Polygala tenuifolia Willd.  (PT) that are active against Alzheimer's disease. Sibiricose A5 (A5), sibiricose A6 (A6), 3,6′-disinapoyl sucrose (DSS), tenuifoliside A (TFSA) and 3,4,5-trimethoxycinnamic acid (TMCA) are the main active components of  To compare the pharmacokinetics and bioavailability of five active components in the roots of raw PT (RPT), liquorice-boiled PT (LPT) and honey-stir-baked PT (HPT).50) was evaluated through acute toxicity test. The pharmacokinetics of five components after oral administration of extracts of RPT, LPT, HPT  and 0.5% CMC-Na solution (control group) were investigated, respectively, in Sprague-Dawley rats  using UHPLC-MS/MS. In addition, the absolute bioavailability of A5, A6, DSS, TFSA and TMCA after oral administration  and intravenous injection  in rats (n = 6) was studied. The median lethal dose , A6  and DSS . The absolute bioavailability of A5, A6, DSS, TFSA and TMCA was 3.25, 2.95, 2.36, 1.17 and 42.91%, respectively. The LD The pharmacokinetic and bioavailability parameters of each compound can facilitate future clinical studies. Polygala tenuifolia Willd.  (PT) is a common traditional Chinese medicine that has been listed as a nootropic, anti-inflammatory and anti-psychotic medicine to cure insomnia  administration. It was expected that the study would provide a scientific basis for the processing mechanism of PT and guide reasonable clinical applications of processed PT.A5 and A6 were supplied by Chengdu Biopurify Phytochemicals Co., Ltd. . The reference standard of TFSA was supplied by Shanghai Yilin Biological Technology Co., Ltd. . DSS and TMCA were obtained from Chengdu Chroma Biotechnology Co., Ltd. . The internal standard  was provided by Shanghai Yuanye Biological Technology Co., Ltd. . The purity of each reference standard was higher than 98%. HPLC-grade water was obtained using a Milli-Q water purification system . In addition, other reagents, such as acetonitrile, methanol and ammonium acetate, were all of HPLC grade and were supplied by Merck Co., Ltd. .ad libitum throughout the experiment.Adult Sprague-Dawley (SD) rats weighing 180–220 g were obtained from Pengyue Experimental Animal Centre . The study protocol was approved by the Committee on Animal Care and Usage of Shandong University of Traditional Chinese Medicine  and SDUTCM20190409001, approval date (9 April 2019)). Animals were kept in an environmentally controlled breeding room  for 7 consecutive days. Despite fasting overnight prior to administration, water was provided All RPT samples (five batches of samples) were purchased from Shandong Baiweitang Chinese Medicine Pieces Co., Ltd. in October 2018  and were authenticated by Li Feng, a professor at Shandong University of Traditional Chinese Medicine . Five batches of samples were collected from Shandong and Shanxi provinces. Five voucher specimens  were deposited at the Herbarium of Traditional Chinese Medicine, School of Pharmacy, Shandong University of Traditional Chinese Medicine. Each batch of samples was divided into three groups: one group was RPT, and the other two groups were used for the preparation of LPT and HPT.LPT and HPT were prepared in the laboratory. The processing procedure of LPT samples was as follows: the liquorice was boiled twice in water for 0.5 h each time. Then, the two filtrates were combined and concentrated to 0.1 g of liquorice per 1 mL of decoction. Subsequently, 150 g of PT was moistened for 1 h with 90 mL of liquorice decoction and 500 mL of water and subsequently boiled for 5 h until it absorbed the liquid decoction completely. Then, LPT samples were dried under vacuum at 50 °C. The HPT samples were obtained as follows: first, 150 g of PT was mixed with 30 g of refined honey and a moderate amount of water and then moistened for 1 h and stir-heated until the PT was dark yellow.Supplementary Figure S1.Raw and various processed products of PT were smashed into powder (40 meshes). One hundred grams of powder from each sample was accurately weighed and extracted by reflux three times with 50% ethanol of 10, 8 and 8 times the volume of the samples, each time for 0.5 h. The combined filtrate was concentrated and dried in vacuum at 50 °C. The five components in the raw and various processed products of PT extracts were quantitatively determined by HPLC as previously established by our team . All oral administration samples were mixed with 0.5% CMC-Na (w/v). The test groups were subjected to one-time intragastric administration of different doses of raw and various processed products of PT extracts at the following dose ranges: RPT was from 4.15 to 13.00 g/kg body weight, LPT and HPT were from 7.99 to 25.00 g/kg body weight. Control rats were treated with the vehicle alone (volume: 10 mL/kg). Then, the rats were observed for 14 days to record the mortality, body weight changes, and signs of toxicity. The median lethal dose (LD50), 95% confidence interval (CL) and probit-log (dose) equations were calculated by SPSS analysis system .A total of 160 SD rats were randomly divided into 16 groups  was used for chromatographic separation. An aqueous 10 mM ammonium acetate solution (A) and acetonitrile (B) were selected as the mobile phase components. The optimal gradient program involved the following steps: 5% B (0–1 min), changing to 85% B (1–3.5 min), 85% B (3.5–4.0 min), changing to 5% B (4.0–4.1 min) and 5% B (4.1–5.0 min). The flow rate was set to 0.4 mL/min, the autosampler was maintained at 4 °C and the injection volume was 5 μL.m/z 517.1 → 174.9 for A5, m/z 547.0 → 204.9 for A6, m/z 753.2 → 205.2 for DSS, m/z 681.3 → 443.3 for TFSA, m/z 236.8 → 103.2 for TMCA and m/z 136.9 → 92.9 for IS. All data were acquired by using AB Sciex Analyst software (version 1.6.3).UHPLC-MS/MS was conducted by an Applied Biosystems (AB) Sciex Exion LC system  connected to a 6500 + triple quad tandem mass spectrometer with an electrospray ionization source. All detection and quantifications were carried out in positive multiple reaction monitoring (MRM) mode. The optimized parameters were