diff --git "a/deduped/dedup_0256.jsonl" "b/deduped/dedup_0256.jsonl"
new file mode 100644--- /dev/null
+++ "b/deduped/dedup_0256.jsonl"
@@ -0,0 +1,42 @@
+{"text": "We conducted the present study to determine the usefulness of routinely inserting a pediatric airway exchange catheter (PAEC) before tracheal extubation of adult patients who had undergone maxillofacial or major neck surgery and have risk factors for difficult reintubation.A prospective, observational and clinical study was performed in the 25-bed general intensive care unit of a university hospital. Thirty-six adult patients who underwent maxillofacial or major neck surgery and had risk factors for difficult reintubation were extubated after insertion of the PAEC.Four of 36 (11.1%) patients required emergency reintubation after 2, 4, 6 and 18 hours after tracheal extubation, respectively. Reintubation of these patients, which was thought to be nearly impossible by direct laryngoscopy, was easily achieved over the PAEC.The PAEC can be a life-saving device during reintubation of patients with risk factors for difficult reintubation such as laryngeo-pharyngeal oedema due to surgical manipulation or airway obstruction resulting from haematoma and anatomic changes. We therefore suggest the routine use of the PAEC in patients undergoing major maxillofacial or major neck surgery. Maxillofacial and major neck surgery has a considerable risk for postoperative laryngo-pharyngeal oedema and airway obstruction due to surgical manipulation or haematoma . When paHence, acute respiratory distress can develop after extubation and mandate emergency tracheal reintubation. Mask ventilation and tracheal intubation may be difficult or impossible. Considerable time and an experienced physician are needed to secure a difficult airway with the use of alternative methods such as fibre-optic bronchoscope, retrograde] intubation or cricothyroidotomy. Re-establishing the airway in these patients can be extremely challenging, and often results in considerable morbidity and mortality . In the The aim of this study was to determine the usefulness of routinely inserting the PAEC before tracheal extubation of adult patients undergoing major maxillofacial or neck surgery Fig. .Thirty-six patients admitted to our intensive care unit (ICU) after maxillofacial or major neck surgery between January 2001 and May 2002 were routinely extubated with the use of a no. 11 PAEC , with the approval of the Institutional Review Board. Patients included in the study consisted of 13 post-operative patients with maxillofacial trauma, 14 patients who had undergone neck surgery (5 with hugely enlarged thyroid gland or tumor and 9 with larynx or tongue cancer), and 9 patients who had undergone maxillofacial cancer surgery.Written consent for publication of the photos of the patients was obtained.2) of 0.4, a positive end expiratory pressure of less than 5 cmH2O and pressure support of less than 8 cmH2O. In addition, the haemodynamic status of the patients had to be stable before the decision to extubate was made. The PAEC was carefully inserted through the existing ETT before extubation, avoiding carinal irritation by placing it at the same depth as the ETT tip . The PAEC was not inserted against a resistance. After the ETT had been removed and the PAEC had been secured, humidified oxygen with a low flow of 1\u20132 l/min was insufflated via the lumen of the PAEC. Signs of respiratory failure and tolerance were also assessed. The PAEC was removed when it became clinically apparent that the need for tracheal reintubation was unlikely. We considered the ability of patients to manage secretions including cough and swallow functions in making the decision about extubation of the PAEC. A stable O2 saturation and the extent of surgery were also important factors in this decision. The timing of removal of the PAEC was therefore different depending on various characteristics of patients and surgery. When patients failed to respond to tracheal extubation, the PAEC was used to facilitate the reintubation.A no. 11 PAEC is 83 cm in length and has a 4 mm external diameter and a 2.3 mm internal diameter with a hollow lumen. It is semi-rigid and made of radio-opaque polyurethane; there are six sideports in the distal 3 cm of the catheter. The patients were extubated when they became conscious and had normal body temperature and normal blood gases with an inspired oxygen concentration (FiOTwenty-eight patients (77.8%) were men, and 8 (22.2%) were women. Ages ranged from 19 to 76 years, with a mean age of 52.6 \u00b1 10.8  years. An oral ETT was in place in 18 patients (50%) and a nasal ETT in 18 (50%). All patients had a cuff leak test before tracheal extubation. The median duration of endotracheal intubation after the operations was 1.2 days (range 2 hours to 10 days). After tracheal extubation with the PAEC, 4 of 36 patients (11.1%) required reintubation  and his oxygen saturation decreased to 85%. He was reintubated orally over the PAEC with the assistance of a laryngoscope within a few seconds by using an 8 mm ETT. During observation with a laryngoscope, reintubation of this patient by direct laryngoscopy was thought to be nearly impossible because the glottis could not be seen as a result of the anatomic abnormality caused by haematoma. He was extubated again using the PAEC 24 hours after his second operation; the PAEC was removed again 6 hours after insertion.The second patient , who had also undergone neck surgery , was intubated with difficulty using a Fasttrach  because of anatomical abnormalities, which developed as a result of previous operations and radiotherapy. He was extubated 4 hours after the operation in accordance with the criteria mentioned above. However, he required emergency reintubation 18 hours after extubation because he suffered acute respiratory distress following aspiration and bronchospasm. We found out from the history obtained from his relatives that the patient had already had a swallowing disorder before the operation and suffered from aspiration. Thus, we prolonged the presence of the PAEC. Reintubation of this hypoxic patient was urgently achieved over the PAEC with the assistance of a laryngoscope using a 7.5 mm ETT under sedation and neuromuscular relaxation. During laryngoscopic observation we could not see the glottis. In this patient, a surgical tracheotomy was performed later because of recurrent aspiration and the need for tracheal suction.2 pressure increased, and she became confused as a results of hypoxaemia. She was reintubated nasally with a 7 mm ETT over the PAEC, with intravenous midazolam 0.05 mg/kg and fentanyl 1 \u03bcg/kg, without cutting the archbar. She was extubated with the use of the PAEC 2 days after her reintubation, and the PAEC was left in place for 8 hours. She did not need intubation again after the PAEC had been removed.This patient  was admitted to the ICU after she operation for maxillofacial trauma. She had been intubated nasally by direct laryngoscopy using a Magill forceps; she could not open her mouth after the operation because of inter-maxillary fixation Fig. . Six houThe fourth patient, a male, underwent maxillofacial reconstructive surgery for cancer after he had been intubated nasally over a flexible bronchoscope because of anatomical abnormalities in the oral route. He became hypoxic 4 hours after his extubation and required immediate reintubation. A serious pharyngeo-laryngeal oedema was thought to be the reason for hypoxia. His reintubation was easily achieved over the PAEC, with intravenous midazolam 0.05 mg/kg. He was extubated with the use of the PAEC 3 days after reintubation, and the PAEC was left in place for 6 hours. He did not require intubation again after removal of the PAEC.During the perioperative period, serious respiratory events due to inadequate airway management can develop, which can cause severe brain damage or death. Rosenstock and colleagues  reportedIn previous studies, reintubation rates of 5\u201319% have been reported in surgical ICU patients -8. In ouin situ ETT. The primary use of the PAEC  has been as a tube exchanger in the critical care setting. It has been also used before the extubation of patients with a known difficult airway [The PAEC is a long, flexible and hollow tube designed to facilitate the exchange of an t airway . In the t airway , the useVarious methods have been used to facilitate the reintubation of these patients such as a fibre-optic bronchoscope , rigid EIn patients whose reintubation was considered a risk and who were known to present difficult tracheal reintubation, elective tracheotomy has even been performed in many institutions . BesidesAlthough the PAEC is rigid enough to facilitate tracheal reintubation, not all patients' tracheas may be easily reintubated. Forceful insertion of the ETT should be avoided to minimize trauma to vital airway structures and to avoid kinking the PAEC. Direct laryngoscopy may also relieve the obstruction and identify its cause. We also used the assistance of the laryngoscope during the reintubation of two patients over the PAEC both to facilitate the intubation and to evaluate the anatomical structure of the upper airway with regard to the possibility of laryngoscopy. Gentle rotation of the ETT while trying to insert it may release the tip . The PAEIn a study of patients requiring tracheal reintubation, 87% (34 of 39) required reintubation within the first 4 hours after extubation . In our The routine use of a PAEC in patients who have undergone maxillofacial or major neck surgery facilitates reintubation when necessary, and can be a life-saving method. It allows a safer trial of tracheal extubation and therefore can shorten the duration of intubation. We suggest that after these surgical procedures a PAEC be used routinely before tracheal extubation because it is difficult to predict which patients will require reintubation.\u2022 \tThe PAEC is a long, flexable and hollow tube designed to facilitate the exchange of an in-situ endotracheal tube.\u2022 \tThe routine use of the PAEC in patients who underwent maxillofacial or major neck surgery facilitates the reintubation when necessary, and can be a life-saving method.The authors declare that they have no competing interests.ETT = endotracheal tube; ICU = intensive care unit; PAEC = pediatric airway exchange catheter"}
+{"text": "Typical advice on the design and analysis of cluster randomized trials (C-RCTs) focuses on allowance for the clustering at the level of the unit of allocation. However often C-RCTs are also organised spatially as may occur in the fields of Public Health and Primary Care where populations may even overlap.We allowed for spatial effects on the error variance by a multiple membership model. These are a form of hierarchical model in which each lower level unit is a member of more than one higher level unit. Membership may be determined through adjacency or through Euclidean distance of centroids or in other ways such as the proportion of overlapping population. Such models may be estimated for Normal, binary and Poisson responses in Stata (v10 or above) as well as in WinBUGS or MLWin. We used this to analyse a dummy trial and two real, previously published cluster-allocated studies  to investigate the extent to which ignoring spatial effects affected the estimate of treatment effect, using different methods for defining membership with Akaike's Information Criterion to determine the \"best\" model.The best fitting model included both a fixed North-South gradient and a random cluster effect for the dummy RCT. For one of the real RCTs the best fitting model included both a random practice effect plus a multiple membership spatial term, while for the other RCT the best fitting model ignored the clustering but included a fixed North-South gradient. Alternative models which fitted only slightly less well all included spatial effects in one form or another, with some variation in parameter estimates (greater when less well fitting models were included).These particular results are only illustrative. However, we believe when designing C-RCTs in a primary care setting the possibility of spatial effects should be considered in relation to the intervention and response, as well as any explanatory effect of fixed covariates, together with any implications for sample size and methods for planned analyses. Few examples of properly designed & analysed C-RCTs exist before 1978, but since then with developments in methodology and software such designs have become increasingly common [It is well-recognised that a proper analysis of C-RCTs must allow for the clustered nature of the data to reduce the risk of a type 1 error. However other methodological biases may arise in such trials ,3 and deOne aspect which is frequently overlooked is the intrinsically spatial distribution of general practices and other community-based clusters, which have populations that are not only adjacent (and therefore likely to have shared exposures) but which actually overlap. In the context of GP Practices the overlap would be in the sense that a patient registered with one GP Practice may live next door to a patient registered with a completely different Practice (which is distinct from the kind of overlap that might occur if a single patient were registered with multiple Practices). The effect of proximity and possibly overlap and how to account for it has received little attention in the literature on C-RCT design and reporting -7.In a time series, variations in incidence over time may be represented by a smooth \"deterministic\" trend combined with random noise which is likely to be autocorrelated - that is, observations which are closer together in time are more similar than observations further apart . SpatialAn additional complication familiar to geographers and others interested in modelling the spatial distribution of disease is that in spatial models the observations are typically not evenly distributed (unlike the regular periodic observations of many time series). One way to allow for spatial autocorrelation is through a spatial error model, one example of which is a multiple membership model.A multiple membership model is therefore a form of hierarchical model in which each lower level unit can be a member of more than one higher level unit. Figure The extent of such membership may be defined in different ways, depending on the data available and the purpose of the analysis. One way might be on the percentage of total in-patient stay; another might be on proximity. For spatial models in which the units of interest are geographically defined, membership may be defined in terms of adjacency or through Euclidean distance of centroids or in other ways such as the proportion of overlapping resident population. Such models may be estimated in WinBUGS or MLWin and also  in Stata (version 10 or above).Three data sets were used: one dummy RCT and two real cluster-allocated studies for which one of us (DK) was chief investigator. The first of these allocated general practices within a single City and the other allocated general practices within a single County.The dummy RCT was based on the Scottish male lip cancer data set . This daThe analysis of this data set by means of a Poisson multiple membership model presented by Browne  was firsWe then randomly allocated each County district to one of two nominal treatments and the observed number of cases in those districts receiving one of the treatments was doubled to reflect the effect of a hypothetical intervention. It is important to remember that this not a real trial so that the actual nature of the outcome, the intervention and the covariate do not matter. The data are being used simply to provide realistic statistical data.As implemented by Browne, the multiple membership function was defined as follows. For a given index district each adjacent district was assigned a membership weight of 1/n where n was the total number of such adjacent districts. The index district itself was not included thus enabling it to be represented by a separate random effect.For our dummy RCT this procedure would not be feasible because some of the districts chosen might have no adjacent neighbours in the sample. However the influence of neighbouring Districts might not require actual adjacency. We therefore based membership on an ad hoc function of Euclidean distance between the District centroids. The function used to transform Euclidean distance to proximity was:Where Distance is measured in Standard Normal Deviate units, \u03a6 denotes the standard Normal cumulative distribution function, and k is a parameter estimated by minimizing Akaike's Information Criterion (AIC) over a range of fitted models. Figure Figure For each index district in turn, membership of the other districts was defined as the proximity divided by the sum of all the proximities to the index. Again, the index district was omitted from the summation.We then applied this function to the full Scottish lip cancer data set to identify firstly the value of k that gave the closest AIC to Browne's method, and secondly to identify the value that minimized the AIC.While hypothetical in terms of the allocated \"treatment\", the resulting dummy C-RCT incorporates real covariate and proximity data as well as the response in the control arm . Such a C-RCT based on geographically defined clusters  might be performed in a Public Health context for road traffic measures or water fluoridation.For each model  the value of including a spatial gradient as a fixed effect was also assessed, and each model included the percentage of sun exposed workers as a covariate.The first trial  evaluateThe second trial  evaluateThe default analysis for the two trials was on an individual patient basis, with allowance for the clustering by means of a practice level random effect.Because practice centroids derived from practice populations were not available, one of us (DK) converted participants postcodes into grid references using a file for East Midlands postcodes supplied by East Midlands Public Health Observatory. She then centred these values by subtracting the mean easting and northing respectively and then averaged these values over the patients within each practice to give a centroid for the practice based on participants (arguably perhaps more relevant), before passing these to PS for analysis. As with the Scottish data these centroids were used to estimate both distances between each practice and general east-west and north-south fixed effects.All analyses were performed using Stata v10, and model selection was based on minimizing the AIC. The incomplete availability of postcode data was ignored for our purposes, because our interest lay in comparing estimated treatment effects with and without allowance for spatial effects rather than obtaining a \"correct\" reanalysis of the original trials. In addition we ignored the matching in the home safety intervention study partly because the original analysis did so and also because the matching variable could not be found in what remains of this now fairly old dataset.given by the data in hand (these are not significance-testing procedures). Benchmark values for likelihood ratios of 8 and 32 have been suggested [For a given dataset and range of models, an AIC greater by four units or more than the minimum AIC is evidence of considerably less statistical support (as measured by the likelihood of the model), while differences in the range 0-2 indicate substantially the same support . Becauseuggested  to distiFigure Initially we repeated Browne's analysis of the Scottish Lip Cancer data using Stata. The only variables for which there was any material difference were the the (common) random effect variance for the neighbours forming the multiple membership set, and the variance of the random intercept for districts themselves. MLWin gave values of 1.204 (0.493) and 0.056 (0.059) respectively, while corresponding Stata estimates were 0.980 (0.392) and 0.059 (0.055), values in brackets being the standard errors of each estimate. The fixed effect estimates and standard errors were similar even though Stata uses an approximate method to integrate out the random effects. In terms of choice of the parameter k we found that a value of 4.03 gave a model with AIC closest to the AIC of the adjacency membership model, albeit with rather different fixed parameter estimates . For the dummy RCT we used a value of k = 1 which minimized the AIC at a value two units less than that for the adjacency multiple membership. This value of k gave a slope for percent sun-exposed very similar to Browne's although the intercept was 27% smaller in magnitude. We justify our choice by observing that the original adjacency membership function is not itself based on any a priori argument, nor on any formal model comparison and need not be considered necessarily \"right\".Results for the dummy CRCT are displayed in Table Tables In the baby walker study Table  by contrThe results of this study can only of course be illustrative and will not necessarily apply to all C-RCTs, however, we believe that the possibility of spatial effects should be considered when designing any C-RCT in a primary care setting or planning its analysis.For this study we have also assumed that the primary interest is in the treatment fixed effect and its standard error, for which the Laplacian method used by Stata is fast but may underestimate random effects which might cause a problem if treatment by centre interactions are to be studied. However if necessary this problem can be avoided by use of WinBUGS or MLWin.On the other hand it is useful to be able to evaluate different forms of proximity weighting and random effects models rapidly within a familiar computing environment without the need to learn new, infrequently used, packages.For our dummy RCT representing a C-RCT performed in a population of about 5 million, a random treatment effect combined with a fixed North-South gradient performed best and there was a moderate sensitivity to model choice of the estimated treatment effect. For the home safety intervention study the minimum AIC model was one that included both a random practice and a multiple membership term, and although addition of a fixed spatial gradient had relatively little effect on the AIC, the estimated treatment effect was very sensitive to model choice, even to the extent of reversing its sign.By contrast in the baby walker study the estimated treatment effect was relatively insensitive to model choice and the best fitting model ignored random effects altogether, although it did include a fixed spatial term.In all three studies both the point estimate and its standard error were affected by the model choice; while the best-fitting (minimum AIC) models did not have the smallest standard errors, these were not the largest either, and this benefit was apparent even if the range of point estimates was not large.We are not sure how we would have allowed for the matching in the home safety trial in addition to the spatial effect, had it been available . Because this was pair-matching, use of a fixed effect dummy variable to denote each matched pair would have been inadvisable, so we would probably have treated the matching variable as an additional random effect.Other rules could be used for defining the membership function, but we have not explored them here. One topic of potential interest would be the extent to which other ways of defining spatial relationships in C-RCTs based in primary care influence conclusions, and in particular the effect of using individual participant grid references as opposed to those of Practices.It is of some interest to speculate on the reasons why a North-South effect may have been important in the baby walker study but not in the home safety intervention study, although this is of necessity no more than a post-hoc rationalization. One possibility is that the baby walker study included a wider and possibly more diverse population, whereas the home safety intervention study included practices within a single city, where the north/south gradient in terms of deprivation would be less marked.In addition, random effects models (of which a MM is one kind) can be used as a catch-all for various unmeasured effects and conceivably if these could be identified in advance they could be handled as fixed effects and make the random effect less relevant - and the fixed effect more specific, if for instance the North-South gradient could be replace by a more direct measure of deprivation. A further improvement could be obtained if individual-level covariates were included. Lastly both the kind of intervention and the nature of the response variable may influence whether allowance for spatial effects is likely to be important, and therefore allowed for in sample size estimation and in the statistical analysis plan.In brief we conclude that:1. spatial effects may well need to be allowed for in the design and analysis of C-RCTs.2. the optimum analysis method may involve either a multiple membership and or a fixed spatial covariate and the choice should be identified by a pre-specified process of model selection.3. ignoring spatial effects may affect model fit, estimated parameter values and their standard errors.Paul Silcocks - None. Denise Kendrick - was Chief investigator of the two previously published Trials used in this paper.PS conceived the study, performed the analyses and drafted the manuscript. DK provided data on the studies used, generated the anonymous analysis data set used by PS and revised and jointly approved the final version of the manuscript."}
+{"text": "The O=C\u2014C=C\u2014N portion is essentially planar [maximum deviation = 0.046\u2005(2)\u2005\u00c5] and is aligned at dihedral angles of 22.6\u2005(1) and 78.9\u2005(1)\u00b0 to the phenyl and pyridyl rings, respectively. The N\u2014H and O=C groups are linked by an intra\u00admolecular hydrogen bond. In the crystal, C\u2014H\u22efO hydrogen bonds and C\u2014H\u22ef\u03c0 inter\u00adactions occur.The reaction of 3-C \u00c5                           b = 10.5851 (13) \u00c5                           c = 12.7122 (14) \u00c5                           \u03b2 = 99.111 (17)\u00b0V = 1362.6 (4) \u00c53                                                   Z = 4                           K\u03b1 radiationMo \u22121                        \u03bc = 0.08 mmT = 295 K                           0.21 \u00d7 0.14 \u00d7 0.11 mm                                 Enraf\u2013Nonius CAD-4 diffractometeret al., 1968T                           min = 0.965, T                           max = 0.987Absorption correction: \u03c8 scan (North 2821 measured reflections2668 independent reflectionsI > 2\u03c3(I)1833 reflections with R                           int = 0.029                           3 standard reflections every 200 reflections  intensity decay: none                                 R[F                           2 > 2\u03c3(F                           2)] = 0.050                           wR(F                           2) = 0.140                           S = 1.04                           2668 reflections174 parametersH-atom parameters constrainedmax = 0.19 e \u00c5\u22123                        \u0394\u03c1min = \u22120.15 e \u00c5\u22123                        \u0394\u03c1                                 CAD-4 Software (Enraf\u2013Nonius, 1989CAD-4 Software; data reduction: XCAD4 (Harms & Wocadlo, 1995SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008PLATON (Spek, 2009WinGX (Farrugia, 1999Data collection: 10.1107/S1600536810041127/ng5038sup1.cif            Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536810041127/ng5038Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:"}
+{"text": "In the crystal, mol\u00adecules are stacked along the a-axis direction by C\u2014H\u22ef\u03c0 inter\u00adactions.In the title compound, C For dep al. 1964; Harkins al. 1956; Haga  \u00c5b = 15.4102 (17) \u00c5c = 20.953 (2) \u00c5\u03b2 = 95.363 (8)\u00b0V = 1522.6 (3) \u00c53Z = 4K\u03b1 radiationMo \u22121\u03bc = 0.09 mmT = 296 K0.2 \u00d7 0.2 \u00d7 0.2 mmBruker APEXII CCD diffractometerTmin = 0.984, Tmax = 0.984Absorption correction: multi-scan 2355 reflections with Rint = 0.027R[F2 > 2\u03c3(F2)] = 0.038wR(F2) = 0.103S = 0.933133 reflections208 parameters1 restraintH-atom parameters constrainedmax = 0.14 e \u00c5\u22123\u0394\u03c1min = \u22120.17 e \u00c5\u22123\u0394\u03c1APEX2  used to solve structure: SHELXS97  I, global. DOI: 10.1107/S1600536814005947/bq2393Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536814005947/bq2393Isup3.cmlSupporting information file. DOI: 992223CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:"}
+{"text": "An intra\u00admolecular C\u2014H\u22efN hydrogen bond is observed. In the crystal, mol\u00adecules are stacked along the a axis by weak C\u2014H\u22ef\u03c0 inter\u00adactions.In the title compound, C For the al. 1997; Wong &  al. 1997, Kelland al. 1997. For rel al. 2007, 2014 \u25b6.24H25N3CMr = 355.47Monoclinic, a = 5.3470 (3) \u00c5b = 21.0622 (12) \u00c5c = 17.0379 (9) \u00c5\u03b2 = 97.699 (3)\u00b0V = 1901.50 (18) \u00c53Z = 4K\u03b1 radiationMo \u22121\u03bc = 0.07 mmT = 296 K0.25 \u00d7 0.20 \u00d7 0.15 mmBruker APEXII CCD diffractometerTmin = 0.982, Tmax = 0.989Absorption correction: multi-scan 3056 reflections with Rint = 0.026R[F2 > 2\u03c3(F2)] = 0.052wR(F2) = 0.150S = 1.043741 reflections254 parametersH-atom parameters constrainedmax = 0.24 e \u00c5\u22123\u0394\u03c1min = \u22120.17 e \u00c5\u22123\u0394\u03c1APEX2  used to solve structure: SHELXS97  I, global. DOI: 10.1107/S1600536814007934/rz5115Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536814007934/rz5115Isup3.cmlSupporting information file. DOI: 996309CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:"}
+{"text": "The loss of tumour suppressor gene function is a hallmark of malignant transformation and can occur by a variety of genetic and/or epigenetic alterations. We have previously characterised p38\u03b4 mitogen-activated protein kinase (MAPK) as a tumour suppressor in oesophageal squamous cell carcinoma (OESCC) and outlined how loss of p38\u03b4 MAPK expression promotes increased proliferation and migration, as well as reduced chemosensitivity. Our aim was to investigate the underlying molecular causes of loss of p38\u03b4 MAPK expression in OESCC. Sequence analysis of DNA from p38\u03b4 MAPK positive and p38\u03b4 MAPK negative OESCC cell lines was used to investigate potential loss of function causing mutations. Epigenetic control of p38\u03b4 expression in OESCC was examined using methylation-specific PCR and sequencing of bisulfite-converted DNA. We did not identify any mutations in the MAPK13 sequence in OESCC cell lines which lack p38\u03b4 MAPK expression. However, we identified a differential pattern of methylation between p38\u03b4 MAPK positive and p38\u03b4 MAPK negative cell lines. We outline here for the first time differential MAPK13 promoter methylation in OESCC. Our results suggest that epigenetic alterations are responsible, in part, for the suppression of p38\u03b4 MAPK expression and promotion of tumourigenesis in OESCC. We have previously documented the differential expression of p38\u03b4 mitogen activated protein kinase (MAPK) in oesophageal squamous cell carcinoma (OESCC) and the consequences of p38\u03b4 MAPK expression on OESCC tumourigenicity and response to cytotoxic drugs. Loss of p38\u03b4 MAPK expression contributes to a pro-oncogenic phenotype in OESCC and affords a level of resistance to conventional cisplatin and 5-fluorouracil treatment ,2. Re-inAt the genomic level, point mutations, insertions, and deletions of genetic material are common mechanisms by which expression of tumour suppressor genes are inactivated. The somatic mutation rate for OESCC is higher than in breast carcinoma, with C.G \u2192 T.A transitions the most common, followed by C.G \u2192 G.C transversions ,5. Pointi.e., CpG dinucleotides and is essential for normal maintenance of tissue-specific gene expression [INK4b and p14ARF, are frequently hypermethylated [Cancer genomes are also characterised by epigenetic changes such as aberrant DNA methylation, in addition to mutations and chromosomal abnormalities ,10,11. Dpression . In comppression ,14,15. Tpression . Hypermepression ,18,19,20thylated ,16. Notathylated ,22. Restthylated .In this study mRNA and gDNA sequences from p38\u03b4 MAPK protein positive and negative cell lines were compared in order to identify any of the genetic abnormalities discussed above, which may explain the differential loss of p38\u03b4 MAPK expression. Possible epigenetic regulation of p38\u03b4 MAPK expression was investigated using methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) analysis of CpG islands in the MAPK13 promoter region.p < 0.01) faster than the p38\u03b4 positive cell lines (p < 0.01) faster than their p38\u03b4 positive counterparts (C).We have previously identified that p38\u03b4 MAPK plays a key role in OESCC cell proliferation and migration using a small number of cell lines . We havell lines A,B. Of nterparts C.p < 0.01) difference between the two cell lines (p < 0.001) down-regulated in p38\u03b4 MAPK protein negative cell lines KE-3, KE-8, and KYSE-70 when compared with both KE-6 and KYSE-450 p38\u03b4 MAPK protein positive cells (p < 0.001 and ** p < 0.01) decreases from KE-6 and KYSE-450 expression  in KE-3, KE-8, KYSE-70, and OC-1 cells (Bisulfite sequencing PCR (BSP) was used for further validation and analysis of MAPK13 promoter methylation density in OESCC. The methylation status of four individual CpG sites within a MAPK13 promoter region CpG island was analysed. Comparison of DNA sequence reads of PCR products with in silico bisulfite-converted MAPK13 reference sequence (NC_000006.12) identified differential CpG methylation in OESCC B. UnmethOf the four MAPK13 promoter region CpG sites analysed by BSP, only one was found to be methylated in KE-6 and KYSE-450 p38\u03b4 MAPK protein positive OESCCs B,C. ConvCell culture- The KE oesophageal cancer cell lines  [, Japan) ,24,25 as, Japan) . OC-1 an, Japan)  were culProliferation assay\u2014Cells were plated at a density of 3 \u00d7 104 cells/well in a six-well tissue culture plate. Cell viability was assessed by trypan blue (0.4% w/v) exclusion assay at the indicated times [ed times .Boyden chamber cell migration assay\u2014Cells were plated in starvation medium at a density of 3 \u00d7 104 cells/well into a 96-well plate of the upper chamber. The bottom chamber contained 10% FCS as the chemoattractant. Cells were left migrate for 24 h through the matrigel filter (8 mm). Migrated cells were treated with MTT -2,5-diphenyltetrazolium bromide) (5 mg/mL) and absorbance read at 540 nm to calculate viable cell numbers as previously described [PCR\u20141575bp and 1605bp fragments of p38\u03b4 MAPK mRNA, corresponding to positions 111\u20131686 and 82\u20131686 respectively of NCBI Reference Sequence NM_002754.4 were amplified from cellular cDNA using oligonucleotide primers P003F (5\u2032-CGAGATCGGGTGCCCGGGAT-3\u2032) or P002F (5\u2032-CCGGAAAAAGGGCTTCTACAA-3\u2032) and P001R (5\u2032-CCGCCACAAGCTAAAAAGAG-3\u2032).Quantitative rt-PCR\u2014p38\u03b4 MAPK mRNA expression was quantitated using a Qiagen QuantiFast\u00ae MAPK13 Probe Assay with one-step rt-PCR and simultaneous detection of GAPDH according to manufacturer\u2032s instructions.Methylation analysis\u2014The methylation status of DNA was determined using sodium bisulfite conversion with an EpiMark\u00ae Bisulfite Conversion Kit  according to manufacturer\u2032s instructions. Methylation-specific PCR (MSP) is a method of assessing the methylation status of a group of CpG sites using individual pairs of primers specific for methylated (M) versus unmethylated (U) DNA [ (U) DNA . MSP pri (U) DNA . For bisTumour suppressor gene function can be lost in a variety of ways in order for cancer cells to gain the capacity for uncontrolled proliferation, migration, and escape from apoptosis. These include somatic mutations, including insertions, deletions or point mutations, and/or epigenetic changes such as DNA methylation or histone deacetylation. p38\u03b4 MAPK displays tumour suppressor functions in OESCC\u2014its loss promotes proliferation, migration, anchorage-independent growth, and resistance to conventional chemotherapy ,2. p38\u03b4 The results presented here indicate that this loss of protein expression cannot be attributed to a genetic mutation or deletion. p38\u03b4 MAPK mRNA is expressed in all OESCC cell lines, regardless of p38\u03b4 MAPK protein status. However, differential mRNA expression levels were observed between p38\u03b4 MAPK protein positive and protein negative cell lines. The highest levels of p38\u03b4 MAPK mRNA expression were observed in p38\u03b4 protein positive cell lines KE-6 and KYSE-450. Significantly lower levels of p38\u03b4 MAPK mRNA were detected in p38\u03b4 MAPK protein negative cell lines. This suggests that the absence of p38\u03b4 MAPK protein expression in these cells may be attributed to p38\u03b4 MAPK mRNA expression failing to reach the threshold required to achieve a detectable level of protein synthesis.p < 0.001), a decreased incidence of DNA methylation was detected by both MSP and BSP. OC-1 cells also expressed significantly higher (*** p < 0.001) levels of p38\u03b4 MAPK mRNA than KE-3, KE-8, and KYSE-70 cells despite the fact that they are also p38\u03b4 MAPK protein negative. Interestingly, however, the MAPK13 promoter was also found to be hypermethylated in this cell line when compared with p38\u03b4 MAPK protein positive cells (two CpG sites methylated versus one CpG site methylated). The significance of these results is that there appears to be an inverse correlation therefore between MAPK13 promoter methylation and p38\u03b4 MAPK mRNA expression in OESCC. Of note, methylation of the specific CpG sites examined here may not be the critical determinants of MAPK13 expression but rather these results reflect a general pattern of MAPK13 promoter methylation. This would explain why the OE21 cell line does not appear to fit this model of p38\u03b4 MAPK epigenetic regulation\u2014the loss of p38\u03b4 MAPK expression in the OE21 cell line may be caused by MAPK13 promoter methylation at a critical CpG site not included in this analysis.Our results show that the downregulation of p38\u03b4 MAPK mRNA expression in KE-3, KE-8, KYSE-70, and OC-1 cells is most likely associated with MAPK13 promoter hypermethylation. The MAPK13 promoter regions of the OESCC cell lines with the lowest levels of p38\u03b4 MAPK mRNA expression  are highly methylated, as demonstrated by both MSP and BSP analysis. Conversely, in p38\u03b4 MAPK protein positive cell lines KE-6 and KYSE-450, in which p38\u03b4 MAPK mRNA expression was significantly higher  is used as a treatment for myelodysplastic syndromes and leukaemia ,30. PoteIn conclusion, promoter hypermethylation of the MAPK13 gene in KE-3, KE-8, KYSE-70, and OC-1 OESCC cells inhibits transcription of p38\u03b4 MAPK, resulting in down-regulation of p38\u03b4 MAPK mRNA expression. This decrease in p38\u03b4 mRNA correlates with a loss of p38\u03b4 MAPK protein expression that is associated with increased proliferation and migration in OESCC."}
+{"text": "While the p38 MAPK family as a whole has been implicated in a wide variety of biological processes, a specific role for p38\u03b4 MAPK in cellular signalling and its contribution to both physiological and pathological conditions are presently lacking. Recent emerging evidence, however, provides some insights into specific p38\u03b4 MAPK signalling. Importantly, these studies have helped to highlight functional similarities as well as differences between p38\u03b4 MAPK and the other members of the p38 MAPK family of kinases. In this review we discuss the current understanding of the molecular mechanisms underlying p38\u03b4 MAPK activity. We outline a role for p38\u03b4 MAPK in important cellular processes such as differentiation and apoptosis as well as pathological conditions such as neurodegenerative disorders, diabetes, and inflammatory disease. Interestingly, disparate roles for p38\u03b4 MAPK in tumour development have also recently been reported. Thus, we consider evidence which characterises p38\u03b4 MAPK as both a tumour promoter and a tumour suppressor. In summary, while our knowledge of p38\u03b4 MAPK has progressed somewhat since its identification in 1997, our understanding of this particular isoform in many cellular processes still strikingly lags behind that of its counterparts.p38 It was isolated as a 38\u2009kDa protein rapidly tyrosine phosphorylated in response to lipopolysaccharide stimulation [\u03b2 MAPK in the same year, p38\u03b3 MAPK in 1996, and lastly p38\u03b4 MAPK in 1997 [ S. cerevisiae HOG1 gene, an important component of osmoregulation and the cell cycle, was found to be a homologue of p38 MAPK [\u03b3) arose from a tandem duplication of MAPK11 (p38\u03b2) on chromosome 22 while MAPK14 (p38\u03b1) and MAPK13 (p38\u03b4) subsequently resulted from a single segmental duplication of the MAPK11-MAPK12 gene unit on chromosome 6 [\u03b1 and p38\u03b2 MAPKs, each isoform is an important kinase in its own right with distinct cellular functions. This review aims to highlight components of the previously neglected p38\u03b4 MAPK signalling pathway and emphasises recent progress in our understanding of p38\u03b4 MAPK involvement in diverse physiological as well as pathological processes.The first and now archetypal member of the p38 MAPK family, p38mulation  and as a in 1997 \u20139. The pp38 MAPK . This co\u03b1 and p38\u03b2 MAPK signalling, functions, and substrates. Both p38\u03b1 and p38\u03b2 MAPK are highly sensitive to inhibition by SB203580, SB202190, and newer compounds such as L-167307 [\u03b4 MAPK is insensitive to inhibition by pyridinyl-imidazole compounds has hindered its study in cellular events [\u03b1 and p38\u03b2 MAPK has been identified as the major determinant for imidazole inhibitor specificity as it orientates the drug to interact with His107 and Leu108 thereby preventing ATP binding [\u03b4 MAPK is a methionine (Met), the large side chain of which prevents binding of these inhibitors. In fact, substitution of Met106 in p38\u03b4 MAPK with Thr was found to confer some sensitivity to inhibition by SB203580 [\u03b1 MAPK mutants in which Thr106 is replaced with Met displayed reduced sensitivity to inhibition by SB203580 [\u03b4 MAPK specific inhibitor has been identified to date. Although the diaryl urea compound BIRB796 allosterically inhibits p38\u03b4 MAPK at high concentrations, it is also a powerful inhibitor of p38\u03b1, -\u03b2, and -\u03b3 MAPK [\u03b4 MAPK specific signalling pathways, the possible influence of the other p38 MAPK isoforms, in particular p38\u03b3 MAPK, must be considered when interpreting any results.The use of pyridinyl-imidazole inhibitors has largely driven the advancement in our understanding of p38L-167307 . In cont binding . The equSB203580 . ConversSB203580 . It is u\u03b4 MAPK shares highly similar protein sequences with the other p38 MAPK isoforms. It displays 61%, 59%, and 65% amino acid identity to p38\u03b1, -\u03b2, and -\u03b3 MAPKs, respectively [\u03b4 MAPK and the other p38 MAPK family members can be observed in the ATP binding pocket. This has consequences for inhibitor sensitivity and contributes to substrate specificity. On the other hand, the greatest sequence similarities lie in the highly conserved kinase domains, where the four isoforms share >90% amino acid identity [\u03b4 MAPK possesses a TGY dual phosphorylation motif which is the hallmark of p38 MAPKs and is conserved among all known mammalian p38 isoforms [\u03b4 MAPK has a distinct distribution profile in human tissue that is relatively limited compared to that of p38\u03b1 and p38\u03b2 MAPK isoforms which are largely ubiquitously expressed [\u03b4 mRNA have been detected in endocrine tissues such as salivary, pituitary, prostate, and adrenal glands, while more modest levels are expressed in the stomach, colon, trachea, pancreas, skin, kidney, and lung [\u03b4 MAPK activation in these cell types, distinct from that of the other p38 family members.Unsurprisingly, p38ectively . Differeidentity . Within isoforms \u20139, 18. p\u03b4 MAPK amino acid sequence is 92% identical to the human sequence and the adult mouse displays a broadly similar pattern of p38\u03b4 MAPK expression to that seen in human tissue, that is, lung, testis, kidney, and gut epithelium [\u03b4 MAPK expression varies at different stages in the developing mouse embryo. At 9.5 days it is primarily expressed in the developing gut and septum transversum, while by 15.5 days its expression expands to most developing epithelia [\u03b4 MAPK has a role in embryonic development. However, knock-out of p38\u03b4 MAPK results in mice which are both viable and fertile and exhibit a normal phenotype [\u03b2- and p38\u03b3-null mice as well as p38\u03b3/p38\u03b4 double knockout (KO) mice are also phenotypically normal [\u03b1 MAPK is embryonic lethal at day 10.5\u201311.5 [\u03b1, -\u03b2, or -\u03b3 compensating for the loss of p38\u03b4 MAPK activity during development. However, it appears that p38\u03b1 MAPK plays a critical role in early development where its loss cannot be overcome.The murine p38ithelium . Murine pithelia . This suhenotype . Moreovey normal , 20, gen0.5\u201311.5 . Functio\u03b1, -\u03b2, and -\u03b3 MAPK isoforms are activated by alterations in the physical and chemical properties of the extracellular environment with diverse triggers including environmental stress signals, inflammatory cytokines, and mitogenic stimuli [\u03b4 MAPK, a similar activation profile has been defined for p38\u03b4 MAPK [\u03b1, and IL-1. Despite their similar activation profiles, differences in the levels of activation of p38\u03b4 MAPK and the other p38 MAPK isoforms have been reported. For example, while hyperosmolarity appears to stimulate both p38\u03b1 and p38\u03b4 to a similar degree, under hypoosmotic conditions, p38\u03b1 MAPK is more strongly activated than p38\u03b4 MAPK [\u03b2 activated kinase 1), ASK1 , TAO (thousand-and-one amino acid), DLK1  MEKKs, and ZAK1 (leucine zipper and sterile-\u03b1 motif kinase 1)  . To datederstood \u201328. Furtderstood , 30. Thi\u03b4 MAPK is unique as it can be activated by four separate MKKs: the p38 MAPK specific MKK3 and MKK6 and also the JNK MKKs-4 and -7 [\u03b4 MAPK activation in different cell types and under diverse conditions is lacking. Current evidence suggests that activation of p38\u03b4 MAPK is significantly influenced by both the nature and the strength of the stimulus as well as the cell type involved. This may be the result of varying levels of expression of upstream components of the MAPK signalling cascade in different cell types. For example, MKK3 is the major direct activator of p38\u03b4 MAPK phosphorylation in response to UV radiation, hyperosmotic shock, and TNF\u03b1 in mouse embryonic fibroblast (MEF) cells [\u03b4 MAPK activation in response to transforming growth factor-\u03b21 (TGF-\u03b21) as MKK3 deficiency impairs endogenous p38\u03b4 activation by TGF-\u03b21 in murine glomerular mesangial cells [\u03b4 MAPK in KB (HeLa) cells subjected to IL-1, anisomycin, or osmotic stress [\u03b4 MAPK in 293T cells under peroxide stress, mediated by the scaffolding action of islet brain-2 [\u03b4 MAPK activation is the likelihood that, in some cases, the cooperation of two MKKs may be necessary. While MKK4 preferentially phosphorylates JNK on Tyr, MKK7 preferentially phosphorylates JNK on Thr [\u03b4 MAPK on both the Tyr and the Thr residues. Interestingly, two reports outline a MKK-independent mechanism of activation for p38\u03b1 MAPK via autophosphorylation [\u03b4 mutants [\u03b4 MAPK isoform.The upstream direct activators responsible for dual phosphorylation of the p38 MAPK TGY motif are the MAPK kinases (MKKs). p384 and -7 , 18. HowF) cells . It alsoal cells . On the c stress . Further brain-2 . FurtherK on Thr \u201336. Therrylation , 38. Whi mutants , 40, no \u03b4 MAPK phosphorylation is the strength and duration of the activation signal. p38\u03b4 MAPK activation is largely transient with activation and downregulation occurring within minutes of stimulation [\u03b4 MAPK in HEK293FT cells, it does not interact with p38\u03b4 MAPK in the NIH3T3 cell line [\u03b4 MAPK phosphorylation as okadaic acid (OA), a PP1/PP2A inhibitor, causes increased p38\u03b4 MAPK activity in human epidermal keratinocytes [An important factor in determining the biological consequences of p38mulation . This isell line , 42. Theinocytes .\u03b4 MAPK substrate specificity overlaps to some extent with that of p38\u03b1, -\u03b2, and -\u03b3 MAPKs, there are a number of notable differences. Common substrates of p38 MAPKs include MBP, PHAS-1, and transcription factors ATF2, SAP1, Elk-1, and p53  [\u03b4 MAPK is responsible for driving the translation of proteins associated with stress responses. Consistent with this hypothesis is the observation that the MKK3/6-p38\u03b4 MAPK-eEF2K pathway in myeloid cells is implicated in the production of the proinflammatory cytokine TNF\u03b1 in bacterial LPS induced acute liver disease [\u03b4 MAPK can potentially be significantly different to those of the other p38 MAPK isoforms.In response to anisomycin stimulation, p38 (eEF2K) . Phospho (eEF2K) . This su disease . These d\u03b4 MAPK in 1997 it has been implicated in a range of diverse physiological events, namely, differentiation, apoptosis, and cytokine production , calcium, OA, or green tea polyphenol corresponds with increased involucrin promoter activity, mRNA, and protein expression, as well as increased levels and activity of AP1 and C/EBP transcription factors [\u03b1/\u03b2 MAPK inhibitor. In addition p38\u03b3 MAPK is poorly expressed in keratinocytes [\u03b4 MAPK. Involucrin expression can also be further upregulated in keratinocytes coexpressing p38\u03b4 MAPK and PKC\u03b7, -\u03b4 or \u03b5 isoforms [\u03b1 MAPK [\u03b4 MAPK in keratinocyte differentiation was recently identified. p38\u03b4 MAPK can regulate expression of ZO-1, an epidermal tight junction membrane protein associated with keratinocyte differentiation [\u03b4 MAPK results in depletion of ZO-1 protein in calcium induced differentiating keratinocytes while other junction proteins remain unaffected [\u03b4 MAPK plays in keratinocyte differentiation, it is no surprise that aberrant p38\u03b4 MAPK signalling has been implicated in the pathogenesis of psoriasis. Expression of the MAPK13 gene is commonly upregulated in psoriasis [\u03b4 (as well as -\u03b1 and -\u03b2) MAPK activity has been detected in psoriatic lesions compared to nonlesional psoriatic skin. After treatment for psoriasis, phosphorylated p38 MAPK levels return to those of uninvolved skin [A number of different studies have identified a role for p38ntiation \u201360. p38\u03b4 factors , 59, 61. [\u03b1 MAPK , 63. Thintiation . Inhibitaffected . Psoriassoriasis . Furtherved skin .\u03b4 MAPK is also implicated in hematopoiesis. In human primary erythroid cells, p38\u03b4 MAPK mRNA is only expressed in late-stage differentiation where along with p38\u03b1 MAPK it is increasingly activated [\u03b4 MAPK in erythrocyte membrane remodelling and enucleation. Interestingly, an increase in p38\u03b4 MAPK mRNA and protein expression is observed as blood monocytes differentiate to macrophages [\u03b4 MAPK in functions gained by mature macrophages. A possible candidate is phagocytosis given that the microtubule associated protein stathmin is such a strong p38\u03b4 MAPK substrate.Further to its role in keratinocyte differentiation p38ctivated . This marophages . This su\u03b4 MAPK has been identified as a component of differentiation in bone repair [\u03b4 MAPK protein and mRNA is highly expressed in monoosteophils compared to undifferentiated monocytes. Monocytes from p38\u03b4 MAPK KO mice are incapable of this differentiation, suggesting a critical role for p38\u03b4 MAPK in this process [Most recently, p38e repair . In bone process .\u03b4 MAPK has also been identified as a regulator of keratinocyte apoptosis. This dual functional role may be attributed to the overlap of differentiation and apoptosis signalling pathways [\u03b4 MAPK enhances this OA driven apoptotic morphology. This response is specific to p38\u03b4 MAPK activation as it occurred in the presence of the p38\u03b1/\u03b2 MAPK inhibitor SB203580 [\u03b4 MAPK coexpressed with either MEK6 or PKC\u03b4, both upstream p38 MAPK activators, elicited an apoptotic response similar to that induced by OA but in the absence of an external stimulus. This was also independent of SB203580, again ruling out a contribution from other p38 MAPK isoforms [\u03b4 MAPK activation and inactivation of the proproliferative MAPK ERK1/2 were observed with OA stimulation and PKC\u03b4/p38\u03b4 MAPK coexpression [\u03b4/p38\u03b4 MAPK mediated apoptosis [\u03b4 MAPK is essential in determining keratinocyte fate. In regulating this balance, p38\u03b4 MAPK and ERK1/2 form a complex that is translocated to the nucleus upon stimulation by PKC\u03b4. This nuclear localisation facilitates ERK1/2 inactivation by nuclear phosphatases, while maintaining p38\u03b4 MAPK activation [As well as its significant role in keratinocyte differentiation, p38pathways . As wellpathways , OA simupathways . OverexpSB203580 . Furtherisoforms . Interespression , 61, 71.\u03b4 MAPK in apoptosis has recently been demonstrated in vivo using p38\u03b4 MAPK KO mice. Mice deficient in p38\u03b4 MAPK displayed a fivefold lower rate of pancreatic \u03b2 cell death in response to oxidative stress than WT mice and are afforded protection against insulin resistance induced by a high-fat diet [\u03b4 MAPK to the pathogenesis of diabetes mellitus, a disease characterised by reduced insulin sensitivity and a decrease in insulin-producing pancreatic \u03b2 cells [\u03b4 MAPK specifically has also been implicated in the regulation of insulin secretion. Phosphorylation by p38\u03b4 MAPK negatively regulates the activity of protein kinase D1 (PKD1), a known positive regulator of neuroendocrine cell secretion [\u03b2 cells lacking p38\u03b4 MAPK. As p38\u03b4 MAPK is normally quite highly expressed in the pancreas this can contribute to heightened insulin secretion and improved glucose tolerance in p38\u03b4 MAPK-null mice [\u03b4 MAPK plays in integrating insulin secretion and survival of pancreatic \u03b2 cells makes it an attractive potential therapeutic target for the treatment of human diabetes.A role for p38fat diet . This wo \u03b2 cells . Increas \u03b2 cells , 74. p38ecretion . Thus, pull mice . The piv\u03b1 MAPK was discovered was via its identification as a regulator of proinflammatory cytokine biosynthesis [\u03b4 MAPK KO mouse models has focused on identifying specific roles for p38\u03b4 MAPK in inflammation. A study of p38\u03b4 MAPK KO mice as well as myeloid-restricted deletion of p38\u03b4 MAPK in mice has shown that p38\u03b4 MAPK is required for the recruitment of neutrophils to sites of inflammation [\u03b4 MAPK and its downstream target PKD1 conversely regulate PTEN activity to control neutrophil extravasation and chemotaxis. The accumulation of neutrophils at inflammatory sites is known to trigger inflammation-induced acute lung injury (ALI) which can cause acute respiratory distress syndrome (ARDS), a condition with a high mortality rate [\u03b4-PKD1 signalling may play an important role in both ALI and ARDS in humans.One of the pathways by which p38ynthesis . Thus, iammation . p38\u03b4 MAity rate . Therefo\u03b4 MAPK  is expressed in the synovium of rheumatoid arthritis patients its level of activation is lower than that of the four other p38 MAPK isoforms [\u03b4 MAPK as an essential component of joint damage in a collagen-induced model of arthritis. p38\u03b3/\u03b4\u2212/\u2212 mice displayed reduced arthritis severity compared to WT mice [\u03b2 and TNF\u03b1 as well as a reduction in T cell proliferation, IFN\u03b3, and IL-17 production. Lack of either p38\u03b3 or p38\u03b4 MAPK alone yielded intermediate effects, suggesting significant roles for both isoforms in arthritis pathogenesis.Rheumatoid arthritis is a typical example of an inflammatory disease involving chronic synthesis of proinflammatory cytokines which result in synovial hyperplasia and joint destruction . While pisoforms . Despite WT mice . The dec\u03b4 MAPK has been implicated in the signalling pathway responsible for controlling IL-13 driven excess mucus production. Increased MAPK13 gene expression is evident in the lungs of patients with severe COPD [\u03b4 MAPK blocked mucus production by IL-13 in human airway epithelial cells [\u03b4 MAPK inhibitors become clinically available.Proinflammatory cytokines also play a significant role in the pathogenesis of inflammatory airway diseases, including asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. While increased mucus production is linked to the morbidity and mortality of such diseases the underlying molecular mechanisms remain somewhat unclear . The criere COPD . Novel ial cells . Thus, i\u03b1 MAPK, has been identified as both a tumour promoter [\u03b4 MAPK in cancer development and progression. Like p38\u03b1 MAPK, p38\u03b4 MAPK would also appear to have both pro- and antioncogenic roles, depending on the cell type studied.In recent years, the function of the p38 MAPK signalling pathway in malignant transformation has been intensively studied. As a result, the best characterised isoform, p38promoter \u201386 and apromoter \u201389. Rece\u03b4 MAPK as a potential tumour promoter is based on the evidence that p38\u03b4 MAPK expression and activation are significantly increased in a variety of carcinoma cell lines such as human primary cutaneous squamous carcinoma cells [\u03b4 MAPK was first shown to promote a malignant phenotype (over eight years ago) in head and neck squamous cell carcinoma (HNSCC) [\u03b4 MAPK impaired the ability of cutaneous HNSCC cells to implant in the skin of immunodeficiency mice as well as inhibiting the growth of xenografts [Interest in p38ma cells , head anma cells , cholangma cells . p38\u03b4 MA (HNSCC) . It was  (HNSCC) , 91. Mornografts .\u03b4 MAPK-null mice have been utilised to demonstrate that p38\u03b4 MAPK is required for the development of multistage chemical skin carcinogenesis in vivo. When compared with WT mice, p38\u03b4 MAPK-deficient mice displayed reduced susceptibility to 7, 12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate induced skin carcinoma with a significant delay in tumour development [\u03b4 MAPK promotion of proliferation via Stat3 may be a significant mechanism in the promotion of carcinogenesis by p38\u03b4 MAPK. Similarly, p38\u03b4 MAPK KO mice have reduced susceptibility to development of K-ras driven lung tumorigenesis. Compared with WT mice, p38\u03b4\u2212/\u2212/K-RasG12D+/\u2212 mice displayed significantly decreased tumour numbers, average tumour volume, and total tumour volume per lung [\u03b1 MAPK-deficient mice which display hyperproliferation of lung epithelium and increased K-Ras-induced lung tumour development [p38elopment . Furtherelopment . This deelopment . The ERKelopment , 94. Staelopment . It is aelopment , 97. Theper lung . This iselopment . This hi\u03b4 MAPK in promoting cancer progression has most recently been demonstrated in cholangiocarcinoma (CC) [\u03b4 MAPK expression is upregulated in CC when compared with normal biliary tract tissue. Knockdown, however, of p38\u03b4 MAPK expression by siRNA transfection significantly inhibited motility and invasiveness of CC cells. In contrast, overexpression of p38\u03b4 MAPK in these cells results in enhanced invasive behaviour. Significantly, p38\u03b4 MAPK may prove to be a useful marker for the differential diagnosis of CC over hepatocellular carcinoma where it lacks expression [Further evidence for a specific role of p38oma (CC) . p38\u03b4 MApression .\u03b4 MAPK as a tumour promoter an increasing number of reports since 2011 outline its activity as a tumour suppressor. The first indication of a tumour suppressive role for p38\u03b4 MAPK was observed in mouse embryonic fibroblasts (MEF). p38\u03b4\u2212/\u2212 (and p38\u03b3\u2212/\u2212) MEFs displayed increased cell motility compared to WT cells [\u03b4\u2212/\u2212 MEFs continued to grow, forming foci rather than a monolayer [\u03b4 MAPK in the control of oesophageal squamous cell carcinoma (OESCC) migration, invasion and contact inhibition, processes which are crucial for the progression of primary tumours to distant metastases [\u03b4 MAPK into OESCC cells which lack endogenous expression significantly impaired cell proliferation, migration, and invasion as well as significantly reducing the number of colonies formed on soft agar compared to WT. These effects were further enhanced in cells transfected with a constitutively active form of p38\u03b4 MAPK [\u03b4 MAPK expression appears to influence the chemosensitivity of OESCC to apoptosis. Our recent study indicates that OESCC cells expressing p38\u03b4 MAPK are significantly more sensitive to cisplatin and 5-fluorouracil combination therapy than p38\u03b4 MAPK-deficient cells [\u03b4 MAPK may be a useful predictor of response to chemotherapy in OESCC patients. Further supporting the hypothesis that loss of p38\u03b4 MAPK confers a survival advantage, p38\u03b4 MAPK expression was found to be downregulated in brain metastases of triple-negative breast cancer (TNBC). Abolition of p38\u03b4 MAPK expression in TNBC induced cell growth, while overexpression of p38\u03b4 MAPK in brain metastases reduced growth rates [In contrast to the relatively well characterised role of p38WT cells . Furtheronolayer . This deonolayer . Our owntastases . Reintro38\u03b4 MAPK . Furthernt cells . These fth rates .\u03b4 MAPK mRNA and protein expression. Melanoma cell lines displaying MAPK13 gene promoter methylation do not express significant levels of p38\u03b4 MAPK when compared to fibroblasts, melanocytes, and melanoma cell lines with unmethylated MAPK13 promoters. Furthermore, treatment of melanoma cells with the demethylating agent 5-aza-2\u2032-deoxycytidine significantly increases the expression of the MAPK13 gene [\u03b4 MAPK expression in melanoma cells with MAPK13 hypermethylation suppresses cell proliferation. The effect was further enhanced upon expression of a constitutively active form of p38\u03b4 MAPK. Interestingly, however, overexpression of p38\u03b4 MAPK or its constitutively active form in cells in which MAPK13 was not epigenetically silenced only marginally affected proliferation [Cancer genomes are increasingly associated with epigenetic alterations whereby tumour suppressor genes exhibit promoter hypermethylation. Interestingly, hypermethylation of the MAPK13 gene promoter region has recently been characterised in both malignant pleural mesothelioma  and primK13 gene , 105. Imferation .\u03b4 MAPK is a unique stress-responsive protein kinase. It is mainly activated by environmental stresses, including UV radiation, osmotic shock, and oxidative stress, to illicit an adaptive response within the cell. This is mediated through phosphorylation of substrates involved in cytoskeleton organisation such as tau and stathmin, as well as transcription factors responsible for the expression of stress-responsive genes [\u03b1 MAPK (and to an extent p38\u03b2 MAPK) studies cannot be automatically applied to the p38\u03b3 and p38\u03b4 MAPK isoforms due to their different expression patterns, substrate specificities, and sensitivity to chemical inhibitors. Studies carried out in the last few years have led to some small advances in our knowledge of the regulation of p38\u03b4 MAPK and its physiological roles. In particular, the development of p38\u03b4 MAPK KO mouse models has yielded a greater understanding of the consequences of p38\u03b4 MAPK signalling in vivo. Roles for p38\u03b4 MAPK in important cellular processes such as differentiation and apoptosis have been identified [\u03b4 MAPK is now implicated in a variety of pathological conditions including inflammatory diseases, diabetes, and cancer [\u03b4 MAPK may now be considered as a potential therapeutic target for the treatment of these disorders.p38ve genes , 107. Sive genes . Researcentified , 72. As d cancer , 92, 98.\u03b4 MAPK in a wide range of human diseases should strengthen future research interest in this isoform. The main limiting factors to the further study of p38\u03b4 MAPK functions, however, are the lack of specific inhibitors and activators. Fuelled by the prospect of therapeutic benefit for patients with diabetes or inflammatory disease, for example, the search for more potent and specific inhibitors of p38\u03b4 MAPK is ongoing [\u03b4 MAPK functions in the absence of involvement from other p38 MAPK isoforms. This in turn may identify other diseases where p38\u03b4 MAPK could be a potential therapeutic target. In this review we also present important and interesting observations which suggest that focusing on identification of specific p38\u03b4 MAPK activators is also warranted. This may in the future translate to the development of novel therapeutic strategies for patients with OESCC or melanoma. Whether considering the possible therapeutic benefits of p38\u03b4 MAPK inhibitors or activators it is important to heed the diversity and important role(s) of p38\u03b4 MAPK signalling in normal physiological processes. In conclusion, uncovering some of the physiological as well as pathological roles of p38\u03b4 MAPK since its discovery almost twenty years ago has been somewhat successful. However, based on our current knowledge continued focused research on this particular isoform is necessary if p38\u03b4 is to translate into a novel therapeutic target for a range of diverse human diseases.The implication of p38 ongoing . These m"}
