Background: Resveratrol (RES) has a protective effect on acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). Our purpose was to conduct a meta−analysis to investigate the efficacy of RES for ALI/ARDS in animal models.Methods: PubMed, EMBASE and Web of Science were searched to screen relevant preclinical trials. The standardized mean difference (SMD) was used to compare the lung injury score, lung wet−dry weight ratio (W/D ratio), tumor necrosis factor−α (TNF−α), interleukin−1β (IL−1β), IL−6, IL−10, the number of neutrophils in bronchoalveolar lavage fluid (BALF) and the total protein in BALF between the treatment and control groups. SYRCLE’s risk of bias tool was used for quality assessment.Results: A total of 17 studies published from 2005 to 2021 were included in our study to calculate the SMD with corresponding confidence interval (CI). As compared with controls, RES significantly decreased the lung injury score  and W/D ratio . RES also reduced the number of neutrophils in BALF  and the total protein in BALF . Furthermore, RES was found to downregulate proinflammatory mediators such as TNF−α , IL−1β  and IL−6 . But RES had little effect on the anti−inflammatory mediators such as IL−10 . Sensitivity analysis and stratified analysis were performed for the outcome indicators with heterogeneity.Conclusion: RES treatment is effective on reducing the severity of ALI. However, more animal studies and human trials are needed for further investigation. Our study may provide a reference for preclinical and clinical studies in the future to some extent. ALI/ARDS is a clinically critical illness caused by severe infection and trauma, and its pathological manifestations include acute diffuse lung inflammation, alveolar damage, increased pulmonary capillary permeability, hemorrhage, pulmonary interstitial edema and massive inflammatory cell infiltration . In clinResveratrol  was found in different fruits such as grapes, raspberries, mulberries, blueberries and groundnuts . PreviouThe systematic review and meta−analysis were conducted by following the Preferred Reporting Items for Systematic Reviews and Meta−Analysis (PRISMA) guidelines . PubMed,The inclusion criteria were set up based on the PICOS−scheme . Published studies were included if they met the following criteria: 1) ALI animal model; 2) setting up the RES experimental group; 3) setting a control group with placebo; 4) the effectiveness of RES on the ALI animal models were measured; 5) results were expressed or could be converted to mean and standard deviation; 6) Other criteria: published in English.The exclusion criteria were as follows: 1) review articles, letters and case reports; 2) repeated publications and abstracts without full text; 3) not reporting the number of animals in each group; 4) animals treated with RES analogues; 5) studies that only tested the effects of RES combined with other chemicals or drugs (such as nanoparticles).All of the data were extracted independently by two reviewers (Tang and Fu). Any disagreements were resolved by consensus with a third reviewer (Wei). The information collected from each study included the following details: 1) author and year of publication; 2) rodent models, including species, gender, age and modeling methods; 3) anesthetic agents and route of administration; 4) RES treatment, including intervention dosage , time point of treatment and route of administration; 5) assessment time; 6) outcome measurement; 7) methodological quality score. If a study comprised multiple experimental groups distinguished by different dosage, time point of treatment and assessment time that were compared with the control groups, these experimental groups would be considered as independent comparisons. For continuous variables, mean and standard deviation were used for statistical calculations. For graphical data, the GetData Graph Digitizer software (version 2.25) would be used to measure values for mean and standard deviation from highly magnified images. For standard deviation without direct report, we would calculate it by multiplying the reported standard error of mean (SEM) by the square root of the group size.Two reviewers (Tang and Fu) independently evaluated the quality of each study according to the SYRCLE’s risk of bias tool (an adaptation of the Cochrane Risk of Bias Tool) . Any dis2) statistic. I2 < 50% indicated low heterogeneity and the fixed−effects model was used, while I2 ≥ 50% indicated high heterogeneity and the random−effects model was used  5.4 software  and STATA 12 software . The SMD was used to compare the effect of RES on ALI between the treatment and control groups . Heterogwas used . Sensitiwas used . We condwas used . p < 0.0The flow diagram of the literature search process is shown in n = 7), Sprague–Dawley (SD) rats (n = 5), Wistar rats (n = 1), C3H/HeJ mice (n = 1), BALB/c mice (n = 1) and ICR mice (n = 2). These studies involved male animals (n = 12), female animals (n = 4) and one study did not report the gender of animals. Adult animals were used in all studies. Further, these studies chose cecal ligation and puncture (CLP) (n = 3), intraperitoneal (i.p.) injection of LPS (n = 4), intratracheal (i.t.) instillation of LPS (n = 5), administration of staphylococcal enterotoxin B (SEB) (n = 2), severe acute pancreatitis (SAP) (n = 2) and traumatic spinal cord injury (n = 1) to induce the ALI model. In all of them, RES was administered before ALI (n = 9), after ALI (n = 7) or before and after ALI (n = 1). RES was administered by i. p. (n = 9), intragastric administration (n = 6), injection through the vena dorsalis penis (n = 1) and one study did not report the way of administration. And single dose (n = 12) or multiple doses (n = 5) of RES were used in all studies. The detailed characteristics of the included studies are presented in The animal models were established in C57BL/6 mice . RES reduced the W/D ratio by an SMD of −1.92 . RES downregulated proinflammatory mediators IL−1β by an SMD of −2.51 . RES downregulated IL−6 by an SMD of −2.26 . So, we did further stratified analysis from animal race, animal gender, methods to induce ALI model, time point of RES treatment, RES treatment route, number of doses and total dosage of RES in lung injury score, W/D ratio, IL−1β and IL−6.The RES could significantly reduce the lung injury score by an SMD of −2.06 . RES upregulated anti−inflammatory mediators IL−10 by an SMD of 2.80 . RES reduced the total protein in BALF by an SMD of −5.59 . So, we did further sensitivity analysis in TNF−α, IL−10 and total protein in BALF. The treatment of RES had a favorable effect on the number of neutrophils in BALF by an SMD of −3.03 . Therefore, further stratified analysis was not performed.Furthermore, RES was found to downregulate proinflammatory mediators TNF−α by an SMD of −2.02 , the time point of RES treatment (p = 0.98) and RES treatment route (p = 0.67). Significant differences among subgroups were found in animal race (p = 0.0006), animal gender (p = 0.001), number of doses (p = 0.04) and total dosage of RES (p = 0.004). Among them, there was a clear difference in therapeutic effect by the number doses of RES and animal gender. Compared with single dose , the effects size of multiple doses  was less than single dose. In addition, the effect size was greater in males  than in females   .p = 0.08), the time point of RES treatment (p = 0.42), RES treatment route (p = 0.05), number of doses (p = 0.23) and total dosage of RES (p = 0.05). Significant differences among subgroups were found in animal race (p = 0.0005) and ALI induction methods (p = 0.0001)  .p = 0.55), ALI induction methods (p = 0.73) and RES treatment route (p = 0.55). Significant differences among subgroups were found in time point of RES treatment (p = 0.02), number of doses (p = 0.003) and total dosage of RES (p = 0.01). Among them, there was a clear difference in therapeutic effect by the number doses of RES. The effect size was greater in single dose  than in multiple doses   .p = 0.19), ALI induction methods (p = 0.16), the time point of RES treatment (p = 0.62) and RES treatment route (p = 0.27). Significant differences among subgroups were found in animal gender (p = 0.03), number of doses (p = 0.06) and total dosage of RES (p = 0.002). Among them, there was a clear difference in therapeutic effect by the number doses of RES. The effect size was greater in single dose  than in multiple doses   .A sensitivity analysis was conducted to evaluate the stability of our results. The number of neutrophils in BALF, lung injury score, W/D ratio, IL−1β and IL−6 were not significantly affected by any study for the pooled SMD . But TNFp = 0.000) and W/D ratio (p = 0.002). Under the circumstances, to estimate the missing studies and recalculate effect estimates, the trim−and−fill analysis was conducted for lung injury score and W/D ratio. The results of lung injure score  and W/D ratio  were consistent, indicating no “missing” studies (p = 0.07).We performed publication bias analysis for outcome measures with more than 10 included studies. Conspicuous publication bias for lung injure score and W/D ratio were suggested by visual inspection of the funnel plot . Then, t studies . The fun studies , and no RES is a promising lung injury protector that has shown beneficial outcomes in animal models of ALI . To our Over the past few decades, researches have sought for various potential natural compounds to treat ALI, while RES became a popular choice for many studies . RES conIn this meta−analysis, RES had significant protective effects in reducing