programmed as follows: ion source temperature, 500 °C; curtain gas pressure, 30 kPa; ion source gas 1 pressure, 55 kPa; ion source gas 2 pressure, 55 kPa; and ion spray voltage, −3500 V. The chemical structures and ion mass spectra of targeted compounds are shown in Stock standards of A5, A6, DSS, TFSA, TMCA and IS were prepared in methanol at a concentration of 1 mg/mL. A series of working standard solutions of the targeted compounds ranging from 10 to 20,000 ng/mL and the IS solution at 600 ng/mL were prepared by diluting the stock solutions with methanol and maintained at −20 °C. Calibration standards were freshly prepared by spiking the series of standard solutions (30 μL) including the IS into blank rat plasma (270 μL) to achieve concentrations of 1, 10, 50, 100, 500, 1000, 1600 and 2000 ng/mL. Quality control (QC) samples were prepared independently at low (2 ng/mL), medium (600 ng/mL) and high (1600 ng/mL) concentrations following the same method for the five compounds.g for 10 min. Finally, 200 μL of the obtained supernatant was transferred to a fresh autosampler vial, and then 5 μL of the sample was injected for analysis.After thawing the frozen blood samples at room temperature, a 100 μL aliquot of plasma was spiked with 50 μL of the IS working solution in 2 mL EP tubes, and 750 μL of methanol was added into each of the tubes. Then, these tubes were mixed on a multi-tube Vortexer for 0.5 min before centrifugation at 11,800×The UHPLC-MS/MS method validation was performed in accordance with an internal standard operation procedure based on the US Food and Drug Administration (FDA) guidelines (US Food and Drug Administration n = 6 in each group), including the RPT group, LPT group, HPT group and control group. All oral administration samples were mixed with 0.5% CMC-Na (w/v) and then given to rats at a dose equivalent to 1.9 g/kg of RPT extract. The control group was orally treatment with equal volume of 0.5% CMC-Na solution. After a single administration, all blood samples were collected from the orbital vein into heparinized centrifuge tubes before administration (0 h) and at 0.083, 0.17, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 5, 8 and 12 h after administration and then centrifuged at 4 °C and 11,800×g for 10 min. The separated supernatant samples were preserved at −80 °C until they were measured.Male SD rats were randomly divided into four groups (n = 6 in each group). The oral doses of A5, A6, DSS, TFSA and TMCA groups were 7.40, 11.60, 16.00, 50.00 and 3.11 mg/kg, respectively, and the corresponding i.v. doses were 0.74, 1.16, 1.60, 5.00 and 0.31 mg/kg, respectively. Blood samples were collected from the suborbital vein into heparinized tubes before and 0, 0.083, 0.17, 0.25, 0.5, 1, 2, 3, 5 and 8 h after administration. The treatment method of blood samples was the same as that mentioned above.The SD rats were randomly divided into oral and i.v. groups for A5, A6, DSS, TFSA and TMCA administration , MRT (the mean residence time), Cmax (peak plasma concentration) and Tmax , t1/2   was evaluated as follows:The elementary pharmacokinetic parameters of plasma samples were analyzed by DAS 2.0 pharmacokinetic software. One-way analysis of variance (ANOVA) was used for statistical comparison for pharmacokinetic and interaction studies. A The contents of five components in the raw and various processed products of PT extracts are summarized in 50 of RPT, LPT and HPT was 7.79 g/kg (CL: 5.11–9.38 g/kg), 14.55 g/kg (CL: 8.03–18.21 g/kg) and 15.99 g/kg (CL: 10.74–20.23 g/kg), respectively. The LD50 regression equation of RPT, LPT and HPT for oral administration was y = −7.695 + 8.632 × log (d), y = −8.904 + 7.658 × log (d) and y = −8.035 + 6.675 × log (d), respectively.Acute toxicity test was conducted to preliminarily evaluate the safety of extracts from raw and various processed products of PT. The death in the acute toxicity test is reported in Representative chromatograms of blank plasma, blank plasma spiked with five reference compounds at the LLOQs, and rat plasma obtained 15 min after oral administration are displayed in y was used to express the peak-area ratio of each analyte to that of the IS, and x was used to represent the plasma concentration of each analyte). The calibration curves were fitted by a weighed least squares linear regression (1/x2) method. The method possessed good linearity for the five compounds over concentrations ranging from 1 to 2000 ng/mL with a high correlation coefficient (r > 0.994). All acquired values are described in Supplementary Table S1. The LLOQs for the five compounds were lower than 1 ng/mL, which would contribute to obtaining accurate pharmacokinetic parameters in rat plasma.Linearity was expressed by plotting the peak-area ratio of each compound/IS versus the concentration  and relative standard deviation , respectively. All data indicated that the intra- and inter-day accuracies were within acceptable limits of −4.6 to 5.3% and −7.1 to 6.8%, respectively, at all tested concentrations, and the corresponding precisions ranged from 2.5 to 6.9% and 0.9 to 7.4%, respectively. All the obtained values were within acceptable criteria (±15%), indicating that the established method had high accuracy and precision.The results on a single day (intra-day) and three consecutive days (inter-day) were utilised to determine the precision and accuracy analysis of three concentration levels of QC samples, which are listed in Supplementary Table S3. The extraction recoveries of the five analytes were within the range of 85.2–103.4%, and the RSDs were no more than 6.9%, while the matrix effects were within 85.5–103.3%, and the RSDs were less than 7.2%, which indicated that methanol was an ideal precipitation agent and that there was no significant matrix effect in the analytical method.The extraction recovery was measured by comparing the peak area of each analyte in pre-extraction samples with those in post-extraction spiked samples. The matrix effect was evaluated by comparing the peak area of each analyte in spiked