentration, the crude product was purified by silica gel column chromatograph using a methanol in dichloromethane gradient (dichloromethane: methanol = 60:1\u20135:1) yielded compounds 10s\u2013v.To a solution of (1-Benzylpiperidin-4-yl) methanamine derivatives (N-((1-Benzylpiperidin-4-yl)methyl)-4-methylbenzenesulfonamide (10s). (1-Benzylpiperidin-4-yl) methanamine , TsCl , Et3N , DCM (9 mL) White solid, m.p.:78\u201379 \u00b0C, yield: 80%, 1H NMR  \u03b4 7.68 , 7.36\u20137.24 , 3.60 , 2.93 , 2.67 , 2.39 , 2.09 , 1.68 , 1.44 , 1.28\u20131.13 . 13C NMR  \u03b4 143.22, 137.71, 136.09, 129.69, 129.36, 128.05, 127.39, 126.67, 62.59, 52.72, 35.66, 28.79, 20.11. HRMS (ESI): calcd. For C20H26N2O2S [M + H]+ 359.1788, found 359.1802.N-((1-(2-Chlorobenzyl)piperidin-4-yl)methyl)-4-methylbenzenesulfonamide (10t). (1-(4-Chlorobenzyl)piperidin-4-yl)methanamine , TsCl , Et3N , DCM (9 mL) White solid, m.p.: 111\u2013112 \u00b0C, yield: 73%, 1H NMR  \u03b4 7.69 , 7.44 , 7.37\u20137.31 , 7.23 , 3.62 , 2.89 , 2.67 , 2.39 , 2.06 , 1.65 , 1.37 , 1.20 , 1.15 . 13C NMR  \u03b4 143.42, 137.49, 135.59, 133.53, 131.79, 130.10, 129.48, 127.74, 127.35, 126.68, 126.68, 20.13. HRMS (ESI): calcd. For C20H25ClN2O2S [M + H]+ 393.1398, found 393.1397.N-((1-(4-Methoxybenzyl)piperidin-4-yl)methyl)-4-methylbenzenesulfonamide (10u). (1-(4-Methoxybenzyl)piperidin-4-yl)methanamine , TsCl , Et3N , DCM (9 mL) White solid, m.p.: 76\u201377 \u00b0C, yield: 87%, 1H NMR  \u03b4 7.61 , 7.25 , 6.85 , 3.80 , 3.70 , 3.10 , 2.63 , 2.44 , 2.32 , 2.05 , 1.73 , 1.52 , 1.27\u20131.19 . 13C NMR  \u03b4 160.41, 143.33, 137.60, 131.82, 129.42, 126.66, 113.87, 60.67, 54.47, 51.88, 34.72, 27.47, 20.10. HRMS (ESI): calcd. For C21H28N2O3S [M + H]+ 389.1893, found 389.1894.4-Methyl-N-((1-(4-(trifluoromethyl)benzyl)piperidin-4-yl)methyl)benzenesulfonamide (10v). (1-(4-(Trifluoromethyl)benzyl)piperidin-4-yl)methanamine , TsCl , Et3N , DCM (9 mL) White solid, m.p.: 87\u201388 \u00b0C, yield: 88%, 1H NMR  \u03b4 7.76 , 7.71\u20137.66 , 7.61 , 7.57\u20137.51 , 7.40\u20137.31 , 3.58 , 2.79 , 2.67 , 2.40 , 1.97 , 1.69\u20131.59 , 1.40 , 1.15 . 13C NMR  \u03b4 143.14, 137.67, 133.70, 131.73, 130.36, 130.33, 130.03, 129.28, 127.36, 126.84, 126.60, 125.27, 113.19, 59.65, 58.14, 53.26, 52.97, 48.19, 48.15, 48.12, 48.06, 47.95, 47.89, 47.78, 47.72, 47.61, 47.44, 47.32, 47.27, 47.10, 35.94, 35.86, 29.45, 20.03. HRMS (ESI): calcd. For C21H25F3N2O2S [M + H]+ 427.1662, found 427.1660.11  and substituted (2-romoethyl)benzene derivatives (12a\u2013b)  were dissolved in 20 mL acetone. Then, anhydrous K2CO3  and catalytic amount KI were added. The reaction mixture was refluxed for 4 h. After completion of the reaction, acetone was concentrated, and the residue was dissolved in water (60 mL) and extracted with ethyl acetate (60 \u00d7 3 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuum. The obtained oil was used in further synthesis without purification yielded compounds 13a\u2013b (Yields were 67% and 72%). Then, 4 mol/L KOH (2.5 equiv.) was added to the solution of compounds 13a\u2013b in C2H5OH: H2O = 5:1(6 mL). The reaction mixture was stirred at room temperature for 7 h. After completion of the reaction, the reaction mixture was evaporated to dryness after neutralization with dilute hydrochloric acid solution. Poured into ethyl acetate to deposit the solid, after cooling off, the mixture was filtered and washed with cold ethyl acetate to give compounds 14a\u2013d.Ethyl 2-piperidin-4-ylacetate 14a\u2013b) (1.2 equiv.), PyBOP (1.2 equiv.) and DIPEA (1.5 equiv.) were added to 6 mL DMF and stirred at room temperature for 20 min. Then, intermediates (15a\u2013b) (1.0 equiv.) was added and stirred at room temperature for 4 h. After completion of the reaction, the reaction mixture was quenched with saturated NaCl solution. The aqueous phase was extracted with DCM. The DCM layer was combined and washed with brine solution. The organic layer was dried over anhydrous Na2SO4 and the solvent was removed under reduced pressure. After concentration, the crude product was purified by silica gel column chromatograph using a methanol in dichloromethane gradient (DCM:methanol = 60:1\u20135:1) yielded compounds 16a\u2013d.Finally, intermediates (N-(1H-Indol-5-yl)-2-(1-phenethylpiperidin-4-yl)acetamide (16a). 2-(1-Phenethylpiperidin-4-yl) acetic acid , 1H-Indol-5-amine , PyBOP , DIPEA , DMF (6 mL). Yellow solid, m.p.: 165\u2013167 \u00b0C, yield: 80.70%, 1H NMR  \u03b4 7.74 , 7.28 , 7.20 , 7.15 , 6.37 , 3.60 , 3.26 , 3.08\u20133.01 , 2.39 , 2.21\u20132.13 , 2.04 , 1.74\u20131.60 , 1.27 . 13C NMR  \u03b4 170.66, 136.38, 133.81, 129.79, 128.59, 128.42, 127.97, 126.86, 125.29, 115.83, 115.76, 112.47, 110.75, 101.07, 72.24, 70.09, 60.80, 57.69, 53.42, 52.37, 41.90, 31.68, 31.23, 29.38, 28.89, 22.34, 13.05. HRMS (ESI): calcd. For C23H27N3O [M + H]+ 362.2227, found 362.2242.2-(1-(2-Chlorophenethyl)piperidin-4-yl)-N-(1H-indol-5-yl)acetamide (16b). 2-(1-(2-Chlorophenethyl)piperidin-4-yl)acetic acid , 1H-Indol-5-amine , PyBOP , DIPEA, DMF (6 mL). Yellow solid, m.p.: 172\u2013173 \u00b0C, yield: 30.41%, 1H NMR  \u03b4 7.75 , 7.39\u20137.29 , 7.28\u20137.20 , 7.19 , 7.16 , 6.37 , 3.45 , 3.15\u20133.09 , 3.08\u20133.01 , 2.79 , 2.36 , 2.10 , 2.00\u20131.92 , 1.60 . 13C NMR  \u03b4 171.22, 135.56, 133.78, 133.57, 130.84, 129.85, 129.30, 128.28, 127.96, 127.17, 125.21, 115.80, 112.48, 110.73, 101.06, 56.91, 52.68, 42.55, 32.24, 29.95, 29.32, 29.01. HRMS (ESI): calcd. For C23H26ClN3O [M + H]+ 396.1837, found 396.1838.N-(1H-Benzo[d]imidazol-5-yl)-2-(1-phenethylpiperidin-4-yl)acetamide (16c). 2-(1-phenethylpiperidin-4-yl)acetic acid , 1H-Benzo[d]imidazol-5-amine , PyBOP , DIPEA, DMF (6 mL). Red solid, m.p.: 167\u2013168 \u00b0C, yield: 45.92%, 1H NMR  \u03b4 8.09 , 8.03 , 7.52 , 7.29\u20137.12 , 3.07 , 2.84\u20132.78 , 2.64\u20132.58 , 2.32 , 2.17 , 1.93 , 1.81 , 1.42 . 13C NMR  \u03b4 171.84, 141.67, 139.67, 133.93, 128.33, 128.21, 128.20, 125.93, 116.32, 60.33, 53.19, 47.89, 43.30, 33.27, 32.51, 31.15. HRMS (ESI): calcd. For C22H26N4O [M + H]+ 363.2179, found 363.2197.N-(1H-Benzo[d]imidazol-5-yl)-2-(1-(2-chlorophenethyl)piperidin-4-yl)acetamide (16d). 2-(1-(2-Chlorophenethyl)piperidin-4-yl)acetic acid , 1H-Benzo[d]imidazol-5-amine , PyBOP , DIPEA, DMF (6 mL). Red solid, m.p.: 176\u2013177 \u00b0C, yield: 40.33%, 1H NMR  \u03b4 7.95 , 7.70 , 7.66 , 7.48 , 7.35 , 7.24 , 7.19 , 6.90 , 3.62 , 3.57 , 3.37 , 2.95 , 2.10 , 1.75 , 1.66 , 1.60 , 1.39 . 13C NMR  \u03b4 171.90, 141.65, 137.34, 134.98, 133.95, 133.58, 130.73, 129.20, 127.73, 126.97, 121.27, 116.28, 58.31, 58.27, 53.17, 53.06, 50.62, 43.39, 40.15, 33.39, 32.59, 31.29, 31.15, 30.24. HRMS (ESI): calcd. For C22H25ClN4O [M + H]+ 397.1790, found 397.1797.The inhibitory activity of the target compound against AChE (from Electrophorus electricus (eeAChE), Sigma-Aldrich, Munich, Germany) and horse serum BChE  were measured by Ellman\u2019s method . In 96-w50) value was calculated according to the inhibition curve and the data were shown in the layout of mean \u00b1 SEM by GraphPad Prism 6.0.Changes in absorbance were measured at 412 nm by using microplate reader . The measurement of each concentration for each compound was detected in triplicate. GraphPad Prism 6.0  was used for data processing. The inhibition curve was fitted by plotting the logarithm of the concentration of the tested compounds with the percentage of enzyme activity (reference to 100%). The  concentrations of 90, 150, 226, 452 and 904 \u03bcM. The concentration of compound 8i. The three protein structures are pretreated  by the \u201cprepare protein\u201d module in DS to provide the structures suitable for docking. The \u201cprepare ligand\u201d module in DS is used to test the structural preparation of the compound. The native ligand in the crystal structure was used to define the binding site. The binding site was defined as the site sphere ((in 10 \u00c5 radius) around the original ligand in the co-crystal structures. The docking program CDOCKER encoded in DS 2016 was applied to identify the potential binding of compound 8i to eeAChE, huAChE, and huBChE. Other CDOCKER parameters were set to default values. Compound 8i was chosen for molecular modeling as the most active compounds in the series  , huAChE e series . Compoun2. To carry out the experiment, cells (6 \u00d7 103 cells/well) were seeded in 96-well plate in complete medium. After 24 h, the culture medium was removed and the cells were exposed to increasing concentrations of compounds 6b, 6c, 6d, 8i, 10s, 10v or Tacrine  in DMEM for further 24 h. Cell survival was measured by 3--2,5-Diphenyltetrazolium Bromide (MTT) assay [Pheochromocytoma-derived cell line (PC12 cells) were grown in Dulbecco\u2019s Modified Eagle Medium (DMEM) supplemented with 10% FBS at 37 \u00b0C in a humidified atmosphere containing 5% CO6a\u2013h, 8i\u20131, 10s\u2013v, and 16a\u2013d were synthesized and evaluated, together with the known analogs 11a\u2013f, for their inhibitory activities towards AChE and BChE. The results show that most of the compounds have AChE and/or BChE inhibitory activity. Compound 8i showed the strongest inhibitory effect on both AChE (eeAChE IC50 = 0.39 \u03bcM) and BChE (eqBChE IC50 = 0.28 \u03bcM). Compared with compound G801-0274, compound 8i has comparable inhibitory activity against two ChEs, so that it can exert an anti-ChEs effect in a balanced manner. Kinetic studies indicated a mixed-type inhibition of compound 8i, including competitive inhibition and non-competitive inhibition. Subsequently, molecular docking was performed to evaluate the interaction mechanism between compound 8i and enzymes. Enzyme inhibition kinetics and molecular modeling studies have shown that compound 8i bind simultaneously to the PAS and the CAS of AChE and BChE. Therefore, compounds 8i may be promising scaffold for treatment, and further modifications have been made to obtain novel AChE and BChE dual-target inhibitors.In this paper, a series of novel compounds"}
{"text": "Haloterrigena turkmenica and Haloterrigena salina and has been denominated Haloterrigena sp. strain SGH1. Strain SGH1 grew at 20\u201340\u00b0C (optimum 37\u00b0C), at salinities between 15 and 30% (w/v) NaCl (optimum 25%) and growth was improved by addition of 50 mM KCl and 0.5% w/v casamino acids. Growth was severely restricted at salinities below 15% NaCl and cell lysis is avoided at a minimal 10% NaCl. Maximal concentrations of magnesium chloride and sodium or magnesium perchlorates that supported SGH1 growth were 0.5 and 0.15M, respectively. Haloterrigena sp. strain SGH1 accumulates bacterioruberin (BR), a C50 xanthophyll, as the major carotenoid. Total carotenoids in strain SGH1 amounted to nearly 400 \u03bcg BR per gram of dry biomass. Nearly 80% of total carotenoids accumulated as geometric isomers of BR: all-trans-BR (50%), 5-cis-BR (15%), 9-cis-BR (10%), 13-cis-BR (4%); other carotenoids were dehydrated derivatives of BR. Carotenogenesis in SGH1 was a reversible and salt-dependent process; transferring BR-rich cells grown in 25% (w/v) NaCl to 15% (w/v) NaCl medium resulted in depigmentation, and BR content was recovered after transference and growth of unpigmented cells to high salinity medium. Methanol extracts and purified BR isomers showed an 8\u20139-fold higher antioxidant activity than Trolox or \u03b2-carotene. Both, plasma membrane integrity and mitochondrial membrane potential measurements under acute 18-h assays showed that purified BR isomers were non-toxic to cultured human THP-1 cells.An extreme halophilic archaeon, strain SGH1, is a novel microorganism isolated from endolithic microbial communities colonizing halites at Salar Grande, Atacama Desert, in northern Chile. Our study provides structural, biochemical, genomic, and physiological information on this new isolate living at the edge of the physical and chemical extremes at the Atacama Desert. SGH1 is a Gram-negative, red-pigmented, non-motile unicellular coccoid organism. Under the transmission electron microscope, strain SGH1 showed an abundant electro-dense material surrounding electron-lucent globular structures resembling gas vacuoles. Strain SGH1 showed a 16S rRNA gene sequence with a close phylogenetic relationship to the extreme halophilic archaea  The Atacama Desert is considered to be the driest and oldest dryland on Earth . The micLithobiontic colonization can be considered as one of the most successful strategies for microbial survival under the extreme desiccation and high solar radiation on the Atacama Desert. Lithic habitats provide one of the most efficient niches to sustain microbial life by acting as a filter to solar and ultraviolet radiations, as a liquid water harvesting device from fogs and dew events, by salt deliquescence and capillary condensation . In AtacHaloterrigena sp. strain SGH1 and it is an extreme halophilic archaeon with a high content of the carotenoid bacterioruberin . Collection and further work were carried out aseptically with all materials previously sterilized.Salar Grande, with an approximate surface of 500 km3, 50 mM KCl, with NaCl at concentrations between 0 and 30% w/v (medium Z8-NK). Compatible organic solutes  were also added to the Z8-NK medium at 10 mM final concentration. Cultures were incubated at 30\u00b0C, in a rotary incubator (Zichen ZWWY-100B) at 120 rpm, under constant illumination with white fluorescent light (34 \u03bcmoles m\u20132 s\u20131). After 2\u20133 weeks, growth was observed in cultures growing in Z8-NK containing 25% NaCl and 10 mM L-proline, as an evident increase in turbidity. Aliquots were transferred to 3% agar plates prepared in Z8-NK plus 25% NaCl and 10 mM L-proline and incubated until colonies were observed. Light microscopy observations, before or after Gram staining, confirmed the presence of only one type of microorganism in the isolated colonies. The isolated microorganism was denominated strain SGH1. SGH1 growth improvement was obtained after L-proline was replaced by 0.5% w/v casamino acids. This new medium, Z8-MOD at 25% NaCl, was used for maintenance and biomass production. Strain SGH1 was conserved using glycerol 25% at \u221280\u00b0C.Halites samples were broken open to expose the endolithic colonization which can be observed as a greenish line that runs parallel at few mm below the rock surface . Using aAliquots of liquid cultures and agar colonies were fixed 3 h at 5\u00b0C with 3% glutaraldehyde (EMS grade) in 0.1 mM cacodylate buffer. Samples were washed several times by centrifugation and pelleted in fresh sterile agar. Pellets were post-fixed with 1% (w/v) osmium tetroxide for 5 h at room temperature. Dehydration included a step with 70% ethanol with 2% uranyl acetate to increase contrast of intracellular components. Samples were infiltrate with LR-White resin and polymerized using gelatin capsules at 60\u00b0C for 24 h. An Ultracut E ultramicrotome (Reichert-Jung) was used for preparation of ultrathin 80 nm sections that were placed on copper grids and contrasted with lead citrate. Grids were examined at 80 kV with a Leo 910  transmission electron microscope equipped with a Gatan BioScan 792 camera .660: 0.1) was cultured in 25 mL of liquid medium Z8-MOD at different final salinities from 0 to 30% w/v NaCl. Once the optimal salinity was determined, the SGH1 cultures were grown at various pH (from 6 to 10) and temperatures (from 20 to 50\u00b0C) in order to define optimal growth conditions. SGH1 growth was followed as increments in turbidity at 660 nm and doubling time were computed at the exponential phase of growth. Tolerance of strain SGH1 to MgCl2 x 6H2O (50 to 500 mM), to sodium or magnesium perchlorate (50 to 300 mM) was conducted in independent growth experiments, in triplicates, in medium Z8-MOD. The specific growth rate for each condition was computed after 96 h of growth.Strain SGH1  and finally stored in amber bottles at 4\u00b0C.Strain SGH1 grown in Z8-MOD medium was harvested at the early stationary phase of growth by centrifugation . Cells were washed with fresh growth medium at a final concentration of 10% (w/v) NaCl and, cells were recovered by centrifugation . The cell pellet was lyophilized and the dried biomass was exhaustively extracted with 100% methanol (HPLC grade) at 4\u00b0C, under dim light, in order to obtain a fraction enriched in total carotenoids. The methanolic extracts were clarified by centrifugation  and the uncolored cell residues were discarded. The methanolic extract was dried by N\u20131 cm\u20131) (Absorption spectrum (350\u2013600 nm) of carotenoid extracts were obtained in a Shimadzu spectrophotometer UV-1601 and absorbance values at 494 nm were used to compute total SGH1 carotenoids concentration in SGH1 methanol extracts, using the following equation and the extinction coefficient for BR in methanol  :2 and dissolved in 100% methanol. Each recovered HPLC fraction (10 \u03bcL) was injected in a Thermo Scientific Dionex Ultimate-3000 UHPLC system with a C18 column ; the mobile phase for elution was a methanol/acetonitrile/water gradient  for 2 min, 30:70:0 (v/v) for 10 min, 50:50:0 (v/v) for 15 min, and 90:10:0 (v/v) for 20 min, at 25\u00b0C, at a flow rate of 1.0 mL per min. Data analysis was managed with the Xcalibur 2.3 software (Thermo Fisher Scientific). Absorbance spectra were conducted at 494 nm. The UHPLC chromatographer was coupled to a high-resolution mass spectrometer Q Exactive (Thermo Fisher Scientific) controlled by the Q Exactive 2.0 SP 2 software (Thermo Fisher Scientific). Carotenoid ionization was conducted with an Electrospray Ionization Source at 400\u00b0C and 2,500 V, under nitrogen (Genus NM32LA Peak Scientific) to generate ionized fragments. The scanning of positively ionized fragments was conducted at 100 to 1,000 m/z. Caffeine, N-butylamine, buspirone, sodium dodecyl sulfate, and taurocholate served as standards (Sigma-Aldrich). The scanning of positively ionized fragments was conducted at 100 to 1,000 m/z.ethanol extracts (50 \u03bcL) were injected to a Shimatzu Hitachi CL-20A HPLC system containing a C18 reverse phase column  as stationary phase. For elution, the mobile phase used was methanol/acetonitrile/water  at a flow rate of 1.0 mL/min during 12 min. The collection of fractions was conducted after programming the HPLC system software to start and finish at specific retention times to avoid contamination among the collected peaks. The more abundant HPLC fractions were recovered, dried under NHaloterrigena sp. SGH1 was conducted by data analyses of Raman spectroscopy, chromatographic migrations, UV-Vis spectra, spectral fine structure and MS fragmentation patterns  using a 25% w/v NaCl solution. Next, the samples were dried at room temperature for 24 h in darkness. Micro-Raman analyses of upper region of the dried samples were performed on a multichannel Raman DXL Thermo Fischer spectrometer coupled with a Peltier-cooled (\u221250\u00b0C) CCD detector and equipped with 10x objective. The grating with 900 lines/mm was used. Excitation was provided by the 523 nm Argon laser. Spectrograph slit aperture was 25 \u03bcm. To achieve enhanced signal-to-noise ratios, 44 scans were accumulated, each of 2.70 s exposure time with a laser power of 8.0 mW at the samples. The background exposures were 512. Spectra were recorded with a spectral resolution of 1.9 cmThree assays were employed to evaluate antioxidative activity of the methanolic extracts and fractions purified by HPLC chromatography.2O2 in the presence of ferrous ions and, oxidative damage to DNA can be observed by a differential electrophoretic mobility of relaxed and supercoiled molecules (2O2 plus 15 \u03bcL 100 \u03bcM FeCl2). Oxidized DNA decreases its electrophoretic mobility when compared with intact non-oxidized supercoiled DNA. Protection of plasmid DNA to oxidation was evaluated with 5 \u03bcL of Trolox (50 \u03bcg/\u03bcL), 5 \u03bcL of \u03b2-carotene (50 \u03bcg/\u03bcL) or 5 \u03bcL of SGH1 extract . The assays were incubated at 37\u00b0C during 30 min and stopped by adding 1.0 \u03bcL of 5 mM EDTA. Electrophoresis of intact or damaged plasmid DNA was carried out at room temperature on 0.8% agarose gels for 40 min at 80 mV. The gels were stained with ethidium bromide, dried and bands densitometry was estimated by ImageJ, an open source image processing program.1This assay involves the formation of hydroxyl radicals from Holecules . The ass*+. This mix was diluted with 70% ethanol until the solution reached an absorbance of 0.7 \u00b1 0.02 at 734 nm. The assay included 1.98 mL of the ABTS*+ solution plus 0.02 mL of a carotenoid solution and it was incubated during 7 min at room temperature, in darkness. A decrease in absorbance at 734 nm was used to compute the antioxidant activity of the carotenoid samples, expressed as percentage of inhibition of the oxidation of ABTS*+. The antioxidant activity of SGH1 carotenoids was finally expressed as TEAC  and IC50  (A solution of 7 mM ABTS dissolved in water was mixed with 2.45 mM potassium persulfate in a 1:1 (v/v) ratio during 16 h to generate the radical ABTS ABTS*+) . Each in+3  which was prepared with 10 mM TPTZ, 20 mM FeCl3 x 6 H2O and 0.3M acetate buffer, pH 3.6, at a ratio 1:1:10 (v/v). The assay was conducted during 30 min at room temperature, in darkness. Absorbance at 593 nm was computed to determine the antioxidant activity of carotenoids with respect to Trolox (Albiochem) (The reducing power of SGH1 carotenoids was done mixing SGH1 carotenoid samples (100 \u03bcL) with a freshly prepared solution containing TPTZ-Febiochem) . The ant\u00ae 30-2001) derived from the blood of a 1-year-old boy with acute monocytic leukemia  containing 10% fetal bovine serum (Gibco Cat. 11875085) at 37\u00b0C, in an atmosphere of 5% CO2 in air. HP-1 cells were selected for these studies since cytotoxicity and other cellular responses can be easily and rapidly conducted by flow cytometry; also, they are non-adherent easily cultured cells and, as bloodstream circulating cells, they are a reasonable experimental model for exposure to high concentrations of novel compounds that could eventually be used in biomedical applications (THP-1 cell line (ATCCleukemia  was usedications .5 cells/mL) were starved for 24 h in FluoroBrite DMEM medium (Gibco Cat. A18967-01), free of fetal bovine serum and glutathione. Next, cells were incubated with 0.5% (v/v) methanol , methanol extract (500 \u03bcg/mL), purified Fractions I, II, III, IV, or V (500 \u03bcg/mL), 0.5% (v/v) ethanol  or Antimycin A (30 \u03bcM), as final concentrations in the assays. Eighteen hours after treatment, cells were incubated with 2.4 \u03bcM PI during 15 min. PI positive cells were measured by flow cytometry using an excitation laser beam at 488 and 585 nm detector. Results were expressed as percentage of viable cells. The assays were done in four independent experiments.Cell viability was evaluated by dye exclusion using a membrane impermeant compound. Propidium iodide  is a membrane impermeant fluorogenic dye generally excluded from viable cells . PI bindTM Cat. 564697) is a cationic, lipophilic and fluorescent dye that is readily sequestered by active mitochondria of healthy cells, but not within mitochondria that have lost its \u0394\u03a8m (5 cells/mL) were starved for 24 h in FluoroBrite DMEM medium (Gibco Cat. A18967-01), free of fetal bovine serum and glutathione. Next, cells were incubated with 0.5% (v/v) methanol , methanol extract (500 \u03bcg/mL), purified Fractions I, II, III, IV, or V (500 \u03bcg/mL), 0.5% (v/v) ethanol  or Antimycin A (30 \u03bcM), as final concentrations in the assays. Eighteen hours after treatment, the cells were incubated with 0.8 \u03bcM MTRed during 15 min and MTRed positive cells were quantified by flow cytometry using an excitation laser beam at 640 nm and emission detector at 660 nm. Based on the MTRed intensities of the cell, they were distributed in three different populations nominated as THP-1 cells with low, middle or high \u0394\u03a8m. Results were expressed as percentage of cells distributed in each \u0394\u03a8m level. The assays were done in four independent experiments.Cell viability was also evaluated by measurements of the mitochondrial membrane potential (\u0394\u03a8m), which can be lost in cells undergoing apoptosis or necrosis . MitoSta its \u0394\u03a8m . CellulaMN410435) against the sequences of different groups of Haloarchaea Class that were retrieved from NCBI. Multiple sequence alignment was created by ClustalW in MEGA 7  or t-test .Statistical data analysis was done by ANOVA with Bonferroni correction  against NCBI non-redundant nucleotide collection showed a 99% of identity to Haloterrigena turkmenica  NaCl at 45\u00b0C . Strain 2 x 6 H2O). SGH1 cells were also able to grow at 150 mM sodium or magnesium perchlorate with a decrease in its specific growth rate close to 40% (The effect of magnesium chloride (50 to 500 mM) and sodium or magnesium perchlorate (50 to 300 mM) on SGH1 aerobic growth was considered as a measure of tolerance of strain SGH1 for these salts, and results are shown in e to 40% . Higher SGH1 accumulated nearly 400 \u03bcg of BR per gram of dry biomass at late exponential phase of growth at 25\u201330% NaCl and 25\u201335\u00b0C . Strain Haloterrigena sp. SGH1 was based on the data analyses of their chromatographic migrations, UV-Vis spectra, spectral fine structure and MS fragmentation patterns (\u20131) that were in close proximity with those informed for extracts from other haloarchaea  were used to discriminate among geometric isomers and anhydrous derivatives of BR in strain Haloterrigena sp. SGH1 (absorbance ratios%III/II and%DII/II)  .Haloterrigena sp. strain SGH1 cells contain six C-50 molecules related to the BR family: four geometric isomers  and two dehydrated derivatives  and, one the metabolic intermediate . Also, all-trans-BR was the major BR isomer accounting for nearly 65% of all BR-type carotenoids in SGH1. Fractions FI and FII are enriched in all-trans-BR and 5-cis-BR, respectively, and 9-cis-BR is the major carotenoid in FIII. Carotenoid composition plus UHPLC-MS and spectrophotometric data are summarized in Mass spectrometry provided the corresponding profiles for positively ionized fragments and their m/z for each major subfraction as shown in Haloterrigena sp. SGH1. First, the protective role of SGH1 extracts against oxidative damage of DNA was tested using plasmid pUC19. Incubation of plasmid DNA with the Fenton\u2019s reagent rendered a relaxation of the original supercoiled DNA with a decreased electrophoretic mobility  to H. turkmenica 4kT  NaCl  NaCl , which iHaloterrigena sp. strain SGH1 is a reversible, salt-dependent metabolic process and the maximum carotenoid content was obtained in cells grown at 25\u00b0C and 25% NaCl. Comparatively, a lower carotenoid content has been registered for other halophilic archaea; e.g., 45 \u03bcg g\u20131 of dry biomass in Halococcus morrhuae  in a hyperpigmented, genetically modified Haloferax volcanii strain HVLON3 is higher than in other haloarchaea. The red-pigmented strain SGH1 accumulates BR when growing at high salinity (25% NaCl) but the BR content decreased nearly 20-fold after the SGH1 cells are transferred and adapted to grow at low salinity (15% NaCl). Depigmentation is a reversible process in which the BR biosynthesis resumed after the unpigmented cells are transferred and grown at high salinity, reaching the maximum BR content observed in SGH1 cells adapted to grow at high salinity. Thus, BR content correlates reversibly with the salinity of the growth medium, an adaptive process not previously reported in other Haloterrigena species. Several studies on the reversible effect of salinity on carotenoid content are available; for example, (i) two extreme halophilic archaea, Halobacterium cutirubrum and Halobacterium mediterranei, showed that their C40 and C50 carotenoids content was also salt-modulated in an opposite fashion, that is, carotenoids in H. mediterranei decreased severely at high salinity, while high carotenoid content was observed when H. cutirubrum were grown at 20\u201335% salinity  to high salinity (20% NaCl) showed a twofold increment on the expression and a threefold increase in specific activity of HMG-CoA reductase, an enzyme involved in isoprenoide biosynthesis leading to carotenoids in haloarchaea, but a 1.5-fold decrease in carotenoid content  Kuechanism  and,  and the three major Raman displacement signals  observed in the methanolic extracts , two dehydrated derivatives  and one metabolic intermediate (cis-mono-anhydrous-BR). Among them, all-trans-BR was the most abundant carotenoid accounting for 65% of total carotenoids in strain SGH1 and all geometric isomers of BR represented nearly 80% of total carotenoids in SGH1. The presence of minor carotenoids  was not investigated. In addition, the use of a C18 column in the HPLC system showed limitations in the separation of fractions; for example, -cis-BR. In a similar way, the major signal in Fig. FIII.2 has a RT of 11.71 min, and it was identified as 9-cis-BR in Methanolic extracts of isolate SGH1 were chromatographically separated into 17 fractions. Those fractions with the highest relative abundance were selected for further analyses. The identification of the carotenoids was based on their UV-Vis spectra, spectral fine structure and MS fragmentation patterns. We found that BR molecules from 50 carotenoids have been reported in other halophilic archaea, as expected for studies carried out with different genera and species. Abundant C50 carotenoids and minor contents of C30, C40, and C51 carotenoids were found in H. turkmenica DMS-5511 , five different cis-isomers of BR and 10 different dehydrated isomer derivatives of BR, and all-trans-BR (Different CDMS-5511 . Extracttrans-BR . The pretrans BR isomers have a linear arrangement of its doble bonds but cis-BR isomers are bended molecules (cis-isomers have higher absorbance than trans-isomers at the cis-peak (388 nm) which becomes more intense as the cis double bond gets closer to the center of the molecules  NaCl at a maximal concentration of 150 mM magnesium or sodium perchlorate, with a 40% drop in its specific growth rate, suggesting the existence and expression of SGH1 genes involved in perchlorate tolerance.Lithobiontic microbial consortia thriving within halite pinnacles from Salar Grande are in a \u2018foggy environment\u2019 . These eoarchaea . The halte salts . Similard medium . EvidencHaloterrigena sp. strain SGH1 to magnesium was investigated by adding increasing concentrations of MgCl2 until it was equivalent to a 2,500-fold increment from 0.2 mM MgCl2, the concentration in the growth medium. A drop of 25% on its growth rate was observed when isolate SGH1 was grown in 25% (w/v) NaCl plus 0.5M magnesium chloride. The magnesium concentration within the endolithic colonization zone in halites from Salar Grande is 0.4 mg kg\u20131 nearly 10 times lower than the concentration in the growth medium. Thus, isolate SGH1 is tolerant to chaotropic agents such as high magnesium concentrations during growth in a medium at high salinity (25% NaCl). This agrees with growth at a maximal concentration of 0.42M MgCl2 and 2.76M NaCl, of halophilic isolates from a MgCl2-dominated hypersaline lake at the Mediterranean Sea  were similar or higher than their original methanolic extracts, depending upon de antioxidant assay used. This is the first report of antioxidant activity of purified isomers of BR. Our results are in clear correspondence with previous reports using extracts of halophilic archaea from various origins; for example, TEAC values for carotenoid extracts from two halophilic archaea, H. morrhuae and Halobacterium salinarium, were 5.1\u20135.3, with IC50 of 0.8 \u03bcg/mL  in comparison with control cells (80% viability). Then, BR isomers and derivatives are not toxic to cultured human cells. The addition of these antioxidant and non-toxic molecules improved the viability of starved THP-1 cells for at least 42 h (24 h of starving plus 18 h of treatment).We report here the effect of BR isomers and derivatives on human THP-1 monocytes. HP-1 cells are bloodstream-circulating, non-adherent cells and a good model to be exposed to high concentrations of novel compounds that eventually may be used in biomedical applications . All celHaloterrigena sp. strain SGH1 decreased the mitochondrial membrane potential of THP-1 cells at a non-toxic level. The mechanism for this effect requires more investigation and we propose that the activation of mitochondrial uncoupling proteins may be involved. The existence of a feedback loop between ROS and the inner mitochondrial membrane proton leak has been proposed . A parallel increase in the percentage of middle \u0394\u03a8m-cells was observed, and Fractions II and Fraction III were most effective . Neither the methanolic extract nor all the purified fractions increased the percentage of cells showing low-\u0394\u03a8m, in contrast to the effect of 30 \u03bcM Antimycin A (46% increase), which is used as a positive control for a partial disruption of \u0394\u03a8m by inhibiting Complex III at the mitochondrial electron transport chains. In consequence, BR molecules from proposed : ROS wouproposed . Since pproposed , we propArthrobacter agilis 50Cyt, containing all-trans-BR, was shown to be not phototoxic to cultured murine fibroblasts; however, the glycosylated BR molecules were highly phototoxic and cytotoxic to these cells  showed maximal BR content, but growth at low salinity (15%) rendered unpigmented cells. Growth of strain SGH1 at 25% (w/v) NaCl, was limited by magnesium chloride and sodium or magnesium perchlorate at maximal tolerable concentrations of 500 and 150 mM, respectively; thus, isolate SGH1 can be considered a new experimental model in which to study genes and enzymes associated to aerobic/anaerobic perchlorate metabolism and its applications on biological removal of perchlorates from nitrate-rich fertilizers and natural waters. Also, this is the first report that shows that the antioxidant activity of purified isomers of BR is higher than Trolox, beta-carotene, and astaxanthin. In addition, we report the first evaluation of BR toxicity on THP-1 cells concluding that purified BR isomers are not toxic to cultured human cells and open the opportunity for new applications in pharmaceutical, food and medical products.Lithobiontic microbial consortia in Atacama halites form biofilms with a surprisingly complex diversity that includes unculturable members of the three domains of life and viruses. MN410435.Nucleotide sequence used in this work can be found in NCBI accession number NF conducted extraction, purification and identification of carotenoids and evaluated their biochemical properties. SH isolated and purifies strain SGH1. NF, MV, and AG implemented and evaluated the antioxidant activity of extracts and purified fractions. LZ, FF, and BP conducted cytotoxic assays. CS-A applied HPLC to extracts and collected fractions. CA, JW, and VS-E conducted TEM and JW the Raman studies. SH, AG, CV, and JA conducted genomic studies. RB-G analyzed results and reviewed critically the manuscript. BG-S conceived and designed the studies and wrote the manuscript. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