lung injury score, W/D ratio, TNF−α, IL−1β, IL−6, the total protein and number of neutrophils in BALF. But the heterogeneity was statistically significant among lung injury score, W/D ratio, IL−1β and IL−6 groups. Thus, we performed stratified analysis from animal race, animal gender, methods to induce ALI model, time point of RES treatment, RES treatment route, number of doses and total dosage of RES.First, our stratified meta−analysis of lung injury scores and W/D ratio found that animal race was a source of heterogeneity. The researchers selected C57BL/6 mice, SD rats, Wistar rats and ICR mice for the study. Among them, Wistar rats has the largest effect size, but only one study chose the Wistar rats. A small number of studies might lead to an overestimate on the efficacy of RES. Furthermore, we found that most studies selected C57BL/6 mice and SD rats for their studies. Second, we found that animal gender was a source of heterogeneity through our stratified meta−analysis of lung injury scores and IL−6. We found that most studies selected male animals, which also had larger effect sizes. Previous studies have reported that male mice are more likely to develop ARDS . HoweverOur stratified meta−analysis of W/D ratio found that the source of heterogeneity was related to the methods to induce ALI model. Among them, CLP had the largest effect size, but it didn’t mean that CLP was the most suitable modeling method of ALI. Furthermore, we found that most of the studies selected LPS i. p. And LPS i. t. for study. A previous study found that the ALI model induced by intratracheal instillation of LPS was more effective than the ALI model induced by CLP . HoweverFirst, our stratified meta−analysis found that the route of RES treatment was not a source of heterogeneity. Second, the time point of RES treatment was found to be a source of its heterogeneity through stratified meta−analysis of IL−1β. We found that more studies gave RES after ALI modeling, but these included smaller effect sizes. Therefore, we can’t confirm when it is better to give RES. Third, our stratified meta−analysis of lung injure score, IL−1β and IL−6 found that the number of doses and total dosage of RES were a source of heterogeneity. We found that most studies selected single dose, which also had larger effect sizes. In our included studies, the total dosage of RES was 0.1–−700 mg/kg. Among them, most of the studies selected 10–−50 mg/kg for study, which had the largest effect size. Previous studies have shown, increasing the dose of RES can reduce lung injury scores and inflammatory factor levels . But higIn our meta−analysis, we have made great efforts to obtain objective results. First, to provide the most complete evidence, we searched for reports relevant to the field. Then, to reduce potential publication bias of included studies, two reviewers independently assessed and extracted all data, and differences were handled reasonably. Finally, our meta−analysis showed that RES treatment had positive outcomes in ALI animal models. Sensitivity analysis confirmed stable results of the number of neutrophils in BALF, lung injury score, W/D ratio, IL−1β and IL−6, while stratified analysis detected heterogeneity in the result of lung injury score, W/D ratio, IL−1β and IL−6 from methodological differences. To some extent the results suggested positive effect of RES treatment, indicating RES may serve as a new therapeutic strategy for the clinical ALI patients.There are several limitations in our meta−analysis. 1) Our study only included data published in English and studies that had been published, and some negative results were less likely to be published. Therefore, this meta−analysis may have exaggerated the effect size. 2) Our study included comparatively small number of published studies with a highly significant heterogeneity. Although a further stratified analysis was conducted, the differences among most subgroups were still not significant. These results may be related to insufficient sample size. Therefore, sufficient evidence needs to be provided in the studies with large sample sizes in the future. 3) We did not analyze the timing of outcome assessment. Different time of assessment will also have different results. For example, at 48 h after RES intervention, the inflammatory factor level of TNF−α was found to be lower than that at 3 h. IL−6 levels were found to decrease at 3 h after RES intervention, but increase at 48 h . TherefoOur systematic review and meta−analysis revealed that RES treatment had an improvement in pathology, pulmonary edema and inflammatory markers in ALI animal models. Limitations of the experimental design should be considered when interpreting the results. The results of this study may provide a reference for preclinical and clinical studies in the future to some extent with great significance for human health.


The pDTT derivatives studied include polymers with simple thiohexyl end-caps or modified with AQ or methyl groups by Steglich esterification. All polymers were shown to be depolymerized using catalytic amounts of electrons delivered by AQ•−. For pDTT, as little as 0.2 electrons per polymer chain was needed to achieve complete depolymerization. We hypothesize that the reaction proceeds with AQ•− as an electron carrier (either molecularly or as a pendant group), which transfers an electron to a disulfide bond in the polymer in a dissociative manner, generating a thiyl radical and a thiolate. The rapid and catalytic depolymerization is driven by thiyl radicals attacking other disulfide bonds internally or between pDTT chains in a chain reaction. Electrochemical triggering works as a general method for initiating depolymerization of pDTT derivatives and may likely also be used for depolymerization of other disulfide polymers.We report the use of electrogenerated anthraquinone radical anion (AQ• First, 55 μM AQ•− was electrogenerated by applying an electrolysis potential of −1.07 V. The stability of AQ•− is observed to be relatively good until a degassed solution of pDTT, HTpDTT, or (Me)pDTT (200 μM resulting concentration) is added (defined as time zero). For pDTT and HTpDTT, AQ•− is consumed within 1–2 s, while, for (Me)pDTT, a sharp decrease in [AQ•−] to ~25 μM is detected within seconds, whereafter a slow decrease is observed, reaching ~15 μM after 20 s and zero after 70 s , dimethyldisulfide (DMDS), and 2,2′-dithiodipyridine (DTDP) in the presence of AQ. These compounds nicely correspond to or mimic the end product, the backbone, and the end-caps of the polymers.−) and −1.7 V vs. SCE (AQ•− to AQ2−), respectively (−) still appears Nernstian. In contrast, the second wave at −1.7 V (AQ•− to AQ2−) exhibits a noticeable increase in the peak current, coming along with a diminishing current of the oxidation wave at −1.5 V (AQ2− to AQ•−) on the anodic sweep. The anodic peak at −0.8 V (AQ•− to AQ) is of similar size as before but begins to overlap with the oxidation of the thiolates at −0.2 V. This would suggest that transfer of electrons to (Me)pDTT from the more potent electron donor, AQ2−, can take place, while AQ•− has no such ability on the time scale of a voltammetric cycle. Furthermore, the additional reduction peaks observed at −2.7 V indicate that, besides direct reduction of the polymer itself, other disulfide-containing species, originating from the depolymerization, may contribute to these signals.Reduction of AQ takes place as two Nernstian one-electron transfer processes at −0.9 V vs. SCE (AQ to AQ•ectively a. The voectively a. On the−) looks similar to that of AQ alone, while the second reduction wave comprising two peaks shifts in a positive direction. The reduction wave of the polymer itself shifts towards a more negative potential and becomes larger in the presence of AQ. For HTpDTT the electron transfer pertaining to the first reduction peak at −0.9 V (AQ to AQ•−) is Nernstian, while the second reduction peak shifts to a less negative potential. The reduction peak for the polymer itself shifts to a more negative potential (−2.7 V vs. SCE) and becomes broader, but with little change to the peak current.Cyclic voltammograms of pDTT and HTpDTT with and without AQ can be found in the − to AQ and dihydroanthraquinone , wi, wi®  is more profound. Earlier studies on indirect reduction of simple disulfides demonstrated that electrogenerated AQ•− can be used to reduce disulfides to thiolates on a time scale of min/h , together with cDTT, contain hydroxyl groups, which may protonate AQ•emically . Both th of cDTT , which d of cDTT . In thiseresting  as all hRegarding the three model compounds, both DMDS and cDTT comprise aliphatic disulfide bonds, thus mimicking the end-caps of HTpDTT as well as the dialkyl disulfide bonds along the backbone of all polymers. Note that cDTT also contains hydroxyl groups and is cyclic, yet without ring tension. Likewise, DTDP is meant to serve as a model compound mimicking pyridyl disulfide end-caps. Acknowledging that the four polymers, pDTT, (Me)pDTT, (AQ)pDTT, and (AQ)(Me)pDTT, contain a mixed alkyl pyridyl disulfide end-cap, the reduction potential of this end-cap should thus be located somewhere between those of DMDS and DTDP.kDMDS = 2 × 102 M−1 s−1, kDTDP = 105 M−1 s−1), because of a greater extent of electron delocalization onto the pyridyl groups. This decreases significantly the reorganization energy of the stepwise dissociative electron transfer  from Fischer Scientific  and EDC•HCl from Fluorochem . All chemicals were used without further purification. The polymers pDTT, (Me)pDTT, (AQ)pDTT, and (AQ)(Me)pDTT