Law and Human Behavior, 36(3), 247–255), to data from a new experiment and to eight recent studies comparing simultaneous and sequential lineups. We found that although responses tended to be more conservative for sequential lineups there was little or no difference in underlying discriminability between the two procedures. We discuss the implications of these results for the diagnostic feature detection theory and other kinds of sequential lineups used in current jurisdictions.Debate regarding the best way to test and measure eyewitness memory has dominated the eyewitness literature for more than 30 years. We argue that resolution of this debate requires the development and application of appropriate measurement models. In this study we developed models of simultaneous and sequential lineup presentations and used these to compare these procedures in terms of underlying discriminability and response bias, thereby testing a key prediction of diagnostic feature detection theory, that underlying discriminability should be greater for simultaneous than for stopping-rule sequential lineups. We fit the models to the corpus of studies originally described by Palmer and Brewer (2012,  Sequential lineups are currently used by police jurisdictions in the USA, Canada and the United Kingdom. Contrary to prior consensus, recent research employing signal detection measures has reported that simultaneous lineups may be superior to sequential lineups. This is consistent with diagnostic feature detection theory (DFDT), which attributes this difference to the greater ability of witnesses presented with a simultaneous lineup to compare different items and to isolate features that are uniquely shared by the perpetrator and the target item. If the sequential lineup is inferior, this has important implications for procedural fairness in those jurisdictions that currently rely on it. In addition, to the degree that this supports theories such as DFDT, these can be used to develop improved lineup procedures that maximize performance. We developed a set of formal models based on signal detection theory and applied them to comparative data drawn from historic and contemporary studies in order to compare underlying memory performance between simultaneous and sequential lineups. Our results revealed little to no simultaneous advantage in underlying discriminability, although the effect may be smaller than our study could detect, and a substantial shift in response bias in that eyewitnesses given sequential lineups require more evidence to identify an item. We show that the reason our results differ from some that have been published previously is due to the way in which eyewitness performance is measured in those studies where they are susceptible to distortion by structural features of the procedures. We also provide supplemental materials for fitting a signal detection model to simultaneous lineup data.A major goal of eyewitness research is to develop procedures that maximize correct identifications and minimize incorrect identifications by eyewitnesses. The sequential lineup has been proposed as one such procedure  or reject the lineup, indicating that no item is a suitable match. In a sequential lineup, as originally proposed by Lindsay and Wells , each lirelative to the other items. Such a strategy would tend to lead to a high false positive rate because there is a basis for identification even when memory of the perpetrator is poor or the target is not a member of the lineup. An absolute judgement is said to occur when an identification judgement does not depend on the similarity of other lineup items to the witness’ memory of the target. Such a strategy would tend to lead to lower false positive rates because witnesses have a basis to reject the lineup when memory of the target is poor or if the target is not present. Lindsay and Wells  (the TID rate) to the proportion of incorrect innocent suspect identifications (SIDs) . A TID is made when the witness correctly identifies the target in the lineup. An SID is made when the target is not a member of the lineup and the witness incorrectly identifies the innocent suspect. On this measure of performance, an identification made from a lineup procedure that reliably generates a higher diagnosticity ratio is to be preferred to one that does not.An alternative performance measure is based on signal detection theory  and develop a comparable model of the simple stopping rule version of the sequential lineup task (SDT-SEQ). We then apply these models to extant and new data to estimate memory strength across the two procedures.n-item lineup. In a target present (TP) lineup, one item is the target (a picture of the perpetrator) and the remaining items are foils or fillers (pictures of other people). In a target absent (TA) lineup, one item may be designated as the innocent suspect with the remaining items being foils. The participant is required to judge whether the lineup contains the target and, if they believe it does, to identify the corresponding item. We assume that each lineup item is associated with a familiarity value that reflects its similarity to the participant’s memory of the perpetrator. Each familiarity value is considered a random draw from one of several distributions - a target distribution if the item is a target, an innocent suspect distribution if it is an innocent suspectdt and standard deviation st, both of which can be estimated from the data. Because st may not equal one the model is called the unequal variance signal detection model. In addition, because the innocent suspect may be distinct from the remaining foils, the suspect distribution has mean ds and standard deviation ss.The starting point for all the lineup models we consider is the unequal variance signal detection (UVSD) model. The UVSD model accounts well for data in laboratory-based recognition memory tests  be the maximum familiarity value associated with item m. The decision rule is this: if xm < c1 then reject the lineup, otherwise choose lineup item m with confidence level l where cl is the largest element of the set, {ci ∈ C : xm ≥ ci}.SDT-MAX, also known as the independent observations model Duncan, , is perhϕ be the normal probability density function and let Φ be the normal cumulative distribution function evaluated at x ∈ ℝ. Recall that the foil distribution takes the form of the standard normal distribution with µ = 0 and σ = 1. In this case, we write ϕ = ϕ(x) and ϕ = ϕ(x). Let c ∈ C be a decision criterion and let PTID(c) be the probability of a correct target identification with confidence greater than or equal to c. ThenPSID(c) be the probability of an incorrect suspect identification with confidence greater than or equal to c. Then, if there is a designated innocent suspect,As detailed in Additional file X) < c1 then reject the lineup, otherwise choose lineup member m with confidence level l where cl is the largest element of the set, {c ∈ C : sum(X) ≥ c} Let t is the position of the target item and μx and σx are the mean and standard deviation, respectively, of the standard normal distribution truncated at the upper limit of x. The equation is not exact because it assumes that the sum of truncated distributions is approximately normal . Similarly, if there is a designated innocent suspect, thenThe equations for the probability of a correct identification and probability of a false identification under the SDT-INT model are summarized below  should be greater for simultaneous lineups. Based on the conclusions reached by Palmer and Brewer (c1 (and possibly other criteria) should be greater for sequential lineups.Predictions were preregistered on the Open Science Framework, available at d Brewer , sequentχ2 value, p value and parameter estimates from each fit. Further detail on the simulation process and expanded results are available in Additional file We have described three models that we propose to fit to data. This is motivated in part by the idea that there are differences between the models that determine how well they fit different kinds of data. This means that if data are simulated from a model, while this model should fit the data well, other models should fit relatively poorly. In order to investigate this question, we conducted a cross fitting and parameter recovery analysis. First, we randomly generated 100 sets of parameter values for a 6-item lineup and then used each of these to generate 100 simulated datasets from each model. To avoid issues with low cell counts, we set the number of TP and TA lineups to 10,000, giving 20,000 simulated observations for each dataset. We then fit each model to its own sets of data and to those generated by the other models, recording the p < .05. It shows that when a model is fit to data generated by any other model, it is highly likely to be rejected. In other words, the models are, in principle, distinct - given sufficient statistical power, if the data are consistent with one model then they should be poorly fit by any of the remaining models.Figure We measured parameter recovery by examining the correlation between generating and recovered parameter values for each model fit. Scatterplots and tables of correlations are available in Additional file r > = .90 for generating versus recovered parameter values. SDT-MAX recovers the generating parameters perfectly when fit to its own data, but both SDT-SEQ and, to a lesser extent, SDT-INT, recover a small number of outliers, affecting the correlation coefficients. These are most likely due to the presence of local minima, which can be avoided by starting parameter search from different initial values. It is evident from the scatterplots in Additional file When fit to their own data, the models generally recover their own parameters well, with dt is poor. This suggests that if SDT-SEQ is a good representation of the sequential lineup task, then fitting SDT-MAX or SDT-INT to sequential lineup data may lead to inaccurate estimates of dt. Recovery of st was poor for all models when fit to data they did not generate, while recovery of the decision criteria  was generally good for all fits, with r > = .80.When SDT-MAX and SDT-INT are fit to data generated by SDT-SEQ, recovery of N = 3871, simultaneous n = 1952, sequential n = 1919) that compared simultaneous and stopping-rule sequential presentation procedures using the “full diagnostic design” inclusion criteria described in Steblay et al. . Accordingly, we specified that st = ss = 1, as was also assumed by Palmer and Brewer.The simultaneous lineup data from each study were fit by both SDT-INT  and SDT-MAX. The corresponding sequential lineup data were fit by SDT-INT  and SDT-SEQ. Each model was fit using the Matlab® fmincon function. Because each study required participants to make a single choose-no choose decision, there are not enough degrees of freedom to fit all of the model parameters, specifically ds, the mean of the suspect distribution, otherwise we stipulated that ds = 0, the same as the mean of the foil distribution. In addition, studies differed in the probability of a target (and suspect if relevant) appearing at different sequential lineup positions. When specified, this information was used in fitting the SDT-SEQ model  = .40, p =.69. We re-ran the analysis, excluding datasets that the models failed to fit, but this did not change the result. This result is consistent with the conclusion reached by Palmer and Brewer  = .08, p = .94, or sequential presentation, t (35.22) = -.26, p = .79.Table ds for studies that employed a designated innocent suspect selected to resemble the perpetrator more closely than foils, where previous analyses assumed that the innocent suspect and the foils were drawn from the same distribution with a mean of zero and standard deviation of one. In the case where ds is greater than zero, DFDT does not predict a strong simultaneous advantage, because the features uniquely shared by the innocent suspect and the perpetrator will cause the innocent suspect to be identified at a higher rate in the simultaneous procedure compared to the sequential procedure. For this reason, we examined whether there was a simultaneous advantage in the subset of studies that did not use an innocent suspect. We found that the mean weighted difference in underlying discriminability between simultaneous and sequential presentation as estimated by SDT-MAX and SDT-SEQ respectively was less for the 8 studies that used an innocent suspect (M = −.23) compared to the 14 that did not (M = .09). However, the Welch two-sample weighted t test indicated that the difference between these means is not significant, t (9.78) = − 1.61, p = .14.Our estimates of mean weighted underlying discriminability shown in Table c. Analysis of mean weighted c values show that these are greater (indicating more conservative responding) for sequential than for simultaneous lineups, Welch two-sample weighted t test, t (35.83) = − 3.88, p < .01. Once again, excluding the datasets that the models failed to fit did not change the result.Visual examination of Fig. The re-analysis of the Palmer and Brewer  corpus oIn addition to a lack of statistical power, two other methodological issues limit the utility of the corpus for investigating differences in underlying discriminability. First, a designated innocent suspect was selected to resemble the perpetrator in some studies, which may