{"text": "Despite substantial evidence for sex differences in addiction epidemiology, addiction\u2010relevant behaviors and associated neurobiological phenomena, the mechanisms and implications of these differences remain unknown. Genetic analysis in model organism is a potentially powerful and effective means of discovering the mechanisms that underlie sex differences in addiction. Human genetic studies are beginning to show precise risk variants that influence the mechanisms of addiction but typically lack sufficient power or neurobiological mechanistic access, particularly for the discovery of the mechanisms that underlie sex differences. Our thesis in this review is that genetic variation in model organisms are a promising approach that can complement these investigations to show the biological mechanisms that underlie sex differences in addiction. Sex differences in addiction are well documented in prevalence, trajectory and treatment outcomes. Human genetic studies require tremendous sample sizes to identify the biological basis of these effects. Control over environmental influences and drug exposure combined with the ease of neuro chemical and neuro anatomical access make model organisms especially suited to reveal the mechanisms underlying sex differences in addiction. Although human genetic studies remain the standard for establishing genetic etiology for a complex neuropsychiatric condition like addiction, studies attempting to investigate sex\u2010specific differences in processes of addiction largely lack the phenotypic breadth, power and neural tissue access needed to discover the underlying molecular mechanisms. In contrast, genetic studies in model organisms benefit from lower sample size requirements, and a wealth of in vivo technologies for research into mechanisms. Model organism studies have showed or corroborated observations of sex and gender differences in addiction. Therefore, there is potential for genetic mapping of these phenomena to identify mechanisms of sex differences in addiction.In this review, we highlight the epidemiological evidence for sex differences in SUDs, the influence of the environment on such sex differences and the promise and challenge of genetic analysis for discovering the biological mechanisms that underlie sex differences in addiction through the use of human and model organism discovery genetics. We highlight the major gender differences in phenomena related to SUDs, and the progress made toward studying these phenomena in human genetic and conventional model organisms studies. The literature on these phenomena is quite uneven with respect to the classes of drugs investigated across species and genetic backgrounds but show some general phenomena across classes of drugs. In other cases, the magnitude and direction of effects vary across strains and species. We have chosen examples from the literature that reflect this diversity.Overall, few human genetic studies have succeeded in identifying sex\u2010specific loci for addiction because of inadequate statistical power. Although human genetic studies have been attempted for each major class of drugs, these studies are few in number and sex differences have been studied in even fewer. We have summarized these studies to call attention to this dearth of results. We argue that model organism studies show many sex differences in addiction\u2010related phenomena, and that with recent improvements to genetic analysis methods in model organisms, these are now amenable to genetic investigation. Model organisms have benefits of (a) increased minor allele frequency, (b) controlled genetic background variation, (c) controlled environments, (d) fully ascertained drug exposure histories and (e) access to neurobiological intermediate phenotypes that serve to increase the observed effect size of genetic loci, revealing previously undiscovered causal mechanisms underlying sex differences that can be further interrogated.2There are many documented differences in drug use and addiction between men and women. The Treatment Episode Data Set provided by the Substance Abuse and Mental Health Services Administration indicates sex differences in prevalence of substance use, the particular substances involved, the age at which drug use is initiated and drug use patterns.3Males and females exhibit differences in the substances they use and in patterns of drug use. For example, men are more likely to smoke marijuana, while women are more likely to use alcohol and prescription drugs, including benzodiazepines and sedatives.Subjective effects of drugs in women are affected by the stage of the menstrual cycle.Treatment outcomes also differ between the genders. Men and women face unique challenges in cessation of drug use. Women tend to enter treatment sooner after becoming substance dependentIn summary, gender differences in human addiction are likely the result of sex\u2010specific biological mechanisms that interact with sociocultural influences and life stressors that affect individuals of different genders differently. The complex biopsychosocial interactions underlying addiction make discovery of the biological basis of sex differences in humans challenging, yet identifying these biological mechanisms is critical for the development of more precisely tailored preventative and therapeutic interventions.44.1It has long been appreciated that a family history of SUD is one of the strongest risk factors for the development of drug addiction.A small number of these studies have assessed the heritability of substance use by gender of the subjectsGenome wide linkage studies, genome\u2010wide association studies (GWAS), whole genome sequencing and exome sequencing have begun to identify specific genes and genetic variants that explain the heritability of SUDs, although few such studies have reported gender specific genetic effects Table .4.2Early genome\u2010wide linkage studies4.3For highly complex diseases like addiction that are influenced by vast numbers of genetic variants, population\u2010based genome\u2010wide association approaches are better suited to identify risk loci with relatively small effects compared with family\u2010based genetic studies,A recent GWAS5Some GWAS studies have incorporated the stress\u2010related risk factors that contribute to the development of drug addiction and in addiction relapse susceptibility in GWAS. As noted above, the psychosocial and cultural factors that influence addiction epidemiology are largely attributable to stress. Stress increases vulnerability to drug addiction,6Genomic studies examine the abundance of all expressed genes under various conditions in the postmortem human brain. The transition from recreational substance use to addiction is accompanied by drug\u2010induced neuroadaptations brought about by long lasting expression changes in neural genes.Integrating genome\u2010wide genetic findings with tissue\u2010specific gene expression genetics could reveal additional biological mechanisms underlying substance dependence. Using this approach, Huggett and Stallings have identified a SNP associated with cocaine dependence and detected three genes (two loci) underlying this predisposition that displayed robust enrichment in numerous brain regions, including the hippocampus.7In summary, human GWAS have been making inroads into the discovery of genetic influences on addiction. However, identifying the genetic components that influence sex\u2010specific vulnerability to addiction using GWAS has been particularly challenging because of the high heterogeneity of the population in terms of the environmental factors that influence addiction, from drug exposure and use trajectories to the involvement of interacting histories of stressful life events and other confounding influences of social, economic and cultural factors. As a result of this heterogeneity, the common variants identified for SUD in humans by GWAS have modest effect sizes. The sample size requirements necessary to achieve adequate statistical power to detect the numerous polygenic and small effect sizes associated with complex and highly heterogeneous phenotypes8Despite the inherent challenges of modeling psychiatric disorders like addiction in animals,Model organisms, particularly rodents, have many conserved neurobiological and physiological features of humans and display specific facets of addiction\u2010related behavior and neurobiological endophenotypes. As such, they can be used to identify causal mechanisms in brain\u2010behavior relationships, including neurobiological and behavioral consequences of chronic substance exposure.Animal studies allow greater choice over the population's extent of genetic heterogeneity and each population has specific advantages in noise reduction for detection of small effect alleles or a broad survey of very high genetic heterogeneity with variation in nearly every gene in the genome. Minor allele frequencies in even the most complex mouse populations such as the Diversity Outbred (DO) are theoretically 12.5%, providing greater power for detection of variants that influence complex traits in substantially smaller sample sizes than required in human populations.9https://phenome.jax.org) in 2017, 47.5% of all behavioral measures are on males only, 5% on females only and 47.5% on both sexes. Strain by sex differences were observed in 42% of behavioral measures where males and females were included. Of these behavioral measures annotated to the Vertebrate Trait term \u201cresponse to addictive substance trait,\u201d 39% exhibited strain by sex differences. As this number grows, and as genetic mapping studies in these populations are performed, discovery of mechanisms of addiction\u2010related sex differences will be increasingly possible.Females have been systematically understudied in neuroscience and biomedical research.Reservations about including females have been based on the assumption that female hormonal cycles introduce substantial \u201cnoise\u201d and complicate experimental studies relative to male\u2010only studies,In recent years, the number of studies that have included female subjects has increased. However, studies that have explicitly investigated sex differences10There are several reviews addressing sex differences in SUD and addiction.Sex differences in behavioral effects of addictive drugs are widely observed, but the extent and magnitude of these differences vary across species, strains and even vendors Table . For exaAlthough the magnitude and direction of the differences may vary in different species and strains, this is a \u201cfeature,\u201d not a \u201cbug.\u201d It is precisely this variation that we are harnessing in the use of genetic variation to discover the biological mechanisms of sex differences. The genes and variants detected are likely to interact with stress, as employed in the laboratory and as perceived by the subject. Stress effects vary in their direction with variation in the magnitude of the stressor. Therefore, stress\u2010related genes may have similar roles across species but the magnitude and direction of the effects may vary. Such was the case observed in studies of melanocortin 1 variation in mice and humans, originally detected in rodent genetic studies of stress\u2010induced analgesia.11Gene expression analyses in reward\u2010related regions over time following drug exposure show mechanisms that underlie drug response, drug\u2010seeking and drug\u2010taking.12Despite the general consensus that females are more sensitive to many drug effects, some studies report either no difference13Rodent studies allow the evaluation of the nature of sex differences and to what extent they are attributable to chromosomal or hormonal influences. Sex differences are influenced by multiple separable and/or interacting sex\u2010biasing factors.Quantitative trait locus (QTL) mapping is one of the primary genetic strategies used to show mechanisms underlying sex differences. Modern behavioral QTL studies of sex\u2010specific loci are readily performed in recombinant inbred mouse strains C57BL/6JxDBA/2J (BXD), experimental crosses of closely related strains (C57BL/6J (B6) and C57L/J (C57)),Minor allele frequencies are typically higher in mouse populations than human populations, rendering possible the detection of small effect alleles. Selective breeding for behavioral phenotypes that correlate with drug\u2010seeking behaviors enriches and genetically fixes risk alleles.Gene expression is also influenced by sex by genotype interactions, and expression QTL that represent genomic loci responsible for differential transcriptional regulation have been identified. Through correlational analysis, we have been able to move beyond sex\u2010specific QTL and identify sex\u2010specific gene expression networks.14Model organisms allow the study of mechanisms of the interplay among, environmental and genetic interactions that contribute to sex differences in addiction vulnerabilities. Modulation of the interactions among stress\u2010 and drug\u2010related traits by sex has been investigated in rodents for alcohol use15In conclusion, genetic variation in humans and model organisms can be exploited in complementary ways to reveal the biological mechanisms that underlie sex differences in addiction. The genetic influence of sex differences in addiction\u2010related behavior can be detected but not readily identified in human genetic studies because of lack of statistical power at current sample sizes, and perhaps more importantly, the tremendous variability in drug exposure, lifetime history of stress and other environmental influences that contribute to human heterogeneity. However, there is now ample evidence for the existence of sex differences, and abundant evidence for genetic differences in stress\u2010related effects, known to often mediate or modulate sex differences in addiction\u2010related behaviors. Rodents exhibit many addiction\u2010related behaviors and sex and strain x sex differences are present in drug\u2010related phenotypes and predisposing traits such as vulnerability to stress effects on these behaviors. Sophisticated genetic mapping populations, neurobiological and molecular analysis tools are more readily deployed in rodent populations and sex by genotype analyses are more adequately powered as a result of the higher minor allele frequencies present in these populations. Therefore, the biological mechanisms of sex differences in many different processes of addiction are more readily discoverable using model organism genetics. The challenge remains in clearly establishing the meaning of the model\u2014what human traits, including endophenotypes, are conserved? What elements of the biological mechanisms are conserved and which are not? Clearly, the precise genetic variants harbored by one human population or another are not readily found in a rodent population, but many elements of the molecular pathways are. These serve as valuable pointers to the mechanistic basis of sex differences in addiction and their implications for clinical applications in the prevention and treatment of SUDs."}
{"text": "Krishnan et al. use FISH to show that specific forms of RB induce changes in the organization of euchromatin and heterochromatin domains. These changes are visible under the microscope, occur after cell cycle arrest, are separable from senescence, and represent an E2F-independent activity of RB. RB restricts G1/S progression by inhibiting E2F. Here, we show that sustained expression of active RB, and prolonged G1 arrest, causes visible changes in chromosome architecture that are not directly associated with E2F inhibition. Using FISH probes against two euchromatin RB-associated regions, two heterochromatin domains that lack RB-bound loci, and two whole-chromosome probes, we found that constitutively active RB (\u0394CDK-RB) promoted a more diffuse, dispersed, and scattered chromatin organization. These changes were RB dependent, were driven by specific isoforms of monophosphorylated RB, and required known RB-associated activities. \u0394CDK-RB altered physical interactions between RB-bound genomic loci, but the RB-induced changes in chromosome architecture were unaffected by dominant-negative DP1. The RB-induced changes appeared to be widespread and influenced chromosome localization within nuclei. Gene expression profiles revealed that the dispersion phenotype was associated with an increased autophagy response. We infer that, after cell cycle arrest, RB acts through noncanonical mechanisms to significantly change nuclear organization, and this reorganization correlates with transitions in cellular state. The best-known molecular function of RB (the protein product of the retinoblastoma tumor susceptibility gene) is the regulation of E2F-dependent transcription . E2F conPerhaps less well known is that ChIP-seq experiments show an extensive distribution of RB-binding sites that extends far behind the conventional set of E2F-regulated genes . These sTo answer these questions, we used FISH probes and took advantage of oligopaint technology  to look To assess chromatin organization, we took a visual approach and performed FISH experiments to detect large chromosomal regions. We used probes against two euchromatin regions on chromosome 19  and two heterochromatin regions . These gave two clearly separated signals in nuclei of WT RPE1 cells , but eacTo examine the hypothesis that active RB influences the organization of large chromosomal regions, FISH was performed on cells induced to enter senescence. We chose this cellular context because previous studies have shown that oncogene-induced senescence is RB dependent and have also described reorganization of large chromosomal regions to form senescence-associated heterochromatic foci (SAHF) in some cell types . IR was To ask whether similar changes in organization occur in other forms of RB-mediated arrest, we examined RPE1 cells treated with the CDK4/6 inhibitor palbociclib . PalbociTo test whether active RB is not just necessary but sufficient to cause dispersion, we used a recently described set of RPE1-derived cell lines engineered such that addition of doxycycline (DOX) induces the knockdown of endogenous RB and its replacement by FLAG-tagged versions of RB . With thTo ask whether similar effects of RB activation are visible at other genomic loci, we used two euchromatic probes (19q13.42 probe and 19q13.2) and a second heterochromatin probe that detects the \u03b1-satellite regions on chromosome 6. Measurement of skeleton dot lengths revealed that \u0394CDK-RB had a consistent effect in all four regions examined . \u0394CDK-RBExamination of RB ChIP-seq data revealed an important difference between the euchromatic and heterochromatic regions probed here. While the euchromatic probes 19q13.2 and 19q13.42 contain 119 and 54 RB peaks, respectively (2.52 and 1.99 RB bound loci per 100 kb), the regions targeted by the chromosome 6 and 7 \u03b1-satellite probes contain 0 RB peaks and 0 E2F1 peaks . This suSince IR, palbociclib, and \u0394CDK-RB expression all cause G1 arrest, it was plausible that chromatin organization might fluctuate during the cell cycle and that the measured effects might reflect a state that is prevalent during G1. To explore this, FUCCI-RPE1 cells  were useDuring the time course of IR-induced senescence in IMR-90 fibroblasts, we noted that dispersion of the FISH signal was not an immediate event. Western blots demonstrate that the kinetics and extent of RB dephosphorylation varies between sites . Since RAlthough, expression of all mP-RBs increased G1 , the mP-We infer that all of the regions examined can be organized into states that have different degrees of compaction/dispersion/scatter. In asynchronously dividing cells, these regions typically form compact foci during S/G2. A minor fraction of cells have a more diffuse organization during G1 or in G1-arrested cells. However, the expression of unphosphorylated RB (\u0394CDK-RB), or specific mP-RBs, greatly increased the dispersion/scattering of these regions, effects that were more extensive and more prevalent than the changes seen in other G1 cells. Hence, the diffuse, dispersed, and scattered organization of these chromosomal regions induced by \u0394CDK-RB is not a universal feature of G1 cells but increases under specific conditions and is driven by specific forms of RB.\u0394CDK-RB increases the skeleton dot lengths of both heterochromatin and euchromatin regions, and visually it enhances the punctation, branching, and length of both kinds of chromatin. Previous studies showed that euchromatin regions tend to contract during replicative senescence, whereas heterochromatin regions expand . Thus, tThe four regions examined by FISH lack any well-studied E2F-regulated cell cycle genes or components of the RB loss gene signature , but theIn addition to E2F, RB interacts with many transcription factors and recruits various activities to chromatin . We usedRB has also been found to associate with type II topoisomerases (TOPIIs) and to facilitate the processing and repair of TOPII-induced double-strand breaks during alterations of DNA topologic states . Interesd-ribofuranosylbenzimidazole (DRB), a general inhibitor of transcription  and used chromatin conformation capture (3C), a proximity-based ligation assay, to look for effects of \u0394CDK-RB on intrachromosomal interactions. An interaction map of the region  was usedCollectively, these molecular studies show that the activation of RB changes the patterns of interactions between some RB-bound loci. 3C assays are focused in nature, and a more complete assessment of chromatin interactions will be required to identify the global effects of RB on TAD boundaries and between loci bound (or unbound) by RB. Nevertheless, these results confirm that \u0394CDK-RB causes changes in chromatin organization. \u0394CDK-RB\u2013induced dispersion does not appear to be driven by classic E2F targets, but it requires HDACs and topoisomerases, two chromatin regulatory activities previously shown to associate with RB.To ask whether the nuclear changes extend beyond the four regions examined by FISH and 3C, the effects of \u0394CDK-RB were assessed using whole-chromosome probes. As expected, probes for chromosomes 17 and 19 gave FISH signals that were more extensive than the regional probes, yet they clearly scattered further following \u0394CDK-RB expression . QuantifA second feature of the chromosome 19  and 17  FISH sigThese results show that RB does not simply cause reorganization of a few small regions. Instead RB has extensive effects on nuclear organization that are evident across whole chromosomes and even impact chromosome positioning within the nucleus. Using a complementary biochemical method to look for global changes, we treated chromatin with micrococcal nuclease (MNase) and found that \u0394CDK-RB expression increased the resistance of chromatin to digestion . Genomewr > 0.6). In contrast, only three transcripts showed a negative correlation , including genes that are required for autophagosome formation and maturation  and the increase in their levels was not due just to defects in autophagosome clearance, we costained for LAMP2  as well. We compared \u0394CDK-RB and mP-RB 356, two forms of RB that strongly induce dispersion with mP-RB 780 (largely defective for dispersion) and mP-RB 252 (an intermediate euchromatin dispersion phenotype). Cells were stained after treatment with or without bafilomycin, a vacuolar H+/ATPase inhibitor that prevents lysosomal acidification and allows autophagosomes to accumulate, making it possible to quantify autophagy levels. Time course samples  showed a progressive increase in LAMP2-colocalized LC3B-II puncta in cells expressing \u0394CDK-RB and mP-RB 356  increased expression specifically in cells presenting high to medium chromatin dispersion , in a time-dependent manner, upon RB activation  to suppress dispersion. The number of LC3B-II puncta was reduced significantly by etoposide treatment . ConversIn summary, these results show that chromatin dispersion induced by active RB was associated with increased autophagy flux and increased expression of a panel of autophagy-related genes. RB is typically linked to the regulation of cell cycle genes, and this association is notable because it is an unusual signature for an RB-induced phenotype.In this study, we have visualized changes in chromosome and nuclear organization driven by the activation of RB. For years, we and others have speculated that RB might control aspects of chromosome structure, but to date, it had not been possible to observe RB performing this role. Here, using FISH probes, we show that expression of unphosphorylated RB (and specific mP-RBs) causes changes in the organization of euchromatin and heterochromatin domains that are visible under the microscope.Between 48 and 72 h after the induction of active RB, regions of heterochromatin changed in appearance, moving from a compact focus into an undefined shape with multiple domains, fingers, and clusters of foci. In a similar time frame, regions of euchromatin that already had a visibly noncompact organization became more diffuse, with increased numbers of foci that became spaced farther apart. These changes, which we call RB-induced dispersion, were apparent with multiple probes, were seen across whole chromosomes, and when quantified, caused a two- to threefold increase in the mean skeleton dot length of FISH signals. Accompanying these changes, chromosome 19 appeared to alter position so that it localized near to nucleolar structures. This was not a random change, since it was not seen with chromosome 17 probes. Together, these results show that RB-induced arrest involves extensive changes in nuclear organization. Such effects are consistent with the idea that RB is a master regulator of cell proliferation , and theStrikingly, RB-induced dispersion was evident in chromosome domains that are rich in RB-bound loci, as well as those that lack RB-binding sites. RB ChIP-seq data showed that the two euchromatin regions examined here are highly enriched in RB peaks, whereas the two tested heterochromatin regions lacked any mapped RB-binding sites. The fact that all of these regions showed RB-induced dispersion can be interpreted in three different ways. One interpretation is that the reorganization of chromatin domains following the activation of RB is not a direct effect of RB action but an indirect consequence that is triggered by active RB. Thus, dispersion occurs in chromosome domains regardless of the number of RB-binding sites that they contain. An alternative explanation suggested by the work of Dick and colleagues  is that Regardless of the model, the bottom line is that the RB-induced changes in organization are a noncanonical activity of RB . The chaThe finding that some mP-RBs strongly promote dispersion, while others are almost completely deficient for this activity, adds to an emerging view that there is not a single form of functional RB, but many variations of RB that have distinct properties . These fr > 0.6) have RB-binding sites in their promoter or enhancer sequences , and antibiotics . IMR-90 , a human diploid lung fibroblast cell line, was cultured in modified Eagle media supplemented with 10% tetracycline-free FBS and antibiotics (100 units/ml penicillin and 100 \u00b5g/ml streptomycin). To serum-starve RPE1 cells, we cultured them for 3 d in DMEM supplemented with 0.1% tetracycline-free FBS, 2 mM L-glutamine, and antibiotics (100 units/ml penicillin and streptomycin).Telomerase-expressing nontransformed human retina epithelial cells (RPE1) were kindly provided by Dr. David Pellman\u2019s laboratory . RPE1 cells were cultured in DMEM  supplemented with 5% tetracycline-free FBS , 2 mM RB1 knockdown or replacement of endogenous by exogenous RB protein in RPE1 cells transduced with pINDUCER11-shRB1 and/or pINDUCER20-FLAG-RB constructs was induced by 2 \u00b5g/ml DOX  for 72 h. The above constructs and cell lines were described before by For autophagy time course experiments, \u0394CDK RB, WT, and mP-RB cells were induced with DOX for 24, 48, 72, and 96 h. To accurately determine autophagy flux, 100 nM bafilomycin was added to the medium 3, 24, 24, and 24 h, respectively, before fixing for immunofluorescence (IF) experiments. For autophagy epistasis experiments, 100 nM bafilomycin was added 3 h before fixing for IF experiment.Cells were seeded 16 h before siRNA transfection. Cells were transfected with siRNAs against TOPII\u03b2, TOPII\u03b1, CAPD3, WAPL . The sequences of siRNA pool used for knockdown were TOPII\u03b2, 5\u2032-GAA\u200bGUU\u200bGUC\u200bUGU\u200bUGA\u200bGAG\u200bA-3\u2032, 5\u2032-CGA\u200bAAG\u200bACC\u200bUAA\u200bAUA\u200bCAC\u200bA-3\u2032, 5\u2032-GAU\u200bCAU\u200bAUG\u200bGGA\u200bUGU\u200bCUG\u200bA-3\u2032, and 5\u2032-GGU\u200bGUA\u200bUGA\u200bUGA\u200bAGA\u200bUGU\u200bA-3\u2032; TOPII\u03b1, 5\u2032-CGA\u200bAAG\u200bGAA\u200bUGG\u200bUUA\u200bACU\u200bA-3\u2032, 5\u2032-GAU\u200bGAA\u200bCUC\u200bUGC\u200bAGG\u200bCUA\u200bA-3\u2032, 5\u2032-GGA\u200bGAA\u200bGAU\u200bUAU\u200bACA\u200bUGU\u200bA-3\u2032, and 5\u2032-GGU\u200bAAC\u200bUCC\u200bUUG\u200bAAA\u200bGUA\u200bA-3\u2032; CAPD3, 5\u2032-GAG\u200bAUA\u200bAGG\u200bUCA\u200bUCA\u200bGUU\u200bG-3\u2032, 5\u2032-GAG\u200bUCA\u200bCCC\u200bUGG\u200bUAC\u200bCUU\u200bA-3\u2032, 5\u2032-UAU\u200bGUU\u200bUGA\u200bUCG\u200bUUG\u200bCUU\u200bA-3\u2032, and 5\u2032-GUA\u200bCAC\u200bUGG\u200bUGG\u200bCAU\u200bUUU-3\u2032; and WAPL, 5\u2032-GGA\u200bGUA\u200bUAG\u200bUGC\u200bUCG\u200bGAA\u200bU-3\u2032, 5\u2032-GAG\u200bAGA\u200bUGU\u200bUUA\u200bCGA\u200bGUU\u200bU-3\u2032, 5\u2032-CAA\u200bACA\u200bGUG\u200bAAU\u200bCGA\u200bGUA\u200bA-3\u2032, and 5\u2032-CCA\u200bAAG\u200bAUA\u200bCAC\u200bGGG\u200bAUU\u200bA-3\u2032). After 24 h, transfection medium was removed and replaced with DOX-containing medium to induce either WT RB or \u0394CDK-RB expression. After another 24 h, cells were split into new wells. Cells were transfected once more with siRNAs after 60 h of DOX addition. Cells were harvested or fixed after 72-h DOX induction was completed. The final concentration of siRNAs used was 20 nM for all the above gene targets.RPE1 cells were spotted onto slides using cytospin  and fixed with methanol/acetic acid (3:1) solution for 15 min, followed by two washes with 1\u00d7 PBS for 5 min each. Slides were dried and then washed in 50% formamide (Sigma-Aldrich)/2\u00d7 saline-sodium citrate buffer (SSCT) at 78\u00b0C for 2 min, followed by 50% formamide/2\u00d7 SSCT at 60\u00b0C for 20 min. Probe mix , 2\u00d7 SSCT, 10% dextran sulfate, and 1 \u00b5g RNaseA  was added to the samples, and coverslips were affixed and sealed using rubber cement. Slides were denatured at 78\u00b0C for 2 min and incubated at 37\u00b0C for 42\u201346 h. Coverslips were removed, and slides were washed in 2\u00d7 SSCT at 60\u00b0C for 15 min, followed by 2\u00d7 SSCT and 0.2\u00d7 SSC washes for 2 min each. Slides were mounted then with Vectashield  and sealed.Coverslips with adhered RPE1 cells were washed with PBS and fixed by sequentially incubating in 70, 85, and 100% ethanol for 2 min each. Coverslips were dried, and probe mix was added for chromosome 7 \u03b1-satellite  or chromosome 6 \u03b1-satellite . Coverslips were affixed to slides and sealed using rubber cement. Slides were denatured at 75\u00b0C and incubated at 37\u00b0C for 16 h. Coverslips were removed and washed in 0.25\u00d7 SSC at 72\u00b0C and 2\u00d7 SSC (with 0.05% Tween-20) for 2 min each. Coverslips were mounted using Vectashield (with DAPI) and sealed.Coverslips with adhered RPE1 cells were washed with PBS and fixed by sequentially incubating in 70, 85,and 100% ethanol for 2 min each. Coverslips were dried, probe mix for chromosome 19 or 17 was added , and coverslips were affixed onto slides and sealed using rubber cement. Slides were denatured at 75\u00b0C and incubated at 37\u00b0C for 16 h. Coverslips were removed and washed in 0.4\u00d7 SSC at 72\u00b0C and 2\u00d7 SSC (with 0.05% Tween-20) for 2 min each. Coverslips were mounted using Vectashield (with DAPI) and sealed.Coverslips with adhered RPE1 cells were washed with PBS and fixed with methanol for 10 min at \u221220\u00b0C. Coverslips were washed with PBS two times for 2 min. Cells were permeabilized using permeabilizing buffer (PBS and 0.5% Triton X-100) for 3 min on ice. Coverslips were washed twice with PBS for 2 min. Blocking buffer (1% BSA and 0.05% Tween-20) was added for 1 h. Primary antibodies  were diluted in blocking buffer,added on coverslips, and incubated overnight. Coverslips were washed three times with PBS for 5 min each. Secondary antibodies  were diluted 1:500 in blocking buffer and incubated for 1 h at RT. Coverslips were washed three times with PBS for 5 min and stained with DAPI. Coverslips were mounted using Vectashield  and sealed.A Zeiss LSM 710 Laser Scanning Confocal Microscope and Zeiss Zen software were used to acquire FISH images. Images were captured using parameters for each probe that remained consistent across samples and experiments. Images were acquired at RT, and a Plan-Apochromat 63\u00d7/1.40 Oil DIC M27 was used to capture Z-stacks (6\u201312 sections of 0.5-\u00b5M thickness). FITC/Alexa Fluor 488 filter was used to acquire images for chromosome 7 \u03b1-satellite, 19q13.42, and whole-chromosome 19 and 17 probes. Alexa Fluor 568/Texas Red filter was used to acquire images for chromosome 6 \u03b1-satellite and 19q13.2 probes. Maximum projections of Z-stacks were used for image analysis. No other image processing was performed. Images were analyzed using Fiji (ImageJ) software. To effectively quantify the dispersion phenotype, we used a skeleton dot length plugin, and macros were made to execute the steps outlined in For IF images, a Zeiss LSM 780 Laser Scanning Confocal Microscope and Zeiss Zen software were used to acquire images. A Plan-Apochromat 63\u00d7/1.40 Oil DIC M27 was used to capture snaps. Images were quantified using Fiji (ImageJ). Cytoplasmic LC3B-II puncta were counted using Fiji (ImageJ). Four steps were performed to quantify the puncta: (1) individual cells were cropped/segmented out, (2) images were converted into binary scale using the default or moments dark threshold function, (3) background outliers (punta <1 \u00b5M) were removed (if any), and (4) puncta were counted by analyze particles function. Macros were made to execute these steps.4, 50 mM ascorbic acid, and 2.5 \u00b5M Alexa Fluor 647-azide ) for 30 min and 3 \u00b5M DAPI  in staining buffer  for 15 min. FACS analysis was performed using a LSR II flow cytometer (BD Biosciences), and data were analyzed using FlowJo software.After 3 d of DOX induction, RPE1 cells were incubated with 20 \u00b5M 5\u2032-ethynyl-2\u2019- deoxyuridine (EdU) for 2 h, fixed with 3.7% formaldehyde in PBS for 15 min, blocked with 3% BSA in PBS for 1 min, permeabilized with 0.5% Triton X-100 in PBS for 30 min, and stained with ClickIT reaction . Lysates were boiled for 10 min and vigorously vortexed in between every 3 min. Lysates were analyzed using Bio-Rad gel and transferred to PVDF membranes . Primary antibodies were diluted in 5% BSA  in TBS containing 0.1% Tween 20 (Bio-Rad). The following primary antibodies were used: WAPL (D9J1U) rabbit monoclonal antibody , TOPII\u03b2 rabbit polyclonal antibody , TOPII\u03b1 rabbit monoclonal antibody , CAPD3 rabbit polyclonal antibody , RB1 (4H1) mouse monoclonal antibody , tri-methyl-histone H3 (Lys27) rabbit monoclonal antibody , anti-histone H3 rabbit polyclonal antibody , phospho-histone H2A.X , and anti-FLAG . Secondary antibodies were obtained from Cell Signaling Technologies  diluted in 5% milk at 1:5,000. Molecular weight in the figures was estimated by comparing to molecular weight markers.The assay was performed using Cellular Senescence Assay Kit . Culture plate wells with adhered RPE1 cells were washed with 1\u00d7 PBS and fixed with 1\u00d7 fixing solution for 15 min. Cells were washed twice with 1\u00d7 PBS. 1\u00d7 SA-\u03b2-galactosidase detection solution was added to the cells and incubated overnight in dark at 37\u00b0C. Cells were washed with 1\u00d7 PBS, and the stained cells were imaged under bright-field light microscopy using an ECHO Revolve microscope.RNA was extracted using RNeasy kit  and DNase treatment of RNA was performed before any downstream applications . cDNA was prepared using Taqman Reverse Transcription Reagents . qPCR was performed using Lightcycler 480 SYBR Green I Master  and was run using a Roche LightCycler 480 system. \u03b2-Actin qPCR was run as internal control for each sample/time point. qPCR primers used for IMR-90 senescence time course were \u03b2-actin_F, 5\u2032-CAC\u200bCAT\u200bGTA\u200bCCC\u200bTGG\u200bCAT\u200bTG-3\u2032; \u03b2-actin_R, 5\u2032-GTA\u200bCTT\u200bGCG\u200bCTC\u200bAGG\u200bAGG\u200bAG-3\u2032; LMNB1_F, 5\u2032-AAG\u200bCAT\u200bGAA\u200bACG\u200bCGC\u200bTTG\u200bG-3\u2032; LMNB1_R, 5\u2032-AGT\u200bTTG\u200bGCA\u200bTGG\u200bTAA\u200bGTC\u200bTGC-3\u2032; ICAM1_F, 5\u2032-ATG\u200bCCC\u200bAGA\u200bCAT\u200bCTG\u200bTGT\u200bCC-3\u2032; ICAM1_R, 5\u2032-GGG\u200bGTC\u200bTCT\u200bATG\u200bCCC\u200bAAC\u200bAA-3\u2032; HDDC2_F, 5\u2032-GGG\u200bCAG\u200bCTC\u200bAAG\u200bAGA\u200bGTC\u200bC-3\u2032; HDDC2_R, 5\u2032-GGC\u200bGTA\u200bCAC\u200bATC\u200bGGT\u200bCTT\u200bTGT-3\u2032; CCND1_F, 5\u2032-CAA\u200bTGA\u200bCCC\u200bCGC\u200bACG\u200bATT\u200bTC-3\u2032; and CCND_R, 5\u2032-CAT\u200bGGA\u200bGGG\u200bCGG\u200bATT\u200bGGA\u200bA-3\u2032. qPCR primers used to measure expression of autophagy genes were \u03b2-actin_F, 5\u2032-CAC\u200bCAT\u200bGTA\u200bCCC\u200bTGG\u200bCAT\u200bTG-3\u2032; \u03b2-actin_R: 5\u2032-GTA\u200bCTT\u200bGCG\u200bCTC\u200bAGG\u200bAGG\u200bAG-3\u2032; LC3B_F, 5\u2032-ACG\u200bCAT\u200bTTG\u200bCCA\u200bTCA\u200bCAG\u200bTTG-3\u2032; LC3B_R, 5\u2032-TCT\u200bCTT\u200bAGG\u200bAGT\u200bCAG\u200bGGA\u200bCCT\u200bTCA\u200bG-3\u2032; p62_F, 5\u2032-GAC\u200bTAC\u200bGAC\u200bTTG\u200bTGT\u200bAGC\u200bGTC-3\u2032; p62_R, 5\u2032-AGT\u200bGTC\u200bCGT\u200bGTT\u200bTCA\u200bCCT\u200bTCC-3\u2032; ATG14_F, 5\u2032-GCG\u200bCCA\u200bAAT\u200bGCG\u200bTTC\u200bAGA\u200bG-3\u2032; ATG14_R: 5\u2032-AGT\u200bCGG\u200bCTT\u200bAAC\u200bCTT\u200bTCC\u200bTTC\u200bT-3\u2032; WIPI1_F, 5\u2032-AAC\u200bAGG\u200bTCT\u200bATG\u200bTGC\u200bTCT\u200bCTC\u200bT-3\u2032; WIPI_R, 5\u2032-CTC\u200bATG\u200bGGC\u200bAGC\u200bAAT\u200bAGT\u200bGC-3\u2032; ATG4B_F, 5\u2032-ATG\u200bGAC\u200bGCA\u200bGCT\u200bACT\u200bCTG\u200bAC-3\u2032; ATG4_R: 5\u2032-TTT\u200bTCT\u200bACC\u200bCAG\u200bTAT\u200bCCA\u200bAAC\u200bGG-3\u2032; VCP_F: 5\u2032-CAA\u200bACA\u200bGAA\u200bGAA\u200bCCG\u200bTCC\u200bCAA-3\u2032; VCP_R, 5\u2032-TCA\u200bCCT\u200bCGG\u200bAAC\u200bAAC\u200bTGC\u200bAAT-3\u2032; PSAP_F, 5\u2032-ATG\u200bCAA\u200bAGA\u200bCGT\u200bTGT\u200bCAC\u200bCG-3\u2032; PSAP_R, 5\u2032-GGG\u200bAGG\u200bTAG\u200bGAG\u200bTCC\u200bACT\u200bATC\u200bT-3\u2032; SEC23B_F, 5\u2032-GCT\u200bGGA\u200bGGC\u200bTAC\u200bAAG\u200bAAT\u200bGGT-3\u2032; and SEC23B_R, 5\u2032-AAC\u200bCTG\u200bACA\u200bAAG\u200bTGG\u200bGTT\u200bGAG-3\u2032.PLA assay was performed using a Duolink In Situ PLA GREEN kit from Sigma-Aldrich and following manufacturer instructions. WT and \u0394CDK-RB RPE cells were incubated at 37\u00b0C for 24 and 48 h in the presence of 2 \u00b5g/ml DOX on glass coverslips (EdU incubation was performed at 37\u00b0C for 30 min in the presence of 10 \u00b5M). Cells were then washed twice with cold PBS and fixed with 3.7% formaldehyde in PBS for 15 min at RT and permeabilized for 10 min at RT with 0.5% Triton X-100 in PBS. Blocking was performed with 2.5% BSA in PBS. EdU was detected using Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 647 . Primary antibody incubation was carried at RT for 2 h using mouse-\u03b1-RB  and rabbit-\u03b1-TOPII\u03b2 . Cells were then incubated with \u03b1-rabbit PLA plus probe and \u03b1-mouse PLA minus probe for 1 h at 37\u00b0C. Ligation reaction was then performed for 30 min at 37\u00b0C followed by amplification reaction for 100 min at 37\u00b0C. Finally, coverslips were mounted using SlowFade Diamond Antifade Mountant with DAPI . Images were taken with an ECHO Revolve microscope; images were analyzed and fluorescence was quantified with Matlab software. For analysis, in brief, cell nuclei were segmented using a custom-made image-processing pipeline that can distinguish them from the background. The pipeline identifies the nuclei based on DAPI staining, size, and circularity. Nuclei that were close to image borders or too close to each other and could not be individually segmented were automatically removed by the software. PLA foci numbers and PLA foci and EdU fluorescence intensities were quantified by the software only within the segmented nuclei. Edu-positive cells were classified as S phase cells, and Edu-negative cells were classified as either G1 or G2 cells .p) values to the average Cp values obtained across all tested loci for a particular sample. Fold-changes were calculated using origin ligation product  as the control. Fold-change was calculated using the formula 2(Cp \u2212 CpORIGIN), where pC is the normalized qPCR signal obtained for a specific locus, and pORIGINC is the qPCR signal obtained for the origin self-ligation product. Three biological replicates were performed for each sample, and mean fold-change was calculated and plotted. P values were calculated using multiple t test and Holm\u2013Sidak multiple comparison with GraphPad Prism 6.0 software.5 million to 10 million cells were used per experiment, and 3C was done according to the protocol in To determine interactions between TAD boundaries, qPCR primers were designed around HindIII sites located at the edge of TADs. All TAD boundary interactions were measured with respect to the origin, which was TAD4-reg1. The following primers were used: TAD4-reg1 (origin primer), 5\u2032-CAT\u200bCAG\u200bACA\u200bAGC\u200bCCT\u200bCCC\u200bTC-3\u2032; TAD4-reg1, 5\u2032-TTG\u200bGGC\u200bTTT\u200bCCT\u200bGTG\u200bCTT\u200bCT-3\u2032; TAD4-reg2, 5\u2032-TTG\u200bTCT\u200bAAG\u200bTCG\u200bCTG\u200bCTG\u200bGG-3\u2032; TAD4-reg3, 5\u2032-ACA\u200bGGT\u200bGCT\u200bTAT\u200bGTT\u200bTGC\u200bATT\u200bCT-3\u2032; TAD3-reg1, 5\u2032-GTG\u200bCAG\u200bGGG\u200bTAC\u200bAGA\u200bACA\u200bGA-3\u2032; TAD3-reg2, 5\u2032-CCA\u200bGCC\u200bACC\u200bGAC\u200bAAT\u200bTCC\u200bTT-3\u2032; TAD2-reg1, 5\u2032-TAT\u200bTGG\u200bATG\u200bCCA\u200bGCA\u200bGAG\u200bGC-3\u2032; TAD2-reg2, 5\u2032-ACG\u200bGTA\u200bTCC\u200bCTC\u200bTTC\u200bCCC\u200bTT-3\u2032; and TAD2-reg3, 5\u2032-GGA\u200bGAG\u200bACA\u200bGGA\u200bGAT\u200bGGG\u200bGA-3\u2032. For interactions between RB bound sites located in TAD loops, qPCR primers were designed around HindIII sites located within RB binding sites or closest to binding site. All loop interactions were measured with respect to the origin, which was KMT5C. The following primers were used: KMT5C (origin primer), 5\u2032-TGG\u200bGAC\u200bAGC\u200bTCC\u200bTCT\u200bTTC\u200bCA-3\u2032; KMT5C, 5\u2032-GGC\u200bTTT\u200bCTC\u200bCCT\u200bCCT\u200bGTG\u200bG-3\u2032; UBE2S, 5\u2032-CCG\u200bTCT\u200bGCT\u200bCAC\u200bAGA\u200bGAT\u200bCC-3\u2032; U2AF2, 5\u2032-CAC\u200bAGG\u200bCCA\u200bCAG\u200bAAT\u200bGGT\u200bCT-3\u2032; ZNF524, 5\u2032-ACG\u200bTAC\u200bTTG\u200bCCC\u200bAGA\u200bCAT\u200bGC-3\u2032; SSC5D, 5\u2032-TGA\u200bAAT\u200bTGC\u200bAAC\u200bTGA\u200bGGG\u200bGG-3\u2032; HSPBP1, 5\u2032-GGA\u200bCCC\u200bTAT\u200bGAC\u200bGAG\u200bCAC\u200bAG-3\u2032; PPP6R1, 5\u2032-GGG\u200bCAG\u200bAGT\u200bTAG\u200bGGT\u200bTAC\u200bAGT\u200bG-3\u2032; PTPRH, 5\u2032-TGG\u200bGGG\u200bAAT\u200bTTC\u200bTAG\u200bGGG\u200bCT-3\u2032; and PRPF31, 5\u2032-TTT\u200bTAA\u200bGCC\u200bCCT\u200bCTC\u200bCCC\u200bAG-3\u2032.To determine the effect of DNA damage on dispersion, damage was induced in WT RB and DCDK RB\u2013expressing cells using 10Gy ionizing radiation and camptothecin. We chose these damaging agents because they have the ability to generate damage in cells arrested in G1 as well as later stages. Cells were treated with IR after 72-h DOX induction of \u0394CDK-RB or WT RB (control). Cells were fixed/harvested 2 h after IR treatment. 