plasma samples against those of the corresponding reference standard solutions. The extraction recovery and matrix effect of each analyte were determined at three QC levels with six replicates. The relevant data are summarized in Supplementary Table S4. The obtained results demonstrated that the developed method had good stability for routine analysis.Stability studies included short-term stability , freeze-thaw stability (three consecutive freeze-thaw cycles from −20 °C to room temperature), autosampler stability (24 h storage in the autosampler at 4 °C) and long-term stability (4 weeks storage at −80 °C). The stability data of all analytes are presented in The UHPLC-MS/MS method was sensitive enough to analyze A5, A6, DSS, TFSA and TMCA in rat plasma after a single administration. The mean concentration-time profiles of five components in rat plasma after oral administration of extracts from raw and various processed products of PT are described in AUC0-tvalues of A5, A6 and DSS, along with the Cmax value of A6, were obviously elevated (p < 0.05) in the LPT group and HPT group  in the other two processed products of PT groups. Moreover, the MRT values of A5 and A6, along with the t1/2 value of A5, were still remarkably improved (p < 0.05) in the LPT group compared to those of the RPT group. Additionally, the MRT and t1/2 values of DSS were significantly elevated (p < 0.05) in the HPT group compared with those in the RPT group.Double peaks were observed from the curves of A5, A6 and TFSA in the RPT, LPT, and HPT groups, and additional peaks were detected from the curves of DSS in the RPT, and LPT groups see . ComparePT group . In addit1/2 values of A5 were prolonged compared with those of A6 after oral and i.v. administration, which indicated that the elimination of A5 was slower than that of A6. Moreover, A5 showed a higher AUCΔ0-t  and CΔmax than A6. Comparing the pharmacokinetic parameters of DSS with those of A6, we found that the AUCΔ0-tvalues of A6 were approximately three times larger than those of DSS after both routes of administration, with a higher absolute bioavailability of 2.95%, despite its relatively lower absorbed ratio. Notably, the TFSA was rapidly absorbed and reached its peak plasma concentration approximately 0.19 h after oral administration, with a bioavailability of 1.17%. However, the AUCΔ0-t and CΔmax values of TMCA were much larger than those of TFSA after oral and i.v. administration, with a higher absolute bioavailability of 42.91%.The mean concentration–time profiles of five compounds following oral and i.v. administration are depicted in In our preliminary experiments, the oral lethal potency of extracts from raw and various processed products of PT was RPT > LPT > HPT and their main toxic target organs were the stomach and small intestine. The acute toxicity test results indicated that processing technology could reduce the toxicity of RPT and improve its safety. Meanwhile, the results showed that the doses given to rats in the pharmacokinetic study are safe.in vivo, which may be the other reason for the double peak phenomenon.The double peak phenomenon of A5, A6, DSS and TFSA has been reported in previous studies (Lin et al. AUC0-t and Cmax values with the contents of target components in the RPT and various processed PT products, we found that these parameters did not change proportionally. After processing, the contents of A5, A6 and DSS in PT decreased to different degrees, whereas the AUC0-t or Cmax values were significantly increased in the LPT group and HPT group, indicating that liquorice boiling and honey stir-baking could enhance the bioavailability of A5, A6 and DSS in PT. Additionally, the content of TFSA in PT decreased during processing, but the two processing methods had no significant effect on the absorption of TFSA in vivo. It is worth noting that the content of TMCA in RPT was the lowest, while it was mostly increased in the processed PT products, and the absorption in vivo was better in these products than in RPT. The results demonstrated that processing technology could promote the bioavailability of TMCA.Combining the changes in t1/2, AUCΔ0-tand CΔmax of A5 were larger than those of A6, indicating that the molecular weight might have an important impact on their pharmacokinetic parameters. The absolute bioavailability of TMCA was higher than that of TFSA, which clearly showed that the transformation of the proglucoside to its aglycone form would be conducive to increasing absorption in vivo.As far as we know, the absorption degree of oral drugs in animals is closely related to their molecular weight, molecular spatial structure and polarity. The molecular weights of A5, A6, DSS, TFSA, TMCA, and IS are 518.47, 548.49, 754.68, 682.23, 238.24 and 138.12 amu, respectively. As shown in In summary, we conclude that the synergistic mechanism of processing PT might be related to some components in liquorice and honey that could promote the absorption of certain oligosaccharide esters and organic acids, such as A5, A6, DSS and TMCA, and the hydrolysis of the oligosaccharide esters into aglycones is beneficial for their absorption by the body. In the future, our team will conduct further verification in combination with pharmacodynamic experiments.A fast, accurate, and precise UHPLC-MS/MS method was successfully applied to compare the pharmacokinetic characteristics of A5, A6, DSS, TFSA and TMCA in rats after oral administration of extracts from raw and various processed products of PT, as well as to study the absolute bioavailability differences between the five compounds. This study showed that liquorice boiling and honey stir-baking could enhance the bioavailability of A5, A6 and DSS in PT. It also revealed that the absolute bioavailability of TFSA was significantly lower than that of its aglycone TMCA. This pharmacokinetic study might provide a scientific basis for the clinical application and processing mechanism of processed products of PT.Click here for additional data file.Click here for additional data file.