attenuate any simultaneous advantage in underlying discriminability. Second, the target was fixed to appear in certain positions in many of the sequential lineup studies. While our modelling approach accounted for fixed target positions, there is some evidence to suggest that underlying discriminability may increase with target position  and target presence (TP versus TA).Participants were 600 Amazon Mechanical Turk (AMT) workers who were compensated US$1.00 for the 5–10-min experiment. There were 11 participants excluded for failing attention-check questions relating to the content of the stimulus video, leaving 589 participants  for the eventual analysis.https://osf.io/p2hck/.This study employed a pool of 16 female lineup members, drawn from the Adelaide Lineup Database. This consists of a video and accompanying head-and-shoulders photographs taken front-on, at 90° side-on, and approximately at 45° for each of 194 persons. Only front-on photos were used in this study. In each video, the actor wears a black shirt T-shirt with a white University of Adelaide logo to remove the identifying potential of coloured clothing in the lineup phase. The scene opens with an actor (each of the 194 persons in turn) seated at a computer with their back to the camera. After a few seconds during which they type on the computer keyboard, the actor picks up a mobile phone placed on the table to their left and turns to face the camera while looking at the phone. The actor then stands and walks towards the camera while looking at the phone, glancing up briefly to the camera as they pass by. Each video is approximately 10–15 s in duration. An example video can be found at n = 76) were compensated US$1.30 to rate 45 pairs of faces on a Likert scale from 0 (most similar) to 10 (least similar). Each participant rated a different subset of the possible face pairs to reduce participant burden and ensure timely collection of the data. The average number of ratings per similarity pair was 5.92, minimum 1, maximum 10. This resulted in a similarity matrix with each cell containing the mean rating of similarity between each pair of faces.In order to minimise the potential for stimulus effects, rather than a single target and set of foils, we used a pool of lineup members that could all act as both targets and foils. The starting point for selecting the pool members was similarity ratings previously collected for front-on photographs of 90 female faces in the Adelaide Lineup Database. AMT workers  estimated by SDT-INT are spread wider than those estimated by both SDT-MAX and SDT-SEQ. This is because they are scaled according to the detection decision variable for SDT-INT, the sum of the familiarity of all lineup items. Consequently, the decision criteria estimated by SDT-INT are not directly comparable to those estimated by SDT-SEQ (or SDT-MAX). In contrast, the decision variables of SDT-SEQ and SDT-MAX are both based on “untransformed” signal strengths and are therefore directly comparable.These difficulties in comparing the parameter estimates of SDT-INT to SDT-SEQ mean that SDT-INT is not well suited to testing our hypothesis. As a result, we employ SDT-MAX as the simultaneous lineup model and SDT-SEQ as the sequential lineup model in all subsequent analyses.Table dt and st did not differ significantly between the simultaneous and sequential conditions.Table g for the difference between simultaneous and sequential underlying dt is small, g = .06.The lack of a significant difference in underlying discriminability between simultaneous and sequential lineups is consistent with our previous re-analysis of the Palmer-Brewer database. It suggests that this result is not easily attributable to non-random target position in sequential lineups or the use of a designated innocent suspect selected to resemble the target to a greater extent than the foils. We also attempted to address the lack of statistical power in many of the studies in the Palmer-Brewer database. Despite increasing the number of participants compared, we did not observe a statistically significant difference in underlying discriminability. This suggests that if there is a simultaneous advantage, it is small and therefore difficult to detect. The effect size as measured by Hedge’s ensemble model based on the idea of comparing diagnostic features.2(8) = 6.96, p = 0.54. However, we again found no statistically significant difference between its estimate of dt and the estimate from the SDT-SEQ model, χ2(1) = 0.29, p = 0.59.Additionally, our conclusion rests on the assumption that the SDT-MAX model is an appropriate model of the simultaneous lineup data. Recently, Wixted et al.  proposedc2, …, c5) are comparable between simultaneous and sequential lineups for each criterion except c1, which separates lineup identification and rejection decisions (the choose/no choose threshold). Table c1 was significantly larger in the sequential condition, supporting our hypothesis and conforming to previous literature  did not differ between simultaneous and sequential presentation. The stvalues displayed in Table st = ss = 1 did not significantly worsen the fit for SDT-MAX, χ2(1) = .28, p = .60, or SDT-SEQ, χ2(1) = .61, p = .43. This indicates that equal-variance models adequately capture these data, in contrast to long-standing findings of unequal target and lure distribution variance reported in the literature on basic recognition memory  model of the yes/no task to estimate show up dt = Φ− 1(H) – Φ− 1(F) and c = Φ− 1(1 – F), where H is the Target ID rate, F is the TA Foil ID rate and Φ− 1 is the inverse normal cumulative distribution function. Because there are no degrees of freedom, this model necessarily fits perfectly. The estimated parameter values were, dt = 1.58 and c = 1.49. We fit the SDT-MAX to the simultaneous data with the constraint that st = 1. It fit these data well, χ2(1) = 2.54, p = .11, with estimated parameter values, dt = 1.98 and c = 1.18. Although underlying discriminability appeared to be greater for the simultaneous lineup, this difference was not significant, χ2(1) = 1.87, p = .17. Responding was significantly more conservative for sequential position 1, χ2(1) = 5.79, p < .05, consistent with previous findings at the aggregate level.We fit the EVSD model to the show-up data. In this case, it has an analytic solution given by, 1 was relatively small (N = 28) which means that the analysis may not have sufficient statistical power. Nevertheless, it is possible to conclude that if there is an advantage for simultaneous presentation it is likely to be a relatively small effect.Despite previous studies that have reported a simultaneous advantage in underlying discriminability over show ups  provides more power to detect a simultaneous advantage in underlying discriminability than the Palmer and Brewer corpus .We failed to find an underlying discriminability advantage for the simultaneous lineup compared to the sequential lineup in a corpus of studies published prior to Steblay and Phillips  and in odt, c and, where relevant, ds for each study by fitting SDT-MAX to the simultaneous data and SDT-SEQ to the sequential data. We then calculated mean discriminability (dt – ds) and response bias (c) weighted by sample size for simultaneous and sequential presentation. For most of the studies, we estimated parameters separately for each experimental condition, rather than collapsing over conditions other than presentation format. This led to thirteen simultaneous versus sequential datasets from the eight studies. For Carlson and Carlson  for simultaneous and sequential presentation, weighted by sample size. The Welch two-sample weighted t test indicated no significant difference in mean weighted discriminability, t (21.43) = 1.14, p = .27 or mean weighted response bias, t (20.72) = 0.08, p = .94, between presentation formats. As for the Palmer and Brewer corpus and our experiment, this does not support the hypothesis that underlying discriminability is greater for simultaneous presentation. Unlike our previous analyses, the hypothesis that responses are more conservative in the sequential procedure was not supported.Model-fit statistics and parameter values for each dataset are available in Table The present study sought to compare performance between the simultaneous lineup and sequential stopping-rule lineup in order to test the central prediction of the diagnostic feature detection hypothesis; that underlying discriminability is greater when lineups are administered simultaneously rather than sequentially . Second, we found a shift to a more conservative response bias in sequential lineups. As the studies in the database did not collect or report post-decision confidence estimates, we were unable to estimate all the parameters specified in our models, leaving more nuanced aspects of the simultaneous versus sequential presentation question unexplored. Most studies also had relatively small numbers of participants and so lacked statistical power to detect a small effect, they selected designated innocent suspects designed to resemble the target and they did not randomise the position of the target in sequential lineups. For this reason, we conducted a more powerful experiment that elicited multiple confidence judgements, did not employ a designated innocent suspect and randomised the position of the target on sequential lineups. We found no significant difference in underlying discriminability and more conservative responding for the sequential lineup, consistent with the Palmer and Brewer re-analysis. Finally, we analysed a corpus of data containing eight recent lineup studies that compared simultaneous and sequential presentation. The results were consistent with the previous findings in that there was no significant difference in underlying discriminability, but we did not find more conservative response bias for sequential presentation.We first fit SDT-MAX, SDT-INT and SDT-SEQ to the Palmer and Brewer  databaseOur analyses provide estimates of the difference in underlying discriminability between simultaneous and sequential lineups across a total of 36 separate studies or conditions within studies. While many features of these studies  vary considerably, each provides a point estimate of the difference in underlying discriminability. These estimates are plotted in Fig. Our results are not consistent with a key prediction of diagnostic feature detection theory (DFDT), that the greater opportunity to compare lineup items in the simultaneous lineup should improve underlying discriminability compared to the sequential lineup. However, the lack of an easily detected difference in underlying discriminability between simultaneous and sequential lineups does not necessarily militate against the processes proposed by the DFDT. All things being equal, it is possible that the greater detectability of diagnostic features in simultaneous lineups may lead to a performance advantage. However, this is a critical caveat - there may be other differences between the procedures that serve to counteract this effect. One obvious difference is the size of the choice set. In a simultaneous lineup, the target (if present) is one of several alternatives while in a sequential lineup, on each trial only a single item is presented. It is well-known that the probability of correct target detection declines with the increasing size of the choice set Swets, . On the simultaneous show up in which the suspect was presented along with five fillers, none of which could be identified as the target. Based on ROC analysis, they found that the opportunity to compare the suspect to other similar faces in the simultaneous show-up procedure improved empirical discriminability. Because the structural characteristics of the standard and simultaneous lineups are essentially the same - both require a decision to be made about a single item - the difference in empirical discriminability suggests a corresponding difference in underlying discriminability. If so, then the results reveal the kind of advantage predicted by the DFDT.The foregoing analysis suggests that if relevant differences between simultaneous and sequential lineups could be reduced then the effects of diagnostic feature detection may be revealed. A recent study by Colloff and Wixted  bears onIn a series of studies, Seale-Carlisle and colleagues have investigated the empirical and underlying discriminability of the UK ) lineup procedure (Seale-Carlisle et al., Seale-Carlisle and Mickes  found thThe results of Seale-Carlisle et al.  are, to Consistent with previous studies (Carlson et al., This study introduced a new model of the sequential lineup task, SDT-SEQ, and in conjunction with models of the simultaneous lineup task, SDT-MAX and SDT-INT, tested a key prediction of the diagnostic feature detection theory that underlying discriminability should be greater in a simultaneous lineup. In both our re-analysis of the Palmer and Brewer  databaseAdditional file 1. Model Equations.Additional file 2. Model Simulations and Cross Fits.Additional file 3: Table S1.dt, ds, c and C for each dataset in the Palmer and Brewer (Table S2. Fit statistics and dt, ds and c for each dataset in the post-2011 corpus, estimated by SDT-MAX and SDT-SEQ for simultaneous and sequential presentation respectively.d Brewer  corpus, Additional file 4: Supplement 1. Fitting SDT-MAX to Simultaneous Lineup Data.