1 \u00b5M camptothecin was added to \u0394CDK-RB or WT RB (control) cells between 48 and 72 h of DOX induction. Cells were fixed/harvested after the completion of 72-h DOX induction. Lysates for Western blot were prepared as noted above, and FISH for the chromosome 7 \u03b1-satellite was performed as stated above.2, 0.5 mM dithiothreitol, 1 mM PMSF, and protease inhibitor cocktail ). Cells were allowed to swell by keeping them on ice for 10 min. 400 \u00b5l of ice-cold Buffer A + 0.4% NP-40 was added and incubated on ice for 10 min. Cells were centrifuged at 300 g for 5 min at 4\u00b0C to pellet nuclei and other fragments. The isolated nuclei were washed with 1 ml PBS without disturbing the pellet and centrifuged at 2,800 g for 5 min at 4\u00b0C. The pellet was resuspended in 300 \u00b5l of 1\u00d7 Micrococcal Nuclease Buffer (supplemented with 100 \u00b5g/ml BSA) and either 1,500 or 40 units of Micrococcal Nuclease  and incubated at 37\u00b0C on a shaker (300 rpm) for various time intervals. 45 \u00b5l of reaction was taken out at every time interval, and digestion was stopped by adding 5 \u00b5l of 0.5 M EGTA and 200 ng RNase A. Reactions were incubated at 37\u00b0C for 1 h and supplemented with 1% SDS and 10 \u00b5g of proteinase K  at 55\u00b0C for \u22652 h. To extract DNA, an equal volume of phenol/chloroform/isoamyl alcohol  was added to the samples and vortexed vigorously. Samples were centrifuged for 5 min at 16,000 g, and the upper aqueous layer was transferred to a new tube. To precipitate the DNA, glycogen (20 \u00b5g), NH4OAc , and 100% (2.5 volume) ethanol was added to the aqueous phase. Precipitated DNA was washed twice with 70% ethanol and resuspended in H2O. DNA was quantified using Nanodrop, and 1 \u00b5g of DNA per lane was loaded onto an 0.8% agarose gel.The MNase assay was performed using 2 million RPE1 cells per sample. Cells were trypsinized and resuspended in 400 \u00b5l of ice-cold Buffer A   for 30 min at RT and 1% formaldehyde  for 10 min at 37\u00b0C. Chromatin was fragmented to a size range of 200\u2013700 bases with a Branson 250 sonicator. Solubilized chromatin was immunoprecipitated with FLAG rabbit monoclonal antibody  or E2F1 rabbit polyclonal antibody  overnight at 4\u00b0C. Antibody\u2013chromatin complexes were purified with Dynabeads Protein G . After cross-link reversal and RNase A and proteinase K treatment, immunoprecipitated DNA was extracted with AMP Pure XP beads  and analyzed by SYBR-Green real-time qPCR, along with the input DNA using the Roche LightCycler 480 system . ChIP reU test, and exact P values were reported. For multiple comparisons, datasets were analyzed by nonparametric Kruskal\u2013Wallis\u2013Dunn\u2019s multiple comparison test, one-way ANOVA\u2013Dunnett\u2019s multiple comparison test, and multiple t tests. P values are displayed as ns, P > 0.05; *, P \u2264 0.05; **, P \u2264 0.01; ***, P \u2264 0.001; and ****, P \u2264 0.0001.Figures of quantifications were assembled, and statistics were run in GraphPad Prism 6. For comparisons between sample pairs, datasets were analyzed by the nonparametric two-tailed Mann\u2013Whitney r > 0.6) with chromatin dispersion and RB binding status.Table S1shows the results of GO analysis of transcripts positively correlating with dispersion.Click here for additional data file.Table S2r > 0.6) with chromatin dispersion and RB binding status.lists genes positively correlating (Click here for additional data file.SourceData FS1contains original blots for Fig. S1.Click here for additional data file.SourceData FS3contains original blots for Fig. S3.Click here for additional data file.SourceData FS4contains original blots for Fig. S4.Click here for additional data file."}
{"text": "Eighty-five percent of multiple sclerosis cases begin with a discrete attack termed clinically isolated syndrome, but 37% of clinically isolated syndrome patients do not experience a relapse within 20\u2009years of onset. Thus, the identification of biomarkers able to differentiate between individuals who are most likely to have a second clinical attack from those who remain in the clinically isolated syndrome stage is essential to apply a personalized medicine approach. We sought to identify biomarkers from biochemical, metabolic and proteomic screens that predict clinically defined conversion from clinically isolated syndrome to multiple sclerosis and generate a multi-omics-based algorithm with higher prognostic accuracy than any currently available test. An integrative multi-variate approach was applied to the analysis of cerebrospinal fluid samples taken from 54 individuals at the point of clinically isolated syndrome with 2\u201310\u2009years of subsequent follow-up enabling stratification into clinical converters and non-converters. Leukocyte counts were significantly elevated at onset in the clinical converters and predict the occurrence of a second attack with 70% accuracy. Myo-inositol levels were significantly increased in clinical converters while glucose levels were decreased, predicting transition to multiple sclerosis with accuracies of 72% and 63%, respectively. Proteomics analysis identified 89 novel gene products related to conversion. The identified biochemical and protein biomarkers were combined to produce an algorithm with predictive accuracy of 83% for the transition to clinically defined multiple sclerosis, outperforming any individual biomarker in isolation including oligoclonal bands. The identified protein biomarkers are consistent with an exaggerated immune response, perturbed energy metabolism and multiple sclerosis pathology in the clinical converter group. The new biomarkers presented provide novel insight into the molecular pathways promoting disease while the multi-omics algorithm provides a means to more accurately predict whether an individual is likely to convert to clinically defined multiple sclerosis. Probert et al. use an integrative analysis approach to identify novel CSF biomarkers from biochemical, proteomics, and metabolomics assays for the prediction of clinically conversion to multiple sclerosis in patients recruited at clinically isolated syndrome onset. The proposed multivariate algorithm significantly outperforms oligoclonal band status for prediction of clinical conversion.  Clinically isolated syndrome (CIS) is the first manifestation of multiple sclerosis (MS) in 85% of patients.,Currently, MS diagnosis relies upon exclusion of other possible diagnoses followed the interpretation of a combination of detailed clinical evaluation, magnetic resonance imaging (MRI), and CSF analysis according to the latest McDonald Diagnostic criteria.,2 weighted MRI lesions,,,,Recognised risk factors of clinically defined conversion from CIS to MS have been identified including younger age of disease onset, male gender,To identify novel predictive biomarkers for the transition to MS we collected baseline CSF samples from 54 patients with CIS with 2\u201310\u2009years of follow-up to determine clinical conversion. This study represents the first comprehensive multi-omics investigation of CIS to include simultaneously clinical chemistry, metabolomics and proteomics analysis of baseline CSF samples coupled with extensive clinical follow-up and to investigate the power of such biomarkers at predicting clinically defined conversion in an individualized manner. Here we report the capacity of novel biochemical and protein markers to predict the transition from CIS to clinically defined MS. The future use of simple multivariant biomarker algorithms in the clinical care pathway has the evident potential to facilitate the personalization and optimization of therapy for individuals with CIS.CSF samples were collected in the Department of Neurology of the University Hospital Basel, during routine diagnostic measures, as indicated by the treating physicians. Inclusion criteria were as follows: (i) the presence of a monophasic clinical episode suggestive of MS (CIS), not attributable to other diseases Supplementary Fig.Written informed consent was obtained from all patients according to the Declaration of Helsinki. Ethical approval was obtained by the local ethics committee.n\u2009=\u200922 in the converter group would be sufficient.Prior to analysis, a power calculation (PPCA model using the R package MetSizeR) was carried out. This confirmed that a sample size of 40 (20 non-converters and 20 converters) would be sufficient to achieve an FDR cut-off of 0.05 assuming the significance of 10% of variables. These assumptions are in line with our previous omics analysis of MS patient cohorts and indicated that g for 10\u2009min at room temperature, and the cell-free supernatant stored at \u221280\u00b0C within 2\u2009h of collection.3), and total protein concentration (mg/dl). Serum samples were collected at the same visit to calculate the CSF/serum albumin ratio . The integrity of the blood\u2013CSF barrier was determined by calculating the CSF/serum ratio for albumin .CSF samples were centrifuged at 400\u20092O (pH 7.4) containing 1\u2009mM maleic acid as an internal reference standard. Samples were briefly centrifuged at 3000 \u00d7 g for 5\u2009min before transferring to a 5-mm NMR tube.On the day of metabolomics analysis, CSF samples were thawed at room temperature and 100\u2009\u00b5l was then diluted with 450\u2009\u03bcl of 75\u2009mM sodium phosphate buffer prepared in D1H [13C/15N] TCI cryoprobe . The noesygppr1d  pulse sequence was used to acquire 1H NMR spectra with a 2\u2009s presaturation, 32 data collections, a spectral width of 16\u2009ppm, and an acquisition time of 1.46\u2009s. All spectra were preprocessed in Topspin 2.1 ; zero filled by a factor of 2 and multiplied by a 1D exponential corresponding to a 0.3\u2009Hz line broadening. All spectra were baseline corrected with a fifth-degree polynomial and referenced to the lactate doublet at 1.33\u2009ppm. Following visual inspection for errors in baseline correction, referencing, spectral distortion or contamination, the processed spectra were exported to ACD/Labs Spectrus Processor Academic Edition 12.01 , whereby regions of the spectra between 0.83 and 8.47 were split into 0.02-ppm-wide bins. The residual water resonance region (4.13\u20135.22\u2009ppm) was removed from the analysis. The integral of each spectral bin was calculated and exported as a .csv file for statistical analysis. Metabolite assignment was performed by referencing to literature values,,All nuclear magnetic resonance (NMR) spectra were acquired at 310\u2009K using a 700-MHz Bruker AVIII spectrometer operating at 16.4\u2009T equipped with a \u00ae 8000 modular analyzer  and the Gluc3 and LAC2 assays, respectively. There was a significant correlation between the NMR-determined concentration and the laboratory chemistry determined concentration for both glucose  and lactate  . In order to validate the quantification of the metabolites by NMR, the glucose and lactate levels in all CSF samples were measured using a Cobas< 0.001) and B and< 0.001) and D.\u00ae platform from SomaLogic Somalogic Inc, Boulder, Co).Protein biomarker profiling in CSF samples was performed using the SomaScan\u00ae is multiplexed proteomic tool that measures more than 5000 protein analytes including 4783 SOMAmers (slow off-rate modified aptamers) that recognize 4137 distinct human gene targets. The SOMAmers are constructed with chemically modified nucleotides that expand the physicochemical diversity of the large randomized nucleic acid libraries from which the SOMAmer reagents are selected. The SomaScan\u00ae assay measures native proteins in complex matrices by transforming each individual protein concentration into a corresponding SOMAmer reagent concentration, which is then quantified in customized DNA microarrays. The assay takes advantage of SOMAmer reagents\u2019 dual nature as both protein affinity-binding reagents with defined three-dimensional structures, and unique nucleotide sequences recognizable by specific DNA hybridization probes.,SomaScan\u00ae analysis.The CSF samples were stored at \u221280\u00b0C and shipped to SomaLogic  on dry ice for SomaScan\u2018omics datasets (as used in other similar aforementioned studies), such as those studied here, because it helps reduce the dimension of such datasets by extracting a subset of most salient biomarkers (described as a linear equation) that can subsequently be used to predict the class of interest\u2014in this instance clinical converters and non-converters. OPLS-DA models were validated using an external 10-fold cross-validation strategy with repetition coupled with permutation testing as previously described.R2 and Q2 values are then used to assess the model performance  on the training data. The model generated is then applied to the test set (to which the OPLS-DA model is blinded) to determine the predictive accuracy, sensitivity, and specificity of the model . This process of model training and testing is repeated for a total of 1000 times, thereby creating an ensemble of models. It has been shown that in cases where the sample sizes are small, one can achieve a prediction accuracy of up to 70% or higher by chance alone in differentiating two-classes.P-value 0.05 or less), then the discriminatory variables responsible for the observed class separation are extracted by inspection of the average variable importance (VIP) scores. The VIP score of a given variable represents the mean decrease in accuracy which occurs when that variable is removed from the model. Thus, a variable which is highly significant and plays a large role in the diagnostic accuracy of the model will result in a large decrease in accuracy when removed from the model resulting a large VIP score. Conversely, variables which do not play a role in discriminating between groups have very little effect on model accuracy when removed and have a low VIP score.Multivariate orthogonal partial least squares discriminant analysis (OPLS-DA) was performed in R software ,Elastic Net feature selection was applied to the proteomics data using the glmnet packageIn order to identify the combination of clinical chemistry, metabolomics and proteomics variables with the highest predictive accuracy a combined multi-omics strategy was applied to the selected discriminatory variables  by applying the OPLS-DA cross-validation strategy (described above) to every combination of one to six variables and performing a ROC analysis on each model to assess the performance. The linear combination of metabolites which resulted in the highest performing model  are then reported. There was no significant increase in AUC or accuracy between five and six variables and so linear combinations of variables containing greater than 6 were not pursued.t-tests were used for continuous variables while Chi-square tests were used for categorical variables as appropriate. A Bonferroni correction, to account for multiple comparisons, was applied throughout. Two-tailed P-values < 0.05 were considered statistically significant. Receiver operator curves (ROC), area under the curve (AUC), 95% confidence intervals, optimal thresholds for diagnosis and P-values (relative to a null distribution ROC curve with AUC = 0.5) were calculated for each discriminatory variable using the pROC package.All analysis was performed in R software . Two-sample http://metascape.org].P-value < 0.01, a minimum count of 3 and an enrichment factor (the ratio of observed counts to the counts expected by chance) >1.5 were collected and grouped into clusters based on their membership similarities. Kappa scoresPathway enrichment analysis was performed on the discriminatory proteins identified by the multivariate analysis (described above) using Metascape  and followed up for up to 10\u2009years. Twenty-two patients converted to clinically defined MS (hence forth referred to as \u2018converters\u2019) while 32 patients had no signs of further relapses during follow-up (hence forth referred to as \u2018non-converters\u2019). The median time to sample collection in both the converter and non-converter groups was 3\u2009weeks and there was no significant difference in the means  still the majority (69%) of the non-converters also tested positive at onset . As a realb. There was no significant difference in the Qalb or total CSF protein concentration between converters and non-converters at onset. Twenty-nine patients (54%) had \u2018normal\u2019 (< 4 cell/mm3) CSF leukocyte cell counts at baseline. The highest leukocyte cell count was 28.7 cell/mm3 and 13 (24%) patients (nine converters and four non-converters) exhibiting a cell count above 10 cell/mm3 at onset. The leukocyte cell count was increased (>4 cell/mm3) in a larger proportion of converter patients (72%) than non-converters (41%) . Interestingly, the mononuclear cell sub-population was elevated in the converter group relative to non-converters, while the PMN subset was not significantly altered  and weekly correlated with PMN although this correlation did not reach significance following correction for multiple comparisons . As expected, a strong correlation  between total protein levels and Qalb was observed  cell counts], total CSF protein levels and Q altered . Indeed, altered . Mononucobserved .P-value < 0.001) we then investigated how each of these metabolites would perform in isolation, to determine if measuring a single biomarker could produce sufficient predictive accuracy for use in a clinical setting. Both myo-inositol and glucose CSF levels showed greater specificity for predicting the occurrence of a second clinical attack resulting in improved overall accuracy and AUC compared to OCGB status alone . The multi-variate analysis uncovered a panel of 89 proteins driving the discrimination between converter and non-converter in CIS CSF samples, 72 of which predict occurrence of a second attack with greater AUC (>0.66) than OCGB alone  with an AUC of 0.94; the addition of further variables provided no significant increase in accuracy. The variables selected by the multivariate model with greatest accuracy included MUSK, Ribosomal protein S6 kinase alpha-5 (RPS6KA5), Dynein light chain Tctex-type 1 (DYNLT1), CSF myo-inositol and mononuclear cell levels. While inclusion of mononuclear cell counts gave the highest performance, replacing this measure with leukocyte cell counts resulted in a decrease in accuracy of only 2%. The predictive accuracy of the multivariate multi-omics model greatly outperforms the accuracy of each identified biomarker in isolation . InteresProtein pathway enrichment analysis revealed that the top 89 discriminatory variables are linked through several pathways and physiological functions. The discriminatory proteins upregulated in the converter group relative to the non-converter group are consistent with perturbations in cytokine, TNF, and interferon-gamma signalling pathways along with leukocyte proliferation, leukocyte mediated immune response and chemotaxis . In contThis study provides a detailed exploration of predictive biomarkers in a prospective cohort of 54 individuals with CIS. Due to differences in past McDonald diagnostic criteria and the fact that the 2017 McDonald criteria have low specificity, the occurrence of a second attack was used to define clinical conversion in this cohort. This not only ensures that the predictive biomarkers identified are applicable to the \u2018gold standard\u2019 definition of MS, but also ensures clinical utility as those individuals who have a second clinical attack are more likely to develop severe permanent disability than those with \u2018silent\u2019 demyelinating events and, thus, should be treated as early as possible.In total, only 41% of the patients recruited had a second attack and converted to clinically defined MS over the course of follow-up, which is somewhat lower than figures reported in a 20-year follow-up study where 63% of patients experienced a second clinical event.Previous reports suggest that between 61 and 68.9% of patients with CIS test positive for OCGB3) in up to 60% of MS cases3) are known to have increased annualized relapse rates.3 predicted transition to clinically defined MS with an accuracy of 65%, although ROC analysis revealed that a threshold of 3.5\u2009cells/mm3 was optimal (CSF leukocyte cell count was moderately elevated (>4 but < 30 cells/\u00b5l) in 54% of the cohort at baseline. A larger proportion of the patients who went on to have a second clinical attack (72%) exhibited elevated leukocyte cell counts at baseline when compared to those who did not clinically convert over the follow up period (41%). As a result, a significant elevation in leukocyte cell counts was observed in converter CSF samples relative to non-converters, which is consistent with an increased immune response at baseline in these individuals. Interestingly, when the total leukocyte population was evaluated at the cell-type level, a significant elevation in the mononuclear, but not PMN cells, was observed in the converter group suggesting that the elevated leukocyte levels are dominated by changes in mononuclear cells. Leukocytes are known to be moderately elevated , dynein light chain Tctex-TYPE 1 (DYNLT1), and natural cytotoxicity triggering receptor 1 (NCR1) with AUC values of 0.84, 0.84 and 0.83, respectively. Partial loss of XRCC1 renders brain cells vulnerable to oxidative damageThe extensive biomarker discovery employed here successfully identified clinical chemistry, metabolite, and protein biomarkers of conversion to clinically defined MS, each of which perform well in isolation and provide novel insight into the different pathological mechanisms in converters and non-converters at baseline. Future work, on larger prospective cohorts, will investigate whether the biomarkers identified here could be included in the McDonald criteria to improve specificity when identifying patients at high risk of second clinical attack. In particular, the data presented suggest that a larger follow-on study investigating the impact of leukocyte and mononuclear cell counts on prediction of clinical conversion would be particularly attractive as these measures are routinely available. Ongoing work will validate the identified biomarkers in the further independent cohorts, develop a method to measure the identified biomarkers using a single, cost-effective, assay for use in a clinical setting, and compare this test with other recently identified predictive biomarkers including baseline MRI findings, clinical variables, NfL and IgG/M levels.Brain Communications online.F.P. received funding from the Multiple Sclerosis Society UK (grant 59) and the Medical Research Council (MC_PC_15029). T.Y. is supported by the Ministry of Health, Singapore through the National Medical Research Council Research Training Fellowship (NMRC/Fellowship/0038/2016).Y.Z., M.S., S.A., T.D.W.C., R.H., J.O., D.L. and D.C.A. declare no competing interests. S.A. declares no competing interests. J.K. received speaker fees, research support, travel support, and/or served on advisory boards by ECTRIMS, Swiss MS Society, Swiss National Research Foundation, (320030_160221), University of Basel, Bayer, Biogen, Celgene, Merck, Novartis, Roche, Sanofi. FP has received travel awards from ECTRIMS, Merck, and the Multiple Sclerosis Society UK. T.Y. has received travel grants from UCB, Merck and PACTRIMS, and travel awards from ECTRIMS, ACTRIMS and Orebro University. J.P. is partly funded by highly specialized services to run a national congenital myasthenia service and a neuromyelitis service. She has received support for scientific meetings and honorariums for advisory work from Merck Serono, Biogen Idec, Novartis, Teva, Chugai Pharma and Bayer Schering, Alexion, Roche, Genzyme, MedImmune, EuroImmun, MedDay, Abide ARGENX, UCB and Viela Bio and grants from Merck Serono, Novartis, Biogen Idec, Teva, Abide, MedImmune, Bayer Schering, Genzyme, Chugai and Alexion. She has received grants from the MS society, Guthrie Jackson Foundation, NIHR, Oxford Health Services Research Committee, EDEN, MRC, GMSI, John Fell and Myaware for research studies.fcab084_Supplementary_DataClick here for additional data file."}
{"text": "Two patients experienced post-operative complications. At the one-year follow-up, difference in skin pigmentation was reported in 10 cases, a depressed contour of the ear was found in 4 cases, and moderate ear asymmetry was found in 11 cases. No patient needed a secondary procedure. In conclusion, the proposed reconstructive algorithm represents a reconstructive indication that is simple and characterized by low complication rates and good outcomes for both the patient and the surgeon.Ear reconstructive surgery aims to solve the deformities caused by cancer excision. Despite the numerous surgical procedures described, recreating the complex anatomy of the ear still represents a challenge, particularly for young surgeons. The purpose of this exploratory pilot study is to review our experience with single stage reconstruction of the partial defects of the auricle, and propose an algorithm based on defect size, location, and characteristics. We retrospectively reviewed patients who underwent ear reconstruction after cancer excision at our institution between February 2018 and November 2020. The data collected included patients\u2019 demographics, defect characteristics, reconstructive technique used, complications, and outcomes. The patients were evaluated at a minimum follow-up time of 12 months. Forty-six patients were included in the study. The most common cause for ear reconstruction was basal cell carcinoma. The mean area of defect was 4.3 cm The reconstruction of the auricle represents a complex challenge for the plastic surgeon. The different etiologies, the wide range of reconstructive options available, and the patient\u2019s expectations require a global evaluation of the appropriate surgical technique . MoreoveEar reconstructive surgery aims to resolve the deformities generated by cancer excision, using various techniques capable of restoring the original shape of the auricle. However, some of the surgical procedures described in literature require multiple stages, and this might be an issue for elderly patients ,3. In the present work, we reviewed our experience with single-stage reconstruction of the partial defects of the auricle after cancer excision. The aim of this exploratory pilot study is to show how principles of single-stage ear reconstruction can be applied to most partial defects of the auricle with satisfying results for both the surgeon and the patient, as it allows for a quick recovery with acceptable outcomes. Moreover, we propose an algorithm based on defect size, location, and characteristics to guide the surgeon approaching this topic.In this study, we retrospectively reviewed patients treated with ear reconstruction at our institution between February 2018 and November 2020. The inclusion criteria were auricle reconstruction after cancer excision and a minimum follow-up time of one year. The exclusion criteria were smaller defects treated with primary closure, larger defects requiring major ear reconstruction and multiple-stage procedures, patients who did not attend the follow-up, and lack of documentation. The study was performed with respect to the ethical standards of the Declaration of Helsinki, and all patients signed an informed consent form comprising a photo release section. The data collected from patients included in the study were patients\u2019 demographics , histological diagnosis and defect characteristics , reconstructive technique used, and cancer recurrence and complications. Photographic documentation was also collected. At a minimum follow-up time of one year, post-operative pictures were taken, and patients were reassessed, evaluating aspects such as difference in skin pigmentation between the reconstructed site and adjacent areas, donor site morbidity, altered and depressed contour of the reconstructed site, constriction of the external auditory canal, and ear asymmetry.Intraoperative antibiotic prophylaxis with amoxicillin was administered in all cases. Most procedures were performed under local anesthesia by injecting carbocaine and ropivacaine with epinephrine in the subcutaneous plane. In the case of the local flap, the donor area was also injected. We applied the same protocol even in the case of patients undergoing general anesthesia to prevent massive bleeding and post-operative pain. After surgery, patients were evaluated for early complications.The surgical reconstructive procedures performed in this study included full thickness skin grafts and local flaps. The Antia-Buch flap was performed in the case of defects of the helix greater than 1.5 cm with cartilage involvement. This flap requires incisions to be placed along the helical sulcus inferiorly, superiorly, or in both directions. Incisions should avoid damaging the skin of the posterior aspect of the ear, as this ensures the vascular supply to the flaps. Further mobilization of the upper segment can be achieved through a V-Y advancement flap of the helix root, which allows the advancement of the upper flap to be extended, and, therefore, was used for larger defects. The preauricular flap is designed to exploit the skin laxity of the preauricular area and its design should not exceed a 4:1 length to width ratio. The flap can be harvested thinner in its distal aspect to match the thickness of the skin of the auricle, and thicker at the level of the pedicle to ensure blood supply to the tissues. It can reach the defect at the ear level directly or by creating a tunnel under healthy skin according to reconstructive requirements. In our study, it was used for antihelix, tragus, antitragus, and lobule defects. The retroauricular flap was used for the reconstruction of the marginal defects of the helix without cartilage involvement. The flap is designed on the posterior aspect of the ear and advanced to cover the defect. It provides an excellent skin match without the need for reduction in the size of the auricle. The \u201crevolving door\u201d flap was used to reconstruct the anterior auricular defects of the scapha and concha when the cartilage was removed. The surgical technique involves the rotation of the flap from the retroauricular to the preauricular surface on a vertical axis represented by the neurovascular subcutaneous peduncle at the level of the retroauricular sulcus. The lobular flaps were used for the reconstruction of the lower aspect of the ear  when an excess of skin was found at the level of the lobule. This is usually possible in elderly patients.p < 0.05.Statistical analysis was performed with SPSS software . The relationships between patients\u2019 demographics and complication rates were analyzed by chi-square tests. The statistical significance was defined as From February 2018 to November 2020, a total of 49 patients underwent auricle reconstruction, but three of them were excluded from the study as they died during follow-up, due to circumstances not related to the cause of reconstruction. Among the 46 patients included in the study, 36 were males and 10 were females. The average patient age was 79.9 years (range: 61 to 94 years). Regarding additional patients\u2019 demographics, 14 patients were smokers (30.4%), none of the patients were diabetic, and 16 patients were affected by cardiovascular disease and were on anticoagulants (34.8%). The most common cause for ear reconstruction was represented by basal cell carcinoma (20\u201343.5%), followed by squamous cell carcinoma (16\u201334.8%) and precancerous lesions (10\u201321.7%) .The standard excision limits for the primary lesions were \u22650.5 mm. Among patients affected by squamous cell carcinoma, 11 lesions were 2 cm or less in greatest dimension and 5 lesions were between 2 and 4 cm in greatest dimension. No patients showed lymph node involvement prior to tumor excision nor during follow-up.2. Regarding defect location, in 24 cases the helix was affected, in 14 cases the antihelix was involved, 4 cases required lobule reconstruction, in 2 cases the tumor affected the tragus and antitragus, and in 2 cases the concha was involved (The mean area of defect was 4.3 cminvolved .p > 0.05). The most commonly performed reconstructive procedures were Antia-Buch flap (30.4%), followed by wedge excision (26.1%), preauricular flap (13.0%), retroauricular flap (8.7%), lobular flap (8.7%), full thickness