+{"text": "We determine that prestin itself possesses a stretch-sensitive, non-selective conductance that is largest in the presence of thiocyanate ions. This conductance is independent of the anion transporter mechanism. Prestin has been modeled, based on structural data from related anion transporters (SLC26Dg and UraA), to have a 7\u2009+\u20097 inverted repeat structure with anion transport initiated by chloride binding at the intracellular cleft. Mutation of residues that bind intracellular chloride, and salicylate treatment which prevents chloride binding, have no effect on thiocyanate conductance. In contrast, other mutations reduce the conductance while preserving NLC. When superimposed on prestin\u2019s structure, the location of these mutations indicates that the ion permeation pathway lies between the core and gate ring of helices, distinct from the transporter pathway. The uncoupled current is reminiscent of an omega current in voltage-gated ion channels. We suggest that prestin itself is the main regulator of intracellular chloride concentration via a route distinct from its transporter pathway.Prestin in the lateral membrane of outer hair cells, is responsible for electromotility (EM) and a corresponding nonlinear capacitance (NLC). Prestin\u2019s voltage sensitivity is influenced by intracellular chloride. A regulator of intracellular chloride is a stretch-sensitive, non-selective conductance within the lateral membrane, G Outer hair cell (OHC) electromotility is at the heart of cochlear amplification, a mechanism responsible for the mammal\u2019s ability to hear sounds of extremely low intensity134678111214metL, has been observed in the lateral membrane of OHCs, and awaits molecular identification1719202122233\u2212/Cl\u2212 exchange at a 2:1 ratio generating small transporter currents1415metL that operates independently of its putative transporter pathway.Intracellular chloride anions play a critical role in prestin\u2019s function17\u2212 ions that correlates with the expression of prestin. We find that expression of prestin induces currents in the absence of SCN\u2212, as well. In the presence of Cl\u2212, we observed a current in tet-inducible, prestin expressing HEK cellsmetL) observed within the lateral membrane of OHCsSch\u00e4nzler and Fahlke+ . Each of these ion replacements is known to cause a shift of up to 50\u2009mV in the NLC voltage operating range+ and Na+ with other ions  did not affect the size of the current or its reversal potential  while 300\u2009mOsm extracellular osmolarity was maintained with sucrose. The reversal potential shifted by \u221215\u2009mV\u2009+/\u2212\u20092\u2009mV with 14\u2009mM extracellular KCl, and is consistent with a calculated permeability ratio of 0.45 for Cl\u2212 relative to K+ . Consistent with the current being carried by a non-selective \u201copen pore\u201d, we note that cells expressing prestin show significant depolarization, relative to controls, in resting membrane potential, determined immediately after establishing the whole cell configuration under current clamp , was \u221229\u2009\u00b1\u20092\u2009mV. We calculate a permeability ratio of 0.27 for Na+ and 0.11 for Cl\u2212 relative to SCN\u2212. These permeability ratios are underestimates, and most probably they are discordant due to the background conductances that are also seen in control HEK cells (Similar to the Sch\u00e4nzler and Fahlke observation1415EK cells .\u2212 substitution (data not shown). The shape of the I-V relationship is similar to that observed with Cl\u2212 as the dominant intra and extracellular anion, with increasing slope upon both depolarization and hyperpolarization. This I-V relationship would be expected from a channel having a large central energy barrier and whose currents are sufficiently small to exclude diffusion limitation. A symmetrical, single-barrier channel model, which yields a hyperbolic sine I-V relationship, fits the data well  from prestin expressing cells demonstrate an increase in average current of +11.94\u2009pA (+50\u2009mV) and \u221212.56\u2009pA (\u221250\u2009mV), and +7.73\u2009pA (+50\u2009mV) and \u221210.36\u2009pA (+50\u2009mV) over HEK cells in the presence of thiocyanate and chloride, respectively.Another approach to determining the size of unitary currents is fluctuation analysis. Sch\u00e4nzler and Fahlke\u2212 and SCN\u2212 we also observed fluctuations with a power-law frequency dependence, approximately 1/f, whose magnitude clearly depended on membrane potential . We int pathway . We note\u2212 currents with charge neutralizing mutations of several charged residues in prestin\u2019s transmembrane domains  in individual cells the conductance increases with prestin expression and is proportional to NLC. 2) the conductance appears with the same time course as NLC upon release of Golgi block. 3) Mutations in prestin affect the current. An alternative possibility that a prestin associated protein contributes to the current could account for the first two observations. However, the third observation strongly suggests that the current is carried by prestin itself. Moreover, two other SLC26 family members have been demonstrated to have an uncoupled current in the presence of SCN\u2212\u200914Previous studies have characterized an ionic conductance in the SLC26 transporter family in the presence of SCN\u2212 transport pathway in prestin. Evidence for this is: 1) mutation of residues that are modeled to bind intracellular Cl\u2212, the first essential step in the transporter cycle, do not affect the size of the current. 2) salicylate, which in non-mammalian homologs has been shown to block the transporter current while simultaneously blocking NLC, has no effect on the size of the current. 3) single point mutations have effects on the size of the current or on NLC but not both. Since mutation of F137, that we speculate based on homology to UraA to be involved in the transporter cycle, changed the reversal potential in the presence of SCN-, it is possible that the alternative leakage pathway shares some commonality with the transporter pathway. In this context, owing to data that prestin and many similar transporter proteins form dimers and since the charge mutations that affected current are in proximity to its dimer interface, it is possible that the leakage pathway may in fact lie within the dimer interface4546474849Our second major finding is that the conductance is carried by a pathway different from the ClThe unusual current-voltage relationship can be explained by a channel having a high central energy barrier to ion transport. Consistent with this possibility we note small unitary currents in the fA range. We conclude that prestin forms at least part of an independent pathway for ions to cross the membrane. Mutations suggest that the pathway is located between the core and gate domains of the 7\u2009+\u20097 inverted repeat structure. In this way the pathway is reminiscent of the omega current pathway in voltage gated channels. There a nonselective cation current is carried by voltage-sensor domains, in a structure entirely separate from the ion-selective pore of these channels.\u2212 and SCN\u2212 differed between whole cell recordings and excised patches. These discrepancies between recording approaches could very well be explained by the effects of an increase in membrane tension in excised patches, given the conductance\u2019s tension dependence . We also note that the relative conductances for Clpendence  and F.The non-selective nature of the prestin associated currents and its small size is somewhat at odds given our current understanding of the selectivity filters in ion channels. Perhaps the mechanical model gleaned from structural data of the unrelated EAAT transporter that has a similar leakage conductance  has bearing\u2212 in OHCs. A similar conductance in the lateral membrane of OHCs has been demonstrated, and Cl\u2212 ions have been shown to affect the voltage sensitivity and affect the speed of transitions from expanded to contracted states of prestin. Although the current carried by individual molecules of prestin is small, the presence of large numbers of prestin in the lateral membrane of OHCs (estimated to be upwards of 106 per OHC) likely makes this current important. Consistent with this possibility, expression of prestin at high levels in HEK cells has a major effect on its resting potential. A similar role has been established in the leakage conductance of EAAT1/2 in glial cells in the cerebellum, that show developmental expression, reducing intracellular chlorideOur data suggest that currents carried by prestin\u2019s leakage conductance could affect Cl2 incubator (5%).Chinese hamster ovary (CHO) cells were cultured in Hams-F12 medium (high glucose), containing 50\u2009U/ml each of penicillin and streptomycin, 10% fetal bovine serum, 2\u2009mM L-glutamine at 37\u2009\u00b0C in a CO2 incubator (5%).HEK293 cells were cultured in Dulbecco\u2019s modified Eagle\u2019s medium  containing 50\u2009U/ml each of penicillin and streptomycin, 10% fetal bovine serum at 37\u2009\u00b0C in a COThe tetracycline-inducible, highly-expressing monoclonal prestin HEK 293 cell lines were reported previouslySingle amino acid substitutions were generated using QuickChange II site-directed mutagenesis kit  with a gerbil prestin (genbank AF230376\u00ae CCL-61\u2122) was done using Fugene 6  according to the manufacturer\u2019s instructions, in 24-well plates. Cells were recorded 24\u201372\u2009h after transfection.Transfection of constructs into CHO cells  to the base solution (in mM): NaCl 100, NaSCN 100, NaOCN 100, NaIO3 100, Na Methanesulfonate 100, Na2Malate 60, or Na2Oxalate 60. Similarly, for cation substitutions the following were added separately to the base solution (in mM): KCl 100, NMDG-Cl 100, or Tris-Cl 100, separately. The pipette solution contains (in mM): NaCl 100/NaSCN 100, CsCl 20, EGTA 5, MgCl2 2, Hepes 10, pH 7.2. Osmolarity was adjusted to 300\u2009\u00b1\u20092\u2009mOsm with dextrose. After whole cell configuration was achieved in extracellular NaCl and a ramp protocol recorded to confirm baseline NLC and currents. Extracellular anion and cation substitutions were then made by local extracellular perfusion of each individual cell followed by recording a ramp protocol.Prestin stably expressed in a tetracycline inducible HEK line or transiently transfected into CHO cells were recorded using a whole-cell configuration at room temperature using an Axon 200B amplifier , as described previouslyCommand delivery and data collections were carried out with a Windows-based whole-cell voltage clamp program, jClamp , using a Digidata 1322A (Axon Instruments).A continuous high-resolution 2-sine voltage command was used, cell capacitance and current being extracted synchronously. Capacitance data were fitted to the first derivative of a two-state Boltzmann function:whereQmax is the maximum nonlinear charge transfer, Vh the voltage at peak capacitance or half-maximal nonlinear charge transfer, Vm the membrane potential, Clin linear capacitance, z the unitary charge (a metric of voltage sensitivity), e the electron charge, k the Boltzmann constant, and T the absolute temperature. Qsp the specific charge density, is the total charge moved (Qmax) normalized to linear capacitance. Capacitive currents (Icap) generated by our ramp protocol were removed to reveal ionic currents. In order to estimate Icap (from both linear and NLC), we modelled the patch clamp-cell in Matlab and extracted membrane capacitive currents arising from averaged values of linear capacitance and NLC. In all figures, individual I-V plots were corrected based on averages from the inclusive cells. Reversal potentials, thus, arise from ionic current contributions only. Separately, in specific experiments currents were also determined after voltage steps (50\u2009ms duration) from \u2212150\u2009mV to 150\u2009mV, with 20\u2009mV step increments. Where cations and anions were substituted, local perfusion of the cells were estimated to give rise to small junctional potentials . These varied from +1.1\u2009mV (SCN-) to \u22125.4\u2009mV (Methanesulfonate) with anion substitutions. Cation substitutions also were estimated to give rise to small junctional potentials varying from \u22120.8\u2009mV (KCl) to +6.8\u2009mV (NMDG+). Since these numbers were small no corrections were made to the IV plots. For fluid jet experiments we used a QMM perfusion system . The manifold\u2019s output tip was 200\u2009\u03bcm placed 1\u2009mm from the cell, and the flow rate increased by an applied pressure of approximately 20\u2009kPa. Significance of the size of currents at different voltage steps from experimental perturbations were determined using a mixed model analysis of correlated measures (MMACM). Statistical analysis was done with SAS software .2 2, Hepes 10, pH 7.2. Bath solution contains (in mM):): NaCl/NaSCN 100, TEACl 20, CsCl 20, CoCl2 2, MgCl2 2, Hepes 5, pH 7.2. Osmolarity was adjusted to 300\u2009\u00b1\u20092\u2009mOsm with dextrose. Inside-out patches were obtained from control HEK cells or the tetracycline-induced prestin stable line. Data acquisition was done with the same interface and amplifier described above. Continuous 10\u2009second commands at desired holding potentials were delivered during recording.Thick wall glass pipettes were pulled using same puller as described above, and coated with sylgard. The initial resistances of the pipettes are 13\u201315\u2009mOhm. Pipette solution contains (in mM): NaCl 100, CsCl 20, EGTA 5, MgCl\u2212 conditions 5 for symmetrical chloride of HEK cells and 5 for chloride/SCN\u2212 conditions in HEK cells. Spectra were taken for each individual patch recording , averaged and plotted out to 3\u2009kHz. Data are presented following subtraction of baseline spectra at 0\u2009mV. Variance was determined by integration of spectra between 0.1\u2009Hz and 1\u2009kHz after subtraction of calculated shot noise (2ie0). Analyses were made with a custom written MATLAB application.PSD analyses of excised patch currents from prestin transfected cells, filtered with a 10\u2009kHz, 4 pole Bessel filter and Hamming windowed, were derived from 10\u2009second recordings held at \u221250, 0, and +50\u2009mV potentials. In some cases, voltages up to +/\u2212120\u2009mV were analyzed. Some patch records were excluded because of seal loss during recording. Number of patches from separate cells: 11 for symmetrical chloride condition; and 11 for chloride/SCNg is the slope conductance at E\u2009=\u20090, q is the apparent charge, is the electrical distance of the barrier, and P is the relative permeability of ions carrying outward current compared to those carrying inward current; if the current were entirely anion selective P would be the permeability ratio PSCN/PCl. For Cl\u2212 as the external anion the values were P\u2009=\u20091, \u03b4\u2009=\u20090.467, q (e0)\u2009=\u20090.75 and g (pS/pF)\u2009=\u200946.3. For SCN\u2212 as the anion the values were P\u2009=\u20096.18, \u03b4\u2009=\u20090.339, q (e0)\u2009=\u20090.75 and g (pS/pF)\u2009=\u200992.8. The values that differ for the two anion species, and the value of q smaller than the elementary charge, are consistent with a distribution of barrier locations, for example distinct barrier locations for anions and cations.where How to cite this article: Bai, J.-P. et al. Current carried by the Slc26 family member prestin does not flow through the transporter pathway. Sci. Rep.7, 46619; doi: 10.1038/srep46619 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."}
+{"text": "Syncope is difficult to definitively diagnose, even with tilt-table testing and beat-to-beat blood pressure measurements, the gold-standard. Both are qualitative, subjective assessments. There are subtypes of syncope associated with autonomic conditions for which tilt-table testing is not useful. Heart rate variability analyses also include too much ambiguity. Three subtypes of syncope are differentiated: vasovagal syncope (VVS) due to parasympathetic excess (VVS-PE), VVS with abnormal heart rate response (VVS-HR), and VVS without PE (VVS-PN). P&S monitoring  differentiates subtypes in 2727 cardiology patients , serially tested over four years . P&S monitoring noninvasively, independently, and simultaneously measures parasympathetic and sympathetic (P&S) activity, including the normal P-decrease followed by an S-increase with head-up postural change (standing). Syncope, as an S-excess (SE) with stand, is differentiated from orthostatic dysfunction  as S-withdrawal with stand. Upon standing, VVS-PE is further differentiated as SE with PE, VVS-HR as SE with abnormal HR, and VVS-PN as SE with normal P- and HR-responses. Improved understanding of the underlying pathophysiology by more accurate subtyping leads to more precise therapy and improved outcomes. The current standard for diagnosing syncope is a positive tilt-table test performed according to one of the currently acceptable methods \u20135. PresyA more quantitative and universal method of assessing autonomic state is applied to both improve the diagnostic yield of syncope and provide a simpler (quantitative) diagnostic criteria, especially for the nonspecialist. This method improves the differentiation between the parasympathetic and sympathetic (P&S) activity. This method, when used in response to postural change or standing, helps to differentiate four etiologies underlying dizziness and lightheadedness: (1) parasympathetic excess , (2) sympathetic withdrawal (SW) associated with orthostatic dysfunction , 24, (3)SE\u2009+\u2009PE, which is VVS due to PE (VVS-PE), is defined as the presence of SE upon standing with PE demonstrated during rest, Valsalva, or stand, regardless of the HR response to stand.SE\u2009+\u2009abn-HR, which is VVS with abnormal HR response (VVS-HR), is defined as the presence of SE upon standing with an abnormal HR response to stand (stand HR compared with resting HR).SE , which is VVS without PE , is defined as only the presence of SE upon standing. In these cases, the patients do not demonstrate an abnormal HR response to stand nor PE.Defined herein are three of the subtypes of SE as manifested in VVS and NCG syncope. They are based on the differences in pathophysiology present in the P&S nervous system responses. Improved understanding of the underlying pathophysiology demonstrates how this more accurate subtyping leads to more precise medical therapy and thus improved patient outcomes. In general, an S-excess (SE) response to the stand challenge is associated with syncope. Abnormal sympathetic responses to stand differentiate syncope (SE) from orthostatic dysfunction  , 24. TheUsing these subtype definitions, the P&S measurements from patients diagnosed with VVS-PE and VVS-HR are presented.A database of 3670 consecutive, serial patients was followed in a large cardiology practice drawing from both urban and suburban populations. P&S function was assessed noninvasively using the ANSAR Medical Technologies, Inc.  software (ANX 3.0 autonomic function monitor). The ANX 3.0 computes simultaneous, independent measures of P&S activity based on continuous, time-frequency analysis of HRV with concurrent, continuous, time-frequency analysis of respiratory activity (RA). Time-frequency analyses employ a normalized CMORL wavelet with a Q of 5 and a spectral update of 4 seconds.While this method facilitates reading P&S responses in the presence of arrhythmia , to permHRV-alone analyses compute mixed measures of P&S activity. For example, spectral HRV analyses result in a low frequency (LF) and a high frequency (HF) term , 13. LF To eliminate the need for assumption and approximation required by LF and HF, independent spectral analyses of RA are added to spectral analyses of HRV . This seThe clinical study employed to determine P&S activity includes four well-known autonomic challenges, separated by resting baseline periods. These six periods are labeled in the figures as (A) resting baseline, (B) deep breathing, (C) baseline, (D) Valsalva maneuvers, (E) baseline, and (F) stand . The stand challenge, in the clinical study used in this article , is a pop \u2264 0.05.The time requirement, as well as the safety factor, is further improved with the implementation of a spectral analysis technique that eliminates the time-frequency compromise: the wavelet transform , 22\u201329. The instantaneous SE, as seen in the trends plot of These differences are also reflected in the heart rate response plots Figures  and 3. TWithin this cohort at baseline, 38.6% of patients complain of lightheadedness . Of the cohort, 31.4% (81.3% of the LH patients) were diagnosed with some form of orthostatic dysfunction, including POTS. Orthostatic dysfunction is associated with sympathetic insufficiency, or sympathetic withdrawal, upon standing (a decrease in S-activity from baseline (rest) to stand ). Of the remaining 7.2% of those complaining of LH, 3.9% were diagnosed with syncope (tilt-positive) or presyncope from other clinics. From the entire cohort, 5.2% of the patients demonstrate SE upon stand. All of these patients complained of LH, and all of the patients diagnosed with (pre)syncope elsewhere demonstrated stand SE.From One of the most difficult forms of syncope to diagnose is VVS. Often tilt-table testing itself causes patients worry, anxiety, or stress. This stress (an S-stimulus) changes the patient's typical physiological response, the Vagal (or P-) excess, associated with VVS. In effect, the patient is temporarily treated by being placed on the tilt-table. As a result, the tilt-test may be falsely negative . From thSE upon standing is hypothesized as a result of the patient's brain becoming hypoperfused, which in turn causes an increase in S-activation in an attempt to normalize brain perfusion. The oscillations in the instantaneous S-activity demonstrated in the trends plots of Figures Given the difficulty of differentiating VVS from POTS, adding another parameter improves this differentiation. Again, stand SE is associated with syncope and SW is associated with orthostatic dysfunction . In thisBTB analyses, including heart rate variability (HRV), may be quantified with spectral analyses and other methods , 13. HowDifferentiating the underlying abnormalities of the autonomic nervous system into specific subtypes based on pathophysiology significantly aids in therapy planning. In cases of VVS-PE, it has been found that PE should be treated as the primary autonomic disorder to effectively treat symptoms and underlying autonomic dysfunction. It is known that the parasympathetics set the threshold around which the sympathetics react. By treating the parasympathetics as the primary autonomic dysfunction and normalizing them, often the reactionary sympathetics  is naturally relieved, followed by BP or HR. When total relief is not experienced, what remains is a function of end-organ disorder (including vestibular) and typically requires less therapy.In patients who are diagnosed with autonomic neuropathy or autonomic dysfunction and also heart diseases, hypertension, CAD, heart failure, or post-MI, the recommended therapy to treat both PE with SE and the cardiology diagnoses is carvedilol . CarvediIn summary, traditional testing modalities  are confounded due to their measures mixing both P- and S-activity in a single parameter. Frequency analysis of standard btb-cardiac activity (with HRV or btbBP) in response to the stand challenge is further compromised by the nature and definition of Fourier transforms or fast Fourier transforms (FFT), including short-term FFT (st-FFT) . FourierDuring the course of the first five minutes after a head-up postural change (including sitting to standing), there are several physiologic changes that affect the stand response, including (1) the response to the gravitational challenge, (2) the response to the exercise reflex, and (3) the recovery from both. Even a normal gravitational response comes and goes over a 30 second period, invalidating the use of Fourier transforms. An abnormal response to any one of these three physiologic changes may underlie LH and lead to syncope. As a result of these changes, the stand challenge is not stationary (quiescent), even st-FFTs with a 32-second analysis window, as per the standard practice , 13. TheVVS-PE is perhaps the most common subtype of syncope. Further differentiating syncope by identifying the autonomic components helps to improve differential diagnosis, which improves therapy planning, resulting in improved outcomes. Independent P&S monitoring provides more specific data regarding the pathophysiology of VVS. Improved subtype differentiation allows for more precise therapeutic modalities and improved symptom management. In the case of VVS-HR, ruling out VVS-PN may lead to lower doses of medication prescribed while still improving patient outcome."}
+{"text": "This study was aimed at finding out the exposure level of bisphenol A (BPA), a well-known endocrine disruptor, in relation to dietary factors using a data representing the Korean general population.This study was performed on 5402 adults aged 19\u00a0years and older based on the Second Korean National Environmental Health Survey (KoNEHS 2012\u20132014). The data analyzed urinary BPA concentration in relation to socio-demographic variables, health behavior-related variables, and dietary factor-related variables. Odds ratio (OR) was calculated through a logistic regression analysis after dividing the participants into high BPA exposure group and low BPA exposure group based on the top 75 percentile concentration. The logistic regression analysis was carried out considering the appropriate sample weight, stratification, and clustering of the second KoNEHS sample design.The group drinking bottled water at home and the group using zip-top bags/plastic bags showed significantly higher urinary BPA concentration in female. OR tends to increase as the intake frequency of frozen food increased and OR of frozen food consumption of more than once a week was 1.48  1.02\u20132.24) for male and the group drinking bottled water showed significantly higher OR of 1.45 (95% CI 1.06\u20132.17) after adjusting the related factors for female.BPA levels were high in female using bottled water and in male consuming frozen food, and therefore bottled water and frozen food need to be avoided to reduce BPA levels. Bisphenol A (BPA) is synthesized by the condensation of two phenols with one acetone and used as a monomer . After BBPA is mainly used to make polycarbonate plastic, epoxy resins, and thermal paper . PolycarBPA, a well-known endocrine disruptor, interacts with female hormone receptors and activates the female hormones, showing weak estrogenic effects . Such efIn a study describing estimated BPA daily intake based on urine BPA concentration data in 30 countries worldwide, the estimated BPA daily intake was especially high for adult population of Daejon, Korea in an areal analysis . In anotDirect contact with products containing BPA is the main source of BPA exposure with the main exposure route being diet via food and drink , 7. BecaEven though a nationwide biomonitoring of BPA exposure was conducted on the Korean general population, there is still not enough number of studies exploring the relation between BPA exposure level and dietary factors. Therefore, the purpose of this study was to analyze the relationship between urinary BPA concentration and dietary factors using data representing the Korean general population.This study was analyzed based on the Second Korean National Environmental Health Survey (KoNEHS 2012\u20132014). The second KoNEHS is designed to understand the Korean population\u2019s exposure level of environmental pollutants and explore the influencing factors. The KoNEHS is conducted every 3\u00a0years by the National Institute of Environmental Research under the Ministry of Environment.The second KoNEHS area consisted of 16 cities and provinces nationwide and for the survey sample size the enumeration district of the National Population and Housing Census 2010 was used as the population with the initial stratification classified by local administration. The secondary stratification was classified by socio-economic factors, using the stratified multistage cluster sampling applied with the proportional allocation of square root of population to extract 400 sample enumeration districts. Around 15 people from each sample enumeration district were surveyed targeting total of 6478 Koreans aged 19\u00a0years and older. The survey consists of an environmental exposure-related questionnaire, clinical tests, and analysis of environmental harmful substances in biological samples.In this study, 5402 subjects , excluding 928 subjects whose urinary creatinine concentration exceeded the proper range of 0.3\u20133.0\u00a0g/L and 215 subjects with missing urinary BPA concentration data, were included among the total participants of 6478 people. This study was approved by the Institutional Review Board (IRB) of Kyung Hee University Hospital (KHUH 2017\u201303-049). And this study only used the published data of the second KoNEHS.Urinary BPA concentration was measured by Ultra Performance Liquid Chromatography - Mass Spectrometry (UPLC-MS) using spot urine. After hydrolyzing the urine sample with \u00df-glucuronidase/aryl sulfatase-degrading enzyme, the metabolites of BPA were extracted with ethyl ether and measured. The principle of analysis figures out the value of sample concentration using the calibration curve constructed by the Standard Addition Method which adds a fixed amount of standard solution to the sample . The outThe variables related to potential BPA exposure among the variables associated with dietary factors were reclassified in the following manner for this study. The type of drinking water at home was classified into filtered water, tap or boiled water, bottled water, and mineral or ground water while the frequency of canned food consumption and frozen food consumption were classified into rarely consumed, consumed less than once a week, and consumed more than once a week. Food containers used in refrigerator were classified into glass/metal/porcelain ware, plastic ware, and zip-top bags/plastic bags.2, normal weight for BMI of 18.5\u201325\u00a0kg/m2, and overweight for BMI of greater than or equal to 25\u00a0kg/m2, whereas the level of educational attainment was classified into less than high school, graduated from high school, and graduated from junior college or more. The marital status was classified into single, married, and others (divorced/death of spouse/separated) while the household income was classified into four groups according to the quartile. Alcohol consumption was classified into non-drinker for participants who did not drink at all or drank in the past but not anymore, light drinker for participants drinking less than heavy drinker, and heavy drinker for participants drinking 3 times and more a week and drinking more than 7 glasses per occasion for male . The exercise status was classified into exercising group for participants exercising more than 3 times a week over 20\u00a0min and sweating during workout and non-exercising group for participants who do not fall under the exercising group. Smoking was classified into smoker for participants currently smoking and non-smoker for those who never smoked or quit smoking.The study participant\u2019s socio-demographic variables and health behavior-related variables were classified in the following manner. Among the socio-demographic variables, the age was classified into units of 10\u00a0years from the age of 19. Body Mass Index (BMI) was classified into underweight for BMI of less than 18.5\u00a0kg/mp\u00a0<\u00a00.05.The final data analysis of this study was analyzed by applying the weights presented in the original publicly open final dataset in accordance with the KoNEHS analysis guideline. The frequency and proportion of each variable were presented after gender stratification to figure out the general characteristics of the participants and chi-square tests were carried out to analyze the difference in the distribution of each variable. The geometric mean (GM) of urinary BPA was calculated by converting the natural logarithm because the urinary BPA level shows right deviation. Analysis of variance (ANOVA) was applied to compare the geometric mean value of metabolites in relation to dietary factors and Analysis of covariance (ANCOVA) to compare the geometric mean value with adjustments made to each variable. Odds ratio (OR) was calculated by using logistic regression by dividing the participants into high exposure group and low exposure group based on the top 75 percentile concentration . The logistic regression analysis was carried out considering the appropriate sample weight, stratification, and clustering of the second KoNEHS sample design. After gender stratification, in Model 1, no adjustments were made while age, BMI, socio-economic variables, health behavior-related variables and dietary factors were adjusted in Model 2. The IBM SPSS (version 19 for Windows) was used for the statistical analysis and the statistical significance level was set at p\u00a0<\u00a00.001). For age, urinary BPA level increased with age for both male and female showing a decreasing tendency after the age of 50s for male and the age of 40s for female. For male, urinary BPA concentration increased significantly as alcohol consumption increased with the heavy drinker group showing the highest concentration of 1.58\u00a0\u03bcg/g creatinine. For female, a statistical significant difference was observed in relation to the educational attainment with the high school graduates showing the highest concentration of 1.91\u00a0\u03bcg/g creatinine, followed by junior college graduates or more, and junior high graduates. For household income, urinary BPA concentration increased significantly as the income increased with the highest income class showing the highest concentration of 2.05\u00a0\u03bcg/g creatinine in female.Table\u00a0p\u00a0=\u00a00.011). The concentration increased as the intake frequency of canned food and frozen food increased. In addition, users of zip-top/plastic bags showed the highest concentration of 1.81\u00a0\u03bcg/g creatinine (p\u00a0=\u00a00.036).Urinary BPA concentration according to dietary factors after gender stratification was presented in Table\u00a0The logistic regression was conducted based on the top 75 percentile BPA concentration in Table\u00a0We analyzed the relationship between urinary BPA concentration and dietary factors using national survey data. The main finding of the present study is that high BPA concentration was associated with the use of bottled water in women and frozen food intake in men.It was confirmed that BPA can leach out of plastic beverage bottles, and exposure to BPA increased when these beverages were consumed . Adult gp\u00a0=\u00a00.036). Therefore, use of these containers for frozen food storage can also increase BPA exposure as a result of frozen food consumption.Frozen food intake and BPA exposure can be associated with packaging material for frozen food, cooking method and food container. Plastic wraps used in food packaging can cause BPA migration, and urinary BPA concentrations decreased when consumption of plastic packaged food were limited for 3\u00a0days compared to when on a general diet , 20. It In many studies, BPA was detected in commercial canned foods , 24. AndAlthough the present study analyzed the level of BPA exposure from food container in refrigerator, there was no statistical significance in OR. The reason for that is that BPA is included in polycarbonate plastic or polyvinyl chloride plastic used in food storage products, and that migration levels of BPA from containers vary for foods with different pH , 24. FurIn the study of temporal trends in BPA exposure conducted in NHANES (2003\u20132012), female, daily smoker, and low household income group showed a significantly higher concentration . SimilarThe limitations of this study are as follows. Firstly, the impact of dietary factors outside the home on BPA concentration could not be determined as only dietary factors were included in the analysis. Secondly, a more detailed content analysis on the dietary factors, such as the effect of amount of daily water intake, types of canned food and frozen food, and storage period, and type of food stored in the food containers for refrigerator could not be performed. To resolve this problem, an in-depth analysis needs to be carried out by adding detailed questionnaire later on based on the study performed abroad on exposure factors. Thirdly, only the data on BPA concentration measured in random spot urine was analyzed in this study. The sampling method needs improvement, such as multiple sampling or 24-h urine collection because of BPA\u2019s short half-life and variation in a day, and an analysis needs to be performed not only on urine samples but also on blood samples , 29.Despite these limitations, this study explored BPA exposure levels of Korean adults using the KoNEHS representing the Korean general population and found out the association between urinary BPA and dietary factors.BPA levels were high in female using bottled water and in male consuming frozen food. According the result of the present study, bottled water and frozen food need to be avoided to reduce BPA levels. Further research should be needed to investigate detailed dietary factors associated with BPA concentration."}
+{"text": "The Hedgehog (Hh) signalling pathway plays important roles during embryonic development and in adult tissue homeostasis, for example cartilage, where its deregulation can lead to osteoarthritis (OA). microRNAs (miRNAs) are important regulators of gene expression, and have been implicated in the regulation of signalling pathways, including Hh, thereby impacting upon development and disease. Our aim was to identify the function of miRNAs whose expression is altered in OA cartilage. Here we identified an increase in miR-324-5p expression in OA cartilage and hypothesised that, as in glioma, miR-324-5p would regulate Hh signalling. We determined that miR-324-5p regulates osteogenesis in human mesenchymal stem cells (MSCs) and in mouse C3H10T1/2 cells. Luciferase reporter assays demonstrated that miR-324-5p directly regulated established targets GLI1 and SMO in human but not in mouse, suggesting species-dependent mechanism of Hh pathway regulation. Stable Isotope Labelling with Amino acids in Cell culture (SILAC), mass spectrometry and whole genome transcriptome analysis identified Glypican 1 (Gpc1) as a novel miR-324-5p target in mouse, which was confirmed by real-time RT-PCR, immunoblotting and 3\u2032UTR-luciferase reporters. Knockdown of Gpc1 reduced Hh pathway activity, and phenocopied the effect of miR-324-5p on osteogenesis, indicating that miR-324-5p regulates Hh signalling in mouse via direct targeting of Gpc1. Finally, we showed that human GPC1 is not a direct target of miR-324-5p. Importantly, as well as identifying novel regulation of Indian Hedgehog (Ihh) signalling, this study demonstrates how a miRNA can show conserved pathway regulation in two species but by distinct mechanisms and highlights important differences between human diseases and mouse models.  Ptch1 and Hhip [The Hedgehog (Hh) signalling pathway is important in embryonic development and adult tissue homeostasis and is aberrantly activated in many human diseases, including osteoarthritis (OA) . The patand Hhip . During and Hhip ,4 by, foand Hhip . Conversand Hhip . In humaand Hhip  and Acroand Hhip , two disand Hhip .OA is the most common musculoskeletal disease and is epitomised by the loss of articular cartilage, thickening of subchondral bone and the formation of osteophytes . The bioMicroRNAs (miRNAs) are small (approximately 22\u202fnt), single stranded, non-coding RNAs that modulate gene expression through base-specific interactions within the target gene 3\u2032untranslated region (UTR) causing translational inhibition and mRNA degradation . NumerouHere we initially screened for differential microRNA expression in normal  and OA cartilage, identifying miR-324-5p as upregulated in the diseased tissue which had previously been associated with regulation of Hh signalling. Next we used quantitative proteomic and transcriptomic microRNA target identification to investigate the mechanisms by which OA-associated miR-324-5p regulates the hedgehog signalling pathway in humans and mice.To identify miRNAs differentially expressed in OA, a TaqMan\u00ae low density array of 365 miRNAs was performed on cartilage RNA. Samples were obtained from total hip replacements for either OA or fracture to the neck of femur (NOF). Data were normalised to the mean expression value of the miRNAs profiled, using a previously published method . A numbeFerretti et al.  previousThe Hh signalling pathway is known to be involved in bone formation . We therSince the miR-324-5p sequence is perfectly conserved between humans and mice, we also used the mouse pluripotent cell-line C3H10T1/2 to test the functional activity of miRNA, since under the correct stimuli these cells have the ability to undergo osteogenic differentiation . Gli1, aTo test if miR-324-5p regulated IHH signalling via direct targeting of Gli1 and Smo in mouse we generated mouse specific 3\u2032UTR luciferase constructs to recapitulate the effect miR-324-5p has in the human system. However, miR-324-5p did not repress activity from either reporter A and B, We used stable isotope labelling with amino acids in cell culture (SILAC) quantitative proteomics  to identOverexpression of miRNAs followed by transcriptome profiling (RNA hybridisation microarray) has also previously been used to screen for miRNA targets . TherefoIn total, 2086 genes were identified in both the transcriptome and proteomic screens, 176 of which contain a known miR-324-5p binding site in their 3\u2032UTR E. For 18Targeting of Gpc1 mRNA and protein by miR-324-5p was confirmed in mouse C3H10T1/2 cells A and B. To test if Gpc1 is important for Hh signalling, C3H10T1/2 cells were depleted of Gpc1 using siRNA transfection and stimulated with Ihh, again Gli1 transcript and protein was used as a readout for Hh pathway activation. As with miR-324-5p transfection C and D, Analysis of the human GPC1 3\u2032UTR identified one centrally located putative miR-324-5p seed binding site, though this was a relatively poor \u20187mer\u2019. A human GPC1 3\u2032UTR luciferase construct was then created, expression was surprisingly unaffected by miR-324-5p A, suggesHere we show miR-324-5p regulation of Hh signalling is conserved in humans and mice, yet the regulatory mechanism is not. In order to regulate Hh signalling in humans, miR-324-5p targets SMO and GLI1, but not GPC1, whereas in mice, miR-324-5p targets Gpc1, but not Smo or Gli1 B and C.Initially to identify miRNAs differentially expressed in OA, we screened end-stage OA articular cartilage and used healthy articular cartilage from Neck of femur fracture (NOF) patients as controls. These sample types have been previously used to identify differences between OA and healthy cartilage ; includiGiven the role of Hh signalling in OA , the datMiRNAs often have many targets, with both miRNA and target requiring temporo-spatial co-expression for effective regulation. Therefore to identify miR-324-5p targets involved in Hh regulation, C3H10T1/2 were stimulated with Ihh, ensuring potential target mRNA important for Hh signalling regulation were expressed. Since the majority, of miRNA targets are decreased at an mRNA and protein level , we combDrosophila are described as being involved in Hh signalling [Members of the glypican family have been reported to regulate Hh signalling. For example, Gpc3 in mice and Dally-like protein in gnalling ,46. Datagnalling  and impognalling . Glypicagnalling ,49. HSPGgnalling . Thus itgnalling . Glypicagnalling , potentiHS is a well-established regulator of cartilage development and maintenance ,53. ConsGpc1 is unlikely to be the sole mechanism by which miR-324-5p exerts any effects on the nervous system, and in fact we found that miR-324-5p can also inhibit App expression , was performed and analysed as previously described .l-glutamine, 1% non-essential amino acid solution, 100\u202fIU/ml penicillin and 100\u202f\u03bcg/ml streptomycin. Human MSC were cultured in mesenchymal stem cell growth medium (Lonza Biosciences) supplemented with 5\u202fng/ml fibroblast growth factor-2 . For all experiments involving C3H10T1/2 mouse pluripotent mesenchymal cells, with the exception of SILAC experiments, cells were cultured in Minimum Essential Medium, supplemented as described for SW1353 cells. All cells were cultured in vented T75\u202fcm2 flasks at 37\u202f\u00b0C and 5% (v/v) CO2.SW1353 were cultured in Dulbecco's Modified Eagle's medium (Sigma), supplemented with 10% foetal bovine serum , 2\u202fmM SpeI/HindIII) to enable cloning into the pMIR-Reporter vector (Ambion). Mutation of the miRNA seed binding sites was performed using QuickChange II (Agilent). Mouse Gpc1 overexpression plasmid was generated by infusion cloning of Gpc1 cDNA clone (Source Bioscience) into N-terminal HaloTag\u00ae expression construct (Promega).Potential target 3\u2032UTRs were amplified from genomic DNA using PCR primers (Supplementary Table 2) for In-Fusion\u00ae cloning (Clontech) or double-restriction enzyme digestion  were transfected using Dharmafect transfection reagent 1 . Expression and reporter plasmids (500\u202fng/ml) were transfected using FugeneHD. For miRNA and reporter plasmid co-transfection incubated lipid complexes were added to cells simultaneously, essentially as previously described [SW1353 chondrosarcoma cells (for human 3\u2032UTRs) or mouse C3H10T1/2 cells  were seeded to reach ~50% confluence after 24\u202fh, representing 1.8\u202f\u00d7\u202f10escribed . TransfeHuman MSCs were cultured and transfected with either miCon or miR-324-5p in 96 well plates as described above. After 48\u202fh media was replaced with OsteoMax medium (Merck Millipore) in the presence or absence of Recombinant Indian Hedgehog (Ihh)  and BMP2 . Fresh OsteoMax medium was added after 4\u202fdays. After 7\u202fdays cells were fixed in 10% formalin and mineralisation assessed by incubation with Alizarin Red solution (Sigma)  for 30\u202fmin. For quantification the staining was extracted with 10% (w/v) cetylpyridinium (Sigma) solubilised in 10\u202fmM sodium phosphate buffer (pH\u202f7) and the absorbance measured at 620\u202fnM.C3H10T1/2 cells (with or without transfection) of 80\u201390% confluence were serum-starved for 16\u202fh and stimulated with either Ihh or BMP-2 for the time and concentration indicated in each figure.Total RNA was extracted using Cells-to-cDNA II Kit . Complementary DNA (cDNA) was synthesised from total RNA using MMLV reverse transcriptase and random hexamers according to the manufacturer's instructions (Life Technologies). mRNA levels were determined using TaqMan\u00ae real time PCR. Assay primers and probes are listed or numbered as in the universal probe library (Roche) in Supplementary Table 2. For miRNAs, cDNA was synthesised from total RNA using TaqMan\u00ae MicroRNA Reverse Transcription (Life Technologies) according to the manufacturer's instructions. Real-time PCR was carried out using Life Technologies miRNAs assays according to the manufacturer's instructions.3VO4, 10\u202fmM 2-glycerophosphate, 50\u202fmM NaF, 5\u202fmM sodium pyrophosphate, 1% (v/v) Triton X-100, 1\u202f\u03bcM microcystin-LR and 0.1% (v/v) 2-mercaptoethanol. Particulates were removed by centrifugation at 13,000g for 5\u202fmin, supernatants were frozen at \u221280\u202f\u00b0C until needed. To determine protein concentration, Bradford reagent (Biorad) was added to the lysate, absorbance measured at 595\u202fnm with a plate reader (TECAN) and protein concentrations equalised. Proteins (5\u202f\u03bcg per sample) were separated by SDS-PAGE electrophoresis, transferred to nitrocellulose membrane and probed with antibodies against Gli1 , Gpc1 , GAPDH  or \u03b2-tubulin (1/1000). Expression was monitored by chemiluminescence  using HRP conjugated secondary antibodies (1/2000).Cells were lysed using ice cold lysis buffer containing: 50\u202fmM Tris-HCl, 1.2\u202fM glycerol, 1\u202fmM EGTA, 1\u202fmM EDTA, 1\u202fmM Nap-nitrophenylphosphate, Sigma) added. Active alkaline phosphatase (produced p-nitrophenol) was measured at 405\u202fnm, using a TECAN plate reader and Xflor 4 software.C3H10T1/2 cells were cultured and transfected as described above in 24 well plates. After 48\u202fh the cells were stimulated as indicated C in seru13C l-Lysine-2HCl and 13C 15N l-Arginine-HCl (heavy), or normal Lysine and Arginine (light). Both heavy and light media contained dialysed FBS . After 7\u202fdays of culture, the isotopic incorporation of the heavy amino acids was assessed. More than 99% of the peptides assessed by mass spectrometry contained heavy Arginine and Lysine (data not shown). Both heavy and light cells were seeded in 6\u202fcm dishes to reach around 50% confluence after 24\u202fh. Cells were then transfected using DF1 with 100\u202fnM of either miCon2 (non-targeting miRNA mimic) or miR-324-5p mimic in the light and heavy cells, respectively. After 24\u202fh of transfection, cells were serum-starved for a further 16\u202fh and then stimulated with 2\u202f\u03bcg/\u03bcl recombinant Ihh for 48\u202fh. Cells were then lysed using 150\u202f\u03bcl of lysis buffer, as described for immunoblotting. The Heavy and Light lysates were then mixed at a ratio of 1:1. Protein was boiled and separated using SDS-PAGE with nanopure H2O used in all gels and buffers. Gel preparation, protein digestion, mass spectrometry and initial processing of data was performed by NEPAF, Newcastle University. Gels were cut into 12 segments, each digested with trypsin and analysed by mass spectrometry. Data were normalised and analysed using MaxQuant software as described previously [C3H10T1/2 cells were cultured for more than five population doublings (7\u202fdays of culture), in DMEM containing either isotopically labelled eviously .4 cells per well for 24\u202fh. Cells were then transfected using DF1 and 100\u202fnM of either miCon2 or miR-324-5p for 24\u202fh. Cells were then serum starved for 16\u202fh before being stimulated for a further 48\u202fh as described. Cells were then lysed and RNA extracted using the Qiagen RNeasy mini kit according to the manufacturer's protocol. Microarray analysis used the Illumina mouse ref8 v3 bead array and was performed by The Genome Centre, Queen Mary, University of London. Raw expression data were analysed using Agilent GeneSpring GX 11 . Raw data were normalised with a quantile algorithm and the baseline was transformed to the median of all samples prior to further analysis.C3H10T1/2 cells were cultured as described and seeded into 6 well plates at a density of 8.2\u202f\u00d7\u202f10Enrichment analysis was performed by calculating the fraction of genes containing miR-324-5p binding site within the 3\u2032UTR, for decreased (FC\u202f<\u202flog2 \u22120.2), increased (FC\u202f>\u202flog2 0.2) and unaltered transcripts and proteins. The list of genes whose 3\u2032UTR contained a miR-324-5p binding site was obtained from TargetScan.Cumulative fraction plots were generated by plotting FC gene expression on the x axis against the fraction of all genes whose 3\u2032UTR contain (or does not contain for control) a miR-324-5p binding site on the y axis for all genes whose expression decreased more than that value on the x axis. The list of genes containing non-conserved and conserved miR-324-5p binding sites was obtained from TargetScan .t-test performed for single comparisons. Enrichment was calculated using Chi-squared test. *p\u202f<\u202f0.05, **p\u202f<\u202f0.01, ***p\u202f<\u202f0.001, ns\u202f=\u202fp\u202f>\u202f0.05. Statistical test is indicated within figure legends.Statistical differences were calculated using a one- or two-way analysis of variance followed by Bonferroni post hoc test for multiple comparisons or Student's"}
+{"text": "Caenorhabditis elegans CL4176 model of AD, as shown in our previous study. Therefore, this study was aimed at exploring the neuroprotective effect of DhHP-6 in the APPswe/PSEN1dE9 transgenic mouse model of AD. DhHP-6 reduced the diameter and fiber structure of A\u03b21-42 aggregation in vitro, as shown by dynamic light scattering and transmission electron microscope. DhHP-6 exerted its neuroprotective effect by inhibiting A\u03b2 aggregation and plaque formation, and by reducing A\u03b21-42 oligomers-induced neurotoxicity on HT22  and SH-SY5Y (human neuroblastoma) cells. In the AD mouse model, DhHP-6 significantly ameliorated cognitive decline and improved spatial learning ability in behavioral tests including the Morris water maze, Y-maze, novel object recognition, open field, and nest-building test. Moreover, DhHP-6 reduced the deposition of A\u03b2 plaques in the cerebral cortex and hippocampus. More importantly, DhHP-6 restored the morphology of astrocytes and microglia, and significantly reduced the levels of pro-inflammatory cytokines. Our findings provide a basis for considering the non-toxic, peroxidase mimetic DhHP-6 as a new candidate drug against AD.Alzheimer\u2019s disease (AD) is the most common neurodegenerative disorder in the elderly, which is characterized by the accumulation of amyloid \u03b2 (A\u03b2) plaques, oxidative stress, and neuronal loss. Therefore, clearing A\u03b2 aggregates and reducing oxidative stress could be an effective therapeutic strategy for AD. Deuterohemin-AlaHisThrValGluLys (DhHP-6), a novel deuterohemin-containing peptide mimetic of the natural microperoxidase-11 (MP-11), shows higher antioxidant activity and stability compared to the natural microperoxidases. DhHP-6 possesses the ability of extending lifespan and alleviating paralysis in the A\u03b21-42 transgenic  Alzheimer\u2019s disease (AD) is an age-related progressive neurodegenerative disorder mainly affecting people over 65 years of age. AD patients exhibit senile dementia characterized by a progressive deterioration in memory, cognition, and behavioral patterns ,2. The mSeveral \u03b2-sheet breaker polypeptides and antioxidants ,17,18 haCaenorhabditis elegans by 19%, and improves its survival rate during acute heat stress (35 \u00b0C) and paraquat-induced acute oxidative stress, indicating the potential anti-aging effects of DhHP-6 as well -2,5-diphenyl tetrazolium bromide (MTT) cell viability assays. DhHP-6 was non-toxic to HT22 and SH-SY5Y cells at the concentration range of 50\u2013300 \u03bcM after 24 h of incubation A. Studien = 8/group): Low Tg (treated with 0.3 mg/kg DhHP-6), High-Tg (treated with 3 mg/kg DhHP-6) and NaCl Tg . A negative control group comprising of wild-type male mice of the same age that were mock-treated with the same volume of saline was also included (NaCl-Wt). The mice were intraperitoneally treated with the respective reagents for 12 weeks, and the behavioral tests were started from the 10th week onwards until the mice were euthanized at the end of the 12-week period . Compared to NaCl-Tg, the High-Tg group showed significantly shorter duration of escaping latency , indicating a significant spatial memory disorder. The High-Tg mice spent more time in the platform quadrant and passed over the platform site more times compared to the NaCl-Tg mice  mice using established behavioral tests. Six-month-old male transgenic mice were divided into the following three groups  in terms of entering into the three arms consecutively (p < 0.05). The exploration and anxiety-related behaviors were evaluated using the open field test [###p < 0.001, ** p < 0.01 and *** p < 0.01).Spatial memory loss caused by amyloid deposition was also evaluated by the Y-maze test . Both Locutively I,J. Furteld test , and NaCNon-maternal nest building performance is sensitive to hippocampal damage and used to evaluate the murine models of psychiatric disorders ,40. DuriAmyloid deposition in the brain of both AD patients and animal models is an important biomarker of AD pathogenesis and treatment response . Since c#p < 0.05 and ###p < 0.001), and DhHP-6 at high doses significantly restored the length of dendrites (*** p < 0.001). A\u03b2 deposition exacerbates neuroinflammation, resulting in neuronal atrophy. The clustering of microglia and astrocytes around A\u03b2 plaques is a pathological hallmark of AD . Activatp < 0.05 and High-Tg, *** p < 0.001) and hippocampus   D. Taken A\u03b2 deposition in the brain induces an inflammatory response via the activation of microglia and astrocytes, which produce pro-inflammatory factors such as tumor necrosis factor-\u03b1 (TNF-\u03b1), interleukin-1\u03b2 (IL-1\u03b2), or IL-6, and growth factors and extracellular matrix proteins . TherefoC. elegans model of AD (strain GM101). It inhibits A\u03b2 aggregation, scavenges ROS, reduces A\u03b2, and maintains full-length A\u03b21-42 expression in the nematode, thereby increasing the lifespan, and abrogating paralysis and plaque deposition. AD is a common progressive neurodegenerative disorder characterized by cognitive decline, memory impairment, and behavioral anomalies. Several hypotheses have been proposed to explain the molecular and pathological mechanisms of AD, and among them, the amyloid cascade hypothesis is the most widely accepted. The extracellular aggregation of the insoluble proteinaceous amyloid \u03b2 plaques and/or the soluble toxic oligomers of A\u03b21-42 exacerbate oxidative stress and trigger mitochondrial dysfunction, leading to neuronal death . Increas3 U/\u03bcM  that was firstly designed to treat cataracts [C. elegans and retards the aging process by enhancing the activity of the upstream transcriptional regulator DAF-16 [sod-3), transmembrane tyrosine kinase (old-1), metallothioneine (mtl-1), and small heat-shock proteins [C. elegans strain CL4176 expressing A\u03b21-42, and resulted in improved survival, and a reduction in paralysis and plaque deposition (data not shown). To determine the underlying mechanism of the potential neuroprotective effect of DhHP-6 in the mammalian system, we used the APPswe/PSEN1dE9 transgenic mouse model of AD. DhHP-6 is a mimetic of the natural MP-11 with a high peroxidase activity of 3.9 \u00d7 10ataracts . DhHP-6 r DAF-16 , which aproteins ,53. On tThe A\u03b2 aggregates are typically characterized by the highly ordered \u03b2-sheet conformation . DhHP-6 APPSwe/PS1dE9 (APP/PS1) transgenic mice overexpressing the human mutated APP with Swedish mutation (APP695Swe), and mutant human presenilin 1 with exon 9 deletion (PSEN1dE9), are characterized by age-related memory deficit, anxiety, hyperactivity, and impaired social interaction . APP/PS1The deposition of A\u03b21-42 positive plaques in the cerebral cortex and hippocampus is the strongest evidence of AD, and is used to monitor the therapeutic response . We obseAt present, amyloid cascade targeting drugs such as aducanumab (BIIB037) , ALZT-OPDhHP-6 was designed and synthetized in our laboratory on solid phase, as previously described . ThioflaPeroxidase mimetic deuterohemin-AlaHisThrValGluLys (DhHP-6) was synthesized on rink-amide resins by solid-phase peptide synthesis. Crude peptides were obtained by cleavage solution (trifluoroacetic acid:triisopropylsilane:water = 95:2.5:2.5) followed a standard protocol, purified by high-performance liquid chromatography System  equipped with a C18 column, and characterized by MALDI-TOF/TOF mass spectrometer .A\u03b21-42 peptide (10 \u03bcM) and DhHP-6 incubated with A\u03b21-42 (10:10 molar mass ratio) were dissolved in phosphate buffer  with thioflavin T (Th-T) at 37 \u00b0C. Fluorescence intensity was measured using a microplate reader  at various incubation times and the 450-nm excitation wavelength according to a standard thioflavin T assay protocol .Pure A\u03b2 and A\u03b2 mixed with DhHP-6 (10 \u03bcM:10 \u03bcM molar ratio) were incubated at 37 \u00b0C for five days. All of the samples were dispersed in milliQ water and then transferred to a cuvette, and the hydrodynamic particle size was analyzed at 25 \u00b0C by dynamic light scattering (DLS) technique using a Malvern Zeta sizer Nano ZS Instrument NANO ZS90  ,61.Pure A\u03b2 and A\u03b2 mixed with DhHP-6 (10 \u03bcM:10 \u03bcM molar ratio) were dissolved in phosphate buffer (pH 7.4) and incubated at 37 \u00b0C for three days. Then, an aliquot of 10 \u03bcL from each sample was placed onto TEM grids, dried, and viewed using a JEM-2100F microscope with a field emission gun operating at 200 kV. 2. A\u03b21-42 oligomer (A\u03b2O) was prepared as previously described [SH-SY5Y and HT22 cells were maintained in DMEM , 100 units penicillin and 100 \u00b5g/mL streptomycin) and cultured at 37 \u00b0C with 5% COescribed . Pure A\u03b25 cells/mL) were seeded in a 96-well cell culture plate at 100 \u03bcL/well and cultured for 12 h; then, they were exposed to 50\u2013300 \u00b5M of DhHP-6 for 24 h. As regards the neuroprotective test, cells were pre-treated with DhHP-6 (100 \u00b5M) for four hours, and then treated with A\u03b2O (30 \u03bcM) for 24 h. Subsequently, MTT solution  was added, and cells were incubated for 4 h at 37 \u00b0C. Next, MTT was removed, DMSO (150 \u03bcL/well) was added, and the absorbance was read at 490 nm by an ELISA reader  [As regards the cytotoxicity test, SH-SY5Y and HT22 cells  .Two-month-old male transgenic mice, B6C3-Tg (APPswe/PSEN1dE9)/Nju (APP/PS1) and B6C3F1-Wt (Wild-type), were provided by the Model Animal Research Center of Nanjing University in China. All of the animals were housed in standard cages under 12 h/12 h light/dark cycles, with free access to food and water. The animal experimental procedure was approved by the Animal Care Committee of Jilin University (License No.: 20160518) at 18 May 2016 and conformed to the Animal Ethical Standards and Use Committee at Jilin University .Six-month-old male transgenic mice were divided into the following three groups (n = eight/group): Low-Tg (treated with 0.3 mg/kg DhHP-6), High-Tg (treated with 3 mg/kg DhHP-6), and NaCl-Tg . Wild-type male mice of the same age were treated with the same volume of saline as the negative control (NaCl-Wt). Mice were intraperitoneally treated with the respective reagents for 12 weeks, and the behavioral tests were started from the 10th week onwards until the mice were euthanized at the end of the 12-week period. All of the tests were conducted by an individual who was blind to the treatment assignment. Mice were weighed and subjected to visual tests before the behavioral tests to exclude treatment effects and individual differences. The sensorimotor tests, including the rotating bar and grasping tests, were used to measure general motor functions, coordination, and balance. The behavioral testing, including Morris water maze, Y-maze, novel object recognition, open-field, and nest-building tests, were used to assess short-term memory as well as working memory, spatial memory ability, anxiety, social interaction capacity, and hippocampus damage relative cognitive behavioral changes. All of the instruments were purchased from ZS Dichuang, China. Mice movement tracking was recorded by an automated video tracking system .Mice were placed on a 30-mm diameter rod of the rotaryrod fatigue tester to keep the balance for 60 s, and trained for 10 min under a speed of five rpm on the first training day, 10 rpm on the second training day, and 20 rpm on the third training day. Then, on day four (the testing day), the speed of the rod was set up to 40 rpm. Mice falling time and rod speed were recorded (within 10 min) when the mice fell from the top of the rotary rod. Skimmed cotton was placed under the rod to prevent injury from falling.Limb strength and body movement-related neural lesion were measured by the grasping test. During the test, mice were placed on the surface of a 230 mm \u00d7 250 mm grasping test plate. The grasping force was tested by the sustained and stable tension of each mouse for three trials. The maximum pulling force was considered as the grasping mice force.The Morris water maze, as the typical assessment of cognitive capacity, was used for evaluating learning and memory ability . The watThe Y-maze apparatus consisted of three radial 30-cm long arms  originating from the central space to form a \u201cY\u201d shape. Mice were placed into the starting arm to explore the maze based on the rodent\u2019s innate curiosity to explore novel areas. Briefly, mice were placed into starting arm to explore and locate the novel arms freely by spatial clues for 10 min (training period). After a 2-h interval, mice were placed into the Y-maze again as part of the training period protocol to evaluate spatial memory. Time, distance, enter times, and movement tracks were recorded by an automated video tracking system.Novel object recognition is widely used to measure short-term memory and learning, the preference for novelty, and the influence of the hippocampus area in the process of recognition in rodents . It was The open field test measures the exploration of a new environment and anxious behavior, which is based on the idea that mice naturally prefer to be near a protective wall rather than exposed to danger out in an open field ,67. It wThe nest-building test was used to detect hippocampus damage and evaluate murine models of psychiatric disorders . Each moAfter mice sacrifice, brains were excised, fixed in 4% paraformaldehyde, and embedded in paraffin. Serial coronal brain sections were cut along the entire rostro-caudal brain axis and stained for IHC analysis using a standard protocol . After bAfter mice sacrifice, brains were excised and immediately frozen in liquid nitrogen. Then, the brain samples were homogenized in 0.9% normal saline containing a protease inhibitor cocktail , followed by ultrasonication for 10 min. After centrifugation, the supernatant was collected to measure cytokine levels using mice IL-1\u03b2, IL-6, IL-17A, TNF-\u03b1 quantikine ELISA kits (R&D systems) according to the manufacturer\u2019s instructions.t-test for two independent samples, and ANOVA analyses of Tukey\u2019s multiple comparison test for studies comparing four groups in behavioral tests. All of the statistical analyses were performed using GraphPad Prism 5  for testing the significance of the results. Each experiment was performed in triplicate; data are presented as mean \u00b1 SD; and a p value < 0.05 was considered statistically significant.Image-Pro Plus 6.0 software  was used to analyze plaques, GFAP (astrocytes biomarker), and Iba-1 (microglia biomarker) in the cerebral cortex and hippocampus. Statistical analysis was carried out using the DhHP-6 significantly reduced A\u03b2 aggregation and size in vitro, and ameliorated the cognitive decline and hippocampal damage in a murine AD model by inhibiting the deposition of A\u03b2 plaques. Mechanistically, DhHP-6 restored the morphology of astrocytes and microglia, and significantly reduced the levels of pro-inflammatory cytokines. Therefore, DhHP-6 is a promising therapeutic candidate for AD. Moreover, recently studies proposed A\u03b2 could reduce sirtuin 1 and its downstream signaling, resulting in increased intracellular ROS accumulation and mitochondrial dysfunction. It is worthwhile to conduct further studies and tests."}
+{"text": "ARs) are the major mediators of synaptic inhibition in the brain. Aberrant GABAAR activity or regulation is observed in various neurodevelopmental disorders, neurodegenerative diseases and mental illnesses, including epilepsy, Alzheimer\u2019s and schizophrenia. Benzodiazepines, anesthetics and other pharmaceutics targeting these receptors find broad clinical use, but their inherent lack of receptor subtype specificity causes unavoidable side effects, raising a need for new or adjuvant medications. In this review article, we introduce a new strategy to modulate GABAeric signaling: targeting the intracellular protein interactors of GABAARs. Of special interest are scaffolding, anchoring and supporting proteins that display high GABAAR subtype specificity. Recent efforts to target gephyrin, the major intracellular integrator of GABAergic signaling, confirm that GABAAR-associated proteins can be successfully targeted through diverse molecules, including recombinant proteins, intrabodies, peptide-based probes and small molecules. Small-molecule artemisinins and peptides derived from endogenous interactors, that specifically target the universal receptor binding site of gephyrin, acutely affect synaptic GABAAR numbers and clustering, modifying neuronal transmission. Interference with GABAAR trafficking provides another way to modulate inhibitory signaling. Peptides blocking the binding site of GABAAR to AP2 increase the surface concentration of GABAAR clusters and enhance GABAergic signaling. Engineering of gephyrin binding peptides delivered superior means to interrogate neuronal structure and function. Fluorescent peptides, designed from gephyrin binders, enable live neuronal staining and visualization of gephyrin in the post synaptic sites with submicron resolution. We anticipate that in the future, novel fluorescent probes, with improved size and binding efficiency, may find wide application in super resolution microscopy studies, enlightening the nanoscale architecture of the inhibitory synapse. Broader studies on GABAAR accessory proteins and the identification of the exact molecular binding interfaces and affinities will advance the development of novel GABAAR modulators and following in vivo studies will reveal their clinical potential as adjuvant or stand-alone drugs.\u03b3-aminobutyric acid type A receptors (GABA ARs) are the principal mediators of phasic and tonic inhibition in the human brain, being a vital part of the molecular machinery that creates cognition, behavior, and consciousness  domain carrying proteins. These showed that through modulation of receptor-scaffolding protein interactions a variety of responses could be achieved, ranging from disruption of glutamate signaling to neuroprotective effects in ischemic brain damage  is a guanine nucleotide exchange factor for Cdc42, a gephyrin binding partner (Kins et al., AR subtypes and GABAAR scaffolds. Neuroligin dysfunction has been implicated in autism (Pettem et al., AR, remain uncharacterized. These molecular insights could greatly contribute to our understanding of the development of the inhibitory synapse, as well as the underlying molecular causes of developmental diseases. Neuroligin family members exert distinct roles in the formation and stabilization of inhibitory and excitatory synapses and display distinct cellular and subcellular distributions. Accordingly, molecules that interfere with their isoform-specific interactions could act as highly cell-type selective modulators of neurotransmission.Proteomic studies (Kang et al., ARs. The loss of gephyrin clusters following the loss of the GABAAR \u03b32 subunit (Essrich et al., AR clusters upon gephyrin deficiency (Kneussel et al., AR subunits could be confirmed (Tretter et al., in-silico studies (Pennacchietti et al., AR complexes and demonstrated that at least the GABAAR \u03b11\u20133 and GlyR \u03b2 subunits bind to an overlapping site within gephyrin in a mutually exclusive fashion (Maric et al., ARs and GlyRs (Specht et al., Gephyrin is a prime candidate for the role of master regulator of neuronal function at inhibitory sites (Tyagarajan and Fritschy, AR diffusion and contributing to input-specific adaptations at postsynaptic sites (Chen et al., AR synapse dynamics remain to be explored in a comprehensive approach that includes an extensive alternative splicing and complex post-translational modification patterns of this region. Identification of the targeted binding pockets and insights into the binding affinities of the modified and unmodified peptide regions within the central region of gephyrin could shed light on the enigmatic molecular mechanisms of gephyrin multimerization, degradation and the tuning of its ligand binding affinities. Additionally, gephyrin isoforms are tissue-specific (Paarmann et al., Gephyrin itself is dynamically regulated, affecting GABAvia gephyrin was achieved more than a decade ago using intrabodies (Zacchi et al., ARs. One such example is artemisinins [AR signaling. Kasaragod et al. (ARs clustering, making artemisinins the first small molecule lead compounds for a new class of inhibitory neurotransmission modulators. Strikingly, the druggable artemisinin-binding pocket overlaps with the universal receptor binding region of gephyrin, which is critical for the interaction with GABAA and glycine receptors (Kasaragod et al., A and glycine receptors that bind to gephyrin. Biomimetic optimization of the \u201chotspots\u201d amino acid sequence, enhanced the affinity of the resulting peptide ligands 46,000-fold compared to the corresponding native peptides (Maric et al., in vitro applications of these new super binder peptide reduced GABAAR \u03b12 conductivity and clustering, providing evidence that GABAAR-associated proteins can be successfully targeted with modified peptides to modulate fast synaptic inhibition (Maric et al., Gephyrin\u2019s crucial role in glycinergic and GABAergic transmission made it a major pharmacological target. The modulation of synaptic responses d et al.  identifiAR trafficking is pivotal for the plasticity (Luscher et al., AR cycling is involved in various neurological disorders (Smith and Kittler, AR sites, that are involved in the trafficking of the receptors, has been identified to control receptor numbers and their concentration at synaptic sites (Comenencia-Ortiz et al., AR trafficking, endocytosis, degradation or recycling, is a promising pharmacological strategy. The proposed direct protein interactors are numerous, among them are muskelin (Heisler et al., GABAAR interaction rapidly modulates synaptic GABAAR numbers, inhibitory synaptic strength, neuronal excitability, and notably, affects animal behavior (Kittler et al., AR subunits (AR derived peptides, that effectively compete with AP2 binding, were successfully used to block the receptor internalization in hippocampal neurons, increasing surface concentration of GABAAR clusters by 50% (Smith et al., AR interactions with its intracellular trafficking partners is an alternative way to influence GABAergic signaling.The AP2-GABAsubunits . Short GAR clustering, like artemisinins or \u201csuper binding peptides,\u201d could reduce the GABAAR synaptic concentration and function. On the other hand, peptides hampering receptor interaction with AP2 trafficking protein increased the synaptic receptor levels. In theory, these approaches could be applied together to achieve bi-directional modulation of inhibitory neurotransmission, promoting a shift in the dynamic equilibrium from phasic to tonic neuronal response.Ongoing research uncovered original, seemingly contrasting, strategies of GABAergic signaling modulation. On the one hand, ligands disrupting gephyrin-GABAAR associated scaffold or trafficking proteins could be applied wherever abnormal GABAergic activity or regulation is involved in pathogenesis. In status epilepticus patients develop a time-dependent pharmacoresistance to GABAergic agents, probably, due to GABAAR internalization (Naylor et al., ARs (Nicholson et al., ARs and both could potentially be alleviated by targeting GABAAR-associated proteins. Stabilization of the gephyrin-receptor scaffolds at inhibitory postsynapses with molecules that mimic the stabilizing action of CB (Saiepour et al., AR loss and preserve inhibitory neurotransmission, alternatively, applying AP2 inhibitors could reduce GABAAR internalization and reverse the loss of postsynaptic GABAARs. Those examples illustrate the potential of GABAergic modulators as adjuvants ameliorating the effect of existing potent drugs, whereas in epilepsy or other diseases involving deregulation of inhibitory neurotransmission they could be applied as stand-alone therapeutics.Those new strategies of GABAergic neurotransmission modulation possess an untapped clinical potential. Agents targeting GABAAR intracellular interactors, accelerated by in-silico predictions and high throughput approaches, will lead to the discovery of novel GABAergic modulators. Affinity, selectivity, bioavailability and immunogenicity of these compounds would have to be optimized for clinical applications, where peptide-based ligands could be further evolved by the introduction of unnatural amino acids, cyclization and other chemical modifications.We expect that the study of GABAMicroscopy is an additional intriguing application of these molecules. The enhanced affinity and specificity of the engineered peptide-based compounds allowed to pioneer their use as fluorescent probes [AR associated proteins could prove to be a versatile pharmacological strategy with clinical potential. Further, we suggested that when combined with state-of-the-art super-resolution microscopy methods, the peptide-based fluorescent probes may resolve the nanoscale architecture of synapses in unprecedented detail. We anticipate that the discovery of additional GABAAR interactors could open the way for the development of new imaging tools and alternative pharmacological approaches.Here, we discussed how the targeting of GABAVK and HM wrote the manuscript and prepared the figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "During 12 weeks, THG performed 2-3 60-min recreational team handball matches weekly (average: 2.2 \u00b1 0.7), and CG maintained an inactive lifestyle. Average heart rate (HR) during matches was 80 \u00b1 7%HRmax, with peak values of 91 \u00b1 6%HRmax. A time-by-group interaction was shown in aerobic performance (p=0.016), postural balance (p=0.019), maximum oxygen uptake (VO2max) (p=0.023), resting HR (p<0.001), high-density lipoprotein (HDL) cholesterol (p=0.048), and fasting blood glucose (p=0.052) in favor of THG. THG improved aerobic performance , VO2max , and postural balance . Decreases in resting HR  and fasting blood glucose  and increases in HDL cholesterol  were found in THG. Recreational team handball practice shows positive physical fitness and health-related adaptations, with high attendance, which may contribute to the reduction of the risk of developing lifestyle diseases.Recreational team handball is an intermittent high-intensity exercise mode with physiological demands in the range of those found to enhance health and physical fitness of sedentary adults. We examined the effects of a short-term team handball-based training programme on physical fitness and metabolic and cardiovascular health of sedentary 33-55-year-old former male team handball players. Twenty-four participants were divided into team handball (THG; In modern society, the adoption of sedentary lifestyles results in low physical fitness levels, one of the most important risk factors for chronic-degenerative diseases, especially those related to the cardiovascular system , 2. EpidIn the last decade, recreational soccer has led the way in the field of using team sports as an alternative exercise mode in the treatment and prevention of lifestyle chronic diseases . Positivmax, and for 71% of total match time  the HR was above 80%HRmax and for 24% above 90%HRmax . The imposed cardiovascular strain is within the range of that considered sufficient to cause marked improvements in cardiorespiratory fitness, systolic blood pressure, and glucose tolerance, increasing the overall health profile ), capillary blood samples , a marker of arterial stiffness, was assessed in a quiet, dimmed room by a single trained cardiopneumology technician with a portable device  with the participants placed in a supine position. After 5\u2009min of rest, the carotid-femoral distance was assessed as the distance of suprasternal notch to the umbilicus and from there to the measuring point at the femoral artery plus the suprasternal notch to the measuring point at the carotid artery. Electrocardiogram registry was performed simultaneously, allowing the software to calculate the time from the peak of the R-wave to the foot of the pulse wave at the carotid and femoral arteries, respectively. The digital volume pulse waveform had to fill 2/3 of the display with little or no noise and artifact to be considered and three 10-seconds measurements of PWV were performed and averaged for analysis.2max), the participants performed an incremental treadmill  test until voluntary exhaustion  were 3.7 \u00b1 1.5 (1.3-6.8) mM and 4.4 \u00b1 1.3 (2.7-6.8) mM, respectively. The outfield players' postmatch RPE was 7.3 \u00b1 0.9 (2-10) arbitrary units .For 45% of total match time (27 \u00b1 10\u2009min) the outfield players' HR was between 81 and 90%HRmax), respectively. The goalkeepers' cardiovascular load was between 81 and 90%HRmax for 25% of the total match time and only 7% above 90%HRmax, while HRs were equal to or below 70%HRmax for 40% of total match time. Average [Blac] was 1.1 \u00b1 0.1 (0.9-1.2) mM and postmatch RPE was 6.3 \u00b1 2.1 (1-9) AU (hard). Match demands were significantly different between goalkeepers and outfield players (p<0.050). When considering all participants in the THG , the average HR during the matches was 80 \u00b1 7%HRmax, with peak values of 91 \u00b1 6%HRmax.Jumps were the actions most frequently performed by the goalkeepers (37 \u00b1 7), followed by changes of direction (28 \u00b1 5), defense actions (22 \u00b1 3), and stops (17 \u00b1 7). Peak and mean HRs were 149 \u00b1 29 and 127 \u00b1 29\u2009bpm  with the THG showing a marked 80% (p<0.001) increase in YYIE2 at 12 weeks (351 to 652\u2009m) and a 27% (p=0.018) drop in the number of falls in the postural balance test  and the THG had half the falls in the balance test .A time-by-group interaction was shown in YYIE2 performance p=0.016;  and post2max  with the THG showing a 14% (p<0.001) increase in VO2max (40.1 to 44.8\u2009mL/min/kg) and a 16% (p<0.001) decrease in resting HR (62 to 52\u2009bpm) at 12 weeks . At 12 weeks, MAP was lower for the THG than for the CG .A time-by-group interaction was shown in VOp=0.023;  and rest12 weeks . No sign12 weeks ; Table 2p=0.048; ; Table 2p=0.048; p=0.052) in favor of the THG. In this group, HDL cholesterol increased by 11% (p=0.002) at 12 weeks and fasting blood glucose decreased by 7% (p=0.015). A time effect was shown in total cholesterol, LDL cholesterol, total cholesterol/HDL ratio, total cholesterol/LDL ratio, and LDL/HDL ratio . Nonetheless, only the THG showed a significant increase in HDL cholesterol (p=0.002). The THG showed a medium to large significant decrement in fasting blood glucose, blood glucose, and plasma insulin after the two-hour oral glucose tolerance test  with previous experience with this sport. This training intervention showed that an average of 2.2 sessions per week of 60-min per day of recreational team handball practice, during 12 weeks, induced mainly moderate to large positive improvements in physical fitness and health-related variables in the participants. These findings confirmed this study work hypothesis and line up with the effects on fitness and health, previously demonstrated in training interventions using soccer, basketball, and floorball in similar populations , 23, 24.2max associated with aging is considered of importance in preventive medicine [2max corresponding to approximately 5\u2009mL/min/kg . These results are of clinical significance as it is known that increments of 1 MET correspond to a 13% reduction in the risk of mortality [2max in the range of 8-13% as effect of 12 weeks of recreational soccer, performed ~2 times per week, in 20-43 year-old healthy untrained men. It has been suggested that the time spent at high-intensity during the recreational soccer matches, considered as match HRs above 90% of HRmax, was the possible cause of the reported similar improvements in VO2max [max resulted in moderate to large improvement in aerobic fitness [2max were largely correlated with the time spent with HRs >90%HRmax . In fact, during the intervention the outfield players spent 21% or their match time with HRs >90%HRmax, a percentage in the upper range of that reported for enhancing VO2max in recreational soccer interventions (12-30%) [2max was higher  than that reported by Randers et al. [2max found in this study may be due to the nature of team handball that, differently from soccer, involves a remarkable use of the upper limb muscles beside the legs [Maximal oxygen uptake is considered a valid measure of cardiorespiratory and aerobic metabolism efficiency and a strong predictor of overall mortality , 26. Thumedicine . The recortality , 29. Ourortality . Indeed,ortality  and Randortality  reportedn VO2max . Further fitness , 32. Int(12-30%) . The reps et al.  in untrathe legs . This coA drop in resting HR has been reported as a reflection of functional adaptations in the autonomic nervous system induced by aerobic fitness improvements . Cooney 2max (40.1 \u00b1 8.4 versus 40.0 \u00b1 6.2\u2009mL/min/kg) [2max values between the studies. The YYIE2 performance was reported to be largely associated with individual VO2max level in untrained individuals, although the test heavily stresses the anaerobic pathway [2max, resting HR, and YYIE2 large improvements) provide evidence to support the effectiveness of recreational team handball practice in improving individual cardiovascular and anaerobic fitness. Given the interest of the possible cause-effect relationship between recreational team handball and physical fitness, future mechanistic training studies are warranted.The 12-week period of recreational team handball practice was successful in producing a remarkable 80% change  in players' YYIE2 performance. The achieved change in YYIE2 performance was largely higher than those reported in recreational soccer studies  after similar intervention durations and addressing different populations . Interes/min/kg) . However/min/kg)  reported pathway . GloballRecreational team sports interventions in the form of soccer, floorball, and basketball played as small-sided games were reported to induce significant changes in health-related variables, such as blood pressure and blood profile , 23, 24.Regular aerobic exercise has been shown to positively affect blood profile by lowering circulating cholesterol . Total cThe PWV constitutes an indirect method to assess arterial stiffness considered as a variable which reflects alterations of vessels structure and function . UpregulBlood glucose concentration was considered as an indirect marker of insulin resistance and affected by aerobic and resistance exercise , 48. ThePostural balance is considered a physical ability related to neuromuscular health and associated with the risk of falling , 50. RecHandgrip strength has been described as a general maker of body strength and its reduced values have been reported to be associated with increased risk of cardiovascular and all-cause mortality across age and gender, in socially and culturally diverse populations . Team haThe changes shown in most of the physical fitness and metabolic and cardiovascular variables were similar between outfield players and goalkeepers, indicating that if the participants are previously trained in team handball, the beneficial effects may occur, regardless of the playing position. Nevertheless, one can speculate that if the participants are not specifically trained as goalkeepers, the demands would be quite lower due to lack of expertise , 52\u201354. The CG showed no significant changes in all of the evaluated variables, with the exception of cholesterol , the ratios: total cholesterol/HDL, total cholesterol/LDL, and LDL/ HDL, and fasting plasma insulin, underlying the effectiveness of recreation team handball as a training intervention exercise mode.In summary, short-term recreational team handball played as small-sided or formal games  is a high attendance team sport for 33-55-year-old sedentary former team handball players, which positively impacts on participants' physical fitness and cardiovascular and metabolic health. Training interventions aimed at analyzing the effects of recreational team handball practice on participants of either gender with little or no experience in this sport and also the physical and physiological demands of playing different game formats are warranted."}