Facial trustworthiness and feedback information of trustees can influence trustors’ investment behavior in trust games. This study investigated the temporal features of outcome evaluation  and how they influence the processing of facial trustworthiness. A total of 25 college students participated in a decision-making task in which feedback was presented prior to a face stimulus. The decision of participants to continue investing was evaluated. We observed that trustors were more inclined to keep investing in trustworthy trustees or those appearing after positive feedback (gains). Event-related potential (ERP) results revealed that in the face presentation stage, trustworthy faces with losses induced more negative feedback-related negativity (FRN) than did trustworthy faces with gains and untrustworthy faces with losses. Further, faces that did not meet expectations induced more negative FRN. Trustworthy faces with gains induced more positive late positive component (LPC) than did trustworthy faces with losses and generated more motivated attention. Bottom–up and top–down processes were integrated for facial trustworthiness perception at different stages. In sum, top–down processing exerted a greater impact during the early stage of facial trustworthiness perception, both top–down and bottom–up processing were involved in the medium term, and bottom–up processing exerted a greater impact in the later stage. Facial trustworthiness perception is a face-based trait inference process that refers to the evaluation of others’ trustworthiness based on their faces. The perception of facial trustworthiness is a fast process; indeed, individuals can judge the trustworthiness of a face when it appears for 33 ms  affects facial trustworthiness judgments. Our study aimed to explore the dissociation between bottom-up and top-down mechanisms in facial trustworthiness processing. Leng et al.  reportedf = 0.25, α = 0.05, power = 0.80), and the sample size should be more than 24. A total of 28 college students were recruited via advertisements; three participants were excluded due to excessive artifacts in their electroencephalography (EEG) data. Therefore, 25 participants (10 men and 15 women) were included in the final analysis . All participants were right-handed and had normal or corrected-to-normal vision. This study was approved by the institutional research ethics committees of Liaoning Normal University. All participants signed a written informed consent prior to the study.We calculated the sample size using G Power software  = 31.021, p < 0.001, Cohen’s d = 4.06). The scores for trustworthy faces (4.87 ± 0.48) were significantly higher than those for untrustworthy faces (2.87 ± 0.51).The face images  used in the experiment were the same as those used by Leng et al. . NeutralE-Prime software  onset. The data were then baseline corrected according to the 200-ms pre-feedback (pre-face) period. Based on previous studies  was used to collect the EEG data. The cap covered 64 scalp sites with tin electrodes arranged according to the 10-20 international placement system. All electrode impedance was maintained below 5 kΩ. EEG signals were sampled at 500 Hz/channel. FCz was used as the reference electrode, and vertical electrooculogram was recorded using an electrode located under the right eye. The bilateral mastoid process was used for re-reference during off-line treatment. The Gratton and Coles ocular correction algorithm of BrainVision Analyzer 2.1 software was used to analyze the EEG data. Channels were marked as artifacts if the signal variation exceeded ±80 μV and were filtered with a low pass of 20 Hz. For analyses of variance (ANOVAs), F = 56.537, p < 0.001, F = 139.747, p < 0.001, F = 14.108, p < 0.001, t(24) = 12.537, p < 0.001, Cohen’s d = 2.506). When the trustee did not reciprocate, the proportion of participants continuing to invest in trustworthy faces (42.6 ± 25.5%) was significantly higher than that for untrustworthy faces  = 7.661, p < 0.001, Cohen’s d = 1.531).Two-way repeated measures ANOVA was performed with the proportion of participants continuing to invest in four conditions as the dependent variable, [2  × 2 ]. Both the main effect (t(24) = 0.790, p = 0.437). The ERPs of different feedback results (gain or loss) at electrode sites Fz, FCz, Cz, CPz, and Pz are shown in P200 and FRN were induced during the feedback presentation stage. The average trial numbers in the gain and loss conditions were 113 ± 8 and 113 ± 9 (M ± SD), respectively. The number of trials per condition did not differ  = 16.282, p < 0.01, F = 7.768, p < 0.01, F = 1.350, p = 0.268).Two-way repeated measures ANOVA was performed with the average amplitude of P200 (150–250 ms) as the dependent variable [2  × 5 ]. A significant main effect of feedback was observed, whereby loss (−0.982 ± 2.624 μV) induced a more positive P200 than that of gain  = 3.15, p = 0.089, F = 3.878, p = 0.039, F = 0.553, p = 0.554).Similarly, the ANOVA on the FRN amplitude found that the main effects of feedback were marginal significance, whereby loss (−5.166 ± 2.344 μV) induced a more negative FRN than that of gain  = 0.958, p = 0.415).N1, P200, FRN, and