skin graft (8.7%), and \u201crevolving door\u201d flap (4.3%). In terms of post-operative complications, one patient (2.2%) developed a post-operative infection treated with antibiotics, and another patient (2.2%), who was treated with a skin graft and experienced partial graft loss, resolved with secondary healing not requiring additional procedures . The relAt a minimum follow-up time of 12 months , the characteristics related to the outcomes of the reconstruction were evaluated. In particular, a difference in skin pigmentation of the reconstructed site compared to the adjacent areas was reported in 10 cases (21.7%). For none of the cases in which a donor site was used for reconstruction, relevant issues were identified with the donor site itself. A depressed contour of the ear at the level of the reconstruction was highlighted in four (8.7%) cases. No cases of a constriction of the external auditory canal were recorded. In 11 cases (23.9%), however, a moderate ear asymmetry with the contralateral was found. Secondary surgeries were not performed in any cases, neither for enhancing cosmetic outcomes nor due to recurrence of disease .In this study, the defects were reconstructed according to location, size of defect, and tissue involved . In partIn the upper aspect of the ear, it is appropriate to consider cartilage involvement when considering defects affecting the helical region. If it was possible to spare the cartilage, we performed a retroauricular advancement flap by lifting the flap from the retroauricular region and advancing it horizontally until full coverage of the defect was achieved . On the other hand, if the cartilage was involved, the size of the defect was considered. For lesions smaller than 1.5 cm, we performed a wedge-shaped excision of the lesion or its variants (star resection) to reduce the tension resulting from juxtaposing the edges of the defect . For defects larger than 1.5 cm, but less than 40% or the whole length of the auricle, we performed the Antia-Buch flap . If needed, a V-Y advancement flap was performed at the helix root to obtain an additional advancement . Still considering the upper aspect of the ear, if the scapha and antihelix region was involved but not the helix, and the perichondrium was intact, a full thickness skin graft was used to cover the defect. On the other hand, if the cartilage was removed, we performed a \u201drevolving door\u201d island flap .Regarding the middle aspect of the ear, the same considerations presented before were applied to the helix area. If the defect involved the antihelix but spared the ear margin, a local lobule flap was performed . If the perichondrium was intact, the defects of the concha were treated with a full thickness skin graft. Otherwise, the \u201drevolving door\u201d island flap was used. Preauricular flaps were used to reconstruct the defects of the tragus, antitragus, and the lobule .Recreating the complex anatomy of the auricle after cancer excision still represents a challenge, particularly for young plastic surgeons. A wide range of procedures have been described to approach this topic, but it is not always easy to determine which is the most suitable for any specific situation. We retrospectively evaluated 46 patients treated at our institution for partial defects of the auricle. The most common cause for ear reconstruction was represented by basal cell carcinoma followed by squamous cell carcinoma. Patients in which the defect was caused by excision of the precancerous lesions were also included, as the reconstructive procedure followed the same principles. Even though conservative treatment of precancerous lesions is usually performed, in our cases the excision was suggested after thorough assessment by a dermatologist due to characteristics of the lesion which could not exclude its malignant nature . In thisAs ear reconstruction after cancer excision is often performed in elderly patients (mean age of 79.9 in our study), it is advisable to keep in mind that a simple yet effective reconstructive procedure, with low risks of complication, is usually the best choice. Interestingly, Sanniec et al. found no statistically significant difference in complication rate in elderly patients compared with younger patients ,9. At a long-term follow-up, we evaluated the quality of reconstruction by analyzing the different factors, such as difference in skin pigmentation between the reconstructed site and adjacent areas, donor site sequelae, altered and depressed contour of the reconstructed site, constriction of the external auditory canal, and ear asymmetry. In terms of the difference in skin pigmentation, this was noticed in 10 cases (21.7%) which were reconstructed with full thickness skin grafts and preauricular flaps. Skin grafts represent a simple and reliable procedure when only the skin is resected and the underlying perichondrium is intact, but they usually determine color mismatch and a longer healing time ,11. WhenIn four cases (8.7%), a depressed contour of the ear at the level of the reconstruction was noticed. It must be stated that these were cases reconstructed with the Antia-Buch flap for defects greater than 2.5 cm. According to a cadaveric study performed by Calhoun et al., the helical rim advancement flaps provide satisfactory closure of helical rim defects up to at least 2 cm, but also longer in some cases, preserving the normal anatomic appearance . This isIn our study, no case of constriction of the external auditory canal was recorded. This is a complication which may affect the reconstruction of the defects involving the concha . In our Regarding ear asymmetry, even though a mild asymmetry was noticed at the follow-up of 11 patients, this was not found to be an issue as the vertical height of the helix root was maintained, hence allowing the patients to wear glasses. As no secondary procedures were performed to enhance cosmetic outcomes, we believe that our tailored approach to partial defects of the ear was successful in addressing common issues in auricle reconstruction. The selection of a single stage procedure was enthusiastically accepted by the elderly patients and, in our opinion, represents a valuable solution, especially during the COVID-19 pandemic, as it reduces patient access to hospitals ,24,25,26The aim of this paper is to present the authors\u2019 experience with the tailored reconstruction of the complex structures of the face, which carry both an aesthetic and functional burden, as previously discussed in studies and reviews on the topic ,29,30,31The high anatomical complexity of the skin and cartilage of the auricle is an issue when addressing ear reconstruction. Since these procedures are often required in elderly patients, a simple yet effective and reliable approach is advisable. The goal of this exploratory pilot study is to propose a single-stage reconstructive approach for the elderly patients which is easy to apply, with low complication rates and good outcomes for both the patient and the surgeon. For this reason, we propose an algorithm to choose the most suitable technique for each specific partial defect based on size, location, and tissue involvement."}
{"text": "Late-night overeating (LNO) is associated with several cardiovascular disease (CVD) risk factors. Limited data exist regarding the association between late-night (LN) systematic food consumption, LNO, and LN poor food quality with subclinical vascular damage (SVD) which precedes the onset of CVD. This study aimed to investigate the above associations with SVD in a large sample of adults, free of established CVD, with one or more CVD risk factors. In total, 901 adults  underwent anthropometric, dietary  and vascular assessment. LN systematic eating was defined as consumption of food after 19:00 h in both dietary recalls and LNO was defined as systematic consumption of >40% of daily total energy intake (dTEI) after 19:00 h. Systematic LN food consumption was inversely associated with diastolic blood pressure (DBP) ) after adjusting for age, sex, hypertension, diabetes, dyslipidemia, smoking, BMI and dTEI. LNO was positively associated with existence of carotid plaques ), while LN increased consumption of red meat, refined grains and wine and low consumption of whole wheat grains was positively associated with Aix (Augmentation Index) ), after adjusting for all the mentioned confounders. Systematic LN eating is associated with lower DBP while systematic LNO and consumption of poor-quality food late at night, is associated with SVD. Further research is needed to define more accurately the impact of LN eating habits on vascular health. Late-night overeating is considered the consumption of a large amount of daily total energy intake (dTEI) late in the day and/or during the night. However, so far there is no standardized and widely acceptable definition for late-night overeating. Different approaches have been suggested in relevant studies, such as intake of >25% of dTEI between 19:00 to 4:59 h or \u226533% at night, an occasion of eating in between sleep, consumption of dinner within two hours before bedtime, and intake of >35% of dTEI after 20:00 h ,7,8.Subclinical vascular damage (SVD) precedes the onset of CVD; therefore, its detection can contribute to identifying potential risk factors and improving timely prevention strategies . VasculaLate-night overeating has been associated with several CVD risk factors, such as obesity, hypertension, glycemic and lipid profile disorders and increased CRP ,12,13,14The present study has a cross-sectional design, and it was conducted at the Laiko University Hospital, Athens, Greece. The study is in accordance with the Declaration of Helsinki and its later amendments, it was approved by the Bioethics Committee of the University Hospital and all participants signed a written consent form before entering the study.The study sample consisted of 901 adults  with one or more CVD risk factors  but free of established CVD. Individuals with cancer, liver disease, gastrointestinal health issues or eating disorders were excluded from the study. All patients abstained from food, drink, or any medication for 12 h prior to their visit in the laboratory for the conduction of anthropometric measurements and vascular assessment.2) (Kg/m2) and used as a marker of obesity. Family history of premature CVD was defined as the presence of coronary heart disease in a 1st degree relative under the age of 55 years for males and 65 years for females. Hypertension was defined by the use of antihypertensive drugs and/or office blood pressure measurement higher than 139/89 . Dyslipidemia was defined on the basis of treatment with lipid modifying drugs or low-density lipoprotein cholesterol level > 160 mg/dL. Current smoking was defined by the use of at least 1 cigarette per day each day of the week; ex smoking was defined as discontinuation for more than 6 months. Body mass index was calculated as weight/, height (by stadiometer SECA 213) and BMI were measured.The study\u2019s participants underwent vascular assessment, performed by the same examiner. BP measurement was conducted three sequential times, after a 10-min rest of the volunteers in the supine position. The average of the three measurements was used as the final BP value. Central pressures, pressure wave reflections by AIx and carotid to femoral PWV were measured by tonometry methods in order to assess aortic stiffness, as described previously . SubclinFollowing vascular assessment, two 24 h dietary recalls were conducted (one weekday and one weekend day) with an interval of at least one week in between, by trained dieticians. Data were analyzed in food groups, where separation was based on food equivalents with further separation in subgroups where needed. Analysis of energy content, macronutrients and micronutrients was also conducted by using the Nutritionist pro Software . Regarding late-night overeating, food consumption data for the evening hours were recorded separately. Energy intake, macro- and micronutrients consumed after 19:00 h and their percentage in dTEI were calculated. Systematic late-night food consumption was defined as food intake after 19:00 h in both dietary recalls and non-systematic late-night food consumption was defined as food intake after 19:00 h in 0 or 1 dietary recall. Systematic late-night food consumption was further divided into systematic late-night eating (consumption of <40% of dTEI after 19:00 h) and systematic late-night overeating (consumption of >40% of dTEI after 19:00). Quality of dinner among systematic late-night food consumers was performed using 3 dietary patterns, which statistically emerged a posteriori (description below) based on the food consumed after 19:00 h.p \u2264 0.05 for all analyses.Statistical analyses were conducted using statistical package 21.0 . Level of statistical significance was set at t-test analysis was applied to address differences between non-systematic late-night food consumption and systematic late-night food consumption, and between systematic late-night eating and systematic late-night overeating. Multiple linear and logistic regression analysis was performed to determine the independent associations between vascular biomarkers and (i) systematic late-night food consumption (vs. non-systematic), and (ii) systematic late-night overeating (vs. systematic late-night eating). Results are presented as standardized beta coefficients and 95% confidence intervals (CI)  or as odds ratio (OR) expressed as ExpB and 95% CIs . Three different models were applied to assess the above associations: Model 1: adjusted for age and sex;Model 2: adjusted for age, sex, hypertension, diabetes mellitus, dyslipidemia, smoking and BMI;Model 3: adjusted for age, sex, hypertension, diabetes mellitus, dyslipidemia, smoking, BMI and dTEI.The normality of distribution of variables was checked using the Kolmogorov\u2013Smirnov test. Categorical variables are presented as relative frequencies (%) and continuous as mean \u00b1 standard deviation (SD). Paired Principal Components Analysis (PSA) was applied to identify dietary patterns (DP) among study participants with systematic late-night food consumption. The Kaiser\u2013Meyer\u2013Olkin (KMO) criterion was applied, and it was equal to 0.592. The orthogonal rotation (varimax option) was used to derive optimal non-correlated components (DPs) and the Bartlett\u2019s method was used to estimate factor scores. The selection of the optimal number of components was based on an eigen value > 1 (Kaizer criterion) and it was further corroborated by visual assessment of the scree plot, retaining only components on the steep slope. If one of the initial variables correlated with more than one component, it participated in the interpretation of the DP in which it displayed the highest coefficient value. Three DPs emerged after PCA. Multiple linear and logistic regression analysis were applied to determine possible associations between DPs and subclinical vascular biomarkers. Results are presented as standardized beta coefficients and 95% confidence intervals (CI)  or as odds ratio (OR) expressed as ExpB and 95% CIs . The aforementioned models that were applied in food quantity analyses, were also applied in this analysis, in order to assess the above associations.2 with at least one risk factor for CVD. One hundred and seventy-eight participants were non-systematic late-night food consumers and 723 were systematic late-night food consumers. Systematic late-night food consumers were divided into two subgroups: 572 systematic late-night eaters (<40% of dTEI) and 151 systematic late-night overeaters (>40% of dTEI). After an unadjusted T-test comparison, systematic late-night food consumers present with significantly better values of several vascular biomarkers  compared to non-systematic late-night food consumers. Systematic late-night food consumers of <40% of dTEI had significantly better values of HDL and LDL cholesterol, but higher values of PWV and AIx compared to late-night overeaters. Descriptive and clinical characteristics of study\u2019s participants are presented in The study sample consisted of 901 CVD free adults , with mean age 52.4 \u00b1 13.81 years, mean BMI 28.0 \u00b1 5.53 Kg/mt-test comparisons. No statistically significant differences were observed between dTEI (p = 0.172) and macronutrients intake expressed as percentage of dTEI  between systematic vs. non-systematic late-night food consumers. However, in systematic late-night food consumers lower consumption of legumes (p = 0.006) and higher consumption of cheese (low (p = 0.012) and full fat (p = 0.027)) was observed. On the other hand, systematic late-night overeaters had significantly higher dTEI compared to systematic late-night food consumers of <40% of dTEI , but lower percentage of carbohydrates . Moreover, systematic late-night overeaters tended to have worst diet quality compared with systematic late-night food consumers of <40% of dTEI, consuming less low-fat dairy products , fruits and fresh juices and more refined grains , dressings and soft drinks with sugar .Dietary intake of the study participants is presented in The associations of subclinical vascular biomarkers with systematic food consumption late at night compared to non-systematic food consumption late at night, and with systematic late-night eating compared to systematic late-night overeating, are presented in p = 0.094) after adjusting for age, sex, hypertension, diabetes mellitus, dyslipidemia, smoking and BMI. However, after adjustment for dTEI, DP3 was positively associated with AIx , p = 0.028). In further analysis, after adjusting for age, sex, BMI, dyslipidemia, diabetes mellitus, hypertension, smoking and total energy intake after 19.00 (instead of dTEI), the positive association with Aix remained unaltered , p = 0.019) (data are not presented in tables).Finally, In the present study, we aimed to examine the potential associations of eating late at night with SVD, by taking into consideration two major parameters of eating, i.e., total energy intake (food quantity) and dietary patterns  in adults with CVD risk factors. Our findings suggest that systematic late-night overeating (consumption of >40% of dTEI late at night), non-systematic late-night food consumption and low-quality food choices late at night is associated with SVD. Regarding food quantity, a negative association between systematic late-night food consumption and DBP, and a positive association between late-night overeating with the existence of carotid plaques was observed. Furthermore, regarding food quality, there was a positive association between the dietary pattern characterized by high consumption of red meat, wine, refined grains and low consumption of whole wheat grains with AIx.To begin with, there was no difference in dTEI between systematic or non-systematic late-night food consumption. However, dTEI was significantly higher in systematic late-night overeaters compared to systematic late-night food consumers of <40% of dTEI. Our findings are in accordance with the results of another cross-sectional study, where it was also found that late-night overeating was associated with high total caloric intake . RegardiNon-systematic compared to systematic late-night food consumers had higher BP levels ; however, this might be attributed to the high percentage of hypertension in the study\u2019s participants. In a retrospective cohort study, Kakamu et al. found that eating before bedtime could be a risk factor for hypertension in normotensive elderly , but theLate-night overeating was positively associated with the existence of carotid plaques. There is only one available study examining late-night overeating and its association with SVD, but their analysis showed a positive association only with PWV . There aRegarding the quality of food in systematic late-night food consumers, it was found that a DP characterized by increased consumption of red meat, wine, refined grains and decreased consumption of whole grains was positively associated with AIx, regardless of various confounders, such as age, BMI, dTEI, and energy intake of any meal after 19:00 h. Since the presented association is independent of dTEI and energy intake after 19:00 h, quality of food after 19:00 h, independently of quantity is associated with AIx. Although, there is no available study examining consumption of the above DP with AIx, components of the DP have been studied separately. Red meat consumption in young students was positively associated with AIx . ExcessiThe present study is one of the few available studies to examine the associations of (i) systematic vs. non-systematic late night food consumption, (ii) systematic late-night food consumption of <40% of dTEI vs. systematic late-night overeating and (iii) the quality of food consumed late at night among systematic late-night food consumers, with different SVD biomarkers. In terms of nutritional evaluation, the 24-h recalls which were applied in our study, are based on participants\u2019 memory, thus they are prone to recall bias. However, in our study, we use the multiple pass method during the dietary assessment, in order to minimize the potential error. Furthermore, 24-h recalls remain a valuable tool for dietary evaluation in studies, as they not only allow the assessment of dietary intake of the population, but they also provide the advantage of additional information recording, regarding the overall quality of nutrition. It is also important to note, that dietary intake analysis was conducted by Nutritionist Pro, a program that mainly contains American foods, and there may be variation in food composition from country to country. However, a variety of common greek foods and their nutritional analysis were registered into the program, to minimize possible declinations. Furthermore, regarding vascular assessment, it was conducted by the same examiner with the same equipment, thus reducing any possible measurement error. Another possible limitation of the study is that the majority of the participants were late-night eaters. However, this reflects a very typical dietary behavior in Greece as late-night overeating is very common among Greeks. Lack of data on participants\u2019 physical activity is also a limitation of the present study. Finally, although the cross-sectional design of this study does not allow to establish a causative relationship, we performed a statistical analysis with detailed adjustment for all potential confounders, in order to limit potential bias, as possible.In conclusion, both quantity and quality of food late at night are associated with SVD. Regarding the quantity of food, late-night overeating was positively associated with the existence of carotid plaques and non-systematic late-night food consumption was associated with higher DBP levels. In terms of food quality of systematic late-night food consumers, a dietary pattern consisting of increased red meat consumption, wine, refined grains and reduced consumption of whole grains was positively associated with AIx, therefore with arterial stiffening. As eating late at night concerns many people due to the modern way of life, further investigation is necessary in order to identify dietary behaviors and/or food choices late at night which will be possibly associated with improved vascular health and consequently will enable CVD prevention."}
{"text": "Danio rerio) for accelerating our understanding of human skeletal genomics and outlining the remaining gaps in knowledge. We provide an overview of zebrafish skeletal morphophysiology and gene homology, shedding light on the advantages of human skeletal genomic exploration and validation. Knowledge of the biology underlying osteoporosis through animal models will lead to the translation into new, better and more effective therapeutic approaches.The advancement of human genomics has revolutionized our understanding of the genetic architecture of many skeletal diseases, including osteoporosis. However, interpreting results from human association studies remains a challenge, since index variants often reside in non-coding regions of the genome and do not possess an obvious regulatory function. To bridge the gap between genetic association and causality, a systematic functional investigation is necessary, such as the one offered by animal models. These models enable us to identify causal mechanisms, clarify the underlying biology, and apply interventions. Over the past several decades, small teleost fishes, mostly zebrafish and medaka, have emerged as powerful systems for modeling the genetics of human diseases. Due to their amenability to genetic intervention and the highly conserved genetic and physiological features, fish have become indispensable for skeletal genomic studies. The goal of this review is to summarize the evidence supporting the utility of Zebrafish ( Osteoporosis is the most prevalent bone condition in the ageing population. It is a disease that weakens the bones, making them highly susceptible to sudden and unexpected fractures . It is eGenome-wide association study (GWAS) and whole genome sequencing (WGS) analyses have transformed the field of genetics of complex diseases in general, and for osteoporosis (OP) in particular. Although a range of modifiable factors contribute to age-related bone loss, including diet, exercise, medications, and comorbidities, primary OP is largely mediated by genetic factors, making it a promising field for exploration by GWAS and WGS .Bone mineral density (BMD) remains the strongest predictor of fracture risk and is highly heritable . Over thHeritability of OP-associated traits may have quite a range\u2014from 10 to 80%\u2014but is always significantly different from zero. Despite the strong heritability associated with osteoporosis, the identification of causal genes remains complex. Historically, studies in mice have added functional evidence to accompany GWAS. More recently, zebrafish have been used as alternative models to explore variants harbored in protein-coding regions. In this review, we will address current challenges faced by researchers to bridge the wide gap between association and causality in osteoporosis. In addition, we will demonstrate the power of functional studies using small teleost zebrafish to underpin the genetics of osteoporosis and the biology behind this condition that currently affects over 200 million people worldwide.In 2007, the first GWAS of the then commonly measured osteoporosis-related phenotypes was published . Since tStudies conducted on fracture risk accumulated large sample sizes, including data from the GEFOS consortium and UK Biobank . An imponew loci  was released to be used by the wider scientific community [MEPE associated with heel bone density. To date, exome data from 200,643 UKBB enrollees are now available, which include ~10\u2009million exonic variants. Interpreting results from human association studies requires bridging the gap between genetic association and molecular function. A systematic functional investigation is necessary to interpret significant variants/genes they regulate, to decipher the exact disease-causing genes, and the cells in which they act [The UK BioBank (UKBB) has recently sequenced whole exomes of >200 thousand participants, which were made available to the Exome Sequencing consortium. A subset of that enormous sample \u2014rare subtrochanteric or diaphyseal fractures\u2014are regarded as side effects of bisphosphonates (BPs). Zhou et al. summarized the most recent knowledge about the genetics of AFFs . AFFs hareatment . WES wasreatment . WES anaFF cases ,33. BispFF cases . RecentlFF cases .The genes regulated by SNPs can be located tens or hundreds of kilobase-pairs away and mingled among other genes, making the discovery of the actual regulated gene a tremendous challenge . IntuitiIt was shown that the distance between the lead SNP and the neighbor gene is a strong predictor of causality, yet there are examples of causal genes at GWAS loci that lie hundreds of kilobases away from the lead SNP ,38. The Since the majority of GWAS hits cannot be easily linked to a candidate causal gene, multiple methods and software are dedicated to the purpose of variant annotation, as depicted in recent reviews ,41. Of nWith WES being increasingly applied to large population-based settings, its annotation seems more straightforward (since these SNPs are coding). The American College of Medical Genetics and Genomics and the Association for Molecular Pathology have released standards and guidelines for the interpretation of sequence variants . AccordiFTO locus, which has been strongly associated with the risk of obesity [IRX3 and IRX5 but not those of IRX6 or FTO [Irx3 and Irx5 mice, the group showed the presence of an anti-obesity phenotype in Irx3 knockout and Irx5 heterozygous mice. Their studies confirm that variants in multiple enhancers within FTO obesity-associated regions regulate the expression of multiple genes (IRX3 and IRX5) in at least two obesity-relevant tissues (adipose and brain). In another study, Chesi et al. efficiently combined open chromatin landscape  with chromatin interactions data (Capture C) and BMD GWAS loci to map informative SNPs [CPED1, WNT16, and SFRP4, using cell cultures. They showed that knockdown of two novel genes (ING3 and EPDR1), not previously associated with BMD but highlighted through their chromatin analysis, affected osteoblast and adipogenic differentiation [The high complexity of the gene regulatory landscape also adds to the challenge in interpreting GWAS findings. Recent works have demonstrated that the genetic architecture of disease-associated loci may involve extensive pleiotropy . GWAS va obesity . Sobreir6 or FTO . By geneive SNPs . SNP prontiation . TogetheGiven the recent expansion of sequenced genomes, it became possible to intersect datasets for evolutionarily conserved genes, regulatory elements, or entire pathways with the evolution of homologous traits in a relatively unbiased fashion ,56. WhilIt has been postulated that cis-regulatory sequences and other non-coding elements may be a primary substrate for evolutionary divergence . IndeedAncient CNEs that arose in the vertebrate ancestor usually have only a small core of sequence conservation in fish species, while mammals often exhibit much broader conserved flanks. This observation inspired Madelaine et al. to look for GWAS-originated SNPs that fall in deeply conserved CNEs, from humans down to zebrafish, that also preserve gene synteny . SyntenyIRF6 enhancer-binding site in cleft lip and palate [RET enhancer in Hirschsprung disease [Phenotypic screening of genetically diverse species can reveal CNEs with specific trait associations. Such associations allow us to identify CNEs altered in human diseases, for example, the disruption of an d palate  and a mu disease . Non-codLBX1, previously identified as responsible for scoliosis [microRNA-9, rather than MEF2C as predicted by the original GWAS. Under miR-9 depletion, the zebrafish retinal vasculature formation was compromised [GWAS-derived SNPs embedded specifically in gene regulatory regions conserved to zebrafish are further expected to lie next to the orthologous gene(s) in both human and zebrafish, based on synteny. To exemplify the relevance of syntenic regions, Madelaine et al. identified SNPs associated with human diseases, such as rs11190870, mapped in a CNE close to coliosis . Madelaipromised . This woTaking advantage of synteny to predict causality, Cl\u00e9ment et al. extensively mapped CNEs on the entire genome-scale and predicted enhancer-gene interactions using a method previously applied to only the human chromosome X, called PEGASUS . PEGASUSHDAC9) protein-coding sequence are associated with disruption of TWIST1 regulatory elements that reside within the HDAC9 sequence. Based on SVs within the HDAC9-TWIST1 locus, they defined the 3\u2032 HDAC9 sequence (~500 Kb) as a critical TWIST1 regulatory region, encompassing craniofacial TWIST1 enhancers and CCCTC-binding factor (CTCF) sites. Deletions of either Twist1 enhancers (eTw5\u20137\u0394/\u0394) or CTCF site (Ctcf\u0394/\u0394) within the Hdac9 protein-coding sequence in mice led to decreased Twist1 expression and altered anterior/posterior limb expression patterns of Shh pathway genes. The authors found that Twist1 frequently interacts with several regions encompassing enhancer candidates. Then, they tested these candidates for functional activity using zebrafish enhancer transgenic assay, in the craniofacial tissues of zebrafish embryos. The zebrafish enhancer assay showed that some candidate enhancers drove specific GFP expression in branchial arches and other facial bones. By this, Hirsch et al. provided an insight into the spatiotemporal regulatory network that controls Twist1 expression in the developing craniofacial tissues [A relevant recent work by Hirsch et al. from Birnbaum\u2019s lab showed that structural variants (SVs) disrupting protein-coding sequences can also function as cis-regulatory elements . They sh tissues . These sOne of the goals of in silico and in vitro methods is to narrow down candidates and translate findings to in vivo models. Animal models have contributed to our understanding of mechanisms of human diseases, such as how the genetic analysis of diversity in outbred mice facilitates the identification of novel loci in humans . HoweverMost recently, Swan et al. explored data from 3823 mutant mouse strains for BMD . A totalglo1-/-) fish. Thus, validated diabetic induction methods are suggested to help researchers confirm their proposed models [Hyperglycemic zebrafish models are used as well in diabetes-related studies . Hypergld models .To date, even with the advanced Mouse KO and phenotyping collaborations, such as the International Mouse Phenotyping Consortium  and the To complement the usability of fish to model skeletal diseases, a recent review summarized the rapid emergence of zebrafish models of rare skeletal diseases during the last 10 years . Many avZebrafish have emerged as model organisms in bone research within recent decades, being choice models to confirm gene discovery and to perform functional analysis of human variants, often providing viable alternatives to mouse lethal alleles. One of the most fascinating advantages of zebrafish is their transparency A. Zebrafsp7. Remarkably, these findings in zebrafish precisely recapitulated those observed in a reported patient affected by osteogenesis imperfecta recessive that carried a homozygous loss-of-function mutation in SP7 [zic1, atp6v1c1a, atp6v1c1b, prkar1a, and prkar1b in zebrafish, unprecedently showing that loss-of-function of each of these genes could lead to craniofacial malformation and abnormal BMD, thus, exemplifying the use of zebrafish to elucidate genes playing a role also in developmental craniofacial conditions.In 2016, Kague et al. took advantage of the transparency offered by zebrafish to show the complete development of the cranial sutures in vivo for the first time . The samn in SP7 . TherefoWith the transparency gradually lost during development, the adult zebrafish endoskeleton becomes covered by musculature and mineralized scales. While in vivo imaging can still be used to study bony elements located superficially and permanently accessible in fish  A, to anaZebrafish passed through an additional whole genome duplication. Zebrafish gene duplicates (orthologs to mouse/human genes) may functionally balance each other, so single knockouts might be viable at least during embryonic and early larval periods, allowing basic analysis of skeletal development. Zebrafish orthologs sometimes manifest sub-functionalization, so that viable loss of function alleles of a specific paralog can be obtained and studied ,81. EachAlthough the evolutionary distance between zebrafish and humans date back to 450 million years, about 71% of zebrafish genes are conserved with humans. Moreover, when considering only genes involved in diseases, zebrafish have ~80% of disease-causing genes conserved with humans. For this reason, zebrafish have been an attractive model in forward genetic screening using Ethyl-N-nitrosourea (ENU)-driven chemical mutagenesis. In addition, in reverse genetics by targeting specific genes using gene-editing techniques such as zinc-finger nucleases (ZFINs) ,139,140,PLOD2) and osteoporosis (LRP5), and were able to reproduce similar phenotypic characteristics, such as low BMD, as those of mutants [We and others demonstrated that G0 knockouts reliably recapitulate complex mutant phenotypes, such as osteopenia and osteogenesis imperfecta ,144. Mos mutants ,144. ZebThe identification of genes involved in skeletal diseases can result in novel treatments for osteoporosis, as was the case for denosumab, an anti-RANKL monoclonal antibody ,147 and alk8-/-) [clemizole to treat patients with Dravet syndrome. Dravet syndrome is a catastrophic childhood epilepsy with early onset of seizures, caused primarily by mutations in SCN1A. Zebrafish scn1 mutants recapitulated Dravet syndrome and were thus used in a phenotypic screening resulting in the identification of Clemizole. Clemizole binds to serotonin receptors; its antiepileptic activity can be mimicked by other drugs acting on the serotonin