+{"text": "Interpretation of bowel sounds (BS) provides a convenient and non-invasive technique to aid in the diagnosis of gastrointestinal (GI) conditions. However, the approach\u2019s potential is limited by variation between BS and their irregular occurrence. A short, manual auscultation is sufficient to aid in diagnosis of only a few conditions. A longer recording has the potential to unlock additional understanding of GI physiology and clinical utility. In this paper, a low-cost and straightforward piezoelectric acoustic sensing device was designed and used for long BS recordings. The migrating motor complex (MMC) cycle was detected using this device and the sound index as the biomarker for MMC phases. This cycle of recurring motility is typically measured using expensive and invasive equipment. We also used our recordings to develop an improved categorization system for BS. Five different types of BS were extracted: the single burst, multiple bursts, continuous random sound, harmonic sound, and their combination. Their acoustic characteristics and distribution are described. The quantities of different BS during two-hour recordings varied considerably from person to person, while the proportions of different types were consistent. The sensing devices provide a useful tool for MMC detection and study of GI physiology and function. For centuries, auscultation has been used as a non-invasive technique in medicine . The mosDoctors have also listened to gut noises to diagnose conditions such as bowel obstructions and paralytic ileus based on the nature or absence of bowel sounds (BS) . In 1905BS generation is linked with gastrointestinal motility and a key physiological phenomenon underlying motility, the migrating motor complex (MMC) . The MMCThe relationship between MMC and BS was first proposed by Tomomasa et al. in 1999 . They foMicrophone-based sensors are popular for BS recording ,18,19,20The proposed device can be evaluated by comparing the observed BS with previous studies. Different BS classification methods have been investigated, although no standard definitions or classes of BS have been established. In 1967, Watson and Knox described three different types of BS by analyzing the sounds from three participants , includiThis study presents a low-cost and straightforward piezoelectric acoustic sensing device that can effectively allow detection of BS and MMC cycle with a personal computer and commercial software Audacity. This offers the potential of greater understanding of the physiology of the gastrointestinal tract and possible clinical use. BS from ten participants were recorded for two hours for identification and characterization of BS types. The observed BS were classified in a fundamental way with two physical indexes, waveform, and spectrogram. Five different types of BS were systematically analyzed including the duration, spectral flatness, spectral bandwidth, and mean-crossing ratio. The quantities of different types of BS were also counted from these long-term recordings, and their quantities proportion of different types within each participant were found to be stable. Also, in eight- and four-hour recordings from a healthy participant, several MMC cycles and different phases were observed under the fasting state using the sound index, and longer BS are more likely to occur in Phase III using sound duration as another index. As expected, the cycles disappeared when the participant consumed food.A low-cost and straightforward piezoelectric sensing head that connects to a sound recorder  was used for BS recordings. This piezoelectric sensing head consisted of a solid housing, a membrane, a piece of foam and a piezoelectric disk. The piezoelectric disk is composed of a PZT ceramic circle on the top of brass circular base, with diameters of 20 mm and 27 mm, respectively. The thickness of the disk is 0.52 mm and the resonant frequency and impedance are 4200 Hz and 300 \u03a9, respectively. This piezoelectric disk is commercially available, and the reader could buy it online. The sensor head configuration is shown in Two experiments were conducted for BS verification. An anechoic chamber recording on the body was conducted to ensure that the types of sounds that we had identified as coming from the abdomen were not actually background noise from our quiet recording room. The sounds were present in both locations, which indicated that the sounds were indeed BS. Another preliminary recording in our quiet room was executed and a potential BS library was established for two medical doctors to verify. The BS in the library was collected from the participants\u2019 recordings with 18 BS containing all different types and eight irrelevant and environmental noises. Two medical doctors participated in this blind test. 100% of the BS and noises identified in this study matched the doctor\u2019s judgement based on their experience. Hence, we systematically validated the recorded BS as sounds typically regarded as GI in origin by clinicians.Two experiments were designed to record BS passively for BS identification and MMC detection and observation, with the study approved by the UWA Human Research Ethic Office (study no. RA/4/1/8893).First, two-hour recordings were taken from each of ten participants (participant No. 1 to 10) with 44.1 kHz sampling frequency. This quantity of participants was selected because it was found to be useful in previous studies of the individual characteristics of BS ,27,29,30Second, a total of twelve hours of BS recordings were taken from participant L, primarily for study of the MMC. Recordings were made using two sensor heads, one placed on the upper abdomen and one placed on the lower abdomen, simultaneously. Participant L was asked to fast overnight, skip breakfast and recorded under fasting conditions for eight hours to see the MMC cycle. Both the sound duration and sound index over the eight hours of recording were documented in this stage. Another four hours recording after a meal was conducted to investigate how the MMC changed after the meal.Using the 20 h of recordings from ten participants, we identified five typical types of BS with our proposed sensing device. They were identified according to their time and spectrogram information expanded based on short time Fourier analysis . These fThe most frequent type of BS in the recordings was the SB, which is a simple pulse probably caused by a single contraction of the bowel muscle ,28,29. AIn MB can be described as a repetitive SB with a shorter interval time between adjacent components. The CRS is shown in Another typical BS is the HS, which is a whistling-like sound and is presented in In addition to the above four separate types of BS, many detected BS often appear as a combination of the types described above. As presented in Although the quantity ratios across these four types of BS vary, their proportions were consistent. The consistency can be represented by CV, which is the ratio between the standard deviation and the mean value. It is clear that the CV of proportion is much smaller than the CV of quantities. Several typical acoustic features were studied for different types of BS including the duration, spectral bandwidth, spectral flatness, and mean-crossing ratio. The BS acoustic features from first and third quadratic are shown in For the duration, it is common that the SB is typically short and the duration of the other four types of BS have a similar distribution. However, the CRS has some extra-long BS beyond 4000 ms. The second longest type of BS is the MB, which can last up to 3000 ms. In this study, the longest duration observed for the HS was 1800 ms. The spectral centroid is the \u201ccenter of mass\u201d of the spectrum, where all five types of BS share similar distribution and values. The spectral bandwidth is defined as the wavelength interval in which a radiated spectral amplitude is not less than half its maximum value. The mean value of spectral bandwidth decreases from MB, SB, HS to CRS. The spectral bandwidth distribution of the CRS type of BS is the narrowest at low spectral bandwidth. The spectral flatness is a measure used in digital signal processing to characterize an audio spectrum to show how tonal the signal is. It is interesting to note that the CRS has the smallest spectral flatness. As with spectral bandwidth, the mean values of spectral flatness decrease from MB, SB, HS to CRS. The SB and HS share the same distribution with different mean values, and the CRS has the narrowest distribution again. This order is observed in the spectral bandwidth and mean-crossing ratio. The mean-crossing ratio value is the number of times the waveform crosses its mean value. The distributions of these five types of BS have the most significant difference among all other characteristics. The MB tend to have a higher value with narrower distribution. The lowest mean value is that of the HS with wide distribution.The BS sound index was calculated in a long recording after verifying the performance of our acoustic sensing device and identifying the BS. The presented sound index values are scaled by the maximum values in From In addition, the MMC pattern was also observed in the other ten participants from their two-hour recording based on the sound index. As shown in We also studied the mean BS quantities before and after the meal over the eight- and four-hour recording. The means were 3.09 and 7.12 BS per minute for the upper and lower abdomens respectively, from the first eight hours of recording. From the recording after meal, there were on average 4.59 and 45.88 BS per minute detected at the upper and lower abdomen, respectively. These quantities indicate a significant increase in gut activity in the lower abdomen after eating.A low-cost and straightforward acoustic sensing device was proposed in this paper to enable long duration BS recordings. The sensor head consisted of only a piezoelectric sensor, housing, membrane, and a piece of foam. The performance of the device has been tested with two hours of recording from each of the ten participants and an eight- and four-hour recording from one participant. We undertook analysis of BS from ten volunteers with the proposed sensing device. Our simple sensor heads were able to pick up previously described BS types. However, we were also able to improve characterization of BS types and produce a standard categorization system. Based on our analysis, we found five different characteristic types of BS based on their waveform and spectrogram. The first four separate types of BS comprise the single burst, multiple bursts, CRS, tone sound, harmonic sound, and the last one is their combination. Their behavior and characteristics were described, and the quantities of different types recorded from each participant were counted. These quantities varied significantly from person to person, from 0.04 to 0.7 per second. However, the proportions of different types of BS were reasonably stable. The mean proportion of SB sounds was 87.8% \u00b1 5.4%, mean multiple bursts proportion was 7.8% \u00b1 3.5%, mean continues random sound proportion was 3.5% \u00b1 2.0%, and the mean proportion harmonic sound was 0.9% \u00b1 0.5%. Also, from the eight- and four-hour recording, six MMC cycles were successfully observed based on the sound index with clear phases identifiable under the fasting state, and, as expected, the MMC disappeared once the participant consumed food. The MMC cycle was also observed from the two-hour-long recordings taken from ten other participants. This study also shows that when the gut enters MMC Phase II and III, longer BS were generated due to the increased gut activity by looking at the sound duration. This result shows that this simple and low-cost acoustic sensing device can effectively detect the MMC cycle by using the sound index as its biomarker. This work will help in further bowel sound studies with long recordings by providing guidance on the design of an effective acoustic sensing device and may aid in the development of new methods for understanding GI physiology."}
+{"text": "Pain levels evaluated using VAS within 1\u2009min  and 1\u2009h after oocyte aspiration  were lower in the TEAS group than in the mock TEAS group. Nausea assessment revealed a significantly lower VAS score in the TEAS group within 1\u2009min . Serum \u03b2-endorphin levels were significantly higher in the TEAS group than in the mock TEAS group  after retrieval. Serum \u03b2-endorphin levels were higher in the TEAS group after the procedure than baseline . Oocyte retrieval causes pain and discomfort, but TEAS is effective and safe for suppressing the pain and alleviating nausea associated with the operation.Acupuncture has pain-relief effects, but no data were available on the use of transcutaneous electric acupoint stimulation (TEAS) in pain relief during oocyte retrieval. This study was designed to examine the effect of TEAS for pain relief in women undergoing transvaginal ultrasound-guided oocyte aspiration. This single-blinded, multicenter, randomized controlled trial was performed in China between May 2013 and May 2015. The subjects were randomized to mock TEAS and TEAS. TEAS or mock TEAS was administered 30\u2009min before oocyte retrieval until the end of the operation. The primary and secondary endpoints were the pain measured using the visual analog scale (VAS) within 1\u2009min and 1 hour after oocyte retrieval, respectively. Serum  Transvaginal ultrasound-guided oocyte aspiration is a standard procedure in the process of in vitro fertilization and embryo transfer (IVF-ET). Oocyte aspiration is associated with pain and negative emotions. The level of pain perceived during oocyte aspiration varies widely from one individual to another and is often associated with the level of menorrhagia , 2.Conscious sedation is the most commonly used method to reduce pain during oocyte aspiration (or ovum pick-up (OPU)) . ConventKwan et al. showed that the simultaneous use of more than one method seems to achieve better pain relief than any modality alone . AcupuncWe hypothesized that TEAS could be used to alleviate pain during oocyte retrieval. The aim of this single-blinded, multicenter, randomized controlled clinical trial was to examine the pain-relief effect of TEAS during OPU. The results could provide new analgesia methods for OPU, with a smaller use of drugs.ChiCTR-TRC-13003952, Principal investigator: Jisheng Han, Date of registration: 2013-11-25).This clinical trial was approved by the Institutional Review Board of Peking University (IRB00001052-13003) and the four participating centers, and it conforms to the provisions of the Declaration of Helsinki (as revised in Tokyo 2004). Written informed consent was obtained from all subjects. The trial was registered prior to patient enrollment at the Chinese Clinical Trial Registry  at the reproductive centers of Peking University People's Hospital, Peking University Third Hospital, Beijing Obstetrics and Gynecology Hospital, and Shandong University of Traditional Chinese Medicine Second Affiliated Hospital. The study was completed between May 2013 and May 2015. Information regarding the study was provided to all patients during their initial IVF consultation. This manuscript adheres to the applicable CONSORT guidelines.The inclusion criteria were (1) infertile women of 20\u201345 years of age; (2) those undergoing microstimulated superovulation, natural cycle; and (3) those with five or fewer oocytes (diameter >10\u2009mm) retrieved. The exclusion criteria were (1) use of sedative or analgesic drugs or (2) use of a pacemaker, any device to assist the heart or lungs, Holter monitoring, or any implanted device sensitive to microwaves.Participants were randomly assigned (in a 1\u2009:\u20091 ratio) to receive either TEAS or mock TEAS. Randomization was stratified according to the different centers. A third-party statistician produced a randomization table with stratified blocks of four using SAS 9.13  and prepared sequential sealed opaque envelopes. The women undergoing OPU and the physician who evaluated the levels of pain, nausea, and vomiting were blinded to grouping. The evaluation was performed by one physician at each center who received unified training. The operators who did TEAS treatment were not blinded since the intensity of the stimulation in the treatment group needs adjustment, and small muscle trembling near the stimulation sites were visible.All patients received a standard microstimulated superovulation protocol. On the third day of the menstrual cycle, 300 units HMG225 was used to promote the ovum and was continuously used for 8 to 10 days. Follicle growth was evaluated by ultrasound before hCG administration. Intramuscular injection of human chorionic gonadotropin  was performed when the maximum follicle reached at least 18\u2009mm in diameter. Oocyte retrieval was scheduled 36\u2009h later.For women allocated to the TEAS treatment group, TEAS  was administered 30\u2009min before OPU and continued during the OPU procedure till the operation was completed.LI 4 (Hegu at the part between the thumb and index figure) and PC 8  on one side, PC 6 (Neiguan), and SJ 5 (Waiguan) on the wrist of the other side received sticking skin electrodes of 4\u2009\u00d7\u20094\u2009cm. The TEAS was administered at alternating frequencies , with a pulse width of 0.6\u2009ms at 2\u2009Hz and 0.2\u2009ms at 100\u2009Hz and an intensity of 10\u201320\u2009mA (two times the threshold intensity) [For women in the mock TEAS group, 2/100\u2009Hz intermittent (10\u2009s on and 20\u2009s off) TEAS at 5\u2009mA was given (slightly above the threshold). The acupoints used were the same as for the TEAS group. The mock TEAS represents a weak , but still, sensible stimulation, which has been demonstrated as a successful placebo  treatment modality in a previous smoke abatement trial .Body temperature, respiratory rate, heart rate, and blood pressure were measured before OPU. If the patients changed their minds before the operation and demanded the use of analgesics, those patients were naturally excluded. Intraoperatively, if patients reported unbearable pain, propofol 2\u2009mg/kg was immediately administered. The specific conditions of this event for those patients were recorded; they were not included in the final statistical population. Patients with postoperative pain received sedatives or analgesics (such as morphine pump) immediately after the first postoperative evaluation; they were included in the statistical analysis.The primary endpoint was the visual analog scale (VAS) for pain within 1\u2009min after OPU. The secondary endpoint was the VAS for pain at 1\u2009h after OPU. The occurrence of nausea and vomiting within 1\u2009min and 1\u2009h after OPU was evaluated as safety endpoint using a VAS, where 0 indicates no pain or nausea and 100 indicates the worst pain or nausea ever imagined. Vomiting was also recorded. The reproductive outcomes were exploratory endpoints.\u03bcL aliquots, and immediately frozen at \u221280\u00b0C. Serum concentrations of \u03b2-endorphin were measured using a commercially available human \u03b2-endorphin ELISA kit .Blood tests were performed for the first 50 patients in each group at Shandong Center. Samples were collected by trained nurses before and within 1\u2009min after OPU. Patients were fasted overnight and allowed for only moderate amounts of drinking water. Venous blood (2\u2009mL) was collected into clotting tubes. The samples were placed at room temperature for 30\u2009min and then centrifuged at 1600\u2009\u00d7\u2009g for 15\u2009min at 4\u00b0C. The serum was isolated, divided into 600\u2009Adverse events (AEs) were followed by the investigators until 1 week later after the intervention.\u03b1 was set 0.05, and the power was set 0.8. According to the formula NC=NT=(2(Z\u03b11\u2212+Z\u03b21\u2212)2\u03c32)/(\u0394\u03b4\u2212)2, at least 157 patients were needed in each group according to 1\u2009:\u20091. The estimated dropout rate was 20%, and then the sample size of each group was 196 patients or 392 patients total.The sample size was estimated based on data collected from a pilot study in which the VAS score was 3.0\u2009\u00b1\u20091.2 for the TEAS group and 4.9\u2009\u00b1\u20091.5 for the control group. t-test. The paired t-test was used for intragroup analyses. Continuous data with a skewed distribution were presented as medians (range) and were analyzed using the Mann-Whitney U test. Categorical data were presented as frequencies and were analyzed using the chi-square test. The primary endpoint compared between the two groups was using the Student t-test. Two-sided P values <0.05 were considered statistically significant.Data were analyzed using SPSS 19.0 . Continuous data were tested for normal distribution using the Kolmogorov\u2013Smirnov test. Normally distributed continuous variables are presented as means\u2009\u00b1\u2009standard deviation and were analyzed using the Student n\u2009=\u200912). A total of 392 subjects were randomized, and 390 subjects were analyzed, including 194 in the mock TEAS group and 196 in the TEAS group. All patients were followed up. The intent-to-treat (ITT) principle was used to analyze the data. As shown in A total of 430 subjects were recruited between May 2013 and May 2015 . SubjectP < 0.01) and 1\u2009h  after OPU.As shown in P=0.033). There were no significant differences between the two groups in nausea 1\u2009h after OPU   than in the mock TEAS group  , which iSix patients in the mock TEAS group and two patients in the TEAS group vomited within 1\u2009min after OPU, without differences in VAS scores between the two groups (data not shown). No patient vomited 1\u2009h after the procedure.After 1 week of follow-up, no severe AEs were found.\u03b2-endorphin levels were significantly higher in the TEAS group compared with the mock TEAS group  after OPU. Serum \u03b2-endorphin levels were higher in the TEAS group after the procedure than before . No change was found before and after oocyte retrieval in the mock TEAS group.As shown in There were no significant differences between the mock TEAS and TEAS groups regarding the reproductive outcomes  and nausea (within 1\u2009min after OPU) were significantly alleviated by TEAS compared with the mock TEAS intervention. The novelties of the present study were to use TEAS instead of EA and to try TEAS in the setting of a multicenter study. Concerning the design of the control group, since TEAS stimulation would produce subjective feeling, it is very difficult to design a satisfactory placebo group. Based on a previous study in smoking cessation , we usedIndeed, previous studies showed that EA or TEAS reduces patient-controlled analgesia (PCA) requirement and incidence of nausea, vomiting, dizziness, and pruritus , 19 and The output parameters used in EA or TEAS therapy appear to be critical for achieving the optimal efficacy of TEAS. Enkephalins are preferentially released in the brain in response to a 2\u2009Hz stimulation, and dynorphin release is promoted preferentially by a 100\u2009Hz stimulation in the spinal cord. Therefore, the alternating 2 and 100\u2009Hz stimulation with a dense disperse pattern has been demonstrated to be most effective in activating the endogenous opioid system , 21 explA better analgesic effect of using the alternating 2/100\u2009Hz stimulation for pain relief was supported by two recent studies , 25. TheThe selection of acupoints used in the present study was based on reports in the literature that LI 4 (Hegu) is an acupoint most commonly used for analgesia  and PC 6Several underlying mechanisms have been proposed for the EA/TEAS-induced reduction of postoperative nausea and vomiting. In a clinical setting, TEAS apparently relieves nausea and vomiting during and after cesarean section , and the\u03b2-Endorphin is a hormone mainly used by the human body to control stress and pain and is involved in reward recognition [\u03b2-endorphin levels were significantly increased by TEAS and were higher in the TEAS group compared with the mock TEAS group after OPU. \u03b2-Endorphin probably played a role in the reduction of pain by TEAS. This effect of TEAS on \u03b2-endorphin levels was observed by the early studies of TEAS [ognition . In the  of TEAS \u201337.TEAS is generally considered to be a safe procedure , 38, 39.\u03b2-endorphins were measured, but additional biomarkers of pain and stress could be measured in future studies. The conventional medical analgesia (CMA) group would have been useful for fully evaluating the efficacy of TEAS. Unfortunately, a CMA group was not included in the original design of this randomized controlled trial. Since the trial is completed, it cannot be provided. Such a group will be included in future trials.The present study has limitations. Considering the characteristics of the TEAS procedure, the TEAS operator cannot be blinded. Only the patients and evaluators were blinded. Only In conclusion, the present study demonstrates that the pain associated with OPU was less in the TEAS group compared with the mock TEAS group. The nausea was also less severe in the TEAS group than in the mock TEAS group. These findings provide strong evidence for the clinical efficacy of TEAS in reducing pain and discomfort associated with OPU."}
+{"text": "BackgroundGiardia lamblia is a pathogenic intestinal flagellate transmitted by the ingestion of\u00a0contaminated water or food with the cyst stage of the parasite. Giardiasis can cause severe acute diarrhea and malabsorption or may persist as a chronic infection. Effective treatment and control measures depend on proper laboratory diagnosis using diagnostic methods with high sensitivity and specificity.ObjectiveG. lamblia in clinical human fecal samples.To compare the sensitivity and specificity of direct smear, Ritchie sedimentation technique, two brands of rapid chromatographic immunoassay test, and real-time polymerase chain reaction (PCR) for the detection of Materials and methodsUnpreserved 100 stool specimens were collected in clean plastic containers and labeled with the patient\u2019s information and examined through light microscopy, immunochromatographic\u00a0test (ICTs), and real-time PCR.ResultsGiardia deoxyribonucleic acid (DNA) in (42) samples followed by ImmunoCard STAT! (31) samples , direct smear (23) samples, CerTest (19) samples , and Ritchie technique (17) samples. Real-time PCR was the most sensitive for the diagnosis of G. lamblia in comparison to the other techniques.Out of 100 fresh stool samples obtained from workers analyzed, real-time PCR targeting the SSU rRNA gene was able to detect ConclusionsG. lamblia in stool samples. Further studies are recommended using multiplex real-time PCR assay in order to increase the possibility of the presence or absence of the infection.All the techniques investigated were sensitive for the detection of  Giardia lamblia. The life cycle is simple and direct, including the motile trophozoite and nonmotile cyst stages\u00a0while the ingestion of cysts in contaminated water or food  is the main mode of transmission [Giardia has been included in the \"neglected diseases initiative\" [Giardia in many people is asymptomatic\u00a0while others suffer from acute watery diarrhea, abdominal cramps, nausea, epigastric pain, malabsorption, flatulence, and weight loss [G. lamblia (2%-20%) among various populations [One of the most important causes of parasitic diseases is a flagellated protozoan called smission . The Wortiative\" -3. Infecght loss . In someght loss . Previouulations -9. Giardia are classified into eight genotypes and the studies of these genes have indicated evidence of cryptic infection, suggesting the use of molecular diagnostics [The early accurate diagnosis of giardiasis is important for the successful treatment and prevention of diseases. The routine laboratory diagnosis is performed for the detection of trophozoites or cysts by microscopic examination of at least three stool samples collected independently -11. Thisgnostics .G. lamblia in clinical human fecal samples.The aim of the present study was to compare the sensitivity and specificity of several diagnostic methods: direct smears, Ritchie sedimentation technique, two brands of ICTs, and real-time PCR\u00a0for the detection of Specimen\u00a0collectionStool specimens were collected in clean plastic containers from workers recently arrived in Jeddah, Saudi Arabia, during their screening visit to get a residence permit .Questionnaire formThe data of patients were collected by interviews in a standard questionnaire that was designed for this study. It included questions on demographic information  and some general information about intestinal parasitic infections.Laboratory examinationG. lamblia, Cryptosporidium parvum, and Entamoeba histolytica. Real-time PCR\u00a0was used to detect the small subunit ribosomal RNA gene (SSU rRNA) of G. lamblia.Laboratory microscopic examination of feces was accomplished by using direct smears, the Ritchie technique, Para-Pak trichrome stain. Two brands of rapid chromatographic immunoassay tests were performed according to the manufacturer's instructions. ImmunoCard STAT! CGE  and CerTest  are designed to identify Direct smearsAbout 2 mg for each stool sample was mixed with a drop of normal saline (0.9%) and iodine on a clean glass slide (75 X 26 mm). A cover glass (22 X 22 mm) was placed carefully and then examined by light microscopy using (10x) and (40x) objective lenses.Ritchie  sedimentation techniqueAbout 2 gm of each stool sample was emulsified in 10 ml of 10% formal-saline. It was then filtered through two gauze layers fitted in\u00a0a funnel and centrifuged for 5 min at 2000 rpm. The supernatant was discarded, and the sediment was re-suspended in 10 ml of formal saline (10%) and mixed very well. After that, 3 ml of diethyl ether was added, shaken vigorously for 10 sec, and then centrifuged for 5 min at 2000 rpm. Four layers were formed: the top layer of ether, the\u00a0plug of the debris, 10% formal saline, and the sediment layer at the bottom of the tube. The top three layers were removed, and a drop of iodine was mixed with the sediment and then examined using (10x) and (40x) objective lenses .Para-Pak trichrome stainStool samples were fixed in polyvinyl alcohol (PVA). Stool smears were prepared and stained by Wheatley trichrome stain according to the manufacturer's instructions, . Smears were air-dried then stained with trichrome stain for 6-8 min, placed in acid ethanol for 5-10 sec, then two quick dips in (95%) ethanol. The slides were immersed two times in (95%) ethanol for 5 min, then in (96-100%) ethanol for 3 min, and finally placed in xylene for 3 min. Finally, each slide was mounted with a coverslip (22 X 40 mm) and examined using an oil immersion (100x) objective lens.ImmunoCard STAT! CGEC. parvum antigens. In the presence of G. lamblia antigens, a red/pink color is observed at band 2. A green band appearing at position 3\u00a0is related to E. histolytica antigens.For each specimen, 1 ml of the diluent was transferred into a 15 ml centrifuge tube. Thereafter, approximately 50 mg of each stool specimen was added and mixed gently, then centrifuged for 5 min at 3000 rpm. Next, 125 \u00b5l of each prepared specimen was placed into the sample port of the device and the results were interpreted after 10 min. A purple band appears at the control line (C), indicating that the test was completed successfully. The blue band at position 1 on the device frame\u00a0is indicative of the presence of CerTestOne-hundred twenty-five (125) mg was added into the diluent collection tube,\u00a0mixed very well, and then shaken vigorously for seconds. Four drops of the prepared specimen were dispensed in each of the circular windows. The test was interpreted after 10 min with a green band-of-control line. The formation of a red band at the test line is indicative of the presence of the investigated parasite.DNA isolationStool samples were subjected to DNA extraction using the QIAamp DNA Stool Mini Kit  protocol. Each DNA sample was eluted in a final volume of 100 \u03bcl and stored at -80\u00b0C until needed.Real-time PCR amplification G. lamblia speci\ufb01cally. To select the speci\ufb01c Giardia sequence, alignments were made of 31 GenBank sequences that included 16 Giardia and 15 sequences of other parasites and bacteria\u00a0(non-pathogenic). The Giardia-speci\ufb01c primers and probe set consisted of the forward primer Giardia F, the reverse primer Giardia R, and the Giardia specific double-labeled probe Giardia T  [Giardia F , Giardia R  and Giardia T .The target gene of the real-time PCR assay is the SSU rRNA gene. DNA was ampli\ufb01ed and detected forStock primers and probeAccording to the manufacturer's instructions, to reach 100 pmol/\u00b5l, 250 \u00b5l of nuclease-free water was added for each primer and 280 \u00b5l was added for the probe.Working primers and probeFifty \u00b5l from each stock primer was added to 50 \u00b5l nuclease-free water to prepare 50 pmol/\u00b5l. From this, 10 \u00b5l was mixed with 90 \u00b5l of nuclease-free water. Thirty \u00b5l from the stock probe was added to 70 \u00b5l nuclease-free water to prepare 30 pmol/\u00b5l. From this, 10 \u00b5l was mixed with 90 \u00b5l of nuclease-free water. All working primers and probes were stored at -20\u00b0C until needed.\u00a0Real-time PCR master mix preparationThe real-time PCR master mix was prepared according to QuantiTect\u00ae Probe PCR Kit instructions  in a 1.5 ml microcentrifuge tube, vortexed very well, and then briefly centrifuged at full speed. After that, the reaction mix was dispensed in a volume of 15.5 \u00b5l using a standard PCR plate containing 96 wells strips tubes. Then, 9.5 \u00b5l of the template DNA was added to the individual wells containing the reaction mix. The plate was then transferred into Applied Biosystems 7500 Fast cycler , to start the cycling program.Real-time PCR data analysisData analysis was performed with the Applied Biosystems 7500 Fast System\u00a0by calculating the threshold cycle number  that presented the positive amplification of gene in a real-time cycler number.Statistical analysisCategorical data were reported as frequency and percentage (%). Data were analyzed using SPSS . A P-value of < 0.05 value was accepted as statistically significant ). Sensitivity, specificity, PPV, NPV, and accuracy were calculated to test the diagnostic yield.G. lamblia, 18 samples for E. histolytica, nine samples for C. parvum, three samples for E. coli, three samples for hookworms, two samples for Trichuris trichiura, two samples for Enterobius vermicularis, and one sample for each of the following: Endolimax nana, Hymenolepis nana, and Taenia spp.Out of 100 fecal samples analyzed, 48 were positive for Table G. lamblia, real-time PCR was able to detect Giardia DNA in 42 samples followed by ImmunoCard STAT! CGE (31), direct smears (23), CerTest (19), and the Ritchie sedimentation technique (17).Out of the 100 samples screened for Card STAT! CGE revealed the highest sensitivity followed by direct smears  sensitivity with the Ritchie technique Table . ImmunoCG. lamblia in clinical human fecal samples.Intestinal parasitic infections are among the major public health problems in tropical and sub-tropical parts of the world, particularly in rural communities with poor sanitation and poor personal hygiene behaviors. The goal of our study was to compare the sensitivity and specificity of three diagnostic methods: light microscopy, ICTs, and real-time PCR for the detection of Light microscopy is still considered the reference standard access to the fecal parasitological laboratories but there is growing interest in alternative detection methods to overcome the current limitations of microscopic examination. We accomplished a microscopic examination by using direct smears, and the Ritchie technique. The Para-Pak trichrome stain was used to confirm the morphological features for trophozoites and cysts for protozoa parasites. Meanwhile, DNA detection by PCR is offering a better turnaround time and ICTs are commercially available and widely used in clinical laboratories, with limited capacities for diagnostic complexity .Stool samples were collected from non-Saudi workers applying for a residence permit (Iqama). Non-Saudis represent around 30% of the population in Saudi Arabia . All subG. lamblia detection, 42 were found positive by real-time PCR, 31 by ImmunoCard STAT! CGE, 23 by direct smears, 19 by CerTest, and 17 by the Ritchie technique. In 2016, Ahmed and others [E. histolytica, G. lamblia, and Cryptosporidium spp. among children in Bangladesh. Regarding the variation in their study subject, the prevalence of giardiasis was (50.6%), (26.7%), and (3.3%) by using real-time PCR, ICTs, and direct microscopy respectively. The authors also observed that the positive samples by real-time PCR but negative by the other techniques showed higher Ct values due to the small number of parasites in those samples that fell below the other methods limit of detection. Our finding is in agreement with that observation. The variation in our results between microscopic examination using direct smears and the Ritchie technique is due to the ability of each technique for the detection of cyst and trophozoite. Direct smears detect both cyst and trophozoite stages\u00a0while the Ritchie technique is applicable to the cyst stage only [Out of the 100 fecal samples analyzed for A study conducted in Mozambique for the detection of intestinal parasites in fecal samples showed that the sensitivity of real-time PCR for the detection of each of the parasite species was higher than direct smears, Ritchie technique, Kato smear, Baermann, and coproculture . The worNominating real-time PCR as a gold standard, ImmunoCard STAT! CGE showed a higher sensitivity (59.5%) than both direct smears and the Ritchie technique (52.4% and 40.5%), respectively , respectively (Table Giardia in fecal samples in Central Africa [G. lamblia [Using the Ritchie technique as the reference method, the sensitivity for all the diagnostic tests has reached 100%\u00a0while the specificities ranged from 69.9% to 97.6%\u00a0Table . Anotherl Africa . The lowl Africa . A simil lamblia , and theG. lamblia in stool samples. The real-time PCR assay was revealed as the most accurate diagnosis of giardiasis. ICTs proved to be practical, easy to perform, time-saving, and beneficial to analyze a low number of samples. It can be a valuable diagnostic tool in case microscopic expertise is limited. Wet mount microscopy and the Ritchie technique are still important methods for the detection of gastrointestinal parasites when a skilled technologist tests the samples.In conclusion, with some variations, all the investigated techniques were applicable for the detection of"}
+{"text": "Ascaris suum . Some NITs had anthelmintic activity across the phylogenetic diversity of the Nematoda. The whole-worm motility inhibition assay quantified anthelmintic activity, but worm responses to NITs in relation to pathology or affected molecular pathways was not acquired. In this study we extended this research to more comprehensively determine in whole larval A. suum the cells, organ systems, molecular targets, and potential cellular pathways involved in mechanisms of toxicity leading to cell death. The experimental system utilized fluorescent nuclear probes , NITs, an A. suum larval parasite culture system and transcriptional responses (RNA-seq) to NITs. The approach provides for rapid resolution of NIT-induced cell death among organ systems , discriminates among NITs based on cell death profiles, and identifies cells and organ systems with the greatest NIT sensitivity . Application was extended to identify cells and organs sensitive to several existing anthelmintics. This approach also resolved intestinal cell death and irreparable damage induced in adult A. suum by two NITs, establishing a new model to elucidate relevant pathologic mechanisms in adult worms. RNA-seq analysis resolved A. suum genes responsive to treatments with three NITs, identifying dihydroorotate dehydrogenase (uridine synthesis) and RAB GTPase(s) (vesicle transport) as potential targets/pathways leading to cell death. A set of genes induced by all three NITs tested suggest common stress or survival responses activated by NITs. Beyond the presented specific lines of research, elements of the overall experimental system presented in this study have broad application toward systematic development of new anthelmintics.In research focused on the intestine of parasitic nematodes, we recently identified small molecule inhibitors toxic to intestinal cells of larval   \u2022A unique rapid cell death assay was developed for parasitic nematodes.\u2022A. suum.Multiple drug-like molecules cause widespread cell death in many organs of \u2022Multiple cell and organ systems were validated as targets for anthelmintics.\u2022Potential drug targets/pathways were implicated in activating cell death processes. These pathogens also reduce food production in livestock and plants that in turn differentially restrict nutritional resources of the global poor. Anthelmintic treatments play a major role in the control of nematode pathogens. However, the limited selection of efficacious anthelmintics and emerging pathogen resistance to anthelmintics  identifiA. suum L3 and L4. This progress was accomplished using a de novo approach involving intestinal multi-omics databases, coupled with pathway and drug database analysis to identify druggable targets and related small molecule inhibitors, respectively. Several NITs were also efficacious against phylogenetically diverse nematode pathogens (Brugia pahangi and Trichuris muris), indicating broader application of those findings. Although NITs include drugs approved for use in humans, and thus may represent anthelmintic candidates, they also represent valuable tools to obtain basic information underlying toxic effects, which has relevance in more general terms to anthelmintic research.As one example, our research has focused on the nematode intestine due to its apparent hypersensitivity to some anthelmintics and because it has relative tractability to experimental approaches compared to other tissues . In receA. suum system provided compelling evidence that NITs can cause tissue damage inclusive of cell death, which is a specific end point with important implications for anthelmintic research. For instance, two major mechanisms of cell death dominate research in C. elegans, apoptosis and necrosis  to idennecrosis , and whinecrosis . Of imponecrosis , increasnecrosis ).A. suum L3 and L4 stages with fluorescent nuclear probes  and provide a rapid resolution of cell death among organ systems conferred by NIT treatments , while comparing the performance of NITs in causing cell death among cells and organ systems (PI labeling profiles). The approach also identified cells susceptible to several existing anthelmintics, and when extended to adult A. suum, NIT-induced cell death was documented in freshly dissected intestine. Thus, a method was developed to inventory cell and organ system targets of any of a number of toxins/toxicants of interest in whole parasitic nematodes, while also demonstrating previously unrealized potential of many different organs as targets for anthelmintics. The pathological profiling was complemented with molecular profiles, using RNA-seq based transcriptional profiling of L3 treated individually with several NITs leading to identification of cellular pathways and targets that may represent antecedents to cell death illuminated in PI assays. The results show that the approach successfully discriminated performance among NITs in relation to their toxicity for cells and organ systems.There currently are no existing methods that support rapid, comprehensive monitoring of live cells or cell death in intact parasitic nematodes, and this capability would have high value for experimental dissection of mechanisms related to this outcome. Thus, the objective of the current study was to devise an experimental approach that can illuminate all cells in live 22.1All animal experiments were carried out under protocols approved by Washington State University Institutional Animal Care and Use Committee, protocol 4097. The protocols meet requirements of AVMA Guidelines for the Euthanasia of Animals: 2013 Edition; Guide for the Care and Use of Laboratory Animals: 2011 Edition, National Research Council, and USA Animal Welfare Act and Animal Welfare Regulations: 2017 Edition (AWA), US Department of Agriculture.2.2A. suum lung-stage L3 were obtained as described before . Eggs stripped from the last 3\u00a0cm of A. suum uterus were washed in PBS  then decorticated using 0.25% hypochlorite until decortication was observed . Decorticated eggs were rinsed in 50\u00a0mL double distilled water 3 times, and eggs were then cultured to the infective stage at 20\u00a0\u00b0C for 60 days in 0.1\u00a0M H2SO4  with a loosened screw cap for 3 days, and medium was replaced daily with fresh medium. L3 molt to L4 by day 3. L4 generated in this way were dispensed into wells of 96 well plates used for experiments described here and incubated as for L3.Third-stage larvae (L3) were obtained from lungs  and trac2.3A. suum to ingest fluorescent probes, two function-based fluorescent probes were used in feeding assays: 1) beta-ala,lys-AMCA (BAL-AMCA), 200\u00a0\u03bcM  (To demonstrate the ability of L3 and L4 zerland) ; and 2) zerland) . L3 and 2.4Fluorescent labelling was monitored using a Nikon Diaphot 300 inverted microscope equipped with epifluorescence capabilities (UV-2A filter (blue), BB, BAL-AMCA; G1A, PI; B2A, DQ-BSA) and recorded with a Nikon D5100 digital camera. Most observations were made using the 20\u00d7 microscope objective (200\u00d7 magnification). To better resolve cells and organs in which BB stained nuclei reside, larvae were viewed using a Nikon Optiphot compound microscope equipped with differential interference contrast (DIC) filters, epifluorescence capabilities and a Nikon D5100 digital camera. To optimize resolution of DIC/fluorescence details, images were captured in movie mode, and then selected screen shots were copied and used to produce final digital images.2.5A. suum L3 and L4   were use3 and L4  and was 3 and L4 . Excepti3 and L4 , and ure2.6L4 larvae were used for these assays because toxic effects on L3 can induce the larvae ensheathed with deformation (LED) phenotype , which i2.72 for 2\u00a0h. To reduce equilibration of media with ambient atmosphere during pH measurements, wells of 96 well plates were loosely fitted with film designed to seal wells for PCR analysis . Loose fitting permitted equilibrium with incubator atmosphere. After the 2-h incubation period, and prior to removal from the chamber, the film was pressed to seal wells. Wells were maintained at 37\u00a0\u00b0C on a hot plate  then a slit was created in the film over each well just prior to insertion of a pH microprobe . Three replicate wells were measured for each treatment and compared to media control, with and without 1% DMSO. Once a range of pH was determined for culture conditions, buffering ability was determined for RPMI media buffered by bicarbonate (R8758), Sigma-Aldrich, St. Louis MO, or HEPES , Sigma-Aldrich, St. Louis MO, with starting pH up to 9.0. HEPES, but not bicarbonate, buffered media maintained the pH near the highest pH achieved with NITs and other treatments (see results). Next, PI labeling of L4 cultured in complete medium buffered by 25\u00a0mM HEPES with a starting pH of 8.0, 8.5 or 9.0 was assessed after incubation for 2 days at 37\u00a0\u00b0C in 5% CO2. Wells were run in duplicate, each containing approximately 40 larvae. PI assessment (first 20 L4) and pH measurements were made for each well using our standard methods.Assays were conducted to control for pH effects on PI labeling independent of toxins/toxicant effects. pH of RPMI media buffered by bicarbonate and containing antibiotics was determined after addition of NITs and other treatments (at the highest concentration used for each) and then incubated at 37\u00a0\u00b0C in 5% CO2.8Area, length and width measurements were made for EGCN of L4 cultured for 2 days with staurosporine (25\u00a0\u03bcM), sunitinib (500\u00a0\u03bcM) or control treatments each containing BB to document resultant atrophy of EGCN. The first 10 L4 encountered with lateral lines facing up and down (EGCN at widest profile) were photographed under epifluorescence for identical exposure times. Area measurements on digital photographs were determined by outlining BB labeled EGCN with the drawing tool in Image J  and dete2.9A. suum using two different approaches. One involved injecting BB to achieve 10 \u03bcg/ml (1% DMSO) into the pseudocoelom and incubating for 1\u00a0h, after which nuclear labeling was assessed either directly through the cuticle (EGCN) or in dissected tissues. The volume of BB  for injection was based on mass measurements of adult worms. Injection of live worms depended on movement that would distribute BB throughout the body. However, after treatments described below, movement could not be relied upon for this purpose and then tissues dissected from worms were incubated in PBS with BB and PI for 30\u00a0min to label nuclei for assessment. The PI assay focused on the mid-portion of intestine that lies free in the pseudocoelom because this segment could be dissected with minimal potential tissue damage from handling. Experiments to determine effects of staurosporine (25\u00a0\u03bcM) and leflunomide (500\u00a0\u03bcM) on intestinal cells were conducted on adult A. suum obtained as described above from swine processed for market. Adult worms were washed extensively in PBS, 37\u00a0\u00b0C, then single adult worms were placed in 250\u00a0mL culture flasks, Falcon Brand (353084), Becton Dickinson, Franklin Lakes NJ, containing 50\u00a0mL of PBS and staurosporine or leflunomide with 1% DMSO. Control worms were maintained in PBS and 1% DMSO. Worms were cultured for 2 days at 37\u00a0\u00b0C, 5% CO2, and movement was recorded on each day. At the end of this treatment duration, intestinal tissue was dissected and stained with BB and PI under standard conditions, and then evaluated using light and epifluorescence capabilities of the inverted microscope. Images were captured as described for the L4.BB was used to label nuclei in adult 2.10A. suum L3s were treated with NITs , collected 2 and 4\u00a0h after treatment and rinsed 3 times in ice cold phosphate buffered saline . Following sedimentation in 1.5\u00a0mL microfuge tubes, pelleted larvae were overlaid with 50\u00a0\u03bcl TRIzol reagent , then stored frozen at \u221280\u00a0\u00b0C. RNA was extracted by homogenizing larval pellets as they thawed using a microfuge pestle, and then processing extracts according to the manufacturer's instructions. Ethanol pellets of isolated RNA were shipped to Washington University for further processing and sequencing. cDNA libraries were prepared from RNA samples using the Clontech SMARTer universal low input RNA kit to maximize yield and processed cDNA was sequenced on the Illumina NovaSeq S4 platform (paired-end 150bp reads). After adapter trimming using Trimmomatic v0.36  using t. WBPS14  (v2.7.3a. WBPS14 ), and co. WBPS14  with defate [FDR ] correct2.11A. suum genome .Functional annotations for genes were assigned using InterProScan v5.42  InterPr (KEGG), m genome  to the cm genome  by ident2.12Differential expression and functional enrichment analysis (described above) all utilize FDR-based correction for multiple testing. Two-tailed T-tests to compare measurements of EGCN assumed unequal variance. For PI profiles, since all tests were performed with N\u00a0=\u00a020, significant positive detection of PI was determined to correspond to 25% PI detection , according to Fisher's exact test when control worms have zero PI detection. However, PI profiles were analyzed and compared qualitatively rather than requiring strict significance cutoffs.3Ascaris suum larval parasite culture system and iv) RNA-seq transcriptional response to NITs, to conduct rapid pathologic and molecular assessment of NIT toxicity relative to many cells and organ systems in the whole worm. The approach was designed to discriminate performances among inhibitors in relation to their toxicity for cells and organ systems.Frequently, whole-worm motility inhibition assays are used to evaluate effects of drugs and drug-like compounds on parasitic nematodes. Rapid resolution of individual cells and organs affected by these treatments in whole worms while in culture would greatly enhance details of likely relevance to the anthelmintic effects at play. In this investigation we integrate the use of i) fluorescent nuclear probes , ii) a set of 6 small molecule inhibitors recently demonstrated to target the nematode intestine , iii) a3.1A. suum in feeding assays using two function-based fluorescent probes: 1) beta-ala,lys-AMCA (BAL-AMCA), a fluorescent dipeptide that enters nematode intestinal cells by receptor mediated transport . Nuclei that comprise the lateral lines run nearly the full length of the body and consist of a longitudinal series of single file pairs of lateral and hypodermal cell nuclei with an intervening, central line of single seam cell nuclei , a cell permeable DNA binding dye to establish a map of nuclei localization for various cells and/or organ systems within healthy larvae A\u2013B. Thisbjective C\u2013F. We rbjective D, and thbjective   with an inverted, fluorescence microscope.DIC (60\u00d7 objective) coupled with BB fluorescence validated assignments of nuclei to organ systems made by using standard microscopic methods, and also resolved nuclei in somatic muscle and esophageal cells . Thus, n3.4A. suum L4 were first allowed to ingest BB and PI for 4\u00a0h prior to addition of NITs  signal in organ systems A  indicate a real potential of many specific cells and organs affected by NITs as targets in anthelmintic strategies, b) identify specific experimental treatments that confer toxicity to these cells and organs, and c) identify the intestine and cells anterior and posterior to the nerve ring as relatively sensitive to the NITs tested compared to other cells and organ systems assessed.Dilutions were next conducted to determine concentration-dependent PI profiles for selected NITs A. Leflun3.6per se, the range of concentrations tested for ivermectin, pyrantel and albendazole overlap with those tested on A. suum L4 in motility assays  or binding to tubulin and depolymerizing microtubules . Dilution experiments were conducted with ivermectin, pyrantel, albendazole-SO starting at 500\u00a0\u03bcM to allow comparison to NITs, while levamisole was tested beginning at 10\u00a0mM because this concentration is used to anesthetize nematodes in vitro . The nony assays . With thy assays B. Thus, 3.7We next tested effects conferred by NITs that may be independent of intended pharmacological effects C. These Osmotic and chaotropic effects. The possibility that osmotic (hypertonic) or chaotropic effects of NITs could account for PI labeling profiles was tested in dose response to sorbitol (osmotic) or urea (osmotic and chaotropic) beginning at 1\u00a0M concentrations. At high concentrations, widespread nuclear PI labeling ensued, which rapidly decreased to negligible levels, with sparse PI labeling restricted to the intestine at the lower concentrations. Even considering unexpectedly high dissociation of NIT compounds in solution, the maximum NIT concentrations used (500\u00a0\u03bcM) are not expected to exceed 1\u20132 mOsm. Thus, neither osmotic nor chaotropic effects are sufficient to explain the PI labeling effects documented for the NITs. Nevertheless, relative sensitivities to these treatments could be ordered as intestinal cells\u00a0>\u00a0cells anterior and posterior to the nerve ring\u00a0>\u00a0hypodermal and seam cells, tail cells\u00a0>\u00a0other cells.A pH effect. When the highest concentrations of NITs, urea and sorbitol used here were added to media, mean pH values taken immediately after removal from culture chambers were found to vary from approximately 7.4 (\u00b10.10) to 7.79 (\u00b109). We tested pH effects on larvae cultured from pH 7.46 to 7.74, which required change from a bicarbonate to HEPES (25\u00a0mM) buffered media to achieve the highest pH value. No effect leading to significant PI labeling of L4 was observed at any pH tested . In contrast, no labeling was observed at either treatment time point for media controls, and all larvae showed widespread labeling in the staurosporine positive controls on both assessment days. Consequently, although lack of nutrients can lead to minimal PI labeling by 5 days in culture, this level is insufficient to account for the PI labeling induced by NITs at the initial concentrations tested in assays reported above (2 days after treatment).This series of experiments collectively dispel concerns that non-specific physical effects or indirect impedance of nutrient ingestion account for PI labeling induced by NITs. On the other hand, osmotic and/or chaotropic treatments led to PI nuclear staining, with intestinal cells and cells anterior and posterior to the nerve ring displaying the greatest sensitivity, albeit at concentrations orders of magnitude above those relevant to the NIT concentrations investigated here.3.8Independent of PI labeling, BB labeling of nuclei supported detection of altered nuclear morphology and abnormal spatial distributions of nuclei in intestinal tissue of unfixed whole larvae in culture following NIT treatments . LikewisIn combination, our experiments demonstrate toxic effects of NITs on EGCN based on atrophy (BB labeling) and PI labeling, each fluorescent marker assessing different cellular properties. Consequently, we sought to assess relationships between EGCN atrophy and PI labeling. In a separate double labeling experiment, EGCN from ten staurosporine-treated or control L4 were assessed for EGCN area and PI labeling. All EGCN measured in the staurosporine treatment had areas that fell below the 95% confidence interval for control larvae. In contrast to the consistency of nuclear atrophy, only 4 out of 10 of the staurosporine-treated EGCN showed PI labeling, indicating cell death. Thus, cell death indicated by PI nuclear staining was observed in only a subset of EGCN that showed atrophy. This result may indicate independent processes, e.g. one leading to atrophy of the EGCN and another related to excretory cell membrane damage leading to PI labeling, or that atrophy precedes EGC death.3.9A. suum produced rapid staining of nuclei after 1\u00a0h in several examples chosen to highlight the application  and with staurosporine . Matched controls for each experiment  showed full movement at day 2. Intestine dissected from 2 day-treated worms and then incubated with dyes showed staining of extensive patches of nuclei after treatment with both NITs  that lacked PI staining. However, other areas of the intestinal tube, with an otherwise intact basal lamina, had PI-labeled patches of cell with no adjacent intact cells (no BB-only labeled nuclei), indicating denudation of intestinal cells from the basal lamina and more extensive tissue degeneration.A. suum; second, treatment with each of these NITs led to immotility of adult A. suum in culture; third, treatment with each NIT caused extensive damage to intestinal cells, inclusive of cell death, observable in whole dissected and unfixed intestine. These findings validate a new system involving treatments and experimental outcomes that can be used to investigate mechanisms by which NITs, and other treatments, cause irreparable damage to intestinal cells, and potentially other tissues.These results demonstrate the following: first, rapid high resolution of normal and damaged cells in unfixed tissues from adult 3.10A. suum model described. Cell targets and pathways disrupted by NITs represent points at which cell death processes can be initiated, and thus can provide information of importance to general anthelmintic strategies. Gene response profiles induced by toxic treatments can inform about cell targets and pathways affected  ortholog and an NHR-3 ortholog  were among 41 genes commonly upregulated in response to five different benzimidazole drugs . In C. fication . A totalricoides ) and a dle drugs .3.11C. elegans is required for locomotion and pharyngeal pumping . In humans, leflunomide targets only dihydroorotate dehydrogenase (DHODH) at lower doses, with some inhibition of tyrosine kinases at higher doses (\u22124) of the 18 KEGG pathways significantly enriched among the 44 genes lower with leflunomide at 2\u00a0h  than at 4\u00a0h . Only 3 genes were significant at both 2 and 4\u00a0h, including: (i) a NEP-1 ortholog , which in  pumping , (ii) a er doses . The UPPer doses . Additioer doses A, possiber doses . The mose at 2\u00a0h  was \u201cpyre at 2\u00a0h C. WithinCID1067700 has been identified as a Rab GTPase inhibitor . Rabs diA. suum. However the gene response profile induced by staurosporine may provide information on the terminal process of cell death. In human cell lines, staurosporine is known to induce apoptosis via caspase-9 activation in the intrinsic apoptosis signaling pathway, independent of Apaf-1 and apoptosome formation . Among tion \u22124; . In micetion \u22124; . Taken t4A. suum, and ii) interrogation of transcriptional profiles resulting in identification of hypothetical targets of the drug-like compounds. The assays directly identify the constellation of cells and organ systems that undergo cell death with respect to each of multiple toxins/toxicants. Susceptibility to inducible cell death adds rationale to investigate the cells and organs identified in anthelmintic research, and advances established here provide new experimental methods to do so. We also identify, for some NITs, cellular pathways and molecular targets that are potential antecedents to irreparable pathologic outcomes. Our original derivation of NITs involved a de novo approach (This study presents an interdisciplinary approach designed to intersect i) technical advances that comprehensively and rapidly resolved in whole unfixed parasites pathologic damage induced by NITs among most cells and organ systems that comprise life cycle stages of approach , which w10.13039/100006150PI labeling routinely supports monitoring of viability in many tissue culture cell types  and can rapidly focus research on specific organs, cells, and potentially molecular targets and mechanisms of pathology for investigation by other approaches. When coupled with DIC, and presumably confocal microscopy, greater detail is attainable, although at a cost to efficiency of assessments. The detail obtained at lower magnification with less sophisticated methods identifies a system with substantial versatility to meet a range of research goals.There is no other in culture whole-worm assessment with comparable cellular resolution of cell death for parasitic nematodes. ll types  and has athology . Generalaffected . HoweverA. suum, the issue of PI access is markedly reduced or eliminated.A caveat in interpreting PI labeling is that it provides assessment of cells to which PI is accessible. General body distribution of PI is likely to occur if it gains access to the pseudocoelomic fluid, which bathes most or all organs and cells of the nematode body. Breaching by PI of the internal gut  layer which is continuous with the outside environment, or the external cuticular-hypodermal-muscular layers, might be sufficient to accomplish this access. In this vein, the ability to deliver PI to the intestine pre-treatment is likely to increase sensitivity of the assay. Under conditions of positive PI signal, the assay has value for identifying affected cells and to conduct comparisons among experimental treatments. The interpretation of negative staining may vary, however, according to cell location. Lack of PI staining in cells at external (hypodermis) or internal (intestine) surfaces likely indicates cell viability, if ingested PI is available at the internal surface. In contrast, cells that die in internal organs, absent access of PI could go unnoticed in this assay. For dissected tissues, as with adult As another caveate, we chose a single time point to assess treatment effects here in order to demonstrate utility of the method. Longer exposure may demonstrate cell death for lower treatment concentrations at which this outcome was not observed at 2 days. However the optimal time course will likely depend on both concentration and the specific toxin/toxicant under investigation. Additionally, although PI labeling provided for objective determination of nuclear labeling, subjectivity in scoring among the categories of cell, organs and regions varied to some extent, as might be expected, and consultation with BB labeling proved helpful in making assignments. Also, variation within treatments is reflected by percentage labeling for each category of cell, organ, or region assessed, rather than quantitative assessment of numbers of cells (at least hundreds for each larva), which would have made the analysis far too cumbersome. Consequently, non-parametric measures made sense for feasibility of this analyses. Nevertheless, the approach was adapted with these caveats in mind and addressed the specific questions under investigation in this study.PI labeling produced different cell and organ profiles for different NITs and other treatments, which indicates properties of individual NITs that may have bearing on mechanisms of action. For example, leflunomide and staurosporine produced the most inclusive PI labeling across cells and organ systems, suggesting inhibition of processes by these NITs that lead to cell death in many cells and organs. In contrast, CID1067700 and alvocidib produced more intestinocentric labeling, which may indicate inhibition of target/pathways preferentially expressed in intestinal cells, or a greater sensitivity of intestinal cells once inhibition is achieved. Additionally, BB staining of the EGCN identified morphologic atrophy that was most pronounced for sunitinib and staurosporine treatments. When coupled with knowledge of predicted or actual cellular targets, perhaps contributed by RNA-seq data (see below), knowledge of specific cells affected may aid in dissection of mechanisms involved.Overall comparisons among the different NITs and anthelmintics tested identified cells located in the intestine and adjacent to the nerve ring as most frequently showing PI staining. Reports that intestinal cells are relatively sensitive to benzimidazole anthelmintics in some, but not all , parasitA. suum L4 induced by albendazole-SO are relevant. Combretastatin is not an approved anthelmintic, and although having distinct contacts, it also binds to the colchicine binding site of beta-tubulin  would have produced significant PI signatures at some of the lower concentrations also cannot be excluded. In contrast to the neurotoxic anthelmintics, benzimidazole microtubule inhibitors are known to cause cell degeneration in nematodes. In this regard, the broad range of cells demonstrating PI labeling in -tubulin , as do b-tubulin  suggest A. suum cell death. As such, toxic compounds that initiate processes leading to cell death provide tools to investigate mechanisms of importance in anthelmintic research.PI labeling indicates irreparable cellular damage and cell death, and apoptosis and necrosis are two major mechanisms known to mediate cell death in nematodes . Two sigA. suum. Support for this general view comes from the irreparable damage produced in intestinal cells of A. suum and Haemonchus contortus by benzimidazole treatments, which also disrupted intestinal vesicle transport  and cell death assessments (48\u00a0h), and likely comprise a complex picture to untangle. Overall, the system described offers tools to investigate changes that unfold later, as well as acquire potentially insightful information regardinng A. suum cellular targets and pathways affected by other NITs.In context of initiator pathways, early gene expression responses to leflunomide treatment generated the greatest insight here and identified DHODH and uridine synthesis pathway inhibition as a possible entry point into a process leading to cell death. Although other targets and pathways may contribute to the leflunomide effects, the effects on genes in the primary uridine synthesis pathway, and then secondary energy generating pathways, was remarkably consistent with predictions for DHODH as a target for leflunomide. CID1067700 is a RAB GTPase inhibitor, and gene responses to this treatment differed from that of leflunomide, demonstrating specificity, and included cytoskeletal components that may reflect disruption of vesicular transport pathways. The strength of these findings is somewhat muted by limited information on pathways that respond to RAB GTPase inhibition in other organisms and cells. Nevertheless, inhibition of RAB GTPases and disruption of vesicular transport pathways may represent initiators of processes leading to cell death in ransport . Despiteransport . It is pC. elegans. Because inhibitors of stress response proteins can enhance effects of anthelmintics, including parasitic nematodes, it will be of interest to determine if this gene response is common to other toxic perturbations in A. suum and other parasitic nematodes.Finally, we identified four genes that were upregulated by all three NITs investigated, which are of interest because they may reflect a common response to stress. In this context, 2 of the 4 genes encode glutathione S transferase and NHR-3, homologues of both of which are tied to stress responses in 5A. suum parasite. This advance was complemented with gene response profiles, resulting in the identification of hypothetical targets of the NITs and cell pathways impacted by treatments that may reflect initiators of cell death processes. There is no other in culture whole-worm assessment with comparable cellular resolution of pathology for parasitic nematodes, thus the approach resulted in significant findings, especially because it implies that the pathological and molecular patterns are NIT-specific. The observed discriminatory nature of the profiles has broad applicability, including the identification of drugs able to induce similar pathologies, thus potentially having similar targets and/or mechanisms as well as identifying similar pathologies and molecular responses of new small molecule inhibitors. The results support the overall approach to have high value toward identifying cells and pathways that, when perturbed, can initiate processes leading to cell death in nematode cells.In this study we made technical advances that rapidly and comprehensively resolved pathologic damage induced by NITs among most cells and organ systems that comprise the The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper."}