LPC were observed during the face presentation stage. The average trial number of trustworthy faces in both the gain and loss condition was 59 ± 2. The average trial numbers of untrustworthy faces in the gain and loss conditions were 59 ± 1 and 59 ± 2, respectively. No significant differences were observed in the average number of trials among the four cases  are shown in F = 10.662, p < 0.001, F = 5.401, p < 0.05, t(24) = 2.978, p < 0.01, Cohen’s d = 0.595); when the feedback was a loss, the N1 amplitude of trustworthy faces (−5.094 ± 3.254 μV) was not significantly different from that for untrustworthy faces  = 0.031, p = 0.976). No other significant main effect or interaction was noted. The ANOVA on the P200 amplitude showed that neither the main effect nor interaction was significant.A three-way repeated measures ANOVA was performed with N1 amplitude (80–150 ms) as the dependent variable [2  × 2  × 3 ]. A significant main effect of electrode was noted, whereby the electrode located at the Fz/FCz site exhibited a higher amplitude in N1 .The ANOVA on the FRN amplitude revealed that both the main effect of face type  = 6.496, p < 0.05, t(24) = −1.366, p = 0.185); when the feedback was a loss, the FRN amplitude of trustworthy faces (0.531 ± 4.355 μV) was significantly lower than that for untrustworthy faces  = −4.614, p < 0.01, Cohen’s d = 0.920). The FRN amplitude for trustworthy faces with losses (0.531 ± 4.355 μV) was significantly lower than that for trustworthy faces with gains  = 2.307, p < 0.05, Cohen’s d = 0.463). No significant difference between the FRN amplitude for untrustworthy faces with losses (2.230 ± 5.479 μV) and untrustworthy faces with gains (1.811 ± 5.329 μV) was observed (t(24) = −1.376, p = 0.181).A significant interaction between face type and feedback was observed  = 26.048, p < 0.001, F = 88.961, p < 0.001, t, and the electrode located at the Pz site exhibited a higher amplitude in LPC [Pz (7.217 ± 4.412 μV) > CPz (5.314 ± 4.613 μV) > Cz (3.265 ± 4.576 μV) > FCz (1.538 ± 4.343 μV) > Fz (0.031 ± 4.159 μV)].Similarly, the ANOVA on the LPC amplitude found that both the main effect of face type  = 7.637, p < 0.05, t(24) = 3.276, p < 0.01, Cohen’s d = 0.654; there was no significant difference between the LPC amplitude of the untrustworthy faces with gains (4.099 ± 4.616 μV) and the untrustworthy faces with losses (4.688 ± 5.112 μV), t(24) = −1.264, p = 0.219; there was no significant difference between the LPC amplitude of the untrustworthy faces with gains (4.099 ± 4.616 μV) and the trustworthy faces with gains (3.138 ± 4.076 μV), t induced more positive LPC than the trustworthy faces with losses  = −5.954, p < 0.001, Cohen’s d = 1.190).A significant interaction between face type and feedback was noted, The main findings of our study were that facial trustworthiness and feedback information collectively affected trust behavior. Trustors were more inclined to keep investing in trustworthy trustees or those appearing after positive feedback (gains). This is consistent with previous research, i.e., both initial impressions and previous interactions affect people’s trust in their partners  exerted a greater impact during the early stage. Trustworthy faces with losses induced a more negative FRN compared to that for trustworthy faces with gains and untrustworthy faces with losses. Outcomes that did not meet expectations induced more negative FRN, suggesting that top–down and bottom–up processing are integrated in the medium term. Untrustworthy faces induced more positive LPC compared to that for trustworthy faces and generated more motivated attention; trustworthy faces with gains induced more positive LPC than did trustworthy faces with losses and generated more motivated attention. Regardless of previous feedback, no significant difference was noted in the amplitude of LPC induced by untrustworthy faces, implying that bottom–up processing  exerted a greater impact during the later stage.With regard to face stimuli, a degree of synthesis was employed even though some original natural faces were utilized. Future experiments should construct larger face stimulus libraries to generate more ecologically valid experimental materials. In our study, participants were instructed to imagine that they had invested 100 yuan. Future studies should also consider actual investment in individuals and present the faces of investors after a period of time. For the ERP analysis, we would try to use principal components analysis to better compare with other studies and increase the sample size to verify the stability of the results.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The studies involving human participants were reviewed and approved by institutional research ethics committees of Liaoning Normal University. The patients/participants provided their written informed consent to participate in this study.ZJ conceived this study. HL participated in performing the study and writing the manuscript. YL, QL, QW, and DL participated in editing the manuscript. All authors contributed to the article and approved the submitted version.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.