This indicates that hCSF possesses properties that are not matched by the aCSF, which is generally used for most electrophysiological recordings. To evaluate the possible significance of using hCSF as an electrophysiological recording medium, also for human brain tissue, we compared the network and single-cell firing properties of human brain slice cultures during perfusion with hCSF and aCSF. For measuring the overall activity from a majority of neurons within neocortical and hippocampal human slices, we used a microelectrode array (MEA) recording technique with 252 electrodes covering an area of 3.2 × 3.2 mm2. A second CMOS-based MEA with 4225 sensors on a 2 × 2 mm2 area was used for detailed mapping of action potential waveforms and cell identification. We found that hCSF increased the number of active electrodes and neurons and the firing rate of the neurons in the slices and induced an increase in the numbers of single channel and population bursts. Interestingly, not only an increase in the overall activity in the slices was observed, but a reconfiguration of the network could also be detected with specific activation and inactivation of subpopulations of neuronal ensembles. In conclusion, hCSF is an important component to consider for future human brain slice studies, especially for experiments designed to mimic parts of physiology and disease observed in vivo.Human cerebrospinal fluid (hCSF) has proven advantageous over conventional medium for culturing both rodent and human brain tissue. In addition, increased activity and synchrony, closer to the dynamic states exclusively recorded  The activation of G-protein coupled receptors are postulated to be the reason for the enhanced activity. In addition, clear advantages utilizing hCSF as the culturing medium for human organotypic brain slices have previously been shown by our group . CSF can be found in the space between the brain surface and the skull, the subarachnoid space, in all the ventricles as well as in and around the spinal cord. CSF is a fluid characterized by a low percentage of protein and high percentage of salts and is in constant contact with the interstitial fluid of the parenchyma, the fluid located between neurons and glial cells all over the brain. The close connection with the interstitial fluid gives the CSF a potential to act as a vessel for different neuromodulatory signals , 2010 anur group , 2019. Tur group . Using ro system .Ex vivo measurements of single cells and small networks open interesting avenues for research, but the translation of the results back to the in vivo state remains challenging. It is therefore of great importance to design ex vivo experiments mimicking the in vivo conditions as closely as possible. Usage of hCSF, as the recording solution, might be therefore an interesting alternative option for specific experimental questions.As stated above, changes in the activity of neuronal networks were described for rodent tissue and cells, but if hCSF affects the function of human neurons and modulates the level of activity is still an open question. The aim of our present study is to investigate whether perfusion with hCSF could modulate the neuronal activity differently, on the single cell and network level, compared to perfusion with the commonly used aCSF. We hypothesized that, just as was seen in the rodent studies, the firing activity of neurons will increase with perfusion of hCSF compared to aCSF. We performed extracellular recordings using high-density microelectrode arrays (MEAs) to study both the response from individual neurons and how their activity contributes to the overall network activity. en bloc to ensure tissue integrity, directly transferred into ice-cold aCSF  equilibrated with carbogen , and immediately transported to the laboratory. Tissue was kept submerged in cool and carbogenated aCSF at all times. For hippocampal slices, the tissue was trimmed to give a flat glue surface in the coronal plane, glued onto the slicing platform, and then sliced into 250-μm-thick slices using a Microm HM 650 V vibratome (Thermo Fisher Scientific Inc.). Cortical slices were prepared as follows. After removal of the pia, tissue chunks were trimmed perpendicular to the cortical surface and 250–350-μm-thick acute slices were prepared using a Microm HM 650 V vibratome (Thermo Fisher Scientific Inc.). Subsequently, the slices were transferred onto culture membranes  and kept in six-well culture dishes (BD Biosciences) . We received and pooled hCSF of several patients who needed to undergo a tap test either by lumbar puncture or lumbar drain as part of the diagnostic workup for NPH. It is well established and known from daily clinical practice that hCSF of NPH patients exhibits physiological hCSF parameters  from the ethics committee of the University of Tübingen together with written informed consent from all patients allowed spare tissue from resective surgery to be included in our study. Hippocampus and cortex were carefully micro-dissected and independently resected ciences) . HCSF waciences) , 2019. T2  118 NaCl, 3 KCl, 1.5 CaCl2, 1 MgCl2, 25 NaHCO3, 1 NaH2PO4, 30 D-glucose, and equilibrated with carbogen  and heated to 34°C for an acclimation period of 30 min. After acclimation, aCSF was perfused for an additional hour (we name this period aCSF) followed by 1 h of hCSF perfusion (we name this period hCSF) and then a final hour with aCSF (we name this period washout)  . To avoiwashout) . In four2∗6H2O, 10 EGTA, 2 MgCl2∗6H2O, 4 Na2ATP, and 10 HEPES, pH 7.2, 300 mOsm. The intracellular whole-cell patch-clamp pipette solution contained biocytin (5 mg/mL) to allow for post hoc identification of the location and morphology of recorded neurons. Whole-cell current-clamp recordings were obtained from cortical neurons using either the visual-patch or blind patch technique and sampled at 20 or 100 kHz with a low-pass filter of 5 or 30 kHz. Recordings were performed with unpolished patch electrodes manufactured from borosilicate glass pipettes with filament (Science products). Patch-clamp experiments were performed with a patch-clamp amplifier (Multiclamp 200B) or an NPI Bridge Amplifier (Model BA-01X), a digitizing interface (Digidata 1440A or 1550A Digidata), and pClamp 10 software (Molecular Devices). The junction potential was calculated and subtracted offline to correct the membrane potential in current clamp mode.Slices were positioned in a submerged-type recording chamber , continuously superfused with aCSF and visualized with a BX61WI Microscope (Olympus) or a Leica stereomicroscope. Recordings were performed using recording electrodes with a resistance of 3–5 MΩ and filled with an intracellular whole-cell patch-clamp pipette solution containing the following components (in mM): 140 K-gluconic acid, 1 CaClThe analysis of recordings from 256-MEAs was performed using Python . Recordings were bandpass-filtered  and spikes were detected as described  using a Single electrode bursts were defined as having at least three consecutive spikes with a maximum of 100-ms inter-spike interval. This definition was adapted from a previous report  and the The population bursts were defined as described previously . In briehCSF - parameter aCSF)/(parameter hCSF + parameter aCSF). This ratio is 1 if cells are active only in hCSF and -1 if cells are activity only in aCSF.In addition to the absolute parameter values , we calculated the relative change when switching from recording solution A to recording solution B. The relative change was calculated as the  and differences between groups were considered significant when aCSF, 95.42 ± 15.58 during hCSF, and 59.08 ± 20.69 during washout, which was significantly different between the groups (p = 0.0062). In Dunn’s multiple comparison test, we found that the increase in number of active electrodes between aCSF and hCSF (p = 0.0239) and the decrease in number of active electrodes between hCSF and washout (p = 0.0128) were both significantly different, while the number of active electrodes was not significantly different between aCSF and washout (p > 0.9999), altogether demonstrating reversibility of hCSF-mediated activity increase . Dunn’s multiple comparison test found the increase in firing rate when comparing aCSF and hCSF to be significant (p = 0.0016) as well as the decrease between the hCSF and the washout (p = 0.0239) while the spike rate remained stable between aCSF and washout (p > 0.9999)  . In addip-values). This discrepancy is explained by data obtained from one cortical slice, which was clearly going against the trend, together with the relatively low number of slices (n = 6). This single slice showed a decrease of number of active electrodes and of spike rate when washing in hCSF  was detected. These data indicate that individual neurons or subnetworks in the slices respond differently when hCSF is washed in, with some neurons decreasing action potential firing (decreased activity detected via 9.3 ± 1.86 electrodes), but with the majority of neurons responding with increased action potential firing (increased activity detected via 58.7 ± 8.88 electrodes per slice)  decreased firing rate, (ii) increased firing rate, and (iii) no change in firing rate. Via an average of 9.3 ± 1.86 electrodes, a decreased firing rate in each slice was detected and through 5.2 ± 1.35 of the electrodes per slice, no spikes during r slice) .n = 13), no change in the waveform could be observed between aCSF and hCSF , we performed a detailed analysis of the specific activation based on identified cellular activity using CMOS-MEAs. Recordings were performed under similar experimental conditions as with the 256-MEAs . These rand hCSF . Howeverand hCSF . The aveand hCSF . We idenand hCSF . In summn = 6, ∗p < 0.05), which was reversible by a washout  of cells by hCSF, we performed whole-cell patch-clamp experiments in current clamp in a small sample of human cortical neurons n = 6, . In five washout .aCSF was 11,637 ± 7616  and that it was 16,597 ± 9561 during hCSF, and 12,258 ± 7839 during the washout. These values were significantly different between the groups (p = 0.0052). In Dunn’s multiple comparison test, the number of bursts recorded in aCSF was significantly lower compared to hCSF (p = 0.0322). The decrease between hCSF and the washout was also statistically significant (p = 0.0092), while the number of bursts was not significantly different between aCSF and washout (p > 0.9999)   on individual active MEA electrodes. We found that the average number of bursts in the slices during  0.9999) . The dur 0.5580) .aCSF, 190 ± 76.3 during hCSF, and 33 ± 23.1 during washout, which were significantly different (p = 0.0148). Using Dunn’s multiple comparison test, we found that the decrease in the number of population bursts comparing hCSF and washout was significantly different (p = 0.0201), but the number of population bursts was not significantly different between aCSF and hCSF (p = 0.3765) as well as aCSF and washout (p = 0.7158) , which decreased by 71 ± 16% (p = 0.002) when switching back from hCSF to aCSF.To evaluate the effect hCSF has on synchronous network activity, we continued