+{"text": "We analyze projected changes in climate extremes (extreme temperatures and heavy precipitation) in the multimodel ensembles of the fifth and sixth Coupled Model Intercomparison Projects (CMIP5 and CMIP6). The results reveal close similarity between both ensembles in the regional climate sensitivity of the projected multimodel mean changes in climate extremes, that is,\u00a0their projected changes as a function of global warming. This stands in contrast to widely reported divergences in global (transient and equilibrium) climate sensitivity in the two multimodel ensembles. Some exceptions include higher warming in the South America monsoon region, lower warming in Southern Asia and Central Africa, and higher increases in heavy precipitation in Western Africa and the Sahel region in the CMIP6 ensemble. The multimodel spread in regional climate sensitivity is found to be large in both ensembles. In particular, it contributes more to intermodel spread in projected regional climate extremes compared with the intermodel spread in global climate sensitivity in CMIP6. Our results highlight the need to consider regional climate sensitivity as a distinct feature of Earth system models and a key determinant of projected regional impacts, which is largely independent of the models' response in global climate sensitivity. Changes in climate extremes as a function of global warming are quasilinear and determine a \u201cregional climate sensitivity\u201d in CMIP5 and CMIP6The regional climate sensitivity of climate extremes is found to be very similar in CMIP5 and CMIP6, unlike global climate sensitivityModel spread in regional climate sensitivity in CMIP6 contributes more to uncertainty of projected extremes than global climate sensitivity These global features are regularly reported prominently in first analyses of new multimodel experiments, such as when the first simulations of the sixth phase of the Coupled Model Intercomparison Project  climate sensitivity in different generations of Earth system models (ESMs)   projections running from 2006 to 2100  projections . TXx and TNn are derived from daily maximum (\u2018tasmax\u2019) and daily minimum (\u2018tasmin\u2019) surface air temperatures, respectively. Rx1day is derived from daily precipitation data (\u2018pr\u2019). Global means and regional means are calculated from the original grid resolution; for the maps, the data are aggregated to a common 2.5\u00b0 x 2.5\u00b0 grid, using second\u2010order conservative remapping Jones, .2.2Calculate latitude\u2010weighted global\u2010mean annual\u2010mean temperatureConcatenate historical data and projections Subtract the mean of the climatological period (1850 to 1900)Calculate 20\u2010year centered running meanFind the first year in which the running mean exceeds the desired warming level . From this, the period starts 10\u00a0years before the \u201ccentral year\u201d and ends 9\u00a0years later.The years corresponding to the given global warming levels are determined as the first 20\u2010year period where the global\u2010mean annual\u2010mean surface air temperature (Tglob) exceeds the targeted global mean temperature relative to 1850 to 1900 . Here, we present results for global warming levels of +1.5\u00b0C, +2.0\u00b0C, and +4.0\u00b0C. The period for each warming level, model, and scenario is calculated as follows . We thus have several simulations from the same model (but for different emissions scenarios) which are used in the statistics. This may slightly bias the results if the different runs by the same models are correlated. However, we show that this is not the case  AR6 regions Figure\u00a0 in the SOverall, these analyses further confirm that the regional climate sensitivity of the analyzed extreme indices is very similar in the CMIP5 and CMIP6 multimodel ensembles, with few limited exceptions Figure\u00a0. The scaThe regional climate sensitivity of TXx as a function of global warming is also generally linear, but not identical to that of Tmean Figure\u00a0. This shIn the case of Rx1day, there are also very few differences found in the regional scaling relationships derived in the CMIP5 and CMIP6 ensembles. One region with larger differences, WAF, is shown in Figure\u00a03.3Figure\u00a0The results show an early emergence of regional climate signals for TXx while Rx1day requires higher temperatures for signal emergence. The TXx signal emerges at less than 0.5\u00b0C of global warming for most regions while for Rx1day, a global mean warming of more than 0.6\u00b0C is required. The regions that require the largest warming levels, however, tend to be either small, for example, the Caribbeans (CAR) or very dry, for example,\u00a0the Arabian Peninsula (ARP). In line with our other results, the temperature of emergence from CMIP5 and CMIP6 are very similar. The differences between CMIP5 and CMIP6 are no larger than 0.3\u00b0C for TXx and 0.5\u00b0 C for Rx1day, with the exception of the SAH. The results are qualitatively consistent with those of Kirchmeier\u2010Young et al.  for a su3.4UGTCS, with that resulting from the uncertainty of the regional transient climate sensitivity for the considered extreme, referred to as URTCS.An important and yet unaddressed question is the respective contribution of the regional versus global climate sensitivity for regional changes in extremes. Indeed, while much effort has gone into constraining global climate sensitivity  of the anomaly in the considered climate extreme Vextr\u2032 as a function of the mean global warming Tglob(MMM)\u2032at +1.5\u00b0C: We assume that changes in a given climate extreme of interest p Figure\u00a0, consistglobT\u2032 in the CMIP5 or CMIP6 ensembles when their respective multimodel mean reaches +1.5\u00b0C of global warming. The respective values are referred to as globT\u2032MMMn) at +1.5\u00b0C, UGTCS(1.5), is then estimated as follows: We then determine the uncertainty in the estimated global mean warming from the minimum and maximum response in extrV\u2032 resulting from the regional climate sensitivity, URTCS(1.5), is estimated for the minimum and maximum responses in extrV\u2032 when globT\u2032 reaches +1.5\u00b0C, which are referred to as Vextr\u2032 and Vextr\u2032, respectively: The respective uncertainty in UGTCS(1.5) and URTCS(1.5) values for the regional projected changes in TXx in CMIP5 and CMIP6 in Figures\u00a0URTCS(1.5)) is often found to be larger than that resulting from global climate sensitivity (UGTCS(1.5)) in both CMIP5 and CMIP6. In the case of CMIP6, the contribution of URTCS(1.5) is particularly large and constitutes the dominant driver of uncertainty in a majority of regions. This highlights regional climate sensitivity as a key determinant of projected uncertainty in climate extremes and the at least equal importance of constraining regional climate sensitivity and global climate sensitivity to reduce uncertainties in projections of impacts at higher global warming levels.We display the URTCS versus\u00a0UGTCS would be similar at different global warming levels if UGTCS remained more or less constant independently of global warming. This assumption is, however, not fulfilled at higher global warming levels, as seen in Figure\u00a0globT\u2032 is approximately 1.1\u00b0C at +1.5\u00b0C of global warming in CMIP5 and CMIP6  climate sensitivity, the regional climate sensitivity in the analyzed climate extremes  is very similar in the CMIP5 and CMIP6 ensembles. This highlights regional climate sensitivity as a distinct feature compared with global climate sensitivity.Reasons for the different responses in regional climate sensitivity versus global climate sensitivity are likely related to the differing underlying processes driving them. Regional climate sensitivity has been shown to be strongly affected by regional land processes such as soil moisture and snow feedbacks  climate sensitivity in determining uncertainty of projections. These results highlight that better constraining processes affecting regional climate sensitivity could strongly reduce uncertainty in extremes' and impacts' projections, potentially even more so than further refinements of global climate sensitivity. It will be of high relevance that more investigations focus on regional climate sensitivity as an individual quantity to assess in the evaluation of ESMs, on equal footing with global climate sensitivity.Supporting Information S1Click here for additional data file."}
+{"text": "In the dominant livestock systems of Sahelian countries herds have to move across territories. Their mobility is often a source of conflict with farmers in the areas crossed, and helps spread diseases such as Rift Valley Fever. Knowledge of the routes followed by herds is therefore core to guiding the implementation of preventive and control measures for transboundary animal diseases, land use planning and conflict management. However, the lack of quantitative data on livestock movements, together with the high temporal and spatial variability of herd movements, has so far hampered the production of fine resolution maps of animal movements. This paper proposes a general framework for mapping potential paths for livestock movements and identifying areas of high animal passage potential for those movements. The method consists in combining the information contained in livestock mobility networks with landscape connectivity, based on different mobility conductance layers. We illustrate our approach with a livestock mobility network in Senegal and Mauritania in the 2014 dry and wet seasons. These movements often cause conflicts with farmers, especially during the wet growing season, when animals can invade cultivated plots6. Livestock trade mobility is also a key driver in spreading animal diseases. Indeed, on their way, livestock may cross areas with a high prevalence of mosquitos , which are vectors of diseases. The contact between animals when herds meet each other, is also conducive to disease transmission. Mapping movement patterns is thus essential for improving many aspects of livestock management at regional and national level, such as the management of natural resources, the positioning of borehole installations, the reduction of conflicts, and the control of animal diseases. However, the intrinsic complexity of livestock mobility paths makes it extremely tricky to map them.Every year in West Africa, millions of animals move from the Sahelian semi-arid regions, where they were bred, towards southern regions looking for better grazing areas, or to be sold on consumption marketset al.7 disaggregated census data taken at administrative level to produce risk maps for Rift Valley fever and Napp et al.8 used buffer areas to disaggregate their static data. Fourni\u00e9 et al.9 used densities derived from human demographic data, aggregated at village level, to study the transmission of Peste des Petits Ruminants. However, these approaches are limited to a static vision and do not enable animal movements to be explicitly taken into account.One way of mapping livestock spatial distribution consists in working from a census or estimation of the number of animals at different resolutions. Some recent work improved the mapping of static livestock distribution by disaggregating census counts of animals, but provided no information about their actual movements. For instance, Tran 12. Such methods have been tested in many African countries18. It consists in describing livestock movements as a directed and weighted spatial network, where nodes represent villages, markets or premises and each link between two nodes represents at least one animal moving from one site to another. The weight of a link is equal to the total number of animals exchanged. In some ideal cases, the spatial pathway of the links is known, thanks to GPS tracking of animals19, but in Sahelian areas such data are rarely available and have only been tested on a few cattle21. Thus, the majority of livestock network analysis studies do not explicitly spatialize animal pathways between two nodes; the flows of the graph only provide information about the direction, distance and volume of movements.We recently witnessed the emergence of network-based approaches to study livestock movementsHere we propose a way of mapping livestock movements that combines the information contained in livestock mobility networks with a landscape connectivity-based approach. The method consists in producing a conductance map representing the ease of livestock movements, to be linked with the mobility network in order to produce a map of potential paths. We illustrate our approach with a livestock mobility network in Mauritania and Senegal during the 2014 dry and wet seasons. The next section presents the proposed framework and the data used to illustrate our approach. The results are then presented, demonstrating the capacity and robustness of our approach in identifying potential paths for livestock movements in Sahelian Africa. Lastly, we discuss the advantages and limitations of our approach.22. In Mauritania, rangelands are predominant, with agricultural areas being limited to irrigable or flooded areas along the Senegal River and in oases. In Senegal, livestock farming is mostly located in Ferlo, a region of 70,000\u2009km2 in the North east of the country, where climatic conditions do not allow the development of agricultural activity. A large share of the cattle spend the wet season in this rangeland area of Mauritania and northern Senegal, then moves towards the markets, or towards the crop residues of the central and southern regions, especially in the groundnut basin of Senegal. This animal trade mobility network between Mauritania and Senegal involves up to 1.9 million bovines16. Fewer than 20% of these animals are conveyed by vehicles, mostly commercial requests for religious feasts, with the rest traveling on foot, over a distance of one to three hundred kilometers16. Conveyance on foot enables the cattle to benefit from the pastures and crop residues of southern regions in order to continue fattening along the way. Animals traveling on foot often cross large areas before arriving at their final destination. At the border, large cattle herds will cross at official passage points, but the majority of herders use non-official points to avoid paying taxes, or because they are more accessible16, increasing the difficulty of mapping their paths.Our study area encompasses Senegal and Mauritania, where a recent report estimated the total number of cattle to be between 2 and 3 millionLivestock mobility data are collected by field Veterinarian Services in Senegal, Gambia and Mauritania. In those countries, a certificate system based on sanitary movement permits (Sanitary Laissez-Passer or LPS) has been set up to keep track of animal mobility and map the main axes of movements in the area. Every time herders move their herds towards markets, or to other grazing area, a certificate is issued declaring, among other things, the date, the location of origin, the location of destination, the species and number of head, and the means of transportation. In this article, we consider only information relative to cattle movements, on foot, in 2014. We aggregated our data on a timescale of one month, providing a representation of the mobility dynamics over the year. This mobility information is represented by a weighted and directed livestock mobility network where the nodes correspond to the origin and destination locations , the in- and out-strength , and the betweenness. The betweenness of a node is proportional to the number of shortest paths (weighted by the distance) going through this node.As depicted in Fig.\u00a02.http://www.fao.org, last accessed 14/06/2019). The original classification has been aggregated in 14 land-use types available in Fig.\u00a0A walking layer based on land use and land cover information provided by the FAO . A map of the road network is available in Supplementary Information . The border crossing points  to 1 (high conductance/low resistance). It is important to note that a pixel with no value  to any pixels of \u03b4R\u2009\u2208\u2009. The value Ci of a pixel i on conductance map C according to R and \u03b4R is given by:Finally, the last level of information is given by the administrative border line. To adjust the permeability of the border line to pixels that are not border crossing points, we introduced the parameter 23 using Circuitscape software (v4) . This approach has been widely used in wildlife corridor design25, movement ecology27, and epidemiology28.The last step consisted in assigning a potential route between every pair of nodes of our livestock mobility network using the conductance map described in the previous section. To do so, we conducted a connectivity analysis based on concepts from electronic circuit theory23. This map informs us about the potential for each pixel to be crossed during a livestock movement from the market of origin to the market of destination. We then normalized the map by its highest pixel value.For each pair of locations, represented by two pixels on the conductance map, Circuitscape computes a map of the total movement resistance accumulated from the origin and destination based on the electronic circuit theory applied on the conductance mapThen, we multiplied each normalized connectivity map by the ratio of animals concerned . We finally summed all the maps. We obtained a final map of the potential path for livestock, presented in the next section, where the highest values indicate the highest potential for livestock movements.https://gadm.org/index.html, last accessed 18/02/2020). We thus obtained a distribution of values informing us about the level of activity within each administrative unit based on the potential for each 500\u2009\u00d7\u2009500 m2 pixel to be crossed during a livestock movement. To facilitate the interpretation, the level of activity has been normalized by its maximum value and used to rank the different administrative units. We can also compute the level of normalized activity in each administrative unit based on the information provided by the livestock mobility network to compare the different approaches. In this case the activity is based on the total number of animals transiting in the administrative unit (sum of the in- and out-strength of the nodes located in the administrative unit).In animal health programs, land-use planning, or management of conflicts between farmers and herders, it is essential to be able to prioritize intervention zones. To do so, we need to spatially aggregate the information contained in the maps of potential paths for livestock movements in order to identify high potential areas. In this study, we spatially aggregated the maps of potential paths at regional level for Senegal, Gambia and Mauritania, using data downloaded from the GADM web platform  or the results obtained for different seasons, the distance between distributions of normalized activities (i.e rankings) can be assessed with the Kendall\u2019s \u03b4W and \u03b4R used to combine the different layers and the weights used to model the land use conductance . The reference for the land use weights are displayed in Table\u00a0\u03c4 coefficient to compare the ranking of administrative units obtained with the reference distribution of activity with the ones obtained with different parameters and land use weight values. The two sources of uncertainty have been evaluated independently. For the parameters \u03b4W and \u03b4R, we generated 25 rankings obtained with different pairs of values ranging between 0 and 1 by step of 0.25. For the land use weights, we changed one-at-a-time the weight of the different land use types by adding or subtracting an amount \u0394\u2009=\u20090.05 or \u0394\u2009=\u20090.1 from the original value.There are two main sources of uncertainty in the mapping of potential paths for livestock movements: the parameters ce Table\u00a0. We usedFigure\u00a0Table\u00a0The role played by the different locations slightly changes from one season to another. Figure\u00a0We plotted in Fig.\u00a0\u03c4 coefficient as described in the Material and methods section. Table\u00a0\u03c4\u2009=\u20090.84) between the rankings obtained with the landscape connectivity approach in wet and dry seasons. It is interesting to note that this correlation falls to 0.66 when comparing the network approach in the wet and dry seasons.We plot in Fig.\u00a0\u03b4W values is globally high with a Kendall\u2019s \u03c4 coefficient ranging from 0.8 to 1. The similarity decreases slowly when \u03b4W decreases below the reference value, we observe a break of this trend when \u03b4W\u2009=\u20091. In this particular case, the results are no longer driven by the road network, leading to a modification in the potential movement patterns on a global scale. Note that since \u03b4R has almost no impact on the activity at a global scale . Our results demonstrate the robustness of our approach in identifying and ranking areas according to their potential for livestock movement. Other applications from our methodology are now possible. For example, we could cross the information contained in our potential maps with risk factors for the spread of diseases like Rift Valley feverIt needs to be kept in mind that our approach is highly dependent on the data being used and their resolution. The resolution of the conductance map, at 500 meters in our study, depends on the resolution of the land cover map and might not enable the consideration of very fine paths. Our results showed that the potential map was mostly driven by the road network, which can also be a major source of uncertainty.Many factors drive mobility dynamics: landscape configuration, road quality, need for food, need for watering points, border crossing, religious feasts, etc. The conductance map has to include all these mobility-driven factors. For this study, we were able to collect most of the geographical layers for each of these factors, except that of the watering points (boreholes and ponds). Consequently, the maps obtained in this study do not take into consideration the need to pass through watering points, especially during the dry season. This is an important drawback counterbalanced by the fact that Senegal and Mauritania have a very dense grid of boreholes.Another difficulty is the reliability of the mobility data. Mobility data were collected using two different approaches in Senegal and Mauritania. For the Mauritanian case, a synthetic survey was conducted by the National Livestock and Veterinary Research Centre (CNERV) and compared with health certificates collected by Veterinarian Offices. In the case of Senegal, paper copies of sanitary movement permits (LPS) were collected by ad-hoc activities. These certificates provided information about origins and destinations, and we do not know if the composition of the herd changed during the journey due to animal sales. Furthermore, there was no proof that the herds actually reached their destination. Another bias in the data was linked to the fact that this data set did not include undeclared movements (for herds that did not have a sanitary movement permit).Lastly, construction of the conductance map, which is the basis of the proposed methodology, relies on resistance weights given by experts. It should be noted that the main purpose of this article was to propose a methodology and we did not try to increase the number of experts. Nevertheless, we showed that small variations applied one at a time to the land use weight values have no significant effect on the rankings. To use the presented method for operational purposes, concerted thought needs to be given to the weights to be assigned, and a multivariate sensitivity analysis of these weights needs to be integrated into the approach.The identification of high potential for livestock movements is a core issue for decision-makers, whether in the field of animal health or territorial planning. Our approach opens up some interesting perspectives for modeling potential animal passage in semi-arid regions experiencing a lack of specific data on livestock movements. It is, however, important to note that a large share of livestock remains in its zone of origin. These sedentary animals are often in contact with transhumant animals that cross their territory. This information should be added, to complete the map of the potential for livestock movements provided in this study.Supplementary information."}
+{"text": "Aspergillus terreus strain and pretreatment of sugarcane bagasse as carrier substrate for bulk production of lovastatin, a cholesterol-lowering drug, in solid state fermentation. Sugarcane bagasse was treated with alkali (1\u20133% NaOH) for the conversion of complex polysaccharides into simple sugars for better utilization of carrier substrate by microorganism for maximum lovastatin production. Ethidium bromide (time of exposure 30\u2013180 min) was used to induce mutation in Aspergillus terreus and the best mutant was selected on the basis of inhibition zone appeared on petri plates. Fermented lovastatin was quantified by high-performance liquid chromatography. The fermented lovastatin, produced by parent and mutant Aspergillus terreus strain, was checked on body weight, blood glucose and serum cholesterol, ALT, AST, HDL-C, LDL-C, TG and TC levels of rats for their cholesterol lowering capacity. Our results indicate that selected strain along with 2% NaOH treated sugar cane bagasse was best suitable for bulk production of lovastatin by fermentation and fermented lovastatin effectively lower the cholesterol level of rats.The aim of this project was to improve the  Aspergillus terreus is the most reported microorganism for the production of lovastatin [Lovastatin belongs to an important class of statins and is frequently used as a cholesterol- lowering drug. Fungally-derived lovastatin is generally considered as a \u201csafe drug\u201d. vastatin ,2. As covastatin . Lovastavastatin . LovastaThe lactone ring after hydrolysis has structural similarity to the tetrahedral intermediate synthesized by reductase to bind the drug with HMG-CoA reductase . The douAspergillus terreus and hydrolysis of complex polysaccharide of sugarcane bagasse by alkali treatment for bulk production of and comparison of both strains of Aspergillus terreus; parent and chemically improved along with synthetic atorvastatin to estimate their hypocholesterolemic efficiency in rats.Pretreatment of lignocellulosic biomass is a very effective way of degrading biomass into simpler molecules before enzymatic hydrolysis. There are few effective pretreatments that significantly enable cellulosic fraction of lignocellulosic biomass cell wall for enzymatic hydrolysis of cellulosic degradation into simpler monomers molecules ,8. Acid-In the present study the compositional analysis of sugarcane bagasse revealed that sugarcane bagasse consists of 42% cellulose, 21.25% hemicelluloses, 22.76% lignin, 2.97% total sugars, 7.19% reducing sugars, 0.86% ash and 7.3% moisture content. However, Javed et al.,  found 40\u22121 was characteristic of hemicellulose and lignin due to the stretching of C-O bond [\u22121 has shown a deformation of lignin CH2 and CH3, and band around 1.508 cm\u22121 represents the C=C stretching of the aromatic ring in lignin [\u22121 exhibits the stretching of the C=O and C=C lignin aromatic rings. A band around 1.733 cm\u22121 is characteristic of C=O stretching of unconjugated hemicellulose. The region between 3.801 and 3.676 cm\u22121 indicates the crystalline structure of cellulose. This region constitutes the sum of the vibration of valence bands of the H-bond of the OH group and the bands of intramolecular and intermolecular H-bonds [\u22121 clearly shows peaks after alkali hydrolysis indicating the removal of hemicellulose. In the region of 1.245 cm\u22121, the removal of hemicellulose is also evident. A comparison of the Fourier transform infrared (FTIR) spectra of native and NaOH treated sugarcane bagasse is shown in C-O bond . A band n lignin ,19. The  H-bonds . Figure \u22121, 1.733 cm\u22121, and 1.580 cm\u22121 and cellulose-containing regions around 2.918 cm\u22121 and 2.850 cm\u22121 have shown noticeable changes after alkaline hydrolysis. It is evident from these changes that a delignification process has occurred in the sugarcane bagasse.Alkaline hydrolysis promotes removal of lignin moieties. The lignin macromolecule regions around 1.868 cmCandida albicans grown on agar plates (From ethidium bromide (EB) treated plates, 10 hyperproducing mutants were selected on the basis of their efficiency on plates. A total of 10 positive mutants were picked predominantly on the basis of their bigger zone of inhibition on r plates . The mutr plates . Aspergillus terreus along with parent strain were used for production, using sugarcane bagasse in solid state fermentation (SSF). Among these, the maximum production (91 \u00b1 1.77 mg/L) with dry cell mass (4.49 \u00b1 0.81 g/L) was noticed for the mutant ATE-120 using 2% NaOH treated sugarcane bagasse (Aspergillus terreus is a best lovastatin (55 mg/L) producing strain out of 110 fungal strains. Mangunwardoyo et al. [Aspergillus flavus UICC 360. Moreover, Lakshman and Radha [P. ostreatus.These mutants were also evaluated through still culture flask experiments. The mutants of  bagasse . The reso et al.  detectedBesides, the blood glucose levels of high cholesterol dietfor fermented parent derived lovastatin (PL), mutant derived lovastatin (ML), and atorvastatin groups were crucially higher than in control group during the whole experimental period after administration of high cholesterol diet in rats . ConcentThe findings of the present study also demonstrated that the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were remarkably increased in high cholesterol diet (HCD) than normal and fermented lovastatin (PL & ML) diet. Serum total cholesterol (TC), triglycerides (TG) and low density lipoproteins (LDL) levels were also notably increased after 20, 40 and 60 days. Thus, HDL-cholesterol level reduced in high cholesterol diet (HCD) group as compared to normal rats. These findings are in close agreement with the Stepherd  work who\u22121 with a resolution of 4 cm\u22121 per minute and room temperature detector .Sugarcane bagasse was obtained from Shakarganj Sugars Mills Jhang  were made and 2 g of powdered bagasse was added to each concentration. These samples were kept at 80 \u00b0C and neutral pH in microwave for 10 min then filtered and rinsed with tap water. The pretreated samples were dried at 65 \u00b0C . The natAspergillus terreus strain. A stock solution of 0.5 mg/mL ethidium bromide (w/v) was prepared. The stock solution of EB  was prepared. It was mixed with Vogel medium (9 mL), which contained 1 \u00d7 107 spores mL\u22121 of Aspergillus terreus and kept it in a water bath  at 37 \u00b0C. One milliliter sample was withdrawn after a specific time interval, and cell pellet was washed thrice with normal saline solution. The sample was centrifuged at 12,000 rpm for 1 min using centrifuge machine , to separate out the mutagen from sample [Ethidium bromide was used to induce mutation in the l medium  mL, whicCandida albicans was used for the selection of best mutant. It was grown on PDA dishes at 28 \u00b0C for 12 h. 50 \u00b5L of fermented lovastatin extract were added onto a 6 mm diameter paper disk. These paper disks were placed on the surface of a 90 mm diameter C. albicans plate. The maximum distance of 15mm was kept between lovastatin impregnated disks on a plate. The plates were incubated for 6 h and zones of inhibition were recorded. A large diameter of the inhibition zone implied a high titer of lovastatin [vastatin .Aspergillus terreus and the flasks were subjected to SSF under still culture conditions for 72 h.Lovastatin was produced in solid state fermentation (SSF) by the method as described by Javed et al. . Experim10% 1N HCl was added to the fermentation broth after 3 days of fermentation. The acidified broth was extracted with equal volume of ethyl acetate at 70 \u00b0C temperature and 180 rpm for 2 h. The Whatman filter paper No. 40 was used for the filtration of broth. The filtrate was centrifuged at 3000 rpm for ten mins and the organic phase was collected. 10 mL of 1% trifluoroacetic acid was mixed in the one ml of organic phase of filtrate for lactonization. The extract was heated at 80 \u00b0C to evaporate the moisture, diluted with one mL acetonitrile and filtered for high-HPLC analysis.\u22121.The identification and quantification of lovastatin was carried out by HPLC method . The samGroup 1 contains normal rats on normal dietGroup 2 contains rats with high cholesterol diet (HCD)Aspergillus terreus strain (PL)Group 3 HCD+ fermented lovastatin derived from parent Aspergillus terreus strain (ML)Group 4 HCD+ fermented lovastatin derived from mutant Group 5 contains rats which are on synthetic statinThirty male albino rats were selected for the induction of lovastatin. These male rats of 200\u2013230 g of weight were obtained from National Institute of Health . These albino rats were group into 5 groups each containing 6 rats and caged into separate cages under suitable temperature of 25 \u00b1 5 \u00b0C. Proper ventilation system is maintained and standard feed and tap water is provided according to their weight. According to the method explained by Kamal and Thanaa  with higThe efficacy of fermented lovastatin on serum of rats AST, HDL-C, ALT, LDL-C, TC and TG level of rats was determined by the method of Kamal and Thanaa,  and Javep \u2264 0.05 was considered to denote a statistically significant difference [Statistical significance of the differences between mean values was calculated by ANOVA under CRD and DMR test using Minitab 2000 version 13.2 statistical software . A probability value of fference .Aspergillus terreus using ethidium bromide and pretreatment of sugarcane bagasse using NaOH for hydrolysis of complex polysaccharides for better utilization of substrate by fungal strain were effective strategies to hyper- produce lovastatin and successfully treat hypercholesterolemia. The present data would certainly help to ascertain the ability of fermented lovastatin as the potential drug for hypercholesterolemia to be used in the pharmaceutical industry.It can be inferred from the present study that strain improvement of"}
+{"text": "N\u2009=\u2009406), we assessed how OC, anxiety and depression symptoms changed throughout the first pandemic wave in a sample of the general UK public. We further examined how these symptoms affected pandemic-related information seeking and adherence to governmental guidelines. We show that scores in all psychiatric domains were initially elevated, but showed distinct longitudinal change patterns. Depression scores decreased, and anxiety plateaued during the first pandemic wave, while OC symptoms further increased, even after the ease of Covid-19 restrictions. These OC symptoms were directly linked to Covid-related information seeking, which gave rise to higher adherence to government guidelines. This increase of OC symptoms in this non-clinical sample shows that the domain is disproportionately affected by the pandemic. We discuss the long-term impact of the Covid-19 pandemic on public mental health, which calls for continued close observation of symptom development.Increased mental-health symptoms as a reaction to stressful life events, such as the Covid-19 pandemic, are common. Critically, successful adaptation helps to reduce such symptoms to baseline, preventing long-term psychiatric disorders. It is thus important to understand whether and which psychiatric symptoms show transient elevations, and which persist long-term and become chronically heightened. At particular risk for the latter trajectory are symptom dimensions directly affected by the pandemic, such as obsessive\u2013compulsive (OC) symptoms. In this longitudinal large-scale study ( The global coronavirus SARS-CoV-2 (Covid-19) pandemic has created a situation of severe uncertainty and isolation, fuelled by disruptions in finance, politics, social life and healthcare. As such, it constitutes an immense psychological challenge, especially for people\u2019s mental health.3 and obsessive\u2013compulsive disorder (OCD)4 were reported to experience a pandemic-related increase in symptoms. Likewise, the general public reported worsened mental health with a rise primarily seen in anxiety and depression levels10.First studies have reported adverse psychological consequences of the pandemic among people with and without pre-existing mental-health conditions. Patients with anxiety, depression, bipolar disorders, schizophrenia11 as many of them revolve around contamination, infectious illnesses, and causing harm12. First evidence suggested a worsening of symptoms in OCD patients during the pandemic16, although the results are somewhat inconsistent (cf.18), which may be driven by the heterogeneity of the disorder or differences in underlying comorbidities. How OC symptoms developed in non-patient populations during the pandemic is, however, to the best of our knowledge, unknown. Research conducted on previous health crises, such as the HIV/AIDS epidemic in the 1980s has shown that health campaigns could influence the occurrence of epidemic-related OCD-symptomatic behaviour in individuals with no reported history of psychiatric disorders21. These findings raise the concern that Covid-19 campaigns may have a similar detrimental effect on the general public.Obsessive\u2013compulsive (OC) symptoms are likely to be disproportionately affected by the pandemic23, are common. Decades of research demonstrated that mental health worsens after stressful events25. However, importantly, psychiatric levels usually improve after a reasonable time without inflicting long-term impairments28. This adaptation process has been attributed to coping and (re-)appraisal strategies29. A failure of this process, however, may lead to long-term adverse consequences and chronic mental-health problems27. It is thus critical to not only assess momentary increases in psychiatric symptoms but to also investigate the long-term trajectories of these symptoms.In addition, elevated levels of psychiatric symptoms following a stressful time, such as the pandemic34. We show that rising OC symptoms in the general public are linked to pandemic-related information seeking, which in turn promotes higher guideline adherence.Here, we conducted a large-scale, non-clinical, longitudinal study investigating OC, anxiety and depression symptoms measured as self-reported dimension scores during the Covid-19 pandemic. We tracked these psychiatric scores over a period of several months and show that OC symptoms in this general UK public sample increase, whilst anxiety levels plateau and depression levels diminish. Moreover, we investigated how these symptoms impacted participants\u2019 Covid-related information seeking and adherence to governmental guidelines, based on the hypothesis that OC symptoms are associated with excessive information seeking35, anxiety, and depression symptoms36 using standardised questionnaires. In addition, we recorded participants\u2019 Covid-19-related information seeking with a new scale (cf. below) as well as whether they had received a positive Covid-19 test or/and had essential worker status (as defined by the UK government) during or before data collection. At the first time point (T1), participants also reported their baseline news and social media consumption and completed a cognitive ability assessment37 serving as an IQ estimation. At the second time point (T2), we additionally measured people\u2019s adherence to Covid-19 guidelines as instructed by the UK government.We conducted a longitudinal online study of the general public, collecting data at two time points throughout the first pandemic wave  until the 7th of May 2020. This was approximately at the peak of the first pandemic wave in the UK . All subjects were over 18 and gave their informed consent before starting the study. There were no other recruitment restrictions to gain a representative sample of the general public. The study was approved by University College London\u2019s Research Ethics Committee.We recruited participants living in the UK via the Prolific recruiting service .A total of 446 participants completed the study at T1. We excluded 30 participants because of missing data or failing at least one of two attention checks (instructed questionnaire answers) and 10 due to a self-reported OCD diagnosis. We excluded the latter to not bias our non-clinical spectrum approach as most recent findings showed distinct psychiatric score developments in patients versus non-patients during the pandemicMage\u2009=\u200935, SDage\u2009=\u200912.623; cf. Supplementary Table t-tests (OC symptoms: t(404)\u2009=\u20091.588, p\u2009=\u20090.113; anxiety: t(404)\u2009=\u20091.790, p\u2009=\u20090.074; depression: t(404)\u2009=\u20091.498, p\u2009=\u20090.135). As only one participant indicated a positive Covid-19 test at T1 and two at T2, we did not exclude these subjects or controlled for this status in the subsequent analysis as the small number of subjects is unlikely to alter our results.We re-invited participants to also take part at T2. Of the final T1 sample, 315 completed this second time point, of which we excluded additional 19 participants because of missing data or failed attention checks. The final T2 sample consisted of 296 participants .We implemented all questionnaires using a web API programmed with React JS libraries . Control analyses based on this score should ensure that our results were not merely driven by pandemic-related adaptive behaviours captured by PI-WSUR items evolving around hygiene and disease 36. We did so because these dimensions show substantial overlap in the general population40, and they are often comorbid in patients with OCD12. The HADS consists of two subscales (anxiety and depression) that are intended to be evaluated separately. We chose the HADS scale as it solely focuses on psychological anxiety and depression symptoms in contrast to other popular scales42, which include items related to physical symptoms (e.g. insomnia) that may be caused by physical illness (such as Covid-19). The HADS also has a high internal consistency and test\u2013retest reliability43.We further measured anxiety and depression using the Hospital Anxiety and Depression Scale (HADS)To investigate information seeking during the pandemic and across different psychiatric domains, we developed a new Covid-19 information-seeking questionnaire. The questionnaire entailed five items with a 5-point Likert scale with lower scores indicating less information seeking. Items asked about information exchange and seeking via different social (and) media channels  for further data analyses.We pre-processed and analysed data in MATLAB 2020a (MathWorks) and used SPSS Statistics  and R, version 3.6.2 via RStudio version 1.2.5033  was larger than 0.75. For comparability between scores, we first z-scored all psychiatric scores across both time points. To further compare the changes of psychiatric scores and information seeking between time points, we conducted two-sided paired t-tests using the stats package in R44, supplemented with paired permutation tests using the CarletonStats package45.To investigate whether psychiatric scores changed differently over time, we conducted a repeated-measures ANOVA with two within-subject factors , the 46 in R with a bisquare method to down-weight outliers and assessed their p values using the rob.pvals function of the clickR package47. To investigate changes in the association between information seeking and psychiatric scores over time, we also conducted a random intercept mixed-effects model using the lme4 package48. The model entailed main effects for time point (coded as 0 (T1) and 1 (T2)) and all z-scored psychiatric scores as well as interactions between time point and psychiatric scores and demographic control variables. In the syntax of the lme4 package, the model was specified as follows:To evaluate the association of Covid-19-related information seeking and psychiatric scores for each time point separately, we computed robust multiple regression models using the rlm function of the MASS packageInformation seeking\u2009~\u2009time point\u2009*\u2009(OC symptoms\u2009+\u2009anxiety\u2009+\u2009depression)\u2009+\u2009gender\u2009+\u2009age\u2009+\u2009IQ\u2009+\u2009education\u2009+\u2009essential worker status\u2009+\u2009(1|subject)https://github.com/canlab/MediationToolbox), which adapts the standard three-variable path model50. All results refer to two-tailed bootstrapped p values, and the procedure was repeated 10,000 times with replacement producing a null hypothesis distribution. In our mediation model, the total OC symptoms score at T1 was taken as a predictor, guideline adherence at T2 as the dependent variable, and information seeking at T1 as the mediator  combined with a significant indirect effect (path ab) is interpreted as a full mediation.Finally, we conducted a mediation analysis to examine whether information seeking was associated with guideline adherence. For this analysis, we used the Mediation Toolbox in MATLAB  and ordinal  general public UK sample longitudinally at two time points during and after the first pandemic wave.SD\u2009=\u200922.103; Fig. 55. We restrained from quantitative comparisons due to unequal sample sizes and other potential confounding factors such as the large contextual differences (pandemic vs. non-pandemic) between the studies, which also make clinical inferences challenging. However, overall our sample seemed to score between patient and non-patient samples with some subscores being similar to pre-pandemic OCD patient samples  or whether they rose furtherF\u2009=\u200919.848, p\u2009<\u20090.001), extending a significant main effect of time \u2009=\u20094.957, p\u2009=\u20090.027).To do so, we ran a two-way repeated-measures ANOVA with within-subject factors time (T1 vs. T2) and psychiatric domain . We found a significant time by psychiatric domain interaction (t(295)\u2009=\u20095.294, p\u2009<\u20090.001; Fig. t(295)\u2009=\u20092.824, p\u2009<\u20090.001), suggesting that the observed increase in OC symptoms throughout the lockdown cannot be prescribed to adaptive protective behaviours during the pandemic and was present across multiple OC domains \u2009=\u2009\u22120.436, p\u2009=\u20090.025; Fig. t(295)\u2009=\u2009\u22120.025, p\u2009=\u20090.903; Fig. This increase in symptoms was unique to the OC domain. In fact, we saw a significant decrease in depression scores from T1 to T2 \u2009=\u2009\u22122.108, p\u2009<\u20090.001; Supplementary Fig. We thus examined whether people engaged in information seeking using a newly developed and validated scale cf. . We asserT1(404)\u2009=\u20090.235, p\u2009<\u20090.001, rT2(294)\u2009=\u20090.249, p\u2009<\u20090.001). A similar, but smaller association was found for anxiety scores (rT1(404)\u2009=\u20090.182, p\u2009<\u20090.001, rT2(294)\u2009=\u20090.149, p\u2009=\u20090.010). We only found a weak association between depression and information seeking at T1 (rT1(404)\u2009=\u20090.100, p\u2009=\u20090.043), which did not hold at T2 (rT2(294)\u2009=\u20090.103, p\u2009=\u20090.076). All associations, except for the one with depression at T1, further remained when controlling for demographic variables .We next investigated whether information seeking was associated with any of the psychiatric symptom dimensions. We found that OC symptoms indeed were linked to increased information seeking at both time points (rT104\u2009=\u20090.23rT1(404)\u2009=\u20090.583, p\u2009<\u20090.001, rT2(294)\u2009=\u20090.620, p\u2009<\u20090.001; OC symptoms and depression: rT1(404)\u2009=\u20090.387, p\u2009<\u20090.001, rT2(294)\u2009=\u20090.462, p\u2009<\u20090.001; anxiety and depression: rT1(404)\u2009=\u20090.684, p\u2009<\u20090.001; rT2(294)\u2009=\u20090.703, p\u2009<\u20090.001; Supplementary Fig. These consistent correlations between psychiatric symptoms and information seeking were not surprising considering the psychiatric domains themselves are known to be related and are thus expected to show similar links  or depression . The same effects were found when investigating T2 scores . Again, including baseline news and social media consumption as covariates did not change the results . We further replicated these effects when combining the data from both time points into one mixed-effects regression model \u2009=\u20090.143, p\u2009= 0.014; information seekingT2: r(294)\u2009=\u20090.271, p\u2009<\u20090.001). To investigate this relationship further, we performed a mediation analysis with all three factors . This analysis showed a significant direct effect of OC symptoms at T1 on guideline adherence at T2  and on information seeking at T1 . It further showed a significant path from information seeking at T1 to guideline adherence at T2 . When then controlling for information seeking, the effect of OC symptoms on guideline adherence was no longer significant . These results suggest that individuals scoring higher on OC symptoms engaged more in pandemic-related information seeking, which in turn reinforced their adherence to governmental guidelines  public across multiple OC domains. Moreover, OC symptoms were inter alia elevated in domains not directly linked to Covid-related themes . This speaks for a generalized and thus striking effect that goes beyond adaptively increased hygiene behaviours that could be expected during the pandemic. This is most likely capturing psychological effects, e.g. elicited by the Covid-related threat or campaigns, rather than neurological consequences of a virus infection.The documented elevations of anxiety and depression scores during the pandemic are in line with previous studies conducted in the general public7, we used a longitudinal design that allowed us to characterize the dynamics of psychiatric symptoms throughout the pandemic. Based on coping and adaptation theories56, initially developed in the field of stress research, we expected a decrease in psychiatric symptoms after the peak of the pandemic, a pattern that has recently been documented for some psychiatric domains9. However, we found an important dissociation in symptom dynamics. While depression decreased and anxiety levels plateaued with the ease of lockdown and the drop of Covid-19 infections, the OC symptom levels further increased. Although some environmental circumstances  might have facilitated this decrease in depression scores and kept anxiety stable, general uncertainty still remained high  and therefore external factors do not seem to explain why one psychiatric domain would change substantially differently than the others.In contrast to previous studies that looked at the psychiatric symptoms (before and) at one time point during the pandemic34. We developed a novel information-seeking measure related to Covid-19 and found that individuals with high OC scores again tended to seek more information, even when controlling for other psychiatric symptoms, the general news and media consumption and demographic variables. Furthermore, this association between OC symptoms and information seeking persisted throughout the pandemic. We further show that Covid-related information seeking was linked to later adherence to governmental guidelines. We found that information seeking mediated the influence of OC symptoms on guideline adherences meaning that individuals with elevated OC symptoms adhered more strongly to guidelines as they extensively sought Covid-related information.We further investigated how OC symptoms related to daily behaviours such as information seeking and adherence to governmental Covid guidelines. Previously, increased information seeking has been reported in subjects with high OC symptoms in abstract laboratory tasks58 self-report questionnaires are also commonly used in clinical contexts and serve as valuable pre-selection instruments for mental-health services59. These OC symptom scores in our general public data complement increased new support requests recently reported by OCD charities60, symptom increases in OCD patients16 and observations during preceding health crises19. We should not make clinical statements on our dimensional data, and future studies should investigate whether OCD diagnoses are on the rise amid the Covid-19 pandemic.It is important to note that an elevation in dimensional scores should not be mistaken for a clinical diagnosis. In contrast to self-report questionnaires, such as the ones used here, clinical assessments and interviews can differentiate between other possible diagnoses and make informed decisions about the impairment and distress, additionally taking into account the given context (e.g. a pandemic). Context variations also constrain comparisons of our data to previously established benchmarks or pre-pandemic samples. Nonetheless, our longitudinal data clearly shows a rise in OC symptoms in the general public throughout the Covid-19 pandemic. Moreover, due to their high predictive validity62. Moreover, even though our study exploits its longitudinal design to report directional effects, given our design, causality cannot directly be inferred.Our study has some unique strengths, such as its large sample size, longitudinal testing, and real-world relevance of the measures. However, it would be interesting to further investigate these associations using additional metrics, such as smartphone geolocation patterns, browser history or in-lab experiments to overcome drawbacks of self-report measures, e.g. constraints on introspective abilityOverall, this study provides a first account of OC-symptom dynamics in the general public throughout the Covid-19 pandemic and how these symptoms translate into other behaviours such as information seeking and adherence to governmental guidelines. The increase and persistence of OC symptoms stress the importance of close monitoring of the public\u2019s mental health and substantial support to alleviate the pandemic-related psychological burden.Supplemental Information"}
+{"text": "Purpose: This observational longitudinal investigation aimed to investigate whether change in bioelectrical regional phase angle (PhA) is a predictor of change in vertical jump performance in elite soccer players. Methods: Fifteen soccer players  competing in the first Italian division (Serie A) were included in this study and tested before the pre-season period and after the first half of the championship. Whole body and lower hemisoma PhA were obtained with a phase-sensitive 50 kHz bioelectrical impedance analyzer and legs lean soft tissue was estimated using specific bioimpedance-based equation developed for athletes. Vertical jump performance was assessed using the countermovement jump (CMJ). Results: The major findings of the study are that changes in lower hemisoma PhA are more strongly related with changes in jump performance  than changes in whole-body PhA , even after adjusting for legs lean soft tissue and for body mass index . Conclusions: These data suggest that changes in lower hemisoma PhA might be used as a tool for evaluating performance related parameters in sports where specific body segments are involved, in preference to the whole-body measured value. In elite soccer players, higher levels of strength and power are now required in order to reproduce intense muscular bursts such as accelerations and decelerations, maximal sprinting (30\u201340 times), turning (>700 times), tackling and jumping (30\u201340 times) . NotablyIt is well known how different physical performance parameters including jumps are related to body composition features ,4. InterRecently, Nabuco et al.  reportedFrom a practical perspective, PhA represents a simple, quick, and non-invasive value that could be used to monitor the overall fitness and body composition features of the players during the competitive season. The assessment of PhA can be considered together with other parameters related to physical performance, such as muscle strength and endurance level, in the search for the player\u2019s optimal physical condition. In addition, since PhA can be measured either whole-body or in the different hemisomes, it is important to study which measure may be more informative in the evaluation of a soccer player. Therefore, the aim of this study was to verify, through a longitudinal pilot study design, the association between changes in lower hemisoma PhA and vertical jump in elite soccer players. Our hypothesis was that change in lower hemisoma PhA could be more strongly related with change in vertical jump performance than that concerning the whole body PhA.2) took part in the study. The players voluntarily decided to participate and provided a signed informed consent after a detailed description of the study procedures. The project was conducted according to the Declaration of Helsinki and was approved by the Bioethics Committee of the University of Milan.Seventeen male professional soccer players of the same team attending the Italian first division (Serie A) were selected to participate in this study. Two subjects were excluded as they received injuries before starting the experimental protocol. Therefore, fifteen players : 23.8 \u00b11.2 kg/mBiolectrical whole body and lower hemisoma PhA (LPhA) were obtained using a phase-sensitive segmental bioelectrical analyzer  at a single frequency of 50 kHz. After cleaning the skin with isotropy alcohol, four low intrinsic impedance adhesive electrodes  were placed on the right hand and foot respecting the standard protocol ,11. PhA In the 24 h period before executing the countermovement jump (CMJ) test, participants did not engage in activity that was considered unduly fatiguing. Each participant executed three maximal jump trials on a portable force platform . Each trial was performed from a standing position with hands placed on the hips. During an individual trial, the participants quickly bent downward and then performed a fast upward push to reach the highest height from the lower limbs action. CMJ height, power output, and strength were recorded within the upward phase by a dedicated software . A recovery of 2 min was allowed between trials. The best performance was used within the analysis. Body composition and jump assessments were performed in the morning at the same time (9.00 a.m.) before the pre-season period and after the first half of the championship.t test for paired data. Effect sizes were calculated as the mean difference standardized by the between-subject standard deviations. Statistical significance was predetermined as p < 0.05.Statistical analysis was performed using IBM SPSS Statistics for Mac OS version 22.0, 2010 . Descriptive statistics (mean \u00b1 standard deviation) were performed for all measurements. All variables were checked for normality using the Shapiro\u2013Wilk test and the results were normally distributed. Regression analysis was used to determine whether change in PhA was a significant predictor of change in jumping performance after adjusting for confounding variables (LLST and BMI). Changes in bioelectrical, body composition, and jump parameters were compared with the student\u2019s p < 0.05) increased from the pre-season phase to after the first half of the competitive period in the participants, whereas no change was assessed for whole body PhA, BMI and LLST, as shown in Bioelectrical LPhA and jump performance significantly . After adjusting for LLST and BMI, change in LPhA remains a significant predictor in the model , as shown in A multiple regression analysis was performed while adjusting the relationship between changes in LPhA and changes in CMJ for confounding variables, as shown in The aim of this investigation was to test the association between changes in lower hemisoma PhA and vertical jump in elite soccer players. As hypothesized, lower hemisoma PhA is a better predictor of vertical jump performance than whole body PhA. In particular, increases in lower hemisoma PhA are associated with an improvement in CMJ capacity. To the authors\u2019 knowledge, this is the first study to investigate the longitudinal association of regional PhA with performance parameters, regardless of their important body components such as lean soft tissue mass and BMI. The result of the regression analysis identified the change in lower heimsoma PhA as a significant predictor of change in vertical jump performance, accounting for ~60% of inter-individual variance. In the study by Nabuco et al.  and thisThe present investigation has some limitations. In fact, our results cannot be compared with those obtained from bioimpedance measurements performed using different technology and sampling frequency than the ones used here. In addition, future studies including a larger sample size and a wider battery of tests are required to investigate the role and usefulness of regional PhA in predicting soccer-specific performance.Many studies report the negative influence of sub-par body composition features  on physical and mental performance, which not only compromise normal daily activities but can negatively affect sports performance . This stIn some sports where particular body segments are predominantly involved, a regional PhA evaluation may be more informative than the same value assessed using a whole-body approach in predicting physical performance. This pilot study, conducted during a competitive Serie A soccer season, suggests that an increase in lower limb PhA appears to be associated with an increase in vertical jump performance in elite soccer players."}
+{"text": "The role of streptozocin-based chemotherapy (STZ CTx) in advanced, well-differentiated pancreatic neuroendocrine tumours (PanNET) and the best sequence of treatments in advanced PanNET are unclear. We examined the outcomes after STZ CTx in patients who had been selected according to the current therapeutic guidelines.Data from 50 PanNET patients consecutively treated with STZ CTx between 2010 and 2018 were analysed. The endpoints of the study were the objective-response rate (ORR), progression-free survival (PFS), and overall survival (OS).p\u2009=\u20090.001, log-rank test) for subsequent lines. Bone metastases negatively impacted survival .STZ CTx was the first-line treatment in 54% of patients. The PanNET grades were as follows: 6% G1, 88% G2, and 6% well-differentiated G3. The ORR was 38%. Stable disease was the best response in 38% of patients and 24% showed progressive disease. Treatment was discontinued because of toxicity in one patient. Median PFS and OS were 12 , 8.5\u201315.5) and 38 months , respectively. In the Kaplan-Meier analysis, the median OS was 89 months  for STZ CTx as first-line therapy compared with 22 months (95% CI, 19.3\u201324.7; In patients selected according to current guidelines, PFS, and OS after STZ CTx were lower than previously reported, whereas ORR was unchanged. First-line treatment was positively associated with OS and the presence of bone metastases was negatively associated with OS. Pre-treatment with targeted or peptide-receptor radionuclide therapy did not alter ORR, PFS, or OS. Pancreatic neuroendocrine tumours (PanNET) are rare neoplasms with an annual incidence of 0.48/100,000 . StreptoIn 2010, a unified PanNET assessment was established and novel treatment options have been developed since then. The World Health Organisation (WHO) introduced a new classification system that abolished the distinction between well-differentiated endocrine tumours of benign behaviour and endocrine carcinomas, highlighting the malignant potential of all neuroendocrine tumours (NET) based on the proliferation marker, Ki-67 . The valSunitinib, a multikinase inhibitor, approved by the European medicines agency (EMA) in 2010 and the food and drug administration (FDA) in 2011, was the first licensed therapeutic alternative to STZ CTx for progressive, well-differentiated PanNET. Approval of sunitinib was based on a double-blind, randomised study demonstrating an increase in PFS from 5.5 to 11.4 months . In the Since their approval, sunitinib and everolimus have competed with STZ CTx in the treatment of well-differentiated PanNET. However, the importance of the individual substances within the therapy algorithm has not been established because of the lack of comparative studies. Furthermore, peptide receptor radionuclide therapy (PRRT) is commonly used in PanNET, but whether prior PRRT influences the outcome of STZ-CTx is currently unknown , 10. In The aims of the present study were thus (1) to determine the outcome of STZ CTx in a well-defined patient population, treated according to current guidelines, in which the current therapeutic alternatives are available; (2) to assess the influence of previous targeted therapy and PRRT on objective response rate (ORR), PFS, and overall survival (OS), and (3) to detect factors influencing therapeutic outcomes.Patients with histologically confirmed, well-differentiated and locally advanced or metastatic PanNET, who received STZ CTx between January 2010 and January 2018, were identified from our prospective database at the European Neuroendocrine Tumour Society (ENETS) Centre of Excellence at the University Hospital of Essen. The follow-up period was extended until April 2020. Patients with hereditary tumours (multiple endocrine neoplasia type 1 or von Hippel\u2013Lindau disease) were excluded. To ensure consistency, indication for therapies was determined according to ENETS guidelines by an experienced, multidisciplinary tumour board (MTB) , 12. AllThe STZ CTx consisted of 500\u2009mg/m\u00b2 of streptozocin in 100\u2009ml of 0.9% NaCl IV infusion given over 30\u2009min, followed after 1\u2009h by 400\u2009mg/m\u00b2 of fluorouracil (5-FU) infusion in 100\u2009ml of 0.9% NaCl given over 30\u2009min. Adequate peri-interventional hydration was ensured by administering at least 1000\u2009ml of 0.9% NaCl IV infusion. A 5-hydroxytryptamine (5-HT3) antagonist was administered 30\u2009min before the start of therapy. Dexamethasone (8\u2009mg) was administered per os at the beginning of each chemotherapy day. The therapy was implemented over five consecutive days, with a cycle length of 42 days. In case of impaired performance status or toxicity, a delay of up to 2 weeks was provided. The first staging was performed using computed tomography (CT) after three cycles. Patients who did not show progression received the intended number of six cycles, unless unacceptable toxicity occurred.68Ga DOTATOC positron emission tomography (PET)/CT) was included at the initial presentation and at least every 12 months within the surveillance schedule. After 1 year of SD, partial remission (PR) or complete remission (CR), follow-up intervals were extended from 3 to a maximum of 6 months, according to the MTB decision. Response to treatment was evaluated using the international criteria proposed by the Response evaluation criteria in solid tumours (RECIST) committee. At each scheduled time point, chromogranin A (CgA), hematologic, renal, hepatic, endocrine, and coagulation parameters were measured and clinical symptoms were recorded according to common terminology criteria for adverse events (CTCAE), version 3.0.A baseline CT scan was performed within 4 weeks before starting STZ CTx. After three completed treatment courses, the first evaluation of therapeutic response  was scheduled. In case of stable disease (SD) or remission, STZ CTx was continued until the planned number of six cycles. Within 4 weeks after the last cycle and at 3-month intervals, follow-up examinations (CT or MRI) were performed. Hybrid imaging .Response and tumour characteristics were compared using Fisher\u2019s exact test. PFS was recorded as the time between the start of treatment and radiological progression (based on RECIST 1.1) or death. Survival rates were calculated using the Kaplan-Meier method. OS from diagnosis was defined as the time between PanNET diagnosis and death or the last follow-up. OS from the start of chemotherapy was defined as the time between the start of treatment and death or the last follow-up. Univariate and multivariate Cox regression analyses were performed to identify prognostic factors. Statistical differences in PFS and OS between patient groups were estimated using the log-rank test. A n\u2009=\u20092) and 30% (n\u2009=\u20091), corresponding to NET G3. Stage-IV disease was present in 96% of patients and the majority of these patients had liver metastases. Distant organs (\u22652) were involved in 50% of patients.The cohort consisted of 50 consecutive PanNET patients with well-differentiated morphology. All patients were accessible for analysis Table . Forty-on\u2009=\u200927, 54%) were administered STZ CTx as the first-line treatment. The median time from diagnosis to the onset of chemotherapy was 6.5 months . Of those patients with prior systemic treatments (n\u2009=\u200923), the majority had received long-acting somatostatin analogs  or PRRT (n\u2009=\u200913); nine of the PRRT-treated patients received combination treatment with SSA. Seven patients were pretreated with targeted therapy . Four patients received other CTx (temozolomide-based or carboplatin/etoposide) and one patient received prior treatment with interferon. Twenty-three patients underwent surgery of the primary tumour (46%). At baseline, 66% of patients had radiologically proven progressive disease (PD). Another 17 subjects (34%) had clinically PD, characterized by new-onset or worsened abdominal pain (n\u2009=\u200915), nausea (n\u2009=\u20095), or ascites (n\u2009=\u20093). Four patients showed radiological signs of peritoneal carcinomatosis.The median age at the start of therapy was 61 years . Approximately half of the patients . Prior targeted therapy, previous PRRT, SSA, Ki-67 index, number of distant metastases, or the progression status at baseline did not significantly affect ORR. A detailed analysis of the response to STZ CTx is summarised in Table CR and PR as best responses were observed in 1 2%) and 18 (36%) patients, respectively, corresponding to an ORR of 38%. SD as the best response, including mixed response in one case, was documented in 19 cases (38%), accounting for an overall disease control rate  of 76%. PD was noted in 24% of all cases. Among the functional syndromes, the ORR was 22.2% (2/9), and the DCR was 77.8% (7/9). The four patients with gastrinomas each had SD and PD twice, and the three subjects with insulinomas showed PR once and SD twice. At PTHrPoma PR and at VIPoma SD was the best response. In 47 patients, the complete biochemical course of the tumour marker CgA was evaluated before, during and after STZ CTx. A decrease of CgA levels of more than 30% during therapy was associated with a significantly superior ORR , 8.5\u201315.5 months; Fig. The median survival from the start of treatment with STZ CTx was 38 months in the whole patient group . The OS rate was 57.6%  at 2 years and 33.9%  at 5 years, according to Kaplan-Meier analysis    Fig. . The prest) Fig. . PFS wasAdverse effects were documented in 49 of the 50 patients. The most common side effect reported was constipation in 28 patients (57%). This could be controlled with laxatives and enemas without restricting the activities of daily living (ADL). Fatigue occurred in 11 patients (22%) without limiting the ADL. Nausea and vomiting occurred in eight patients (16%) and were mild to moderate. In two patients (4%), hospitalisation was prolonged by persistent nausea despite adequate therapy. At the onset of chemotherapy, 19 patients (39%) had anaemia with haemoglobin (Hb) levels between the specific lower limit of normal and 10\u2009g/dl; eight patients (16%) had anaemia with Hb values between 10 and 8\u2009g/dl. A decrease in the initial Hb value, corresponding to CTCAE grade 1, was found in 11 patients (22%) during STZ CTx. No patient showed a Hb-level decrease of grade 2 or higher. In two patients (4%), leukopenia occurred, one case of leukopenia led to discontinuation of STZ CTx after four cycles owing to persistent fever. Eight patients had pre-therapeutic thrombocytopenia. In two patients (4%), thrombocytopenia first appeared on therapy (CTCAE grade 2). Liver enzymes  increased in 21 patients (43%) while on therapy, corresponding to CTCAE grades 1 and 2. Renal function could be assessed in 48 patients using creatinine levels and glomerular filtration rate (GFR) calculated according to the Modification of Diet in Renal Disease (MDRD) formula. Prior to STZ CTx, 41 patients had normal renal function and seven patients had a slightly reduced function. Chemotherapy resulted in a mostly mild deterioration of renal function in 13 patients (27%)  had a WHO G1 tumour; thus, a much smaller proportion of our patients had G1 tumours than that in the other studies  from 1973 to 2015 showed a bone metastases rate of 5.7% in stage-IV patients . In contIn contrast to previous studies, we detected no effects of primary tumour resection, the Ki-67 index, or the number of metastatic sites on median OS , 15. SurThe safety profile of STZ CTx in our study was consistent with the previous experience in advanced PanNET. The most frequent toxic reactions were of grade 1 or 2 severity and included renal insufficiency, anaemia, fatigue, and nausea; the frequencies were similar to those reported previously \u201316.The majority of patients with dynamically progressive PanNET benefit from STZ CTx. The toxicity is low. Compared with those in targeted therapies or PRRT, remissions occur more frequently. First-line STZ CTx is associated with prolonged survival. Patients with bone metastases require intensive therapy."}