were synthesized following previously established protocols with a few modifications  = 2 mM, [DMDS]/[DTDP][RSSR] = 5/5/3.2 mM, half peak potentials of AQ locked at −0.87 and −1.61 V vs. SCE at sweep rate = 1 V s−1  = 2 mM, [DMDS]/[DTDP] = 24 mM, half peak potential of AQ locked at −0.87 V vs. SCE at sweep rate = 1 V s−1  = 3 mM, and ~1.5 mg mL−1 of polymers were added, providing polymer concentrations for pDTT (0.60 mM), HTpDTT (0.41 mM), (Me)pDTT (0.39 mM), (AQ)pDTT (0.39 mM), and (AQ)(Me)pDTT (0.31 mM) as listed. The catholyte solution was purged with argon for at least 10 min prior to recordings. A cyclic voltammogram was recorded just prior to electrolysis to determine the cathodic peak potential of AQ (or pendant AQ); electrolysis was conducted at a potential 150 mV more negative than this potential and stopped when a specific charge in the range of 0.50–0.96 C was consumed (corresponding to ~3 electrons per polymer chain in each case).Electrolysis was conducted using a CH Instrument (601D) potentiostat in a two-necked two-chamber H-cell with 0.03 M Bu−1 of polymer, i.e., HTpDTT (0.27 mM), (Me)pDTT (0.26 mM), (AQ)pDTT (0.26 mM), and (AQ)(Me)pDTT (0.21 mM), and stopped after consumption of 0.055–0.072 C. Finally, electrolysis of pDTT passing 0, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, and 3.0 equiv. of electrons per polymer chain was conducted with pDTT (0.40 mM); volume of catholyte = 15 mL and [AQ] = 3 mM. Aliquots for SEC were collected after the specific amount of charge was consumed.Likewise, electrolyzes with passing of 0.5 electrons per polymer chain were conducted using 1 mg mL4NBF4/DMF purged with argon. With a dip-in probe inserted in the catholyte, spectra of AQ•− were recorded and compared with those reported in literature [− was monitored at the wavelength 556 nm with a time resolution of 0.1 s. Lambert–Beer law was used to calculate [AQ•−] from the absorbance at 556 nm and by employing −1 cm−1. Solutions of AQ•− were prepared by electrochemical reduction of 2 mM AQ in 0.1 M Bu4NBF4/DMF. The electrolysis was stopped once the absorbance at 556 nm reached 0.6 corresponding to [AQ•−] = 55 μM. While recording the absorbance at 556 nm, 100 μL of a degassed polymer solution of pDTT, HTpDTT, or (Me)pDTT was injected to achieve [polymer] ≈ 0.2 mM. The decay of [AQ•−] was traced from this point on, defined as t = 0.UV–Vis spectra were collected from the cathode chamber of an H-cell using an Agilent Cary-60 UV–Vis absorption spectrometer with a dip probe from C-Tech with pathlength of 1 cm. The cathodic compartment was filled with 3 mL solution containing 2 mM AQ in 0.1 M Buterature . Absorba−. Only a catalytic amount of electrons was needed to achieve full depolymerization, which would be in line with an intermolecular chain reaction from a mechanistic point of view. The reaction is rapid (within seconds) and applicable to all derivatives regardless of end-cap and whether they contain an internal proton donor or not. Triggering can be completed using either AQ in solution (intermolecular) or pendant AQ moieties (intramolecular). We propose a mechanism where dissociative electron transfer from AQ•− to a polymer disulfide bond generates a thiyl radical and a thiolate. The former may be engaged in reaction with other disulfide bonds, creating a new disulfide and a thiyl radical, eventually leading to smaller disulfides. The proposed mechanism explains both the catalytic nature and rapidity of these depolymerizations.We successfully synthesized poly(dithiothreitol) derivatives by exchanging end-caps and/or introducing various pendant groups via Steglich esterification. All polymers were shown to be depolymerized once triggered by electrogenerated AQ•


Wnt signaling pathway is an evolutionarily conserved pathway responsible for neurogenesis, axon outgrowth, neuronal polarity, synapse formation, and maintenance. Downregulation of Wnt signaling has been found in patients with Alzheimer’s disease (AD). Several experimental approaches to activate Wnt signaling pathway have proven to be beneficial in alleviating AD, which is one of the new therapeutic approaches for AD. The current study focuses on the computational structure-based virtual screening followed by the identification of potential phytomolecules targeting different markers of Wnt signaling like WIF1, DKK1, LRP6, GSK-3β, and acetylcholine esterase. Initially, screening of 1924 compounds from the plant-based library of Zinc database was done for the selected five proteins using docking approach followed by MM-GBSA calculations. The top five hit molecules were identified for each protein. Based on docking score, and binding interactions, the top two hit molecules for each protein were selected as promising molecules for the molecular dynamic (MD) simulation study with the five proteins. Therefore, from this in silico based study, we report that Mangiferin could be a potential molecule targeting Wnt signaling pathway modulating the LRP6 activity, Baicalin for AChE activity, Chebulic acid for DKK1, ZINC103539689 for WIF1, and Morin for GSk-3β protein. However, further validation of the activity is warranted based on in vivo and in vitro experiments for better understanding and strong claim. This study provides an in silico approach for the identification of modulators of the Wnt signaling pathway as a new therapeutic approach for AD.The online version contains supplementary material available at 10.1007/s11030-022-10395-8. Wingless and Integrated (Wnt)  compriseContinuous generation of new cells occurs throughout adulthood; however, there is a limited capacity for the regeneration of neuronal cells in the adult brain. Recently, the discovery of stem cells in the restricted areas of the brain  has brouWnt signaling pathway can be broadly classified into canonical and non-canonical depending on the involvement of β-catenin. Canonical Wnt signaling begins with the binding of Wnt protein to F-class G protein-coupled transmembrane seven helical frizzled receptor and coreceptor lipoprotein receptor-related protein 6 (LRP6). This leads to hyperphosphorylation of disheveled (Dvl) causing the disassembly of the destruction complex comprising of Axin, Adenomatosis polysis coli (APC), and GSK-3β –11. The The extracellular protein Dickkopk-1 (DKK1) prevents the interaction of LRP6 with Wnt thereby negatively modulating the canonical Wnt signaling pathway. Similarly, Wnt inhibitory factor (WIF1) is known to inhibit the Wnt signaling as it directly binds to different canonical and non-canonical Wnt ligands Wnt3a, Wnt4, Wnt5a, Wnt7a, Wnt9a, and Wnt11. WIF1 also plays a critical role in the embryonic developmental stage during axis formation. During the early stages of zebrafish brain generation, significant down-regulation of WIF1 and activation of canonical Wnt signaling was observed .Alteration in Wnt pathway leads to age-related diseases like osteoporosis, Parkinsonism, Alzheimer’s disease (AD), and colon cancer . IncreasThe significance of the computation-based designing of the drug has been proved for several diseases including neurodegenerative disease –20. NatuAll computational-based studies were performed on the Maestro platform, involving LigPrep, the protein preparation wizard, GLIDE (Grid-based Ligand Docking with Energetics), and Desmond tools by Schrodinger Inc.In structure-based molecular modeling, the use of accurate protein structure is important. Downregulated Wnt signaling has been linked to AD. In the present study, proteins linked with Wnt signaling pathway have been selected to explore the binding affinity, interactions of the selected phytomolecules and virtually evaluate their potential to upregulate Wnt signaling. RCSB PDB id: 2YGO , 3S2K 2, 1Q5K 2, and 4M0WIF1 is known to inhibit the Wnt signaling as it directly binds to different canonical and non-canonical Wnt ligands. The PDB id 2YGO which represents human WIF domain-EGF-like domain 1 of WIF1 in complex with 1,2-dipalmitoylphosphatidylcholine was selected for identifying molecules with good interaction and possible WIF1 inhibiting potential. Increased expression of Dkk-1 is seen around the amyloid plaques in the brain of AD patients and the neurons expressing p53 resulting in the Wnt signaling loss in AD . InhibitThe X-ray crystallographic structures of the above-mentioned proteins  were imported, pre-processed, and minimized using the protein preparation wizard. In these steps, missing side chains and amino acids were filled, heavy atoms and water molecules were removed, and restrained minimization was done to generate the lowest energy protein structure at neutral pH. All the important amino acids are retained in protein structure during protein preparation. For protein GSK-3β, AChE receptor grid was generated based on the co-crystallized ligand using the ‘Receptor grid generation panel’ from GLIDE  module, Phytomolecules (1924) from the Biogenic subset of the ZINC database  were preAfter ligand preparation, molecular docking was performed using high throughput virtual screening (HTVS), followed by Standard precision (SP) and extra precision (XP) mode, using the GLIDE module of Maestro, Schrodinger suite . DockingThe relative binding energy of 20 XP docked protein–ligand complexes was calculated with each selected protein, using Prime-Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) –37. PrimThe binding free energy of all the selected proteins–ligand complex is calculated using the formula:∆G bind = G complex–(G protein + G ligand).where G = MME (molecular mechanics energy) + GSGB  + GNP (nonpolar solvation).The