by detecting the number of population bursts  and the number of electrodes participating in the detected population bursts. Population bursts were detected in nine  of the 12 slices, with the number of population bursts being 45 ± 29.8 during  0.7158) . In addiaCSF, 84 ± 18.6 during hCSF, and 32 ± 18.1 during washout. When testing the three conditions with the paired, non-parametric Friedman test, a significant difference was found (p < 0.0001), and Dunn’s multiple comparison test found the increase in number of active electrodes during population bursts seen when comparing aCSF and hCSF significant (p = 0.0286) as well as the decrease between the hCSF and the washout (p = 0.0005), but the aCSF compared to the washout (p = 0.7158) was found non-significant  in high potassium. The significant change, however, was due to one recording  could mimic the activity recorded in hCSF. The number of active electrodes and the number of burst identified on single electrodes did not change in the two conditions. The average spike rate showed a relative increase of 31%  and robust . In a first step, we averaged these parameters for each slice recorded, and on average, the wash in of hCSF induced a significant increase in the number of active channels, spike rate, and number of busts followed by a fast decrease back to baseline when hCSF was washed out and replaced by aCSF. As a previous study demonstrated in rodent tissue, the general increase in activity was not explained by increased concentrations of potassium or other ions in the hCSF compared to aCSF . SimilarAs mentioned above, the increase of the activity in the presence of hCSF was most likely not induced by an alteration of the ionic composition. However, hCSF is in addition composed of a broad spectrum of neuromodulatory substances such as amino acids, peptides, and lipids . All of in vivo have been characterized by narrower waveforms, higher spike rate, and distinct bursting behavior compared to principal neurons (n = 49) identified only in hCSF had indistinguishable waveforms from the average. Thus, we cannot conclude any cell-type-specific activation by hCSF. Future work will reveal if in brain slices electrophysiological parameters are able to separate neuronal populations, which seems impossible in cell cultures . The patients/participants provided their written informed consent to participate in this study.JW and AC performed microelectrode array recordings. NS, BU, NL, JH, TW, HK, and JW did tissue preparation and culturing. BU, NL, and HK did patch-clamp recording. JW, AC, and GZ did data analysis and writing. All authors contributed to proofreading and study design.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The purpose of this study was to investigate the association between drinking habits and subjective symptoms of the oral cavity based on Japanese national statistical data.  The subjects were 8,698 respondents of the Japan National Livelihood Survey 2013, in their 30s to 60s. The association between drinking habits and each survey item was investigated by contingency table analysis and binary logistic regression analysis. p < 0.01), chewing difficulty (p < 0.05), subjective impression of health (p < 0.01), smoking habit (p < 0.01), and income level (p < 0.01). Analysis indicated an association between drinking habits and hospital visits (p < 0.01) and smoking habits (p < 0.01) in women. When comparing the response rates of symptoms and presence of disease stratified according to drinking habits, inflammatory and sensory system symptoms were common in those who had drinking habits, as were diabetes and gout. Binomial logistic regression analysis with drinking habit as the objective variable indicated statistically significant odds ratios for smoking habit , difficulty in chewing , and working hours . This study identified a correlation between drinking habits and masticatory disorders, suggesting that the effects of drinking as a risk factor for dental diseases should be examined in greater detail in the future. The proportion of people with drinking habits was 55.3% among men and 20.3% among women, and the proportion of men with drinking habits above the age of 40 years was high. Contingency table analysis indicated an association between drinking habits and the following items in men: subjective symptoms ( The purpose of this study was to investigate the association between drinking habits and subjective symptoms related to the oral cavity based on Japanese national statistical data. It is internationally recognized that habitual consumption of alcoholic beverages increases the risk of developing heart disease, cerebrovascular disease, malignant neoplasms, and liver disease . In receIn Japan, although there have been many studies on the effects of alcohol consumption , few stuIn this study, in order to comprehensively examine the effects of drinking habits on symptoms related to the oral cavity, an exploratory analysis was conducted using the Japanese national statistical database.An anonymized datasheet of the 2013 National Livelihood Survey Questionnaire  was obtained from the Ministry of Health, Labour and Welfare through routine procedures. 8698 survey respondents  aged 30 to 60 years were included in the current study. The reason for limiting the subjects to those over 30 years old is this period being when the loss holder rate increases according to a dental disease fact-finding survey  conducteIn order to take advantage of the large scale of the data of national surveys and to explore the characteristics of factors related to drinking habits, this study was analyzed in two stages, as shown in As the first stage, contingency table analysis was conducted according to sex groups with and without drinking habits. Drinking habits were defined based on the frequency of drinking, using a cutoff frequency of 3-4 times or more per week, and the amount of drinking. Regardless of the frequency of drinking, those who consumed more than the high-risk amount of liquor  specified in the Japanese National Nutrition Survey  were conThe items evaluated in the analysis were history of illness (disease name from ICD-10), lifestyle consciousness, subjective symptoms , awareness of worries/stress, and working hours, among other items, as shown in n = 1740). A path model was constructed to explore the relationships between the final model variables.As the second stage, binomial logistic regression analysis was performed by the stepwise method, with drinking habit as the objective variable, other variables as the explanatory variables, and those with valid responses to all analysis items as the analysis targets  and Excel Statistics 2012  were used for the aggregate analysis. To test for statistical significance, the The data included in this study were obtained from the database of a national survey conducted in accordance with the survey ethical rules of the Ministry of Health, Labour and Welfare, and the anonymization process was also conducted by the Ministry of Health, Labour and Welfare. This study was conducted with the permission of ministries in accordance with the provisions of Article 36 of the Japanese Statistics Act.This research was conducted with the approval of the Research Ethics Committee of the Nippon Dental University, School of Life Dentistry at Niigata (license no. ECNG-R-398).p < 0.01), dental symptoms (p < 0.05), subjective health (p < 0.01), smoking habits (p < 0.01), and household income (p < 0.01). In women, presence or absence of hospital visits (p < 0.01) and smoking habits (p < 0.01) were related to drinking habits.We examined whether there were differences in symptoms and diseases according to the presence or absence of drinking habits, based on their response rates (see Tables p < 0.01) in the frequency of both symptoms and diseases in heavy drinkers versus occasional/nondrinkers ranking trends.Comparison of the average rank difference in response rates of each symptom and disease showed a significant difference , difficulty in mastication , and weekly work hours  were significantly higher than the adjustment variable (gender) see .The model that only included significant path coefficients is shown in It has been pointed out that Japanese drinking habits tend to be different from those in Europe and the US , making Cooper et al.  proposedIn this study, there were many people who had a feeling of well-being as well as people who had an adequate income or higher and still had a drinking habit. This suggests that most people consume alcohol for social purposes or as a mood elevator.On the other hand, there are some items that are associated with drinking behavior for negative reasons. There are similarities between smoking and “difficult to bite” symptoms, as they explain compensatory reasons. Copeland and Brandon  reportedMany previous studies on drinking habits pointed out the relationship between stress and drinking –16 althoAccording to the WHO Global status report on alcohol and health, although there are regional differences, there is a trend towards increased alcohol consumption, which will lead to an increase in the incidence of health issues, such as liver dysfunction . MantheyRegarding the relationship between dental issues and drinking, according to the WHO's 2007 evaluation , alcoholSankaranarayanan et al.  pointed Given the lack of evidence about the relationship between drinking and oral disease, the fact that the relationship between alcohol consumption and masticatory disorders was confirmed in this study is considered to be a remarkable achievement. Masticatory function is affected by blood flow and muscle activity. This basic inadequate evidence was insufficient.On the other hand, there is a growing social issue related to increasing alcohol consumption by young women in Japan . It has This study has certain limitations, such as that the data were obtained from a cross-sectional survey, and hence, some points cannot be evaluated universally such as lifestyle habits assessment items. Thus, certain countries' alcohol consumption cannot be compared. In addition, due to the restriction in the survey items in the National Livelihood Survey, the number of survey items related to dentistry was limited.In future, from the viewpoint of promoting the utilization of national statistical data, it is necessary to review the survey items and promote the integration of multiple national statistical databases.From the results of this study, items such as chewing disorders and smoking were clarified as factors related to drinking habits. Our study identified different trends in the response rates of symptoms and diseases in subjects with and without drinking habits.Binomial logistic regression analysis with drinking habit as the objective variable indicated that the odds ratios were significant for smoking habit, masticatory disorders, and working hours, confirming the relationship between these items and drinking habit.The observed correlation between drinking and chewing disorders suggests that this relationship needs to be examined in greater detail in future, including the basic lifestyle background mechanism.