signaling pathway . Griffin et al. tested lorcaserin in Dravet patients and showed reductions in seizure frequency and/or severity, similar to Clemizole in the fish [Interestingly, the first small-molecule inhibitor of BMP signaling, dorsomorphin, was one of the first compounds identified from a zebrafish chemical screening aiming to identify compounds that phenotypically reproduce loss of BMP signaling pathway  . Currentthe fish . These tthe fish ). Besidethe fish ,152. Likthe fish .Interestingly, for some drugs, zebrafish are able to recapitulate the effects observed in humans better than mouse models do. This is the case for thalidomide, mentioned above, which safety was originally tested in mice, but when administered to pregnant mice, the drug does not reach teratogenic levels in embryos, thus not causing birth defects as in human . It was rankl-induced OP (medaka) to validate their anti-resorptive and anabolic activities [Glycyrrhiza glabra roots) has a positive effect on osteoblast differentiation, as well as halts osteoclast activity in vitro. Carnovali et al. tested liquiritigenin in zebrafish, revealing increased bone formation rates in a dose-dependent manner and reduced TRAP staining in zebrafish scales of glucocorticoid-induced OP [The zebrafish models of damaging environmental exposures exist for tens of years, most prominently for toxicology research. Specifically for osteoporosis, several non-genetic models were established by the iron-stress , by predtivities . For exaduced OP . A similduced OP . Zebrafiduced OP , which aAlthough the zebrafish skeleton has a reduced requirement for resisting mechanical load due to residing within an aquatic environment, several studies have demonstrated that swim training can influence the timing of skeletogenesis in zebrafish larvae , as wellAlso, an important work by Witten et al. reminds us about the commonalities and specifics of small teleost biology in response to their environments . ZebrafiOsteoporosis is characterized by an imbalance between bone formation and bone resorption, where osteoclast activity exceeds that of osteoblasts. The behavior and interaction of bone cells that are commonly studied using cell cultures, can be easily studied in fish because they are see-through during a relatively long period of their development. Larvae zebrafish are often used to analyze osteoblast behavior, bone matrix deposition and mineralization . In mammHere it should be noted about the zebrafish exoskeleton  for the needs of comparative anatomical understanding. As part of the dermal skeleton, these structures harbor a mineralized matrix and serve as sources for rapid evaluation of bone formation and mineral deposits ,170. In sp7-/- and lrp5-/- [Adult zebrafish models to study osteoporosis have only been developed recently, alongside the study of the ageing zebrafish spine ,173,174. lrp5-/- ,93, etc. lrp5-/- ,108. Tabwnt16 or bmp1a1 [bmp1a fractures are prone to non-union [The endpoint of osteoporosis is the occurrence of a fracture. While the prediction of fractures in zebrafish is not comparable to that of humans, where FRAX (fracture assessment tool) is used; genetically modified zebrafish offer alternative ways to study fracture occurrence, prediction and fracture healing . The zebr bmp1a1 ,176. Moron-union ,107,108.WNT16 locus has been explored using zebrafish by several groups, serving as a good example of GWAS findings followed by functional validation in fish. Since the GWAS signal on 7q31 was discovered [WNT16, FAM3C, or CPED1 [WNT16 in the regulation of cortical bone thickness through increased resorption and reduced periosteal bone formation [wnt16 loss-of-function, incrementing our current knowledge about the gene in bone and its pleiotropic effects. Firstly, McGowan et al. showed that caudal fins of wnt16 zebrafish mutants were highly susceptible to fractures, with a 10 fold increase in numbers of calluses in mutants when compared to WT of a similar age [wnt16 mutant, reporting reduced bone density, curved spines and abnormal jaw [wnt16 in dermomyotome and sclerotome. By performing cell tracing experiments, they demonstrated that somatic wnt16+ cells are muscle precursors that contribute to muscle fibers. Lastly, they performed an analysis of lean mass in wnt16 mutants, which exhibited an altered distribution of lean mass and altered myomere morphology. This study suggests that wnt16 exerts a pleiotropic effect on bone and lean mass. Together, all three wnt16 zebrafish studies are complementary in concluding that wnt16 is necessary for bone mass and/or morphology, serving as a showcase for zebrafish functional studies to interpret findings from GWAS.The scovered ,177,178,or CPED1 . The orior CPED1 ,180. Theormation . In 2021ilar age . The autilar age . While Mrmal jaw . The samrmal jaw . Finallyrmal jaw . The autWhen it comes to the functional validation of bone-disease-related candidate genes highlighted through different venues, including WGS and GWAS, small teleost fish provide an unprecedented speed and precision not achievable with mammalian in vivo systems. Zebrafish are able to recapitulate human bone diseases and offer advantages for functional validation and deep phenotypic studies of osteoporosis-related genomic findings to an extent which they are preferable models, in many of the cases. It is expected that studies incorporating the fresh-water teleost model will continue increasing. We highlight key areas where we foresee the use of zebrafish in the osteoporosis research field.The in vivo aspect of how cells behave under normal conditions, or in diseases and after pharmacological interventions is an important area of research that will optimize future osteoporosis treatments . This haIt is worth mentioning the use of zebrafish exoskeleton and fins for supplementary skeletal studies. With the amenability of scales to be cultured in vitro and the regenerative capacity of fins, these dermal bones have been of interest to test osteoblast anabolic factors and are capable of relatively high-throughput experiments . Togetheklotho zebrafish mutants [Despite a few studies performed in the ageing zebrafish skeleton ,173,174, mutants , telomer mutants  and prog mutants  however  mutants , also reGiven the high number of loci identified to play a role in osteoporosis, we consider this specific gap in knowledge one of the top beneficiaries of what zebrafish can currently offer. The high efficiency of genomic editing in zebrafish using the CRISPR/Cas9 system, as mentioned above, allows us to accelerate our genetic validations using the first zebrafish generation (crispants) that are able to phenotypically recapitulate knockouts. This would allow us to implement the closest to high-throughput genetic screenings that a living vertebrate can achieve . With thFinally, non-coding variants regardless of their conservation within fish clades can be tested using zebrafish ,192. It We have mentioned a few areas where zebrafish are being implemented to add power and speed to the functional evaluation of osteoporosis-associated genes reinforcing them as a choice model organism to support bone-GWAS and help to bridge the current gap between association and causality. Having said this, it is notable that the legislators and the anti-animal-research activists are frustrated that, despite a legal requirement to use methods that do not involve animals, their development and uptake remains slow. Recently, the EU Parliament urged its constituents to accelerate the transition to a research system that does not use animals, pushing for alternative testing methods and phasing out the use of animals in research and testing. This is in line with the previous decade\u2019s conventions and efforts focused on reducing, refining and replacing (3R) procedures on live animals for scientific purposes, as soon as it is scientifically justifiable and \u201cwithout lowering the level of protection for human health and the environment\u201d. It is advised that the scientists must be trained in using advanced non-animal models and in sharing best practices regarding the 3Rs. Thus, for example, both positive and negative indicators of animal welfare must be reported. As such, there is a pressing need for studies of welfare to more fully assess negative and positive factors to truly capture an animal\u2019s experience of change in their health environment. Both the future of small-fish modeling in general and osteoporosis genetic research would benefit from this knowledge."}
{"text": "Danio rerio) is an alternative vertebrate model as a useful tool for the mechanism elucidation and drug discovery of various eye disorders, such as cataracts, glaucoma, diabetic retinopathy, age-related macular degeneration, photoreceptor degeneration, etc. The genetic and embryonic accessibility of zebrafish in combination with a behavioral assessment of visual function has made it a very popular model in ophthalmology. Zebrafish has also been widely used in ocular drug discovery, such as the screening of new anti-angiogenic compounds or neuroprotective drugs, and the oculotoxicity test. In this review, we summarized the applications of zebrafish as the models of eye disorders to study disease mechanism and investigate novel drug treatments.Visual impairment and blindness are common and seriously affect people\u2019s work and quality of life in the world. Therefore, the effective therapies for eye diseases are of high priority. Zebrafish ( Until 2020, an estimated 295 million people suffer from the moderate and severe visual impairment worldwide, and among them, about 43.3 million people are even blind . The lea(Danio rerio) is a kind of common aquarium fish originating from India and has become a prominent vertebrate model for studying diseases [Zebrafish diseases ,5. Zebradiseases . ImportaIn this review, we highlighted the use of zebrafish in modeling eye diseases by (i) introducing the characteristic anatomy and development of zebrafish eye, (ii) summarizing zebrafish models of eye diseases, such as cornea dystrophy, cataract, glaucoma, ocular vascular diseases, and photoreceptor degeneration, and (iii) presenting contributions of these models in the investigation of new drug candidates for eye diseases.Although zebrafish eyes are very small compared with those of humans, they contain almost all the basic structures of human eyes. Firstly, we focused on the anatomy and development of the zebrafish eye to explain why zebrafish is a promising model for human eye diseases.Both of the zebrafish and human cornea contain five major layers: the epithelium, Bowman\u2019s layer, stroma, Descemet\u2019s membrane, and endothelium. In its mature state, the zebrafish corneal epithelium is 12.5 \u03bcm thick with four to six cell layers. The stroma is approximately 6 \u03bcm thick with 34 to 40 layers . The endIridocorneal angle, the region where the cornea meets the iris, hosts cells specialized in maintaining intraocular pressure (IOP) . IOP is Almost all the morphological features of human lens can be observed in the zebrafish eye . The fissws2), ultraviolet (sws1), green (rh2), and red (lws), among which the green and red cones exist as a physically fused double cones [Zebrafish is visually responsive at 72\u2009h post fertilization (hpf), when the retina mirrors adult human retinal morphology and function ,13. Furtle cones . Visual Zebrafish visual acuity is typically measured using the behavioral tests, including optokinetic response and optomotor response. The optokinetic response is a robust behavior in which moving objects evoke the tracking eye movements . It is o(fli1: EGFP) line, which expresses EGFP under the control of fli1 regulatory sequences [The basic vascular biology of the developing zebrafish embryo is analogous to that of other vertebrates, and angiogenesis also plays a vital role in the zebrafish hyaloid vessel, which is similar to the development of retinal vasculature in mammalian embryos ,18. The equences ,23,24.The development of zebrafish eye is strongly similar with that of humans and other vertebrates ,26. TheyThe development of zebrafish eye is fairly rapid. Neurogenesis begins at 28 hpf, and zebrafish embryos possess visual function as early as 72 hpf ,28. The With genetic accessibility, similar characteristics of human ocular development and easy controllability of living environment zebrafish has been used as a popular model to study eye disorders. Over the past decades, many human ocular diseases, such as cataract, glaucoma, DR, and AMD, have already been modeled in zebrafish. Here, we briefly introduced some important zebrafish models of eye diseases from anterior segments to posterior segments.pip5k3, col17a1, and keratocan, also express in the zebrafish cornea. pip5k3 and col17a1 are quite conservative without ocular alteration, while keratocane is significant for corneal transparency and structure [lama1, a gene encoding an important basal membrane protein, leads to focal corneal dysplasia in zebrafish [Corneal dystrophies have a variable age of onset, variable inheritance, and progressive effects on corneal transparency and vision . Due to tructure ,36,37. Lebrafish . Overallebrafish . TherefoCataract, which is characterized by cloudy vision due to lens opacity, mainly includes congenital cataract and age-related cataract (ARC). Genetic studies have identified over 30 causative mutation genes for congenital or other early-onset forms of cataract and only few ARC-associated gene variants . NeverthCRYAB gene, a member of \u03b1-crystalline, results in congenital cataract by activating glucocorticoid receptor signaling [cloche mutant in zebrafish displays cataract related to the insolubility of \u03b3-crystalline and the faulty differentiation of lens fiber cells [\u03b1A-crystalline in the cloche mutant. Therefore, the cloche mutation can be used to investigate the aggregation of lens crystallin and prevent cataract. In addition, the overexpression of Pax6, which is a paired box and homeobox domain protein expressing in the developing nervous system and eye, causes defects in lens fiber cells and human congenital cataract. The Pax6 mutant in zebrafish shows the alterations in the eye size and the abnormalities in lens differentiation [Pitx3 and Foxe3 genes can cause the mesenchymal dysgenesis of anterior segment and cataracts in humans, and the knockdown of Pitx3 and Foxe3 in zebrafish via antisense morpholino results in the lens dysmorphogenesis [Hsf4, a member of the heat-shock transcription factor family, lead to isolated cataracts in humans and an early onset cataract with multiple developmental defects in zebrafish lens by interrupting the terminal differentiation of a lens fiber cell [The use of zebrafish cataract model mainly focuses on the congenital cataract. Among the known gene mutations caused cataracts, mutations in lens crystallins account for the majority, followed by mutations in various growth or transcription factors, connexins, membrane proteins, and lipid metabolism . Hence, ignaling . Similarer cells . Meanwhintiation ,43,44. Togenesis ,47,48,49ber cell ,50. \u03b1-crystallins is considered to be titrated out by the binding of the damaged lens proteins as well as the truncation and insolubilization of the small heat shock proteins; so zebrafish is used to establish a link between in vitro mechanistic models of \u03b1-crystallin chaperone and their roles in lens aging [CRYGC gene  causes a remarkable reduction in the thermal stability of \u03b3C-crystalline and raises the risk of lens opacity when exposed to heat and UV-irradiation stresses, finally resulting in cataract [ARC also has a genetic component, which makes individuals with the variation more vulnerable to environmental insults and aging . Since tns aging ,54. Addicataract . Glaucoma is a kind of optic neuropathy characterized by the progressive and irreversible visual field loss and visual impairment secondary to RGC loss . PrimarySIX6 variants interrupt the development of the neural retina and result in a decreased number of RGC and an increased risk of glaucoma-associated visual impairment [FOXC1, which is one of the few well-established genes related to POAG [FOXC1 has been found as a vital mediator that reacts to oxidative pressure and suppresses apoptosis in cells associated with aqueous humor dynamics [Bugeye mutant, which develops high IOP, enlarged eye globes, morphological abnormalities, and functional deficits in the retina, is identified as the model for myopia and glaucoma [Zebrafish offer great chances to test specific hypotheses associated with glaucoma . For inspairment ,71,72. A to POAG . The tradynamics ,75. Furtglaucoma ,77. ManyNeurodegeneration in the form of RGC death is well documented in glaucoma. The chemical or oxidative stress-induced retinal damage is related to RGC injury and used in zebrafish research . For exaPathological retinal angiogenesis makes a great contribution to irreversible causes of visual impairment at all ages, such as retinopathy of prematurity (ROP), DR, and AMD. Due to the significant similarities of vasculature between zebrafish and human, zebrafish embryos have been used for the identification of genes and mechanisms involved in pathological retinal angiogenesis.The crude prevalence of blindness caused by DR shows a global increase in age-standardized prevalence in 2020 .pdx1 mutants in zebrafish provide the only known model in which hyperglycemia-induced retinal angiogenesis can be studied [Retinal abnormalities of hyperglycemic zebrafish are consistent with those of diabetic patients. After being immersed alternately in glucose solution and water for 28 days, zebrafish has a remarkably thinner IPL and INL . Moreove studied . Therefo studied ,100.(fli1:EGFP) zebrafish with a hypoxia-inducing agent, followed by GS4012 (a VEFG inducer) at 24 hpf; then, the number of sprouts and vascular branches dramatically grow in the central retinal vascular trunks [(fli1:EGFP) zebrafish can also develop severe retinal vascular proliferation [ROP is a kind of retinal vasoproliferative disease in premature infants and one of the leading causes of childhood blindness . As ROP r trunks . In addiferation .von Hippel-Lindau (VHL) gene promotes hypoxia-inducible factor signaling and consequent VEGF expression [VHL knockout zebrafish embryos [VHL mutant zebrafish can be a model for neovascular AMD.AMD, which blur the central vision, is a disorder with multifactorial pathogenesis, including angiogenesis, dysregulation in the complement, lipid, inflammatory, and extracellular matrix pathways ,104. Neopression . Accordi embryos . Hence, gnn mutant displays the AMD-related degeneration of red cones at around 5 dpf [HTRA1, a protein involved in the pathophysiology of AMD, can induce an accumulation of lipofuscin and melanolipofuscin between the photoreceptor and RPE layers in zebrafish [HTRA1 in zebrafish displays certain morphologic changes of the RPE, photoreceptor cell death, and lipofuscin accumulation, which are the features of early AMD [RP1L1 mutant zebrafish using CRISPR/Cas9 genome editing is the first zebrafish model of photoreceptor degeneration with subretinal drusen deposits, which is a hallmark of AMD [Dry AMD, the subtype characterized by RPE disorder, can result in the loss of photoreceptor cells. Interestingly, the zebrafish with the nd 5 dpf . Additioebrafish . The traarly AMD . Recentlk of AMD .Photoreceptor degeneration diseases are exceedingly various, creating the challenges of preventing or reversing vision loss. Due to the similarities in retinal anatomy and function between zebrafish and humans, the zebrafish model has become a predominant model for studying the photoreceptor development and disease. Here, we focused on retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA), which are two major kinds of retinal degeneration diseases .rhodopsin (RHO). Recently, various RHO mutant zebrafish models associated with dominant or recessive RP have been established with progressive rod degeneration [RHO mutants, which is consistent with the features of human RP caused by the RHO mutation [RP is a disease characterized by decreased night vision and loss of peripheral vision due to the progressive photoreceptor cell death and dysfunction of the photoreceptors. The most common cause of human autosomal RP is the mutation in the rod-specific opsin gene, neration ,111,112.mutation ,111. retinitis pigmentosa 2 (RP2), is characterized by the early onset and rapidly progressive vision loss before 40 years old in humans [RP2 in zebrafish results in a small eye phenotype, gradual loss of the photoreceptors\u2019 outer segments (OSs), and defective photoreceptor function, mimicking human X-linked RP [RP2 in the pathogenesis of X-linked RP [X-linked RP, whose major cause is the mutation in n humans . Knockdoinked RP ,115. Furinked RP . AdditioCEP290, RPE65, CRB1, KCNJ13, GUCY2D, AIPL1, CRX, IMPDH1, LCA5, LRAT, RPGRIP1, SPATA7, RD3, RDH12, and TULP1. CEP290 mutant zebrafish displays an intracellular transport delay and a decreased visual perception, which is analogous to human LCA patients [LCA5 in zebrafish using CRISPR/Cas9 technology causes the impaired OS protein trafficking and then cone\u2013rod dystrophy, which mimics the phenotype of cone\u2013rod dystrophy in humans [LCA is a kind of inherited retinal dystrophy disease responsible for early-onset childhood blindness with immense genetic heterogeneity . Presentpatients . Similarn humans .ift28, ift88, and ift172 mutants of zebrafish have the rapidly degenerated photoreceptors and without the developed photoreceptor OS [kif3a (kinesin family 3a) mutant in zebrafish causes photoreceptors to dramatically degenerate and fail to develop OSs, resulting in the extinguished ERG in zebrafish larvae [Mutation genes involved in ciliogenesis initiation and the transport of cilium components can result in LCA or an LCA-like phenotype in mouse models . Intrafleptor OS ,135,136.h larvae ,137,138.Since the drug candidates can be added to the water culture medium rather than injected into the fish, zebrafish has become a promising model for the various successful phenotype-based drug discovery ,139. HerThe chemical testing in zebrafish can screen for new anti-angiogenic drugs for the eye diseases, which is analogous to the in vitro/ex vivo platforms. Therefore, zebrafish has been emerging as an exciting new model organism to discover anti-angiogenic drugs for ocular diseases. For instance, the screening of approximately 2000 compounds reveals that four small molecules affect retinal vessel morphology but do not produce obvious changes in the zebrafish trunk vessels and the retinal neuronal architecture . Similarvhl zebrafish [In a small chemical screen using zebrafish, LY294002, the PI3K inhibitor, is identified as an effective and selective inhibitor of ocular angiogenesis without systemic side effects and diminishing visual function . Additioebrafish ,143. A rebrafish .pde6c zebrafish model of retinal degeneration [Zebrafish models of the photoreceptor disease provide a platform for discovering novel neuroprotective drugs. Zebrafish can be utilized as phenotypes in screening neuroactive compounds for photoreceptor degeneration ,146,147.neration .atp6v0e mutant zebrafish model, a cone photoreceptor degeneration disease, HDAC6 inhibitors successfully reduce the number of apoptotic cells and improve the photoreceptor OS area and visual function [The overactivation of histone deacetylases (HDACs) has been detected in models of photoreceptor degeneration, and HDAC6 inhibition may prevent neurodegeneration . Moreovefunction ,153,154.It is conceivable that many drugs possess oculotoxicity. The prolonged or high-dose exposure to a certain drug may cause eye damage and vision loss. Given that the vertebrate eye is highly conserved, zebrafish can be a useful model for studying the ocular toxicity of drugs . ZebrafiZebrafish provides a convenient animal model for mechanism investigation and drug discovery in ophthalmology due to their similar eye structure with human and accessibility to genetic manipulation. In the last few years, genome editing technologies, particularly based on Crispr/Cas9, have made it fairly easy to generate lines of zebrafish with mutations in targeted genes . Hence, Zebrafish models might be more predominant, powerful, and promising tools for investigating the mechanisms of various human eye diseases and discovering the novel drug therapy in the future."}
{"text": "To determine the prevalence of asymptomatic cardiac autonomic neuropathy and its association with risk factors among patients with Type-2 diabetes mellitus (T2DM)The present case-control study was conducted at Department of Medicine, Civil Hospital, Dow University of Health Sciences (DUHS), Karachi, Pakistan during the period September` 2016 to May` 2017. After taking informed consent, subjects from both genders, 72 healthy controls and 72 clinically diagnosed T2DM diabetic patients, age between 30-65 years were selected by non-probability sampling technique. After taking medical history and demographics, Cardiac Autonomic Neuropathy (CAN) was identified by using Ewing`s cardiac autonomic reflex tests (CARTs) and association of risk factors was also investigated.Severe CAN was identified in 13.9% of T2DM patients while in none of the healthy controls. HR response to deep breathing test was most sensitive (sensitivity= 90%) among all tests. The most common symptoms were Numbness (75.9%) and constipation (69%), resting heart rate and diabetes duration was significantly associated with DCAN.CAN was highly prevalent in diabetic population that may lead to nephropathy and retinopathy in future. It is highly recommended to use sensitive and simple CARTs in clinics for early detection and early treatment of CAN. Diabetes mellitus (DM) is characterized by a high blood sugar level over a prolonged period and its mystifying nature led to pathophysiological alteration in almost all body systems, that causes economic, social and healthcare burden. Diabetes mellitus is a global epidemic that has affected 9.2% of adults globally in 2019.3Diabetes bring a lot of micro and macrovascular complications after its first onset.DCAN is an independent predictor of heart associated mortality and nephropathy, hence it is recommended to perform Cardiac autonomic reflex tests (CARTs) for assessment of subclinical DCAN at first time diagnosis of diabetes and every five years thereafter.There is no case-control study available from our population, only a few cross-sectional studies are available without utilization of five standard CARTs as recommended by American Diabetic Association. We designed this case-control study to identify and compare the frequency of asymptomatic CAN in Type-II diabetics and healthy controls, by utilizing full set of Ewing`s Gold standard CARTs in Type-II diabetics with no other comorbidities from Karachi, Pakistan.The present case-control study was carried out at Department of Medicine, Civil Hospital, Dow University of Health Sciences, Karachi, Pakistan from September` 2016 to May 2017. After taking ethical approval from DUHS IRB and Ethics Committee (IRB-508/DUHS/-14 Dated 9-10-2014). Total 144 study participants were included, 72 were T2DM clinically diagnosed cases while 72 were healthy controls, recruited through non-probability purposive sampling technique. Subjects were well informed for study procedure and informed consent was taken from each participant, detailed medical and family history of diabetes and demographics were measured carefully. T2DM patients who were otherwise healthy and with no history of previous or recent heart surgery, cardiac dysfunction, hypertension, apparent neuropathy/ nephropathy/ retinopathy and definite coronary artery dysfunction, were included in the study and vice versa were excluded. Those who were selected for study underwent Cardiovascular Reflex Tests (CARTs) for evaluation of CAN. Tests were performed by same examiner and on same device to avoid biasness in data collection.After initial rest of five minutes, blood pressure was recorded by using Mercury Sphygmomanometer  and standard 12 lead ECG was recorded by using Power Lab device, Lab Chart Pro-7 software and HRV analysis software module , and resting heart rate was automatically computed from lead II ECG recordings. All five Ewing`s cardiac reflex tests designed for the assessment of cardiac autonomic neuropathy were performed according to the procedure previously described in detail . Heart rHeart rate response to valsalva maneuver: HR change was expressed as the Valsalva ratio , (2) Heart rate response to deep breathing: The mean value of maximum minus minimum heart rates (beats per minute) for six breathing cycles was considered. , (3) Heart rate change on immediate standing: R-R intervals were measured at the 15th and at the 30th beat following beginning to stand. The HR response then expressed as 30:15 ratio , (4) Blood pressure response from lying to immediate standing: The SBP change after two minutes of standing up was measured as the change in response  and (5) Blood pressure response to sustained handgrip exercise: The mean difference highest DBP-value during and before exercise was considered as DPB response to exercise .Ewing`s CARTs consist of (1) Final diagnosis was based on combination of different test results: No CAN; when all five tests were normal or borderline), Early CAN; when only single Heart rate test was abnormal or two border-line, Definite CAN; when any two heart rate tests abnormal, Severe CAN; when any two heart rate tests abnormal plus either of the two Blood pressure tests abnormal.SPSS-20.0 was used for statistical data analysis. Quantitative data were presented as mean\u00b1 SD and qualitative data were presented as frequency and percentage. ANOVA and t-tests were performed for quantitative and Chi square tests were performed to compare the categorical variables. Association of CAN with risk factors was identify by Logistic regression analysis. P-value less than 0.05 was considered statistically significant.2) were normal, yet significantly higher (P<0.05) in cases, with respective values in controls and cases were 22.01\u00b14.3 and 24.78\u00b13.9. Resting HR and resting SBP were significantly while DBP was insignificantly high in cases than controls , from both genders , respective mean age of controls and cases was 49.69\u00b19.1 and 49.72\u00b18.8 years, and this was not a significant difference (P>0.05). In both groups, mean values of BMI  in cases than controls . The oveDiabetes mellitus and its complications contribute to personal misery as well as constitute an economic burden on the resources of the society. The risk of developing cardiac autonomic neuropathy (CAN) among diabetics is associated with duration and magnitude of hyperglycemia. Usually, CAN showed a diverse spectrum of signs and symptoms, however, in case of diabetes, these manifestations are not apparent because autonomic nerve fibers become damaged and less sensitive to autonomic changes, unfortunately, it keeps silent for several years.Although, male participants were more in our study but, frequency of CAN was significantly higher (P<0.05%) in Type-2 diabetic females (44.7%) than males (35.3%). The present study results showed that females are more prone to develop CAN as compared to males. Most recent study from China, also depicted quite high frequency of DCAN, that is 80 out of 386 (20.72%) had DCAN in menopausal T2DM patients.case-control study to identify and compare the frequency of DCAN by utilizing the full set of five CARTs according to the guideline of American Diabetes Association, however, a few cross-sectional studies are available who did not utilize complete set of Ewing\u2019s CARTs. Only one previously published study from Faisalabad, Pakistan, who performed five CARTs in Diabetics to find out the frequency of silent myocardial ischemia in diabetic patients, and it was depicted that even in such a small city of Faisalabad, the prevalence of DCAN was 53.8% with at least two CARTs positive in all patients.16Our study is first attempt from Pakistan to perform Another study from Hyderabad, PakistanThe present study will contribute considerable insight and data regarding DCAN from population of Karachi, Pakistan. The use of CARTs is not yet implemented in our society, we, appreciate the use of these simple and highly sensitive tests at clinics, at first time diagnosis of Type-2 diabetes and every five years thereafter, as recommended by American and Canadian diabetic associations.The strength of our study is that this is a case-control study and results can be compared easily in cases and controls, while in previous cross-sectional studies data from healthy controls was missing. Secondly, we have utilized complete set of Gold standard Ewing`s CARTs recommended by American Diabetes Association, so we are assertive for true results. Though study does not represent whole nation, yet Karachi being a metropolitan city we have data from people of different ethnicities of Pakistan and represented a broader view of this devastating complication of Diabetes.It is single centered study where most of the patients belong to low- or middle-class socioeconomic status, data from upper class is missing. Multicenter study may represent the actual burden of this disease in Karachi, Pakistan.Based on the present study findings, it is concluded that to prevent and reverse DCAN, it is important to use CARTs for early and cost-effective diagnosis, to incorporate lifestyle modifications and available therapeutic approaches. We recommend a multi-centered study at provincial level to find actual status of CAN in Pakistan among diabetic population.TN: Conception, research design, data collection, responsible and accountable for the integrity of the work.NNM: Manuscript preparation, Editing and proofreading, Review and final approval.KA: Research protocol, final drafting and review the manuscript.AS: Updated literature search and Statistical analysis, results computations."}