ADME analysis was calculated for the top 20 hits for each protein, using the ‘QikProp’ module of Maestro, Schrodinger suite . The ‘QiThe ‘Desmond’ module of Maestro, Schrodinger suite, was used to carry out MD simulation of top two hits with all five of the selected proteins. In the Desmond module, the orthorhombic simulation box with specific dimensions using an explicit solvent system simulated under different conditions was used. In the current study, two hits for each protein were selected for MD simulation, based on the results obtained from XP docking, binding interactions, and ADME analysis. MD simulation was done in three steps, namely system builder, minimization, and MD simulation. During the first step, the docked protein–ligand complex was subjected to a system builder using a predefined SPC solvent system. The SPC solvent system is mainly used to create orthorhombic boundary conditions. Also, by the addition of sodium ions, the negative charge was neutralized in the model. In the next step, the generated model was subjected to minimization, which is further balanced at 1 bar pressure using NPT  ensemble and 300 K pressure. The MD simulation was performed for 50 ns, in which for 50-ps frame was captured and saved into trajectory. Overall, 1000 frames were captured during MD simulation.After MD simulation, calculation of RMSD of protein and ligand as well as analysis of the interactions shown by ligand during entire simulation duration was done. RMSD measures the average change in displacement of selected atoms for a particular frame with respect to a reference frame.N is the number of atoms in the atom selection. tref is the reference time, and r׳ is the position of the selected atoms in frame xThe RMSD for frame x is:3 with the druggability score (Dscore) of 1.291 and site score of 1.204 for protein WIF1. The identified pocket for WIF1 protein comprised of the total surface area of 3821.811 Ǻ2 which included hydrophobic region of 526.63 Ǻ2, hydrophilic area of 1969.72 Ǻ2, and hydrogen bond acceptor region of 600.446 Ǻ2. Similarly, for DKK1 protein three druggable sites were identified with the best site as site1 with 307.671 Ǻ3 volume, site score of 0.878, and Dscore of 0.998. The identified site 1 pocket for DKK1 protein comprised of the total surface area of 5811.98 Ǻ2 which included hydrophobic region of 46.31 Ǻ2, hydrophilic area of 3172.79 Ǻ2, hydrogen bond acceptor region of 1624.40 Ǻ2, and hydrogen bond donor region of 1531.69 Ǻ2. Sitemap generated five druggable pockets for protein LRP6 with the best site as site3 of volume 308.7 Ǻ3 with Dscore of 1.042 and sites score of 1.147 containing the residues involved in the binding to DKK1. The identified site 3 pockets for LRP6 protein comprised of the total surface area of 871.75 Ǻ2 which included hydrophobic region of 83.06 Ǻ2, hydrophilic area of 713.35 Ǻ2, hydrogen bond acceptor region of 252.79 Ǻ2, and hydrogen bond donor region of 468.27 Ǻ2. The list of the generated pockets for different proteins with the Dscore, Site scores, and the residues involved in the pocket is represented in Table For three of the proteins, LRP6 (PDB id: 3S2K chain A), DKK1 (PDB id: 3S2K chain X), and WIF1 (PDB id: 2YGO), the co-crystalized ligand structures were not present. Hence, the SiteMap prediction tool was used to predict the druggable binding site. SiteMap prediction led to the identification of a druggable site of volume 573.496 ǺThe validation of the grid generated was done by redocking the co-crystalized ligand using the generated grid followed by superimposition and comparison of the RMSD. The redocking was performed followed by alignment with the aligned poses as shown in Fig. In the current study, we have listed, analyzed, and explained the dock score, and binding interactions of the top five ligands for each protein after docking in XP mode of docking.AChE enzyme is involved in the regulation of the level of the neurotransmitter, acetylcholine mediated through the hydrolysis of acetylcholine into choline and acetate. In AD, the level of acetylcholine neurotransmitters is reduced. FDA-approved AChE inhibitors act by increasing the level of acetylcholine in the brain. The active site for human AChE has shown a deep gorge of 20Ǻ depth with the catalytic triad formed by residues HIS447, GLU334, and SER203 . DonepezScutellaria baicalensis. Hydroxyl group of 7-O-glucuronide of baicalin forms hydrogen bond interactions with ASP74, Similarly, hydroxyl and oxy group of benzopyran ring of baicalin forms H-bond interaction with TYR 341and PHE295 residues, respectively; benzene ring of benzopyran ring is involved in the formation of π–π interaction with TRP286. The docking scores of top molecules were found to be in the range of − 14.205 to − 10.987 kcal/mol. The list of the top two ligands along with their dock score, binding energy, and interactions is shown in Table Currently, in this study, we found the top docking score of − 14.205 kcal/mol shown by Mangiferin. Mangiferin, a xanthone member consisting of 1,3,6,7-tetrahydroxyxanthen-9-one and a beta-D-glucosyl residue at the 6-position, is present at a significant level in the mango fruit, peel, leaves, and kernel. Glucosyl residue of Mangiferin showed the hydrogen bond interactions with amino acid residues SER203, and HIS447 involved in the triad of the active site and xanthone ring formed π–π interaction with TRP286 residue as shown by the standard known AChE inhibitors. Similarly, Baicalin showed the second highest dock score of − 13.224 kcal/mol. Baicalin  belongs to the flavone class of glycoside chiefly found in the herb ., to understand the effect of the single point mutation in LRP6 and its binding affinity with DKK1 suggests the role of specific residues in the binding with DKK1 [LRP6 acts as a coreceptor for Wnt ligands that along with the Frizzled receptor activates the Canonical Wnt/β-catenin signaling pathway. Sequence analysis has revealed that LRP6 proteins are composed of four repeats of extracellular YWTD domain (P1-P4) paired by Epidermal growth factor (EGF)(E1-E4) repeats ranging from amino acid 21 to 1246 followed by three LDLR type domains . The fouith DKK1 . Single-Hydroxyl groups in glucosyl moiety of mangiferin showed the hydrogen bond interactions with ASP668, LEU755, TYR800, and hydroxyl and oxy groups attached to xanthone ring of mangiferin formed H-bond interactions with GLU708, MET710, LEU753, and THR797 indicating that mangiferin binds in the same pocket as that of DKK1. Similarly, compound calystegine showed a docking score of − 10.487 kcal/mol with its hydroxyl groups forming hydrogen bonds with residues LEU667, MET710, LEU753, LEU838, ASP878, and the amine group of azabicycloctane group of calystegine formed a salt bridge with ASP878. The top five compounds showed dock scores in the range of − 10.694 to − 8.913 kcal/mol and free binding energy from − 44.19 to − 22.38 kcal/mol. Top two compounds with dock score, interacting residues, and binding energy are listed in Table DKK1 is an extracellular secretory protein with high affinity for LRP6, and Kremen, promoting endocytosis of LRP6 followed by its degradation making LRP6 unavailable for Wnt signaling . ProteinTerminalia chebula, showed the second highest dock score of − 9.972 kcal/mol. Hydroxyl groups attached to isochroman ring of chebulic acid from H-bond interactions with the residues HIE229, GLN235, ARG236, THR221, carboxyl group formed H-bond interactions with HIS223, CYS239, and salt bridge formation with LYS222. Interaction with residues THR221 and ARG236 was found common in most of the top hit molecules. The detailed information of 2D ligand interaction, docking score, and binding energy of two top hit molecules is listed in Table Currently, in our study, the top site generated by the sitemap tool with the best site score and Dscore including the important residues as suggested by the experimental and computational-based method was selected for the screening of the phytomolecules. Mangiferin has shown a top dock score of − 11.15 kcal/mol among all the molecules with the hydroxyl groups in its glucosyl moiety forming hydrogen bonding with residues SER192, CYS201, and LYS208. Hydroxyl groups and oxy groups of xanthone ring formed H-bond interactions with THR221 and HIE229 residues. Chebulic acid, a phenolic compound isolated from the ripened fruits of The involvement of the GSK-3β has been well established in neuropathological disorders like amyloid deposition, gliosis, and tau hyperphosphorylation. Tideglusib, ATP non-competitive GSK3β inhibitor, has completed phase II of the clinical trial and has been recognized as an orphan drug for the treatment of rare tauopathy by the Food and Drug Administration (FDA). GSK-3β contains two major domains: N-terminal β-strand domain extending from the amino acid residues 25–138 and an α-helical C-terminal domain with residues 139–343. The interface of these two domains contains an ATP-binding site that is connected by a glycine-rich loop and hinge region. The ATP binding pocket involves residues LYS85, GLU97, ASP113, TYR134, VAL135, THR138, ASN186, LEU188, CYS199, ASP200 . The co-Currently, screening of phytochemicals with virtual docking study for GSK-3β enzyme resulted in the identification of mangiferin as the hit molecule. Hydroxyl groups in the glucosyl moiety of mangiferin showed hydrogen bond interaction with amino acid residues LYS183, ASP200, and salt bridge interaction with ASN186 residue with an XP score of − 10.344 kcal/mol. Hydroxyl group of xanthone ring of mangiferin formed H-bond interaction with