+{"text": "Stress is a ubiquitous part of our life, while appropriate stress levels can help improve the body's adaptability to the environment. However, sustained and excessive levels of stress can lead to the occurrence of multiple devastating diseases. As an emotional center, the amygdala plays a key role in the regulation of stress-induced psycho-behavioral disorders. The structural changes in the amygdala have been shown to affect its functional characteristics. The amygdala-related neurotransmitter imbalance is closely related to psychobehavioral abnormalities. However, the mechanism of structural and functional changes of glutamatergic neurons in the amygdala induced by stress has not been fully elucidated. Here, we identified that chronic stress could lead to the degeneration and death of glutamatergic neurons in the lateral amygdaloid nucleus, resulting in neuroendocrine and psychobehavioral disorders. Therefore, our studies further suggest that the Protein Kinase R-like ER Kinase (PERK) pathway may be therapeutically targeted as one of the key mechanisms of stress-induced glutamatergic neuronal degeneration and death in the amygdala. Stress induces the activation of the body's adaptive response system, which plays a critical role in modulating human behavior in all walks of life. In today's fast moving world, stress has become an inevitable and/or integral part of our life. Interestingly, the moderate stress level has been shown to enhance the general adaptability of the body with respect to physical, mental, and emotional performances . HoweverAs a key component of the limbic system, the amygdala plays a crucial role in the onset and progression of stress-induced phobic anxiety and affective disorders , 10. ThePrevious studies have confirmed that the basolateral amygdala (BLA) plays an important role in fear learning and memory and is a key structure in the neural fear circuit which has been widely concerned . BLA conStress leads to activation of the amygdala and simultaneously induces a large increase in the excitatory neurotransmitter glutamate pool, which plays an important role in neural regulation. Studies have shown that the toxic effects of excitatory neurotransmitters can also cause both acute and chronic nerve cell injuries , 17. HowIn this study, we have established an experimental stress model in rats to investigate the pathomechanism of stress-induced behavioral changes. Furthermore, we investigated how functional modulation of BLA might be related to the ER stress, protein kinase-like ER kinase (PERK) pathway, and excitotoxicity of glutamatergic neurons in the amygdala. Our results thus illustrate the morphological basis for the chronic stress-induced glutamatergic neuronal damage in the BLA and its relationship with the underlying pathomechanism.n = 10 rats in each group).Animal experiments were performed in accordance with the experimental animal research program approved by the laboratory animal management committee. Adult male Sprague-Dawley (SD) rats , weighing 220 \u00b1 20\u2009g, were bred in the experimental environment for 7 days. During the adaptive feeding period, the rats were given grasping and touching every day to reduce the degree of basic stress during experimental procedures. According to the principle of random grouping, the experimental animals were divided into the control and stress groups (restraint stress combined with ice water swimming) at 1, 3, 7, 14, and 21 days' timepoints with 10 rats in each group. In order to study how the ER stress affects glutamatergic neurons of the BLA under stress exposure, we enrolled a group treated with the PERK pathway inhibitor Sal after stress exposure  and another group with only Sal treatment for 14 days , two relatively closed arms (50\u2009cm \u00d7 40\u2009cm \u00d7 10\u2009cm), and two relatively open arms (50\u2009cm \u00d7 10\u2009cm) and was placed 50\u2009cm away from the ground. The rats were put into the open arms from the central grid, and their activity tracks were recorded by an electronic pathway system within 5 minutes. After the completion of each rat experiment, the maze was scrubbed with 75% ethanol, and the next animal experiment was conducted after drying and checked odorless. Residence time and displacement of open arm and closed arm were utilized to measure the anxiety-like behavior in these experimental rats. The less the stay time and displacement of the open arm, the higher was the anxiety level of rats and vice versa , 22.\u03bcm thickness were prepared for H&E staining, FJB staining, and IHC analysis. Brain tissues for neurotransmitter analysis were quickly removed and stored at \u221280\u00b0C. BLA was cut according to the stereotactic map of the rat brain to measure the Glu content.The rats were deeply anesthetized before sacrifice, one hour after the behavioral test. The harvested brains were immediately fixed in 4% paraformaldehyde solution. According to the stereotactic map of the rat brain , the bra\u03bcl of BLA homogenate was mixed with 70\u2009\u03bcl of 0.4\u2009M perchloric acid and centrifuged at 12000 xg and 4\u00b0C for 20 minutes. The supernatant was removed for analysis and filtered with a 0.22\u2009mm GV filter . After that, using a temperature-controlled automatic injector (Agilent), 10\u2009\u03bcl of each sample was injected into the Agilent borohill HPH-C18 column . The mobile phase consisted of 10\u2009mM sodium hydrogen phosphate buffer and 10\u2009mM sodium borate buffer solution in water, methanol, and acetonitrile . The flow rate was constantly kept at 1\u2009ml\u00b7min\u22121. The chromatographic analysis was carried out at 35\u00b0C. The working wavelength of the fluorescence detector was 355\u2009nm, and the emission wavelength was 450\u2009nm. Methanol was adopted as an internal standard, and HP ChemStation software  was used to quantify the Glu level.Using HPLC and fluorescence detector , the conBrain tissue sections were dewaxed and incubated in 0.06% potassium permanganate solution for 10 minutes . The treated slices were incubated in 0.0001% FJB  for 10 minutes (room temperature), and strong light exposure was avoided during the reaction. After being sealed with neutral gum, sections were observed and photographed under a fluorescence microscope.\u03b1 , ATF4 , and CHOP . The tissue samples were incubated for 1 hour with a biotinylated secondary antibody and then with horseradish peroxidase for 30 minutes. 3,3\u2032-Diaminobenzidine (DAB) was utilized as the chromogen to detect the target proteins. The tissue sections were then counterstained with hematoxylin.Following the IHC kit manufacturer's protocol, the paraffin sections were pretreated by heat-treated antigen recovery method and then incubated in a solution of 3% hydrogen peroxide in methanol for 30 minutes, then in goat serum for 30 minutes. Next, the tissue sections were incubated overnight at 4\u00b0C with antibodies against p-eIF2Morphological observation and data analysis were performed on 5 rats in each group. According to the stereotactic map, the BLA region of the amygdala was excised. To quantify DAB-positive cells, the microscope-based polychromatic histocytology (MMTC) has been universally used . Using aFor FJB fluorescence staining analysis, each section in the amygdala region was randomly selected 200 times for the bright field-related counting work, and two observers observed the average number after counting.P > 0.1). Results were expressed as mean \u00b1 SEM. Since all samples were normally distributed, one-way ANOVA was used for statistical analysis. The action of the inhibitor Sal was analyzed by two-way ANOVA. When the comparison was limited to two experimental groups, Student's t-test was used. All statistical analyses were carried out utilizing GraphPad Prism 5  and SPSS 21.0. The statistically significant threshold was P < 0.05.By Kolmogorov-Smirnov test, the distribution was normalized  and the central area exercise distance ratio , without affecting the movement ability of the study animals  as measured by one-way ANOVA. As demonstrated in Figures P < 0.05), and the central area exercise distance ratio (P < 0.05) in the stress group were significantly reduced (P < 0.05) compared with that of the control group rats.Open field experiment revealed that stress could induce notable changes in the central area exercise time ratio  and the percentage of time spent in open arms  were significantly altered after stress exposure, as measured by ANOVA. Figures P < 0.05) and the percentage of time spent in open arms (P < 0.01) were significantly reduced (P < 0.01) in the stress group, further suggesting that stress exposure may lead to anxiety in rats.The elevated plus maze test exhibited that the percentage of open arm entries  in stress-induced rats. As shown in th day of stress exposure (564.60 \u00b1 54.56). Notably, the content of Glu exhibited a significant decrease on the 14th day (470.47 \u00b1 40.31) and 21st day (450.94 \u00b1 19.75) compared with that on the 7th day (P < 0.01), but still was at the increased level with respect to the control group .The content of Glu in the BLA was measured by high-performance liquid chromatography (HPLC) analysis. ANOVA revealed that the Glu contents in the BLA were significantly changed  and a high-affinity Fluoro-Jade B (FJB) staining to investigate the stress-induced glutamatergic neuronal degeneration in the BLA.The H&E staining in F = 128.678, P < 0.01). Compared with that of the control group, the degeneration and death of glutamatergic neurons were more prominent at 3 days of stress exposure, and the number of glutamatergic neurons was gradually increased with prolonged stress duration.FJB staining in Previous studies have shown that the ER-PERK pathway is involved in stress-induced neuronal injury. To test whether it was also involved in the stress-induced injury of glutamatergic neurons in the BLA, experimental rats were treated with the ER stress inhibitor salubrinal .F = 199.569, P < 0.001; F = 12.144, P = 0.001) and the percentage of movement distance in the center area , while no remarkable effect on the total movement distance was detected . As demonstrated in this experiment, compared with the stress-exposed group, the Sal treated and stress-exposed  rats exhibited significant increase in the percentage of cumulative duration in the center  and the percentage of movement distance in the center . Consistently, in the elevated plus maze test, as shown in Figures F = 234.87, P < 0.001; F = 26.097, P < 0.001) and the percentage of time spent in open arms . Likewise, compared with the stress-exposed group, the percentage of time spent in open arms  and the percentage of open arm entries  in the stress+Sal group were significantly increased, suggesting that inhibition of the PERK pathway (or ER stress) may alleviate anxiety behavior.In the open field test, as shown in Figures F = 17.462, P = 0.001; F = 223.182, P < 0.001; and F = 11.238, P = 0.004). Post hoc test revealed that the levels of Glu exhibited significant improvement in Sal pretreated stress exposed rats . The post hoc test showed there was an increase (P < 0.01) in the number of FJB staining-positive cells in the BLA after stress treatment. In contrast, stress+Sal treatment resulted in a decrease (P < 0.01) of the number of FJB staining-positive cells in the BLA.Two-way ANOVA of FJB staining-positive  cells re\u03b1 (p-eIF2\u03b1), activating transcription factor 4 (ATF4), and the C/EBP homologous protein (CHOP) was detected in the cytoplasm of glutamatergic neurons by immunohistochemical (IHC) staining . Post hoc test indicated that there was an increase in the immunohistochemical mean intensity of p-eIF2\u03b1, ATF4, and CHOP expressions in glutamatergic neurons after stress treatment . In contrast, Sal treatment alone and the stress+Sal treatment decreased  the immunohistochemical mean intensity of ATF4 and CHOP in the BLA-specific glutamatergic neurons.Two-way ANOVA of p-eIF2The BLA nucleus, the key structure of the brain for learning and memory of fear emotion adapts to stress response by regulating its neuroendocrine functions. However, under the sustained effects of overloaded stress levels, it can lead to neuroendocrine dysfunction and psychobehavioral disorders , 28. OurStress-triggered nerve impulses are transmitted to the excitatory neurons of the basolateral nucleus of the amygdala, resulting in a large amount of Glu secretion to maintain its corresponding functional state , 30. How2+ influx through N-methyl-D-aspartate (NMDA) receptor, resulting in intracellular Ca2+ overload, Ca2+ dyshomeostasis, perturbed ER function, and ER stress induction [\u03b1 inhibits protein translation and synthesis, thereby reducing the load of protein folding at ER as a protective effect on stressed cells [\u03b1 activates the expression of transcription factor ATF4, further promoting the expression of CHOP, thereby leading to neuronal injury and death [\u03b1 in the PERK pathway [Stress leads to sustained and enhanced expression of Glu in the BLA nucleus. Excitatory neurotransmitter amino acids can induce a large amount of Canduction , 36. In ed cells , 38. Butnd death , 40. Prend death , 19, 41. pathway , 43. OurChronic stress can lead to the degeneration and death of glutamatergic neurons in the BLA nucleus, resulting in psychobehavioral disorders and ER stress. Moreover, the ER stress-associated PERK pathway may be one of the key mechanisms in stress-induced glutamatergic neuronal degeneration and death in the amygdala."}
+{"text": "Inherited metabolic disorders (IMDs) are mostly rare, have overlapping symptoms, and can be devastating and progressive. However, in many disorders, early intervention can improve long-term outcomes, and newborn screening (NBS) programmes can reduce caregiver stress in the journey to diagnosis and allow patients to receive early, and potentially pre-symptomatic, treatment. Across Europe there are vast discrepancies in the number of IMDs that are screened for and there is an imminent opportunity to accelerate the expansion of evidence-based screening programmes and reduce the disparities in screening programmes across Europe. A comprehensive list of IMDs was created for analysis. A novel NBS evaluation algorithm, described by Burlina et al. in 2021, was used to assess and prioritise IMDs for inclusion on expanded NBS programmes across Europe. Forty-eight IMDs, of which twenty-one were lysosomal storage disorders (LSDs), were identified and assessed with the novel NBS evaluation algorithm. Thirty-five disorders most strongly fulfil the Wilson and Jungner classic screening principles and should be considered for inclusion in NBS programmes across Europe. The recommended disorders should be evaluated at the national level to assess the economic, societal, and political aspects of potential screening programmes. Inherited metabolic disorders (IMDs) are a large class of rare genetic disorders. IMDs are defined as any primary genetic condition in which alteration of a biochemical pathway is intrinsic to specific biochemical, clinical and/or pathophysiological features . AccuratIn the United States (US), the Recommended Uniform Screening Panel (RUSP) has allowed for expansion and standardisation of NBS across states. While in Europe there are some countries screening newborns for over 20 disorders\u2014such as Italy, Hungary, or Austria\u2014other countries screen newborns for as few as two disorders . In 2016With the desire to pave the way forward for evidence-based expansion of NBS programmes, a new approach to objectively evaluate and prioritise IMDs for inclusion in NBS programmes was recently proposed in the form of an algorithm . This alThe objective of this work is to evaluate and rank a comprehensive list of IMDs using the NBS evaluation algorithm, as described in a previous paper by the authors .A three-step process was used to select which IMDs would be analysed with the NBS evaluation algorithm see . First, The NBS evaluation algorithm  was usedCondition: Information on the natural history and frequency of each IMD was gathered from the following references, sequentially checked in June 2021: GARD database, Orphanet portal, MedlinePlus, and PubMed for relevant peer-reviewed publications. For the frequency of the disorder, European birth prevalence or incidence was used preferentially, and worldwide or US data were used as an alternative when European evidence was lacking  marked or Food and Drug Administration (FDA) approved DBS assay was assigned two points for the Screening \u201cAvailability\u201d category. For disorders where this was not the case, further research was performed using PubMed to find any relevant published evidence of a DBS test in development. PubMed was also used to find performance data of the DBS tests, determining if the DBS test had a low false-positive rate by itself or if additional confirmatory strategies were required and available to improve screening performance, such as second-tier enzyme activity tests performed on the same blood spot or multivariate pattern recognition software.Treatment: A stepwise approach was used to assess the Treatment \u201cAvailability\u201d category. First, the European Medicines Agency (EMA) website was used to search for approved treatments. If no approved treatment was found, a search was performed on the ClinicalTrials.gov database to identify investigational treatments in phase III development. Peer-reviewed publications were identified which documented other available treatment interventions such as diet, hematopoietic stem cell transplantation (HSCT), or bone marrow transplant (BMT). In this paper, \u201ctreatment strategy\u201d includes both EMA-approved and in development treatments, and treatment interventions . Only the highest-scoring treatment strategy was used to assess the Treatment \u201cOutcomes\u201d category. For the pre-symptomatic initiation of treatment criterion, no score was given if there was no available clinical data.Total scores were obtained by adding up the sub-scores for each pillar of the algorithm. The IMDs were then ranked based on their total scores see .Forty-eight IMDs were selected for evaluation, as described in the Methods section. Twenty-one are lysosomal storage disorders (LSD), eight are disorders of organic acid metabolism (DOAM), seven are disorders of amino acid metabolism (DAAM), nine are disorders of fatty acid metabolism (DFAM), three disorders are classified as Other;Nine disorders had a frequency greater than or equal to 1 in 50,000; 10 disorders had a frequency between 1 in 50,000 and 1 in 100,000; seven disorders had a frequency between 1 in 100,000 and 1 in 150,000; eight disorders had a frequency between 1 in 150,000 and 1 in 250,000; 14 disorders had a frequency less than 1 in 250,000;Four disorders are screened for in over 20 European countries, 15 disorders are screened for in 11 to 20 European countries, nine disorders are screened for in at least one, but fewer than 10 European countries, and 17 disorders are not screened for in the European countries covered in Castineras DE et al. 2019 [The 48 IMDs were assessed with the NBS evaluation algorithm and a score was attributed. The highest scoring disorders were carnitine uptake defect/carnitine transport defect (CUD), with a score of 12.5 out of 13, and severe combined immunodeficiency (SCID) with a score of 12. Seven disorders scored 11.5 or 11 points, 14 disorders scored 10.5 or 10 points, nine disorders scored 9.5 or 9 points, and three disorders scored 8.5 points. The remaining 13 disorders scored between 1 and 8 points. Of the 20 LSDs analysed with the NBS evaluation algorithm, eight had a score of 8.5 and higher: Pompe disease, Gaucher disease, lysosomal acid lipase deficiency (LAL-D), metachromatic leukodystrophy (MLD), mucopolysaccharidosis type I (MPS I), Krabbe disease, Batten disease (CLN2), and Niemann Pick A/B (ASM deficiency).Condition, 77% of disorders (37/48) received points for \u201call forms of the disorder are asymptomatic for the first few weeks of life\u201d \u201d  have a treatment strategy (either an EMA-approved treatment or a treatment intervention) that results in better outcomes if initiated pre-symptomatically \u201d see . For theally see . Condition, all 35 top-ranked disorders have a rapidly progressing form and all but one disorder, PKU, can be fatal by adolescence  have a treatment strategy available, either an EMA-approved treatment (14/34 disorders) or a treatment intervention (20/34 disorders)  see . One disCurrently, in the EU, there are great disparities in the number of disorders screened for between countries. Since 2011, when the European Commission published documentation that supports discussion on how to develop policies for NBS for rare disorders, there has been little concrete progress . The NBSWe need leadership to drive a consistent and methodological approach for NBS programmes across Europe. The majority of the European member states have no legislation governing NBS , but cerExpanding screening for IMDs at birth has the potential to reduce the time to diagnosis, and the related psychological impact on families and patients, and to allow for early, pre-symptomatic treatment that may change prognosis. NBS programmes also benefit society and the healthcare system because increased patient identification increases our knowledge on natural history, frequency, and genotype/phenotype correlations, and thus can help to advance diagnosis and treatment options .This paper is intended to present a living list of IMDs that, at present, most strongly fulfil objective and measurable clinical criteria, thereby recommending them for further evaluation for inclusion on national NBS programmes. Nevertheless, to accelerate NBS expansion it is critical that countries work together to leverage each other\u2019s success and evidence, especially in the rare disease space because of the severity and rapid progression inherent to so many of these disorders. There are disorders that can be accurately diagnosed via DBS test and have a treatment strategy that can change prognosis if initially pre-symptomatically. We need to expand NBS programmes now to diagnose and treat patients earlier."}
+{"text": "This study aimed to identify potential novel drug candidates and targets for Parkinson\u2019s disease. First, 970 genes that have been reported to be related to PD were collected from five databases, and functional enrichment analysis of these genes was conducted to investigate their potential mechanisms. Then, we collected drugs and related targets from DrugBank, narrowed the list by proximity scores and Inverted Gene Set Enrichment analysis of drug targets, and identified potential drug candidates for PD treatment. Finally, we compared the expression distribution of the candidate drug-target genes between the PD group and the control group in the public dataset with the largest sample size (GSE99039) in Gene Expression Omnibus. Ten drugs with an FDR\u2009<\u20090.1 and their corresponding targets were identified. Some target genes of the ten drugs significantly overlapped with PD-related genes or already known therapeutic targets for PD. Nine differentially expressed drug-target genes with p\u2009<\u20090.05 were screened. This work will facilitate further research into the possible efficacy of new drugs for PD and will provide valuable clues for drug design. PD places a significant burden on society and the affected individuals, and approximately 6.1 million people worldwide had been diagnosed with PD in 20162. Dopamine depletion leading to hyperactivity of the corticostriatal glutamatergic pathway is thought to be primarily responsible for parkinsonian symptoms such as resting tremors, rigidity, dyskinesia and postural instability4. Levodopa is the gold standard drug that provides symptomatic relief from motor problems but it has some side effects, and its effectiveness is reduced under long-term treatment. In the present context, medication (including dopamine agonists and monoamine oxidase B inhibitors) and invasive surgery (deep brain stimulation) are being used to reduce the shortcomings of levodopa therapy5. These therapies are quite helpful but are not always completely satisfactory. Thus, it is imperative to identify and develop promising drugs for preventing and treating PD.Parkinson's disease (PD) is a pervasive, progressive, disabling neurodegenerative disorder with motor and nonmotor features6. Under such a situation, the repositioning of available drugs for other disorders as potential novel therapeutic agents for PD becomes an ideal approach. A well-known example of drug repositioning against cancer is thalidomide, which was initially used as a sedative drug7. Network pharmacology is a commonly applied strategy that analyzes biological systems and establishes a drug-target-disease network for drug repositioning8. Many computational methods based on transcriptomic data have also been developed. Chuan et al. found 10 drugs that had certain therapeutical effect on PD based on a handful of genes9. Hindol et al. developed a bidirectional drug repositioning method to find out new drugs for PD10. However, most previous studies have mainly focused on certain specific genes and neglected the gene expression signatures. Here, we present an integrated method for the comparisons of gene expression signatures between a disease model and drug-treated condition network, prediction of drug-protein interactions, and large transcriptomic dataset mining.Unfortunately, drug discovery is expensive and time-consuming. New drug development is affected by many factors, and 85.1% of potential drugs for PD tested thus far have failed in the clinical trial phaseThe purpose of this scheme is to predict and identify new related drugs and targets by applying integrated network pharmacology and transcriptome analysis. Our work will facilitate further studies for better preventive strategies for PD.Genes from the five databases were integrated to include as many PD-related genes as possible. Specifically, 249 genes were retrieved from the Parkinson\u2019s disease pathway hsa05012) included in KEGG. By querying the keyword \u201cParkinson\u2019s disease\u201d, 188 genes were retrieved from OMIM, 9 genes from HGMD, 524 from Genotype, and 370 from DISEASE. After filtering, a total of 970 genes were retained as the PD-related genes. The expression levels of these genes in the PD and control subjects (GSE99039) are shown in the heatmap  revealed that PD-related genes were mainly involved in the ATP metabolic process, energy derivation by oxidation of organic compounds, and oxidative phosphorylation Fig.\u00a0a. MolecuWe tidied up the list of proximal drugs and excluded drugs irrelevant to PD by applying a network-based proximity analysis. The density plot showed that the distance distribution of drugs to PD-related genes overlapped but differed significantly from that of the reference data in the range of \u2212\u00a010.0\u2009~\u20095.0 Fig.\u00a0. The oveUltimately, 46 drugs with an FDR\u2009<\u20090.25 were identified. They may significantly influence PD-related genes. The drugs with FDR\u2009<\u20090.1 (ten drugs) and the corresponding targets (fifty-one targets) are shown in Table We took the intersection of DEGs and drug-target genes, and nine differentially expressed drug-target genes with p\u2009<\u20090.05 were screened. Then, we compared the expression distribution of these genes in the two groups and visualized them with a box plot Fig.\u00a0a. In the12. In the current work, we selected a systematic computation framework to explore potential treatment options for PD based on existing data about diseases, drugs and drug targets.Recently, the repurposing of existing drugs has been proposed as a strategy for new drug development15. These findings established the foundation for further mechanistic studies and provided novel targets for therapy. It is well recognized that PD, as a complex disease, may be caused by mutations of multiple genes or by the dysfunction of multiple biological processes. Earlier studies have shown that disease genes tend to interact in cellular networks16. We calculated a score to predict the proximity between the drug targets and PD-related genes by integrating the information in the PPI networks and kept the drugs with high proximity as candidates.Since drugs usually interact with specific targets to exert an effect on biological processes, and drug targets always interact with disease-related genes, we collected PD-associated genes. GO enrichment analysis of all genes showed that the most enriched terms were oxidative respiratory chain, energy metabolism and ion transport, which are consistent with prior findingsThe major features of PD pathology are the loss of dopaminergic neurons from the midbrain and the presence of \u03b1Syn protein inclusions. Hence, we chose profiles of neural cell lines to perform the IGSEA procedure. Eventually, ten drugs were kept after filtration.17. Vorinostat  has recently been reported to be a potential novel candidate for treating PD18. However, there are no reports about the correlations of tucatinib, tazemetostat and avapritinib with PD pathology. Two of these candidates are hormones. To date, few effective treatments for PD have been reported, but a phase 1/2a clinical trial (Identifier: NCT04127578) is ongoing, the aim of which is to characterize the potential efficacy of methylprednisolone for treating patients with PD who have at least one GBA1 mutation, and we look forward to witnessing more promising discoveries. Isosorbide mononitrate (ISMN) is a candidate treatment for cerebral small vessel disease and lacunar ischemic stroke19. Arsenic trioxide showed beneficial effects on patients with acute promyelocytic leukemia20 and systemic lupus erythematosus (SLE) in a mouse model21. Inflammasomes might be involved in the therapeutic mechanism of arsenic trioxide in the above diseases. However, these two drugs have not yet been investigated in PD.Among the ten candidates, six drugs have been approved by the Food and Drug Administration (FDA) for clinical use in cancer patients. For instance, carfilzomib and afatinib are epidermal growth factor receptor (EGFR) inhibitors. Interestingly, EGFR gene polymorphisms were reported to be related to the susceptibility to PD22 found that three beta subunits of immunoproteasome  all colocalized with \u03b1-syn, and PSMB9 knockdown aggravated the accumulation of \u03b1-syn in a cell model of PD. BCL2 overexpression protects dopaminergic neurons against neurodegeneration23 and it may play a role in dopaminergic development and PD24. St\u00e9phane et al. showed that eliminating Jun N-terminal kinases (JNKs) can prevent neurodegeneration and improve motor function in an animal model of PD25. Another study suggested that activating the Akt1-CREB pathway might halt neurodegeneration in PD26. Furthermore, we found no experimental studies that have focused on PD in association with SLC47A2, HDAC8, TXNRD1, PML, and KIT, so this requires more observational data for verification.Nine differentially expressed drug-target genes with p\u2009<\u20090.05 were screened, and PSMB10, SLC47A2, HDAC8, and BCL2 were clustered into the same model, while KIT, TXNRD1, JUN, AKT1, and PML were clustered into another one, suggesting that these nine targets may act through distinct mechanisms. Sun et al.27. In a randomized control trial (RCT), the effects of glial cell line-derived neurotrophic factor (GDNF) in Parkinson's disease were investigated28. MAPK consists of 3 subfamilies, ERK, JNK and P38. Results from the PD model implicate that selective inhibitors of p38 may help preserve the surviving neurons in PD and slow down the disease progression29.Further KEGG pathway analysis revealed that these nine genes were enriched in the apoptosis, neurotrophin, estrogen, MAPK, and PI3K-Akt pathway. Some of them may represent novel targets for therapeutic intervention. Apoptosis is considered the main mechanism of neuronal death in PD, which could be targeted as possible therapies for PD30. Bromophenols is a ligand for both dopaminergic receptors and human monoamine oxidase31. This study may provide new insights for revealing novel potential drugs and targets for multi-target drug screens. Additionally, the present strategy based on repositioning drugs could provide valuable clues for drug design and exploration for the treatment of other disorders.In the last decade, multi-target drugs have attracted considerable interest in the treatment of complex diseases. The multi-target ligands have clear advantages, such as more predictive pharmacokinetics and reduced risk of drug interactions. For example, the multi-receptor approach for the Cannabinoid Receptor Subtype 2 was proposed for cancer and neurodegeneration therapyHowever, there were also several limitations of this study. First, a potential drawback of proximity analysis is that it relies heavily on known information, including genes, drugs and targets, but this information is still far from being completely understood. Second, although some of the drugs extracted appear to be good candidates for further investigation, it is uncertain whether any of them would actually be effective for PD. More investigations are needed to determine the best use of these drugs to minimize side effects and to maximize patient benefit. Additionally, future studies should pay more attention to the novel targets, not just the drugs themselves.In summary, this network-based approach enabled us to identify several novel drug candidates and targets that could been applied in treating PD. Although these results are still in the preliminary stages, they will provide clues for further experimental exploration. Additional investigation of these drugs and gene networks could lead to better preventive strategies for PD.The workflow of this study is presented in Supplementary Fig. 32, which contained 13,680 drug entries and 4875 corresponding targets. The gene expression data were downloaded from Gene Expression Omnibus . The dataset with the largest sample size (GSE99039)33 was selected as the reference group. The platform data is GPL570 Affymetrix Human Genome U133 Plus 2.0 Array, and 233 healthy controls and 205 PD patients are included. After log2\u2010 transformation, the data were centered and scaled for differentially expressed genes (DEGs) analysis by limma Package34 of R software (version 3.6.4)35. DEGs were defined by a p-value\u2009<\u20090.05.The drug\u2013target relationships were constructed based on the DrugBank databasehttps://www.genome.jp/kegg/)37, OMIM (https://www.ncbi.nlm.nih.gov/omim), Genotype (https://www.ncbi.nlm.nih.gov/gap/phegeni), DISEASES (https://diseases.jensenlab.org/Search), and HGMD (http://www.hgmd.cf.ac.uk/). This was done by querying the above databases using the \u201cParkinson\u2019s disease\u201d keyword. Genes from the above five databases were combined and mapped to their corresponding HUGO gene nomenclature committee38 (HGNC)-based official gene symbols. Duplicate genes and genes of unknown function were removed, and all remaining genes were retained as the PD-related genes.Genes associated with PD were retrieved from KEGG 42.Proteins are the molecules that execute most cellular functions and many regulatory processes take place at this level, and biomolecules always achieve certain functions through extensive interactions with other proteins. To evaluate the correlations between the PD-related genes, we adopted the protein\u2013protein interactions (PPI) network-based approach. The PPI data were obtained from the STRING43. The GO annotation and KEGG pathway enrichment analysis were performed using an R package \u201cclusterProfiler\u201d44. Only the terms/pathways with a false discovery rate (FDR)\u2009<\u20090.05 were considered significantly enriched in this work.Functional enrichment analysis is often conducted to investigate the potential mechanism of the gene set of interest. Gene Ontology (GO) annotation and KEGG analysis are the most commonly used methods. GO provides the classification of gene functions, the relationships between genes of interest in three categories . The KEGG analysis is applied to explore potential signaling pathways that genes may participate in45 to calculate the distance between drugs and PD-related genes. Given G, the PD-related genes-set; T, the set of drug targets, the distance d, namely the shortest path length between nodes g (g\u2208G) and t (t\u2208T) in the network, was calculated as below:To uncover the targeting genes of drugs, we also drew upon the new approach from prior studiesw, the weighted-score of a target; w\u2009=\u2009\u2212\u00a0ln(D\u2009+\u20091) if a target is in the PD-related genes-set; if not, w\u2009=\u20090. D, the PPI degree of PD-related genes.P) matched to the number of drug targets was randomly selected in the network. The distance d between these proteins and PD-related genes was computed. We repeated the randomization process 10,000 times and achieved the reference distribution. The mean \u03bcd and standard deviation \u03c3d of the reference distribution were used to calculate a z-score by converting observed distance to a normalized distance, i.e., proximity value:The significance of relatedness between a drug and PD was evaluated using a reference distance distribution corresponding to the drug. Specifically, a set of proteins  with the PD-related genes47, the enrichment score (ES), and the nominal p-value for quantifying enrichment magnitude and statistical significance of the genes. Finally, multiple comparisons among all the expression datasets were performed using the Benjamini\u2013Hochberg FDR method. The gene set with FDR\u2009<\u20090.25 after performing 1,000 permutations was considered significantly enriched for gene expression datasets, and the corresponding drugs were deemed to be potential candidates for PD.The drug-perturbed gene expression profiles were derived from the Library of Integrated Network-based Cellular Signatures (LINCS)A gene that acts as both an effective drug target and a differentially expressed site could emerge as an important therapeutic target for the treatment. So, we took the intersection of DEGs and drug-target genes, i.e., differentially expressed drug-target genes, compared the expression distribution of these genes between the two groups of samples, and visualized with a box diagram.Supplementary Information."}
+{"text": "The separation was made on a Thermoscientific ODS HypersylTM chromatographic column  with oven temperature 35 \u00b0C and UV detection at 235 nm. The optimized assay conditions were validated according to ICH guidelines. Hence, the results clearly showed that QbD approach could be successfully applied to optimize HPLC method for estimation of M-HCl. The method was applied both for the evaluation of M-HCl content in tablets, and for in vitro dissolution studies of M-HCl from conventional and prolonged-release tablets.The current studies entail quality by design (QbD)-enabled development of a simple, rapid, precise, accurate, and cost-effective high-performance liquid chromatographic method for estimation of metformin hydrochloride (M-HCl). Design of experiments (DoE) was applied for multivariate optimization of the experimental conditions of the HPLC method. Risk assessment was performed to identify the critical method parameters (CMPs) using Ishikawa diagram. The factor screening studies were performed using a two-factor three-levels design. Two independent factors, buffer pH and mobile phase composition, were used to design mathematical models. Central composite design (CCD) was used to study the response surface methodology and to study in depth the effects of these independent factors, thus evaluating the critical analytical attributes (CAAs), namely, retention time, peak area, and symmetry factor as the parameters of method robustness. Desirability function was used to simultaneously optimize the CAAs. The optimized and predicted data from contour diagram consisted of 0.02 M acetate buffer pH = 3/methanol in a ratio of 70/30 ( Metformin hydrochloride , at the muscle level, is responsible for increasing insulin sensitivity by intake and using it, but it also delays intestinal glucose absorption .Metformin used alone has been shown to reduce glycosylated hemoglobin  by approIn the literature there are presented different methods for metformin hydrochloride analysis in various pharmaceutical formulations, synthetic mixtures, water samples, human plasma, biota, and others, by microgravimetry, electrochemical methods , spectrofluorimetry, UV\u2013Vis spectrophotometry, capillary electrophoresis, gas chromatography, and HPLC. The HPLC methods are used for the qualitative identification and quantitative determination of M-HCl alone or in combination with other antidiabetic class, such as sulfonylureas derivatives ; thiazolidinediones (glitazones) ; alpha-glucosidase inhibitors ; methylglinides ; dipeptidyl peptidase-4 (DPP-4) inhibitors ; sodium-glucose co-transporter 2 (SGLT-2) inhibitors  or with other therapeutic classes of molecules (hypocholesterolemic) ,32,33,34The quality by design (QbD) approach suggests looking into the quality of the analytical process during the development stage itself. It says that quality should be built into the process design rather than testing final results of analytical process. QbD is defined as a systematic approach to development that begins with predefined objectives and emphasizes product and process understanding based on sound science and quality risk management ,36. In aThe QbD approach is more appropriate for application during method development than during method validation, because method validation is a process which demonstrates that the analytical method is appropriate for its intended use. In the development of analytical methods it is frequent practice to implement the principles of QbD. These principles facilitate the scientific and risk-based understanding of major sources of variation. In this way, a high robustness and an improved performance of the analytical methods are obtained.Regulatory agencies do not define any specific process of analytical QbD; however, a parallel approach can be drawn based on product QbD. Equivalent to process QbD, the outcome of analytical QbD (AQbD) is well understood and fit for intended purpose with robustness throughout the lifecycle. Quality target product profile (QTTP), critical quality attributes (CQA), and design of experiments (DOE) can be interpreted as analytical target profile (ATP), critical method attributes (CMA), such as retention time, peak area, symmetry factor, tailing factor, the resolution between adjacent peaks, plate count, etc., and method operable design region (MODR), respectively ,39.Fundamentally, the QbD analytical approach requires the identification of the ATP before considering analytical technology. The next step is to establish the CMAs. An MODR is required for analytical methods during the development phase for a better improvement as well as for a quantitative understanding of the factors that influence the performance of the method. To find high-risk variables that have a critical impact on analytical performance, the aim is to identify critical method parameters (CMPs) such as material attributes, instrument-related aspects, instrument operating parameters, and method parameters, based on risk assessment and factor screening studies, followed by their optimization using appropriate experimental projects to increase method performance. The optimized chromatographic conditions are thus evaluated in terms of the CMAs based on the knowledge obtained in the development stage and the screening studies ,40,41,42An important aspect for the development of the HPLC method using the QbD approach using preliminary risk assessment experiments is the choice of CMPs and responses. In addition to the preliminary experiments, the Ishikawa (fish bone) diagram was used to identify and evaluate the CMPs that pose a risk to the performance of the method.Each analytical technique has different CMPs. In the case of HPLC methods, the CMPs with high risk assessment are those regarding the material, instrumental parameters, mobile phase , column characteristics and preparation, and the analyst. In general, for risk identification and assessment, Ishikawa fishbone diagram can be used  42,43,44,45,46.In short, the necessary steps in developing an analytical method with the implementation of QbD principles are as follows: Project initiation \u2192 Literature search and initial risk assessment \u2192 Identification of ATP, CMAs, risk assessment \u2192 Method optimization and method development with DOE \u2192 MODR, Control Strategy and Risk Assessment \u2192 AQbD method validation \u2192 Continuous method monitoring.With all these in mind, the aim of this work was to go through the necessary steps, namely, the development of an HPLC method for the quantitative determination of M-HCl and its optimization in accordance with QbD principles. The next steps were to validate the method according to ICH Q2 (R1) Guidelines  and to a3COONa, \u226599.0%) was obtained from Silal Trading SRL , sodium phosphate dibasic  was obtained from Sigma-Aldrich Chemie GmbH , potassium dihydrogen phosphate  was obtained from Utchim SRL , potassium chloride  was obtained from Chemical Company S.A. , orthophosphoric acid  was obtained from Chemical Company S.A. , glacial acetic acid  was obtained from Chimreactiv SRL , hydrochloric acid  was obtained from Chemical Company S.A. , sodium hydroxide  was purchased from Chemical Company S.A. , gradient grade methanol and acetonitrile (Hiposolv Chromanorm) were obtained from VWR International S.A.S. . Ultrapure water (resistivity 18.2 M\u03a9\u00b7cm) was obtained from a local pharmaceutical company. Industrial M-HCl tablets with a content between 500 mg and 1000 mg were purchased from the local market (pharmacy).Metformin hydrochloride (97%) was purchased from Sigma Aldrich Chemie Gmbh , sodium acetate , dissolution apparatus SR 8 Plus Dissolution Test Station , microliter\u2122 Syringes, 20 \u03bcL Hamilton Bonaduz AG , Transferpette\u00ae Dig. 100\u20131000 \u03bcL  and Rotilabo\u00ae-Mikroliterpipette 0.5\u20135.0 mL .Agilent Technologies 1200 liquid chromatograph was equipped with quaternary pump (type G1311A), diode array detector (DAD type G1315B), degasser (type G1322A), oven column compartment (type G1316A), and Agilent ChemStation 32 software (Rev. B.03.02) . The used chromatographic column was a Thermoscientific ODS Hypersyl TM , Lithuania. Other instruments used were pH-meter, inoLAB pH 7110 , water bath Biobase , ultrasonic bath Biobase , analytical balance PIONEERStandard solutions were obtained by dissolving 103.1 mg of M-HCl  in 25 mL of ultrapure water (resistivity 18.2 M\u03a9\u00b7cm) and diluting with ultrapure water after complete dissolution to 50 mL to give a solution with a concentration of 2 mg/mL (2000 \u03bcg/mL). Any further dilutions for standard solutions were also performed with ultrapure water.v/v). To prepare a volume of 1 L of mobile phase, a volume of 300 mL methanol is mixed with 700 mL 0.02 M acetate buffer (pH = 3). After the mixture reached room temperature, the mobile phase was filtered through 0.45 \u03bcm membrane filters by application of vacuum and sonicated for 15 min before introducing into the system.Mobile phases containing methanol, acetonitrile, water, and buffers at different pH were tested in different proportions. Peaks with satisfactory CMAs  were obtained at a flow rate of 1 mL/min with a mobile phase made up of 20 mM acetate buffer (pH 3)/methanol 70/30 . For risk identification and assessment, Ishikawa fishbone diagram was used .2PO4 and CH3COONa acetate buffer with different pH values) and organic modifier (methanol and acetonitrile).In order to obtain an adequate separation of M-HCl with acceptable values of system suitability parameters , the chromatograms obtained by HPLC were recorded for various compositions and mobile phase ratios using various buffer solutions , and secondly, methanol was replaced with acetonitrile.To study the influence of the buffer pH and the ratio between the buffer solution and the organic solvent in the composition of the mobile phase, we choose a central composite design (CCD) in which the star points are at the center of each face of the factorial space ,48,49,500 is the intercept, and \u03b21 to \u03b25 are regression coefficients derived from experimental runs of the observed experimental values of Y. Interaction and quadratic terms, respectively, are represented by the terms AB, A2, and B2. The surface response of dependent variables and the desirability plot were also plotted using MATLAB software.Experimental design , desirability function, and data analysis calculations were performed by using MATLAB and Statistics Toolbox Release 2020a  software, the best-suited response for second-order polynomial exploring quadratic response surfaces ,46:Y = \u03b2The optimized method is checked by the CMAs to see if the method is efficient and operational throughout its life. Thus, for robustness and ruggedness studies, the parameters and performance of the method were evaluated in several deliberately obtained circumstances . The robustness of the method was determined by making some minor changes in the CMPs  and the ruggedness was determined by changing the analyst and the days when analyses were performed. The relative standard deviation (RSD%) acceptance limits for retention time, peak area, and symmetry factor must be less than 2%.Following the development of the method, a control strategy was implemented for the development of which the ATP was established, i.e., a planned set of controls of some parameters to ensure that both the quality of the results obtained and the method performance fall within the established ATP.The HPLC method for M-HCl was validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ), precision, and accuracy according to ICH Q2 (R1) Guidelines .2, the slope and the intercept with ordinate of the regression line), using regression function in Excel.The linearity was determined by preparing a calibration curve for 19 standard solutions with concentrations ranging from 10 to 2000 \u03bcg/mL. Each solution was analyzed in triplicate; from the obtained chromatograms, the peak areas were determined by integration and used to generate the calibration curve using the corresponding concentration of M-HCl. The equation of regression line was determined using the least squares method and mathematical estimates of the degree of linearity  and quantification limit (LOQ) were calculated using the equations The precision of the developed HPLC method was evaluated in terms of repeatability (intra-day precision) and intermediate precision (inter-day precision). For determination of repeatability, we evaluated in triplicate, on the same day, three solutions of M-HCl at three concentrations . Similarly, for the determination of intermediate precision, the above-prepared solutions were analyzed on three consecutive days. The repeatability and intermediate precision of the method were presented as RSD%.MC and RC are the measured and real concentration, respectively.The accuracy of the method has been determined by application of the analytical procedure to recovery studies using the standard addition method. In this, formerly evaluated sample solutions consisting of a known amount of M-HCl were spiked with three different concentration levels. Briefly, to 0.6 mL of 1000 \u03bcg/mL M-HCl solution, 0.4, 0.7, and 1.0 mL of 2000 \u03bcg/mL M-HCl and 1.0 mL, 0.7 mL, and 0.4 mL of water were added to obtain a final volume of 2 mL. In this way, solutions with concentrations of 700, 1000, and 1300 \u03bcg/mL M-HCl were obtained. The accuracy was expressed in terms of percent recovery for final concentrations. The accuracy of the method was also studied at lower concentrations, in this case being calculated in terms of percent relative error (RE%) using the following equation:For the stability study over time of M-HCl solutions, we prepared solutions at concentrations of 1000 and 2000 \u00b5g/mL. These solutions were analyzed initially and then after 24 and 48 h, the solutions were stored in the refrigerator, at room temperature, and in a water bath at 37 \u00b0C. In all cases, recovery of M-HCl was calculated.mM) was determined according to the provisions of the European Pharmacopoeia, 10th edition [mM is average weight of a tablet calculated for 20 tablets (in g), A is the declared content (in mg), a is the quantity of tablet powder (in g), AP is the peak area, and Int and S are the intercept and the slope of the regression line, respectively.Initially, the average weight of one tablet . The pH value of the dissolution media was checked with a pH-meter and, if necessary, the solution was adjusted with concentrated HCl or 1 M NaOH solution, as appropriate. The test sample (tablet) was placed on the bottom of the cylindrical vessel, after which the air bubbles were removed from the surface of the test sample; the apparatus was started, and the rotational speed was adjusted. The dissolution test for conventional release tablets  was performed for 2 h under the following working conditions: dissolution medium 500 mL solution for simulated gastric fluid with pH = 1.2; temperature 37 \u00b1 0.5 \u00b0C; 60 rpm; duration 2 h. In the case of prolonged-release tablets, named CP-1 and CP-5, the dissolution test was performed for a period of 24 h, using specific working conditions, as follows: first 2 h, simulated gastric fluid pH = 1.2; temperature 37 \u00b1 0.5 \u00b0C; 60 rpm. The dissolution medium was replaced with simulated intestinal fluid pH = 6.8; temperature 37 \u00b1 0.5 \u00b0C; 60 rpm. A total of 2 mL of medium was collected; after each sampling, volume was kept constant in the cylindrical vessel by replacing with the same volume of fresh dissolution medium at 37 \u00b0C. In the case of conventional release tablets, we took samples at the following intervals: 5, 10, 15, 30, 45, 60, 90, and 120 min; for prolonged-release tablets, we took samples on time: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 24 h. Samples were collected at the declared time from the distance between the surface of the dissolution medium and the paddle, but also at least 10 mm from the wall of the vessel, then filtered using nylon filters . The samples were analyzed by the described method. If the area of the peak corresponding to M-HCl was greater than that obtained for standard M-HCl (1300 \u03bcg/mL), the solution was diluted twice and reanalyzed, from where a dilution factor (DF) appeared in the equation used for calculations. The dissolution test was performed on six samples (tablets).The dissolution tests were performed at 37 \u00b1 0.5 \u00b0C, using apparatus 2 (paddle apparatus). Two dissolution media were prepared for dissolution studies: for simulated gastric fluid with pH = 1.2  and simulated intestinal fluid pH = 6.8 ;AP = peak area (mAU\u00b7min);Int and S = intercept and slope of the regression line respectively;A = declared content (mg);tx = current sampling time;tx \u2212 1 = previous sampling time.The following equations were used to determine the amount of M-HCl released:At simulated gastric fluid with pH = 1.2, Equation (5) was used for the first sample taken, at the time of 5 min  and 1 h (prolonged-release tablets); for the following samples, Equation (7) was used. In the case of prolonged-release tablets, the dissolution medium was replaced with simulated intestinal fluid pH = 6.8, so that Equation (5) was used for the three hour sample, then Equation (7) for subsequent sampling. The values obtained were added with the final concentration obtained at medium pH = 1.2 (simulated gastric fluid).For the assay and dissolution study of M-HCl from tablets using the HPLC technique with UV detection, the desired ATP was to obtain a retention time of less than 10 min, and the area and symmetry of the peak to be as large as possible. The CMAs requirements are to use a simple mobile phase (buffer/organic modifier) with isocratic elution for an aqueous sample with a concentration around 1000 \u03bcg/mL using a C18 chromatographic column with UV detection with a minimum retention time, with maximum peak area and symmetry of the corresponding peak. A last, but not least, important attribute is that the HPLC separation method can also be used for mass spectrometry (MS) detection.For HPLC analysis, most research studies report the use of a non-polar stationary phase C18) in different chromatographic columns  and a mobile phase that consists of a mixture of two or three solvents , with aqueous phase having a pH = 2.8\u20137.0. The ratio of mobile phases differs greatly, for example, the organic modifier being used in a proportion of 5\u201365%. The flow rate of the mobile phase was generally 1 mL/min, with extreme values of 0.5 mL/min and 1.7 mL/min. The detection was also different (by mass spectrometry or by spectrophotometry in UV). For most research studies, the detection was performed in UV, the wavelength used being between 221\u2013270 nm, most of which were between 220 and 235 nm)  in diffe,32,33,34Regarding the CMPs, from Ishikawa diagram and preliminary experiments that were conducted, CMPs selected for the further study with high risk assessment that can cause variability are the instrument precision and the mobile phase . Different compositions of the mobile phase  were used to study the influence of the mobile phase on the results. To study the precision of the instrument, after the final separation conditions were established, the same solution was analyzed six times and the results were calculated as RSD% of the peak area. The obtained value was less than 2% see .Once the CMPs were identified, the next step was to optimize them in terms of the CMAs. As shown before, in order to better understand the performance of the method and to identify the independent CMPs and their effect on the dependent variables, various preliminary experiments were performed by trial and error.v/v) was tried; the peak was observed to be asymmetric. The further mobile phase tried was 0.02 M KH2PO4 (pH = 3) or 0.02 M CH3COONa buffers (pH = 3)/methanol . It was observed that the improvement of peak shape and symmetry was achieved by adjusting the buffer pH. In both cases, the retention times are close to each other, but when the acetate buffer is used, the peak area increases by about 8% and the peak height decreases by about 7.5%, which leads to an increase in detection sensitivity.Initially, a mobile phase formed by water/methanol  detection, we chose to use acetate instead of phosphate buffer.Next, the nature of the organic modifier in the composition of the mobile phase was studied. For this, we replaced the methanol in the mobile phase with acetonitrile, with the percentage and pH of the buffer solution being kept constant . In this case, there is no significant change in retention time and peak symmetry, but it is observed that a hypochromic effect occurs, decreasing the height and area of the peak, which leads to a decrease in detection sensitivity. As a conclusion, we kept methanol in the mobile phase composition.For the study of the influence of the pH value of the buffer solution and of the ratio between buffer solution and the organic solvent in the composition of the mobile phase, we chose a face CCD. Using the CCD approach, these method conditions were assessed. At the first step, the conditions for retention time, peak area, and symmetry factor were evaluated. For M-HCl, this led to distinct chromatographic conditions. The acceptable value falls within those regions where deliberate variations in the parameters of the method do not affect the quality of HPLC separation. On the basis of the factor screening studies, selection of the CMPs actually affecting the method performance was optimized using a two-factor CCD at three equidistant levels, i.e., low (\u22121), intermediate (0), and high (+1) levels. v/v, and (b) 0.02 M acetate buffer /methanol in a ratio of 70:30, v/v.As an example, The response surface study type, a CCD with 11 runs, was used. The proposed CCD experimental design was applied and the evaluation of mobile phase composition and pH of buffer was performed against the three responses, retention time, peak area, and symmetry factor; the results are summarized in After calculation of a second-order polynomial exploring quadratic response surfaces, in the models below, we have retained only those coefficients \u03b2 that are significant at 95% confidence level. The results are presented in Rt), that is, Rt = 10.209 + 0.38833 \u00d7 A \u2212 0.19623 \u00d7 B + 0.0015567 \u00d7 B2, we see that the coefficient of A is positive (+0.38833). We can interpret this coefficient as follows: if we keep B fixed, then an increase/decrease in A by one unit will determine an increase/decrease in retention time by 0.38833 units. On the other hand, the coefficient of B is negative (\u22120.19623), but this coefficient can no longer be interpreted as we did before, because the term B2 also appears in the formula. We can only say that, for a fixed value of A and the range of B between 70 and 90, retention time is an increasing function of B. This means that a decrease in B will determine a decrease in retention time.From the equation for the retention time (Pa), that is, Pa = 4979.6 \u2212 736.18 \u00d7 A \u2212 15.384 \u00d7 B + 70.254 \u00d7 A2, we see that the coefficient of B is negative (\u221215.384). We can interpret this coefficient as follows: if we keep A fixed, then an increase/decrease in B by one unit will determine a decrease/increase in peak area by 15.384 units. We also see that the coefficient of A is negative (\u2212736.18), but this coefficient can no longer be interpreted as we did for B, because the term A2 also appears in the equation. We can only say that, for a fixed value of B and the range of A between 3 and 5, peak area is a decreasing function of A. This means that a decrease in A will determine an increase in peak area. In other words, if we intersect the peak area response surface by the plane of equation B = c , then the curve of intersection represents a decreasing function of A. Therefore, a decrease in A will determine an increase in peak area.From the equation for the peak area (Sf), that is, Sf = 3.876 \u2212 0.0667 \u00d7 A \u2212 0.0692 \u00d7 B + 0.00038667 \u00d7 B2, we see that the coefficient of A is negative (\u22120.0667). We can interpret this coefficient as follows: if we suppose that B is fixed, then an increase/decrease in A by one unit will determine a decrease/increase in symmetry factor by 0.0667 units. The coefficient of B is negative (\u22120.0692), but this coefficient can no longer be interpreted as we did for A, because the term B2 also appears in the equation. We can only say that, for a fixed value of A and the range of B between 70 and 90, symmetry factor is a decreasing function of B, and, thus, a decrease in B will determine an increase in symmetry factor.Similarly, from the equation for symmetry factor  yielded desirability close to 1.0, along with all the CAAs in the desired ranges. The optimized values for pH and buffer content in the mobile phase and predicted responses are shown in The search for the optimal solution was performed by numerical optimization by \u201ctrading off\u201d various CAAs to achieve the desired objectives, i.e., maximization of peak area and symmetry factor and minimization of retention time, to obtain the desirability function close to 1. The optimized solution showed that the mobile phase composition containing a mixture of 0.02 M acetate buffer (pH = 3)/methanol in a ratio of 70/30 /methanol in a ratio of 70/30, 3COONa (pH = 3)/CH3OH in a ratio of 60/40, the method is faster (retention time decreases) and slightly more sensitive (peak area increases). However, we opted for a 70/30 ratio for three reasons: (i) at a percentage lower than 70% of the buffer solution in the composition of the mobile phase, a negative peak appears in front of the corresponding peak of M-HCl, which makes the integration difficult, and the precision of the peak integration decreases; (ii) reducing costs  reducing pollution (by disposing of as little organic solvent as waste).As a comment, if a higher percentage of organic modifier is used . For the ruggedness study, the determinations were made on different days by another analyst. In both cases, the values obtained for the retention time, peak area, and symmetry factor, respectively, had RSD values less than 2%.As a control strategy, we planned a set of controls of some parameters to ensure that both the quality of the results obtained and the method performance fall within the established ATP .2 = 0.9999). The regression equation obtained was AP = 2.4266 \u00d7 C \u2212 56.361, where AP is peak area and C is concentration of M-HCl in \u03bcg/mL. Linearity of the proposed method was evaluated according to the ICH guidelines . A numbeUsing the peak area for noise (1.51212) and Equation (2), the detection and quantification limits were calculated:To examine the system precision, the same solution containing M-HCl at the concentration of interest (1000 \u03bcg/mL) was injected six times to obtain as many chromatograms. From the chromatograms obtained, the peak areas were measured, calculating the mean value, the SD, and the RSD%. The obtained RSD% value is less than 2% .The repeatability and the intermediate precision were evaluated by injecting three different concentrations  of M-HCl. For repeatability (intra-day) determination, sets of three replicates of the three concentrations were analyzed on the same day. For intermediate precision (inter-day), three replicates were analyzed on three different days. In both cases, the RSD% value is less than 2%, indicating that the method was precise.In order to estimate the accuracy, this was determined by performing the recovery experiments for three different solutions at three concentration levels , and the final recovery was calculated. The mean recovery is 99.75% in the range 98.12\u2013100.93%.The precision and the accuracy of the method are presented in Because, in addition to the quantitative determination of M-HCl from tablets, the application of the method to dissolution studies is also considered, the accuracy was also studied at lower concentrations, being calculated in terms of percent relative error (RE%) using Equation (3). Keeping in account the fact that the calculated value for LOQ is 29.46 \u03bcg/mL, the accuracy was thus calculated for the range 40\u20132000 \u03bcg/mL, with the mean values of three replicates . Since tSolutions with concentration of 1000 and 2000 \u03bcg/mL, respectively, were analyzed under the method conditions at intervals of 0, 24, and 48 h after preparation, with the solutions being stored in refrigerator, at room temperature, and in a water bath at 37 \u00b0C. By integrating the obtained chromatograms, the area of the peaks  correspoFrom this study, it is found that as the temperature at which the aqueous M-HCl solution is stored is increased, its stability is maintained. Thus, for the concentration of 1000 \u03bcg/mL, the recovery decreases by 0.19, 0.3, and 0.35% in 24 h and by 0.58, 1.03, and 1.61%; in the case of the concentration of 2000 \u03bcg/mL, the recovery decreases by 0.17, 0.28, and 0.37% in 24 h and by 0.88, 1.19, and 1.59% if the solution is stored in the refrigerator, at room temperature, or in a water bath, respectively. The best stability is the solution stored in the refrigerator, and as the storage temperature increases, the stability decreases. In conclusion, the method can be applied to determine the M-HCl content of tablets, with the samples being able to be analyzed within a reasonable time. In the case of dissolution studies, due to the large number of samples, the samples collected at different time intervals are stored in the refrigerator and are analyzed chromatographically in a maximum of 24 h.Under the method conditions, the peak of M-HCl has a retention time around of 5.27 min. Confirmation of the identification of the corresponding M-HCl peak was performed by comparing the retention time of the M-HCl peak in the sample chromatogram with that in the chromatogram of a standard. To increase the quality of the identification, the absorption spectrum measured at the apex of the peak from the sample chromatogram was compared with the standard spectrum of M-HCl stored in the spectra library.In order to determine the presence of compounds with similar retention times (co-eluents as impurities or excipients), the study of the spectral purity of the chromatographic peak was performed. From this study, it is found that no co-eluents appear, the purity of the peak being higher than 99%.The experimental results obtained by this method of recovery of M-HCl from different types of tablets are presented in Analyzing the data in Conventional-release tablets (850 mg tablets and 1000 mg film-coated tablets) were used in the dissolution test for tablets, but also for modified-release tablets (500 mg and 1000 mg prolonged-release tablets).In the case of conventional release tablets, at the dissolution test performed at simulated gastric fluid with pH = 1.2, there is a minimum release for M-HCl for CP-2 (63.85%), CP-3 (33.8%), and for CP-4 (47.97%) at the first sampling at 5 min. After 30 min, all three formulas of conventional release tablets released over 80% of the declared amount of active substance , which cIn the case of prolonged-release tablets with 500 mg and 1000 mg M-HCl, it is observed that at the first sampling, simulated gastric fluid with pH = 1.2 has a release of M-HCl from the CP-1 sample of 41.74% and in the case of the CP-5 sample of 23.57%, which corresponds to the acceptance criteria of the European Pharmacopoeia, 10th edition , by prevThe second specification point of the dissolution test will be defined by the release of approximately 50% of M-HCl from prolonged-release tablets. In the case of the first studied formulation, a much faster release profile is observed, so that the second point is achieved after 2 h in simulated gastric fluid with pH = 1.2 (CP-1: 49.71%), unlike formulation CP-5, which released 53.17% of M-HCl at 4 h in simulated intestinal fluid, pH = 6.8. The final specification point stipulated in the conditions of European Pharmacopoeia is the assurance of complete release, which is understood to be at least 80% achieved in a simulated intestinal fluid pH = 6.8. For the CP-1 test, this point is reached after 5 h (81%), with a maximum release of 100.04% at the end of the test; for the CP-5 81.21% is released after 8 h, with a maximum 99.87% after 24 h . The twov/v), a case for which the desirability function was very close to 1 (desirability function = 0.9807).The paper describes the development of an HPLC method for the determination of M-HCl by the QbD approach using a central composite design by studying the interrelationships of two factors regarding the mobile phase (the pH of aqueous phase and the ratio between acetate buffer and methanol) at three different levels. The optimized CMPs were found after calculations of second-order polynomial exploring quadratic response surfaces for retention time, peak area, and symmetry factor as functions of buffer pH and ratio between buffer and methanol. The optimized solution showed the mobile phase composition containing a mixture of 0.02 M acetate buffer (pH = 3)/methanol in a ratio of 70/30 (2 = 0.9999) in the studied range (10\u20132000 \u03bcg/mL) with good detection and quantification limits , good precision (RSD < 2%) and accuracy (mean recovery = 99.75%) in the range 700\u20131300 \u03bcg/mL, and a mean percent error of 1.07% in the range of 40\u20132000 \u03bcg/mL. From the obtained results, it can be concluded that the M-HCl solutions are stable enough at room temperature 24 h after preparations. If the samples were analyzed after more than 24 h, it is recommended that they be kept in a refrigerator.The optimized method was fully validated according to ICH Q2 (R1) Guidelines. The method showed good linearity (rThe optimized and validated method was used for the M-HCL assay from tablets and for dissolution studies. In the case of assay studies, all the pharmaceutical products analyzed have a percentage content of M-HCl in the range 97.78\u201399.20%, for a permissible deviation of \u00b15%. In the case of dissolution studies, for conventional-release tablets, after 2 h a release of over 98% was registered for all the studied formulations. In the case of prolonged-release tablets, the two samples analyzed are compliant and fall into the release profile for extended-release dosage forms, according to \u201c2.9.3. Dissolution test for solid dosage forms\u201d and \u201c5.17. Recommendations on methods for dosage forms testing\u201d from European Pharmacopoeia, 10th edition."}