Our protocol validation may help researchers to measure trace elements and minerals in the mammalian samples fast, easily and accurately.Minerals and trace elements play vital role in the biological functions for all organisms including human and other mammals. Therefore, imbalance in the mineral and/or trace element levels may cause formation of several diseases including cancer, diabetes, cardiovascular diseases, and neurological disorders. ICP-MS (inductively coupled plasma – mass spectrometry) is described as the most sensitive and accurate method. Here we reported an effective and fast protocol as method validation to evaluate trace element and minerals via ICP-MS in the mammalian tissue and serum samples. Our data showed that minimum relative standard deviation (RSD) values with the ICP-MS were observed when we used microwave digestion with the SUPRAPUR® grade nitric acid at the lower dilution rates. Our protocol validation may help researchers to measure trace elements and minerals in the mammalian samples fast, easily and accurately. EMSURE® grade HNO SUPRAPUR® grade is high-purity acid which is suitable for trace element analysis to avoid possible contaminations. The multi-element solution 2A of SPEX CertiPrep was used for the external calibration standard. And the internal standard, which was provided by Agilent, was used for accurate correction due to the non-spectral interferences. It contains 100 ppm 6Li, Sc, Y, In, Tb, and Bi in 5% HNO2.2Rattus norvecigus), weighing 350–450 g were purchased from the experimental animals production center Koc University in Istanbul, Turkey. All rats were housed as described by Aydemir et al. [24–28 weeks old Wistar albino rats  allowed to clot and centrifuged at 400 g for 20 min at 4 °C. Serums were separated from blood samples and stored at −80 °C until analysis.2.43 of EMSURE® and SUPRAPUR® grades. 100–200 μl of the serum samples was used for digestion. The microwave digestion process occurs in three steps. Firstly, the temperature increases to 150 °C in 15 min, and then secondly, the target temperature is constant for 30 min. The method of microwave digestion was created after several trials. When a higher temperature was applied (more than 200 °C) for the acidic digestion of tissue and serum samples, the machine stopped to work. A medium range of temperature and power (500W) is sufficient to digest biological samples. After taking the samples from the machine, they were stored at −20 °C until ICP-MS measurement.Tissue and serum samples were dissolved with the acidic treatment in the microwave digestion system (Milestone START D). 30–80 mg of tissue samples dissolved in 10 ml of 65% HNO2.53. The standard is prepared from high purity single element concentrates of individual elements using Class A laboratory ware to give precise concentrations. It contains 10 ppm of Ag, Al, As, Ba, Be, Ca, Cd, Co, Cr, Cs, Cu, Fe, Ga, K, Li, Mg, Mn, Na, Ni, Pb, Rb, Se, Sr, Tl, U, V, and Zn. 2 ml of 2% HNO3 was used as the blank solution for drawing the abacus origin of the calibration curve.The multi-element solution 2A of SPEX CertiPrep was used as the external calibration standard. The matrix of the solution is 5% HNO2.62O) by serial dilution starting from 1/10 and diluted to 1/20, 1/50, 1/100, 1/200, 1/400, 1/800, 1/1000 and 1/2000. Trace and mineral elements in rat tissue and serum samples were measured by Agilent 7700x ICP-MS . Helium gas is used in the collision/reaction cell in order to remove potential polyatomic interferences. MassHunter Work Station software is used for creating the batch as well as the data analysis. FullQuant analysis method was used for the quantification of the data. The analysis mode was chosen as “spectrum” and for each sample, the peak pattern was drawn from 3 points and the measurements were replicated five times.After microwave digestion, tissue and serum samples were diluted in the ultrapure water .The first measurements of acidic digestion were carried out with EMSURE® grade nitric acid. The data showed inconsistency for the trace elements of copper, zinc and lead in which the concentration did not change even the dilution rate decreased. Firstly, the batch was prepared for reading out of  studies ,11 showe studies . Hsiung  of 66Zn . Therefo2.84OH as described by Lu et al. [For the alkali dilution method, serum and tissue samples were diluted 1:25 with an alkali solution consisting of 2% (w/v) 1-butanol, 0.05% (w/v) EDTA, 0.05% (w/v) triton X-100, 1% (w/v) NHu et al. . The mix2.9https://www.r-project.org). For each tissue versus each trace element and mineral correlation, we calculated the sample mean and the sample deviation over three replicates of the nitric acid (65% EMSURE® and SUPRAPUR® grades) digested samples at each dilution rate. We reported these data as mean ± standard error (SE) in the corresponding figures and tables.Statistical analyses were performed using R statistical software version 3.5.1 (