ASP133 residue. Morin, a pentahydroxy flavone has been proved to possess antioxidant, antihypertensive, neuroprotective activity. The hydroxyl group of morin showed two hydrogen bond interactions with the amino acid residue VAL135. From the binding interactions of the hit molecules with the protein, we observed the residues VAL135 and ASP200 to be common and important. The detailed information of 2D ligand interaction, docking score, and binding energy of the top two hit molecules is listed in Table . have revealed the involvement of both WIF domain and EGF domain in the binding of Wnt. Their mutagenesis study to reveal the involvement of areas in WIF domain for recognition of Wnt3a found that the mutation in residue MET77 was observed to show 10 times decrease in the binding affinity to Wnt3a [WIF1 binds to Wnt proteins thereby, preventing the binding of Wnt to Frizzled receptor and inhibiting the Wnt signaling pathway. WIF-1 consists of N-terminal region, WIF domain (38–177 amino acid residue), five EGF-like domains each with 31–33 residues extending from 178 to 338 residues, and hydrophilic C terminal. Based on site-directed mutagenesis, biophysical and cell-based in vitro assays Malinasuskas et alto Wnt3a .In the present study, docking of natural phytomolecules led to the identification of mangiferin as the hit molecule showing strong binding with WIF1 protein with dock score of − 13.546 kcal/mol. Glucosyl residue of mangiferin hydrogen bonding interaction with amino acid THR167 and the hydroxyl group of xanthone ring of mangiferin formed H-bond with PRO78 residue, π–π interaction with PHE89 and PHE167 residues of WIF1 domain. MET77 residue formed the boundary and nonbonding interaction for the top hit molecules. ZINC103539689 molecule showed dock score of − 12.667 kcal/mol and formed π–π interaction with residue PHE173. Other hit phytomolecules showing stable binding energy of − 34.70 to − 71.13 kcal/mol and docking scores ranging from − 12.680 to − 9.756 kcal/mol. Top two hit molecules with the interacting residues are listed in Table Binding free energy was calculated and tabulated for all the top five compounds from docking simulations. The top molecules for AChE showed binding energy in the range of − 34.53 to − 57.34 kcal/mol. Amorphastilbol (ZINC5158604) showed the highest binding energy of − 57.34 kcal/mol, followed by mangiferin of − 52.65 kcal/mol which is tabulated in Table The success and discovery of a new drug not only depend on its target specificity and selectivity but also depends on pharmacokinetic properties. The selected hit molecules for different target proteins were analyzed using the QikProp module of Schrodinger suite. The selected hit molecules molecular weight was between 130.0 and 725.0. QPlogS is the predicted aqueous solubility in mol dm-3 all the compounds except nicotine have values within the acceptable range of − 6.5–0.5. QPlogPo/w predicted octanol/ water partition coefficient which is acceptable between − 2.0–+ 6.5 all compounds fall well within the acceptable arrange showing hydrophobic and lipophilic balance which is essential for the drug to be absorbed by the body and reach to the target site. QPPCaco stands for predicted Caco-2 cell permeability which signifies gut blood barrier permeability only 5 compounds tridolgosir, butein, scutellarein, curcumin, and resveratrol were in the acceptable range. All the molecules satisfied Lipinski rule of five. QPlogHERG is the predicted IC50 value for blockage of HERG K + channel signifying the potential of the compound to show toxicity. Most of the hit molecules have shown acceptable value for HERG toxicity except ZINC5158604, ZINC100067274, and ZINC103539689 molecules. Mangiferin has an experimental Log P value of + 2.73  that supMD simulation is considered a fundamental computational tool for analyzing dynamic events of ligand–protein complexes. MD simulation has several advantages over XP-docking. It overcomes the problem of the rigid nature of protein in normal XP-docking. In MD simulation, the protein–ligand complex is in dynamic nature, and it also allows conformational changes of ligands within the active site of a protein. This phenomenon also mimics the scenario of a biological system, where the stability of the protein–ligand complex is evaluated in the simulated water boundary. Considering docking score, non-bonding interactions with vital amino acid residues, and binding energy, the top two hits were identified and selected for MD simulation with 2YGO, 3S2K Chain A (LRP6), 3S2K Chain X (DKK1), 1Q5K, and 4M0F proteins. In the current study, an attempt to evaluate its binding potential with other modulators of Wnt signaling and its possible role in AD has been made.nd top hit ligand Figs. The frame was taken every 50 ps for the simulation period of 50 ns which resulted in 1000 frame generation and was saved in trajectory. Based on results obtained from MD simulation, a simulation interaction diagram was generated with root mean square deviation (RMSD), and a Protein–Ligand interaction plot was computed for 2YGO, 3S2K, 1QSK, and 4M0F proteins with the ligand mangiferin and 2The complex 1A was initially stable for 0–24 ns, but the further drift was observed for 24–-50 ns. Average RMSD values for AChE and mangiferin were found to be 2.4 Å and 0.9 Å, respectively. RMSD values of both protein and ligand were found to be within range for complex 1A. In complex 1A, polar interactions with SER203 and HIS447 were retained and lost with THR75. New hydrophobic bond interaction was observed with PHE295, retained with TYR337 and TYR341, and lost with other amino acids as compared to XP docking. The charged negative interactions were retained and were lost with ASP74 and GLU202. New π–π stacking interaction was observed with TYR341 and lost with TRP286. H-bond interactions with SER203 and HIS447 were retained and were lost with TYR72. New H-bond interactions were found with ASP74, PHE295, and GLY122. Complex 1B was stable throughout the simulation of 50 ns RMSD of both protein and ligand which was less than 2 Å. Hydrogen bond interactions like PHE295, ASP74 which were seen in the XP docking pose was retained, and new bonding ASP72 was formed during MD simulation. TYR341 which showed Hydrogen interaction in XP docking pose was changed to a π–π type of interaction in MD. New π–π type of interaction with TRP86, TYR337, and TYR124 was seen during MD simulation.In acetylcholine esterase amino acids TRP86, TYP286 and TYR341, SER293, TYR72, and TYR124 play a critical role in the inhibition of enzyme [In complex 2A, initial drift was observed for 0–8 ns; eventually, ligand–protein complex got stabilized for the simulation period of 50 ns. Additionally, in complex 2A, a drift was observed in between for 13–17 ns. The RMSD value of protein and ligand for complex 2A was observed to be 2 Å within an acceptable range (1–3 Å). H-bond interactions were retained with ASP668, LEU755, TYR480, MET710, and THR797, which showed new interactions with ALA 881, ARG886, VAL712, LEU796, ASP878, MET877, ASN794, ILE798, and THR839, and lost with GLU708 and LEU573 in complex 2. These new H-bond interactions may contribute to the additional stability of complex 2A. The new water bridge interactions were found with amino acids VAL881, ARG886, VAL712, TYR800, MET710, ILE798, THR839, and ASP668, as compared to XP docked poses. The hydrophobic interactions were found with VAL881, retained with MET710, VAL712, LEU755, LEU796, TYR800, MET877, LEU880, and ILE798, and lost with other amino acids. The polar interactions were retained with ASN794, THR797, THR839 and lost with GLU840 and GLU887. The charged positive interaction with ARG886 was retained in MD simulation. The new charged negative interaction was found with GLU708 by retaining other interactions. Several in vivo studies have proved the benefit of mangiferin in memory improvement based on the anti-inflammatory and acetylcholinesterase activity , 50. ComAfter comparing the RMSD plot for both the ligands, we could conclude that the RMSD plot for the mangiferin–LRP6 complex was more stable with less fluctuation indicating the strong and good binding throughout the simulation duration. In LRP6 interactions with amino acids GLU663, ILE681, TYR708, ASP748, SER749, ARG751, TRP767, GLY769, ARG792, ASN794, LEU810, ASP811, ASP830, TRP850, and MET877 are important. However, the occurrence of the interactions with these residues was missing for both ligands in the current MD simulation.Complex 3A was stable for 22–28 ns, 31–36 ns, 38–41 ns, and 46–49 ns, and drift was observed for 0-–22 ns, 28–31 ns, 36–38 ns, 41–46 ns, and 49–50 ns. The RMSD values were found to be 10.5 Å and 7.5 Å, for protein and ligand, respectively. In complex 3A, polar interactions were retained with HIS223 and THR221 and lost with HIS229, SER192, and GLU235. Formation of new H-bonds was observed with HIS223, CYS237, and CYS239, retained with THR221 and lost with SER192, CYS201, LYS208, and HIS229. Hydrophobic interactions were retained with TYR238 and CYS237, whereas lost with other amino acids. New hydrophobic interaction was observed with CYS239 and water bridge-type interaction with CYS237 and CYS239. The charged positive interaction was lost with ARG191, ARG203, LYS208, LYS222, and ARG236, as compared to XP ligand interactions. Complex 3B was stable for 5–15 ns and drift was observed for 0–5 ns and 15–50 ns. Protein and ligand showed RMSD values of 14 Å and 10 Å, respectively. During MD simulation, new hydrophobic and water bridge types of interactions were formed with ILE247 and