Postural control is considered a key variable in team sports, such as handball, which require abilities strongly related to balance. However, postural control and its relationship to the performance of handball skills according to the players’ skill level and age has not been evaluated to date. This study analyzes the relationship between balance ability and team-handball performance according to age and expertise, applying a non-linear approach to balance assessment. Postural control from 114 male team-handball players was analyzed through the center of pressure (COP) during a balance task. Sport performance was measured by the accuracy and speed in throwing. Expert players threw faster, but not more accurately than recreational players. Balance performance was better for 18+ players (older than 18 years old) than those U12 (under 12 years old), but no differences were found according to their skill level. Players who threw with less accuracy showed slower COP velocity during the balance task and their moves were less irregular. Players who threw faster displayed more irregular and less auto-correlated COP movements. In conclusion, experienced team-handball players exhibited better balance performance, and this seems to be related to the maturation of the motor system more than to sport performance level. Nevertheless, non-linear measures of COP excursion revealed an exploratory behavior during balance in expert players, exhibiting more motion adjustments to reduce motor output error. Traditional variables measuring balance performance did not show sensitivity to this motor control process. A non-linear approach to balance assessment revealed functional variability during balance as an intrinsic characteristic of individuals’ motor control according to age and skill level. Studies have indicated that sport training improves sensorimotor performance and postural control ,2. Thus,Studies that have analyzed sports like gymnastics, soccer, or golf have suggested that practitioners of these sports display a higher balance control than their less proficient counterparts or non-athletes ,6,7, butSome studies have reported significant differences between groups with different levels of expertise only on unstable surfaces ,13. It sTeam-handball is a sport that requires abilities  that are strongly related to balance . In addiOwing to the unclear results reported by the literature, the aim of this study was to assess the relationship between balance ability and sport performance (defined as throwing speed and accuracy) in team-handball players of different ages and levels of expertise, with the aim of analyzing the relevance of balance control in throwing performance.One hundred and fourteen right-handed male team-handball players with different levels of experience and ages took part in this study (see Written informed consent was obtained from each participant prior to the experiment. For both U12 (under 12 years old) and U16 (under 16 years old) groups, written informed consent was signed by the players’ guardians. Data were treated anonymously. The experimental procedures used in this study were in accordance with the Declaration of Helsinki and were approved by a University Office for Research Ethics (Ref: DPS.FMH.01.16).Participants performed two different protocols:1) Balance task procedure.Participants were asked to stand “as still as possible”  on an unIn order to assess postural stability, center of pressure (COP) was estimated by a force platform (Kistler 9286AA0) at 20 Hz .2) Team-handball throwing procedure.Before testing, all participants completed a 10 min warm-up consisting of jogging, stretching, and throwing at a submaximal velocity. During the protocol, each player performed 30 throws distributed into three sets of 10 throws. Participants rested for 30 s between sets and 5 s between throws. They were instructed to “throw at the highest speed and with the highest accuracy possible, aiming at the target” . The balTo assess the maximum ball speed during each throw, a Sports Radar SR3600  was placed behind the players pointing at the target . The goa1) Balance task procedure.Before any variable computation, COP signal was processed according to the protocol by Caballero, Barbado . PosturaD − FE computed over the displacement of the COP time series, and FEV − FE computed over the velocity of the COP time series) and detrended fluctuation analysis (DFAD and DFAV). The variables were calculated over the resultant distance (RD) of the COP time series displacement and velocity data, calculated according to Caballero, Barbado [The variables used to assess the variability structure of displacement and velocity of the COP time series were fuzzy entropy  Team-handball throwing procedure.The ball speed (km/h) was computed as the mean maximum speed of the ball. To compute the accuracy of the throw (m), the mean radial error (MRE) of the position of the ball when it touched the net was calculated as the average absolute distance to the center of the target see . Video dage  and skill level  being the between-subjects factors. Statistical significance was set at p < 0.05. Bonferroni adjustment was performed to ascertain differences between balance and sport performance for age and expertise factors. Partial eta squared  were carried out to assess between group differences in all parameters, with 04 small . Pearsonp < 0.001). Regarding team-handball performance variables, significant differences were found only in ball speed between age ranges and expertise level . The resV was the only variable that displayed significantly different values. Recreational players U12 displayed lower FEV values than players under 16 years old (U16) and 18+. This variable also showed significant differences according to the skill levels, as well as DFAD  for players over 18 years old (18+) than for those under 12 years old (U12) , but the as DFAD . Expert D, while ball speed was positively related to FED and FEV and negatively related to DFAD. Players who threw with less accuracy (higher MRE) moved slower during the balance task (lower MVM) and their moves were less irregular (lower FED). On the other hand, players who threw faster  displayed COP movements that were more irregular (higher FED and FEV) and less auto-correlated (lower DFAD).D with MVM appeared only in the 18+ group. The relation between ball speed and FED disappeared, while the relationship between FEV and DFAD appeared only in the U12 group, showing the same trend described above (V) and less auto-correlated (lower DFAV) COP velocity signal during the balance task.When the sample was split by age range, the correlation between MRE and FEed above . Focusined above , in the Previous studies have shown unclear results in the relationship between balance control and sport performance, and in team-handball, this relationship had not yet been assessed. In this study, the relationship between balance ability and sport performance of the team-handball throw in team-handball players of different ages and levels of expertise was addressed.The main throwing performance variable that showed differences according to age range and skill level was the ball speed. The 18+ expert group threw significantly faster than any other group, with values for absolute ball speed (72 km/h) consistent with those reported in previous studies using a standing throwing action  during the balance task were more sensitive to the differences not only between age ranges, but also between skill levels. The players who threw faster displayed higher irregularity and lower auto-correlation in their COP signal. According to some studies, a COP signal with these features indicates a higher number of movement adjustments in a balance task ,28,43. TRegarding correlational analysis, the team-handball performance variables  were not related to each other. Some studies have addressed the relationship between throwing accuracy and speed in team-handball players according to instructions, and it seems that this relationship depends on the instructions and the skill level ,40. In tFocusing on the relationship between team-handball variables and balance performance, the accuracy of the throws of the whole sample showed a slight positive correlation with MVM. Players who had better accuracy in their throws moved faster during the balance task. MVM has been interpreted as an index of the amount of corrections needed to adjust the COP location, increasing neuromuscular effort resulting from participant’s exploratory behaviors . These rD and FEV) and less auto-correlated (lower DFAD). FE and DFA assess the extent to which further motor behavior is dependent on previous fluctuations. Less dependence on previous behavior  has been interpreted as higher flexibility to perform motion adjustments [Finally, the correlational results indicated that there was a relationship between team-handball performance variables and the variability structure of the COP. On the one hand, players who had better accuracy displayed a more irregular COP excursion. On the other hand, players who threw faster showed COP movements that were more irregular , but this increased motor control related to age seems to be owing to the maturation of the motor system more than to their level in sport performance. The relationship between expertise and balance behavior has been found only in the structure of COP variability during the balance task. Expert players exhibited a more irregular and less auto-correlated variability, interpreted as an exploratory behavior, and more motion adjustments to reduce motor output error. These results indicate the significance of using non-linear variables because they provide information about functional variability and movement dynamics during balance tasks, revealing the intrinsic characteristics of the individual’s motor control. Individuals’ intrinsic features seem to be related not only to the maturation of the practitioners, but also to the sport performance. These results are related to previous studies that have related the complexity of the variability structure to a functional exploratory behavior and the ability to adapt.This paper supports the idea that a non-linear approach is a more sensitive option than the traditional approach to quantify and reveal the athlete’s functional variability during balance tasks. This functional variability is an intrinsic characteristic of handball players’ motor control modulated by age and skill level, which is related to the ability to explore the environment in order to optimize motor performance. Practitioners are encouraged to design practice conditions that facilitate functional variability in players’ motor behavior so they will be able to explore all the possible motor solutions to find the best option and achieve a good sport performance. Non-linear measures of variability can be a practical tool to detect the presence of functional fluctuations related to better performance.