We recorded activity via daily steps for 2-week periods. In addition, we measured body composition, grip strength, and assessed nutritional status. Results: Analysis of body composition and daily activity indicated that women who walked more than 4000 steps had lower fat mass and increased muscle mass. Men with >3.0 metabolic equivalents (METs) when walking had lower body fat. Conclusion: For healthy older Japanese individuals in this snowy winter region, walking >4000 steps daily for women and exercise of >3.0 METs for men may indicate health-promoting activities.Background: Despite a long average lifespan, increased life expectancy does not guarantee higher quality of life. Methods: To contribute in understanding some determinants of healthy life expectancies in older Japanese individuals in a snowy winter region, we investigated the indicators of health. Local residents ( Although the Japanese have a long average lifespan, increased life expectancy does not necessarily indicate a higher quality of life. Longer life expectancy may increase the risk of diseases or disability before death . Healthy2/kg body weight per minute. Inactivity among adults including older age groups accounts for 9.4% of all deaths worldwide [One MET is defined as the amount of oxygen consumed while sitting at rest and is equal to 3.5 mL Oorldwide . It is aorldwide , depressorldwide , and resorldwide  have beeorldwide . We beliThe people aged between 65–74 years are defined as “early elderly” and those 75 years and older as “late elderly” in Japan . Many JaIn Japan, a previous study showed that walking decreases during the winter among healthy older people living in snowy areas . The phyThe aim of the study was to investigate age, daily activity level, and body composition of healthy older individuals when physical activity is limited during the snowy winter.Iwamizawa is a city in Hokkaido, the northernmost prefecture of Japan and a snowy, rural area in winter. The city of Iwamizawa has an estimated population of 83,383 people  and a density of 175 persons per square kilometer . This arActivity levels were measured using an activity meter . We asked subjects to wear the activity meter on their waist for 14 days, as much as possible from the time they wake up until bedtime, except while bathing. After the 14-day period, we collected the activity meters and recorded the data. Data included the duration for which the meter was worn, number of steps, and METs when walking, measured as walking exercise recorded as METs × time. Height was measured using a digital height meter . Body composition was measured using a body composition meter . Weight, total body fat, muscle mass, and combined fat and muscle mass per site were measured. Grip strength was measured using a force-measuring instrument .A blood draw was performed using the self-drawn simple blood draw set . Blood specimens were collected and data recorded for total cholesterol, low density lipoprotein (LDL) cholesterol, high density lipoprotein (HDL) cholesterol, neutral fat, creatinine, urea nitrogen, uric acid, and hemoglobin A1c. Participants completed a self-administered Brief Diet History Questionnaire (BDHQ). This questionnaire has been developed for Japanese people. BDHQ assesses amount of nutrients habitually consumed from foods usually eaten (exclude intake from dietary supplements) as a unit of individual, in relatively easy way. BDHQ is designed to obtain each individual’s information of nutrient intake, food intake, as well as information of few markers of dietary behaviors. BDHQ is a 2-pages (A3 size) or 4-pages (A4 size), consisting of 102 questions. BDHQ has been developed to aim for uses in large-scale epidemiologic research and research with topics other than nutrition for a large number of people. The structure of the BDHQ and its validation was written in a published paper ,18. UsinParticipants were divided into two groups according to age: those 65–74 years and those 75 and older. The number of steps was divided into ≥4000 steps group and <4000 steps group based on a standard of approximately 4000 steps, which is the average number of steps taken during 1 day for older Japanese women. Activity intensity was divided into ≥3METs and <3METs based on a standard of 3.0 METs, recommended by the World Health Organization as daily exercise intensity . To testThis study was conducted with the approval of the Ethics Review Committee of the Graduate School of Health Sciences, Hokkaido University . We explained the research objectives and program, emphasizing that participation was voluntary, that there were no disadvantages to declining participation even midway through the study, and ensured that each individual understood to mutual satisfaction that the data obtained were used for research only and that all records were confidential and securely stored.Of the 124 participants aged ≥ 65 years who were analyzed, 84 were women and 40 were men. Average ages for subjects were 70.7 ± 4.75 for women and 72.4 ± 5.86 for men. Average numbers of steps were 4466.5 ± 2536.13 (women) and 5150.5 ± 2636.63 (men). Average METs when walking was 2.7 ± 1.67 METs (women) and 3.3 ± 1.64 METs (men). The body mass index (BMI) values were 22.9 ± 3.56 (women) and 23.5 ± 2.87 (men).As shown in p = 0.03). BMI for those 65–74 years was 22.4 ± 3.48 kg/m2; for those ≥75 years: 24.4 ± 3.44 kg/m2 (p = 0.03). No difference was seen in physical activity, body composition, and blood test between the women’s and the men’s groups.Regarding body composition, there were differences in mean women’s heights between the age groups: for those 65–74, the average was 152.0 ± 4.92 cm; for those ≥ 75, 149.1 ± 4.95 cm .As shown in p = 0.01); water intake for the 65–74 group was 1739.0 ± 486.07 g and that for those in the ≥75 group was 2025.2 ± 654.40 g (p = 0.04); protein intake in the 65–74 group was 69.8 ± 27.29 g and that for the ≥75 group was 96.7 ± 31.31 g (p < 0.01); and fat intake for the 65–74 group and the ≥75 group was 46.9 ± 19.19 g and 64.2 ± 24.88 g (p < 0.01), respectively. For men, there was a difference in water intake: those in the 65–74 age group and the ≥75 age group consumed 1819.9 ± 401.95 g and 2135.9 ± 536.45 g (p = 0.04), respectively.Nutritional conditions showed significant differences between the two age groups for 4 items for women and 1 item for men. For women, these were 1-day total energy intake in the 65–74 group, averaging 1586.2 ± 514.17 kcal and in the ≥75 group, the average intake was 1975.1 ± 563.09 kcal (p = 0.04). The average LDL cholesterol