+{"text": "Sarcopenia is a debilitating skeletal muscle disease that accelerates in the last decades of life and is characterized by marked deficits in muscle strength, mass, quality, and metabolic health. The multifactorial causes of sarcopenia have proven difficult to treat and involve a complex interplay between environmental factors and intrinsic age-associated changes. It is generally accepted that sarcopenia results in a progressive loss of skeletal muscle function that exceeds the loss of mass, indicating that while loss of muscle mass is important, loss of muscle quality is the primary defect with advanced age. Furthermore, preclinical models have suggested that aged skeletal muscle exhibits defects in cellular quality control such as the degradation of damaged mitochondria. Recent evidence suggests that a dysregulation of proteostasis, an important regulator of cellular quality control, is a significant contributor to the aging-associated declines in muscle quality, function, and mass. Although skeletal muscle mammalian target of rapamycin complex 1 (mTORC1) plays a critical role in cellular control, including skeletal muscle hypertrophy, paradoxically, sustained activation of mTORC1 recapitulates several characteristics of sarcopenia. Pharmaceutical inhibition of mTORC1 as well as caloric restriction significantly improves muscle quality in aged animals, however, the mechanisms controlling cellular proteostasis are not fully known. This information is important for developing effective therapeutic strategies that mitigate or prevent sarcopenia and associated disability. This review identifies recent and historical understanding of the molecular mechanisms of proteostasis driving age-associated muscle loss and suggests potential therapeutic interventions to slow or prevent sarcopenia. Skeletal muscle comprises over a third of total body mass in young adults  and is aThe concept of \u201chealthy aging\u201d and maintaining independence has been tied to optimizing muscle mass and strength. While muscle mass is critically important to the quality of life, aging is associated with a gradual reduction in muscle mass that plays a contributory role in the development of age-related diseases and disability ,6. SarcoSarcopenia is usually, but not always ,12,13, aWell-established contributors to sarcopenia include neuromuscular remodeling ,26,27, iSkeletal muscle retains remarkable plasticity by altering cellular size and contractile properties in response to altered stimuli. Muscle fiber size is established as the aggregate result of competition between concomitant rates of anabolic muscle protein synthesis (MPS) and catabolic muscle protein breakdown (MPB). Several anabolic stimuli can enhance MPS, including exercise, dietary amino acids, growth factors, and hormones. Among modulators of MPS, the serine/threonine kinase mammalian target of rapamycin (mTOR) is a critical component of mTORC1 that acts as a central node of regulation . ActivatS6K1). Phosphorylation of 4E-BP1 by mTORC1 results in the disassociation of 4E-BP1 from eIF4E and allows for the formation of the translation initiation complex at the 5\u2032 end of mRNA to facilitate recruitment of the ribosomal machinery and cap dependent mRNA translation [S6K by mTORC1 also boosts protein synthesis through augmentations in ribosomal biogenesis [In addition to prompting the release of insulin, feeding also replenishes intracellular nutrients that can induce mTORC1 activation, in particular amino acids. Cytosolic availability of leucine is a potent signal for mTORC1 activation in large part through the leucine sensor Sestrin2. The availability of leucine reduces Sestrin2 activity, which relieves inhibition of the protein complex GTPase-activating protein activity toward Rags 2 (GATOR2) ,47. Uninnslation . mTORC1 nslation . Activatogenesis , translaogenesis , and eloogenesis .Muscle atrophy occurs when myocytes have reduced protein accretion that results in reduced cellular size as the aggregate result of competition between reduced concomitant rates of MPS and increased MPB. Anabolic resistance refers to a blunted capacity to enhance MPS in response to an anabolic stimulus. Associations between aging and anabolic resistance have been under investigation for decades and have indicated that a perturbed skeletal muscle anabolic response to a nutritional stimulus exists in aged individuals ,56. WhilWhile there is significant support for an aging-suppression of MPS, not all studies have observed a blunted maximal MPS response to high doses of amino acids with elevated ages ,65. FurtThe co-occurrence of age-related muscle loss and obesity, termed sarcopenic obesity, is a growing health concern. Sarcopenic obesity enhances the risk of developing type 2 diabetes mellitus and insulin resistance ,67. The Recent observations have linked the gut-muscle axis as a potential contributor and therapeutic target for aging associated anabolic resistance. While still in its infancy, several studies in rodents have linked alterations to the gut microbiota to aging and sarcopenia ,77. IndeIt is interesting to note that a greater proportion of germ-free mice survived to old age than pathogen-free mice that are housed under standard laboratory conditions, which present with a more pronounced inflammatory state and enhanced gut permeability with increased age . Germ frIt is important to note that while skeletal muscle is modulated by the microbiome, the gut microbiome can be modified by diet, antibiotics, and dietary supplements. The human microbiota has been reported to be similar between young and aged humans but there are larger differences between the young and the very old . FurtherSkeletal muscle necessitates contractile and neural stimuli to maintain tissue integrity ,87. ExteRegulators of mitochondrial volume and function are suppressed during muscle disuse as energy demands and contractile activity diminish. However, it is unclear if mitochondrial impairments during recovery may contribute to age-associated regenerative defects in skeletal muscle and contribute to sarcopenia. It is important to note that some studies have found that the amount of atrophy that occurs during disuse is not exacerbated by age ,112 and Another possibility is that the rate of MPS after disuse may be altered with aging. This is supported by observations that the larger muscles in young animals exhibit a more pronounced drop in MPS during disuse when compared to smaller muscles in aged animals . When MPAlthough mTORC1 is an important regulator of muscle growth, recent evidence has implicated that sustained mTORC1 activation may be a potential contributor to age-associated myopathy . DespiteHow alterations in senescent muscle drive greater basal mTORC1 activity or how sustained mTORC1 activity may differ between young and aged muscle have yet to be determined. Nevertheless, several studies have provided some clues to how mTORC1 could be elevated in aged muscle. For example, data from a transgenic mouse model that allows for the muscle-specific inducible expression of activated Akt has shown that activation of Akt  for three weeks, results in greater muscle mass in young  and old There is a complex regulation of muscle mass in aging because inhibition of chronically active mTORC1 with either rapamycin or low dose rapalogs attenuates aging-associated loss of muscle function and muscle mass in some, but not all, muscles ,117,125.2+ dependent calpain system and caspase-3 [Protein turnover is critical for cellular quality control under both basal and physiologically stressful conditions to maintain the integrity of the proteome. Degradation of cellular proteins occurs primarily through the ubiquitin proteasome system (UPS), autophagy, and to a lesser extent the Caaspase-3 ,128. Inaaspase-3 . The 19Saspase-3 . ProteinUbiquitination of damaged or misfolded proteins is a complex and highly regulated process involving a molecular hierarchy of enzymes involved in ubiquitin activation, conjugation, and ligation . Brn = 2) and E2 (n = ~40) family, there are hundreds of different E3 ubiquitin ligases that confer specificity to the UPS, allowing for tight control of substrate ubiquitination [Relative to the small number of proteins within the human E1  [Importantly, greater mTORC1 activity can impede AKT activation through p70 (IRS-1) , thereby (IRS-1)  showed tSimilar to the UPS, autophagic degradation represents another major arm of protein quality control, to ensure cellular clearance of defective proteins and organelles as well as to recycle substrates during nutrient scarcity. A basal level of autophagy is important for cellular homeostasis, and the autophagic machinery is responsive to several stimuli including fasting ,167 and The initiation of autophagy and autophagosome biogenesis is largely regulated by unc-51 like autophagy activating kinase 1 (ULK1) and to a lesser extent its paralog unc-51 like autophagy activating kinase 2 (ULK2). ULK1 is a serine/threonine kinase that is the core component of the initiation complex alongside autophagy related (ATG)13, Focal adhesion kinase family-interacting protein of 200 kDa (FIP200), and ATG101 ,174,175.While excessive autophagy can contribute to atrophy , in receInvestigations regarding whether skeletal muscle autophagy is reduced or enhanced with age have reached mixed conclusions ,194,195.Measurements of autophagy flux have shown a trend for enhanced basal autophagy in aged muscle ,193,197.in vitro partially via the PKA/LKB1/AMPK pathway [Skeletal muscle AMPK-knockout mice also have defective autophagy and exhibit age-related muscle dysfunction associated with signs of fiber degeneration, diminished force production, and greater mitochondrial size and Parkin accumulation at 18 months of age . These d pathway .Skeletal muscle expression of FoxO in drosophila enhances autophagy and delays the aging associated accumulation of protein aggregates in skeletal muscle . In contS6K1, ribosomal protein S6 (RPS6), and 4E-BP1. Notably, fasting-induced changes were absent in aged mice. Furthermore, mTORC1 phosphorylation of ULK1 (Ser757) was reduced by fasting in young but not aged mice [S6K1 ameliorates the sarcopenic phenotype of skeletal muscle TSC1-knockout mice [S6K1 inhibition and 4E-BP1 activation, only activation of 4E-BP1 in TSC1-knockout muscle improved muscle size, function, and the induction of autophagy by fasting despite no change in ULK1 phosphorylation. TSC1 muscle-knockout mice exhibited greater protein aggregates and histological indications of accumulated mitochondria and lipofuscin; characteristic of blunted mitophagy and lysosome function. Importantly, activation of 4E-BP1 ameliorated signs of mitochondrial and lysosomal dysfunction present with mTORC1 hyperactivation, suggesting a critical role for 4E-BP1 modulation of autophagy that is independent of mTORC1-mediated ULK1 phosphorylation.One potential mechanism by which autophagy may be impaired in aged skeletal muscle is via sustained activation of mTORC1. Comparisons of autophagy and mTORC1 signaling in skeletal muscle of mice that are freely fed revealed little difference between young and old animals. However, upon fasting, skeletal muscle from young mice displayed diminished phosphorylation of AKT and the mTORC1 targets p70ged mice . Mirroriged mice . Indeed,ged mice . A recenged mice  revealedout mice . Despitein situ preparations that maintain mitochondria in their native cellular environment [in situ preparations of human aged skeletal muscle exhibit a respirometric defect and a higher Km for ADP when compared to young muscle [It is well established that mitochondrial dysfunction is a core characteristic of sarcopenia . Whetherironment . This apironment ,209. Nonironment ,210 whicironment . Additioironment , indicatg muscle , suggestg muscle , also ing muscle . Interesg muscle . These d2+ homeostasis. Skeletal muscle function relies heavily on the propagation of signals from the neuromuscular junction to the transverse tubules to the SR, a specialized portion of the ER in striated muscle that mediates the release of Ca2+ ions through the ryanodine receptor to initiate muscle contraction. Disruption of Ca2+ handling by the SR can lead to elevated cytosolic Ca2+ and in turn, exacerbates mitochondrial reactive oxygen species (ROS) production. Oxidation of the ryanodine receptor is greater in aged than in young muscle, which can result in Ca2+ \u201cleakage\u201d from the SR into the cytoplasm [2+ leakage present similar contractile deficits as aged mice [2+ concentration decreases with age in drosophila melanogaster. Furthermore, the decrease of Ca2+ concentration was closely correlated with reduced physical function and not observed in neurons, highlighting a tissue specific dysregulation of Ca2+ homeostasis [2+ ATPase (SERCA) pump activity [2+ from the cytosol to the SR at the end of an electrically evoked stimulus. Interestingly, treatment of aged mice with the allosteric SERCA activator CDN1163 improved indices of muscle function and attenuated mitochondrial dysfunction [-/-) mice, a model of premature aging, also exhibited reduced SERCA pump activity and skeletal muscle contractile deficits that are rescued by CDN1163 treatment [2+, which can amplify mitochondrial dysfunction and contribute to declines in contractile function.Oxidative stress produced by the accumulation of dysfunctional mitochondria can also damage cellular structures that are near the damaged mitochondria. Indeed, in muscle, mitochondria share an intimate role with the ER/sarcoplasmic reticulum (SR) and influence Caytoplasm . Musclesged mice . In agreged mice  found theostasis . In addiactivity . The SERfunction . Furtherreatment . Taken t2+ reuptake, there is evidence that store-operated Ca2+ entry (SOCE) is inefficient in sarcopenic muscle and may contribute to declines in skeletal muscle performance. SOCE is critical to maintain intracellular Ca2+ homeostasis as ER/SR Ca2+ stores are depleted. In brief, reductions in ER/SR Ca2+ are sensed by the Ca2+ sensor stromal interaction molecule 1 (STIM1), which resides on the ER/SR membrane and in response to Ca2+ depletion aggregates to ER/SR locales proximal to the plasma membrane [2+ release-activated Ca2+ modulator 1 (Orai1) [2+ entry into the ER/SR. Indeed, the importance of SOCE for skeletal muscle health is apparent in mice with STIM1 haploinsufficiency, which exhibit markedly greater muscle fatigability [ex vivo contractility assays, it was revealed that inhibition of SOCE reduces contractile activity in skeletal muscle from young but not aged animals, an effect that was most prominent at high frequency stimulation [2+ leakage from the ER/SR, inadequate SOCE may contribute to a dysregulation of Ca2+ homeostasis that compromises function in aged skeletal muscle.In addition to dysfunctional Camembrane . STIM1 i (Orai1) , which fgability . Furthergability . Investigability ,225, altgability . During mulation . A lack mulation  as well mulation . These fThe evidence for non-pharmacological approaches to mitigating or preventing sarcopenia suggests that both exercise ,230 and Branched-chain amino acids (BCAAs) play a crucial role as both signaling molecules and substrates for protein synthesis. Low blood levels of BCAAs are associated with sarcopenia and reduced physical function . IngestiRecently, interest has grown over the use of mTORC1 inhibitors and calorie restriction mimetics as a potential treatment for sarcopenia. Indeed, the use of rapamycin and rapalogs have proven to be beneficial in ameliorating age-associated skeletal muscle defects in preclinical models ,117,122.in vitro administration of urolithin A to the myoblast line of C2C12 cells reduces \u0394\u03c8m and enhances mitophagy. When given to aged mice on either a chow or a high-fat diet, dietary administration of urolithin A potently enhanced skeletal muscle physical function without changes to body mass. Improvements in physical function were concomitant with enhanced AMPK phosphorylation and greater abundance of markers of autophagy and mitophagy [Indeed, mTORC1 inhibitors are not the only pharmaceutical agents with alleged benefits for aging associated muscle defects. Dietary supplementation with the mitochondrial uncoupler BAM15 induced markers of mitophagy and improved muscle mass in a murine model of sarcopenic obesity . Adminisitophagy . These dGiven the association between sarcopenia and hypogonadism, targeting of the androgen receptor via selective androgen receptor modulators (SARMs) and testosterone has been investigated as viable therapeutics for sarcopenia. The administration of exogenous testosterone, while known to substantially enhance skeletal muscle mass , is alsoNotably, there have also been several drug candidates produced to target myostatin, a potent negative regulator of skeletal muscle mass. Administration of the human monoclonal antibody REGN1033 to target myostatin in mice enhanced both muscle mass and force production and prevented muscle atrophy induced by limb immobilization. Treatment of aged mice with REGN1033 led to significantly increased muscle mass and force production without a loss in specific force, indicating a preservation of muscle quality . SubcutaCurrently, there are no approved drugs for the treatment of sarcopenia. Recent and past findings have shown that a finely tuned balance between anabolic and catabolic processes is required to maintain adequate muscle quality and mass in older ages. Questions remain regarding the appropriate degree of caloric restriction to combat age related muscle dysfunction. An important variable to consider when interpreting the impact of caloric restriction is to differentiate between the benefit of an energy deficit vs. the maintenance of a reduced body weight. A perpetual caloric deficit would result in a chronic loss of body mass and exacerbate sarcopenia. Thus, organisms adapt to reduced caloric intake via weight loss and energy sparing processes to reach an equilibrium between calorie expenditure and calorie intake. It is unclear how much of the purported benefit of calorie restriction is due to an incurred energy deficit vs. the maintenance of a healthier lower body weight (primarily reduced fat mass). Additionally, caloric restriction is difficult to execute consistently and effectively in a clinical setting. It is possible that regimens consisting of interspersed periods of caloric restriction with isocaloric diets may activate cellular pathways that confer protection against sarcopenia without compromising absolute skeletal muscle mass. Furthermore, while extensive lifestyle modification may not be a practical and viable option for elderly patients, small amounts of physical activity may improve autophagy/mitophagy and be augmented pharmaceutically to allow for even a greater benefit on muscle quality. A combination of exercise that is coupled with supplementation with the polyphenol resveratrol has also been shown to augment exercise adaptations in older men and women to offset sarcopenia . This suThe primary cause of cellular dysregulation that contributes to sarcopenia development remains elusive. However, recent findings implicate dysregulated proteostasis as an important characteristic of skeletal muscle in aged hosts that may contribute to muscle atrophy and functional decline leading to sarcopenia. Additionally, given the multi-factorial aspect of sarcopenia, effective treatment will likely be best achieved by addressing multiple pathways that contribute to the loss of muscle function and mass with aging. Furthering our understanding of the causes and consequence of dysfunctional proteostasis in aged skeletal muscle will allow for the development of targeted treatments to ameliorate one of the most debilitating geriatric conditions."}
+{"text": "Cardiovascular disease (CVD) is highly prevalent in India, and little is known about the perception of patients and providers about a package of collaborative quality improvement (C-QIP) strategies consisting of provider-focused electronic health records-decision support system (EHR-DSS), non-physician health workers (NPHW), and patient-facing text messages to enhance the CVD care.To explore the barriers and enablers of the C-QIP strategy from the perspective of providers, health administrators, patients, and care givers in India.We conducted a qualitative study using the consolidated framework for implementation research (CFIR) to understand the challenges and facilitators of implementing C-QIP strategy to enhance CVD care in the Indian context. A diverse sample of 38 physicians, 14 non-physician health workers , 4 health administrators, and 16 patients and their caregivers participated in semi-structured interviews. All interviews were audio-recorded, transcribed, translated, anonymised, and coded using MAXQDA software. We used the framework method and CFIR domains to analyze the qualitative data.Barriers perceived from providers\u2019 and health administrators\u2019 perspectives in providing quality CVD care were high patient volume, physician burnout, lack of robust communication or referral system, paucity of electronic health records, lack of patient counsellors, polypharmacy, poor patient adherence to medications, and lack of financial incentives. Low health literacy, high cost of treatment, misinformation bias, and difficulty in maintaining lifestyle changes were barriers from patients\u2019 perspectives. The CFIR identified key enablers for the implementation of C-QIP such as standardized treatment protocol, reduced medication errors, improved physician-patient relationships, and enhanced patient self-care through trained and supported NPHW. Barriers included: heterogenous healthcare settings, diverse patient groups and comorbidities, associated costs of care and interoperability, confidentiality, and data privacy issues around the use of EHR-DSS.Strategies to enhance CVD care must be low-cost, culturally acceptable, and integrated into existing care pathways. Cardiovascular disease (CVD) is highly prevalent in India, with more than 70 million people affected in India, and 523 million globally 23. CVD p245collaborative quality improvement (C-QIP) strategy (combining task-sharing and technology) for chronic care of CVD in India.Quality improvement (QI) strategies involving clinical decision support, audit and feedback reports, and team-based care have been successful in well-resourced settings for improving care . However1112In the Indian context, several factors influence the CVD care gaps such as economic burden on patient and caregivers, low health literacy, and lack of health system infrastructure 15. To im16This qualitative study was designed to inform development and implementation of the C-QIP strategy  to ensure that it addressed the myriad factors that can facilitate, as well as impede, collaborative care programs in India for patients with CVD. A prospective feasibility randomized controlled trial (RCT) will assess the effectiveness and acceptability of C-QIP strategy involving 400 patients with CVD attending four diverse public and private hospitals in India . Since oThis study was conducted among three different groups of stakeholders: 1) health care providers, 2) health administrators, 3) patients, and their caregivers. Health care providers constituted physicians, cardiologists, nurses, community health workers, and pharmacists engaged in providing care for patients with CVD. We used a purposive sampling frame to select an initial sample of participants who had diverse roles and experiences in the care of patients with CVD ranging from cardiologists and physicians to cardiac care unit nurses, pharmacists, community health workers to health administrators and policy makers. We then used a snowballing sampling technique based on the in-depth interviews to recruit additional participants with increasing variability until we achieved theoretical saturation, the point at which no novel concepts seemed to emerge. The four hospitals selected for the C-QIP trial are large , tertiarWe conducted semi-structured interviews in person and over the phone between September 2019 and February 2020 using an interview guide specifically developed for each participant group: healthcare provider, health administrator, patient, and caregiver (see supplement for the interview topic guide). The interview guide broadly focused on three different aspects of collaborative CVD care using CFIR constructs to elicit information on barriers and enablers of the C-QIP strategy in the Indian care setting. First, interview questions were asked about what challenges are faced by providers, administrators, patients, and their caregivers to manage CVD in India. Second, the interview questions delved into understanding how common CVDs are across age, gender, and socio-economic groups, as well as co-morbidities, patients\u2019 needs and understanding of CVD and related self-care, and how providers motivate patients\u2019 self-care. Third, interview questions focused on eliciting views of multiple health system stakeholders on the C-QIP strategy, perceived enablers, and barriers to its implementation in diverse clinical practices , and broader relevance of QI strategies for CVD care in Indian context. Interview questions also probed the relative advantage, self-efficacy, and systems-level challenges or the cultural issues that might affect the implementation of the C-QIP strategy in India.Three members of the study team  conducted the in-depth interviews. In-depth interviews used open ended questions, and probes were used to elucidate emerging themes. All interviews with healthcare providers and health administrators were conducted in English, whereas patient and caregivers\u2019 interviews were conducted in dyads and in the local languages (Hindi and Kannada) by a team member fluent in these languages. Interviews ranged from 30 minutes to more than one hour in length. Interviews were audiotaped, transcribed verbatim, and checked for their accuracy by study team members.An iterative approach was used for data analysis based on the framework method for qualitative research . The fir21Among the three stakeholder groups (N = 72 participants), we interviewed 38 physicians , 14 non-physician health workers , 4 health administrators or policy maker, 100% men with mean age: 54.4 \u00b1 5.2 years, and 16 patients  and their caregivers . Two physicians and a patient refused to participate in the interview due to personal reasons. Physician- and hospital-level characteristics are summarized in high patient volume, time-constraints, and low health education among patients to be greatest challenges for providing quality CVD care. Physicians found insufficient time to provide care in the first place. One cardiologist explained, \u2018Volume is too high that we are not able to spend time with each patient in a proper way.\u2019 Further, there are too few specialists and too many patients that need specialty care. One out of every three providers identified \u2018physician burnout\u2019 to reflect, what they called, a \u2018cultural syndrome\u2019, described by one provider as when clinicians \u2018are no longer interested to be actively involved in patient management\u2019 because \u2018the load is so much, every person has overloaded system, so in an overloaded system a single doctor cannot treat so many cases.\u2019 He went on to explain, \u2018most of the time is going in treating the patient and not in healing. Healing requires both preventive as well as therapeutic (efforts).\u2019 This idea of remedial approaches was common, as many recognized that there is a lack of focus on prevention efforts such as tobacco cessation. This was recognized as an attitude problem of physicians: \u2018The attitude needs to be changed. We wait for the disease to develop and all the efforts of all the corporate hospitals and everybody is just for the disease to occur so that now they can be rectified. There is nobody interested in preventing the disease.\u2019 Lack of robust communication and referral systems, including paucity of electronic health records, emerged as barriers to quality CVD care as described by a health administrator: \u2018I think there is lot of misguidances to the patient, as to how, when and where they should approach which specialty.\u2019 Another physician added \u2018There is no mechanism where we monitor the follow-up of the patient\u2019, emphasizing a gap in care coordination.Further, most physicians recognized that poor patient adherence to medications, polypharmacy and mixed recommendations from other traditional health practitioners influenced chronic care of CVD. For example, a physician stated:\u2018The secondary prevention (of CVD) in whom we advise to continue medicines lifelong, but good number of them (patients) tend to stop their medicines three to six months from the time of the index event thinking that they are normal, especially (those patients) on the governmental schemes get procedures done, tend not to continue medicines.\u2019high cost of treatment and low knowledge/awareness about disease were major barriers. On the other hand, people who are highly educated were thought to be susceptible to unreliable information from online sources according to physicians. The care providers expressed concern that the misinformation epidemic circulating on WhatsApp and other social media platforms mislead patients with chronic conditions. For example, one physician stated: \u2018There is lot of misinformation campaign goes on WhatsApp \u2026 Because of that, lot of mistrust has arisen in patients, and they (patient) just keep on changing doctors, so that misinformation epidemic has to be controlled\u2026that takes lot of time.\u2019 Difficulties in maintaining lifestyle changes, and misconceptions around dietary and exercise habits also emerged as barriers from patient perspectives.Affordability of CVD treatment emerged as a greatest challenge from patients\u2019 perspectives. Particularly for those belonging to the lower income groups, including among individuals with low educational attainment, The C-QIP strategy combines clinical decision supported electronic health records, task-sharing via trained NPHW, and patient-facing text messages with healthy lifestyle advice to improve CVD care quality. Physicians thought that implementing the C-QIP strategy would standardize treatment protocols and reduce medication errors as they will receive alerts or clinical decision support prompts for timely treatment modification. This was best summarized by a cardiologist, \u2018So, given the busy clinic we (physician) miss out on certain essential prescription drugs which need to be there for heart failure patients for example. This DSS prompt can be an alert for the physician if something is missed in the prescription.\u2019it (C-QIP) is worthwhile, it is a beautiful activity. I strongly recommend that. 90% of your problems and recurrence will be stopped if you are able to modify the lifestyle, motivate them (patients) to modify their lifestyle.\u2019EHR-DSS may offer additional value as it will endorse prescription of generic drugs, which can reduce out-of-pocket medical expense, and electronically stored patient data can be used for research purpose  to inform clinical decision making. Further, physicians supported the use of NPHWs to facilitate care for patients and assist in collecting/recording patient data into the EHR, thus allowing more consultation time with patients as described by a physician health administrator, \u2018The disconnect between EHR-DSS developers and end users  emerged as a major deterrent to the use of EHR-DSS by the providers. One physician stated: \u2018Majority of them [EHR-DSS] are designed by non-medicals and they are designed in the IT lead offices who have really not visited the doctors. Their [software developers] advisors are not the real-time doctors, and they have not sat through in the clinic.\u2019 Further, few physicians had concerns about the fidelity of physicians\u2019 use of the EHR-DSS given the varied CVD manifestations, and prognostic factors. Assistance from NPHWs for patient intake and entry into EHR was deemed critical for the success of C-QIP strategy as stated by a physician: \u2018Although some hospitals have introduced all this [EHR-DSS] in the outstation, I believe these physicians just do not use the computer, because to just enter all this data, it takes so much time, I am not going to do it.\u2019Text message-based reminders were thought to be useful and acceptable to both providers and patients as it may increase patients\u2019 retention in care through follow-up visits as well as improve adherence to prescribed therapy. This was best supported by a nurse: \u2018[E]ven a person in village today has a mobile, he [patient] has a WhatsApp, who is capable of reading anything in it.\u2019 And, a patient described that, \u2018If I get morning message that you will have to take care of your health, I will feel very good.\u2019 To improve the usability and acceptability of text messages, key informants suggested to make it available in the local language.All four study sites selected for the feasibility trial lack an EHR-DSS system at baseline, use NPHWs in a minimal capacity, and do not use text messages to support patients\u2019 self-care and management. Physicians had concerns about obtaining legal permission, patient confidentiality, and data privacy issue with the introduction of EHR-DSS as described by a cardiologist: \u2018it is very difficult to manage and convince the \u201cso called\u201d CEO  of this system with several stakeholders that is the biggest challenge (to) implementing it.\u2019However, most key informants described that the hospital leadership strongly supports clinic change efforts to improve quality and will provide necessary support in terms of financial resources, training, staffing, equipment, and materials to cater to patients\u2019 needs and improve quality of care.Almost all providers and administrators believed the C-QIP strategy should address patient barriers  and provide patient choices about various services in response to patient needs as a part of the C-QIP strategy. Stakeholders emphasized that to increase the acceptance of NPHW facilitated care, a cultural shift and sensitization along with policy-level changes related to NPHW scope of practice regulations are needed. Physicians reported understanding the needs of their patients. as expressed by a private clinic physician: \u2018typically, even today patients and relatives do expect the medical doctors to do this job (counselling) for them.\u2019 Another private practitioner reflecting on known barriers to patient care stated, \u2018There could be certain barriers because the patient and his family may not like it (NPHW delivered care). But maybe, we can make it change  and that should be the way.\u2019 If the C-QIP strategy is found to be effective, it can complement the existing infrastructure and activities emanating from the national program for prevention and management of CVD in India.Providers had mixed views about the EHR-DSS adoption and effectiveness but strongly believed in the involvement of NPHWs to provide patient counselling/education and text messages for chronic care of CVD. A few senior consultants also opposed the use of EHR-DSS as quoted by a cardiologist, \u2018Decision support systems are something for people who don\u2019t have their own algorithms in their own mind. For most of the people who have grey hair, they will actually reject them.\u2019Technology-averse attitudes of some physicians, slow typing speed, and less familiarity with computer interface were quoted as barriers to the wider acceptability and adoption of EHR-DSS as concerned by a physician: \u2018They (physicians) would be so resistant to use computers [EHR-DSS] because it takes time for learning.\u2019 Health administrators expressed that dedicated human resources  and information technology and administrative support would be necessary to effectively implement the C-QIP strategy.This formative, qualitative study is part of a multi-step exploration and preparation process that included a scoping review, multi-stakeholder interviews, and an expert consultation meeting to inform the development and implementation of the C-QIP strategy. The C-QIP strategy was perceived as a complex intervention because it involves multi-level implementation strategies, implementation actors, and integration of different disciplines. The complexity of the intervention drives the need for a clear plan to engage all implementation actors, educate them about the intervention, and encourage the adoption of the C-QIP strategy, as captured by a cardiologist:\u2018(In) tertiary care system where there is a lot of disbelief for newer strategies while some people are very forthcoming to the top technologies, there are some who are absolutely against it because that (technology) will take away the human angle from the  care system\u2026\u2019To reduce the complexity of the C-QIP strategy, key informants suggested to provide standard operating procedures and have uniformity in data collection, entry, and execution of DSS plan. The coupling of policy change and the use of NPHWs as intervention champions was perceived as one way to help create an environment that facilitates implementation of the C-QIP strategy. As quoted by a physician administrator:\u2018I sincerely hope that because we have introduced DSS in the National program  so I hope it takes up, but I think there is a lot of push back from physicians to such systems\u2026we need physician assistant to help with that work (manage EHR-DSS) that will help bring these things in a more structured way and address CVD burden more efficiently.\u2019Physicians, health administrators and patients were willing to change and adopt the C-QIP strategy as they believed it will mitigate several structural and systemic barriers to CVD care, such as lack of patient counsellors, poor referral linkages, lack of monitoring systems, and low health literacy among patients.This multi-stakeholder qualitative analysis revealed several barriers to CVD management such as the lack of QI strategies and policies to promote retention, continuity of care, and minimize costs of outpatient care. Further, the most salient modifiable barriers to chronic care of CVD were structural  and educational , which are consistent with previous studies 26. PhysiPrevious research illustrates the critical role of planning and organizing to identify and mitigate potential pitfalls that may hinder QI strategies before implementation 28. We foC-QIP strategy represents an important extension to the existing care models by involving trained NPHWs who can play a larger role than what is currently allowed within their scope of practice. NPHW could obtain recommendations from specialists and convey treatment plans to patients, though the potential for miscommunication exists with additional actors. Whether they provide education to patients for self-care management or more prescriptive activities, NPHWs could serve as the fulcrum of care by ensuring continuity of care for patients through coordination between providers and patients using EHR-DSS and text messages. Patients perceived that C-QIP strategy will empower them for better self-care, improve their knowledge about disease and prevention, and increase their trust in the care providers. However, patients were concerned about the financial viability of such a QI strategy since cost of treatment is a priority (major barrier) than maintaining continuity of care.In this study, hospital administrators believed that the C-QIP strategy can standardize care delivery and enhance patient self-care management. The support of hospital-level leadership and culture plays a critical role in the implementation of QI strategy as also learned in our previous experience of conducting process evaluation of the ACS QUIK trial, which suggest that implementation and acceptability of a QI toolkit were enhanced by hospital-level management support that leveraged available resources to implement the toolkit . Major i3132Through the analysis and interpretation of in-depth interviews with multiple stakeholders, we have provided insights into the current practices, challenges, and opportunities around CVD care in a low resource setting such as India. Further, our results shed light on how the interaction of different CFIR domains  could guide the successful implementation of C-QIP strategy. In particular, the findings of this study described the barriers and facilitators to implementing the C-QIP strategy. A unique strength of this study is that it does not focus on providers and health administrators\u2019 perspective but also include experiences of patients and care givers.This study has few limitations. First, we focused on a breadth of views from diverse health system stakeholders across diverse settings as opposed to studying a single clinical setting. Because providers and patients in these setting face different challenges, the diluting of context within or across clinical settings may cause us to overlook certain facilitators or barriers to a program that might be seen when focusing explicitly on a smaller number of clinical settings. Second, patients, caregivers, and health administrators were selected from the four participating sites in the C-QIP feasibility trial, which might introduce the selection and social desirability biases. However, we utilized a purposive sampling frame to recruit key informants to achieve diverse representation and glean different perspectives on enablers and facilitators of the C-QIP strategy in India. Third, our findings are based on in-depth interviews with little observation using ethnographic methods, which could have provided insights into the actual clinic flow, dynamic interactions between clinicians, nurses, pharmacists, and patients, and process of care measures, so this study is largely based on the self-reported participants\u2019 views and experiences.Our study identified enablers and barriers to implement C-QIP strategy in low resource settings in India. The modifiable barriers were low health literacy of patients, high patient volume, too few specialists, physician burnout and time-constraints, and lack of robust communication, and referral systems. Team-based care involving NPHW, patient education, and EHR-DSS emerged as potentially useful strategies to improve quality of care among patients with CVD. C-QIP strategy has the potential to improve process of care measures, clinical outcomes, and patient experiential quality.Corresponding author has access to all study data. Data will be made available to the external researchers upon request.The additional file for this article can be found as follows:10.5334/gh.1161.s1Supplementary file.COREQ  Checklist."}
+{"text": "Algorithms provide recommendations to human decision makers across a variety of task domains. For many problems, humans will rely on algorithmic advice to make their choices and at times will even show complacency. In other cases, humans are mistrustful of algorithmic advice, or will hold algorithms to higher standards of performance. Given the increasing use of algorithms to support creative work such as text generation and brainstorming, it is important to understand how humans will respond to algorithms in those scenarios\u2014will they show appreciation or aversion? This study tests the effects of algorithmic advice for a word association task, the remote associates test (RAT). The RAT task is an established instrument for testing critical and creative thinking with respect to multiple word association. We conducted a preregistered online experiment  to investigate whether humans had stronger reactions to algorithmic or crowd advice when completing multiple instances of the RAT. We used an experimental format in which subjects see a question, answer the question, then receive advice and answer the question a second time. Advice was provided in multiple formats, with advice varying in quality and questions varying in difficulty. We found that individuals receiving algorithmic advice changed their responses 13 A common feature of these scenarios is the objective nature of the outcomes; in every case, the algorithm is providing advice with the intent of helping the decision maker(s) reach an optimal decision. While the algorithms may be incorrect, or even biased, they are tuned to provide a \u201ccorrect\u201d answer to an underlying optimization problem.With rapid advances in artificial intelligence (AI) and computing power, people are increasingly receiving real-time input from algorithms\u2014which we will refer to as algorithmic advice\u2014in place of or in addition to information from other humans. Current algorithms provide recommendations for well-defined problems with readily available data to a broad array of stakeholders. For example, individuals regularly use platforms such as Spotify, Netflix, or Match.com to find music, movies, or romantic partners. Algorithms can also be used to predict the future price of a stock6 to engage in dynamic problem solving with significant ambiguity.However, this paradigm does not necessarily apply when the task involves creative thinking. It is not clear how algorithmic advice will be received when the objective of a task is to produce and evaluate novel ideas. Here we ask the question, can algorithms help humans to be more creative? More precisely, are humans receptive to algorithmic advice when completing tasks such as brainstorming or word association that require creative thinking? Answering this fundamental question is critical with respect to our society\u2019s increased development and reliance on intelligent technologies. Future businesses, groups, and consumers will be faced with receiving information or advice from intelligent systems or even intelligent teammates8. Divergent thinking is more often associated with creative enterprise as it involves the generation of novel ideas and expansion of the problem space. However, convergent thinking\u2014the evaluation of novel ideas\u2014is also an important component of creative thought. Without convergent thinking, the limitless ideas generated through divergent thinking cannot be assessed for their feasibility or relevance to the problem at hand9. In this study we focus on how algorithms can aid creativity by supporting convergent thinking.There are often multiple ways to solve complex problems, and a degree of creative thinking is required even when there is an optimal solution. To find a satisfactory solution, people engage in a sequence of idea creation and idea refinement, also referred to as divergent and convergent thinking14. People tend to reject algorithmic advice in scenarios that are considered more subjective or sensitive, when they have unreasonable expectations of the algorithm\u2019s ability, or when they lack autonomy over their decisions10. Given the subjective nature of creative tasks such as brainstorming, humans could be predisposed to reject suggestions from an algorithm. On the other hand, there have been well-documented recent examples of machine learning algorithms identifying novel innovations based on analyses of text data15. Further, algorithms are increasingly used to generate text, ranging from simple applications such as auto-complete to more complex programs like GPT-316, BERT17, and Transformers18. Considering the growing prevalence of \u201ccreative\u201d algorithms coupled with their relative efficiency, it is also possible that humans will be biased to accept the advice of algorithms.The extant literature provides mixed answers regarding algorithms and creativity. On one hand, the phenomenon of algorithmic aversion is well documented20, even to the point of potential overconfidence21. Further, decision makers tend to differentiate between good and bad advice, favoring advice sources that are perceived as high quality24. While it is not clear if advice from an algorithm functions differently, prior work has demonstrated a consistent automation bias among humans using intelligent systems, leading to significant complacency28. As such, we anticipate a consistent effect, with humans feeling more confident in decisions supported by algorithmic advice. However, it is unclear how humans will react to low-quality advice, given the tendency to punish algorithms for making mistakes13.Prior literature has demonstrated that receiving advice can significantly boost confidence for a decision maker29. The RAT task is an established instrument for determining convergent and creative thinking with respect to multiple word association32. We specifically tested three research questions. First, to what extent will people incorporate algorithmic advice\u2014compared to advice from other humans\u2014when solving remote association test problems? Second, will advice from algorithms or other humans lead to greater accuracy when solving RAT problems? Finally, do people who receive algorithmic advice express more confidence in their decisions, compared to those who receive advice from other people?In this paper we advance our knowledge of this problem. We conducted a preregistered online experiment  had different effects on behavior and confidence. Participants answer all six questions within one advice condition before moving to the next type. Advice is either of high or low quality (randomized), with half of the questions within each type being high or low quality. RAT questions are either medium, hard, or very hard, and the difficulty is distributed across all conditions in random order. We measure the rate at which participants change their answers after receiving advice, the rate at which they identify the correct response, and the change in their solution confidence. We summarize our experimental format in Fig. All subjects answered 18 Remote Associates Test (RAT) questions. We used the Judge Advisor System task formatOur primary treatment concerned the source of advice, as perceived by participants. There were two conditions. In one condition, participants were told that they were receiving advice from an algorithm trained on similar problems (treatment group). In a second condition, participants were told that they were receiving the consensus guess of other humans (control group). We verified the salience of the condition using manipulation checks. The source of advice was a between-subjects treatment, while all other features of the experiment such as difficulty, advice format, and advice quality were within-subjects factors. See the methods section for additional information on the procedure.Our first objective was to differentiate the effect of algorithmic advice from social advice across various conditions. We determined that participants relied upon advice if they changed their answer from their initial guess. Overall, we find that algorithmic advice prompted changes in initial responses more frequently than social advice, with entclass1pt{minimaWe next dug deeper into the data and calculated these rates while accounting for advice formats, question difficulty, advice quality, whether or not participants correctly responded on their first guess, their level of confidence in their response, and the time it took them to produce an answer. We applied multilevel modeling to account for the nested nature of the data . Because the response variable is binary , we used a mixed-effects logistic regression model with errors clustered on the individuals. The treatment variable is a binary variable indicating whether participants were in the algorithmic advice condition or not.We found that the coefficient for the treatment variable was positive and significant in the main model with all controls  with errors clustered on the individual participants, and the advice source condition the binary treatment variable. We found that individuals who received advice from an algorithm, rather than other people, were significantly less likely to submit a final correct solution  and , which were not significantly different.To test whether the advice source and quality of advice had significant effects on final confidence, we conducted an analysis of covariance (ANCOVA). ANCOVA is an appropriate method given the presence of a continuous covariate  that has a linear relationship with the outcome variable. We find that advice source they believe they are receiving input from an algorithm. We specifically investigate the role of algorithmic advice on a word association task that measures certain elements of creativity, the remote associates test. The RAT task is an established method for assessing both creative and analytic thinking32, and to our knowledge this is the first study to test the effect of algorithmic advice on this type of task. Our experiment reveals that humans behave in two key ways, even after controlling for how advice is given, how difficult the task is, and whether or not the advisor provides accurate advice. First, individuals who received algorithmic advice revised their answers significantly more often than those who received social advice. Second, individuals who received algorithmic advice were significantly less likely to correctly solve a RAT question. This pattern holds even when isolating individuals who started with an incorrect response and changed their answer when receiving advice. In other words, when the advice provided was beneficial, people still performed better when receiving the information from a purported human source. In sum, humans are more likely to change their responses when receiving advice from an algorithm, even though on average they are less successful.In this study, we find evidence that advice coming from an algorithmic source\u2014rather than other people\u2014significantly influences how individuals solve analytic problems. Our approach differs from many modern use cases of algorithmic input\u2014such as movie recommendations on Netflix\u2014in that we explicitly inform participants of the advice source. By doing so, we attempt to highlight how humans behave differently if 12, they also feel generally more confident when their advice is algorithmic, regardless of its quality. A potential rationale for this finding is that humans are willing to overlook mistakes by AI if they are able to modify the solutions13\u2014in this case, use their own answers to the RAT task.We also examined the effect of advice source on the change in reported confidence. Individuals receiving algorithmic advice reported significantly higher confidence after their second guess than individuals in the social condition. They also reported greater confidence when the advice provided was of high quality\u2014i.e., a verifiable correct answer. This difference is particularly salient when the algorithm provides high quality advice; here participants reported the highest confidence levels. However, when an algorithm provides bad advice\u2014an incorrect answer\u2014individuals in the study reported confidence levels on par with correct human advice. This finding suggests that while humans do penalize an algorithm for erring, as shown in prior work35. On one hand, humans are responsive to algorithmic advice. On the other hand, algorithmic advice may not improve human performance. Coupled with the boost in confidence experienced by individuals who received algorithmic advice, we posit that our task elicited a form of automation bias28. Namely, humans rely upon a machine\u2014or a purported algorithm in our case\u2014to perform repeated tasks and over time become complacent. As a result, they may not catch cases where the machine errs, or place undue faith in the machine\u2019s capabilities. Given the increasing prevalence of human-machine collaboration, we argue that designers of these hybrid systems should be cognizant of the potential for this type of automation bias. Further, the results suggest that simply placing a \u201chuman-in-the-loop\u201d may not be sufficient to counter errors produced by algorithms, particularly when the system has a history of high performance.These findings reveal a conundrum in designing cooperative human-machine hybrid systems36, or when making quantitative judgements under uncertainty38. Conversely, aversion is typically strongest in subjective or open-ended tasks39. Even in quantitative tasks, appreciation is not guaranteed13. In fact, the notion of a \u201cspectrum\u201d of objectivity has been noted in a review of human willingness to accept algorithmic advice11. Consequently, we anticipated that a task such as the RAT would arouse some degree of aversion. That we found a consistent appreciation effect suggests to us perhaps word association tasks, while more subjective than tasks such as counting or forecasting, are still relatively analytical in how humans solve them41. Indeed, there is some debate regarding the relationship between the RAT task and creativity. Recent scholarship has suggested that the RAT is a better measure of convergent thinking, i.e., confirming the validity of an answer, rather than divergent thinking which involves generating new possibilities41. However, the RAT task still requires individuals to make free associations between words, and people who are better at the RAT task are less likely to fixate on common words42. Thus, while the RAT test is likely a more analytical task than other creative tasks, it still captures an important part of the creative process40. It is also relevant to some ways in which algorithms are used in practice, such as smart assistants and other text-based automation tools deployed to improve worker productivity. Thus, while our results may not predict algorithmic appreciation (or aversion) in all creative tasks, they do suggest that humans are willing to accept text-based advice from algorithms in certain circumstances, albeit with the risk of automation bias and complacency. Future work is needed to explore how humans react to algorithms for tasks requiring more significant divergent thinking, such as the alternative uses task43.The results also provide further evidence for algorithmic appreciation, rather than aversion, in tasks with measurable outcomes. Individuals have shown a tendency to favor algorithms when solving problems involving logic46 and the role of trust in fostering effective interactions. One particular dimension of trust\u2014performance-based\u2014refers to the confidence that a human develops towards an autonomous agent on account of its demonstrated ability to perform a task successfully. This is akin to what is referred to in psychology as cognitive trust47. Various aspects about the machine\u2019s performance, such as its predictability49, transparency50, and, reliability52 affect the extent to which humans will trust that machine. Other work has suggested that trust is task-dependent; when solving problems involving logic or quantitative judgements, participants trusted the same solution more when it came from an expert system  relative to receiving the solution from a human38. In our experiment, we found that humans seemed inclined to trust the autonomous agent, i.e., they relied on the algorithm for advice, despite having no information regarding its predictability or performance. This finding has important implications for future human-autonomy interactions. For example, when humans are faced with logic-based tasks (not necessarily quantitative), we anticipate that they will be more likely to trust in decision support received from an algorithmic rather than a human advisor. This potential reliance may be problematic since it is disconnected from performance appraisals. In our experiments, people leveraged algorithmic advice even when that advice was incorrect. Depending on the decision-making context, this preference for relying on algorithmic advice in the face of uncertainty may not be in service of optimal performance. Accordingly, individuals or organizations designing human-autonomy hybrid systems should be aware of these inherent biases for a broad range of tasks, including certain types of creative tasks.Finally, we consider the implications for human-autonomy trust. Prior work has explored the antecedents of trust in autonomous agentsThis study was approved by the University of Georgia Institutional Review Board, project 00001012. Subjects gave written informed consent both before and after participation in the study. All methods were carried out in accordance with relevant guidelines and regulations.Participants were recruited from Amazon Mechanical Turk (AMT). All subjects had a greater than 98% lifetime approval rate for prior tasks conducted on AMT, had completed at least 500 tasks on AMT, were located in the United States, and could only participate in the task once. We started with 182 respondents recruited from AMT. Of those, 14 failed the manipulation check, and an additional 14 provided incomplete responses or did not enter a valid MTurk identification value. The analysis is based on the 154 remaining subjects, compared with the preregistered plan of at least 148 subjects. Each subject was paid USD 5.00 to complete the experiment, and an additional bonus of USD 1.00 was given to subjects who correctly answered a randomly selected question in each of the conditions, allowing for up to USD 3.00 of bonuses per subject based on their performance in the RAT questions. Subjects were told the study would take approximately half an hour to complete.Base, Room, and Bowling, the answer would be BALL .All subjects saw 18 RAT questions. In a RAT question, there are three words that are tied together by a fourth word, which can come before or after each of the three words (see Supplementary Information for the list of RAT questions and solutions). For example, participants were shown 53. For each question, the correct answer was the answer offered by the remote associates test. The wrong answer was created by the authors. Each wrong answer fits two of the three given words. Difficulty was predetermined based on the average time it took individuals to solve the RAT question historically. The questions used in this study ranged in difficulty from medium to very hard. For a list of the questions and answers, as well as details on generating wrong answers, please see the Supplementary Information.The fourth word can come before or after each of the three words, and can either form a compound word  or a common two-word expression . We selected remote associates test questions from a well-known repository of remote associates test questions that also includes how difficult each question isAfter each question, participants were asked to rate their confidence in their answer. Then, participants were shown some form of advice, after which they were able to change their answer. They then rate their confidence a second time.Subjects were randomly assigned to the social or algorithmic condition, which was a between-subjects condition throughout the experiment\u2014subjects only received advice from one source throughout all 18 questions. In the instructions, participants in the social condition saw an advice source that read: \u201cthe consensus of other people who have completed this task.\u201d Participants in the algorithmic condition saw an advice source that read: \u201cthe output of an algorithm that has been trained to solve similar word association tasks.\u201d The specific delivery format of the advice was varied across the questions. As a manipulation check, at the end of the survey, we asked subjects \u201cWhat was the source of advice you received in this survey?\u201d with three answer choices: \u201cother people\u201d, \u201can algorithm\u201d, and \u201cI don\u2019t remember.\u201d Fourteen subjects chose either the \u201cI don\u2019t remember\u201d option or the option that did not correspond to the type of advice they received. We excluded those subjects from our analysis.After making an initial guess, participants were then provided with advice, or the opportunity to receive advice. Each subject saw three types of questions: Probability, Standard RAT, and Budget. Subjects saw all questions of one type, then all questions of another type, with no mixing of the types. For example, a subject would see all six Probability questions, then all six Standard RAT questions, then all six Budget questions. Within each advice type, two questions were medium, two were hard, and two were very hard. Before each group of questions, we would provide additional instructions on how the advice would be delivered.For the standard RAT group of questions, subjects always received advice after answering a question. This case follows the typical judge advisor system format. For the Probability group of questions, subjects were asked a RAT question and gave an answer. Then, subjects were shown a probability that their answer was correct; this value was influenced by their first response. For the Budget group of questions, we offered subjects the chance to buy advice for ten cents. This value was significantly lower than the potential bonuses for correct answers, and was thus meant to encourage people to try and increase their likelihood of a higher payout. If they purchased advice, it was then provided in the same way as the standard RAT case. In all cases, the prompt was modified by the algorithmic condition.Within a block of six questions, each subject saw high-quality advice three times and low-quality advice three times. High- and low-quality advice was evenly distributed across advice type and question difficulty, in random order. For high quality advice, participants were shown the correct answer. For low quality advice, the clue fit two of the three words, but not all three. For example, our wrong answer advice for a remote associates test question for the words \u201cCross\u201d, \u201cRain\u201d and \u201cTie\u201d was \u201cHair\u201d because it fit \u201cCross\u201d (Cross hair) and \u201cTie\u201d (Hair tie) but not \u201cRain.\u201d The correct answer is \u201cBow.\u201d We used a third-party dictionary-based website to validate that our wrong answers fit exactly two of the three words.Supplementary Information."}