Paenibacillus polymyxa Kp10 at 22 h during incubation. The effects of several selected protective agents on the viability of P. polymyxa Kp10 after freeze-drying were studied. Response surface methodology (RSM) was used for optimizing formulation for the protective agents. The combination of lactose (10% w/v), skim milk (20% w/v), and sucrose (27.5% w/v) was found to be suitable for preserving P. polymyxa Kp10 during freeze-drying. Further, P. polymyxa Kp10 demonstrated the ability to inhibit fungal pathogens, Colletotrichum truncatum and C. gloeosporioides, at 60.18% and 66.52% of inhibition of radial growth, respectively.Anthracnose is a fungal disease causing major losses in crop production. Chemical fungicides widely used in crop plantations to combat fungal infections can be a threat to the environment and humans in the long term. Recently, biofungicides have gained much interest as an alternative to chemical fungicides due to their environmentally friendly nature. Biofungicide products in powder form can be formulated using the freeze-drying technique to provide convenient storage. Protective agent formulation is needed in maintaining the optimal viable cells of biofungicide products. In this study, 8.10 log colony-forming unit (CFU)/mL was the highest cell viability of  Colletotrichum capsici, C. gloeosporioides, and C. acutatum have been reported as pathogens causing chili anthracnose [Anthracnose is a common disease caused by fungal pathogens affecting crops’ yield and fruit quality . Colletohracnose . Conventhracnose . Therefohracnose . For sucPaenibacillus species has the ability to be applied as an alternative biocontrol agent to the current chemical pesticides against plant pathogens. The species produces substances that can be used as pesticides and utilized for bioremediation agents. For instance, Paenibacillus polymyxa strains possess potential antagonistic activity, where they can secrete antimicrobial compounds that demonstrate a spectrum of activity against fungi, bacteria, and nematodes [P. polymyxa strains were reported to produce a range of antibiotics such as polymyxin, polypeptins, and jolipeptin, which effectively work against bacteria, and also produced gatavalin and fusaricidin against fungi [P. polymyxa strain (HY96-2) isolated from a tomato rhizosphere has proven to control tomato bacteria wilt and provide beneficial effects in promoting tomato plants’ growth [P. polymyxa NSY50 isolated from vinegar waste compost was identified as a potential biocontrol agent for controlling Fusarium wilt, a major destructive soil-borne disease infecting cucumber [Paenibacillus also has the potential as a biocontrol for food-borne bacteria including Salmonella [P. polymyxa into consideration, this species could be the best candidate for biofungicide.ematodes ,5. Furthst fungi ,7. In adst fungi . P. polystrain HY6-2 isolacucumber . Furtherlmonella . PuttingThe success in commercializing bacterial strains as a potential biofungicide is highly dependent on preservation technologies employed by industrial companies. Suitable preservation methods could maintain high cell viability not only during the preservation process but most importantly during the long-term product storage . Since bP. polymyxa Kp10 subjected to freeze-drying using response surface methodology (RSM) to obtain the optimal number of cells after the freeze-drying process and to investigate the biofungicide potential of P. polymyxa Kp10 against fungal phytopathogen which commonly affects crop production.Several factors affect the viability of cells during the freeze-drying process. In general, freeze-drying may cause two different stresses onto the cells. The first is where the cells are frozen and water molecules in the cells are disabled followed by a drying stage where water molecules are removed . Ice forP. polymyxa Kp10 was 8.10 log colony-forming unit (CFU)/mL attained at 22 h of the incubation period. The cell growth started to gradually decrease at 24 h with its cell viability at 8.03 log CFU/mL. According to Keivani et al. [P. polymyxa GBR-1 also has its maximum cell viability between 20–28 h after incubation [Lactobacillus rhamnosus at the stationary phase gave the highest recovery rate (31%–50%) after drying compared to the cells at the early log phase with 14% survival rate [Rhizobia cells from stationary phase achieved higher cell viability compared to those in the log phase [cubation . In geneval rate . Likewisog phase . Thus, iP. polymyxa Kp10 cells in maintaining high cell viability compared to control and Soytone (w/v) provided the highest protection for cell survival at 89.26% followed by lactose (10% w/v) with 87.78% survival rate. Skim milk is one of the most preferable protective agents and widely studied for freeze-drying of various types of bacteria [P. polymyxa Kp10 as the protective agent. Hubalek [w/v) is the most frequently used for microorganism lyophilization. Meanwhile, Ming et al. [w/v) for freeze-drying L. salivarius I 24 and obtained a considerably high cell survival rate of 9.0%.Skim milk was shown as the most suitable protectant agent for  Soytone . Skim mibacteria ,28,29,30bacteria . Sucrose Hubalek  suggesteg et al.  used sucP. polymyxa Kp10 recorded a very low survival rate (62.24%) as there was no protection of the cells during lyophilization. A similar result was observed when Soytone (69.13% w/v) was used as the protective agent in which only a 0.46% survival rate was recorded after freeze-drying. Likewise, Portner et al. [w/v) Soytone gave less optimal protection for Campylobacter jejuni as a protective agent during freeze-drying. Based on this study, skim milk, lactose, and sucrose were the preferred protective agents for P. polymyxa Kp10 and thus they were chosen for the subsequent optimization study.As expected, distilled water that was used as a control for the protective agents’ selection for r et al.  reportedProtective agent combinations were discovered based on a five-level two-variable central composite design as shown in Table 6, which comprised the actual factor level corresponding to coded factor levels. Regression analysis was used on experimental data and several prediction models are shown in p > 0.05), hence defining that the models are significant and can be used for optimization. The F-value for the model was 182.58, which was significant. AB, AC, BC, A2, B2, and C2 were the significant model terms. Model terms of A, B, and C were not significant since their p-value (Prob > F) were more than 0.05. Lack of fit was not significant with the value of 3.52. There was a 9.68% chance that lack of fit value could happen because of noise. In determining the fitness of the model, the model must have the significance of the model (p < 0.05) and the insignificance of the lack of fit (p > 0.05) [2). In this study, R2 was equal to 0.9940, implying low error in the model.ANOVA illustrated that the optimization of protective agent combination was most preferably described to the quadratic model as shown in  > 0.05) . Fitnessw/v), sucrose (27.5% w/v), and skim milk (20% w/v) were used as the combined protective agents for P. polymyxa Kp10. This suggests that the combination of lactose and sucrose could give a synergetic effect in protecting the cells during the process. In addition, it can be seen that the combinations from the selected protective agents provided a synergic effect in maintaining high cell viability after the freeze-drying process. Sucrose was reported to replace water around polar residues in macromolecular, thus stabilizing cell membranes and proteins during desiccation [w/v) and sucrose (27.5% w/v) was 83%. According to Wong et al. [L. salivarius at 83%–85% when a combination of skim milk, trehalose, and sucrose was used as a protective agent [iccation . Sugars iccation . Meanwhiiccation . The surg et al. , 71.65%–ve agent . In thisP. polymyxa Kp10 showed in vitro antifungal activity against the tested pathogens  value (66.52%) was the freeze-dried P. polymyxa Kp10 against Colletotrichum gloeosporioides, while lowest PIRG value (60.18%) of freeze-dried P. polymyxa Kp10 against C. truncatum was recorded. The non-freeze-dried P. polymyxa Kp10 also demonstrated antifungal activity  reduction compared to the fresh culture. Our findings were in accordance with several other studies on the antifungal activity performed by Paenibacillus species [P. polymyxa E681 produced an antibiotic known as polymyxin. Further, Karpunina et al. [P. polymyxa 1460 has the ability to produce metabolites called lectins. Lectins are recognized to enhance cellulose degradation in plant cells and increase β-glucosidase activity in the wheat root cell wall. Moreover, most of the antimicrobial substances produced by P. polymyxa are peptides [P. polymyxa strains WR-2 and SQR-21 produced high β-1.3-glucanase when stimulated by the low concentration of uric acid produced by fungus Fusarium oxysporum [Paenibacillus strain producing a hydrolytic enzyme was also used for attacking a fungal cell wall containing β-1.3-glucan, chitin, and 11% protein [In this study, athogens . As showactivity  with PIRg et al.  reportedalue 60.1% of free species ,40. Jeon species  reporteda et al.  proved txysporum . Furtheralue 60.1% of freeP. polymyxa Kp10, used in this study was provided by Bioprocessing and Biomanufacturing Research Centre (BBRC), Universiti Putra Malaysia, Serdang. The strain was stored at −80 °C in 20% (v/v) glycerol as stock cultures. The strain was grown on an M17 agar plate and incubated for 48 h at 37 °C. A colony from the plate was picked and placed into 10 mL of M17 broth in a 15 mL centrifuge tube, then incubated at 37 °C for 18 h. The culture was used as inoculum culture for cell growth analysis. The culture was inoculated (5%) into M17 broth (100 mL) and incubated at 37 °C for 28 h at 150 rpm. Samples were collected starting at 6 h and sampled for every 2 h after until it stopped at 28 h. The number of cells is recorded as the average of the colony-forming unit (log CFU/mL) which is referred to as cell viability [The bacterium, iability  and was w/v), lactose (10% w/v), sucrose (30% w/v) and Soytone (15% w/v) were screened as the protective agents. The selection of the protective agents was based on the survival rate of the cell after freeze-drying in the preliminary experiments of this study , lactose (5%–15% w/v), and sucrose (15%–40% w/v) were chosen based on the previous preliminary test and several other reported studies [In this study, the considered cell viability of  studies ,28,30,40P. polymyxa Kp10 was used as a response for the experiments. Analysis of variance (ANOVA) was used for regression analysis of variance. ® software version 7.0.0  was used.A five-level two-variable  CCD withv/v) in fresh M17 broth (500 mL) in a 1 L Erlenmeyer flask incubated at 37 °C for 22 h at 150 rpm. Cells were harvested by centrifugation within 22 h at 10,000× g for 15 min at 4 °C. The supernatants were discarded, and the cell pellets were washed once with sterilized 0.02 M phosphate buffer, pH 7.2. The cell pellets were centrifuged again before they were resuspended in different combinations of sterile protective agents. Then, 2 mL of bacterial suspension was placed in a 5 mL sterilized vial in triplicate and kept at −28 °C for 18 h. The frozen samples were desiccated in a pilot-scale freeze-dryer machine  at 0.450 mbar vacuum, with a pre-freezing temperature of −40 °C and drying temperature of 20 °C for 48 h.The strain was inoculated at 5% (−1 to 10−6 CFU/mL) in M17 broth were prepared and plated onto M17 agar plates, which were then incubated at 37 °C for 24 h. Colonies on the plate were counted and represented as colony-forming unit per volume (CFU/mL) [o is the viable cell count before FD (CFU/mL); FD represents freeze-drying.The freeze-dried cell samples were reconstituted to their original pre-freeze-dried volumes by adding M17 broth and incubating at 37 °C for 1 h. For cell viability determination, serial decimal dilutions of each reconstituted sample (10(CFU/mL) . SurvivaP. polymyxa Kp10 was screened for its in vitro antifungal activity against C. gloeosporioides and C. truncatum using a dual culture test based on the percentage of inhibition of radial growth (PIRG) [P. polymyxa Kp10 from 24 h of M17 agar plate culture was taken and streaked in a 3 cm circle from the mycelia agar disc on the same PDA plate and incubated for 5 days. P. polymyxa Kp10 in freeze-dried powder form was tested in the assay. Data for PIRG was recorded during the incubation period by measuring the radius of C. gloeosporioides and C. truncatum agar disc using the following formula:R1 indicates the radial growth of fungal agar disc in the control plate; R2 refers to the radial growth of the fungal agar disc in the dual culture plate [h (PIRG) . A mycelre plate .P. polymyxa Kp10 after freeze-drying could be improved using an optimal concentration of protective agents. The optimal combination was found to be a mixture of lactose (10% w/v) and sucrose (27.5% w/v) that resulted in 5.839 log CFU/mL after freeze-drying. P.polymyxa Kp10 has proved to inhibit fungal phytopathogens through a dual culture test with the potential to be used as a biofungicide. Nevertheless, this study requires further works focusing on P. polymyxa Kp10 as a biofungicide in crop plantation to determine the stability of freeze-dried P. polymyxa Kp10 during field applications.In conclusion, the cell viability of