{"text": "Complex operations will be undertaken by a set of self-learning collaborating robots.\u2022 Knowledge and self-learning algorithms will become an important asset on the meat-packers balance sheet. Existing technology will be disrupted, and first movers will get ahead of their competition: The moment for decision is coming close.The wonderful story about Mr. Henry Ford visiting a Chicago slaughterhouse almost a hundred years ago is truly enjoyable. A meat-packers disassembly line inspired Ford Motor Company to organize its production in continuous assembly lines and sparked the industrial revolution further into higher efficiencies and fewer production flaws. The story makes people in the meat industry proud, and a visit to a modern slaughterhouse clearly demonstrates how far efficiency and productivity can be driven with sophisticated management systems, skilled employees, and highly specialized automation technology.Of course, there are big differences among the various species of animals that are being processed. In general, the level of automation is higher, the smaller the carcasses are. Poultry processors are usually highly automated, whereas beef processors are much less automated. The same observation goes for the line speed, where poultry processors can handle 20,000 carcasses per hour, pig processors 750 per hour, and beef processors 300 per hour.But the classical line production see  that we Another reason is that it is becoming increasingly difficult to attract workers to the meat industry. The work environment is strenuous and often the salary is low. Other sectors offer more attractive jobs, leaving the meat industry with a big recruitment problem.The supporting automation technology is yet another reason why we may see the end of line production in the meat industry . BecauseOn a more theoretical note, it can also be observed that the utilization of the automation technology is low and may be only 30% of the full capacity. This is the nature of line production due to the design. The automation technology is fitted to the production line and is awaiting the carcass to approach before the automation technology can perform the processing. Often the automation technology is standing idle a lot more than the processing time, and from an engineering point of view, this is highly inefficient and adds to production costs.When a production line is running at full speed, it has a high productivity, but experience shows that it is not uncommon that a single incident along the line causes line stops frequently. An uptime less than 80% is not uncommon and sometimes even as low as 50%. This clearly demonstrates the vulnerability of classical line production and the extreme demand for uptime of the automation technology fitted to the line. Keep in mind that any modern slaughterhouse receives animals on an ongoing basis and that breakdown can be detrimental to the inflow of animals and the preceding logistics. Uptime is king.So instead of designing the automation technology to fit the production line, why not design the production setup to fit the automation technology? We need to rethink the production design and it may well mean that we will reinstall the old cutting table where many processes were done by one operator in parallel instead of lines. In fact, this is exactly the development that the Computer Numerical Control (CNC) industry went through in the 1990s and that the automotive industry is going through more recently \u2014migratinIdeally, a production cell is a delimited area where an item is being processed by numerous operations. It can be done by people, but in this context, it is the application of robots that makes it relevant. A robotic production cell can consist of one or more robots that process the carcass with many operations see .The robots will work simultaneously or even collaborate and ideally process an entire carcass including cutting and deboning. A robotic production cell is particularly relevant for larger carcasses due to the labor intensity and at this point lack of cost-efficient available automation technologies on the market.Why are robotic production cells interesting? Part of the answer lies in the fact that it enables much more powerful innovation. Until now, the meat industry and their technology providers have developed costly specialized automation technology, but the application of standardized industrial robots opens the door into the huge innovation storm that takes place in assembling industries . The autIn the pig industry, kill lines are already automated, and it is likely that robotic production cells will be a next-generation choice for technology providers. But a little further downstream, it has been almost impossible to automate the cutting and deboning areas. The product mix is high, production series may be short, and cutting operations are complex. In this situation, the robotic production cell is highly relevant and probably a feasible way to improve efficiency in this area.Robotic production cells will be organized in parallel to match the needed capacity of the slaughterhouse; basically, redundant robotic production cells that can perform the exact same operations. This redundancy implies that the risk of total production stops is much lower compared with the classical line production. If one production cell breaks down, the remaining production cells can still function, and production will continue although with a lower capacity. This is a huge advantage compared with the present situation.Establishing a robotic production cell is a big investment to any meat processor or technology provider. But the explosive development of technology creates a lot of tailwind. Sensors are key to the operation of the robots, and obviously, machine vision is an integral part of the function. The development is rapid, and cameras are becoming cheaper and more powerful year by year. Especially, three-dimensional-,Time-of-Flight-, and multispectral cameras are useful to give input to the robot control system in combination with tactile feedback systems .However, clever design of the tools mounted at the end-effector of the robots will make the entire operation much simpler. The correct choice of materials, design, and operation of the tools is the key to get optimal yields and correct product quality. Although tools are being designed as generic as possible to make sure that they can be used for several operations, they are still specialized to some extent. Therefore, more robots carrying different tools are needed. However, more robots are eating square meters, and there are never sufficient square meters available at the slaughterhouse. Automatic shift of tools is much preferred and will be a way to keep the footprint down of the robotic production cell.In the end, vast amounts of data are being created, and the control algorithms must be able to handle them sufficiently fast to be able to control the robot. Developing algorithms will be the true shift of paradigm, because the algorithms will not only be hard coded for a number of operations, but it will also be self-learning and adaptive. Once in operation, the algorithm will be improved based on the ongoing production. It can learn gradually about how to cut to other specifications, and it can accommodate the biological variation among each carcass, meat cut, or in longer terms, species change over time.The implications of this are huge because this is exactly what is needed to handle small production series and a complex product mix. Ideally, the robotic production cell can operate with an individual cutting pattern optimized for each individual item\u2014a true hyperflexible production.As algorithms are self-learning, they will eventually become adapted to the products and procedures of the slaughterhouse. They will become part of the critical knowledge base that any company tries to protect and that is often part of the company\u2019s competitive edge. Moreover, the algorithms represent a powerful way to transform tacit knowledge into solid documentation. The algorithms can be duplicated and proliferated, and in an international production setup, this is a powerful way to create an aligned and high-quality production, where trained algorithms can be introduced into new robotic production cells anywhere in the world. Furthermore, the algorithms can be interconnected across robotic production cells, across production plants, and across borders\u2014what a powerful learning system!. Just like the roots of a tree. In a future not so far away, the algorithms may be the most important asset on the balance sheet.The concept of robotic production cells may hold the key to a new cell-based production paradigm at the slaughterhouse. However, to turn the idea into reality requires acting. To this end, the development of the world\u2019s first robotic cell for pig production see  at a DanAs cell-based production completely disrupts the traditional line production, it is very challenging to integrate the robotic production cell into existing facilities. If it was possible to introduce cell-based production across the entire slaughterhouse at the same time, these challenges would of course not exist. However, the exploration of robotic production cells has only just begun, and a factory-wide installation is still very far away. A realistic path toward cell-based production will identify and replace parts of the traditional production line, and, therefore, solving problems related to integration is crucial. For this reason, the layout of the robotic production cell as well as the choice of products to be processed and how these will flow in and out have been considered. Six operations have been identified, all of which are carried out at the same location after the cold carcasses leave the chilling area and enter the primal cutting. The six processes are toe cutting, tenderloin extraction, ear cutting, stab wound removal, head cutting, and trisection. These choices make it feasible to install the robotic production cell in the existing production flow at the slaughterhouse.For the tests carried out, the carcasses are brought directly from the production and arrive in the test facilities hanging on rails from the ceiling. The carcasses are taken down onto a conveyor that carries the carcasses into the robotic production cell. After the carcass arrives inside the robotic production cell, it is analyzed with sensors and cameras to extract relevant information. The six operations are then executed by several robotic arms in parallel using dedicated tools developed for each individual task. Everything is orchestrated by a software control system running on a powerful PC next to the robotic production cell. The software is completely modular with key components running on separate threads in parallel to take full advantage of the available computing power. The software structure provides maximal flexibility and makes it easy to be extended with new modules, for instance for additional algorithms, sensors, cameras, tools, or robots.The core idea is to transfer the complexity from dedicated mechanical design to an intelligent software control system. This strategy keeps the cost down on custom machinery and adds the flexibility to reconfigure the system on the fly. It also drastically reduces the development time and facilitates the exploration of more innovative solutions for highly complex manual operations. The ongoing development at the test facilities is focused on intelligent control systems and smart tooling, including a generic fast tool change.One of the main challenges is designing the intelligent part of the control system. The question boils down to how the relevant information is extracted from various sensor and camera inputs to guide the robots. The progress in research and development in artificial intelligence, in particular deep neural networks , has madDesigning robust deep learning algorithms has always been difficult regardless of the application domain. However, the slaughterhouse takes the usual challenges to a new level due to the large variations in the input and the strict requirements on precision and capacity. Experienced butchers are both skilled and fast, and the individual processes are already highly optimized in the existing line production setup. Consequently, the algorithms must be very fast and extremely precise to make the robotic production cell a feasible replacement. The large biological variation and variations introduced from upstream processing by human operators or machines make this task extremely challenging.In the robotic production cell, the algorithms are provided with images of the carcass recorded by several three-dimensional cameras located inside the cell. During initial tests, it was quickly realized that common off-the-shelf deep learning algorithms, for instance, based on semantic segmentation  or objecAlthough several milestones have already been reached, many challenges still lie ahead. The robotic production cell test facility is a first attempt to bring the abstract concepts of multifunctional cell-based production into practice for the meat industry. The task is immense and, even when broken down into parts and solved one at the time, uncharted territory is explored for every step taken.So, what is next? Sometimes it is forgotten that intelligent control systems only provide intelligent solutions at the moment of creation. Automation solutions degrade over time because developers cannot predict what will happen in the future. Will the carcasses increase in size? Or maybe new machinery upstream introduces new physical features on the carcasses? Preparing for the next step, the potential application of a range of online learning algorithms is currently under investigation, for instance, reinforcement learning , which eThe digital development is so fast that even just understanding the new possibilities is a challenge. Everything becomes cheaper and Moore\u2019s law  is also But there is an even more important aspect especially when it comes to capital expenditures and hardware. Hardware updates, new tools, new functionalities, new sensors, and so forth can be retrofitted in the cells. With the natural redundancy in a setup with robotic production cells, the actual retrofitting can be done simultaneously with production, and no stops are needed. A robotic production cell should not be looked upon as a complete machine but rather a versatile platform that continuously can be improved and adapted to new conditions.Most slaughterhouses are sorting carcasses based on value, size, and other parameters. There are millions of dollars hidden in correct sorting of the carcasses, which is basically to match carcass composition with customer specifications and expectations. Modern slaughterhouses have big cooling facilities to handle carcass variability. The reason is that the workers manually must cut to a certain specification based on the type of carcass that is being presented to them, and the only way to do that is to sort the carcasses in a cooling facility before fabrication.But that complexity disappears with the introduction of robotic production cells. In the ideal world, sorting is no longer needed because the production cell can cut the carcass optimally based on the biological variation. It assumes that there are sufficient appropriate customer orders received, but overall, it is a big simplification of the slaughterhouse logistics. A much simpler design is needed and energy for cooling can even be saved.As a bonus, it will be possible to introduce complete traceability down to each cut leaving the robotic production cell; a challenge that remains unsolved today and is currently handled with batch traceability in most cases.It has been demonstrated that it is possible to make a robotic production cell for a pig slaughterhouse. Technologies are available and the business case is very attractive. But of course, there is still a lot of work ahead.Technically there are three main areas that must be solved. First of all, it is necessary to develop a very fast shift of tools may be even on the fly. Robotic production cells will at least in the beginning need to be fitted into existing facilities, so a small footprint is essential. Shift of tools is common in other industries that use robots, but the challenge is the speed.Secondly, a sanitation system must be developed. Probably the most important thing with robotic production cells is that they enable production 24/7. The factory can increase its capacity maybe by 30% without adding any cost of production, and this is where the real business case for this production setup is hidden. This will require that the cells can be cleaned in place (CIP). Multiple robotic production cells create a high redundancy in the production system and, one cell can be cleaned, while the remaining cells are producing. There already exist CIP systems for robot solutions, but they have never been tested in slaughterhouses or in this kind of robotic production cells. Thirdly, logistics around the robotic production cells must be in place. Depending on how many operations the cell will be able to provide, the logistics around the cell will be complex. It is necessary with a simple conveyor design around the cell to make logistics as smooth as possible preferably without any transport boxes. It is easy to underestimate the risk of complex logistics, and in this case, it is not different.In the early days of automation, it was popular to state, that we want so much automation, that we can turn off the light in the factory, meaning that no people were necessary for the production. That statement is not relevant anymore, because robotic production cells still need skilled workers around them. Actually, the role of the workers will radically change. Today most of the work is standardized repetitive operations, where each operator is trained in a particular set of operations. With robotic production cells, the role of the operators is not only to oversee a set of cells but also to be the product quality expert. The skilled butcher will become very important, as their skills will be part of the learning input to the algorithms. If the cell meets a situation, where it does not know what to do, the skilled operator will teach the machinery, and the cell will have achieved a new learning input. The job will change from a blue-collar worker to a skilled technician. Jobs will be much fewer, but they will be better paid and less strenuous.How can the slaughterhouses get access to this kind of technology? As in all aspects of technology development, someone must be the first mover and probably that is the companies with the most urgent need. But beware that there is a dramatic disruptive potential in this technology. As a robotic production cell is basically a standard industrial platform, it opens for many more technology providers than the industry has today. Previous solutions were highly mechanized and integrated tying the slaughterhouse to the particular technology provider, but with this new production paradigm, the field is completely open, and any robot integrator would potentially be able to enter the arena. Moreover, the slaughterhouses will claim their rights to the algorithms, which will lower the bargaining power of the technology providers. Good news for the slaughterhouses and good news for new to the business technology providers, who wants to enter the arena. New technology also means new ways of working. Many slaughterhouses have agreements based on time studies or volumes, and these kinds of agreements will not make any sense with the new technology. As work moves from blue collar to technician type of work, the management approach must change accordingly.So, the first-mover will not only take the risk but also get ahead of their competition if it is a success and the moment for decision making is coming close.Self-learning robotic production cells, which are hyperflexible and which can be added with new features, both physically and digitally, represent a disruptive shift in the production paradigm in the meat industry. The circle is closed, and we are back to the cutting table. Maybe, the automotive industry will be inspired again a hundred years after the visit to Swift and Company\u2019s slaughterhouse in Chicago."}
{"text": "Setting. Ophthalmology Division of San Rossore Medical Center, Pisa, Italy.  To evaluate clinical outcome during 24 months follow-up between small incision lenticule extraction combined with cross-linking (SMILE Xtra) and small incision lenticule extraction (SMILE) only.  Retrospective comparative case series.  The study comprised 70 eyes (35 patients); 40 eyes were corrected using SMILE and 30 eyes were corrected using SMILE Xtra using a low energy protocol. The outcomes were compared at 1, 6, 12, and 24 months postoperatively. p < 0.05). At 24 months the mean SEQs were \u22120.01\u2009\u00b1\u20090.24\u2009D for SMILE and \u22120.15\u2009\u00b1\u20090.33\u2009D for SMILE Xtra (p > 0.05). At 1, 6, 12, and 24 months, there were no statistically significant differences between the SMILE and SMILE Xtra groups in logarithm of the minimum angle of resolution (logMAR) uncorrected distance visual acuity (UDVA), safety, and efficacy index (p > 0.05). The mean average keratometry (K-avg) at 1, 6, 12, and 24 months after surgery did not shown any statistically significant difference between SMILE and SMILE Xtra group (p > 0.05). The mean maximum keratometry (K-max) readings at 1, 6, 12, and 24 months were not statistically significant between SMILE and SMILE Xtra group (p > 0.05). The preoperative mean thinnest point pachymetry (TTP) was 543.90\u2009\u00b1\u200922.85\u2009\u03bcm in the SMILE group and 523.40\u2009\u00b1\u200937.01\u2009\u03bcm in the SMILE Xtra group (p < 0.05). At 1, 6, 12, and 24 months the mean TTP was not statistically significant between the SMILE and SMILE Xtra groups (p > 0.05). At 24 months, the TTP was 408.29\u2009\u00b1\u200938.75\u2009\u03bcm for the SMILE group and 402.22\u2009\u00b1\u200937\u2009\u03bcm for the SMILE Xtra group (p > 0.05). In the preoperative period, the mean maximum posterior elevation (MPE) was 8.63\u2009\u00b1\u20094.35\u2009\u03bcm for SMILE and 8.13\u2009\u00b1\u20092.54\u2009\u03bcm for SMILE Xtra (p > 0.05). After the surgical procedure, both groups showed a statistically significant increase of the MPE (p < 0.05). At 24 months, the MPE was 11.00\u2009\u00b1\u20094.72\u2009\u03bcm for SMILE Xtra and 10.14\u2009\u00b1\u20093.85\u2009\u03bcm for the SMILE group (p > 0.05). In the preoperative period, the means of the root mean square (RMS) of high-order aberration (HOA) were 0.08\u2009\u00b1\u20090.03\u2009\u03bcm for the SMILE group and 0.08\u2009\u00b1\u20090.03\u2009\u03bcm for the SMILE Xtra group (p > 0.05). At 24 months, the RMS of HOA was 0.13\u2009\u00b1\u20090.07\u2009\u03bcm for the SMILE group and 0.14\u2009\u00b1\u20090.07\u2009\u03bcm for the SMILE Xtra group (p > 0.05). In the preoperative period, the root mean square of coma aberration (RMS-Coma) aberration was 0.06\u2009\u00b1\u20090.09\u2009\u03bcm for the SMILE group and 0.04\u2009\u00b1\u20090.03\u2009\u03bcm for the SMILE Xtra group (p > 0.05). At 24 months, the coma aberration of SMILE group was 0.12\u2009\u00b1\u20090.21\u2009\u03bcm and 0.16\u2009\u00b1\u20090.25\u2009\u03bcm for SMILE Xtra group (p > 0.05).  The mean spherical equivalent (SEQ) reduced from \u22127.18\u2009\u00b1\u20091.21\u2009D to \u22120.01\u2009\u00b1\u20090.09\u2009D in the SMILE group and from \u22126.20\u2009\u00b1\u20092.99\u2009D to \u22120.04\u2009\u00b1\u20090.1\u2009D postoperatively in SMILE Xtra ( SMILE Xtra procedure is a safe and simple procedure that can be offered to patients with high corneal ectasia risk because there were no differences in the indices of ectasia compared to the group treated only with SMILE which has a low corneal ectatic risk. The decision for refractive surgery in patients with high myopia, borderline topography, and thinner corneas is challenging. One of the most feared complications is postoperative ectasia, a progressive steepening, and thinning of the cornea \u20134.Collagen cross-linking (CXL) has been proven to be an effective modality to strengthen and stabilize the cornea in keratoconus and ectasia after corneal refractive surgery \u20138. CXL hThe small incision lenticule extraction (SMILE) technique provides a biomechanically stronger cornea since it preserves the integrity of the anterior cornea. Although a large case series has demonstrated an excellent safety profile for SMILE , cornealThe purpose of this study is to assess the 2-year clinical outcome of a series of small incision lenticule extraction combined with cross-linking (SMILE Xtra) cases using a low-energy protocol compared to SMILE alone.This study was a retrospective interventional comparative study. The patients of the current study signed an informed consent before the intervention. The study was approved by the Institutional Ethics Committee and adhered to the tenets of the Declaration of Helsinki. The data were anonymized or maintained with confidentiality.The first group included 30 eyes of 15 patients who underwent SMILE Xtra procedure. The second control group included 40 eyes of 20 patients, who were treated with the SMILE procedure only. Inclusion criteria of the study included moderate to high myopia with baseline spherical equivalent (SEQ) higher than 4 diopters, and all eyes had a emmetropic target refraction.\u03bcm, established keratoconus, family history of keratoconus or eye rubbing [Exclusion criteria included subjects with corneal thickness <450\u2009 rubbing , hyperopAll included patients had preoperative complete ophthalmic clinical examination including slit-lamp examination, intraocular pressure measurement, fundus examination, dry eye assessment, cycloplegic refraction, uncorrected distance visual acuity (UDVA), and corrected distance visual acuity (CDVA) measured with Snellen chart at 6\u2009m, which was converted to minimum angle of resolution (logMAR) value. According to the Randleman Scoring for ectasia risk, eyes were classified into low (score \u22642), and moderate to high risk (score \u22653) . The SMI\u03bcm; cap diameter 7.0\u20137.5\u2009mm; lenticule diameter 6.0\u20136.5\u2009mm with a transition zone of 0.1\u2009mm; cut energy 1.4\u2009J; spot and tracking distance 2.0\u20133.0\u2009\u03bcm. A 2\u2009mm incision located at 10 o'clock position for both eyes was performed. A blunt spatula was used to separate the lenticule, which was then removed by forceps through the incision. Finally, the corneal interface was flushed with balanced salt solution. In SMILE Xtra cases, after lenticule removal, 0.22% riboflavin with saline  was injected through the small incision into the interface and left to soak for 90 seconds, followed by UV-A irradiation at 30\u2009mW/cm2 for 90 seconds  using the Avedro KXL system.A single surgeon performed all the surgeries (M.F.) using an established technique under topical anesthesia. The SMILE procedure was performed using the 500\u2009kHz VisuMax femtosecond laser (Carl Zeiss Meditec). The following parameters were used: cap thickness 130\u2013140\u2009Postoperative medications included ophthalmic topical tobramycin 0.3% and dexamethasone 0.1% suspension four times a day for 1 week and then steroids eye drops were tapered over 1 month. Finally preservative-free artificial teardrops were given for 3 months postoperatively. All patients were examined preoperatively and postoperatively at day 1, 1 week, 1, 6, 12, and 24 months.K-avg), maximum keratometry (K-max), corneal astigmatism, thinnest point pachymetry (TTP), mean maximum posterior elevation (MPE), means of the root mean square of high-order aberration (RMS-HOA), and root mean square of coma aberration (RMS-Coma). The efficacy index was calculated as the ratio of postoperative UDVA to preoperative CDVA, and the safety index was determined as the ratio of postoperative CDVA to preoperative CDVA.The visual acuity (Snellen chart at 6\u2009m) was converted to logMAR value, and refraction was recorded at 1, 6, 12, and 24 months. The corneal tomography , corneal topography  and aberrometry  were performed. The following data were recorded: average keratometry . The parameters between SMILE Xtra and control group were compared with MannWhitney p > 0.05). According to the Randleman Scoring, 30 SMILE Xtra eyes (100%) had an ectasia risk factor score of 3 or higher (moderate/high risk). In the control group, 40 eyes (100%) had a score of 2 or lower (low risk). No intraoperative or postoperative complications occurred in either group. Postoperatively, there was no clinically detectable corneal haze in all cases.A total of 40 eyes for the SMILE group and 30 eyes for the SMILE Xtra group was included for analysis. The baseline demographic and preoperative data are summarized in p < 0.05). At 12 months, the mean errors in the correction in the SEQ were \u22120.01\u2009\u00b1\u20090.21\u2009D for SMILE and \u22120.09\u2009\u00b1\u20090.18\u2009D for SMILE Xtra (p > 0.05). The mean errors in the correction in the SEQ at 24 months were \u22120.01\u2009\u00b1\u20090.24\u2009D for SMILE and \u22120.15\u2009\u00b1\u20090.33\u2009D for SMILE Xtra (p > 0.05)  achieved UDVA 20/20 and only 2 eyes had UDVA 20/25. In the SMILE Xtra group, 29 eyes (96.66%) achieved UDVA 20/20 and only 1 eye had UDVA 20/25. At 24 months, in the SMILE group, 39 eyes (97.5%) achieved UDVA 20/20 and only 1 eye had UDVA 20/25. In the SMILE Xtra group, 29 eyes (96.66%) achieved UDVA 20/20 and only 1 eye had UDVA 20/25 .There was no statistically significant difference in logMAR UDVA at 1, 6, 12, and 24 months after surgery between the SMILE and SMILE Xtra groups (p > 0.05).The safety index and efficacy index of both groups at 1, 6, 12, and 24 months are summarized in K-avg values among SMILE and SMILE Xtra group  . At month 24 after surgery, no statistically significant difference in K values between the two groups  was observed.Preoperatively, there was no significant difference in  > 0.05) . The K-aK-avg at month 1, 6, 12, and 24 months after surgery, did not show any statistically significant difference between the SMILE and SMILE Xtra groups (p > 0.05).As shown in K-max values difference was not statistically significant in preoperative time between the SMILE and SMILE Xtra groups. After the surgical procedure, both groups showed a statistically significative increase of K-max (p < 0.05). The K-max readings at 1, 6, 12, and 24 months were not statistically significant between the SMILE and SMILE Xtra groups (p > 0.05) (The  > 0.05) .\u03bcm in the SMILE group and 523.40\u2009\u00b1\u200937.01\u2009\u03bcm in the SMILE Xtra group (p < 0.05) (p < 0.05). At 1 month, the TPP means were 403.83\u2009\u00b1\u200935.51\u2009\u03bcm for the SMILE group and 399.40\u2009\u00b1\u200940.82\u2009\u03bcm for the SMILE Xtra group (p > 0.05). At 6 months, the TPP means were 407.94\u2009\u00b1\u200937.05\u2009\u03bcm for the SMILE group and 394.45\u2009\u00b1\u200938.00\u2009\u03bcm for the SMILE Xtra group (p > 0.05). At 12 months, the TPP means were 414.03\u2009\u00b1\u200936.28\u2009\u03bcm for the SMILE group and 402.16\u2009\u00b1\u200939.52\u2009\u03bcm for the SMILE Xtra group (p > 0.05). At 24 months, the TPP means were 408.29\u2009\u00b1\u200938.75\u2009\u03bcm for the SMILE group and 402.22\u2009\u00b1\u200937\u2009\u03bcm for SMILE Xtra (p > 0.05) (The means of the postoperative TPP were 543.90\u2009\u00b1\u200922.85\u2009 < 0.05) . After t > 0.05) .\u03bcm for SMILE and 8.13\u2009\u00b1\u20092.54\u2009\u03bcm for SMILE Xtra (p > 0.05) (p < 0.05). At 1 month, the MPE means were 13.22\u2009\u00b1\u20095.86\u2009\u03bcm for the SMILE Xtra group and 12.65\u2009\u00b1\u20095.85\u2009\u03bcm for the SMILE group (p > 0.05). At 6 months, the MPE means were 14.81\u2009\u00b1\u20097.02\u2009\u03bcm for the SMILE Xtra group and 12.47\u2009\u00b1\u20097.33\u2009\u03bcm for the SMILE group (p > 0.05). At 12 months, the MPE means were 11.54\u2009\u00b1\u20095.15\u2009\u03bcm for the SMILE Xtra group and 11.13\u2009\u00b1\u20094.39\u2009\u03bcm for the SMILE group (p > 0.05). At 24 months, the MPE means were 11.00\u2009\u00b1\u20094.72\u2009\u03bcm for the SMILE Xtra group and 10.14\u2009\u00b1\u20093.85\u2009\u03bcm for the SMILE group (p > 0.05)  . After t > 0.05) .\u03bcm for the SMILE group and 0.08\u2009\u00b1\u20090.03\u2009\u03bcm for the SMILE Xtra group (p > 0.05) (p < 0.05). At 1 month, there was no statistically significant difference between the SMILE (0.17\u2009\u00b1\u20090.10\u2009\u03bcm) and SMILE Xtra groups (0.17\u2009\u00b1\u20090.09\u2009\u03bcm) (p > 0.05). At 6 months, the mean RMS-HOA was 0.20\u2009\u00b1\u20090.20\u2009\u03bcm for the SMILE group and 0.19\u2009\u00b1\u20090.14\u2009\u03bcm for the SMILE Xtra group (p > 0.05). At the 12-month follow-up, the mean RMS-HOA was 14\u2009\u00b1\u20090.07\u2009\u03bcm for the SMILE group and 0.12\u2009\u00b1\u20090.06\u2009\u03bcm for the SMILE Xtra group (p > 0.05). At 24 months, the mean RMS-HOA was 0.13\u2009\u00b1\u20090.07\u2009\u03bcm for the SMILE group and 0.14\u2009\u00b1\u20090.07\u2009\u03bcm for the SMILE Xtra group (p > 0.05)  . After t > 0.05) .\u03bcm for SMILE and 0.04\u2009\u00b1\u20090.03\u2009\u03bcm for SMILE Xtra (p > 0.05) (p > 0.05). At 1 month, the mean RMS-Coma aberration was 0.12\u2009\u00b1\u20090.15\u2009\u03bcm for SMILE and 0.16\u2009\u00b1\u20090.18\u2009\u03bcm for SMILE Xtra (p > 0.05). At 6 months, the mean RMS-Coma aberration was 0.14\u2009\u00b1\u20090.34\u2009\u03bcm for the SMILE group and 0.08\u2009\u00b1\u20090.10\u2009\u03bcm for the SMILE Xtra group (p > 0.05). At 12 months, the mean RMS-Coma aberration was 0.06\u2009\u00b1\u20090.07\u2009\u03bcm for the SMILE group and 0.06\u2009\u00b1\u20090.05\u2009\u03bcm for the SMILE Xtra group (p > 0.05). At 24 months, the mean RMS-Coma aberration of SMILE was 0.12\u2009\u00b1\u20090.21\u2009\u03bcm and 0.16\u2009\u00b1\u20090.25\u2009\u03bcm for the SMILE Xtra group (p > 0.05)  . After t > 0.05) .forme-fruste keratoconus [SMILE is a flapless procedure with the theoretical advantage of producing less weakening of the cornea, since the cap thickness contributes to the residual stromal bed. For this reason, SMILE is better option in treating higher myopic errors, thinner corneas, and cases with abnormal topography or atoconus , 18. Desatoconus , 19\u201321.Any corneal laser refractive procedure would certainly decrease the biomechanical stability of the cornea. Combining corneal collagen cross-linking with PRK or LASIK showed good results and has come into practice , 22. In 2 for different durations, with a total energy of 1.8 to 5.4\u2009J/cm2, and all those different regimens proved to be safe and effective [The aim of SMILE Xtra is to deliver the least amount of energy that can stabilize the cornea. Excess energy may cause haze while too little energy may be insufficient to ensure corneal strength. Whereas established CXL protocols for treating keratoconus with long-term follow-up have been shown to be safe and effective , there affective , 18, 22.2 (30\u2009mW/cm2 for 90 seconds). This amount of energy proved to be safe and well tolerated [2 of energy delivered for 75 seconds  [2 for 45 seconds  [2 total energy [In the preoperative time, the SMILE Xtra eyes had a significantly thinner TPP than the control and topographic alteration with an increased risk of ectasia. Our CXL protocol for the SMILE Xtra procedure involves the application of riboflavin directly to the stromal pocket after lenticule extraction followed by UV-A irradiation with a total energy of 2.7\u2009J/cmolerated , as none4\u2009J/cm2) . Ng et a8\u2009J/cm2) . Graue-Hl energy .In our study, both SMILE and SMILE Xtra results showed excellent refractive outcomes. The UDVA were comparable between two groups, with refractive predictability reported in the literature , 29, 30.K-avg and K-max values, with no statistically significant difference from the SMILE group. Before the surgical procedure the TPP between the SMILE and SMILE Xtra showed a statistically significant difference. The SMILE Xtra group showed stable TPP from the 6th to 24th month of follow-up. The difference between the study and control group is not statistically significative at 24 months. Also, the MPE showed stable results during the 24 months of follow-up, with no statistically significant difference with SMILE group. The SMILE Xtra aberrometric data demonstrate no statistically significant differences with SMILE group for both RMS-HOA and RMS-Coma aberration.Regarding keratometry, the SMILE Xtra group showed stable The advantages of the current study are the comparative nature with a control group of SMILE cases and a follow-up period of 24 months. The limitations of our study are the retrospective nature of the study, the requirement of more cases, and need for a longer duration of follow-up to record the long-term effects. Moreover, it was not possible, until now, to evaluate the surgical techniques SMILE vs SMILE Xtra composed of only high-risk subjects in both groups, in order to reduce confounding variables.In conclusion, SMILE Xtra procedure is a safe and effective procedure that can be offered to patients undergoing SMILE with corneal ectasia risk."}