+{"text": "MdHHO3  is upregulated under N (nitrogen) supply. The results of DNA-binding site analysis and electrophoretic mobility shift assays (EMSA) showed that MdHHO3 binds to the motif-containing GAATC. Furthermore, MdHHO3 binds to its promoter sequence and inhibits its activity. In addition, the overexpression of MdHHO3 in apple calli resulted in less accumulation of nitrate in 35S:MdHHO3-GFP calli and downregulated the expression of the nitrate transport-related genes but upregulated the expression of the nitrate assimilation-related genes. Similarly, the expression of the nitrate transport-related genes was downregulated and the expression of the nitrate assimilation-related genes was upregulated in MdHHO3 overexpression Arabidopsis and tobacco plants. Interaction experiments showed that MdHHO3 could bind to the promoter MdNRT2.1 (NITRATE TRANSPORTER 2.1) and negatively regulate its expression. Moreover, the exposure of MdHHO3-overexpressing Arabidopsis and tobacco to nitrate deficiency resulted in an early senescence phenotype as compared to the WT plants. These results show that MdHHO3 can not only negatively regulate nitrate accumulation in response to nitrate but also promote early leaf senescence under nitrate deficiency. This information may be useful to further reveal the mechanism of the nitrate response and demonstrates that nitrate deficiency induces leaf senescence in apples.The regulation of plant gene expression by nitrate is a complex regulatory process. Here, we identified 90 GARP family genes in apples by genome-wide analysis. As a member of the GARP gene family, the expression of  The growth and reproduction of plants are closely related to changes in the environment. Therefore, plants can only grow and reproduce normally once they adapt to changes in the external environment. The soil nutrient status is one of the key environmental factors affecting plant growth and reproduction . In natuNRT2.1, NRT2.2, NRT2.4, and NRT2.5, are involved in the absorption of nitrate at a low concentration, and among these, only NRT2.1 showed a significant correlation between transcript abundance and high-affinity nitrate influx after nitrate supply to nitrate-deficient plants (Arabidopsis high-affinity transport system (HATs) was decreased by 63\u201375% when NRT2.1 was mutated  has been demonstrated to be involved in the nitrate response by encoding a high-affinity transporter gene  play important roles in high-affinity nitrate transport uptake . In the t plants . In addi mutated . Howeverin yield . The rolter gene . Furtherresponse . In addis NRT2.1 , which sNLA (NITROGEN LIMITATION ADAPTATION) plays a key role in the process of leaf senescence induced by N deficiency  with the same growth parameters were planted on foam plates with 20 holes in each plate. Single Malus \u00d7 domestica \u201cGL3\u201d plantlet was planted in each well and cultured with 1/2 Hoagland solution  were grown at 24\u00b0C in the dark and subcultured every 15 days. The Arabidopsis thaliana used in the study was Col-0, and the tobacco was Nicotiana benthamiana; both of these plants were grown at 25\u00b0C/23\u00b0C under long-day conditions (14 h of light:10 h of dark).The tissue culture plantlets of solution  nutrientArabidopsis GARP-type protein sequences were used as a reference to construct the phylogenetic tree of apple GARP-type protein. The GARP-type gene family sequences from apple and Arabidopsis were downloaded from the Plant TF Database v. 4.01 . First-strand cDNA was obtained using a cDNA reverse transcription kit . The primers used for qRT-PCR were designed with NCBI and synthesized by the Sangon Biotech Technology . The primers used are listed in \u2013\u0394\u0394Ct) method. The primers used for qRT-PCR are shown in Total RNA was extracted from apple, calli, The chlorophyll content was determined as described above . In shor2 pieces, placed on MS solid medium, and cultured in the dark at 28\u00b0C for 24 h. The primers used for subcellular localization are shown in MdHHO3 without a stop codon was cloned and inserted into the pZP211 vector. The plasmids harboring 35S:MdHHO3-GFP and 35S:GFP were transformed into the Agrobacterium tumefaciens strain LBA4404 and infected onion epidermal cells. GFP fluorescence was observed under a Zeiss LSM880 microscope  and analyzed using the ZEN lite software.5 The subcellular localization analysis was repeated three times, and the results were similar.The outer skin of the onion was removed, and the inner skin of the onion was peeled with a scalpel on a super clean workbench, cut into small 1-cmMdHHO3 was cloned into the pRI101-AN vector containing the GFP tag sequence to form an overexpression plasmid. The primer is shown in A. tumefaciens GV3101 and LBA4404, and the recombinant vector was then transformed into calli, Arabidopsis, and tobacco using A. tumefaciens cells. Transgenic apple calli, Arabidopsis, and tobacco were obtained as described previously  under long-day conditions (14 h of light:10 h of dark) at 24\u00b0C/22\u00b0C for 4 days. To induce leaf senescence in tobacco seedlings in response to nitrate deficiency, 4-week-old tobacco seedlings were grown in either nitrate-sufficient or a nitrate-free nutrient solution prepared with modified Hoagland\u2019s solution (pH 5.8) for 3 weeks. After observing and recording the phenotype, the leaves of these plants were used to determine the chlorophyll content and the expression of senescence-related genes.MdNRT2.1 promoter fragment (sequence of the fragment containing cis-acting elements) was inserted into the pAbAi vector to construct the reporter gene pBait-AbAi vector, and the full-length CDS of MdHHO3 was cloned into the pGADT7 vector. The primers used in yeast one-hybrid assays are shown in The MdHHO3 was cloned into the overexpression vector pGEX-4T-1 to obtain a recombinant plasmid. The MdHHO3 recombinant plasmid was expressed in Escherichia coli strain BL21  and purified using Glutathione-Sepharose Resin . The synthesis and labeling of the probe were performed by Sangon Biotech Co., Ltd. . The probes used for EMSA are shown in The full-length CDS of MdNRT2.1 gene (including the cis-acting site sequence) was cloned into the pGreenII 0800-LUC vector to generate a reporter vector. The full-length CDS of MdHHO3 was inserted into the pGreenII 62-SK vector to construct the effector vector. The recombinant plasmid was transformed into the Agrobacterium strain GV3101. Tobacco leaves were injected with the Agrobacterium strain, cultivated in the dark for 12 h, and then placed in a light incubator. After 48 h, the injected tobacco leaves were collected and sprayed with D-Luciferin sodium salt (Sangon Biotech), and the fluorescence was then observed using an in vivo imaging system .The promoter fragment of the t-tests .GraphPad Prism 6 software  was used for the drawing, and SPSS 19.0 software  was used for the statistical analyses. The significance of the differences was tested by Arabidopsis GARP-type protein sequences as a reference (Arabidopsis suggests that apple MdHHO3 (MdGARP40) is the closest homolog of Arabidopsis AtHHO3/AtNIGT1.1 (AtGARP17) and AtHHO2/AtNIGT1.2 (AtGARP20).To study the evolutionary relationship between the MdHHOs and AtHHOs genes, we constructed a rootless phylogenetic tree of apple GARP-type proteins using the eference . A totalPhylogenetic tree analyses showed that MdHHO3 was most closely related to PbEFM . In addiMdHHO3 expression induced by nitrate supply, we supplied nitrate to apple seedlings treated with N starvation and performed the quantitative real-time polymerase chain reaction (qRT-PCR) analysis using RNA obtained from roots collected at different times. During nitrate treatment, the expression of MdHHO3 was induced within 1 h, reached a peak approximately 6 h later, and then decreased, as shown in MdHHO3 expression specifically responds to nitrate, we supplied ammonium, phosphorus, glutamate, and trans-zeatin and found that none of these compounds could induce the expression of MdHHO3  and these sites were named P1, P2, P3, P4, and P5 based on the distances between them  and the WT control  was downregulated, whereas that of the nitrate assimilation-related genes (MdNR and MdNIR) were upregulated. In addition, the expression of five genes  that respond to nitrate was found to be upregulated (NRT1.1) and high-affinity transporter (NRT2.1) was downregulated in the MdHHO3 transgenic calli. These results indicate that MdHHO3 may inhibit the absorption of nitrate by inhibiting the expression of high-affinity transporters.The expression of nitrate transporter genes is usually used as a marker for nitrate uptake. To explore how egulated . InteresMdHHO3 in nitrate transport and accumulation, the MdHHO3 overexpression construct was generated and transformed individually into Arabidopsis and tobacco , and these sequences were named P1, P2, P3, P4, and P5 based on the distances between them   . Corresp control . During  , as well as the cytokinin synthesis enzyme genes  in the wild-type and MdHHO3 transgenic lines  were downregulated in the MdHHO3-overexpressing Arabidopsis plants. Under nitrate-free conditions, the expression of the high-affinity transporter genes was upregulated in both the wild-type control and MdHHO3-overexpressing Arabidopsis plants. However, the expression level of these three genes in wild-type control was significantly higher than that in the MdHHO3-overexpressing Arabidopsis. This suggests that MdHHO3 inhibits the expression of the high-affinity transporter genes more significantly under N-deficient conditions. In addition, we examined the expression levels of genes  related to cytokinin synthesis. Under N supply conditions, we did not find the effect of overexpression of MdHHO3 on the expression of the cytokinin synthesis-related genes. However, during N deficiency-induced leaf senescence, the expression levels of these three cytokinin synthesis-related genes decreased in both MdHHO3-overexpressing and wild-type control plants, but more significantly in MdHHO3-overexpressing plants. These results suggest that nitrate deficiency inhibits the expression of the cytokinin synthesis-related genes, and MdHHO3 may play an important role in cytokinin synthesis, but further studies are needed.Considering that bidopsis . We examic lines . The expMdHHO3 overexpression construct to further investigate the function of MdHHO3. As shown in MdHHO3 resulted in lower chlorophyll content, but no significant difference in the chlorophyll content was found in leaves 7\u20139 and 10\u201316  and At1g68670 (AtHHO2/AtNIGT1.2). Previous studies have shown that Arabidopsis AtHHO3/AtNIGT1.1 and AtHHO2/AtNIGT1.2 are induced by nitrate treatment . We speculate that over prolonged treatment, MdHHO3 may bind not only to the promoter of downstream genes but also to its own promoter motif, which results in autorepression. In addition, MdHHO3 was found to act as a potential negative regulator in the nitrate response by regulating its own transcription and the nitrate transporter genes  is involved in the response of plants to nitrate and can bind to the NRT2.1 promoter to enhance its expression  were significantly upregulated during N deficiency-induced leaf senescence (MdHHO3-overexpressing Arabidopsis and tobacco showed early senescence symptoms and a lower chlorophyll level than the WT controls, and a series of genes related to leaf senescence also showed higher expression levels in the overexpressing plants than in the WT controls. The results indicated that MdHHO3 may participate in nitrate deficiency-induced chlorophyll catabolism, but this hypothesis needs further research. In addition, another study found that N supply fails to induce early leaf senescence in Arabidopsis overexpressing MdHHO3 (Leaf senescence is a complex regulatory process that serves as a comprehensive response to endogenous development and external environmental signals . Therefonescence . Furtherg MdHHO3 . These rMdNRT2.1 and inhibits its expression. With prolonged nitrate supply, MdHHO3 binds to its own promoter and inhibits its expression to reduce the nitrate content. Under nitrate deficiency conditions, MdHHO3 promoted leaf senescence induced by N deficiency and the expression of senescence related genes. These results might help us better understand the regulatory mechanisms of nitrate transport and nitrate deficiency-induced leaf senescence.A working model was proposed based on the results of this study . Under nThe original contributions presented in the study are included in the article/LL, QT, and BW designed the study. WD, XG, XC, DL, and XF performed the experiments and analyzed the data. BW wrote and revised the manuscript. All authors contributed to the article and approved the submitted version."}
+{"text": "The [68Ga]Ga-FAPI-04 PET MRI/CT group has a comparatively higher sensitivity in detecting lymph node metastases , peritoneal involvement  in gastric cancer than [18F]-FDG PET MRI/CT group.Five studies, including 148 participants, were analyzed. [68Ga]Ga-FAPI-04 PET MRI/CT in gastric cancer. [68Ga]Ga-FAPI-04 PET MRI/CT was superior to [18F]-FDG PET MRI/CT in detecting the primary tumor, lymph node metastases, and peritoneal metastases. More studies are needed for the sensitivity and specificity of [68Ga]Ga-FAPI-04 PET MRI/CT in different pathological types of gastric cancer.This systematic review confirmed the advantage of [ Gastric cancer is a vital cancer burden globally, with the fifth-highest diagnostic rate and the third-highest mortality rate . Surgery18F]-FDG PET MRI/CT is based on the enrichment of glucose tracers and is related to metabolic activities [18F]-FDG PET MRI/CT in gastric cancer is affected by pathological tumor type and high FDG uptake in the gastric wall, and the detection sensitivity is not ideal [Traditional [tivities . Howeverot ideal .68Ga]Ga-FAPI-04 PET is highly expressed in various cancer types, including cancers with low [18F]-FDG affinity [68Ga]Ga-FAPI-04 PET in almost all normal tissues (including the brain and intestine) was low. Recent studies have shown that [68Ga]Ga-FAPI-04 PET can provide prognostic information, guide treatment choices, and help predict tumor invasiveness [68Ga]Ga-FAPI-04 PET MRI/CT in gastric cancer remains to be studied.Fibroblast activating protein (FAP) is a type II transmembrane protease with dipeptidyl peptidase and endopeptidase activities . It mainaffinity . Moreovesiveness , 8. Howe68Ga]Ga-FAPI-04 PET MRI/CT and [18F]-FDG PET MRI/CT in gastric cancer. We discussed the difference in the detection rate of the primary tumor, lymph node metastasis, and peritoneal cancer metastasis. This article aims to provide a more optimized choice for the screening, condition evaluation and treatment effect monitoring of gastric cancer, and further improve the survival benefit of patients.This article searched the comparative studies comparing Ga-FAPI-04 PET MRI/CT and [18F]-FDG PET MRI/CT, respectively. There was a significant difference in the sensitivity in detecting peritoneal involvement between [68Ga]Ga-FAPI-04 PET MRI/CT and [18F]-FDG PET MRI/CT.   vs. 84.43% (103/122)], lymph node metastases [81.97% (50/61) vs. 67.21% (41/61)] and peritoneal metastases [100.00% (38/38) vs. 44.74% (17/38)].This is the first systematic review and meta-analysis of all relevant comparative studies to compare the detection rates of [e trials \u201313. All 68Ga]Ga-FAPI-04 PET MRI/CT was significantly better than [18F]-FDG PET MRI/CT in detecting primary lesions of gastric cancer. Previous studies have shown that [18F]-FDG PET MRI/CT has limitations in diagnosing gastric cancer. Mukai et al. reported that the detection rate of [18F]-FDG PET in gastric cancer with tumor size less than 30\u00a0mm was 16.8%, and that in early gastric cancer was 25.9% [68Ga]Ga-FAPI-04 PET was higher than that of [18F]-FDG PET in tumors less than 4\u00a0cm (100% vs. 71%) [68Ga]Ga-FAPI-04 PET in signet ring cell carcinoma was significantly higher than that of [18F]-FDG PET Ga-FAPI-04 PET MRI/CT not only has higher diagnostic sensitivity, but also has higher tracer uptake and TBR than [18F]-FDG PET MRI/CT [68Ga]Ga-FAPI-04 PET in the stomach. Recent studies have found that the average SUV max of [68Ga]Ga-FAPI-04 PET in T2-4 tumors is significantly higher than that in T1 tumors (9.7\u2009\u00b1\u20094.4 vs. 3.1\u2009\u00b1\u20091.5), which provides a possibility for noninvasive judgment of the degree of invasion of gastric cancer [68Ga]Ga-FAPI-04 PET scan has potential for gastric cancer staging and can be use as complementary with [18F]-FDG PET scan.In gastric cancer studies, -FDG PET is less sensitive in detecting lymph node metastasis of gastrointestinal tumors [68Ga]Ga-FAPI-04 PET MRI/CT to lymph node metastasis was significantly higher than that of [18F]-FDG PET MRI/CT (81.97% vs. 67.21%). This may be related to the fact that lymph nodes are usually composed of fibroblast reticular cells, which are easier to be detected by fapi. To improve the detection rate of N2 or N3 lymph node metastasis by PET\u2013CT and highlight the specific areas of high metabolic lymph nodes can optimize the surgical decision and treatment plan [68Ga]Ga-FAPI-04 PET MRI/CT in detecting gastric cancer is more helpful in accurately guiding clinical treatment.Radical resection of gastric cancer requires complete resection of the primary tumor and removal of metastatic lymph nodes. Standard gastrectomy involves the removal of at least 2/3 of the stomach and the dissection of D2 lymph nodes  . Lymph nl tumors . Our stuent plan . Therefo18F]-FDG in peritoneal carcinoma is poor, and it is easy to underestimate the degree of peritoneal involvement [68Ga]Ga-FAPI-04 PET MRI/CT was highly sensitive to peritoneal metastasis of gastric cancer (100%). This may be due to the fibrotic reaction of tumor cells invading the peritoneum, and the target of fapi is fibroblast activating protein (FAP). Improving the detection rate of peritoneal carcinoma is helpful to more accurately judge the extent of disease involvement and evaluate the treatment response.Peritoneal carcinoma is common in gastrointestinal tumor metastasis. The extent of its involvement will determine the resectability and healing of the tumor, and further determine the prognosis. Due to the low level of FDG uptake in peritoneal carcinoma, the detection rate of [olvement . Our stu68Ga]Ga-FAPI-04 PET in distant metastasis of gastric cancer. Among the five comparative studies, two compared the detection rates of [68Ga]Ga-FAPI-04 PET and [18F]-FDG PET in patients with gastric cancer with ovarian, liver and bone metastases, and found no significant difference Ga-FAPI-04 PET and [18F]-FDG PET in gastric cancer. Our study has some limitations. First, there are limited articles to evaluate the metastasis of other organs of gastric cancer. We cannot comprehensively and systematically assess the diagnostic value of [68Ga]Ga-FAPI-04 PET MRI/CT and [18F]-FDG PET MRI/CT. Second, the detection methods are not unified. PET CT was used in three articles [68Ga]Ga-FAPI-04 PET MRI/CT and [18F]-FDG PET MRI/CT according to pathological classification. However, each coin has two side. [68Ga]Ga-FAPI-04 PET has some limitations. Because of its high physiological uptake in the uterus and ovary, the detection of uterus or ovary metastasis may not be ideal. However, due to the limited number of patients with uterine or ovarian metastasis in the included cases, further systematic assessment was not possible.To our knowledge, this is the first meta-analysis comparing Ga-FAPI-04 PET MRI/CT had a higher detection rate of primary gastric cancer, peritoneal metastasis and lymph node metastasis than [18F]-FDG PET MRI/CT. 68Ga-FAPI-04 PET provides a possibility for noninvasive determination. The above conclusions need to be confirmed in more extensive cohort studies. More studies are required to explore the role of [68Ga]Ga-FAPI-04 PET MRI/CT in the prognosis of gastric cancer and its sensitivity and specificity in different pathological types of gastric cancer.In conclusion, this systematic review and meta-analysis confirmed that ["}
+{"text": "The purpose of this review was to provide an overview of all the available evidence regarding the use of fibroblast activation protein inhibitor (FAPI) as a tracer for positron emission tomography (PET) in patients with lung cancer. In several studies and case reports, radioisotope-labelled FAPI PET/CT showed a strong diagnostic performance in lung cancer. In addition, the possible prognostic value of FAPI PET/CT in lung cancer is proposed.68Ga]Ga-FAPI PET/CT in lung cancer is presented. [68Ga]Ga-FAPI shows a high uptake  and tumor-to-background ratio (TBR) in primary lung cancer lesions, as well as in metastatic lesions of other tumor types located in the lung and in lung cancer metastases located throughout the body. Where a comparison was made to [18F]FDG PET/CT, [68Ga]Ga-FAPI showed a similar or higher SUVmax and TBR. In brain and bone metastases, [68Ga]Ga-FAPI PET/CT outperformed [18F]FDG PET/CT. In addition to this strong diagnostic performance, a possible prognostic value of [68Ga]Ga-FAPI PET/CT in lung cancer is proposed.Fibroblast activation protein (FAP) could be a promising target for tumor imaging and therapy, as it is expressed in >90% of epithelial cancers. A high level of FAP-expression might be associated with worse prognosis in several cancer types, including lung cancer. FAPI binds this protein and allows for labelling to Gallium-68, as well as several therapeutic radiopharmaceuticals. As FAP is only expressed at insignificant levels in adult normal tissue, FAPI provides a highly specific tumor-marker for many epithelial cancers. In this review, current information on the use of [ Lung cancer is the leading cause of cancer-related death in Europe, accounting for over twenty percent of all cancer-related deaths . HistoloSolid tumors such as these consist not only of malignant cells, but are situated in a Tumor Microenvironment (TME), which contains endogenous stromal cells , held together by the extracellular matrix (ECM) . This unFurthermore, several radiolabeled FAP inhibitors (FAPI) targeting FAP expression in CAFs and characterized by the rapid renal clearance and high tumor-to-background ratio (TBR) have been developed for PET imaging. The derivatives FAPI-02, FAPI-04 and FAPI-46 allow for labelling with the radionuclide Gallium-68 . The che18F]FDG PET performs well in the diagnosis and staging of lung cancer [18F]FDG uptake is variable among lung cancer histotypes and one of the most common organs for lung cancer metastases is the brain, which has high [18F]FDG uptake and, thus, little contrast [68Ga]Ga-FAPI as a tool for diagnosis and staging in lung cancer. In several studies, [68Ga]Ga-FAPI PET imaging has demonstrated an equal or better performance compared to [18F]FDG for the detection of tumors and metastatic lesions. It is also used as a complement to [18F]FDG to improve tumor-delineation for planned surgery or radiotherapy Ga-FAPI PET/CT scans in lung cancer. PubMed library was searched using combinations of the terms \u201cFAPI\u201d \u201clung\u201d \u201ccancer\u201d \u201cFDG\u201d \u201cmalignancy\u201d \u201concology\u201d and \u201cPET\u201d. Articles were selected based on titles and abstracts, and via the references of other relevant articles. All articles that were found describing the use of [68Ga]Ga-FAPI PET/CT in lung cancer are listed in this review.In this review, we aim to explicate the evidence and limitations on the use of Ga-FAPI-46 uptake in PET/CT in 141 patients with fourteen types of solid cancers (SUVmax: r = 0.781 (95%CI 0.376\u20130.936)) [18F]F-FAPI-04-uptake and immunohistochemically determined FAP-expression in six surgical specimens  [Mona et al. confirmed a positive correlation between FAP tissue expression measured using immunohistochemistry scores and [\u20130.936)) . In lung= 0.005) .68Ga]Ga-FAPI-04 PET/CT before treatment and concluded that [68Ga]Ga-FAPI-04 uptake was significantly higher in patients not responding to immune checkpoint blockage therapy Ga-FAPI PET/CT could provide insight in tumor characteristics because FAP-expression in adult normal tissue is low and generally undetectable. [68Ga]Ga-FAPI PET/CT can, therefore, be used in the diagnosis and staging of cancer [68Ga]Ga-FAPI PET/CT for diagnosis and staging of lung cancer is presented.Imaging of FAP-expression by FDG on PET/CT, while ten lesions were immunohistochemically positive for FAP [68Ga]Ga-FAPI PET/CT over [18F]FDG PET/CT. In the following part, currently available research on [68Ga]Ga-FAPI PET/CT in lung cancer is presented.Chen et al. demonstrated that FAP is widely expressed in lung cancer, especially in SCC (100%) and adenocarcinoma (85.7%). In twelve early adenocarcinoma cases, only three lesions were positive for [ for FAP . This in68Ga]Ga-FAPI was studied in multiple cancer types, including lung cancer. In the same center, a specific study targeting patients with fibrotic interstitial lung diseases and lung cancer was conducted. Giesel et al. compared [68Ga]Ga-FAPI PET/CT to [18F]FDG PET/CT in 71 patients with different cancer types, of which nine patients had lung cancer. They used several FAPI subtypes , but did not specify this per patient or cancer type. They showed very low [68Ga]Ga-FAPI-uptake in normal lung tissue, similar to [18F]FDG . The analysis of the uptake in primary tumors was not specified per cancer type, unfortunately. In eight patients with lung metastases, no significant difference between [68Ga]Ga-FAPI and [18F]FDG was found  [68Ga]Ga-FAPI PET/CT and [18F]FDG PET/CT.Only a few studies on FAPI PET/CT in lung cancer have been published. In two studies conducted at the University Hospital Heidelberg in Germany, [= 0.641) . Combine68Ga]Ga-FAPI-04 uptake in eighty patients with 28 different cancer types, and classified the SUVmax as low (SUVmax < 6), intermediate (SUVmax 6\u201312) and high (SUVmax > 12). Lung cancer (n = 25) showed the 6th highest [68Ga]Ga-FAPI-04 uptake and had a SUVmax >12. The average uptake in the background was SUVmax 1.6 for blood-pool and 1.4 for muscle, resulting in a TBR of >7.5 for lung cancer and other cancer types with high uptake. This was a combination of primary tumors and metastases of lung cancer, since patient numbers were not sufficient to analyze these separately [Their colleagues Kratochwil et al. evaluated [parately . Therefo68Ga]Ga-FAPI-46 in PET imaging of fibrotic interstitial lung disease and lung cancer, and showed that both of these conditions can be imaged using this technique. Discrimination between lesions of interstitial lung disease and lung cancer was performed by a multidisciplinary team based on clinical presentation, radiologic pattern on CT and in eight of fifteen patients, on additional lung biopsy. FAPI-46 uptake in fibrosis lesions was comparable to tumor lesions. However, in fibrosis lesions, as well as in the background tissue, SUVmax and SUVmean decreased over time faster than in tumor lesions. This resulted in increasing TBR for the tumor lesions at 10, 60 and 180 min after injection [R\u00f6hrich et al. used [njection .68Ga]Ga-FAPI PET/CT to [18F]FDG PET/CT in 27 primary and recurrent lung cancer lesions and reported a mean SUVmax of 13.7 for [68Ga]Ga-FAPI (subtype not specified) and 10.4 for [18F]FDG (p = 0.02). This resulted in a slightly higher TBR for [68Ga]Ga-FAPI PET/CT  [Wang et al. compared [ = 0.02) .68Ga]Ga-FAPI PET/CT in cancer lesions in lung tissue, as described in this paragraph and complemented by information from case reports that are depicted below, is provided.In 68Ga]Ga-FAPI imaging with [18F]FDG in a patient with locally advanced lung adenocarcinoma and they a showed significantly higher uptake of [68Ga]Ga-FAPI-02 compared to [18F]FDG in metastatic lesions, as well as lower uptake in background tissue, leading to higher contrast. The primary tumor was also clearly visible, but a quantitative comparison to [18F]FDG was not reported [68Ga]Ga-FAPI PET.In 2018, Loktev et al. were the first to compare [reported . After t68Ga]Ga-FAPI PET/CT was performed to identify possible metastases after a lung nodule was discovered using [18F]FDG PET/CT. This showed the presence of brain metastases that could not be detected by [18F]FDG because of the high physiological uptake, but also a higher SUV in the primary nodule (7.64 vs. 6.03). FAPI subtype was not specified Ga-FAPI PET/CT. In this case, suspicion for brain lesions was low based on FDG PET-CT, where limited tumor mass, with only a small T2a primary tumor and one potential N2 lymph node, was shown, without evidence of bone or visceral metastasis. [68Ga]Ga-FAPI-04 PET/CT, however, showed two FAPI-04-positive lesions in the brain that were confirmed by contrast enhanced cerebral MRI [Giesel et al. also describe a case where cerebral metastasis of lung cancer was detected because of [bral MRI .18F]FDG PET/CT showed bone destruction of the left hip with high FDG uptake, as well as multiple FDG-avid areas in the bones, lungs, liver and lymph nodes. A brain lesion was also visible, with an SUVmax of 5.9. In addition, high FDG uptake was observed in the muscles of the right hip and lower leg. [68Ga]Ga-FAPI PET/CT showed that FDG-avid lesions were also FAPI-avid (FAPI subtype not specified). No increased uptake in the muscles was observed, supporting the theory that this increased FDG-uptake was due to overuse of the right leg. Interestingly, the [68Ga]Ga-FAPI PET/CT scan revealed two lesions in the humeri that were not visible on the [18F]FDG PET/CT scan . As expected, the brain lesion showed a higher contrast than in the [18F]FDG PET/CT, despite a lower SUVmax (2.3 vs. 5.9) Ga-FAPI PET/CT in the detection of cerebral metastases of lung cancer. However, in current clinical practice, a brain MRI is part of the standard care for these patient groups.These case reports show the value of [68Ga]Ga-FAPI PET/CT in the detection of lymph metastasis, as they report a patient with SCLC was staged T1bN2M0 based on multiple FDG-avid lymph nodes using [18F]FDG PET/CT. As part of a clinical trial, a [68Ga]Ga-FAPI PET/CT was performed one week later (FAPI subtype not specified). This showed the known primary tumor, with a SUVmax of 7.7, versus 2.7 in the [18F]FDG-scan. However, no increased FAPI uptake was detected in the suspicious lymph nodes. Histology confirmed the absence of malignancy in these nodes and the revised TNM stage was T1bN0M0 [Shang et al. provide an example of the added value of [ T1bN0M0 .68Ga]Ga-FAPI PET/CT is presented by Liu et al. In this case, [68Ga]Ga-FAPI PET/CT (FAPI subtype not specified) aided in diagnosing a mediastinum type lung cancer that could have been easily misdiagnosed, due to the unusual presentation. Additionally, [68Ga]Ga-FAPI PET/CT was well able to distinguish lesions in the mediastinum, while high [18F]FDG uptake in the heart is usually physiological, and thus an [18F]FDG PET/CT might not have revealed the cardiac lesions [Another case emphasizing the clinical significance of [ lesions .68Ga]Ga-FAPI PET/CT in the detection of lung cancer metastases is also supported by the findings of Wang et al., who compared the performance of [68Ga]Ga-FAPI PET/CT with [18F]FDG in 34 patients with stage IV lung cancer [68Ga]Ga-FAPI (subtype not specified) had higher TBR than [18F]FDG in the detection of all types of metastatic lesions, with the largest advantage in brain lesions  and bone lesions . This is especially interesting considering that the nervous system (39%) and bones (34%) are the primary metastasis sites for lung cancer [68Ga]Ga-FAPI outperforms [18F]FDG to a large extent.The added value of [g cancer . In lineg cancer . Togethe68Ga]Ga-FAPI PET/CT in the detection of lung cancer metastasis, as described above, is provided.In 68Ga]Ga-FAPI PET/CT. Tang et al. report a case of a patient presenting with chest pain and cough that was initially diagnosed with lung cancer based on [18F]FDG PET/CT, which showed a soft tissue mass with intense [18F]FDG activity. Lymph nodes in the mediastinum and left hilar region were enlarged, and some of these showed increased FDG-uptake, whereas others did not. [68Ga]Ga-FAPI-04 PET/CT showed intense uptake in the soft tissue mass, but no increased uptake in the enlarged lymph nodes. Biopsy of the mass and the lymph nodes showed inflammatory cell infiltration and fibroblast proliferation, but no signs of malignancy [68Ga]Ga-FAPI-uptake.As described previously, R\u00f6hrich et al. showed that interstitial lung diseases, as well as lung cancer, cause increased FAPI-uptake . Apart flignancy . This de68Ga]Ga-FAPI PET/CT in the detection of lung cancer metastasis is the increased FAPI-uptake in several non-malignant processes, as is substantiated by the following cases.An important limitation of [68Ga]Ga-FAPI-04 PET/CT showed a higher uptake in lung adenocarcinoma than [18F]FDG PET/CT . However, [68Ga]Ga-FAPI-04 PET/CT also showed two FAPI-avid lesions in the vertebral column that were not visible on [18F]FDG PET/CT. An MRI scan revealed no signs of malignancy in the spine and the lesions corresponded to degenerative osteophytes. No FAPI-04-uptake was observed in other osteophytes, indicating that the FAPI activity may be associated with the presence of fibroblasts in early osteophytes FDG PET/CT showed increased uptake in the pulmonary nodule, as well as in several lymph nodules, and slight metabolic activity in the L4 vertebral body. [68Ga]Ga-FAPI PET/CT did not show increased uptake in the lymph nodes, but did show intense uptake in the L4 vertebral body (FAPI subtype not specified). A biopsy of the lung nodule confirmed invasive lung adenocarcinoma. However, the FDG-avid lymph nodes were diagnosed as inflammatory lesions and the vertebral body with intensive FAPI-uptake was concluded to be a fracture caused by the car accident Ga-FAPI uptake in lung cancer is high, whereas uptake in healthy lung- and other tissue is low, resulting in sufficient TBR for application in imaging. Especially in the detection of metastasis, SUVmax and TBR were higher for [68Ga]Ga-FAPI PET/CT than for [18F]FDG PET/CT, indicating a potential added value for [68Ga]Ga-FAPI PET/CT in the staging of lung cancer. A limitation is the increased FAPI-uptake in several non-malignant processes, including organizing pneumonia and interstitial lung disease, which should be considered.Several studies have demonstrated that [68Ga]Ga-FAPI PET uptake is correlated to FAP-expression, we suggest that the relationship between FAP-expression and prognosis should be investigated in future studies using [68Ga]Ga-FAPI PET/CT.In addition, three studies associating high levels of FAP-expression with poor prognosis in lung cancer, and one study correlating high FAP-expression with better prognosis, specifically in SCC, were discussed. As [18F]F-FAPI-04 uptake in PET/CT. They suggested that 18F-labeled FAPI-04-uptake might distinguish different pathological types of lung cancer. They studied thirty patients with lung adenocarcinoma, seventeen patients with SCC and fourteen patients with small cell lung cancer. [18F]-FAPI-04 uptake in both primary tumors and metastases of all three subtypes was extensive. They showed no difference in uptake of FAPI in the different pathological subtypes, but did show that FAPI-04 uptake in metastases from SCC were the highest, as measured by the so-called SUVmax, i.e., maximum standard uptake value (SUVmax 10.41 \u00b1 6.96), followed by ADC (SUVmax 7.03 \u00b1 4.30) and the lowest in SCLC (SUVmax 4.94 \u00b1 2.60) F-FAPI might have good potential for use in lung cancer, as well as [68Ga]Ga-FAPI, as demonstrated by Li et al., who performed a prospective study on the primary staging of lung adenocarcinoma in 34 patients using [18F]F-FAPI-42. They demonstrated similar uptake in 27 primary tumors  and were able to detect more metastases using [18F]F-FAPI than [18F]FDG . For brain lesions, [18F]F-FAPI outperformed [18F]FDG in both detection  and TBR . However, contrast-enhanced MRI was still superior to [18F]FAPI PET/CT, detecting 56 lesions (p = 0.002) [18F]F-FAPI might be more accessible to some hospitals, since the slightly longer half-life of 18F (110 min versus 68 min for 68Ga) allows for it to be labelled by a commercial external party. We expect that [18F]F-FAPI PET/CT, as well as [68Ga]Ga-FAPI PET/CT, will play a role in the staging and evaluation of lung cancer in the future, although more research is needed.[= 0.002) . Dependi90Y]Y-FAPI-46 in nine patients with several progressive, advanced-stage solid tumors, which all had metastatic nodes in the lung. They observed a low rate of attributable adverse events and signs of tumor response [90Y]Y- and [153Sm]-FAPI-46 attained stable disease for eight months [68Ga]Ga-FAPI PET/CT in lung cancer diagnostics, this suggests good potential for the labelling of FAPI to therapeutic radiopharmaceuticals and its application in lung cancer therapy in the future.Furthermore, FAPI can be labelled to therapeutic radionuclides, such as Lutetium-177, Copper-64, Yttrium-90 or Actinium-225 ,46,47,48t months . Combine"}
+{"text": "Their PET/CT images were analysed to quantify the uptake of the two tracers in the form of maximum standardised uptake (SUVmax) values and target-to-background ratio (TBR), which were then compared using Wilcoxon\u2019s signed-rank test. The final changes in the tumour\u2013node\u2013metastasis (TNM) stage of all participants were recorded.This single-center preliminary clinical study (NCT04750772) was conducted at the Peking University Cancer Hospital & Institute and included 61 participants with CRC who underwent sequential evaluation through PET/CT with [68Ga]Ga-FAPI-04 and [18F]F-FDG were 13.3 \u00b1 8.9 and 8.2 \u00b1 6.5, respectively. The SUVmax of [68Ga]Ga-FAPI-04 in signet-ring/mucinous carcinomas (11.4 \u00b1 4.9) was higher than that of [18F]F-FDG (7.9 \u00b1 3.6) (P = 0.03). Both median SUVmax in peritoneal metastases and TBR in liver metastases of [68Ga]Ga-FAPI-04 were higher than those of [18F]F-FDG . Compared with [18F]F-FDG PET/CT, clinical TNM staging based on [68Ga]Ga-FAPI-04 PET/CT led to upstaging and downstaging in 10 (16.4%) and 5 participants (8.2%), respectively. Therefore, the treatment options were changed in 13 participants (21.3%), including 9 with additional chemo/radiotherapy and/or surgery and others with avoidance or narrowed scope of surgery.Of all the participants, 21 were treatment na\u00efve and 40 had been previously treated. In primary CRC lesions, the average TBRs of [68Ga]Ga-FAPI-04 showed potential as a novel PET/CT tracer to detect lymph nodes and distant metastases, which improved CRC staging, thus prompting the optimisation or adjustment of treatment decisions.[ At diag18F]F-FDG) PET/CT, which uses altered glucose metabolism in cancer cells, is a valuable imaging modality in CRC management  is a membrane-anchored peptidase that is highly expressed in cancer-associated fibroblasts (CAFs) in >90% of epithelial tumours, including CRC, and contributes to disease progression and worsening prognosis in various cancers (68Ga)-labelled FAPI, are advantageous in staging and restaging many cancers, including peritonitis carcinomatosis, compared with [18F]F-FDG PET/CT  staging with that of standard-of-care [18F]F-FDG PET/CT imaging in participants with primary and recurrent/metastatic CRC.Non-invasive molecular imaging novel PET tracers is being increasingly used in the field of clinical oncology. Flourine-18 fluorodeoxyglucose . Written informed consent was obtained from all participants who were consecutively recruited from the study institution. The inclusion criteria were as follows: age >18 years, histologically confirmed CRC referral to the Nuclear Medicine Department for both [18F]F-FDG and [68Ga]Ga-FAPI-04 PET/CT scans for staging or restaging to aid optimal clinical decision making and provision of written informed consent. Pregnant or lactating women and those with severe liver or kidney dysfunction were excluded. The final cohort comprised 61 participants with CRC. The diagnosis was confirmed through surgery in 25 participants and through endoscopic biopsy in others. All suspicious metastatic lesions were confirmed by histopathology or follow-up for 3\u20136 months. Histopathology was served as a gold standard reference for the confirmation of the imaging findings by the means of rebiopsy or surgery. If pathological diagnosis was not applicable, conventional imaging  follow-up for anatomical abnormalities of lesions was performed. Lesions were diagnosed of malignant based on any of the following follow-up criteria: 1) typical malignant features demonstrated by other imaging, especially the contrast-enhanced CT/MRI referred to as the first-line imaging. 2) posttreatment shrinkage or expansion of a suspicious lesion on follow-up imaging indicating improvement or progression, periodic plain CT/MRI scan used as the second-line imaging. 3) Density changes of lesions, such as cortical breakthrough for bone metastases. The finial observation of significant malignant features of suspicious lesions was the follow-up imaging end-point. The study flowchart of participant enrolment is presented in This preliminary clinical trial was approved by our Institutional Review Board (no. 2019KT95) and registered on 2.218F]F-FDG was manufactured in accordance with the standard method described by our laboratory using the coincidence [18F]F-FDG synthesis module. The FAPI precursor (DOTA-FAPI-04) was purchased from Huayi Technology Co., Ltd. (China), and synthesis and radiolabelling of [68Ga]Ga-FAPI-04 were performed as previously described  after approximately 1 h. The acquisition was commenced in 6\u20138 bed positions (1 min/bed) covering the area between the top of the skull and upper thigh. Non-contrast-enhanced CT was performed using 100-mA modulation at 120 kV with a 3-mm slice thickness for attenuation correction and anatomical localisation. All data were transferred to the Syngo MultiModality Workplace  and reconstructed using the ordered subset expectation maximum algorithm to construct display images in the coronal, axial and sagittal planes.All enrolled participants underwent routine F-FDG and [68Ga]Ga-FAPI-04 PET/CT images, respectively. Reference information from the other group and all other images and clinical data, including CT, MRI, endoscopic and pathological results, were absent. All differing opinions were interpreted and discussed within the groups until a consensus was reached. The inter-reader agreement within the two groups was expressed using the \u03ba value.All the images were reviewed by two groups of physicians with at least 10 years of experience in nuclear medicine and radiology. The physicians in group 1 (X.C. and M.W.) and group 2 (X.L. and X.W.) independently reviewed the Ga-FAPI-04 PET/CT without any complications. No signs of drug-related side effects were reported during the entire observation period of >5 h.All participants tolerated Ga-FAPI-04 uptake was higher than [18F]F-FDG uptake in the salivary and thyroid glands and the pancreas . The detailed distribution of [18F]F-FDG and [68Ga]Ga-FAPI-04 uptakes is presented in The inter-reader agreement between groups 1 and 2 was nearly perfect, and the 3.4n = 21) and for those who received neoadjuvant treatment (n = 15). Only 1 of these 36 participants had two primary lesions. Therefore, 37 primary lesions were measured. The sensitivity was 100% (37/37) for both [68Ga]Ga-FAPI-04 and [18F]F-FDG PET/CT. The average SUVmax and median SUVmax (range) of all primary lesions were 9.7 \u00b1 5.4 and 9.7 (2.0\u201325.5), respectively, on [68Ga]Ga-FAPI-04 PET/CT and 11.4 \u00b1 7.6 and 10.3 (2.4\u201335.1), respectively, on [18F]F-FDG PET/CT (P = 0.09). The average TBR of all 37 primary lesions was significantly higher on [68Ga]Ga-FAPI-04 PET/CT than on [18F]F-FDG PET/CT . The average SUVmax values for [68Ga]Ga-FAPI-04 and [18F]F-FDG were 12.3 \u00b1 4.6 and 14.1 \u00b1 7.3, respectively, in the treatment-na\u00efve group (P = 0.21) and 5.9 \u00b1 4.1 and 7.4 \u00b1 6.5, respectively, in the neoadjuvant radio-chemotherapy group (P = 0.18). The average TBR of the treatment-na\u00efve lesions was significantly higher on [68Ga]Ga-FAPI-04 PET/CT than on [18F]F-FDG PET/CT , whereas the average TBR was not different between the imaging modalities for the post-treatment lesions .The histopathological data of the primary tumours were available for all treatment-na\u00efve participants (68Ga]Ga-FAPI-04 (11.4 \u00b1 4.9) was significantly higher than that of [18F]F-FDG (7.9 \u00b1 3.6) in signet-ring/mucinous carcinomas . There was also a significant difference in the SUVmax of primary lesions between [68Ga]Ga-FAPI-04 and [18F]F-FDG PET/CT among well-differentiated and moderately differentiated carcinomas . Although the uptake of [68Ga]Ga-FAPI-04 was higher in poorly differentiated carcinomas (12.7 \u00b1 3.7) than in moderately-well differentiated carcinomas (10.8 \u00b1 3.0), no significant difference was noted between them (P = 0.074). Interestingly, in the neoadjuvant chemotherapy group, there was no significant difference in the SUVmax of primary lesions between [68Ga]Ga-FAPI-04 and [18F]F-FDG PET/CT among all carcinoma types (P = 0.182).The analysis of treatment-na\u00efve primary tumours revealed that the avidity of [3.518F]F-FDG PET/CT, [68Ga]Ga-FAPI-04 PET/CT revealed additional findings in 34 of the 61 participants, which led to changes in staging in 15 participants. Particularly, 6 (28.6%) of the 21 treatment-na\u00efve participants were upstaged and 2 (9.5%) were downstaged , the changes were based on the detection of new or additional distant metastases in one or more organ systems. All additional findings were confirmed through biopsy or surgery  or through other conventional imaging modalities . Moreover, in 16 participants, new lymph node metastases were detected but did not lead to changes in the stage  and pelvic lymph nodes  and pelvic lymph nodes  were also significantly higher in [68Ga]Ga-FAPI-04 PET/CT images than in [18F]F-FDG PET/CT images. However, the median uptake  and TBR  of [68Ga]Ga-FAPI-04 were lower than those of [18F]F-FDG in thoracic lymph nodes  and TBR  of [68Ga]Ga-FAPI-04 were higher than those of [18F]F-FDG in peritoneal metastases , but the TBR of [68Ga]Ga-FAPI-04 was significantly lower than that of [18F]F-FDG , the number of positive liver lesions detected using [68Ga]Ga-FAPI-04 PET/CT was higher than that detected by [18F]F-FDG PET/CT because of the lower background SUVmax of [68Ga]Ga-FAPI-04. Further, 30 positive liver lesions detected using [68Ga]Ga-FAPI-04 PET/CT were confirmed as metastases through surgery/biopsy and other imaging modalities. Only 16 of these positive liver lesions were detected by [18F]F-FDG PET/CT . The TBR of [68Ga]Ga-FAPI-04 was significantly higher than that of [18F]F-FDG , and the liver metastases were clearly visible in [68Ga]Ga-FAPI-04 PET/CT images and finally demonstrated using contrast-enhanced MRI , and surgery was avoided or its scope was narrowed in the other participants .468Ga-FAPIs, as newer imaging tracers, present a promising alternative to [18F]F-FDG. This preliminary clinical study investigated the avidity of [68Ga]Ga-FAPI-04 in CRC and explored the potential utility of [68Ga]Ga-FAPI-04 PET/CT as the sole imaging modality for assessing primary and recurrent/metastatic CRC. Our analyses indicated that [68Ga]Ga-FAPI-04 PET/CT improved tumour staging in patients with CRC owing to favourable tumour/background activity and low tracer uptake in the gastrointestinal tract. Moreover, signet-ring/mucinous carcinomas accumulated more [68Ga]Ga-FAPI-04 than [18F]F-FDG, whereas FAPI avidity was lower than FDG avidity in poorly differentiated carcinomas.FAP is an excellent target for tumour stroma, and 68Ga]Ga-FAPI-04 PET/CT is characterised by high tumour/background activity and is more sensitive than [18F]F-FDG PET/CT for identifying primary gastrointestinal carcinoma lesions  located in the pelvic area is affected by high [68Ga]Ga-FAPI-04 uptake, a potential limitation of [68Ga]Ga-FAPI-04 PET/CT imaging.In addition, we found that Ga-FAPI-04 PET/CT in CRC.This study has several limitations. First, the cohort size was small, and the number of participants with brain and bone metastases was low. Second, the cohort was heterogeneous and included participants with different treatment statuses, which could have affected the SUV568Ga]Ga-FAPI-04 PET/CT has several obvious advantages over [18F]F-FDG PET/CT, including the detection of lymph nodes and distant metastases, thereby improving the staging of patients with CRC. This improved staging is helpful for the timely revision of clinical treatment strategies and improvement of patients\u2019 prognoses. Additionally, patients might feel more comfortable during [68Ga]Ga-FAPI-04 PET/CT as it does not require fasting.[The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Institutional Review Board of Beijing Cancer Hospital (no. 2019KT95). The patients/participants provided their written informed consent to participate in this study.XL, XW, ZY, and AW contributed to the study conception and design. Synthesis of tracer and image acquisition were performed by SW and YZ. XL, YL, XC, MW, and HZ processed and analysed the data. The first draft of the manuscript was written by XL. XW, ZY, and AW reviewed and revised the manuscript. All authors read and approved the final manuscript."}
+{"text": "Images of patients with GC who were pathologically confirmed and underwent contemporaneous [68Ga]Ga-FAPI-04 PET/CT exhibited higher sensitivity compared to [18F]-FDG PET/CT for detecting primary tumors , lymph node metastasis , and distant metastases . [68Ga]Ga-FAPI-04 accumulation was significantly higher than that of [18F]FDG in tumors , lymph node metastasis metastases , and distant metastases . Compared to [18F]-FDG PET/CT, [68Ga]Ga-FAPI-04 PET/CT resulted in new oncological findings in 14/25 patients and corrected tumor staging or restaging in 7/25 patients.In total, 25 patients with GC  were evaluated. [68Ga]Ga-FAPI-04 PET/CT on tumor staging highlight the potential of this approach for increasing the accuracy of GC diagnosis, which may facilitate treatment decision-making.Our preliminary results regarding the impact of [ Gastric cancer (GC) is the fifth most common cancer and third most common cause of cancer-related deaths worldwide . Adenoca18F]-FDG)-PET/CT is currently the most frequently used imaging modality for initial tumor staging, evaluation of treatment response, and detection of recurrence in most oncological malignancies. However, [18F]-FDG may not be an ideal imaging agent for GC, as several limitations have been noted. Mucinous, signet ring cell, and poorly differentiated adenocarcinomas demonstrate less [18F]-FDG uptake compared to other histological types of gastric adenocarcinoma  was recently identified to be highly expressed in cancer-associated fibroblasts (CAFs) and is closely related to cancer cell proliferation, tumor immunity, angiogenesis, extracellular matrix remodeling, and metastasis. FAP has low expression levels in normal tissues and organs, which makes it a good molecular target for tumor diagnosis and treatment \u20139. This 4 PET/CT , 12, 13.All procedures performed in this study involving human participants were in compliance with the ethical standards of the Clinical Research Ethics Committee of the Affiliated Hospital of Southwest Medical University  and China Clinical Trials Registry . Written informed consent was obtained from all patients. A retrospective analysis was performed on patients with GC who underwent whole-body PET/CT at the Affiliated Hospital of Southwest Medical University between June 2020 and December 2021.18F]-FDG PET/CT and [68Ga]Ga-FAPI-04 PET/CT for tumor staging and restaging to determine the most appropriate treatment strategy. b. GC diagnosed by pathology or follow-up. Exclusion criteria were as follows: a. pregnant patients or patients under 18 years of age. b. participants and their parents or legal representatives were unable or unwilling to provide written informed consent. A final total of 25 patients were included.Inclusion criteria were as follows: a. patients who underwent [18F]-FDG was manufactured according to the standard method used in our laboratory using the coincidence [18F]-FDG synthesis module . DOTA-FAPI-04 was purchased from MedChemExpress, LLC. [68Ga]Ga-FAPI-04 was prepared according to a previously described protocol . Patients who underwent [68Ga]Ga-FAPI-04 PET/CT examinations did not require special preparation. The intravenous doses of [18F]-FDG and [68Ga]Ga-FAPI-04 were weight-adjusted . Patients were required to drink 1,000 mL of water to fill their stomach before examination and to urinate before the PET/CT scan. 60 minutes after intravenous injection . The location, number, and SUVmax value of positive lesions were recorded. We evaluated primary tumor, lymph node metastasis, and distant metastasis of GC. Based on the guidelines of the American Joint Committee on Cancer (AJCC), T and N pathological staging was performed based on the results of postoperative pathological examination. The lymph nodes were divided into three regions: those around the gastric cavity, perivascular, and perivisceral. Involvement of the retropancreatic, mesenteric root, middle colic, para-aortic, peripancreatic, infradiaphragmatic, paraesophageal, lower thoracic, and other distant lymph nodes was considered distant metastasis (M1). Ovarian metastases were classified separately.Liver lesions with uptake higher than that of the surrounding liver parenchyma were identified as positive. The lesion number and SUVmax of the lesion with the highest pathological tracer accumulation were recorded for each lymph node region or distant metastasis site for both [18F]-FDG and [68Ga]Ga-FAPI-04 PET/CT. The upper limit of the number of regional lymph nodes or distant metastases was set at 20, and numbers > 20 were capped and recorded as 20.Ga-FAPI-04 PET/CT and [18F]-FDG PET/CT for TNM staging of GC was compared using McNemar test. Differences in SUVmax in primary tumor, lymph node metastasis, and distant metastasis were compared between [68Ga]Ga-FAPI-04 and [18F]-FDG PET/CT using the Mann-Whitney U test. Differences were considered statistically significant at P < 0.01.Statistical analysis was conducted using IBM SPSS 26.0 software (SPSS Inc). Continuous variables were evaluated for normal distribution with Shapiro-Wilk test. Categorical variables were expressed as frequencies and percentages. Continuous variables were expressed as means \u00b1 standard deviations (SDs). The diagnostic efficacy of [68Ga]Ga-FAPI-04 and [18F]-FDG PET/CT was performed for restaging due to suspected progressive disease, while the 17 patients with new diagnoses underwent PET imaging for primary staging. Based on pathological type, we identified 16 cases of poorly differentiated adenocarcinoma, , 2 cases of moderately differentiated adenocarcinoma, 2 cases of well-differentiated adenocarcinoma, and 5 cases of adenocarcinoma with unknown differentiation. Patient characteristics are presented in In total, 25 patients  were included in this study. The mean age was 56 \u00b1 12 years (range: 35-79 years). Two patients had primary recurrence with distant metastasis, and two patients had distant metastasis but no primary recurrence. There were no primary recurrences or metastases in four patients. [18F]-FDG and [68Ga]Ga-FAPI-04 PET/CT. PET imaging was well tolerated by all patients. None of the patients reported abnormal symptoms.None of the patients presented with any drug-related side effects during or after [68Ga]Ga-FAPI-04 and [18F]-FDG PET/CT in 19 patients. [68Ga]Ga-FAPI-04 PET/CT exhibited a very high lesion detection rate (positive detection rate: 94.74% [18/19]) in primary gastric tumors, which was significantly higher than that of [18F]-FDG PET/CT (68.42% [13/19]). The uptake of [68Ga]Ga-FAPI-04 in 19 cases of primary GC [SUVmax: 10.28] was significantly higher than that of [18F]-FDG [SUVmax: 3.20] , 83.3% (5/6), 72% (18/25), 92.9% (13/14), and 45.4% (5/11) for [18F]-FDG, respectively.We identified 17 cases of newly diagnosed GC and 2 cases of postoperative primary recurrence of GC. Primary tumors were evaluated using [4 PET/CT 4. One p68Ga]Ga-FAPI-04 or [18F]-FDG PET. Of 77 lymph nodes, 75 were correctly determined by [68Ga]Ga-FAPI-04 PET/CT. In contrast, 32 out of 75 lymph nodes were correctly diagnosed by [18F]-FDG PET/CT . A total of 10 patients had area 1 lymph node metastasis, 3 had area 2 lymph node metastasis, and 3 had area 3 lymph node metastasis. One patient had lymph node metastasis in three regions, two patients had lymph node metastasis in two regions, and nine patients had only one regional lymph node metastasis. The 75 lymph node metastases comprised 53 lymph nodes in areas 1, 11 in areas 2, and 11 in area 3. Among the 75 nodal metastases, 43  were not detected by [18F]-FDG PET  vs. 3.15; U=30, P=0.130). For all metastatic lymph nodes, SUVmax values were significantly higher for [68Ga]Ga-FAPI-04 PET than for [18F]-FDG -FDG, respectively. For the metastatic lymph nodes, there was no evidence of a higher lesion detection rate for [68Ga]Ga-FAPI-04 PET/CT than [18F]-FDG PET/CT.Lymph nodes located around the gastric cavity were defined as area 1, perivascular lymph nodes as area 2, and perivisceral lymph nodes as area 3. In total, 75 positive lymph nodes in 12 of 25 patients were detected either by [-FDG PET 5. The m68Ga]Ga-FAPI-04 PET/CT detected 275 distant metastases  in 12 patients. Based on the reference criteria, only 3 patients with distant metastasis in a single region were identified, including one case of ovarian metastasis, one case of supraclavicular fossa lymph node metastasis, and one case of abdominal lymph node metastasis. Six patients had extensive metastases . The analysis of all distant metastases revealed that SUVmax value was significantly higher for [68Ga]Ga-FAPI-04 PET than for [18F]-FDG PET -FDG PET/CT was used to determine tumor staging in 10/17 newly diagnosed patients with GC and re-staging in 7/8 postoperative patients with GC. [68Ga]Ga-FAPI-04 PET/CT was used to determine tumor staging or restaging in 24 patients. In one patient, the primary tumor was not detected by [68Ga]Ga-FAPI-04, but all distant metastases were detected, indicating that [68Ga]Ga-FAPI-04 PET/CT still correctly defined staging , thus avoiding overtreatment in these patients. Compared to [18F]-FDG PET/CT, [68Ga]Ga-FAPI-04 PET/CT revealed new positive lesions in 14/25 patients. For overall staging or restaging, 5/17 patients  with newly diagnosed GC were upstaged. [18F]-FDG PET/CT failed to detect primary tumors in four patients; perigastric lymph nodes in two patients; and peritoneum, omentum, and mesentery metastasis in one patient. However, [68Ga]Ga-FAPI-04 PET/CT provided the correct staging, thus enabling the patients to receive timely surgical treatment , chemotherapy, and targeted therapy (patients No. 12 and 14).(68Ga]Ga-FAPI-04 and 72% (18/25) for [18F]-FDG, respectively.[ staging Figure\u00a0168Ga]Ga-FAPI-04 is a potential imaging agent for tumor diagnosis that enables visualization of the stroma in the tumor microenvironment. The muscle and blood pool background of [68Ga]Ga-FAPI-04 is very low (SUVmax<2.0), resulting in a high tumor/normal tissue radioactivity ratio  [8], which provides superior image quality compared to [18F]-FDG , nodal metastases (75/77 [97.40%] vs. 32/77 [41.56%]), and distant metastases (275/283 [97.17%] vs. 122/283 [43.11%]) in patients with GC, which is similar to the results of Jiang et\u00a0al.  vs. 3.20; U=59.00, P < 0.01]. Studies have reported that Claudin 18.2, inhibitors of the fibroblast growth receptor 2 pathway, and combinations of anti-angiogenic therapy and immune checkpoint blockade constitute novel targets for the treatment of GC .Lymph node staging is key for the treatment and prognosis of patients with GC. Studies have demonstrated that PET/CT is useful for detecting occult lymph node metastases. The high detection rate of lymph nodes in -FDG was observed in only 38 sites, whereas [68Ga]Ga-FAPI-04 detected all peritoneum, omentum, mesentery metastases. The low detection rate of [18F]-FDG for peritoneal metastasis is due to the relatively low expression of GLUT-1 in signet ring cell and mucinous carcinomas , mediastinum (22 vs. 0), and abdominal pelvic (72 vs. 36) lymph node metastases were significantly higher for [68Ga]Ga-FAPI-04 than for [18F]-FDG (P < 0.01). In this study, both imaging agents detected metastases of lung, accessory, bone, and bone marrow, but there was a higher uptake of imaging agents in the bone marrow, which may be due to the fact that patients with bone marrow metastasis are more likely to develop bone marrow fibrosis.Complete surgical resection of gastric tumors and adjacent lymph nodes with a negative cutting edge is currently the only effective treatment for GC. As resection is not possible for GC with distant metastasis, accurate staging of patients with GC is essential. Due to the limited number of pathological biopsies lesions, the negative predictive value of lesions may be incorrect, especially in distant metastases. Therefore, this study mainly discusses the positive distant metastatic lesions. In our study, peritoneum, omentum, mesentery metastasis was the most common distant metastasis (128/283 lesions). -FDG, while only five bone metastases were detected by [68Ga]Ga-FAPI-04. Accordingly, both imaging agents may be used for diagnosis in patients with bone metastasis of GC in order to avoid missed diagnosis. In addition, the ovarian metastasis of one patient was not detected by [68Ga]Ga-FAPI-04 PET/CT  was small, which precluded subgroup comparisons of GCs with different degrees of differentiation. We analyzed primary tumors, lymph nodes, and distant metastatic lesions of GC in a summarized way and compared tracer uptake and detection rates to obtain diagnostic efficiency. Second, owing to technical and ethical problems, it is impossible to biopsy all lymph nodes and distant metastases, and histopathological confirmation of all positive lesions cannot be used as a reference standard. Thus, the results of morphological and/or follow-up imaging were also used as the reference standard in our investigation.68Ga]Ga-FAPI-04 PET is superior to [18F]-FDG PET for the detection of primary tumors, lymph nodes, and distant metastases in patients with GC. Compared with [18F]-FDG PET/CT, [68Ga]Ga-FAPI-04 PET/CT corrected clinical stage in seven patients. In addition, the negative expression of [68Ga]Ga-FAPI-04 on PET/CT in patients with residual gastritis is helpful for distinguishing it from the high uptake of [18F]-FDG caused by inflammation. [68Ga]Ga-FAPI-04 has evident advantages for the detection of lymph node, peritoneal, omentum, mesentery and liver metastases. However, to confirm the definitive diagnostic value of [68Ga]Ga-FAPI-04 PET/CT for GC, our preliminary results warrant verification in other pathologically confirmed lesions.In summary, [The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by Medical Research Ethics Committee, 2020035; Clinical Trials Registry No.ChiCTR2100044131. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.SZ, WW, TX, LQ and YC contributed to the study conception and design. Material preparation and data collection were performed by SZ, WW, LQ, YC, TX, HD, YL, HL, TD, YZ, LL. SZ, WW, YH processed and analysed the data. The first draft of the manuscript was written by SZ and LQ reviewed and revised the manuscript. All authors read and approved the final manuscript.This study was supported in part by research foundation projects from Luzhou Science & Technology Department , The Affiliated Hospital of Southwest Medical University (20087), and the nuclear medicine and molecular imaging key laboratory of Sichuan province open project (HYX18028).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "High-precision global navigation satellite system (GNSS) positioning and navigation can be achieved with carrier-phase ambiguity resolution when the integer least squares (ILS) success rate (SR) is high. The users typically prefer the float solution under the scenario of having a low SR, and the ILS solution when the SR is high. The best integer equivariant (BIE) estimator is an alternative solution since it minimizes the mean squared errors (MSEs); hence, it will always be superior to both its float and ILS counterparts. There has been a recent development of GNSSs consisting of the Global Positioning System (GPS), Galileo, Quasi-Zenith Satellite System (QZSS), and the BeiDou Navigation Satellite System (BDS), which has made precise positioning with Android smartphones possible. Since smartphone tracking of GNSS signals is generally of poorer quality than with geodetic grade receivers and antennas, the ILS SR is typically less than one, resulting in the BIE estimator being the preferred carrier phase ambiguity resolution option. Therefore, in this contribution, we compare, for the first time, the BIE estimator to the ILS and float contenders while using GNSS data collected by Google Pixel 4 (GP4) smartphones for short-baseline real-time kinematic (RTK) positioning. It is demonstrated that the BIE estimator will always give a better RTK positioning performance than that of the ILS and float solutions while using both single- and dual-frequency smartphone GNSS observations. Lastly, with the same smartphone data, we show that BIE will always be superior to the float and ILS solutions in terms of the MSEs, regardless of whether the SR is at high, medium, or low levels. The key to high-precision global navigation satellite system (GNSS) positioning and navigation is carrier-phase integer ambiguity resolution. The ambiguity-fixed GNSS baseline, as obtained by integer least squares (ILS), is commonly expected to be superior to its float counterpart if the integer ambiguity success rate, i.e., the probability of correct integer estimation, is close to the maximum value of one. On the other hand, the float solution usually becomes the positioning preference when the success rate is too low. Alternatively, the best integer equivariant (BIE) estimator, as introduced by Teunissen , can be Verhagen and Teunissen  proved tThe recent development of smartphone GNSS chipsets, such as Broadcom BCM47755 embedded, makes precise code-based positioning , PPP 2121, and iThis contribution is organized as follows: in In this section, we describe the functional model employed for the single-baseline RTK model while tracking single- and dual-frequency multi-GNSS observations using GP4 smartphones. We also introduce the float, ILS, and BIE estimators.We assume that the two GP4 smartphones track GPS, Galileo, QZSS, and BDS code and carrier-phase frequencies on two frequencies. We make use of broadcast ephemerides for satellite orbits and clocks. The relative ionospheric, tropospheric delays and satellite orbit errors can be assumed negligible, since we employ short baselines. The single-epoch (instantaneous) and linearized double-differenced (DD) system of observation equations reads as follows:he model ,26. For he model .n and To obtain the float solution, denoted with a \u2018hat\u2019 symbol, we estimate the ambiguities and baseline components as real valued parameters in a standard least-squares solution, obtainingBy using the float ambiguities ) method , finallyThe corresponding VCV matrix, provided that the uncertainty in The precision of the fixed ILS baseline When the ILS SR is low, the user will normally prefer the float solution iguities . AssuminNote in Equation (6) that the BIE solution is not always necessarily an integer as it is a weighted sum of integers. We also remark that, for the BIE estimator, no ratio test is needed .The BIE baseline solution can then be derived as instead , which cThis section describes the short-baseline setup configurations of the GP4 smartphones while using (1) external antennas and (2) internal smartphone antennas. In this section, we also outline the stochastic model settings.When evaluating the performance using external antennas, the GP4 smartphones are placed in two separate radiofrequency (RF) shielding boxes to avoid them from receiving the GNSS signals other than from the dedicated reradiating antenna see a\u2013d. The In addition to the short baseline with external antennas, we assessed the positioning performance of the short baseline while using the smartphone internal antennas see e. Note tThe stochastic model was determined by fitting the empirical 95% confidence interval/ellipses to the formal counterparts, as derived from the corresponding VCV matrices of the positions. The empirical VCV matrix was estimated from the positioning errors obtained by comparing the estimated positions to very precise benchmark coordinates, whereas the formal VCV matrix was obtained by the average of all single-epoch formal VCV matrices of the entire observation time span . We usedIn this section, we investigate the BIE estimator and compare the performance to that of the commonly used ILS and float estimators. The benefit of using the BIE estimator is that the MSEs are always smaller than or at least as good as the float and ILS counterparts. For instance, in practice, when the ILS success rate is lower than the desired 100%, the user usually opts for the float solution when, in fact, BIE would be the preferred option. The number of correctly fixed epochs, used below, was determined by the number of epochs where the estimated local east, north, and up coordinate errors were all below or equal to 0.05 m. The ILS success rate was then computed as follows:In the results below, we investigate the BIE performance for GP4 smartphones when using both external and internal smartphone antennas while collecting single- and dual-frequency multi-GNSS data. To further illustrate the optimal performance of the BIE estimator in terms of the positioning MSEs, we depict in To also illustrate the corresponding positioning precisions of the different estimators, Lastly, we can see in In this section, we investigate the corresponding BIE performance when the internal antennas of the smartphones are used. Since, with the smartphone internal antennas, the multipath errors are more significant than when external antennas are used , in thisTo show the above superior performance of the BIE estimator in a different way, To again illustrate the optimal BIE performance, we depict in In this contribution, we analyzed the best integer equivariant (BIE) estimator for real GNSS data collected by Google Pixel 4 (GP4) smartphones and antennas. We compared the instantaneous (single-epoch) positioning performance of BIE to the float and integer least squares (ILS) estimators that are commonly used when the ILS success rate (SR) is different from one and close to one, respectively. Radiofrequency (RF) shielding boxes and reradiating antennas were used to track GNSS signals from external low-cost antennas, consisting of L1 + L5 GPS, E1 + E5a Galileo, L1 + L5 QZSS, and B1 BDS code and carrier-phase observations. The short-baseline real-time kinematic (RTK) performance was also evaluated while using the GP4 internal smartphone antennas. We investigated the BIE performance both when single-frequency and dual-frequency measurements were employed for the combination of GPS + Galileo + QZSS + BDS. We showed that the BIE positioning performance was superior to that of the ILS and float estimators when the ILS SR is different from one. This was demonstrated to be true on the basis of real multi-GNSS data collected by the GP4 smartphones and antennas.Our BIE performance evaluation consisted of comparing the estimated positions to very precise benchmark coordinates, and the optimality of the BIE estimator was further evaluated through its position mean squared errors (MSEs) and standard deviations (STDs). It was shown that the BIE performance resembles that of the float estimator when the ILS SR is very low and was similar to that of the ILS when the ILS SR is very high. For all other cases, we demonstrated that BIE outperformed both the float and the ILS estimators even when on the basis of real GP4 smartphone data while using external and internal smartphone antennas. Future studies could involve evaluating the GP4 smartphone BIE RTK positioning performance for longer baselines, when the relative atmospheric delays need to be estimated ."}
+{"text": "Correction: Discover Oncology (2023) 14:123https://doi.org/10.1007/s12672-023-00713-3The original publication of this article contained several incorrect affiliations. The updated affiliations for the affected authors are shown in this correction article. The original article  has beenGang Chen, Department of Plastic Surgery, The Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, 210029, ChinaGuangqing Zhou & Xinxin Xu, Xuzhou Medical University, Xuzhou, 221002, ChinaYulan Liu & Kai Fang, The Second Affiliated Hospital of Chengdu Medical College, China National Nuclear Corporation 416 Hospital, Chengdu, 610051, China"}