Perceptual video coding (PVC) can provide a lower bitrate with the same visual quality compared with traditional H.265/high efficiency video coding (HEVC). In this work, a novel H.265/HEVC-compliant PVC framework is proposed based on the video saliency model. Firstly, both an effective and efficient spatiotemporal saliency model is used to generate a video saliency map. Secondly, a perceptual coding scheme is developed based on the saliency map. A saliency-based quantization control algorithm is proposed to reduce the bitrate. Finally, the simulation results demonstrate that the proposed perceptual coding scheme shows its superiority in objective and subjective tests, achieving up to a 9.46% bitrate reduction with negligible subjective and objective quality loss. The advantage of the proposed method is the high quality adapted for a high-definition video application. High efficiency video coding (HEVC), also known as H.265, is the latest video coding standard. It was released in 2013 and achieves an average bitrate reduction of 50% for fixed video quality compared with the H.264/AVC which is a block-oriented motion-compensation-based video compression standard. As regards the limited channel bandwidth and storage capacity, its coding efficiency needs further improvement. A perceptual model based on the human visual system (HVS) can be integrated into the video coding framework to achieve a low bitrate with a high perceptual quality.Saliency refers to the indication of the probability of human attention according to the visual stimulus, and the term saliency often appears indistinguishably in two types of studies with different approaches; the “different outputs” approach and the “different evaluations” approach . The firRecently, perceptual video coding (PVC) has attracted lots of attention ,3,4. TheThe main idea, according to the visual attention model, is to encode the importance of different regions with bottom-up or top-down attention. Itti presents a biologically-motivated algorithm to select visually-salient regions for video compression . Gou et In summary, the research related to PVC has made great progress in recent years, but there are deficiencies. For example, there is lack of effective saliency detection computing models for video coding applications. Image-based saliency detection has been extensively studied over the past decades. However, the research related to video-based saliency detection is much less explored. The existing saliency detection models only consider some of the characteristics of the image, and some of the temporal and context information is ignored. Moreover, most of the computation models are based on the pixel level. The computation complexity is too high, making it is unfavorable for real-time video applications.In this paper, we proposed a new saliency detection model for video compression. Firstly, a novel saliency object detection model is proposed to generate a spatiotemporal saliency map. Secondly, the H.265/HEVC-compliant perceptual coding framework is developed based on the spatiotemporal saliency map. This paper has the following contributions: (1) a new superpixel-based salient object model is proposed which incorporates spatial and temporal features. (2) the proposed perceptual coding method achieves higher compression rates with a good balance between subjective and objective quality compared to the H.265/HEVC reference model.For monitoring video, the moving object is the most attractive point of attention for a human. Meanwhile, there are the static features in the video. Thus, the spatiotemporal saliency map generated by the visual attention model and moving object detection model can be explored to guide the bit allocation for video coding.For the input video signal, the spatial saliency and temporal saliency are computed using the spatiotemporal information, respectively. Therefore, according to a certain weight coefficient, the spatial and temporal saliency are merged into the final saliency map.V and E represent the nodes and edges of G, respectively. On the G, each node, including transient nodes and absorbing nodes, is connected to the transient nodes which neighbor it or share common boundaries with its neighboring nodes. Thus, the weight i and j is defined asA single layer graph t nodes are transient nodes and the last r nodes are absorbing nodes, and the affinity matrix A and degree matrix D can be expressed with P on the sparsely connected graph can be calculated from matrix A and D. After that, using the Markov chain theory, the absorbed time for each transient state can be calculated based on the transition matrix P. Finally, the saliency map S can be generated by normalizing the absorbed time.The nodes are renumbered so that the first i.The basic idea of the MC method is to detect the saliency using the time property in an absorbing Markov chain. The virtual boundary nodes are identified as absorbing nodes based on the prior boundary. The saliency is computed as the absorbed time to the absorbing nodes. On the basis of the MC saliency model, the spatial saliency value is represented as A good deal of research effort has already been devoted to saliency models for images. The image saliency models can be used for spatial saliency detection in each video frame. In this paper, the spatial saliency computation is based on the Markov chain (MC) saliency detection model developed by Lu et al. , which iParticularly for videos, the temporal information is a significant hint, and more context exists in the field of video saliency detection. In video saliency detection, the optical flow technique can detect motion accurately. lgorithm . Thus, tFurthermore, the MVA is enhanced asThen, the The temporal saliency reflects the dynamic characteristics of the video, while the spatial saliency reflects the static characteristics of the video. When the spatial and temporal saliency maps are constructed, we use them to get the final video saliency map. Different fusion methods can be utilized, and the linear fusion is used in this paper. The final video saliency map rated bySST(i)= Good detection: the probability of missing real salient regions and falsely marking the background as a salient region should be low. (b) High saliency value: the boundaries of salient regions and region interiors have a high saliency value. The advantages of the proposed work are as follows: (1) In this work, the optical flow technique is used to detect temporal saliency, which can detect moving objects precisely. (2) The Markov chain-based saliency detection method in the proposed work can enhance the region’s interior significantly. Thus, the generated saliency map with the proposed method can increase the coding performance.In the video, the moving objects are regarded as the salient region. Finally, the superposition of spatiotemporal saliency is taken as the final saliency with linear fusion.N is CU size, and In H.265/HEVC standards, the coding unit (CU) is a square region and is a node of the quad-tree partitioning of the coding tree units (CTU) . The quaIn H.265/HEVC standards, a QP is used to determine the quantization step, the setting range of QP is from 0 to 51. In order to associate the block-based saliency value with QP, the saliency value can be normalized to the range . In this paper, the min–max normalization method is used to generate the saliency level for the block-based saliency average value When the Algorithm 1: Saliency-based quantization control algorithmThe proposed algorithm is implemented and verified based on the H.265/HEVC reference model HM 16.0. The quantization parameters are set to 22, 27, 32, and 37, respectively. The configuration profile is low-delay (LD).The performance of the proposed algorithm is evaluated by the average bitrate saving (BS) and Bjontegarrd Delta PSNR (BD-PSNR) according to Reference . The BS The double stimulus continuous quality scale (DSCQS) method is used for subjective evaluation . The subThe subjective evaluation of video quality is evaluated by the difference mean opinion score (MOS) average values Delta MOS (D-MOS), which is calculated asIn the ablation study, the results of spatial-only, temporal-only, and the proposed spatiotemporal saliency-based quantization control scenarios are shown in For our experiments, we selected four test sequences of different resolutions. The four test sequences were also selected by considering the diversity of spatial characteristics and temporal characteristics. The four test sequences were the ‘When the Delta MOS values are smaller, the subjective qualities are closer to the original H.265/HEVC reference software. The fifth, eighth, and eleventh columns in Furthermore, the comparative results with previous work are shown in In summary, from the objective and subjective test results, the proposed perceptual video coding scheme can achieve higher compression rates with the balance between subjective and objective quality compared to the H.265/HEVC reference model.In this paper, we proposed a novel perceptual video coding scheme based on the spatiotemporal saliency model. Simulation results demonstrate that the proposed perceptual coding scheme can save of 5.22% bitrate, while the Bjontegarrd Delta PSNR is only 0.046 dB on average. Moreover, the average absolute Delta MOS value of the proposed method is even smaller than 0.1.


At T2, FMD was significantly higher after isomaltulose administration compared to that after sucrose administration . Pearson correlations between FMD and blood glucose showed a trend for a negative association at T0 and T2 independently of the carbohydrate . Sub-analysis suggested a lower FMD in insulin-resistant (IR) compared to insulin-sensitive subjects. Isomaltulose attenuated the postprandial decline of FMD, particularly in IR persons. These data support the potential of isomaltulose to preserve the endothelial function postprandially and consequently play a favorable role in cardiovascular health.Hyperglycemia is linked to impaired arterial endothelial function (EF), an early sign of cardiovascular disease. We compared the efficacy of low-glycemic index isomaltulose  with that of sucrose in modulating EF, as assessed by flow-mediated dilation (FMD). In this double-blinded cross-over study, 80 overweight mildly hypertensive subjects were randomized to receive 50 g of either isomaltulose or sucrose. On two non-consecutive days, brachial artery ultrasound FMD scans were obtained prior to and hourly (T0–T3) after carbohydrate load. Blood was drawn immediately after scanning. Glucose and insulin levels were analyzed. Overall, the FMD decrease was attenuated by isomaltulose compared to sucrose (ΔFMD = −0.003% and −0.151%;  Endothelial function (EF) plays a pivotal role in cardiovascular health. Impaired EF is a prominent health concern, as it typically contributes to cardiovascular disease (CVD), i.e., hypertension, atherosclerosis, or myocardial infarction ,2. In anEF can be assessed by brachial artery ultrasound imaging. With this completely non-invasive, subject-friendly technique, the shear stress-induced flow-mediated endothelium-dependent vasodilation (FMD) of the brachial artery can be measured . A high Besides insulin resistance itself, accumulating evidence indicates a link between EF and postprandial dysmetabolism such as hyperglycemia and its consequences, i.e., vascular events ,13,14. HThe hypothesis that hyperglycemia causes ED is supported by the observation that overweight and obese individuals and those with diabetes, either type 1 or type 2, are more prone to ED ,11,27,28On the basis of this knowledge and considering the large intake of high-GI foods that characterizes today’s Western diet, low-GI foods might be effective in preventing ED. For instance, a substantial replacement of sugar by low-GI carbohydrates, e.g., isomaltulose , may contribute to improved cardiovascular health, as indicated by Keller et al. . During Both sucrose and isomaltulose are disaccharides consisting of a glucose and a fructose monomer, which provide the same amount of calories. However, they differ in their glycosidic bond, which is α-1,2 for sucrose and α-1,6 for isomaltulose . The bonThe present exploratory study aimed to investigate the