lost other hydrophobic interactions as compared to XP docking. Similarly, new polar interaction was formed with HIS261 and lost other polar interactions. New π–π cation interactions were observed with ARG246 and ARG224 as compared to XP ligand docking interactions. Charged negative interaction was lost with ARG236, and newly charged negative interactions were observed with LYS249, ARG224, ARG259, LYS222, and ARG246. Additionally, water bridge type of interactions was observed with amino acid residues ARG259, HIS261, LYS222, and ILE247. The salt bridge type of interaction with LYS222 is retained in MD simulation as compared to XP ligand docking interactions.Based on previous reports, amino residues CYS217-ARG237 and CYS233-CYS253 compromise the cysteine-rich domain and it is important for inhibition of Wnt signaling pathway . From thIn complex 4A, the initial fluctuation was observed for 0–15 ns, the complex got stabilized for 15–27 ns, and further drift was observed for 27–50 ns. In complex 4A, H-bond interaction was retained with ASP133 and lost with LYS183, ASN186, and ASP200. The new H-bond interaction was found with VAL135 as compared to XP docking. Hydrophobic interactions were retained with VAL70, ALA83, LEU188, and VAL135 and lost with other amino acids. All charged positive interactions were lost in MD simulation as compared to XP docking. Charged negative interactions were lost with ASP181 and ASP200 and retained with ASP133. Complex 4B possesses the RMSD value 2 Å and 1.50 Å. In complex 4B, the difference between RMSD values was not more than 3 Å, which indicates complex 4B is stable. As compared to XP docking, the hydrophobic bond interactions were retained with LEU188, PRO136, VAL135, and ILE62 and lost with CYS199, VAL110, ALA83, LEU132, TYR134, and VAL70. Amino acid residues PRO136 and VAL135 also showed water bridge type and hydrogen bond interactions. The charged positive interactions with ASP200 are retained, and new hydrogen bond and water bridge type of interactions were observed with ASP200 as compared to XP ligand docking interactions. Polar interaction was lost with THR138 as compared to XP docking, and new polar and hydrogen bond interactions were observed with ASN64. Similarly charged negative interaction was retained with ARG141in MD simulation, and amino acid residue ARG141 also showed hydrogen bond interaction as compared to XP docking.In GSK-3β protein, the co-crystallized inhibitor showed that the inhibitor occupies the hinge region along with ATP pocket through the formation of three hydrogen bonds with residue VAL135 . In our RMSD plot of different proteins and mangiferin is shown in Fig. Wnt signaling plays an important role in the cell growth, proliferation, embryogenesis, and organogenesis process. Phytochemical database was considered for screening their potential to modulate markers of the Wnt signaling pathway. Different proteins  linked with Wnt signaling pathway and AChE have been selected to explore the binding affinity, interactions of the selected phytomolecules and virtually evaluate their potential to upregulate Wnt signaling. The inbound ligand was considered for the generation of the grid for docking simulation in the case of proteins GSK-3β and AChE, whereas a possible drug-binding pocket predicted by the sitemap tool was chosen for grid generation for the proteins LRP6, DKK1, and WIF1. Based on the computational molecular docking, the top five molecules were selected for each target protein. The top two molecules were chosen for further analysis of their binding and stability using MD simulation. In all the proteins, mangiferin was found to have the highest docking as well as binding interactions with the key amino acid residues. For AChE protein, although mangiferin has shown a high dock score of − 14.205 kcal/mol with the xanthone ring forming π–π stacking interaction with key interacting residue TRP286, the 2nd hit molecule baicalin with a dock score of − 13.224 kcal/mol has shown a stable RMSD throughout the simulation and exhibited π–π stacking interactions with TRP286 and TRR341 during entire MD simulation period. This shows the possibility of Baicalin to be a potent molecule with strong and stable binding than mangiferin for AChE protein. With DKK1 protein, mangiferin has shown the highest dock score of − 11.155 kcal/mol, but chebulic acid has shown stable interactions in MD simulations with important interactions with key residues ARG224, LYS222, and ARG259. With WIF1 protein, the deviation in the RMSD of ligand and the protein was higher for mangiferin and lower for ZINC103539689 indicating more stable binding in MD simulation for ZINC103539689. For GSK-3β protein morin, with a dock score of − 9.42 kcal/mol has shown stable RMSD throughout the MD simulation as compared to mangiferin although it had a dock score of − 10.344 kcal/mol. In the case of LRP6 protein, mangiferin has shown the highest docking score as well as stable RMSD and interactions with the key residues during MD simulation in comparison to the 2nd hit molecule calystegine. From this in silico-based study, we report that mangiferin could be a potential molecule targeting Wnt signaling pathway modulating the LRP6 activity, baicalin for AChE activity, chebulic acid for DKK1, ZINC103539689 for WIF1, and morin for GSk-3β protein. However, further validation of the activity is warranted based on in vivo and in vitro experiments for better understanding and strong claim.Supplementary file1 (DOCX 1591 KB)Below is the link to the electronic supplementary material.


This study examined the importance of hematological parameters as prognostic markers for people with esophageal cancer receiving radical concurrent chemoradiation.  106 patients with esophageal cancer are included in this study. Cox regression analysis, Kaplan-Meier method, and chi-square test were used to analyze our data. ΔPLR), and the change rate of circulating lymphocyte count (ΔCLC%) were independent influencing factors of OS and DFS. The patients were grouped according to the median of ΔPLR and ΔCLC%, and analysis showed that a higher ΔPLR and a higher ΔCLC% was related to poor OS and DFS . By subgroup analysis, the OS of T1-4N1-2 were better in the low ΔPLR group than the high one . DFS of T3-4N1-2 in the low ΔPLR group were better than the high one . For patients with T1-4N0-2, the OS in the low ΔCLC% group were better than in the high ΔCLC% group . For T1-4N1-2, the DFS were better in the low ΔCLC% group than others .  The median follow-up time for patients was 15.5 months (3-55). Univariate and multivariate analyses showed that age, the change of platelet-to-lymphocyte ratio (ΔPLR and ΔCLC% are independent factors of OS and DFS, and a lower ΔPLR and ΔCLC% are associated with a better OS and DFS. And T3-4N1-2 patients in the low ΔPLR group and low ΔCLC% group have greater survival benefit. China is one of the countries with an high risk in esophageal carcinoma, and esophageal squamous cell carcinoma (ESCC) accounts for 90% of the national cases . RecentlAbove 70% of esophageal carcinoma patients have malnutrition , malnutrNow, the relationship between changes of inflammatory factors during CCRT of esophageal cancer and prognosis has rarely been shown. What our study wants to research is to determine the influence of inflammatory factors on the prognosis of patients with esophageal cancer undergoing radical concurrent radiotherapy and chemotherapy.th edition of AJCC system was used to stage the patient [106 newly diagnosed patients with ESCC, who received radical concurrent radiotherapy and chemotherapy (CCT) between January 2016 and December 2017, were included in this study . The 8thIMRT with 6 megavoltage (MV) photons was given to total patients. The prescribed doses were defined as follows: 60-64 Gy for CTV. Each dose was divided into 30-32 fractions.2, days 1-3) and fluorouracil  which was given to all patients. All patients received two cycles of chemotherapy during radiotherapy.CCT was cisplatin (75 mg/mΔNLR), the PLR (ΔPLR), the platelet (ΔPLT), the circulating lymphocyte count (ΔCLC), change rates during CCRT in the NLR (ΔNLR%), the PLR (ΔPLR%), the platelet (ΔPLT%), the circulating platelet count (ΔCPC%), and the circulating lymphocyte count (ΔCLC%).Eight parameters are the inflammatory factors, namely, changes during CCRT in the neutrophil-to-lymphocyte ratio : ΔNLR = NLR1 − NLR2, ΔNLR% = (NLR1 − NLR2)/NLR1, ΔPLR = PLR1 − PLR2, ΔPLR% = (PLR1 − PLR2)/PLT1, ΔCLC = CLC1 − CLC2, and ΔCLC% = (CLC1 − CLC2)/CLC1.P < 0.05 were included in a multivariate analysis. Subgroup analyses were performed using Chi-square test. The rates of DFS and OS were estimated with the Kaplan–Meier method and compared with the log-rank test. All data were analyzed using SPSS 22.0 software package .The Cox proportional hazard regression model was used to analyze the prognostic factors affecting disease-free survival (DFS) and overall survival (OS). Variables with 106 patients with ESCC were included in the study, including 102 males (96.2%) and 4 females (3.8%), and received radical concurrent radiotherapy and chemotherapy (CCRT) .ΔPLR and ΔCLC% and OS and DFS. After Cox regression univariate analysis, ΔPLR, ΔPLR%, ΔNLR, ΔNLR%, ΔCLC, and ΔCLC% were the independent factors of OS and DFS, ΔPLR, ΔPLR%, and age are the independent factors of OS (The median OS of the patients was 15.5 months (3-55 months), the median DFS was 10 months (1-55 months) in our study. By analyzing the relationship between hematological parameters and OS and DFS, we found that there was a significant correlation between rs of OS .