+{"text": "Journal of Managed Care Pharmacy introduced its readers to disease state management, which attempted to break the siloed culture of the U.S. health care system. Disease state management has been transformed, in part, to population health management. This shift was marked by 3 main inflection points: the rise of the web-enabled smartphone, the Patient Protection and Affordable Care Act (ACA), and the adoption of artificial intelligence (AI). The introduction of smartphones filled the communication gap through improved patient engagement and accessible mobile applications, giving patients access to their clinical data. In addition, through the ACA, bundled payment models moved away from a volume-based to a value-based payment approach and attempted to incorporate population health concerns, such as the social determinants of health. The advancement of AI will allow the health care system to collect comprehensive health data and to predict the population at higher risk. Despite these advancements, some challenges from 25 years ago remain, yet rapid technology advancements may expedite the next wave of change.Twenty-five years ago, the  Journal of Managed Care & Specialty Pharmacy, in which Hadsall and Sargent (1995) first introduced the concept of disease state management to readers,1 we reflect on the evolution of disease state management. We also address 3 key inflection points\u2014the rise of the web-enabled smartphone, the Patient Protection and Affordable Care Act (ACA), and artificial intelligence (AI)\u2014that have collectively transitioned disease state management to population health management.Twenty-five years ago, disease state management sought to improve patient outcomes by maximizing the interrelationship between physicians, payers, pharmaceutical companies, pharmacists, and patients to manage chronic conditions. On the 25th anniversary of the 2 However, the guidelines largely failed to earn support from physicians, who regarded them as \u201ccookbook\u201d medicine that eliminated the role of clinical judgement. In addition, without appropriate financial incentives, guidelines could have meant decreasing the volume of services that were perceived as needed. Despite the initial resistance of physicians toward practice guidelines,3 disease state management, which heavily relied on them, emerged in 1995.Before 1995, practice guidelines were introduced to promote uniformity of care.6 Although short-term savings were observed, long-term projections of such programs remained unclear. The early stage of this system favored relatively low-risk patients, while individuals with complex and costly health conditions were assigned to specialized companies. Although unintended, these specialized interventions added to the fragmentation of care.6 The initial challenges of disease state management were reluctance from physicians, patient retention, and the pharmaceutical companies as early adopters (which was perceived as a marketing strategy).4 In retrospect, disease state management might have succeeded in managing patients with relatively expensive, but manageable chronic conditions. However, the lack of transparency between physicians and managed care organizations hindered this process.7The rise of disease state management stemmed from the cost pressures on managed care organizations.9 Care management has been shown to increase communication between physicians and patients, to use evidence-based care, and to enhance adherence.9 It included the following core competencies: data integration, patient stratification, care coordination, patient engagement, and performance measurement.10 Some observers believed that the success of care management as compared with disease state management was a result of its ability to use technology to identify patients.11In an attempt to unify health care delivery, payers introduced care management programs for complex patients in the outpatient setting.12 Compared with disease state management, population health management attempted to look beyond the rudimentary clinical picture to capture health outcomes at the population level. This comprehensive approach was shaped by the following inflection points: the web-enabled smartphone, the ACA, and AI.Population health is defined as \u201cthe distribution of health outcomes within a population, the determinants that influence distribution, and the policies and interventions that affect the determinants.\u201d13 Concurrently, there was an increase in the use of electronic health record (EHR) systems, supported by the Health Information Technology for Economic and Clinical Health Act of 2009.14 A national survey from the Centers for Disease Control and Prevention showed that the use of any EHR system increased from 18.2% in 2001 to 50.7% in 2010.15 Such expansion allowed physicians to record clinical information electronically and patients to access their own health data remotely. Although access to smartphone data based on socioeconomic and health status are potential barriers, the smartphone is a promising step toward a unified health care system.17The lack of patient engagement was one of the early pitfalls of disease state management. The smartphone was an easy and inexpensive solution to reach patients as most Americans began to own them. By 2010, there were 5,805 health-related mobile applications, with 3% of them pertaining to chronic diseases.The smartphone has revolutionized disease state management by creating a secure and open stream of shared information at a relatively low cost for patients and physicians. At the same time, advanced EHR systems, which included comprehensive health data, helped to change disease state management to population health management.18 Specifically, it focused on improving the quality and efficiency of care through the Centers for Medicare & Medicaid Services innovations models.19 The bundled payment system was created to move away from traditional fee for service and instead to test whether a system focused on episodes of care could decrease health care expenditures. This directly addressed one of the impediments to disease state management success: lack of a financial incentive structure. However, as in disease state management, defining an episode of care for a chronic condition remains a challenge. To address this limitation, bundled payment models are beginning to take a wider view by incorporating population health concerns such as comorbidities and social determinants of health.In 2010, the passage of the ACA boosted population health management through the expansion of health care coverage, improvement of quality of care, prevention and health promotion, and community-based programs.20 With comprehensive health data and the predictive power of AI, risk prediction can further improve current population health management. Risk predictive algorithms used at the population level can support decision making for chronic disease prevention.21 The capability of AI to provide high-resolution clinical data and sharing such data in a collaborative clinical practice environment has the potential to advance efficient and high-quality care. However, leadership from policymakers and engagement of all health care stakeholders is needed for widespread adoption of AI.22Despite the advancement of the current EHR analytical capabilities, most still lack the power to extract clinical information. Yet, as health care evolves toward a comprehensive data-driven environment, AI can maximize our ability to collect information from unstructured narrative texts, such as clinical notes. Natural language processing turns the texts to machine-readable data, which then can be analyzed to supplement and enrich the medical records.Disease state management transformed into population health management around 3 main inflection points: the introduction of the web-enabled smartphone, the ACA, and the adoption of AI. In the last 25 years, this movement progressed into a comprehensive and data-driven approach to care for patients with complex and chronic diseases. Regrettably, some of the health care issues identified 25 years ago persist today, such as system fragmentation and the lack of data transparency. Despite the advances seen in the health care system, the transition from disease state management to population health is not yet complete. Yet, rapidly changing technology suggests that changes to improve patient outcomes may occur faster than in the past."}
+{"text": "Consequently, the use of the SERCA activator will reduce the level of calcium in the sarcoplasm and prevent the negative consequences of muscle unloading. Wistar rats were randomly assigned into one of three groups (eight rats per group): control rats with placebo (C), 7 days of unloading/hindlimb suspension with placebo (7HS), and 7 days of unloading treated with SERCA activator CDN1163 (7HSC). After seven days of unloading the soleus muscle, the 7HS group displayed increased fatigue in the ex vivo test, a significant increase in the level of calcium-dependent CaMK II phosphorylation and the level of tropomyosin oxidation, as well as a decrease in the content of mitochondrial DNA and protein, slow-type myosin mRNA, and the percentage of slow-type muscle fibers. All of these changes were prevented in the 7HSC group. Moreover, treatment with CDN1163 blocked a decrease in the phosphorylation of p70S6k, an increase in eEF2 phosphorylation, and an increase in MuRF-1 mRNA expression. Nevertheless, there were no differences in the degree of fast and slow muscle fiber atrophy between the 7HS and 7HSC groups. Conclusion: SERCA activation during 7 days of unloading prevented an increase in soleus fatigue, the decrease of slow-type myosin, mitochondrial markers, and markers of calcium homeostasis but had no effect on muscle atrophy.Skeletal muscle abnormalities and atrophy during unloading are accompanied by the accumulation of excess calcium in the sarcoplasm. We hypothesized that calcium accumulation may occur, among other mechanisms, due to the inhibition of sarco/endoplasmic reticulum Ca A number of skeletal muscle abnormalities, including prolonged hypokinesia, gravitational unloading, and limb immobilization, as well as prolonged lack of physical activity, can lead to skeletal muscle atrophy due to an imbalance between protein synthesis and protein degradation. During unloading, the content of mitochondrial proteins and DNA in muscles decreases concurrently with the development of atrophy ,2,3. ChaSeveral models of muscle unloading display increased accumulation of calcium in the sarcoplasm ,8,9. Thi2+-ATPase (SERCA) plays a key role in the removal of calcium from the sarcoplasm by pumping calcium ions into the sarcoplasmic reticulum (SR). During skeletal muscle unloading, the function of SERCA is impaired [Sarco/endoplasmic reticulum Caimpaired . Likewisimpaired ,20. It wimpaired ,22,23.Excessive accumulation of calcium in the sarcoplasm can also lead to the accumulation of reactive oxygen species (ROS) and of the disruption of ryanodine channel functioning. This results in the depletion of the SR calcium depot and increased muscle fatigability. Similar changes were described in skeletal muscle during aging, as well as in the muscle of some transgenic animals ,26,27,28We hypothesized that during soleus muscle unloading, there is a decrease in SERCA functions leading to the excessive accumulation of calcium ions in the sarcoplasm. Consecutively, increased calcium activates signaling pathways, triggering proteolysis processes and changes in the myosin phenotype from \u201cslow\u201d to \u201cfast\u201d, resulting in reduced fatigue resistance. In humans, the soleus muscle is involved in walking and running, and it is also responsible for maintaining the vertical position of the body. It is also very sensitive to unloading and shows fast loss of mass and functions . This isThe experiments were performed at the Institute of Biomedical Problems, RAS, Russia. The Committee on Bioethics of the Russian Academy of Sciences reviewed and approved all animal experiments for this study . The internationally accepted regulations in compliance with ARRIVE guidelines  and ruleTwenty-four male Wistar rats  were randomly assigned to one of the three groups : control rats with placebo  (\u201cC\u201d), 7 days of unloading/hindlimb suspension with placebo (\u201c7HS\u201d), and 7 days of unloading with SERCA activator CDN1163  as previously described .Hindlimb suspension was performed using a traction method of noninvasive tail-casting . During The detailed description of how soleus muscle ex vivo function was determined is described in our previous study . BrieflyProtein extraction, gel electrophoresis, and Western blot analysis were performed as previously described . As a brTo study gene expression and mitochondrial DNA content by real-time quantitative PCR, RNA and DNA were isolated from muscle tissue samples. For RNA isolation from skeletal muscles the HiPure Fibrous DNA/RNA Kit  was used according to the manufacturer\u2019s recommendations. RNA and DNA concentration analysis, cDNA synthesis and cDNA and DNA RT-PCRs were performed as previously described . The PCRTransverse sections of frozen muscle 9 \u03bcm thick were made using cryotome . The sections were air-dried and stored at \u221220 \u00b0C. Before immunostaining, sections were thawed and rehydrated at room temperature in phosphate buffered saline (PBS) for 20 min. The protocol of immunostaining for the evaluation of the percent of the slow-type and fast-type myofibers and for the quantification of their cross-sectional areas (CSAs) was previously described . The priThe percent of NFATc1-positive myonuclei relative to the count of total myonuclei was determined using triple staining of the cryosections with anti-NFATc1 and anti-dystrophin antibodies, and with DAPI. DAPI-stained nuclei lying within sarcoplasm below the dystrophin layer were assumed to be myonuclei. After rehydration, muscle cryosections were placed for 1 h into a blocking solution  at room temperature. Primary antibodies against NFATc1  and against dystrophin  in blocking solution were added 150 \u00b5L per slide with overnight incubation at 4 \u00b0C. A negative control (blocking solution without primary antibodies) was used to check for non-specific binding of secondary antibodies to the sections. The next day, the sections were washed with 200 \u00b5L of PBS per a glass slide 3 times for 5 min and 150 \u00b5L of secondary antibodies  diluted in PBS were added at room temperature for 1 h. After incubation, sections were washed for 5 min with 200 \u00b5L of PBS and incubated for 20 min with DAPI . The sections were washed from DAPI with 200 \u03bcL of PBS per glass slide 2 times for 5 min. Then, the sections were dried with filter paper, embedded in Vectashield fluorescence solution, and covered with a cover-slip with varnish fixation. Sections were analyzed using a Leica DMR Upright Microscope equipped with a Leica DC 300F camera . The photographs were analyzed using the ImageJ program.p < 0.05. The data are presented as mean \u00b1 standard error of the mean relative to the control group. For QRT-PCR the data are presented as relative expression with mean value of the control group assigned to be 1.The data were checked for normal distribution by Shapiro\u2013Wilk test. All data were normally distributed, therefore, an ANOVA was used with post hoc Tukey analysis for multiple comparisons. Differences were considered significant when After seven days of unloading, the soleus muscle mass was significantly decreased in both HS and 7HSC groups  when compared with the control rats (109.4 \u00b1 4.3 mg) A. At theSince SERCA regulates the content of calcium ions in the sarcoplasm, we evaluated the leading makers of calcium-dependent pathways: CaMKII phosphorylation, CaN and calpain-1 mRNA expression. There was a three-fold increase in CaMK II\u03b2 (Thr287) phosphorylation A and sigmRNA expression of SERCA1 and SERCA2 in the 7HSC group was significantly higher than in the C and 7HS groups D,E. mRNAThe content of oxidized tropomyosin in the 7HS group was increased by 55% when compared with the C group F. The oxUnloading resulted in a decrease in the number of slow fibers in the soleus muscle of 7HS group (by 60%) and an increase in the number of fast fibers (by 22%) when compared with the C group A,B. TreaThe cross-sectional area (CSA) of both fast and slow fibers was significantly lower in both 7HS and 7HSC groups when compared with the C group C,D. ExamThe mRNA expression of slow myosin (MyHC I\u03b2) in the 7HS group was significantly lower (by 44%) than in the C group A. TreatmNFATc1 is an activator of slow myosin expression in skeletal muscle . The nucMCIP1.4 is a marker of the NFATc1 transcriptional activity . The MCIJNK1/2 and p38 kinases can phosphorylate the NFATc1 transcription factor, causing its removal from the myonuclei and blocking its transcriptional activity ,43. TherPGC1\u03b1 is a critical regulator of mitochondrial biogenesis in skeletal muscle . PGC1\u03b1 mmRNA expression of COX I was decreased by more than 50% in the HS group when compared with the control group B. In themRNA expression of COX II and COX IV was decreased in the HS group when compared with the control group  ubiquitin ligases . The mRNProtein synthesis is regulated by p70S6k and eEF2 phosphorylation . The levThe level of eEF2 phosphorylation in the HS group was increased when compared with the control group B. Treatm\u2212/\u2212 mice and old mice with sarcopenia [Unloading-induced skeletal muscle atrophy is regulated by a number of physiological processes. The current study showed that soleus muscle atrophy during seven days of unloading was not affected by the treatment of rats with SERCA activator CDN1163. Soleus muscle weight and CSA of both slow and fast muscle fibers were decreased after seven days of unloading in groups with and without CDN1163 treatment. Unloading-induced muscle atrophy occurs in both male and female animals . The decrcopenia ,28. The rcopenia . The maxThere are several explanations for the reasons preventing soleus fatigue reduction in the current study after treatment with CDN1163. First, it could be the regulation of sarcoplasmic calcium ions concentrations and consequent regulation of gene expression and oxidative stress. Another contributor could be the regulation of fast/slow myosin expression in unloaded muscle fibers. The third is the regulation of mitochondria in unloaded muscle.Similar to the current study, the increased phosphorylation of CaMK II\u03b2 in skeletal muscle after unloading was reported previously . IntraceAnother calcium-regulated molecule with increased mRNA expression after unloading reported in the current study is CaN. It is a calcium and calmodulin-dependent phosphatase. It was previously reported that CaN content in unloaded muscle is increased simultaneously with the increase in sarcoplasmic calcium concentration . The worSERCA regulates the translocation of calcium ions from the sarcoplasm into the sarcoplasmic reticulum. Treatment with CDN1163 significantly increased the mRNA expression levels of SERCA1 and SERCA2 in unloaded muscle. The increased expression of SERCA could contribute to the increased transport of calcium ions from the sarcoplasm into the sarcoplasmic reticulum. Treatment with CDN1163 upregulated the expression of both SERCA isoforms, suggesting a specific effect in unloaded muscle.\u2212/\u2212 mice [Several previous studies used oxidized tropomyosin as an indicator of the oxidative stress index of carboxylation/decarboxylation of muscle proteins and a marker of oxidative tissue damage ,62,63. I\u2212/\u2212 mice  and in m\u2212/\u2212 mice . It was \u2212/\u2212 mice . Excessi\u2212/\u2212 mice ,65. The Phosphorylation of p38 and JNK1/2 kinases is regulated by CaMK II kinase as well as by oxidative stress ,67. In tTreatment with CDN1163 diminished the calpain-1 mRNA upregulation in unloaded muscle in the current study. Calpain-1 is a calcium-dependent protease; therefore, during unloading it can be regulated by increased calcium concentrations.To summarize, in the current study, treatment with CDN1163 during skeletal muscle unloading downregulated CaMKII phosphorylation, the content of oxidized tropomyosin, mRNA expression of CaN, SERCA1, SERCA2a, and calpain-1.Treatment with CDN1163 diminished the unloading-related changes in myosin composition. This included both the expression of the slow myosin isoform MyHCI\u03b2 and the proportion of slow muscle fibers that are resistant to fatigue. The unloading-induced changes in myosin composition presented here are consistent with the previously reported data . SeveralThe phosphorylation of NFATc1 by JNK1/2 and p38 MAP kinases can result in the decrease of its content in the myonuclei and, therefore, a decrease in its transcriptional activity ,43,75,76In addition to the effects on the slow myosin composition, the treatment with CDN1163 can also increase the resistance of the soleus muscle to fatigue via regulation of the mitochondrial proteins and DNA content. The current study showed a decrease in the content of mitochondrial DNA and TOM20 protein expression, and decreased PGC1\u03b1, COX isoforms, and mitofusins-1 and 2 mRNA expression. TOM20 is a marker of mitochondrial content ,82. ObseExcessive accumulation of calcium in the sarcoplasm increases muscle fatigue due to the increased generation of reactive oxygen species by mitochondria, the subsequent increase in the oxidation of ryanodine receptors, calcium leakage from the sarcoplasmic reticulum, and the depletion of muscle fiber calcium depots . A similPreserved mitochondrial DNA and protein content in unloaded soleus muscle of CDN1163-treated rats in this study can result in a decreased level of reactive oxygen species that cause mitochondrial stress .In addition to the regulation of NFATc1 accumulation in the myonuclei, p38 and JNK1/2 MAP kinases can regulate the increased expression of MuRF-1 ,89. The Observed in the current study, the decrease in the level of p70S6k phosphorylation and the increase in the level of eEF2 phosphorylation in unloaded soleus muscle are consistent with the previous observations ,92.Increased p70S6k phosphorylation leads to the activation of protein translation, while phosphorylation of eEF2 inhibits its activity and, therefore, leads to a decrease in the level of protein translation . TreatmeDespite the observed effects of CDN1163 on protein synthesis and degradation markers in unloaded muscle, it had no effect on soleus muscle atrophy. The observed effect in this study of CDN1163 on mTORC1-dependent p70S6k phosphorylation suggests possible activation of mTORC1 in the unloaded muscle of treated rats. Activation of mTORC1 may lead to improved mitochondrial functions via enhanced mRNA translation encoded by the nuclear genome mitochondrial genes . It is lThis research, however, is subject to several limitations. The duration of the experiments was 7 days, and the myofiber atrophy ratio depends on the time of the muscle unloading, so the results may differ after longer or shorter unloading periods . The resTreatment of rats for seven days with SERCA activator CDN1163 led to the following changes in the unloaded soleus muscle: (1) it downregulated the level of calcium-dependent phosphorylation of CaMK II and tropomyosin oxidation; (2) it prevented changes in muscle fatigue index by maintaining muscle fiber composition, as well as mitochondrial DNA and protein content; and (3) it preserved some markers of protein synthesis but did not inhibit activation of the proteolysis markers and muscle atrophy."}
+{"text": "Remote Photoplethysmography (rPPG) is a contactless method that enables the detection of various physiological signals from facial videos. rPPG utilizes a digital camera to detect subtle changes in skin color to measure vital signs such as heart rate variability (HRV), an important biomarker related to the autonomous nervous system. This paper presents a novel contactless HRV extraction algorithm, WaveHRV, based on the Wavelet Scattering Transform technique, followed by adaptive bandpass filtering and inter-beat-interval (IBI) analysis. Furthermore, a novel method is introduced to preprocess noisy contact-based PPG signals. WaveHRV is bench-marked against existing algorithms and public datasets. Our results show that WaveHRV is promising and achieves the lowest mean absolute error (MAE) of 10.5 ms and 6.15 ms for RMSSD and SDNN on the UBFCrPPG dataset. Heart rate variability is the variation in time between consecutive heartbeats. It is closely related to the autonomous nervous system (ANS), actual heart sound, blood pressure, and mental well-being . TraditiIn recent years, there has been a growing interest in heart rate variability (HRV) estimation using remote photoplethysmography (rPPG), and many researchers have focused on developing robust and accurate algorithms for this purpose. Typically, a pipeline for rPPG-based HRV estimation includes several stages, such as face detection and tracking, skin segmentation, region of interest (ROI) selection, and rPPG construction ,3,4,5. IOne such study by Mitsuhashi et al.  obtainedA wavelet-based approach was proposed by Huang et al.  and He eIn another research, Gudi et al.  sourced A deep learning approach was presented by Song et al. . AccordiA novel HRV algorithm, WaveHRV, based on the Wavelet Scattering Transform technique, followed by adaptive bandpass filtering and statistical analysis of inter-beat-intervals (IBIs);Validation of our algorithm on various public datasets, which achieved promising results;An innovative preprocessing step to filter out noisy ground truth data.All listed HRV algorithms suffer from relatively poor results when compared with ground truth contact-based values. This may be due to limitations in the non-contact measurement techniques used by these algorithms, which can result in inaccuracies and lower overall performance. Additionally, deep learning models require a large amount of data to train on, which can be expensive. Since HRV is highly sensitive to noise, improved algorithms should be devised to decrease the gap between contact and camera HRV. Therefore, in this paper, we introduce the following:The heart rate variability extraction pipeline from a video is presented in Nth order coefficients are given byr(t) is a signal, \u03bb\u03c8 is a wavelet of scale \u03bb, \u03d5 is average pooling, |\u2026| is complex-valued modulus operation and \u2217 is convolutional operation. In this paper, the Kymatio Library Remove data that have IBI \u2209 mean(segmentIBI) \u00b1 0.3mean(segmentIBI), where segment is 20\u201330 IBIsRemove SDNN > 100 ms or RMSSD > 100 msRemove data that have HRV is a sensitive biomarker and even a slight disturbance during the data collection process can alter the outcome dramatically. This paper  shows this paper  reports We reported the results of WaveHRV on publicly available datasets in After filtering out noisy ground truth data according to the criteria mentioned in t-test p-value = 0.05. Similarly, hypothesis testing for RMSSD gives p-value = 0.24. This implies that for a 95% confidence interval (CI), the average WaveHRV SDNN and RMSSD are similar or equal to the average ground truth SDNN and RMSSD. Correlation plots of SDNN and RMSSD for preprocessed UBFC rPPG are demonstrated in Bland-Altman plots of SDNN and RMSSD of three preprocessed datasets namely UBFC rPPG, VIPL-HR, and Stroop are shown in p-values of 0.83 and 0.09 for SDNN and RMSSD respectively. It means that at a 95% CI, average WaveHRV SDNN and RMSSD are not different from ground truth SDNN and RMSSD.Stoop dataset results  indicatep-value = 0.02) and RMSSD \u22121.58 ms . Paired t-test reveals that at 95% CI mean WaveHRV SDNN is different from the mean ground truth SDNN, while the mean WaveHRV RMSSD is equal to the mean ground truth RMSSD. Finally, MAHNOB-HCI has SDNN \u22122.72 ms mean error and RMSSD \u22128.5 ms mean error corresponding to p-values = 0.02 and 10\u22124 respectively. Statistical Analysis at a 95% Confidence Interval implies that average WaveHRV SDNN and RMSSD are different from average ground truth SDNN and RMSSD.Furthermore, looking into Bland-Altman plots of the VIPL-HR dataset in The performance of WaveHRV on physiological stress-related metrics is given in The Baevsky stress index (BaevskySI), also known as the strain index, characterizes a person\u2019s sympathetic nervous system activity (SNS) and is a good indicator of physical and mental load. It has been revealed that both the MAE and SD of VIPL-HR and MAHNOB-HCI datasets have significantly dropped after the implementation of the data preprocessing step mentioned in Furthermore, from Similar conclusions can be attained from statistical analyses and Bland-Altman plots: when the subjects are not under frequent motion and in a well-lit environment like UBFC rPPG and Stroop datasets, the average WaveHRV SDNN and RMSSD are similar to ground truth SDNN and RMSSD. However, for more challenging, real-life scenarios where there is significant motion and poor lighting conditions like VIPL-HR and MAHNOB-HCI, mean WaveHRV results are different from mean ground truth results.In this paper, we have presented WaveHRV, a novel algorithm for HRV extraction from a portable camera. We benchmarked our algorithm against other methods and demonstrated that WaveHRV outperforms other methods on publicly available datasets. Furthermore, we presented a straightforward yet powerful technique to clean ground truth data and highlighted its performance. We also demonstrated the potential for an off-shelf camera to measure stress and mental well-being via the Baevsky stress index. A further direction for this research would include the improvement of HRV algorithms under challenging scenarios such as large head movements and dim lighting to reduce the discrepancy between camera HRV and contact HRV. In addition, work could examine the relationship between HRV and different stress, energy, and productivity metrics."}
+{"text": "In the equal probability sequence, the same consistent and inconsistent characters were presented randomly with identical occurrence probability (p\u2009=\u20090.25). Significant neural representation activities of word frequency were observed in the equal probability sequence. By contrast, neural representations of sub-lexical statistics only emerged in oddball sequences where short-term predictions were shaped. These findings reveal that the statistical properties learned from long-term print environment continues to play a role in current word processing mechanisms and these mechanisms can be modulated by short-term predictions.Statistical learning (SL) plays a key role in literacy acquisition. Studies have increasingly revealed the influence of distributional statistical properties of words on visual word processing, including the effects of word frequency  and mappings between orthography, phonology, and semantics . However, there has been scant evidence to directly confirm that the statistical properties contained in print can be directly characterized by neural activities. Using time-resolved representational similarity analysis (RSA), the present study examined neural representations of different types of statistical properties in visual word processing. From the perspective of predictive coding, an equal probability sequence with low built-in prediction precision and three oddball sequences with high built-in prediction precision were designed with consistent and three types of inconsistent  Chinese characters as visual stimuli. In the three oddball sequences, consistent characters were set as the standard stimuli (probability of occurrence  This statistical structure includes both information at the sub-lexical level, such as orthographic regularities , orthography-to-phonology (O-P) consistency  and orthography-to-semantics (O-S) consistency6, and information at the lexical level such as word frequency  that also captures some of the statistical structure in the mappings from O-P and O-S at the whole-word  level1.Reading is an important social skill that enables us to extract the wisdom of others from a range of symbols. These symbols are visual words with various orthographic features. Proficient reading requires the assimilation of statistical regularities present in the writing system1 (SL), which refers to learning based on some aspect of the statistical structure of input elements, primarily their frequency, variability, distribution, and co-occurrence probability7. SL can occur implicitly and has been observed to be demonstrated across a variety of both linguistic and nonlinguistic contexts13. The investigation into the role of SL in language acquisition originated from the seminal study conducted by Saffran and her colleagues14, which demonstrated that infants possess sensitivity to transitional probabilities (TPs) of syllables within a continuous speech stream. It was seen as providing a viable explanation for identifying word boundaries. In the many hundreds of studies that followed the original auditory TP learning task by Saffran et al.14, researchers often tailored the task\u2019s parameters  to address closely related questions15. TP is a type of conditional probability, which essentially reflects the raw frequency of co-occurrence17. This also makes it limited by the coverage of conditional probability, that is, TP can mainly explain the learning of adjacent regularities . However, SL applies not only to the acquisition of adjacent regularities, but also to the learning of nonadjacent regularities \u2013 regularities that exist over an intervening element . Distributional SL account for the learning of non-adjacent relations, which is termed for sensitivity to those aspects of the statistical structure of the input that capture the frequency and variability of exemplars in the input17. In contrast to conditional SL, which focuses on the acquisition of local statistical structure such as TP learning, distributional SL places greater emphasis on acquiring global statistical structure. These two types of SL may have different contributions to learning different language knowledge, for example, word segmentation depends more on conditional SL, while orthographic and morphological regularities of written words rely more on distributional SL3.The acquisition of such regularity information is considered to depend on statistical learning15. Although there is research evidence to confirm the relationship between these short-term SL effects measured by the classical SL paradigms and reading performance23. The external validity of this evidence is limited because these studies typically deal with learning of a single type of regularity over a short period of time (see refs. 15 for detailed discussion). The complexity of the regularities in a given domain, whether a spoken language or a printed text, is often significantly different from these simplified learning problems. A major review recently highlighted the importance of evidence from tasks that tap regularities characteristic of real-world environments across different domains24. In fact, there has been a study that has made attempts in this direction1. This study used an alternative approach that focused on identifying individual differences in children\u2019s reliance on long-term accumulated statistical regularities as reflected directly in their word naming behavior. The researchers found that the measures of reliance on O-P and O-S had much stronger predictive power than the much weaker correlations observed in correlational studies of \u201ctypical\u201d SL tasks and reading outcomes. The authors argued that these results suggest that these more complex regularities are the ones that play a role in reading acquisition, more so than the simplified regularities typically studied in classical SL paradigms. The study of Siegelman et al.1 considering the long-term accumulated knowledge of statistical regularities in written language is a new attempt, it can help inform researchers about the subtle regularities that humans are able to assimilate \u201cin the wild\u201d.Classical SL experiments represented by TP learning  and artificial grammar learning tasks have typically considered learning on the timescale of minutes. However, SL has a continuous learning trajectory that begins with low-level coding of uncertainty (for single stimulus tokens) and ends with long-term accumulated knowledge of the environment25 (WFE), whereby high-frequency words exhibit processing advantages over low-frequency words across a range of tasks . In addition, the WFE has also been verified by several ERP studies, the typical example is low-frequency words produce larger N400 amplitudes than high-frequency words . At the sub-lexical level, a variety of findings make clear that skilled readers read faster and more accurately words with O-P mappings that are more consistent at multiple grain-sizes . Additionally, several event-related potential (ERP) studies have validated the consistency effects (low-consistency words evoke larger ERP amplitudes than high-consistency words) of Chinese characters within the time windows of several ERP components35. However, these results can be explained by different theories  and therefore cannot be conclusively attributed to the effects of SL. If we can surpass the impact of statistical properties on word recognition and demonstrate that the human brain genuinely decodes these statistical attributes, then we will provide more compelling evidence for long-term SL effects. In order to obtain this critical evidence, we intend to draw momentum from multivariate pattern analysis (MVPA) in cognitive neuroscience. In contrast to traditional univariate analysis techniques , MVPA considers the relationship among multiple variables , which can capture the information that is not detectable in univariate analysis and improves the sensitivity of identifying differences among experimental conditions39. The most popular applications of MVPA are decoding  and, more recently, representational similarity analysis42 (RSA).So far, there is only circumstantial evidence on whether the distributional statistical properties of print have been implicitly learned by proficient readers through long-term exposure experience to print environments. At the lexical level, the typical evidence is the word frequency effect38. By comparing the decodability of all possible pairwise combinations of stimuli, a representational dissimilarity matrix (RDM) is calculated. That is, for each pair of stimuli, the distance between their activation patterns  is computed using one of several distance metrics . Critically, we can calculate a model RDM based on experimental design in the same way, for example, we can calculate pairwise correlations between all words in a word recognition experiment using the frequency (obtained in the corresponding corpus) of each word as its characteristic. Further, by comparing RDMs from brains and models , researchers can know whether brain representations reflect stimulus properties. For data with high temporal resolution such as EEG, a series of RDMs can be created for each time point and used to investigate the temporal dynamics of representations over time.RSA is based on the assumption that stimuli (or manipulated features) with more similar neural representations are more difficult to decode, while those with more distinct representations are expected to be easier to decode3. These orthographic distributional regularities are mainly placed among two elements called radicals, one that provides information about how that character is pronounced , and the other providing information about its meaning . Approximately 80%\u221290% of Chinese characters are compound characters consisting of these two radicals44. For example, in Chinese character \u6e56/hu:2/ (to lake), the phonetic radical \u80e1/hu:2/ reveals its pronunciation (/hu:2/), while the semantic radical \u6c35 indicates its semantic category (water-related concept). In reality, however, many radicals clearly deviate from positional and mapping  regularities in varying degrees. The obvious feature of the positional distribution of characters is that the majority of them are left-right horizontally structured characters . This, in turn, implies the presence of other structures . For mapping regularities, approximately 35% of Chinese characters are phonetic inconsistent characters that differ from the common pronunciations of other characters made up of the same phonetic radicals. Furthermore, 12% are semantic inconsistent characters that differ from the common meaning categories of additional characters that are made up of the same semantic radicals46. This allows us to better manipulate various statistical attributes of words independently. Specifically, characters that are inconsistent in any one dimension may be consistent in the other dimensions . Therefore, we can simultaneously manipulate the consistency of orthographic, phonological, and semantic within a single Chinese character, and construct the appropriate RDM for each consistency feature.The Chinese writing system, which has rich quasi-regularity and distributional properties, may provide excellent material for examining the processing of complex statistical regularities in reading49, then predictive coding emerges as an enchanting framework to elucidate the underlying predictive processes. Within the framework of predictive coding, learning is a continuous optimization of a generative model that reflects the world around us and attempts to explain the causes of the sensory inputs51. This optimization process is achieved by the continuous interaction between top-down flow of predictions and bottom-up flow of prediction errors (the difference between sensory inputs and predictions). Extensive evidence to date indicates that neurophysiological and behavioral responses can unveil musical and linguistic SL effects in the predictive coding framework . In neural response, several studies have examined the role of predictions in regulating the intensity of electrophysiological activities. For example, researchers56 have reported that tones with higher TP  evoked weaker event related potential (ERP) amplitudes compared to tones with lower TP  (see ref. 61 for review). In addition, starting from another aspect, several studies64 focus on the processing of prediction errors due to SL. These studies explored mismatch negativity (MMN), an ERP differential component interpreted as the neural manifestation of prediction error69, and reported a \u201cstatistical MMN\u201d evoked by probabilistic properties  of sounds acquired through SL rather than their acoustical features. The MMN is typically measured with a passive oddball paradigm (employ tasks that are not related to the attributes of the stimuli being explored), in which a series of standard stimuli are interspersed with acoustic deviants . Within this paradigm, the predictable sounds (standards) are subtracted from the surprising sounds (deviants) to obtain the MMN. As in the statistical MMN experiments, by manipulating the feature types of standard and deviant stimuli, researchers can examine prediction errors for different physical or abstract properties. Substantial evidence has accumulated suggesting that prediction error caused by visual deviants can be reflected in visual MMN . At the outset of vMMN research, studies focused on simple physical property deviances ; later vMMN has been investigated for abstract property deviances . To date, no vMMN studies have explored the prediction error response related to probabilistic properties of printed words. This raises two important questions. The first is whether distributional statistical regularities in real-world written language are reflected in the neural processing of visual words by skilled readers. If the answer is yes, the second question is whether the neural activities in response to these statistical properties are continuously modulated by prediction error in the current sensory environment.Assuming that prediction is crucial for SL86. Equal probability sequences were used to provoke the neural activities related to visual words that depended on the participants\u2019 long-term experience. There was no clear predictive evidence in this sequence, and all the characters were presented randomly. Oddball sequences were used to provoke the neural activities related to the same characters when the participants were given clear short-term predictions. In these sequences, consistent characters were repeated with a high probability (p\u2009=\u20090.75) and one of the three types of inconsistent characters occasionally appeared with a low probability (p\u2009=\u20090.25), giving a total of three oddball sequences. To track the neural representations of various statistical regularities, we performed a time-resolved RSA for the electrophysiological activities of the corresponding characters separately in the oddball  and equal probability conditions. Based on the widely reported robust effects of SL on reading in previous studies, we hypothesize that significant neural representations of statistical information (including word frequency and three types of consistency) can be detected even during implicit visual word processing. Starting from the principle that the volatility of the environment will modulate the intensity of prediction error related neural activities88, we propose a second hypothesis that sub-lexical statistical information (consistency) will be detected stronger neural representation activities in the oddball condition than in the equal probability condition.The present study aims to address these questions and considers the real-world statistical regularities of the Chinese writing system. We designed an experiment using the passive oddball paradigm containing equal probability sequences. For the materials, we carefully selected three consistent Chinese characters and nine inconsistent Chinese characters . These inconsistent characters were divided into three categories, each with low consistency only in a particular sub-lexical dimension . This setting of stimuli is similar to the multi-feature paradigm in auditory MMN studies, where properties of multiple dimensions are manipulated simultaneously in the same stimulusThe mean hit rates and mean false alarm rates of the button presses as well as the mean press latencies of correct responses for each experiment are summarized in Table p\u2009<\u20090.05, cluster-corrected significance level p\u2009<\u20090.05). The evidence for representations of frequency  was absent in the EEG signals of oddball condition. In addition, only frequency (150\u2013215\u2009ms) and orthographic consistency (159\u2013217\u2009ms) representations can be detected in the neural activities corresponding to the characters in equal probability condition . For characters in the oddball condition, the RSA results revealed the time course of the representations of orthographic consistency , phonological (220\u2013300\u2009ms) and semantic (193\u2013308\u2009ms) consistency information in oddball condition can be more strongly predicted by neural patterns than those in equal-probability condition Fig. . It is wTo examine the generalizability of the results, we performed RSA analysis based on rating RDMs in the same manner. We obtained the representation dynamics of orthographic consistency (161\u2013263\u2009ms and 268\u2013381\u2009ms), phonological consistency (304\u2013394\u2009ms and 485\u2013551\u2009ms), and semantic consistency (164\u2013267\u2009ms and 304\u2013394\u2009ms) in the oddball condition Fig. . These tThe statistical analysis of the representation differences revealed stronger representation of semantic consistency in oddball condition Fig. . Howeverp\u2009=\u20090.0007, effect size\u2009=\u20090.65; cluster2: sum [t]\u2009=\u2009\u2013643.61, p\u2009=\u20090.0013, effect size\u2009=\u20090.85) and right electrodes  for orthographic-related vMMN. Additionally, a phonological-related vMMN effect was also found in both electrodes that corresponded to a left cluster  and a right cluster . Moreover, a vMMN effect was also discovered for inconsistent semantic characters in the left cluster  and a right cluster . The time range for significant clusters for various vMMNs across different ROI are shown in Table According to a cluster-based permutation test, the vMMN effect was identified for inconsistent orthographic characters across ROIs in the left and right hemisphere, respectively Fig. . The cluThe current work explored two important questions closely related to SL: first, whether the statistical properties of real-world print environments as discerned through long-term experience are reflected in the neural processing of words by skilled readers, and second, whether the neural activities in response to these statistical properties are modulated by short-term implicit predictions. Equal probability sequences and oddball sequences were used in this study to detect the neural processing of Chinese characters based on long-term experience and short-term prediction, respectively. We applied RSA to elucidate the neurodynamic pattern of the multidimensional statistical information processing of Chinese characters. First, we found that word (character) frequency  could be rapidly recovered from EEG response patterns in the equal probability condition. This result answers the first question and confirms the existence of implicit neural representations of statistical properties, derived from the long-term textual environment, in word reading. Second, three types of statistical properties at the sub-lexical level  could be extracted in the oddball condition. In addition, the representation strength of these sub-lexical statistics was significantly stronger in the oddball than in the equal probability condition. These differences in the strength of neural representations are consistent with the significant vMMN activities we obtained. These results answer the second question, revealing the existence of prediction error signals driven by prediction related to sub-lexical statistical properties, and confirming the modulation role of prediction precision in the neural representation of these properties.25 have indicated that the WFE may be part of the indirect evidence for the relationship between SL and reading. Behaviorally, the WFE is a highly replicable and reliable effect that relates to the observation that high-frequency words are processed more efficiently than low-frequency words . In the temporal dynamics of neural processing, most studies94 have found ERP differences between high-frequency (HF) words and low-frequency (LF) words at around 200\u2009ms after word onset. The WFE has also been reported in several recent ERP studies96 focusing on Chinese characters. Wang and Maurer\u2019s study95 found ERP differences at the time interval of 172\u2013253\u2009ms. Another study96 used an implicit color decision task to report divergence between the ERPs evoked in response to HF characters and the ERPs evoked in response to LF characters over a time period of 210\u2013222\u2009ms. The ERP time window corresponding to the WFE is consistent with our RSA results. By breaking through the limitation of univariate analysis , we provide more powerful evidence for the implicit and rapid processing of word frequency information. However, almost no significant neural representation activities in response to sub-lexical level statistical properties were detected in the equal probability condition. This suggests that skilled adult readers may be sensitive to larger-grain size statistical properties  in their daily word processing. In fact, this finding is consistent with prior studies of alphabetic languages. Some studies have reported that adults are particularly impacted by body rime rather than grapheme-phoneme level regularities . It has been reported that, as humans develop, they become increasingly reliant on O-P regularities at larger grain sizes29 .The present study showed that stable neural representations of word (character) frequency could be detected in the equal probability condition in which there were no clear short-term predictions, and these representations were active for periods of about 150\u2013220\u2009ms after the character was presented. The frequency information implied by the characters could only be interpreted by the statistical distribution characteristics of the long-term reading environment. This provides direct evidence for the effect of SL on visual word recognition and reveals that the statistical properties obtained through long-term SL continues to play a role in current word processing mechanisms. Prior studies65. In simple terms, predictive coding is an implicit process that creates an internal model of sensory inputs with the aim of minimizing surprise97 . In order to minimize the cost (free-energy50) of reducing prediction errors, the system assigns different weights to real-time prediction errors according to the variability of the environment , which causes the same external input to evoke different levels of response depending on the environment in which it is located. This precision setting mechanism can be conceptually understood as a form of meta-learning: learning what is learnable or estimating the predictability of new contingencies98. Returning to the current experiment, a lack of effective prediction  was observed within the equal probability sequences. All characters were decoded using the established neural processing patterns, corresponding to neural activities in skilled adult readers that are sensitive to lexical rather than sub-lexical statistical properties. However, the situation changed in the oddball sequences, where the presence of unambiguous prediction  caused any inconsistent information to be evaluated as worthy of learning, thereby activating additional neural activity that was different from the existing processing patterns. This was ultimately reflected in stronger and more explicit neural representations of the various types of sub-lexical statistical properties. In fact, a recent study using the oddball paradigm reported similar findings88. That study investigated the neural mechanisms that underpin SL and volatility attuning, and showed that, in stable conditions, SL  was improved compared to the volatile conditions, prediction errors increased, and there was a greater modulation of neuronal gain, forward connections, and backward connections.Neural representations of various sub-lexical level statistics  become detectable when subjects are exposed to visual inputs that contain clear short-term predictions . In addition, the intensity of these neural representations is significantly stronger than those evoked in the equal probability condition. These findings reveal a short-term plasticity mechanism for neural representations of sub-lexical level statistical properties. We suggest that this plasticity mechanism, as observed in the present experiment, can be appropriately explained by the predictive coding framework100. The prediction error in the current experiment could be clearly attributed to the violation of the consistency category. There are three reasons for this attribution. (1) The vMMNs were calculated via subtraction of the ERPs evoked in response to the equiprobable stimuli from the ERPs evoked in response to the deviant stimuli. The equiprobable and the deviant stimuli were comprised of the same Chinese characters, so that the vMMN cannot be described as involving physical, orthographic, phonological, or semantic differences between them. (2) As the probability of presenting the equiprobable and the deviant stimuli were exactly equal , then the vMMN cannot be explained as a difference in refractoriness between the ERPs evoked by them. (3) The vMMN cannot be explained as a violation of a phonological category or a semantic category since there was no reducible phonetic or semantic category within the deviant or equiprobable characters in the oddball sequence. In summary, we believe that our study defines a class of prediction error responses driven by consistency category violation. These categories of consistency at the sub-lexical level were only available from statistical mappings of a long-term reading environment, so these findings further illustrate the dynamic interaction between short-term plasticity driven by prediction error and long-term experience. We believe that these findings advance the understanding of the mechanisms of SL and provide an interesting perspective on SL from the predictive coding framework.We obtained significant \u201cgenuine\u201d vMMN responses within the active time window of neural representations of various forms of sub-lexical statistical properties. The vMMN responses obtained in the oddball paradigm were interpreted as a neural manifestation of the prediction error signal84. For example, there are hundreds of colors that fall under the category \u201cblue\u201d even though they have different combinations and values of hue, saturation, and brightness. In the field of character processing, the vMMN effects of word meaning101 and phonological83 categorization have been investigated. However, the consistency features of concern in the current study are rather special. Phonological consistency, for example, represents how frequently a phonetic radical represents a given sound by calculating the relative proportion of characters with the same pronunciation among those that share the radical . This means that phonological consistency is essentially a continuous variable ranging from 0 to 1, and the other two types of consistency are essentially the same. However, the consistency effect reported in previous studies reflects a dichotomy. These studies often anchor certain most common  body rime correspondences as consistent words and others as inconsistent words, and found that consistent words are read aloud faster and more accurately than inconsistent words . This consistency effect has also been verified with Chinese characters105. With regard to neural mechanisms, previous fMRI studies106 have reported greater activation in the left inferior frontal gyrus, the left temporoparietal  region, and the left temporal\u2013occipital junction when naming inconsistent characters compared to consistent ones. Additionally, several ERP studies have validated the consistency effects of characters within the time windows of the N170, P200, and N400 components . In fact, the current experimental design perpetuates the above idea of exploring the consistency effect. Although we did not focus on specific ERP components, our results provide new evidence for the rationality of this line of research. Furthermore, from the perspective of predictive coding, the vMMN activities evoked by consistency category violation may reveal the true state of the brain\u2019s SL product . In other words, the rich distributional statistical information obtained through SL can be aggregated and reclassified into consistent and inconsistent features and reflected in different neural representation patterns.Finally, let us consider an additional question about categorization. Categorization is a prerequisite for generating vMMN responses, only when the target characteristics of the deviant and standard stimuli are classified as different types, can the deviants produce violations  of the predictions that are established by the standard. Categorization is a basic process that includes visual perception, and this process goes beyond the physical characteristics of the stimuliWe should also note some limitations in the scope and methodology of the current study. First of all, in terms of the scope of current research, although our study was designed to examine the neural representation of statistical properties contained in print learned through long-term exposure. However, the relevant problems are not included in the scope of traditional SL research, and our study is only an exploratory investigation closely related to SL. In addition, previous MMN studies exploring TP learning usually controlled the absolute pitch of the subjects, but the current study did not consider the characteristics of the subjects\u2019 hearing. As no studies have so far explored the relationship between orthographic regularity learning and these hearing characteristics, there are therefore associated potential limitations to the generalizability of our current findings. Finally, in the experimental design, we draw on previous oddball studies that focus on social category information, so that different oddball sequences have the same short-term predictions (by setting the same consistent characters as the standard stimuli). The underlying assumption of this design is that there are no long-term prediction differences between different types of stimuli . However, as with most category-based oddball studies, there is not much direct proof of this hypothesis, so we consider it as another potential limiting factor in the explanatory power of the current results.In summary, our multivariate RSA study demonstrated the contribution of long-term SL to the neural activity related to current word processing. Importantly, we found that the short-term prediction provided by the visual input environment evoked neural representations of sub-lexical statistical properties. In addition, we also obtained significant vMMNs, which indexed an implicit processing mechanism that operates within the predictive coding framework. These findings reveal the link between predictive coding and SL, and confirm the short-term plasticity of neural activities corresponding to long-term SL.All participants gave oral and written, informed consent in accordance with procedures that were approved by the ethics committee at the School of Psychology, Shaanxi Normal University . The protocols adhered to the Declaration of Helsinki.101 using EEG to investigate implicit character recognition during the oddball paradigm and is comparable to other EEG studies using RSA analysis to explore the representation dynamics of language processing108. Participants were recruited from the undergraduate and postgraduate student population at Shaanxi Normal University and were paid 60 RMB for their participation. All participants reported no speech or hearing problems and had no prior history of neurological or psychiatric abnormalities.Forty-eight healthy young adults were recruited, with three being excluded for excessive EEG artifacts. The final sample of 45 right-handed (via self-report) young adults  had an average of 12.3 years of education, and all had normal or corrected-to-normal vision. The final sample size surpassed that of similar workAn additional group of 33 paid healthy college students  were recruited to rate the orthographic consistency, phonological consistency, and semantic consistency (transparency) of each Chinese character we selected. Take the scoring of semantic consistency, one question was asked to measure semantic transparency: \u201cTo what extent do you think the radical \u201cX\u201d can represent the meaning of the Chinese character \u201cY\u201d?\u201d. For example, for the character \u201c\u6d0b\u201d, the question was \u201cTo what extent do you think the radical \u201c\u6c35\u201c can represent the meaning of the Chinese character \u201c\u6d0b\u201c ?\u201d. In a similar way, each dimension of consistency was measured on a seven-point scale, with 1\u2009=\u2009totally inconsistent and 7\u2009=\u2009totally consistent  of phonograms (45). Semantic radicals usually provide semantic information of characters, while phonetic radicals provide phonological clues. For example, in the Chinese character \u201c\u7272\u201d, the semantic radical \u201c\u725c\u201d is on the left side, while the phonetic radical \u201c\u751f\u201d is on the right. The meaning of character \u201c\u7272\u201d is \u201cdomestic animals\u201d. This means that it can easily be speculated from the semantic radical \u201c\u725c\u201d, which refers to \u201ccattle\u201d. Phonologically, the pronunciation of \u201c\u7272\u201d is \u201csheng\u201d, which is also highly consistent with the sound of the phonetic radical \u201c\u751f\u201d (sheng). Characters like \u201c\u7272\u201d are consistent characters. However, there are some characters  that do not follow the orthographic , phonological and/or semantic rules . Accordingly, these 12 Chinese characters were then divided into three consistent and nine inconsistent characters. The nine inconsistent characters were further divided into three categories, including inconsistent orthographic (IOr) characters, inconsistent phonological (IPh) characters and inconsistent semantic (ISe) characters. The three characters in each category come from different stimulus sets. In other words, all four categories  have the same number of characters and the same phonetic radicals see Fig. . The incSpecifically, compared to the consistent category, IOr characters differ with regards to the structure of characters. That is, phonetic radicals appear on the right side of the character in the consistent category. On the contrary, phonetic radicals in the IOr characters are in the less common position in a character. For example, phonetic radial \u201c\u751f\u201d posits at the right side of a \u201c\u7272\u201d, but it is at the bottom of the IOr character \u201c\u7b19\u201d. Among all characters that are \u201c\u826e\u201d, \u201c\u751f\u201d and \u201c\u7f8a\u201d, they act as phonetic radicals. The probabilities that \u201c\u826e\u201d, \u201c\u751f\u201d and \u201c\u7f8a\u201d appear on the right side of characters are 72.22%, 50.00% and 72.73%, respectively. On the other hand, the percentages of the character positions for \u201c\u75d5\u201d, \u201c\u7b19\u201d and \u201c\u75d2\u201d are 5.56%, 25%, and 18.18%, respectively . However, pronunciation of the IPh character \u201c\u6027\u201d is /xing/, not \u201c/sheng/\u201d. The phonological consistency is 0.39-0.92 for all three CC and is 0\u20130.28 for the three IPh characters  with regards to phonological consistency. The pronunciations of CC are high in phonological consistency of corresponding phonetic radicals, while the phonological consistency is low in IPh characters. The phonological consistency is defined as the proportion of a specific pronunciation among all characters that adopt the same phonetic radicals46. That is, semantic radicals of CC can reflect the meaning of corresponding characters. However, the meanings of the ISe characters cannot be speculated from the semantic radicals. For example, the semantic radical \u201c\u725c\u201d (cattle) is related to the meaning of \u201c\u7272\u201d (livestock). In contrast, the meaning of ISe character \u201c\u80dc\u201d (victory) is much different from that of the corresponding semantic radical \u201c\u6708\u201d (moon). The same as CC, characters in the IPh and IOr categories are high in the transparency of the semantic radical.Finally, the ISe characters differ from the consistent ones with regards to the transparency of the semantic radical. The CC is high in the transparency of semantic radicals, while the transparency in ISe characters is low. The transparency is defined as the connection between the meaning of the semantic radical, and the meaning of the corresponding characterp\u2009=\u20090.25; the number of presentations is divided equally among the three specific characters) across different oddball blocks , while the consistent characters (containing three specific characters) served as standard stimuli   . The task throughout the experiment was to ignore attributes of the character and to press a button with the right thumb as quickly and accurately as possible when red characters (target stimuli) were presented . The deviant stimuli in the oddball blocks would not appear twice in a row, and the target only appeared after one standard trial. Participants sat comfortably in an armchair at a distance of 60\u2009cm from the screen, and were given a break for each block that they completed. Using the E-prime software, the images of words were presented within the central visual field .The experimental procedure consisted of three oddball blocks and an equal probability block. One of the three categories of inconsistent characters (each category contains three specific characters), in turn, served as the deviant stimuli  Fig. . In the 25) Fig. . Within 25) Fig. . MoreoveA participant\u2019s response was counted as a hit if the button was pressed for less than 700\u2009ms after the character color changed. Otherwise, the response was counted as a false alarm. Hit and false alarms (FAs) rates during the color change detection task were analyzed in order to evaluate the degree of commitment to unrelated tasks of the participants.Electroencephalography (EEG) signals were recorded through the use of a 64-channel amplifier  that was mounted on an electrode cap according to the international 10\u201310 system. The online reference electrode during the data collection was CPz. The EEG data was digitized at a sampling rate of 1000\u2009Hz, and impedances were kept below 10\u2009k\u03a9 during the experiment.112. A spatially distributed subset of channels providing whole-head coverage was processed . HAPPE\u2019s artifact removal steps included bad channel rejection, removal of 50\u2009Hz electrical noise through CleanLine\u2019s multi-taper approach113, and participant artifact rejection  through wavelet-enhanced ICA with automated component rejection via EEGLAB and the Multiple Artifact Rejection Algorithm114. The average (SD) number of independent components (ICs) containing artifacts was 9.3 (3.7). Post-artifact rejection, any channels removed during the bad channel rejection were repopulated through spherical interpolation to reduce spatial bias in re-referencing. After filtered with a 0.1\u201340\u2009Hz digital Butterworth bandpass filter with a 12\u2009dB/oct roll-off, the EEG data were then re-referenced to the average reference and mean signal detrended. Epochs were created from \u2212 300\u2009ms pre-stimulus to 700\u2009ms post-stimulus for each trial and baseline corrected using the first 100\u2009ms. Any epochs with retained artifact were rejected using amplitude criteria (\u00b1100\u2009\u03bcV), as in prior research110. After epoch rejection, the average (SD) number of trials retained on inconsistent character trials were 31.56 (5.32), 31.72 (5.94), 30.61 (7.26), 29.72 (6.85), 29.33 (6.34), and 30.22 (5.94) for equiprobable IOr, equiprobable IPh, equiprobable ISe, deviant IOr, deviant IPh, and deviant ISe, respectively.Offline preprocessing, artifact removal, and data quality assessment was carried out via the Harvard Automated Processing Pipeline for EEG (HAPPE) in MATLAB115. First, we created neural representational dissimilarity matrices (RDMs) for each time point in the EEG epochs (10\u2009ms resolution), reflecting the pairwise dissimilarity of the characters\u2019 brain representations. Second, we modeled the organization of the neural RDMs using Spearman rank correlation coefficients116, which allowed us to track when representations are explained by the characters\u2019 lexical (frequency) or sub-lexical  statistical information.To track the representations of individual characters across time, we used RSA117. This resulted in a vector length of 671 features reflecting brain activity spanning 10\u2009ms.At each time point from 100\u2009ms before stimulus onset to 600\u2009ms after stimulus onset, we correlated the EEG activity between trial pairs (for the nine different inconsistent characters), separately for the oddball condition  and equal probability condition. This results in a distance value (1- Pearson correlation) that indicates the dissimilarity between character pairs according to brain activity. By repeating this procedure for each pair of characters we constructed a 9\u2009\u00d7\u20099 neural RDM Fig. . Individ118).We designed two series of model RDMs to explore and validate the representation of different statistical information in the EEG data Fig. . The firTo supplement and validate the results obtained from the predictor RDMs, we constructed another series of RDMs according to the ratings before the formal experiment (from another group of subjects), which resulted in three models of 9\u2009\u00d7\u20099 rating RDMs that corresponded to the orthographic consistency, phonological consistency, and semantic consistency dimensions of our stimuli. Specifically, we calculated the pairwise Euclidean distance between the rating score of each character in each dimension.120. In order to explicitly compare lexical and sub-lexical level statistical information models, lexical (frequency) RDM would be excluded when computing a partial correlation between neural RDM and each sub-lexical  RDMs, and vice versa. We calculated the partial correlation coefficients at each time point for each subject. These partial correlation coefficients served as an indicator of the time course of different statistical information dimensions in the EEG data.The lower off-diagonal of each matrix was extracted as vectors to calculate the Spearman rank correlations between each model and the EEG data. Since some models were correlated, excluding the other models allowed us to separate the contribution of these models from each otherIn addition, in order to detect the difference in the representation strength of different statistical information between the oddball condition and the equal probability condition, we calculated the difference of all partial correlation coefficients of each subject under two conditions .121. Using the maximum cluster size method, significant temporal clusters were defined as adjacent time points that all exceed a statistical cutoff (cluster-inducing threshold). This cutoff was determined through a sign permutation test according to the distribution of t-values from 10,000 permutations of the measured correlation values. The 95th percentile of the t-value distribution was used as the clustering induction threshold of each time point . To identify significant clusters, we determined the 95th percentile of maximum cluster sizes across all permutations . This approach provided us with significant temporal clusters in which correlation showed significant effects.We performed a non-parametric statistical approach for all RSA results which did not depend on assumptions of the data distributions99.To verify the existence of prediction error responses, we examined vMMN activities using a data-driven approach. The differential waveforms of characters with different inconsistent categories were obtained by subtracting the ERPs of the corresponding deviant stimuli from the ERPs of the corresponding equiprobable stimuli. This method allows the comparison of ERPs that are evoked by the deviant of the oddball sequence to the ERPs that are evoked by physically identical stimuli from a sequence without any particular frequent (standard) stimulus123. Activity considered as \u201cgenuine\u201d vMMN  emerges when the oddball deviant evokes a larger negativity than the control stimuli99. Next, a cluster-based permutation test was utilized to search \u201cgenuine\u201d differential activity between the ERPs of deviant and equiprobable stimuli124. We conducted this analysis through the use of the Fieldtrip toolbox125 in MATLAB. We developed grand-averages of differential waveforms across two regions of interest (ROI) that correspond to the left  and right  posterior occipital-temporal electrodes . For each time point (within 0\u2013600\u2009ms) at left or right electrodes, the clusters were formed through two or more neighboring time points whenever the t values (obtained by two-tailed t-test) exceeded the cluster threshold (0.025). The number of permutations was set to 10,000, and the corrected significance level was set to 0.05. That is, when the clustering level error probability of a cluster was less than 0.05, then it was considered that there were significant effects in the corresponding period . We will report the temporal range of the significant negative clusters, their mass  and the effect size of the average over the rectangular shape surrounding a cluster for each inconsistent category.The method of equal probability control was suggested in order to deal with repetition effects due to refractoriness that was assumed to be present in the deviant, minus standard activity that was obtained in classical oddball paradigmsFurther information on research design is available in the Supplementary informationReporting Summary"
\ No newline at end of file