{"text": "The construction of a harmonious society requires college students to coordinate their ideological, political, and moral qualities with social development and the needs of the times. Through the investigation and analysis of the ideological, political, and moral qualities of college students, on the one hand, we can see that the ideological, political, and moral qualities of college students are generally positive and healthy. On the other hand, it also exposes the outstanding problems in the ideological and political aspects of college students and the shortcomings of the ideological and political work in colleges and universities. This paper analyzes the dynamic changes of college students' ideological and political changes and further studies the relationship between various indicators and students' ideological and moral qualities through multiple linear regression analysis. Colleges and universities are an important stage for ideological and political education for young students, and the ideological and political quality of college students is also a major criterion for evaluating the effectiveness of college education. With the continuous improvement of the socialist market economic system, the rapid development of the economy, society and science, and culture, and the integration of Chinese and foreign cultures, many new changes have taken place in the ideological values of current members of society, and some even have contradictions. As the successors and builders of the cause of socialism with Chinese characteristics, the ideological and moral conditions of contemporary college students have also appeared some new changes, new characteristics, and new conditions. In some college students, there are situations such as loss of ideals and beliefs, lack of social responsibility, a weakened spirit of hard work, and a weak concept of teamwork. At the same time, due to the continuous expansion of colleges and universities in my country in recent years and the influence of the global economic recession, new requirements have also been put forward for the ideological and political education work in colleges and universities. In this environment, it is very important and urgent to further strengthen the ideological and political education work of college students. For any society, college students are the most valuable human resources and the hope and future of a country and nation. Do a good job in the ideological and political education of college students, improve their ideological and political level, and train them to be builders and successors of the socialist cause with firm political beliefs and lofty political ideals. This will ensure the healthy and sustainable development of the socialist cause with Chinese characteristics. It has long-term and important strategic significance , 2. The In recent years, with the continuous development of my country's reform and opening up and the gradual improvement of the market economy system, many new situations, new characteristics, and new changes have emerged in the ideological and political quality of contemporary college students , 6. On tAll in all, the ideological and political quality of college students is related to the future and destiny of the country and the nation. Whether they can cultivate qualified and excellent builders and successors who meet the requirements of the cause of socialism with Chinese characteristics, it can be said that the ideological and political work of colleges and universities plays a vital role in it. . Under tStepwise regression: the basic idea of the stepwise regression analysis method is to automatically select the most important variables from a large number of available variables and establish a prediction or interpretation model for regression analysis. The basic idea is that the independent variables are introduced one by one, and the condition of the introduction is that the partial regression sum of squares is significant after testing. At the same time, each time a new independent variable is introduced, the old independent variables should be tested one by one, and the independent variables with insignificant partial regression sum of squares should be eliminated. In this way, it has been introduced and eliminated until neither new variables are introduced nor old variables are deleted. Its essence is to establish the \u201coptimal\u201d multiple linear regression equation. According to the above idea, stepwise regression can be used to screen and eliminate variables that cause multicollinearity. The specific steps are as follows: first is to perform a simple regression on each explanatory variable considered with the explanatory variable and then use the explanation that contributes the most to the explanatory variable. Based on the regression equation corresponding to the variable, the remaining explanatory variables are gradually introduced. After a stepwise regression, the explanatory variables that remained in the model were both significant and did not have severe multicollinearity.MATLAB is commercial mathematical software produced by MathWorks in the United States. It is used in data analysis, wireless communication, deep learning, image processing and computer vision, signal processing, quantitative finance and risk management, robotics, control systems, and other fields. MATLAB is a combination of the words matrix and laboratory, which means matrix factory (matrix laboratory). The software mainly faces the high-tech computing environment of scientific computing, visualization, and interactive programming. It integrates many powerful functions such as numerical analysis, matrix calculation, scientific data visualization, and modeling and simulation of nonlinear dynamic systems in an easy-to-use window environment for scientific research, engineering design, and many sciences that must perform the effective numerical calculation. Realm provides a comprehensive solution and is largely free from the editing mode of traditional noninteractive programming languages , 12  and part-time work (29.7%) are their main social practices. It can be seen from this that contemporary college students have few opportunities for social practice in ideological and political education, and their social practice ability has not been effectively improved, which leads them to place great expectations on social practice. At the same time, most colleges and universities believe that there are problems in the base construction, time arrangement, funding guarantee, and system establishment of social practice. Therefore, it is necessary to pay more attention to and strengthen the social practice work of college students, strive for local support, strengthen the construction of social practice bases, and guide students' social practice activities to develop in a long-term, positional, project-based, and hierarchical direction. Efforts should be made to explore the daily form of social practice activities and use weekends or other spare time to choose the community near the school as a fixed social practice base to carry out social practice activities.The peer groups of college students mainly include classmates, roommates, and members of clubs. Because college students get along with their peers day and night, have the same hobbies, similar life experiences, and have the same troubles and needs, they influence each other infrequent interactions, and especially in specific behaviors, they play a dominant role. The survey data in The influence of peer groups is mainly through good friends. The survey shows that middle school classmates (85.1%), college classmates (76.0%), and roommates (73.5%) are the main objects within the scope of college students' good friends. At the same time, the good friends of college students have also included some new objects, such as fellow villagers (32.7%), members of group organizations or student unions (17.6%), members of clubs (15.0%), netizens (11.7%), class teachers (9.8%), classroom teachers (9.8%), and counselors (9.1%). Once you understand the main scope of good friends for college students, you can start with these objects in a targeted manner and achieve the goal of ideological and political education through the familiarity and example of good friends. At the same time, the dormitory atmosphere is also an important factor affecting the ideological and political education of college students. According to the survey, \u201cstudy\u201d (51.3%) is the eternal theme discussed by college students in the dormitory. \u201cDaily trifles\u201d (32.3%), \u201cemployment prospects\u201d (31.7%), \u201ccurrent affairs news\u201d (31.3%), and \u201cthings happening in the class and school\u201d (29.3%) were also discussed in the \u201csquatting meeting\u201d main content see . Therefox1, x2, ..., xm on y, the factors are introduced into the regression equation one by one from large to small. For the factors that have been introduced into the equation, after the introduction of new factors, they may be eliminated from the equation at any time because they have no significant effect on y. After the variable is introduced, it can also be put back in order to obtain a regression equation with some optimal properties [Stepwise regression is a common mathematical method for selecting regressors in multiple linear regression models , 10. Theoperties  and studoperties . It shouoperties . Many vaoperties . The foloperties  in the MF of the regression model, and the significant probability P associated with the analysis.The parameters of the regression equation are shown in x1), handling affairs (x2), morality and ethics (x3), participation in public welfare social practice (x4), laws and regulations (x5), love marriage (x6), successful employment (x7), and ideal beliefs (x8), were selected into the regression equation, while other variables were eliminated from the original regression equation. Using y to represent the event duration, the established multiple linear regression model isThe 16 variables in Figures It can be seen from the above formula that among the eight independent variables of the equation, ideal beliefs have the greatest impact on ideological and political changes. In addition, two variables, dealing with affairs and love and marriage, also have a large effect on the duration.The constant term in the regression equation is large, which makes the equation predict ideological and political changes, even if the values of all variables are 1, the minimum predicted value is 28.9. That is to say, for small events with small changes, the prediction of the equation will generally be too large.P \u2009=\u20090).As can be seen from In order to examine the validity of the regression model, 170 groups of actual event data are used to test the prediction effect of the regression equation. The results are shown in As can be seen from \u03b1=0.0 and performed a nonparametric one-way ANOVA. The results show that \u2009P=0.0058 < \u03b1, which shows that the multiple linear regression equation has significant statistical significance, and the prediction can basically obtain a satisfactory prediction effect. Finally, we use the above data to prove the feasibility of our model.In order to verify the significance of the prediction of the developed multiple regression model, this study took the significance level This paper conducts a step-by-step regression analysis of the ideological and political change group data of 660 students in a university and establishes the ideological and political dynamics with 8 variables of handling affairs, morality, participation in public welfare social practice, laws and regulations, love and marriage, successful employment, and ideals and beliefs. The multiple linear regression model of change prediction was used, and the prediction accuracy of the prediction model was tested with another 170 sets of data. The correlation coefficient between the predicted value and the actual value was as high as 0.8573. This shows that the multiple linear regression model proposed in this paper can more realistically reflect the duration characteristics of events and can basically obtain satisfactory prediction results. If more and more detailed data can be obtained for the establishment of multiple regression prediction models, it is believed that the prediction accuracy will be further improved. However, due to the extensiveness, randomness, and complexity of the factors that affect the duration of events, the author believes that even the best algorithms cannot predict the duration of events very accurately. It is hoped that the research in this paper can provide some guidance and reference for the development of college students' ideological and political development."}
{"text": "Danaus plexippus), we factorially manipulated dietary macronutrient availability and exposure to zinc, a common metal contaminant in urban habitats that can be toxic but also has nutritional properties. We tested whether (1) the ability to survive zinc exposure depends on dietary macronutrient availability and (2) whether individuals exposed to elevated zinc levels display higher expression of antioxidant genes, given the roles of antioxidants in combatting metal\u2010induced oxidative stress. Exposure to elevated zinc reduced survival only for monarchs developing on a low\u2010macronutrient diet. However, for monarchs developing on a high\u2010macronutrient diet, elevated zinc exposure tended to increase survival. In addition, monarchs exposed to elevated zinc displayed higher expression of antioxidant genes when developing on the low\u2010macronutrient diet but lower expression when developing on the high\u2010macronutrient diet. Altogether, our study shows that organismal survival and oxidative stress responses to anthropogenic zinc contamination depend on the availability of macronutrient resources in the developmental environment. In addition, our results suggest the hypothesis that whether zinc acts as a toxicant or a nutrient may depend on macronutrient supply. Environ Toxicol Chem 2022;41:1286\u20131296. \u00a9 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.Biologists seek to understand why organisms vary in their abilities to tolerate anthropogenic contaminants, such as heavy metals. However, few studies have considered how tolerance may be affected by condition\u2010moderating factors such as dietary resource availability. For instance, the availability of crucial limiting macronutrients, such as nitrogen and phosphorous, can vary across space and time either naturally or due to anthropogenic nutrient inputs . Organisms developing in more macronutrient\u2010rich environments should be of higher overall condition, displaying a greater ability to tolerate metal contaminants. In monarch butterflies ( Anthropogenic activities expose natural populations to a range of chemical contaminants, including pesticides, pharmaceuticals, and heavy metals  generate oxidative stress by removing electrons from water molecules, producing reactive oxygen species (ROS) such as superoxide radicals, hydroxyl radicals, and hydrogen peroxide  to test how variation in dietary macronutrient availability influences heavy metal tolerance. Heavy metals are a persistent threat to insect pollinators such as monarchs, because metal contamination can occur in host plant leaf tissue and nectar in polluted areas  provided in floral wicks. We avoided collecting milkweed plants along roadsides. At eclosion, we transferred butterflies to large mating cages (24\u2009\u00d7\u200924\u2009\u00d7\u200936\u2033) for 5\u20137 days prior to collecting eggs for the experiment . Butterflies in mating and egg collection cages had ad libitum access to 10% honey water provided in sponges. After mating, individual females were transferred to egg\u2010laying cages, each containing a single A. syriaca plant in a water wick and a moist towel to maintain humidity. We replaced plants daily. Each day, we transferred plants containing eggs to 32\u2010oz. plastic cups and stored the cups in a climate chamber . Eggs used for the experiment were collected from the parental females over 1 week in July 2019.All experimental monarch larvae originated from eggs produced by more than 10 wild\u2010collected females reared in common garden conditions. These parental individuals were originally collected as eggs or early instar larvae from the University of Minnesota Saint Paul (USA) campus over a 3\u2010week period in June 2019. To start a population for our rearing experiment, we reared wild\u2010caught individuals in the greenhouse in cages (60\u2009\u00d7\u200960\u2009\u00d7\u200960\u2009cm) with ad libitum access to campus\u2010collected milkweed stalks . On average, the zinc concentration of the elevated zinc diet was approximately 5\u00d7 higher than the control diet . Our elevated zinc treatment was approximately in the range of zinc concentrations observed in plants along highways , and the low\u2010macronutrient diet contained approximately 25% less nitrogen than the high\u2010macronutrient diet . The average carbon\u2010to\u2010nitrogen ratio of the low\u2010macronutrient diet was 7.731 , and for the high\u2010macronutrient diet it was 5.885 . We did not manipulate the milkweed powder portion of the diet, because this serves as a feeding stimulus rather than a nutrient source.We reared monarch caterpillars on a semi\u2010artificial diet designed for monarchs by O.R Taylor at Monarch Watch, validated in previous studies . As expected, monarchs that developed on the elevated zinc diet tended to have higher thorax zinc concentrations, and monarchs developing on the high\u2010macronutrient diet tended to have higher thorax phosphorous concentrations , we transferred larvae with a paintbrush from milkweed to one of the four treatments (40 larvae/treatment). Diet was provided in 16\u2010oz. plastic cups (2 larvae/cup). Throughout larval and pupal development, we housed all individuals in a climate chamber maintained at 25\u2009\u00b0C with a 15:9\u2010h light:dark photoperiod. We positioned all cups on their sides to provide adequate space for butterfly eclosion. To confirm variation in monarch nutrient accumulation across treatments, thorax concentrations of zinc and phosphorous (in mg/kg) were measured in 2) of five randomly selected mature eggs from each dissected female. All dissections were performed using a Leica M165C microscope at \u00d710 magnification while the butterfly abdomen was submerged in a 1\u00d7 phosphate\u2010buffered saline buffer; eggs removed from the ovaries consistently lay on their sides in these conditions, facilitating an area measurement despite the three\u2010dimensional bullet shape of the egg.We calculated monarch development time as the time between the day larvae were transferred to semi\u2010artificial diet treatment and the day of eclosion. Monarch survival was quantified as whether an individual survived to eclosion. After eclosion, we labeled all butterflies on the hindwing with a fine\u2010tipped black sharpie. All females that eclosed with undamaged wings were transferred to a large cage (61\u2009\u00d7\u200961\u2009\u00d7\u200961\u2009cm) in the greenhouse for an egg development period of 7 days before being stored in a \u221220\u2009\u00b0C freezer until ovary dissections. We measured egg production as the number of fully yoked and chlorinated eggs in the ovaries of dissected females. We also measured the average egg area  analyses. First, we selected actin because it is one of the most frequently used reference genes for qPCR in insects and has been shown to be the most stably expressed reference gene across different developmental stages in a broad range of insect taxa , zinc exposure , or their interaction  on caterpillar body mass prior to dissection. We dissected fat body tissue because this organ plays a central role in insect metabolic functions that are highly sensitive to variation in nutrient supply . We extracted RNA from fat body tissue dissected from larvae at the fifth instar (17 days after transfer to each dietary treatment). Although there was no way to account for potential differences in development time across individuals at the time of dissection, analysis of variance indicated that there were no significant effects of macronutrient availability  for complementary DNA synthesis using Invitrogen SuperScript II reverse transcriptase and quantitative real\u2010time (qRT\u2010)PCR. All PCR primers were developed and validated at UMGC; all qRT\u2010PCR was performed using an Applied Biosystems 7900HT real\u2010time PCR instrument. Two technical qPCR replicates were produced for each monarch RNA sample.For statistical analyses, we used R Studio Ver 3.6.3 . No effects of zinc exposure  or an interaction between macronutrient availability and zinc exposure  on egg production were detected. In addition, we detected no effects of macronutrient availability , zinc exposure , or an interaction between macronutrient availability and zinc exposure  on average size of eggs produced by adult females. Across all females, forewing length was not significantly correlated with either egg number  or egg area , so it was not included as a covariate in either analysis.There was a significant effect of macronutrient availability on egg production , had adult forewing lengths that were approximately 4% shorter , and had approximately 15% slower growth rates , on average. There were no effects of zinc exposure or an interaction between macronutrient availability and zinc exposure on development time, forewing length, or growth rate , monarchs in the low\u2010macronutrient/control zinc treatment tended to have lower antioxidant gene expression Figure\u00a0, and expConsistent with our initial hypothesis that tolerance to heavy metal contamination is macronutrient dependent, we found that elevated zinc exposure negatively impacted survival only of monarchs developing on a low\u2010macronutrient diet Figure\u00a0. Given tIn nature, resource dependence may help explain why individuals vary in their abilities to tolerate pollutants across spatial scales. For instance, nutrient availability may vary greatly across landscapes, either naturally or due to differential nutrient inputs from human activities  compared with monarchs on the control treatment Table\u00a0. This isIt is unclear why a given level of zinc exposure might have harmful effects in individuals with low access to macronutrient resources but potential nutritional and regulatory benefits in individuals with high access to macronutrient resources. One hypothesis is that more nutrient\u2010limited individuals have fewer energetic resources available to invest in physiological machinery required to efficiently regulate internal zinc levels. For instance, key regulators of zinc homeostasis are the metallothionein proteins, which sequester zinc, enable its antioxidant properties, and play a central role in detoxification  are not also considered (Sih et al.,\u00a0https://doi.org/10.1002/etc.5305.The Supporting Information is available on the Wiley Online Library at Alexander M. Shephard: Conceptualization; methodology; formal analysis; funding acquisition; investigation; visualization; writing\u2014original draft. Emilie C. Snell\u2010Rood: Conceptualization; funding acquisition; methodology; writing\u2014editorial advice. Noah S. Brown:\u00a0Investigation; writing\u2014editorial advice.This article includes online\u2010only Supporting information.Figure S1.Click here for additional data file.Supplementary information.Click here for additional data file.Supplementary information.Click here for additional data file."}
{"text": "Acute aortic syndromes are life-threatening conditions with high morbidity and mortality. The principal pathological feature is acute wall damage with possible evolution towards aortic rupture. Accurate and timely diagnosis is mandatory to avoid catastrophic consequences. Indeed, misdiagnosis with other conditions mimicking acute aortic syndromes is associated with premature death. In this view, cardiovascular imaging is necessary for the correct diagnosis and management. Echocardiography, computed tomography, magnetic resonance imaging, and aortography allow for diagnosis, guarantee immediate treatment, and detect associated complications. Multimodality imaging is essential in the diagnostic work-up to confirm or rule out acute aortic syndromes. The aim of this review is to highlight the contemporary evidence on the role of single cardiovascular imaging techniques and multimodality imaging in the diagnosis and management of acute aortic syndromes. Acute aortic syndromes (AAS) represent a spectrum of interrelated disorders characterized by the disruption of the aortic integrity, and are associated with high morbidity and mortality. These conditions include aortic dissection (AD), accounting for the majority of AAS (80%), intramural hematoma IMH, ~15%), and penetrating aortic ulcer  5%, and p,3.AD is described as a separation of the aortic wall layers caused by an intimo-medial tear, resulting in the creation of a false lumen that propagates within the medial layer. Thus, AD typically shows the appearance of a dissection flap, an entry tear, and two aortic channels . No modern classifications have substituted the older Stanford and DeBakey ones that remain the most commonly used. AD involving the ascending aorta is defined as a Stanford type A dissection, regardless of distal extension; when the disruption is distal to the left subclavian origin, on the contrary, it is considered to be type B. The DeBakey classification recognizes types I, II, and, III, with type I affecting both the ascending and descending aorta, type II the ascending aorta and the arch only, and type III involving the descending aorta but sparing the arch and the ascending aorta. The Stanford classification is used to guide different treatment options. Indeed, surgery is recommended in type A AD, while medical therapy is suggested in uncomplicated type B AD. IMH is instead a haematoma created within the media layer and secondary to the rupture of the vasa vasorum. Finally, PAU is an ulceration of an atherosclerotic aortic plaque penetrating towards the internal elastic lamina and into the media .The advent of multi-modality imaging in the assessment and management of AAS has multiple applications, not only for diagnosis, but also for risk stratification and planning of the acute intervention, as well as for the evaluation of potential complications and/or the candidacy for intervention or re-intervention in the subacute or chronic phases .In this document, our aim is to critically review the distinctive multi-modality imaging features of AAS, while at the same time providing an update and overview of diagnostic strategies and the current management of these disorders.Echocardiography is renowned for being a non-invasive, safe, and accurate imaging modality used in the clinical evaluation of cardiovascular disease and plays an important role in the diagnosis of aortic diseases. Transthoracic echocardiography (TTE) can be performed at the patient\u2019s bedside, as well as in the emergency and critical care units, and for this reason the European Society of Cardiology guidelines  recommenThe diagnosis of AD by TTE is based on detecting intimal flaps in the aorta. The sensitivity and specificity of TTE range from 77\u201380% and 93\u201396%, respectively, for ascending AD, and varies according to the use of contrast agents . Since sTOE is currently considered as one of the reference techniques in AD diagnosis, with a very high diagnostic accuracy for the detection of both ascending and descending aortic diseases B 12]. I. I12]. IThanks to its high spatial resolution, full assessment of thoracoabdominal aorta, short acquisition time, and wide availability, CT represents the ideal technique for diagnosing AAS.The typical scan protocol first includes a non-contrast CT followed by an arterial acquisition . A late The sensitivity and specificity of CT for AD is approximately 98\u2013100% , and it The classical finding of AD in CT is an intimal flap with a partition between the true and false lumen. Supporting signs are the internal displacement of intimal calcifications, the delayed enhancement of the false lumen, the widening of the aorta, mediastinal, pleural, or pericardial hematoma , and sig2 when gadolinium is used, and the poor assessment of arterial wall calcification.Magnetic resonance imaging (MRI) is a highly accurate, noninvasive imaging modality, with a sensitivity and specificity of almost 98% for detecting AD . AdditioIn a suspected case of AD, the standard MRI examination should begin with rapid spin-echo black blood acquisitions covering the aorta to outline aortic shape and diameter and to rule out alterations in wall structure . In the In the past, aortography represented the gold standard for the diagnosis of AD . In moreAortography visualizes all features of AD: the intimal flap, true lumen , false lumen, craniocaudal extension, indirect visualization of the coronary arteries, possible aortic regurgitation, and aortic rupture E. VariouIMH is classified among AAS and represents about 15% of cases. Specifically, it is defined as Type A when it affects the ascending aorta and the aortic arch and type B when it involves the descending aorta. . IMH is Non-contrast CT from the supraortic vessels to the aortic carrefour is crucial to diagnosing IMH. IMH is visible on unenhanced CT typically as an eccentric, hyperdense (60\u201370 HU), crescentic area of thickening of the aortic wall (>0.5 cm), showing no enhancement after administration of intravenous contrast that extends in a longitudinal, non-spiral fashion A,B. The Other CT findings that can be useful to predict the negative evolution of the IMH include an IMH high mean IMH thickness greater than 10\u201311 mm, compression of the true aortic lumen, a maximal ascending aortic diameter greater than 50 mm, or descending aorta greater than 45 mm, association with PAU, and the presence of pericardial effusion . These hMRI has the unique ability to determine the age of the hematoma based upon T1 and T2 signal characteristics ,48,49. DBreath-hold ECG-gated fast spin echo images acquired with T1 and T2 weighting are used to assess aortic calibre, aortic wall thickness, and aortic wall signal change. These are acquired as contiguous transverse sections from the apexes to the infrarenal abdominal aorta and also as sections paralleling the long axis of the aortic arch. On MRI, the spin-echo black blood images of an IMH show a crescent-shaped area of eccentric thickening, and exhibit the expected signal characteristics associated with the transition of hemorrhage from deoxyhemoglobin (low T1 and T2) in the hyperacute stage to intracellular methemoglobin  in the subacute stage and extracellular methemoglobin (high T1 and T2) in the late stage of IMH A.2 represents a limitation, in addition to the difficulty in monitoring patients in the acute phase.Dynamic SSFP sequences are used to assess aortic valve function and the calibre of the aortic root. These are acquired in both transverse and coronal planes through the left ventricular outflow tract (LVOT). If aortic regurgitation is present, its severity can be quantified via flow sensitive phase contrast sequences. Finally, a gadolinium-enhanced aortic angiography study could be performed. However, the risk of nephrogenic systemic fibrosis with GFR < 30 mL/min/1.73 mA small study suggested three magnetic resonance angiography (MRA) parameters to distinguish IMH from type B dissection: (1) no visualized entry tear, (2) no contrast uptake in aortic lesion on first pass angiography, and (3) no contrast uptake in the aortic lesion on the equilibrium phase T1-weighted sequence . In addiIn the diagnosis of IMH, aortography has a more limited role compared to classic dissection because the intima is intact and, as a result, the lumen of the aorta remains unaffected . The onlTOE is among the methods for detecting PAU and evaluating possible complications B. AlthouCT today represents the most used technique for the diagnosis and follow-up of PAU thanks to its high spatial resolution, which allows for a detailed assessment of atherosclerotic plaques, calcium, and the aortic wall . CT characteristic features of PAU are an aortic out-pouching in the presence of aortic intima calcifications and severe atherosclerotic disease. The edges of the out-pouching are usually irregular or jagged  Figure C. PAU caCT imaging can be helpful in stratifying the risk of patients with PAU to select the appropriate management. In particular, patients with PAU diameters over 13\u201320 mm and more profound than 10 mm and concomitant IMH are associated with a worse prognosis . ChoosinPAU can be well visualized on MRI, as well as the associated IMH. Ulcer craters can be documented on spin-echo images as a signal void within the thickened aortic wall . The immThe diagnostic challenge consists in distinguishing PAU from the ulcerated atherosclerotic plaque and the IMH from the intraluminal thrombus. In 1990 Yucel et al. studied seven patients with acute chest pain and penetrating aortic ulcers by MRI imaging compared with angiography and CT. They demonstrated that MRI, thanks to the ability to image the aortic wall, was superior to angiography in depicting the extent of intramural thrombus and was superior to CT in differentiating acute IMH from atherosclerotic plaque and chronic intraluminal thrombus . As noteCompared to AD, patients with PAU are older and present risk factors for atherosclerosis such as arterial hypertension, dyslipidemia, and smoking . Once agIn an emergency scenario, a rapid comprehensive diagnostic work-up is necessary, including clinical assessment , laboratory data (D-dimer and troponin amongst others), chest X-ray, and ECG in order to expedite the appropriate aortic imaging studies ,64,65,66In this setting, bedside TTE combined with additional TOE have the priority in order to exclude new-onset aortic regurgitation, pericardial effusion, or to visualize proximal dissection. Modern ultrasound equipment is in fact mobile and particularly useful at the bedside for unstable AAS and/or for detecting painless AD . TOE evaGiven the optimal accuracy of all multi-modality imaging modalities, imaging protocols should be adapted to local expertise and availability, the patient\u2019s clinical condition, and the target of interest .CT technology has currently replaced invasive diagnostic angiography for the thoracic and abdominal aorta. CT is rapid, accurate, and has high spatial resolution, but requires patient transportation and, thus, stable haemodynamic conditions .MRI angiography is also capable of providing high-resolution aortic imaging with three\u2013dimensional post-processing; it needs neither iodinated contrast nor ionizing radiation, being particularly useful for patients intolerant to contrast due to allergy or renal failure ,72,73. MAfter an AAS, surviving patients require lifelong follow-up. The imaging approach is crucial in prognostic assessment and to detect evolution and complications. The cardiac imaging techniques utilized for surveillance are echocardiography, CT, and MRI. CT is the most commonly performed for the long-term follow-up after an AAS. However, especially in young patients, this technique must consider radiation exposure. MRI is a valid alternative and avoids the use of iodinated contrast in addition to radiation exposure. Furthermore, in the IMH, it identifies the evolution of temporal bleeding and new hemorrhagic episodes. Cardiac imaging follow-up is indicated both in patients managed with medical therapy alone and in those treated surgically or with TEVAR ,77,78,79A multimodality imaging approach in the AAS is indispensable for accurate and timely diagnosis. In this setting, prompt diagnosis is mandatory considering this life-threatening condition which requires adequate and immediate treatment. In addition, multimodality imaging adds key information about urgency indicators and the associated complications. A correct and high-quality diagnostic work-up improves the poor prognosis in this emergency condition. Advances in cardiovascular imaging techniques have led to a better understanding of the pathophysiology and improvement in diagnosis and management. The appropriate choice of imaging modality is based on the accuracy, advantages, and limitations of the techniques. However, the clinical conditions of the patient and local availability and expertise should be also considered. The correct application of the imaging algorithm and cardiovascular techniques is crucial for the diagnosis and management of AAS."}