ΔPLR, ΔPLR%, ΔNLR, ΔNLR%, ΔCLC, and ΔCLC% are used into multivariate analysis. Age and ΔPLR were the independent factors of OS , and ΔCLC% was an independent influence factor of DFS   .ΔPLR group refer to the absolute value of ΔPLR ≥ 290.72, and the low ΔPLR group was the absolute value of ΔPLR < 290.72. Comparing the clinical characteristics   (ΔPLR group was better than that of the high ΔPLR group (P = 0.027), but there was no significant difference in gender, age, tumor TNM stage, smoking history, drinking history, and radiation esophagitis The high ΔCLC% group was defined as ΔCLC% ≥ 75.51, and the low ΔCLC% group was defined as ΔCLC% < 75.51.The median was used as the cut-off value for grouping. The high eristics  and OS a< 0.001) , and the< 0.001) ). the raΔCLC% group was better than the high one   , and DFS< 0.001) .We make patients into different subgroups by the T stage , N stage (N0-2 and N3), and age (≥61 and <61). TNM staging is closely related to the prognosis, and age in this study was an independent prognostic factor of OS.For age, the OS and DFS were batter in the low group than those in the high group .ΔPLR group were better than the high ΔPLR group       can predict the prognosis of patients with a variety of tumors [Radiotherapy is the indispensable treatment methods of esophageal cancer . In our f tumors –31. Lympf tumors . Increasf tumors –35. In af tumors , 36–40.Platelets contribute to inflammation and immunomodulatory processes. It is reported that the platelet count in cancer patients will increases by about 10–57% . PlateleΔPLR are independent influencing factors of OS, and the OS in the low ΔPLR group is better than the high one ; and ΔCLC% is an independent influencing factor of DFS; the low ΔCLC% group had better DFS . The study of Liang et al. is consistent with ours, in ESCC patients receiving radiotherapy or chemoradiation, NLR, ALC before treatment, NLR and ΔNLR after treatment are all significant for the short-term survival of patients [Our study believes that age and patients . Researcpatients . Researcpatients .A 2015 study that includes 86 esophageal cancer patients who have CRT have the same idea with our study. The high PLR and NLR are related to inferior survival .ΔPLR is related to the pulmonary side effects of patients after CCRT. In the high ΔPLR group, there were 25 patients with radiation pneumonitis 2 and above after CCRT, while the low ΔPLR group had 11 patients; the high ΔPLR patients were more possibly to develop radiation pneumonitis (P = 0.027) (P < 0.001); the study included 379 patients with esophageal cancer; analyzed the relationship between NLR, PLR, MLR, and esophageal fistula; and finally found that PLR is an independent predictor of EC patients receiving CCRT [In our study, we also found that = 0.027) . We temping CCRT . UnfortuΔPLR are independent factors of OS in patients with ESCC treated with CCRT, and ΔCLC% is an independent factor of DFS. And we compared the DFS and OS with ΔPLR and ΔCLC% andfound that lower ΔPLR and ΔCLC% is associated with a better survival. And T3-4N1-2 patients in the low ΔPLR group and low ΔCLC% group have greater survival benefit. Nevertheless, these results are preliminary and need to be validated. The large-scale prospective clinical trials are needed to verify the result.Our study found that age and


Due to an editorial error, “Chantal Pileggi” was listed as a reviewer. The correct second reviewer is “Rumpa Boonsinsukh.”Due to an error, the reviewer comments from reviewer “Rumpa Boonsinsukh” were not implemented in the final published article. The changes have been listed below. The authors and handling editor agree with the changes.Body composition characteristics of community-dwelling older adults with dynapenia or sarcopenia.” The new title is “Body composition characteristics of community-dwelling older adults with dynapenia.”The article title was previously “Schorr et al. (2018) was not cited. The reference details appear below:Biol Sex Differ. (2018) 9:1–10. 10.1186/s13293-018-0189-3”“Schorr M, Dichtel LE, Gerweck AV, Valera RD, Torriani M, Miller KK, et al. Sex differences in body composition and association with cardiometabolic risk. Introduction, paragraph 2. The corrected paragraph appears below.Schorr et al. (2018) has now been added and cited in a new sentence in “Dynapenia, which is mediated by physiological neuromuscular adaptations, is influenced by increases in body fat with consequent infiltration of intramuscular fat; thus, the loss of muscle function is not merely a result of sarcopenia (14–18). A previous study evaluating skeletal muscle characteristics in dynapenia, sarcopenia, and presarcopenia demonstrated that skeletal muscle characteristics of dynapenia vary markedly from those of presarcopenia, which is defined as having low skeletal muscle index but normal muscle function (11). The abovementioned study also indicated that older adults diagnosed with dynapenia or sarcopenia have a lower knee extension torque than both normal older adults and those with presarcopenia (but not those with both dynapenia and sarcopenia) (11). Additionally, a cross-sectional study showed that older adults with dynapenia had decreased thicknesses of the rectus femoris and medial gastrocnemius muscles compared with those without dynapenia (7). Another study reported that older adults with dynapenia have a higher body mass index and fat mass than adults with sarcopenia and presarcopenia (8). The extremities may also have varying muscle and fat masses in dynapenia, sarcopenia, and presarcopenia compared with normal older adults. Moreover, it may be different in men and women, as men have a higher ratio of muscle to fat than women (19). However, appendicular muscle and fat masses of older adults with dynapenia, sarcopenia, or presarcopenia have not been compared with those of normal older adults.”Methods, Section “Statistical analysis and sample size,” paragraph 2, “(11 people in each group)” was missing. The corrected paragraph appears below.In *power 3.1.9.7 was used for this calculation (25). The parameters were set as follows: effect size = 0.15, significance level = 0.05, effect size = 0.80, number of groups = 4, and response variables = 4. The result of the calculation demonstrated that a sample of 44 people (11 people in each group) was required to correctly detect a significant difference among the groups.”“The sample size required to ensure sufficient power in the MANOVA was calculated. GThe publisher apologizes for these mistakes. The original version of this article has been updated.


Materials and Methods, Statistical analysis, Paragraph.In the published article, there was an error in section The effect size (ES) was categorized as follows: small (≤0.2), medium (>0.2 and ≤0.5), and large (>0.5),” it should be “The effect size (ES) was categorized as follows: small (<0.3), medium (≥0.3 and<0.6), and large (≥0.6).”Instead of “Introduction.In the published article, there was also an error in section Basic neuroscience behind VR-based treatment was the finding of mirror neurons (MNs) in the primary motor cortex (M1), dorsal premotor cortex, supplementary motor area (SMA), and M1 from animal studies,” it should be “Basic neuroscience behind VR-based treatment was the finding of mirror neurons (MNs) in the primary motor cortex (M1), dorsal premotor cortex, and supplementary motor area (SMA) from animal studies.”Instead of “The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


Magnetic resonance imaging (MRI) plays a crucial role in diagnosing spinal cord infarction (SCI). However, the findings are often indistinguishable from those of other intramedullary diseases, such as neuromyelitis optica spectrum disorder (NMOSD). Although diffusion-weighted imaging (DWI) is a promising technique, the utility for discriminating SCI from NMOSD remains unclear because the DWI findings of acute NMOSD lesions have not been investigated in detail.Clinical and MRI findings were retrospectively evaluated in 15 and 12 patients with acute SCI and NMOSD, respectively. First, clinical characteristics were compared between the SCI and NMOSD groups. Second, MRI abnormalities were examined to find differences between these groups. Third, in the SCI group, factors influencing T2 and DWI abnormalities were analyzed using the mixed-effects logistic regression analysis.The proportion of female patients was higher in the NMOSD group (92%) than in the SCI (40%). The time from symptom onset to nadir was smaller in the SCI group  than in the NMOSD (252 [162–576]). On T2-weighted images, SCI lesions had smaller length than NMOSD . Focal lesions within the T9–L2 level were found only in patients with SCI. DWI hyperintensity was observed both in the SCI  and NMOSD (60%) groups. On apparent diffusion coefficient (ADC) maps, the hyperintensities of SCI had corresponding hypointensities, whereas those of NMOSD were isointense and a large portion of NMOSD lesions had hyperintense signals. Owl’s eyes sign and pencil-like hyperintensity, typically reported as T2 findings suggestive of SCI, were also found on DWI. Posterior linear hyperintensity was frequently detected on DWI in patients with posterior spinal artery infarction. The presence of MRI abnormality revealing SCI was modeled with the time from symptom onset, imaging sequence and plane, and affected vascular territory.DWI and ADC maps help distinguish SCI from NMOSD. The time from symptom onset, imaging sequence, and imaging plane should be considered when MRI findings are interpreted in patients with suspected