acute effects of isomaltulose versus those of sucrose on endothelium-dependent vasodilation, i.e., FMD, in overweight/obese subjects. The lower blood glucose response following isomaltulose load may allow for enhanced preservation of EF. We therefore hypothesized that, compared to sucrose, isomaltulose would cause lesser postprandial impairment of EF in overweight/obese individuals.Between August 2016 and March 2017, overweight and obese subjects with mild hypertension (systolic/diastolic blood pressure: 130–159/85–99 mmHg) were recruited by Atlantia Food Clinical Trials CRO . After screening, healthy persons aged 25–60 years were included if their body weight was stable (≤5% change over the past 3 months) and their physical activity level was low to moderate . Exclusion criteria were use of medications influencing glucose metabolism  or EF  and chronic or acute diseases. Smokers, heavy coffee drinkers, and pregnant women were further excluded. Throughout the study, subjects were asked to follow their usual diet and exercise routine and to avoid the named medications that could interfere with the study outcomes.clinicaltrials.gov (NCT03986775).The study protocol was approved by the Clinical Research Ethics Committee of the Cork Teaching Hospitals. All participants gave their written informed consent before the study’s start according to the ICH Guidelines on Good Clinical Practice and the declaration of Helsinki. The trial was registered at An outline of the study protocol is depicted in The order of the assessments was exactly the same on both intervention days. Prior to carbohydrate load (T0), a brachial ultrasound FMD scan was performed, with blood drawn shortly thereafter. All subjects were blinded to treatment and randomly assigned to consume either 50 g of isomaltulose  or 50 g sucrose in the form of instant citrus drinks. Therefore, drink powders were dissolved in 500 mL of water shortly before consumption. The subjects were instructed to consume the citrus drink within 10 min after the T0 FMD. At 60 min (T1), 120 min (T2), and 180 min (T3) after the start of the citrus drink consumption, FMD scans and blood sample collections were performed.Ultrasound FMD scanning procedures were strictly standardized and quality-controlled as described in extenso elsewhere ,35. In sThe examinations took place in a quiet room at temperatures between 20 °C and 26 °C and dimmed lighting. For each subject on both test days, initial and follow-up scans were performed by the same technician. Subjects comfortably reclined for 20 min prior to scans. During visits, the subjects’ right arm rested in the arm-supporting cups of the probe-holder arm rest in a fixed position during the 9′ scan procedures. The brachial artery was scanned longitudinally to obtain high-quality reproducible scans of the arterial lumen with the ultrasound probe 5–10 cm proximal to the antecubital fossa, its position documented with a tape measure for accurate repositioning of the probe in the second visit. On the lower arm, a blood pressure cuff was inflated 40 to 50 mmHg above systolic pressure for 5 min to temporarily occlude the brachial artery. According to a fixed ultrasound imaging application protocol, 1 min prior and 3 min after arterial occlusion, electrocardiogram (EKG)-triggered images were recorded in DICOM clips, with each full-frame image in the clip to provide a lumen diameter measurement. For off-line scan analyses, the clips were directly and securely transferred from the ultrasound equipment to the core lab  for immediate in-study quality control and investigational site feedback by the core lab FMD experts. For the treatment efficacy image analyses, per-subject batches of FMD scans were provided to the trained and certified technicians of the core lab. Technicians were blinded to subject demographic, clinical, and treatment data, as well as scan visit order. Validated and FDA-approved image analysis software was used .FMD was calculated as the percentage of change in the brachial arterial diameter:A vein cannula was inserted into the subject’s left arm, and a blood sample (12 mL) was collected shortly after FMD measurement at T0, T1, T2, and T3 during both intervention days. Serum glucose concentrations were measured using the hexokinase method . Serum insulin levels were determined using a chemiluminescent microparticle immunoassay . Homeostasis model assessment (HOMA) index was calculated using the following equation: fasting glucose (mmol/L) × fasting insulin (µU/L)/22.5.Statistical analyses were carried out using SPSS Version 24 and SAS Version 9.3. Sample size was calculated based on primary outcome FMD. On the basis of intra-sonographer repeatability of 1.25%FMD, α of 0.05 (two-sided), and β of 0.1 and on an effect size of 0.9%FMD, the minimal required subject number was 68. To account for drop outs and FMD assessment failures, in total 80 subjects were included.p > 0.05), parametric tests were used. To compare the baseline characteristics of the isomaltulose-first and sucrose-first groups, unpaired t test for continuous variables and χ2 test for categorical variables were applied.Descriptive characteristics are presented as mean ± SD. As the data were normally distributed  with terms for treatment and period was performed to observe differences in endothelial responses following investigational products adjusted for baseline FMD. In this primary statistical analysis, only FMD data of those subjects with fully completed datasets of the four FMD measurements at visit 1 and the 4 FMD measurements at visit 2 were included.t test. In these statistical analyses, subjects were included provided that both FMD measurements at a given time point were available. Pearson correlations were carried out to detect associations between FMD and blood glucose as well as insulin levels at different time points.To compare differences in FMD, blood glucose, and insulin levels for the carbohydrates, an exploratory analysis at each of the time points within subjects between visits was performed using the paired Tx-T0) between IS and IR subjects, unpaired t test was used. Two-sided statistics was performed with the significance level set at p < 0.05.For the evaluation of differences in the endothelial response depending on the insulin sensitivity status, subjects were classified according to their HOMA index on their first intervention day, and a subgroup analysis was performed. Subjects with HOMA < 2.5 were considered to be insulin-sensitive (IS), whereas subjects with HOMA ≥ 2.5 were considered as insulin-resistant (IR). To see differences in the change of FMD from baseline . None of the subjects had a high level of physical activity; 47 subjects (58.8%) had a moderate level and 33 (41.3%) a low level of physical activity. This was not different between the randomization sequences (p = 0.26).The baseline characteristics are presented in Of the 80 subjects, 78 were subjected to the four brachial ultrasound FMD scans at each of the two visits. This amounted to 624 scans, of which 596 provided FMD data (a success rate of 95.5%). For 19 of the subjects, due to anatomic and/or ultrasound imaging reasons, 28 of the brachial scans could not provide FMD data (4.5%).ISO,T1–T3 = −0.003%) and a minor, non-significant, negative change after sucrose administration . The mixed repeated-measures analysis of variance controlling for baseline FMD showed no significant interaction between treatment and period (p > 0.05).For the primary statistical analysis, the available fully completed FMD datasets of 61 subjects were used. The overall FMD change from baseline showed maintenance of the endothelial function after isomaltulose administration  and sucrose , respectively .The consumption of 50 g of carbohydrate led to a decrease in FMD at T1, with changes from baseline FMD (ΔFMD) of −0.40% and −0.74% for isomaltulose . When compared to T2, FMD following isomaltulose administration was almost unchanged at T3, with a change from baseline FMD of +0.19%. In contrast, for sucrose, a rebound was observed at T3, resulting in +0.58% compared to baseline FMD.In further explorative analyses, between-visit, within-subject paired FMD data points of the four brachial ultrasound scan time points T0, T1, T2, and T3 h were available in 73 (94%), 71 (91%), 72 (92%), and 71 (91%) subjects, respectively. After two hours (T2), the absolute FMD was significantly higher following isomaltulose intake compared to sucrose intake (at T2) . For insulin, the response levels were significantly higher after isomaltulose administration compared to sucrose intake (paired t test: p < 0.05), with the exception of T1.As expected, there was a rise in blood glucose and insulin levels after carbohydrate ingestion B,C. Withp < 0.1; data not shown). With respect to insulin response, no associations with baseline or postprandial FMD were observed.Independent of the carbohydrate, a correlation analysis between FMD and blood glucose levels at different time points showed a trend for a negative association at T0 and T2  for IR and IS subjects. Generally, subjects with IR showed a higher decline in FMD at T1 and T2 compared to IS subjects, which was independent of the carbohydrate ingested . When compared to sucrose, the ingestion of isomaltulose showed a lower decline in postprandial FMD at T1 and T2 for both IR and IS subjects . At T3, the consumption of isomaltulose resulted in an FMD which was comparable to baseline FMD for both IS and IR subjects. In contrast, the consumption of sucrose showed an increase in FMD exceeding the baseline value.For this exploratory analysis, fasting blood glucose and insulin levels were available for 70 subjects. On the basis of their HOMA index prior to the first intervention, 79% of the subjects were classified as IS, and 21% as IR. We hypothesized that the decrease in endothelial function caused by food intake could be attenuated by low-GI foods compared to high-GI carbohydrates. The assumption was tested in mildly hypertensive overweight/obese adults using either sucrose (GI = 65) or isomaltulose (GI = 32).t test: p < 0.05, As a novel key finding, our data revealed a preservation of postprandial FMD following the intake of isomaltulose compared to sucrose. Consequently, our results support the hypothesis. The exploratory analysis showed that after two hours, endothelium-dependent vasodilation was significantly higher for isomaltulose ingestion compared to sucrose ingestion  ,38,39. CA second explanation for the link between the GI of foods and CVD involves the action of insulin. Because insulin is a vasodilatory agent, insulin resistance might impair EF. Insulin signaling is important for vasodilation through the activation of endothelial NO synthase. Hence, insulin resistance is accompanied by diminished vasodilatory function due to reduced NO bioavailability . AdditioThirdly, the beneficial effects of GLP-1 on the vascular system in relation to isomaltulose need to be addressed. Due to its slow hydrolysis, isomaltulose enhances GLP-1 secretion compared to rapidly available carbohydrates in both healthy persons ,33 and dTo our knowledge, the present study is the first comparing FMD following the ingestion of sucrose and low-GI isomaltulose. Considering the fact that today’s diet contains high amounts of high-GI foods, which leads to adverse health effects, the present results are of clinical relevance.Nevertheless, the efficacy study was based on the primary outcome FMD. Hence, blood analysis was limited with regard to the number of time points and intervals chosen, i.e., every 60 min. This most likely contributed to the failure to detect actual blood glucose peaks which are known to occur in the early postprandial phase. The inclusion of further time points in blood analyses and FMD scanning might have provided additional mechanistic insight on the potential associations between blood glucose and insulin levels with EF. Another limitation of this study is the sample size of the subgroup analysis. A higher number of subjects might have allowed the detection of significant differences in FMD related to the subjects’ insulin sensitivity status.In our study, we evaluated the efficacy of isomaltulose  and sucrose loads on endothelial function as assessed by brachial ultrasound flow-mediated dilation. Our findings show that low-GI isomaltulose, compared to sucrose, led to a better preservation of the basal, pre-prandial, endothelial function in the postprandial phase. Particularly, persons with impaired insulin sensitivity seemed to benefit from the slow-release carbohydrate isomaltulose. Replacing sugar with isomaltulose may exert beneficial effects on cardiovascular health as a result of the more balanced and sustained blood glucose profile. The impact of continued isomaltulose consumption on endothelial function merits further investigations.