{"text": "Despite major advances in immunosuppression, allograft rejection remains an important complication after heart transplantation, and it is associated with increased morbidity and mortality. The gold standard invasive strategy to monitor and diagnose cardiac allograft rejection, based on the pathologic evaluation of endomyocardial biopsies, suffers from many limitations including the low prevalence of rejection, sample bias, high inter-observer variability, and international working formulations based on arbitrary cut-offs that simplify the landscape of rejection. The development of innovative diagnostic and prognostic strategies\u2014integrating conventional histology, molecular profiling of allograft biopsy, and the discovery of new tissue or circulating biomarkers\u2014is one of the major challenges of translational medicine in solid organ transplantation, and particularly in heart transplantation. Major advances in the field of biomarkers of rejection have paved the way for a paradigm shift in the monitoring and diagnosis of cardiac allograft rejection. We review the recent developments in the field, including non-invasive biomarkers to minimize the number of protocol endomyocardial biopsies and tissue biomarkers as companion tools of pathology to refine the diagnosis of cardiac rejection. Finally, we discuss the potential role of these biomarkers to provide an integrated bio-histomolecular diagnosis of cardiac allograft rejection. Heart transplantation (HTx) remains the most valuable therapeutic option for patients with end-stage heart failure ,2,3. DesOverall, there is a strong and growing literature suggesting a crucial role of invasive and non-invasive biomarkers to better monitor, detect, and refine the diagnosis of allograft rejection after heart transplantation. In this article, we review existing data concerning the use of (i) circulating non-invasive biomarkers of allograft rejection to reduce the number of protocol EMB and (ii) tissue biomarkers as a companion tool for the pathologist to better characterize allograft rejection.During the last two decades, important resources have been allocated to the search for an accurate non-invasive biomarker of allograft rejection. These biomarkers can be classified into two categories: Those reflecting allograft injury and those reflecting the inflammatory and allo-immune processes underlying allograft rejection. Due to the potential consequences of missing and not treating acute cardiac allograft rejection, these biomarkers are required to be highly sensitive for rejection even at the cost of low specificity. Despite dozens of promising studies and candidate biomarkers, only two have been approved by the Food and Drug Administration (FDA) and applied in routine clinical practice, mostly in North America (AlloMap and donor-derived cell-free DNA [dd-cfDNA]) . The oveTroponins are structural proteins of the myocardium, expressed almost exclusively in the heart. They consist of three subunits: Troponin T, C, and I. Troponin T comprises two tissue subunits, one of which is myocardium-specific, cTnT. cTnT is a biomarker mainly used in the diagnosis of acute coronary syndromes and myocarditis. However, its elevation in the blood does not reflect the underlying mechanism, but only the existence of myocardial necrosis. Therefore, the use of this circulating marker of myocardial injury, which is a rapid, non-invasive, and routinely performed assay, has been evaluated as a potential component of the strategy for the non-invasive monitoring of cardiac allograft rejection. Multiple studies, mostly retrospective single-center studies, have reported conflicting results concerning the association between troponins and rejection ,27. TheyIn a longitudinal study including unselected HTx recipients and applying a rigorous statistical approach, the temporal evolution of Troponin T did not predict the occurrence of ACR both in the early and late course of the first year after HTx .Additionally, cTn assay should probably not be used as an isolated non-invasive biomarker of rejection since (i) only ACR \u2265 2R has been evaluated thus neglecting AMR, (ii) only severe rejections with significant allograft injury are expected to increase troponin plasma level, and (iii) no threshold has been defined (importance of time post-transplant and baseline troponin level for each patient).Healthy individuals have a small amount of cfDNA corresponding due to physiological cell death. The majority of this cfDNA is released from hematopoietic cells, with <1% release from the heart. The cfDNA is increasingly used in the medical field in oncology and pre-natal diagnosis.The dd-cfDNA, detected in the blood of transplant recipients, has been proposed as a non-invasive marker of graft injury ,30. EarlThe future developments of dd-cfDNA in heart transplantation include: (i) An ongoing multicenter randomized clinical trial , and (ii) in-depth analysis of dd-cfDNA length and the absolute number of dd-cfDNA in opposition to the relative proportion currently considered.Allomap is a non-invasive test based on gene-expression profiling (GEP) of peripheral blood mononuclear cells, that has been developed for the diagnosis of ACR in HTx. Transcripts of interest reflect diverse immunoregulatory pathways from a variety of immune and non-immune cells. After broad microarray analyses and among 252 pre-selected candidate genes, 11 were finally significantly associated with ACR . A score ranging from 0 to 40 was developed and validated. In two validation cohorts, GEP appeared to detect accurately ACR \u2265 2R on the concomitant EMB. Patients > 1 year post-transplant with scores below 30 were unlikely to have grade \u2265 2R rejection  . In the This test received approval from the FDA in 2008 and the CE marking in 2011. International guidelines support the use of GEP as a non-invasive biomarker to monitor rejection. However, the use of this screening test as an alternative to the EMB in asymptomatic patients suffers from several limitations: (i) AlloMap comprises a limited number of genes expressed by a limited number of cells compared with various pathways and cell types implicated in the pathophysiology of rejection, (ii) GEP has only been evaluated to rule out severe ACR, excluding mild episodes of ACR and AMR, (iii) its high negative predictive value to rule out ACR must be interpreted in the context of a low and declining incidence of significant ACR in HTx, (iv) GEP is not specific of rejection and its positive predictive value is low, (v) the gene signature found in the peripheral blood has never been validated in allograft biopsies, (vi) its use remains limited in Europe due to costs, limited availability, and the absence of a specific economic evaluation.Small and non-coding RNAs called miRNAs have been shown to be involved in gene expression regulation. Although miRNAs are known to be involved in many biological processes, such as development, cell proliferation, differentiation, apoptosis, and oncogenesis, increasing evidence suggests that they may play a critical role in the regulation of immune cell development and in the modulation of innate and adaptive immune responses. Several studies have reported a potential causative role of miRNAs in the pathophysiology of cardiac allograft rejection and distinct miRNA profiles in EMB from patients with or without rejection ,40,41,42In a multicenter retrospective case-control study, among 17 pre-selected candidate miRNAs, four  showed differential tissue and serum expression between rejection and normal heart allografts. There were strong correlations between tissue and serological expression of these four miRNAs. Assessment of these miRNAs in patient sera permitted very high accuracy discrimination between patients with and without allograft rejection. Since then, several observational studies, mostly case-control and single-center studies, have identified various miRNAs as non-invasive biomarkers of rejection, mostly ACR: miR-144-3p , miR-142\u00ae platform , miRNA profiling revealed that human and murine ACR share significant dysregulation of immune genes. Inflammatory miR-155 was the most differentially expressed between ACR and controls in both human and animals. Importantly, the authors of this study demonstrated that absence or pharmacological inhibition of miR-155 attenuated ACR, demonstrating the causal involvement and therapeutic potential of miRNAs.Several studies have reported on the potential interest of miRNA profiling of myocardial tissue. In a study combining human myocardial tissue (case-control) and a murine model of ACR, and performing an unsupervised analysis of miRNA .Based on large, international and deeply phenotyped cohorts, we recently identified five independent predictive variables associated with biopsy-proven AMR during the first year post-transplant and built a clinical risk prediction model for AMR. It showed excellent discrimination in the US test set and in an independent external validation cohort. Simulation analyses suggested that individualizing the EMB protocol according to the predicted probability of AMR may safely reduce the number of EMB performed. A user-friendly web-based interface allowing an open access evaluation of the pre-test probability of AMR on any EMB performed during the first year post-transplant was built  has been used to define the molecular pathways involved in cardiac allograft rejection [Microarray technology  tissue. The main advantages of using FFPE tissue are the ability to perform histopathology and the retrospective access to the blocks of inclusions, which allows longitudinal studies, crucial in heart transplantation (pre-injury diagnosis or response to treatment). Until recently, the main limitation of these techniques had been the relatively small number of targeted genes, which constrained discovery studies and the development of clinically relevant tools. As an example, our group has applied reverse transcription-multiplex ligation-dependent probe amplification (RT-MLPA), a multiplex RT-PCR technique to heart transplantation . We coul\u00ae system allows the specific capture and counting of nucleic acids  [\u00ae allows targeting up to 800 genes organized in the form of a \u201cpanel\u201d of genes. The particularity of the nCounter\u00ae relies on the way it targets mRNA through a fluorescent \u201cbarcode\u201d system. Each barcode is attached to a unique probe specific to a targeted mRNA, which is counted individually and directly without any amplification step. The probes need only 100 nucleotides to recognize their specific target. This sensitivity makes the nCounter\u00ae particularly efficient on samples with degraded nucleic acids and/or in low-quantity samples, such as FFPE tissue. It was recently shown in heart transplantation that a signature of 34 transcripts had a better diagnostic performance than C4d and DSA, and correlated with endothelial activation assessed by electron microscopy [The nCounterd miRNA) . The nCocroscopy .\u00ae technology in solid organ transplantation has been underlined by numerous studies [The relevance of nCounter studies . In the  studies . This ha studies ,68. ThisConfrontation with molecular data could improve the diagnosis of rejection and question the histopathological classifications of rejection. For example, as early as 2005 the Stanford team had shown that biopsies with mild or moderate ACR , which had been included in the 1R category of the 2004 revised ISHT working formulation, significantly differed from a molecular point of view . PangenoA key challenge remains to combine various sources of information in an integrated and more accurate diagnosis. Importantly, the evolution of bioinformatics techniques has contributed to the advancement in searching and predicting biomarkers, pathways, and new target drugs that allow a more precise and less invasive diagnosis .In Once the EMB is performed, on top of standard pathological evaluation, a molecular evaluation may produce valuable information, particularly when there is an uncertain diagnosis, discrepancies between the clinical presentation and pathology , or de novo DSA without pathologic features of AMR. The molecular diagnosis is seen as a companion tool and should not replace pathological evaluation. Finally, integrating pathology with molecular data and non-invasive biomarkers of graft injury may allow the analysis of the molecular activity of rejection and ongoing subclinical allograft injury. This information may be of high interest when discussing the treatment of subclinical rejection. The interest in an integrated bio-histomolecular diagnosis of rejection should be evaluated in dedicated prospective studies, both concerning the patient\u2019s prognosis and the treatment of allograft rejection.Major advances in the field of non-invasive and tissular biomarkers of cardiac allograft rejection have paved the way for a paradigm shift in the monitoring and diagnosis of cardiac allograft rejection. An integrated bio-histomolecular diagnosis of allograft rejection combining non-invasive biomarkers of allograft injury, tissue molecular activity, and pathology may have a major impact on the management of heart transplant recipients."}
{"text": "Objective: The virtual reality (VR)-based path integration task shows substantial promise in predicting dementia risk. However, the reliability and validity in healthy populations need further exploration. The present study investigates the relationship between task indicators and brain structures in a healthy population using a VR-based navigation task, particularly the entorhinal cortex (EC) and hippocampus. Methods: Sixty healthy adults were randomly recruited to perform a VR-based path integration task, the digit span task (DST), and an MRI scan. The indicators of the VR-based path integration task were calculated, including the absolute distance error (ADE), degree of angle deviation (DAD), degree of path deviation (DPD), and return time (Time). The reliability of the above indicators was then estimated using the split-half method and Cronbach\u2019s alpha. Correlation and regression analyses were then performed to examine the associations between these indicators and age, general cognitive ability (DST), and brain structural measures. Results: ADE, DAD, and DPD showed reasonable split-half reliability estimates  and nice Cronbach\u2019s alpha estimates . All indicators correlated with age and DST. ADE and DAD were sensitive predictors of hippocampal volume, and return time was a predictor of EC thickness. Conclusion: Our findings demonstrate that the VR-based path integration task exhibits good reliability and validity in the healthy population. The task indicators are age-sensitive, can capture working memory capacity, and are closely related to the integrity of individual EC and hippocampal structures. Population aging is becoming an increasingly severe global issue. According to the United Nations Population Division, by 2050, the proportion of the elderly in the total population will reach 38% . Mild coHippocampal and entorhinal cortex (EC) dysfunction is a potential biological mechanism of cognitive aging ,5. PreviSpatial navigation function, such as PI, is hard to test using the typical paper-and-pencil neuropsychological test. Innovations in the virtual reality (VR) technique offer a promising opportunity. VR enables the creation of an ecological and immersive spatial environment for testing path integration . Most ofA recent study by Howett and colleagues has made impressive progress . They deThe present study thus investigates the reliability and validity of the VR-based triangle completion task using a healthy community sample. Split-half reliability is a convenient method to estimate the reliability when test\u2013retest information was not available or a stubborn practice effect existed. We estimate the split-half reliability and Cronbach\u2019s alpha for each behavior measure of PI. High-resolution MRI images are obtained for each subject, and brain structure measures, such as volumes and cortex thickness, are calculated. Our results support the reliability and validity of the VR-based path integration task. We recruited nearby community participants by distributing leaflets and posting posters from 2020 to 2022. Sixty-eight participants were recruited. After excluding those who did not complete all tests and had poor MR image quality, 60 people were enrolled (aged 24\u201384 years). All participants had no history of cancer or dementia, were not taking any medications that could affect cognitive abilities, and had no existing psychiatric or neurological diseases. All participants completed the demographic survey, the VR-based path integration task, the digital span task (DST), and the whole-brain MR scans. The behavior test and MRI scanning interval is no more than one week. Participants were informed of the purpose of this study and the privacy policy before data collection. The study was approved by the Ethics Committee of Hefei Cancer Hospital, Chinese Academy of Sciences. 2. When they exceeded the safe range, a red boundary warning would appear in their sight, reminding them to stop moving forward. The task was performed in an open virtual environment, and the boundary clues were projected to infinity. The natural and virtual worlds adopt a 1:1 motion ratio to eliminate vestibular mismatches and reduce nausea and other tolerance issues. The task was written in C# language using the Unity 3D engine  and compiled into an APK file running on the wireless VR headset.All participants wore a wireless VR headset  and walked in a square of 3.5 mParticipants were asked to walk an \u2018L\u2019-shaped outward path to three different locations, numbered as 1 (starting point), 2, and 3 (endpoint), flushing on the head. The number of land markers appeared sequentially, and participants walked following the guide of land markers. When reaching the third land marker, a voice will prompt participants to return to the starting location, and all the landmarks disappear. When participants believed they had reached the location, they pressed the button on the handheld controller to end the trial. Before the formal test, there are three practice trials to ensure that participants adapted to the VR environment. We set up three different scenarios in the formal test, each with unique surface details, boundary cues, and lighting. Specifically, one scene has both mountains and lawn surface features, and the other has only mountains or lawn surface features. Each scenario has nine trials. The environmental cues randomly change in each trial. Four PI indicators, absolute distance error (ADE), degree of path deviation (DPD), degree of angle deviation (DAD), and return time (Time), are calculated to evaluate the performance of participants in the spatial navigation task. ADE is defined as the Euclidean distance between the estimated and actual starting positions. DPD reflects the route distance length deviation, and DAD indicates the angle deviation between the estimated and actual positions. The measure of Time is the time taken from the third marker to the estimated position. DST is a popular neuropsychological tool to evaluate subjects\u2019 attention and short-term memory. The test includes two parts: forward rehearsal and backward rehearsal. Forward rehearsal contains ten items, and backward rehearsal contains nine items. Each span size includes two number sequences. For each trial, the examiner read each number sequence at the rate of one number per second. The subject then repeat it loudly. Each number sequence counts as one point. The test terminates when subjects fail on two subsequent trials of a particular span-size condition. The forward score is the correct forward sequence number, and the backward score is the number of correct backward sequences. The total score equals the sum of the forward score and backward score.All participants underwent whole-brain MR structural image scans during the experiment using a 3.0 T Philips Achieve system and an 8-channel sense head coil. The 3D-T1-weighted images were obtained in the fast acquisition gradient echo sequence prepared by magnetization. Scanning parameters include voxel size = 1.0 \u00d7 1.0 \u00d7 1.0 mm3, TR = 7.0 ms, TE = 3.2 ms, matrix size = 256 \u00d7 256 pixels, slice thickness = 1.0 mm, scanning time = 4 min and 59 s.The whole brain segmentation was performed using FreeSurfer 6.0. The cortex parcellations followed the Desikan-Killiany atlas and subcortical segmentation followed the automatic subcortical atlas in FreeSurfer. We used the Desikan-Killiany atlas because it was the default cortex atlas of FreeSurfer, widely used in clinical studies. Two senior radiologists reviewed the images and manually corrected the offsets. Based on previous studies, we selected the EC and hippocampus as regions of interest (ROIs) ,20. All p < 0.05 was considered significant  to calculate Cronbach\u2019s Alpha, which is a coefficient representing the consistency of \u201citems\u201d (variables or repetitions) across observations (such as participants). Split-half reliability was calculated using R language and the split-half package  using thnificant . GraphPanificant . To deteadjusted p < 0.05).p < 0.05). DPD and DST show a marginally significant association with DST (p = 0.065). p-values of which were corrected for multiple comparisons. r = \u22120.36, adjusted p = 0.013; r = \u22120.31, adjusted p = 0.038) and the right hippocampus volume . DPD was negatively related to the left hippocampus volume . Time was negatively related to the left EC average thickness . See p = 0.074; ADE and right hippocampus volume: p = 0.057; DAD and right hippocampus volume: p = 0.089; DAD and right hippocampus volume: p = 0.043; DPD and left hippocampus volume: p = 0.348; Time and left EC thickness: p = 0.013).We performed a whole-brain correlation analysis of all brain segmentations, the r = \u22120.32, adjusted p = 0.02; r = \u22120.31, adjusted p = 0.04). DAD was negatively related to the right temporal pole cortex and left supramarginal cortex thickness. DPD was negatively related to the left superior frontal cortex thickness, superior temporal cortex thickness, and insula cortex thickness , and right superior frontal cortex thickness, superior temporal cortex thickness, pars opercularis cortex thickness . Time correlated negatively with the left superior temporal cortex thickness, posterior-cingulate cortex thickness, and superior frontal cortex thickness  and right pars opercularis cortex thickness .In addition to the hippocampus and EC ROIs, other brain regions also correlated with VR pathway integration performances. ADE and DAD were negatively related to the right cerebellum-cortex volume , are alsAnother promising future research direction is to examine the relationship between spatial navigation and the sense of agency (SoA). SoA refers to the feeling of control over one\u2019s actions. Previous studies have demonstrated the role of SoA during continuous body movements . In addiThe present study demonstrates that the primary measures of the VR-based path integration task have reasonable split-half reliability and Cronbach\u2019s alpha estimates. These measures are sensitive to age and correlate with general cognitive ability. Moreover, brain structure analysis validates the association between path integration and hippocampal areas. Our results imply that the VR-based spatial navigation test might be a powerful tool in aging research."}
{"text": "Genome compaction and activity in the nucleus depend on spatiotemporal changes in the organization of chromatins in chromosomes. However, the direct imaging of the chromosome structures in the nuclei has been difficult and challenging. Herein, we directly visualized the structure of chromosomes in frozen-hydrated nuclei of budding yeast in the interphase using X-ray laser diffraction. The reconstructed projection electron density maps revealed inhomogeneous distributions of chromosomes, such as a 300\u00a0nm assembly and fibrous substructures in the elliptic-circular shaped nuclei of approximately 800\u00a0nm. In addition, from the diffraction patterns, we confirmed the absence of regular arrangements of chromosomes and chromatins with 400\u201320\u00a0nm spacing, and demonstrated that chromosomes were composed of self-similarly assembled substructural domains with an average radius of gyration of 58\u00a0nm and smooth surfaces. Based on these analyses, we constructed putative models to discuss the organization of 16 chromosomes, carrying DNA of 4.1\u00a0mm in 800\u00a0nm ellipsoid of the nucleus at the interphase. We anticipate the structural parameters on the fractal property of chromosomes and the experimental images to be a starting point for constructing more sophisticated 3D structural models of the nucleus. In the nuclei of eukaryotic cells, individual gene loci are believed to occupy preferential positions with respect to their chromosome territory (CT) and/or other nuclear landmarks such as the nuclear envelopes and nucleoli4. Although the spiral distribution of DNAs stored in a bacteriophage capsid was visualized by transmission electron microscopy (TEM)5, the compaction mechanism of chromatins in the eukaryotic nuclei is probably more complicated than that observed in virus particles. Although computational models proposed regarding\u00a0the spatial organization of chromosomes in the nucleus after genome sequencing6, imaging studies are necessary to elucidate spatiotemporal variations in the organization and three-dimensional (3D) structures of chromosomes and chromatins in the nuclei7.The genetic information recorded in the genomic DNA is implemented in cellular activities through spatiotemporal changes in the organization of a large number of chromatins, assemblies of DNA and proteins, in chromosomes, and the patterns of gene positioning affect the transcription status of the genes8. A TEM study of chemically labeled human chromosomes reported that flexible and structurally heterogeneous chains of 5\u201324\u00a0nm diameter are packed together without any higher-order structures9. Imaging studies using super-resolution light microscopy proposed the presence of nucleosome clutches, which are heterogeneous groups of nucleosomes leading to the formation of chromatin in human nuclei10, a power law between the size and length of chromosomes in Drosophila nuclei11, and compact domains with a diameter of approximately 160\u00a0nm in vertebrate nuclei12. In addition, fluorescence in situ hybridization was used to illustrate the spatial organization of more than two loci13.Various imaging techniques have been applied to understand the structures and dynamics of building blocks in chromatin assemblies at the molecular level. TEM observations of isolated nucleosomes have revealed a variety of higher-order structures depending on the solution conditions, and have led to different ideas on models of nucleosome assemblies15, which provides structural information on the arrangement of chromosomes, such as long-range interactions of chromatins, at each genomic locus16 and at the resolution of nucleosome level19. Studies using the Hi-C techniques have revealed the characteristic features on higher-order folding of chromatins in chromosomes, such as a broad segregation of the genome into compartments20, and topologically associated domains (TADs) of hundreds of kilobases23. Based on the distance geometry information of the loci, the 3D distributions of chromosomes were computationally derived25.An alternative approach to visualize the organization of chromosomes is the high-throughput chromosome conformation capture (Hi-C) technique26, high-voltage cryoTEM tomography27, and focused-ion beam scanning electron microscopy28. In TEM observation, as the contrast of DNA was poor against the other components in nuclei, chromatins were difficult to be identified unambiguously in frozen-hydrated nuclei as pointed out9.Three-dimensional structures of whole cells have been studied using TEM for thin sections29, but fluorescent labels are necessary to visualize specific genomic loci. In addition, only the positions of the labeled molecules are visualized. Regarding the Hi-C technique24, the influence of formamide-crosslinking on the native chromatin organization is still unclear. In electron microscopy observation except high-voltage cryoTEM, specimens are stained by osmium reagent in the presence of acetone28 or sliced after the fixation using epoxy resin26.The techniques described above are useful for studying the structures and dynamics of chromosomes, but require physicochemical modification and/or treatment of the nuclei. Super-resolution microscopy has achieved spatial resolution higher than 100\u00a0nm33. The imaging technique is advantageous to visualize the differences in X-ray absorption of substances of cells. However, as the absorption contrast is poorer than the electron density contrast measurable in X-ray diffraction using short-wavelength X-ray, the visualization of the structural organization of chromosomes in the nuclei regarding X-ray absorption contrast may be still under progress.In contrast, X-ray imaging is a potential technique to visualize the structure of the whole nuclei without sectioning and chemical labeling. Soft X-ray imaging was applied to the structural study of frozen-hydrated eukaryotic cells34, a lens-less imaging technique, helps in the visualization of the shape, size, and internal structure of non-crystalline particles as electron density without the need of sectioning and chemical labeling, owing to the penetration power of X-rays with short wavelengths. XDI has been applied to investigate the structures of frozen-hydrated biological cells with sizes ranging from sub-micrometer to several micrometers using synchrotron X-rays35. The recent implementation of X-ray free electron laser (XFEL), which provides very intense and spatially coherent X-ray pulses at high repetition rates, enables the rapid collection of diffraction patterns from a large number of non-crystalline specimens, such as metal particles37, organelles38, and biological cells39, the sizes of which are smaller than the cross-section of the XFEL pulses. As the electron density contrast of a specimen are reconstructed from the diffraction patterns, XDI structure analysis of the nuclei may be advantageous to visualize the distribution of chromosomes with the highest electron density contrast among the substances in the nuclei.X-ray diffraction imaging (XDI)Saccharomyces cerevisiae at the interphase40 using XFEL-XDI. As the structures of chromosomes in nuclei have been investigated for various biological species of eukaryote, it is interesting whether any structural architectures in S. cerevisiae are common among the species. The budding yeast S. cerevisiae is an attractive model system to study nuclear organization and its functional relevance41. At the interphase, 16 chromosomes with different DNA composition42 45, a single microtubule and the kinetochore complex to a multiprotein complex embedded in the nuclear envelope, and the telomeres are tethered to the nuclear envelope  detectors and automatically processed using a custom-made data processing software suite48. Although a focused XFEL pulse destroys a specimen particle at the atomic level, diffraction occurs from the particle before its destruction49.In XFEL-XDI experiments, diffraction patterns of the nuclei were collected by supplying frozen-hydrated nuclei into the irradiation area of the focused XFEL pulses by scanning the specimen disk Fig.\u00a0\u201d section\u20121 in diffraction space), good visibility of the interference peaks  phase41, which had larger sizes and amounts of DNAs than those of the interphase, were contained in the specimen suspension , the highest resolution achievable by our experimental setup  formed a minor fraction and predominantly appeared in the first experiment only and were slightly inconsistent with the images observed in the other imaging techniques. The elliptic-shaped nuclei may be deformed in blotting procedure in the specimen preparation  and yielded maps of the ARs smaller than 1.4. As the selected 43 diffraction patterns yielded 17 type-\u03b1, 19 type-\u03b2 and 7 type-\u03b3 maps, the shapes of which were circular or semicircular, the averaged profiles excluded structure information from elliptic shaped nuclei, which may be in slight deformation.The electron density fluctuation in the profiles of the projection maps suggested that certain substructures existed in the nuclei at the interphase Fig.\u00a0C. To add\u22121 in diffraction space), indicating that any major and specific arrangements of chromosomes and/or chromatins were poor in the spacing of 400\u201320\u00a0nm. Since each diffraction pattern came from a single nucleus, the averaged profile was substantially different from the diffraction pattern of a pellet of nuclei, in which inter-nuclei interference of X-rays significantly modifies the diffraction patterns from each nucleus.The profile displayed no diffraction maxima in the resolution range of 400\u201320\u00a0nm  was grown at 303\u00a0K in a medium containing 1%(w/v) yeast extract , 2%(w/v) bacterial peptone , and 2%(w/v) D-glucose . When the optical density of the culture medium at 600\u00a0nm reached approximately 0.5, cells were harvested and resuspended in another medium containing 1%(w/v) yeast extract, 2%(w/v) peptone, and 2%(w/v) raffinose 58. After incubation for 16\u00a0h at 303\u00a0K, the harvested cells were suspended in the same medium and incubated further for 1.5\u00a0h at 303\u00a0K.The amount of DNA in the cultivated cells was examined using flow cytometry. Cells from 1\u00a0mL culture were fixed with 70% ethanol. After washing with a buffer containing 0.2\u00a0M Tris HCl , 20\u00a0mM ethylenediaminetetraacetic acid , and 0.1%(w/v) sodium azide  (pH 7.5), the cells were incubated for 2\u00a0h at 310\u00a0K in the presence of 2\u00a0mg/mL RNase . After exchange with phosphate-buffered saline, DNA was labeled with 4',6-diamidino-2-phenylindole (DAPI) . The fluorescence from the labeled cells was measured using CytoFLEX . As a result, we observed that 85% of the cells were in the interphase  sodium azide and 0.5\u00a0mM phenylmethylsulfonyl fluoride (PMSF)  were added to the cell culture. Zymolyase  at a concentration of 10\u00a0mg/mL was used to convert the cells into spheroplasts. After a step-gradient centrifugation of spheroplast lysate, pellets of crude nuclei were suspended in a buffer containing 18%(w/v) Ficoll , 20\u00a0mM PIPES , 5\u00a0mM MgCl2 , and 1\u00a0mM PMSF  (pH 6.5), and again centrifuged at 3000\u2009\u00d7\u2009g for 30\u00a0min to remove unlysed cells, cell wall debris, and entrapped membranes. The average diameter of the isolated nuclei was found to be smaller than 1\u00a0\u03bcm using light microscopy. Although Ficoll was used without monovalent ions in this preparation, little shrinkage of nuclei was observed in reconstructed projection electron density maps  Ficoll may be negligible when viewing at the present spatial resolution.Nuclei were isolated according to a previously reported protocol2 silicon frame with nine 1\u2009\u00d7\u20091-mm2 windows covered by a silicon\u2013nitride (Si3N4) membrane with 100\u00a0nm thickness 46\u00a0. The relative humidity inside the chamber was maintained at\u2009>\u200990% by supplying moist air from a generator HUM-1 . Within a few minutes, the nuclei were adsorbed onto the PLL-decorated silicon nitride membranes. The specimen disk was transferred to a plastic Petri dish, in which the humidity was controlled at 95% with a sponge containing saturated KCl solution.A 30 \u03bcL droplet of suspension of isolated nuclei was placed on the silicon\u2013nitride membranes in a custom-made humidity-controlled chamber2, as assessed by reference observation of fluorescence from nuclei labeled with DAPI. Finally, each specimen disk was flash-cooled using liquid ethane and stored in liquid nitrogen until further use\u00a0. The average number density of nuclei remaining on the membranes was approximately 7/10\u2009\u00d7\u200910 \u03bcmTEM images of the nuclei dispersed on carbon membrane were taken using a JEM-2100 electron microscope  operated at an accelerating voltage of 200\u00a0kV 62.Diffraction patterns were collected using our custom-made diffraction apparatus TAKASAGO-6, the MPCCD-Octal and MPCCD-Dual detectors at the beamline 3 of the X-ray free electron laser facility SACLA42 Fig.\u00a0C. The MP47. The stage was kept at approximately 80\u00a0K during XFEL-XDI experiment. The stage was moved at a maximum speed of 50\u00a0\u03bcm/33\u00a0ms to supply frozen-hydrated nuclei into the focal spot of XFEL pulses. We used the signal from the control system of the SACLA linear accelerator to trigger both the translational motion of the stage and the acquisition of diffraction patterns by the two MPCCD detectors47.Specimen disks stored in a liquid nitrogen bath were transferred to the specimen stage inside the vacuum chamber of the diffraction apparatus without frosting and temperature increaseSITENNO48. After discarding diffraction patterns with respect to the signal-to-noise ratio at a specified resolution, patterns of the two detectors were merged by taking the beam center positions in each detector and the attenuation factor of an aluminum foil placed in front of the MPCCD-Dual detector  and displayed good visibility and centrosymmetry . The maximum resolution of a diffraction pattern was defined as the highest resolution shell, where the signal-to-noise ratios of the speckle peaks were greater than 3. In the two experiments, we obtained 1333 diffraction patterns displayed the maximum resolution beyond 25\u00a0nm (corresponding to a resolution range of 40\u00a0\u03bcm-1 in diffraction space), that was near the highest resolution achieved by the specimen-to-detector distance described above.We selected diffraction patterns, which had speckle peaks with a good signal-to-noise ratio beyond a resolution of 33\u00a0nm . In the first step, we determined the most probable support, i.e. particle shape of each nucleus. PR calculations frequently yield non-realistic maps due to the lack of the very small-angle region by the beamstop and Poisson noise in X-ray detection particularly in the high-angle region. Therefore, the most probable support was extracted from 10 groups of retrieved maps separated by K-means clustering after principal component analysis for 700 independently retrieved projection maps63. In the second step, 500 electron density maps inside the support selected in the first step were independently retrieved from the diffraction pattern trimmed at a resolution of 25\u00a0nm (corresponding to 40\u00a0\u03bcm\u22121 in diffraction space) using the oversampling smoothness (OSS) algorithm66.For each of the selected diffraction patterns, projection electron density maps were reconstructed using two-step phase-retrieval (PR) calculations51 is defined asi-th map. When a pair of maps yields a score of less than 0.2, they are deemed realistic in many cases39.Next, we screened the OSS retrieved 500 maps by referring to the similarity score. The similarity score between a pair of mapsAfter PR calculations for 1,333 diffraction patterns, we extracted maps for illustrating the structures of nuclei through the following two steps. First, we examined whether the frequency distribution of R-factor defined asR-factor of the selected maps was 0.18 (SI Appendix Fig. A set of structure amplitudes calculated from the realistic maps (Supplementary Information."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process. Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process. Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process. Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process. Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process. Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether the authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process.Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}
{"text": "Discrepancies in scopeDiscrepancies in the description of the research reportedDiscrepancies between the availability of data and the research describedInappropriate citationsIncoherent, meaningless and/or irrelevant content included in the articlePeer-review manipulationThis article has been retracted by Hindawi following an investigation undertaken by the publisher . This inThe presence of these indicators undermines our confidence in the integrity of the article's content and we cannot, therefore, vouch for its reliability. Please note that this notice is intended solely to alert readers that the content of this article is unreliable. We have not investigated whether authors were aware of or involved in the systematic manipulation of the publication process. Wiley and Hindawi regrets that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our own Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}