SCI. Spinal cord infarction (SCI) is a rare myelopathy that often causes severe motor, sensory, and autonomic disabilities. In the initial assessments for patients with suspected SCI, the rapidity of symptom progression, neurological deficits that assist lesion localization, and magnetic resonance imaging (MRI) findings are important for the diagnosis , 2. HoweOn T2-weighted images, SCI often presents with “owl’s eyes sign” and “pencil-like hyperintensity” when the infarct exists in the anterior spinal artery (ASA) region. Owl’s eyes sign is a pair of abnormal hyperintensities around the anterior horns, which are observed on the axial plane and may be associated with the vulnerability of motor neurons to ischemic damage . The sigDiffusion-weighted imaging (DWI) is considered a promising technique for diagnosing SCI. Studies have reported that acute SCI was revealed as high-intensity lesions with corresponding low-intensity regions on apparent diffusion coefficient (ADC) maps, indicating diffusion restriction of water molecules in the tissue . The effTo investigate the utility of conventional MRI and DWI in diagnosing ischemic spinal stroke, 27 consecutive patients  were analyzed. First, clinical findings were compared between the SCI and NMOSD groups. Second, the frequency of abnormal MRI findings, including owl’s eyes sign, pencil-like hyperintensity, and LETM ≥ 3 vertebral segments, was examined. Third, in patients with SCI, factors associated with the presence of T2 and DWI abnormalities were evaluated.For this retrospective study, clinical and MRI findings of patients with acute SCI and NMOSD were analyzed. Inpatients hospitalized at the neurology department of a tertiary referral hospital from April 2010 to July 2021 were enrolled. The diagnosis of SCI was based on all of the following items, which were according to the proposed criteria : (1) acu2, minimum b value 0 s/mm2, maximum b value 800–1500 s/mm2; sagittal, FA 90°, TR 2400–3000 ms, TE 79–85 ms, SL 3–4 mm, AC 1, matrix 256 × 144–256 × 192, FoV 300 × 150–320 × 240 mm2, minimum b-value 0 s/mm2, maximum b-value 800–1500 s/mm2. The imaging location, sequence, and plane were selected depending on clinical requirements.MRI examinations were performed on a 1.5 T system  using a spinal array coil. Conventional MRI was usually undertaken with turbo spin-echo sequences and following parameters: T2 axial images, flip angle (FA) 170°, repetition time (TR) 3500–4300 ms, echo time (TE) 82–96 ms, slice thickness (SL) 3–4 mm, number of acquisition (AC) 1, matrix 320, field of view (FoV) 180 mm; T2 sagittal, FA 170°, TR 3200–3500 ms, TE 81–104 ms, SL 3–4 mm, AC 1, matrix 384–448, FoV 280–320 mm; T1 axial, FA 135–170°, TR 550–620 ms, TE 8.7–10 ms, SL 3–4 mm, AC 1, matrix 320, FoV 180 mm; T1 sagittal, FA 140–150°, TR 540–585 ms, TE 9.2–12 ms, SL 3–4 mm, AC 1, matrix 384–448, FoV 280–320 mm. In rare cases, T2 axial images were obtained using a gradient-echo sequence. A single-shot echo-planer DWI sequence was performed in 3-scan trace mode with 2 b-values and following parameters: axial images, FA 90°, TR 3125–4860 ms, TE 84–90 ms, SL 3–4 mm, AC 1, matrix 144 × 256–184 × 256, FoV 130 × 260–180 × 240 mmFirst, clinical characteristics of the SCI and NMOSD groups were compared. The characteristics were age, sex, hypertension, diabetes mellitus, dyslipidemia, atrial fibrillation, time from symptom onset to nadir, motor paresis , sensory disturbance, white blood cell (WBC) count and total protein of the CSF, CSF/blood glucose ratio, and ability to walk independently 1 month after symptom onset. Age, time from symptom onset to nadir, WBC count and total protein of the CSF, and CSF/blood glucose ratio were evaluated as numerical variables and the other items were categorical.Second, on T2-weighted images, craniocaudal lesion length in each patient was compared between the SCI and NMOSD groups. In the situation of a patient having > 1 lesions, the longer or longest lesion was used. The distribution of cord lesions was also examined and compared between the groups when a specific vertebral level was suspected to be differently affected. The frequency of the following findings was examined: owl’s eyes sign, pencil-like hyperintensity, posterior linear hyperintensity , LETM ≥ 3 vertebral segments, and bright spotty lesion.Subsequently, the frequency of DWI and ADC abnormalities was evaluated in both groups. When DWI hyperintensities were observed for the first time in each patient, mean ADC values were measured in the middle position of the hyperintensities and divided by mean ADC values of the adjacent normal cord (ADC ratios for DWI hyperintense lesions). For the NMOSD group, mean ADC values of lesions that were not hyperintense on DWI were also obtained and divided by mean ADC values of the normal adjacent cord (ADC ratios for NMOSD lesions that were not hyperintense on DWI). Moreover, the frequency of owl’s eyes sign, pencil-like hyperintensity, and posterior linear hyperintensity on DWI was examined. Spinal cord atrophy and gadolinium enhancement were assessed on T1-weighted images.Third, in patients with SCI, T2 and DWI signal abnormalities within 7 days from symptom onset were evaluated and analyzed with the mixed-effects logistic regression analysis. MRI signal abnormality  was modeled using the following possible predictor variables: age (measured by years), sex , time from symptom onset (measured by hours), imaging sequence , imaging plane , and affected vascular territory . The mixed-effects logistic regression analysis was performed with the random intercept of subjects and inclusion of predictor variables was determined using the likelihood ratio test and previous knowledge in the literature.P values of < 0.05 were used to denote significance. Statistical calculations were performed with software R 4.1.3 and its lmerTest package ) , imaging sequence , imaging plane , and affected vascular territory . Age and sex did not have significant effects. For clarity, all data used for this analysis are presented as a function of time after classification according to the imaging sequence, imaging plane, and affected vascular territory , and axial plane . On T2-weighted images, focal lesions within the T9–L2 level were observed only in patients with SCI.Diffusion restriction was observed in all patients with SCI; in contrast, no NMOSD lesions showed it. On ADC maps, in patients with NMOSD, the DWI hyperintensities were isointense and often surrounded by hyperintense regions. Although diffusion restriction has been reported as a useful finding for diagnosing SCI, information on other intramedullary spinal cord diseases is scarce. As for NMOSD, acute spinal cord lesions were described to have diffusion restriction in a review article ; moreoveIn this study, NMOSD lesions were largely hyperintese on ADC maps; in addition, only a small part of the lesions, which were hyperintense on DWI, had normal ADC values. Although the reason for the difference is unclear, it may be because not a single mechanism of tissue injury is active in NMOSD lesions. In a study, the lesions can be classified into six types, which are characterized by pathological findings, such as axonal degeneration, clasmatodendrosis, complement deposition, cystic formation, demyelination, granulocyte infiltration, and immunohistochemical reactivity for aquaporins 1 and 4 . ConsideIn patients with ASA ischemia, owl’s eyes sign and pencil-like hyperintensity were observed on DWI. Moreover, PSA ischemia often presented with a characteristic linear hyperintensity on sagittal DWI that was located posteriorly in the spinal cord. These DWI hyperintensities were low on ADC maps. Owl’s eyes sign and pencil-like hyperintensity have been reported as frequent T2 findings in patients with SCI ; howeverIn patients with SCI in the acute phase (within 7 days from symptom onset), DWI more likely detected ischemic lesions than T2-weighted imaging. Furthermore, the axial plane and longer time from symptom onset raised the possibility of detecting lesions. DWI was described to be more useful for detecting early ischemic changes in the spinal cord than T2-weighted imaging , and in Focal lesions within the lower thoracic and lumbar level (T9–L2) were observed only in patients with SCI; moreover, lesion length in each patient was longer in patients with NMOSD than in those with SCI. In previous studies, NMOSD lesions were located mainly in the cervical and upper thoracic cord, whereas SCI often affected the lower thoracic and lumber spinal cord –5, 44. TThis study has some limitations. Although most findings reported in this study were statistically examined, the number of patients was small to obtain definitive conclusions. Another limitation was that no patients with SCI underwent spinal angiography, which was probably associated with the absence of arterial abnormalities near the infarcted cord. The SCI diagnostic criteria of this study included time from symptom onset to nadir and DWI findings; hence, the statistical analyses regarding these items might have been affected by selection bias.In this study, we have demonstrated the utility of DWI and ADC maps. The presence of diffusion restriction indicated SCI rather than NMOSD. Owl’s eyes sign, pencil-like hyperintensity, and posterior linear hyperintensity were observed on DWI with corresponding low-ADC regions. These DWI findings may help diagnose SCI. To enhance the utility of MRI for diagnosing SCI, the time from symptom onset, imaging sequence, and imaging plane should be considered when T2 and DWI findings are interpreted.