for 65–74 year old women was 125.9 ± 22.47 mg/dL and that for women ≥ 75 years was 110.3 ± 34.06 mg/dL (p = 0.03). For men, no significant differences were found between age groups for the remaining data.Although blood analyses data for both men and women were within the standard ranges for Japanese people, women in the 65–74 age group had a higher value for total cholesterol at 222.5 ± 28.74 mg/dL than those in the ≥75 group, with an average value of 204.9 ± 34.34 mg/dL (2), although it was low in that range: 21.8 ± 2.79 kg/m2. For body fat and muscle mass by site for the group who walked more than 4000 steps, fat mass was small: women in the group who walked <4000 steps had fat mass of 16.6 ± 5.55 kg; women in the group who walked ≥4000 steps had fat mass of 13.0 ± 3.84 kg (p < 0.01). The left and right leg muscle mass measures were maintained .Because there was no difference in activity levels between the 65–74 and ≥75 age groups, we examined the other characteristics of older people. Further analyses were performed on pooled data. Participants were divided into two groups based on the standard of approximately 4000 steps, which is the average number of steps taken during 1 day for older Japanese person. These were then compared . For womp = 0.01). For muscle mass, the ≥3.0 METs group had 31.8 ± 2.14 kg overall, while the <3.0 METs group had 34.9 ± 4.12 kg (p = 0.02). For men, there was no significant difference in height and muscle mass; the ≥3.0 METs group showed lower values for body fat (body fat percentage: ≥3.0 METs group had 17.5 ± 6.79% and the <3.0 METs group had 22 ± 4.90% (p = 0.03). Analysis of body fat mass by site revealed that the ≥3.0 METs group had lower values at all sites, the left and right arms ; torso (p = 0.03); left and right legs: left leg: p = 0.04; right leg: p = 0.04.For activity intensity level, we divided subjects into two groups, based on the standard of 3.0 METs recommended for daily exercise intensity by the World Health Organization. The results of the comparison are shown in The average age of our participants was younger than Japan’s average life expectancy and closer to that of the average healthy life expectancy .Age and muscle mass have been reported as factors that influence the number of steps and expended energy during physical activity . AccordiHowever, in this study, a difference according to age was found regarding grip force. It has been reported that there is a decrease in grip force with ageing, reflecting change in muscle strength , corroboThe nationwide average numbers of steps in Japan for men and women aged ≥65 years are 5779 and 4736, respectively . FallingMen generally performed more physical activity than women . In thatAccording to the Physical Activity Standards for Health Promotion 2013 , the sugThe limitations of this study include its relatively small sample size, and a lack of detailed data on certain measures of interest, specifically measures of nutrition and nutritional deficiencies. The small sample size may have been due to the study being conducted during the winter, when walking outside was difficult due to heavy snowfalls. We believe that the analysis that built the regression model is important. However, the variance inflation factor (VIF) was 10 or more. Therefore, the regression model was not considered in consideration of multicollinearity between variables. Future studies should recruit larger samples of older adults and should include more variables, including more detailed data on measures of nutritional status and nutritional deficiencies. Randomized controlled interventions are required for further investigation of the association between of physical activity and body composition in older Japanese adults.The following observations became clear after investigating the activity levels during snowy periods and body composition of older people living in snowy rural areas. First, after comparing the 65–74 age group and the ≥75 age group, no significant difference was found in activity levels among men and women. For body composition among women, there were differences in height and BMI. For men, no difference was found between the two age groups. Second, healthy older women living in snowy areas who walk more than 4000 steps/day had less fat mass and maintained more muscle mass compared to those walking less than 4000 steps each day. Finally, healthy older men living in snowy areas who had activity intensity levels of more than 3.0 METs had lower body fat percentages compared to those in the under 3.0 METs group.


Scedosporium and Lomentospora species are filamentous fungi that cause a wide range of infections in humans. They are usually found in the lungs of cystic fibrosis (CF) patients and are the second most frequent fungal genus after Aspergillus species. Several studies have been recently performed in order to understand how fungi and bacteria interact in CF lungs, since both can be isolated simultaneously from patients. In this context, many bacterial molecules were shown to inhibit fungal growth, but little is known about how fungi could interfere in bacterial development in CF lungs. Scedosporium and Lomentospora species present peptidorhamnomannans (PRMs) in their cell wall that play crucial roles in fungal adhesion and interaction with host epithelial cells and the immune system. The present study aimed to analyze whether PRMs extracted from Lomentospora prolificans, Scedosporium apiospermum, Scedosporium boydii, and Scedosporium aurantiacum block bacterial growth and biofilm formation in vitro. PRM from L. prolificans and S. boydii displayed the best bactericidal effect against methicillin resistant Staphylococcus aureus (MRSA), Burkholderia cepacia, and Escherichia coli, but not Pseudomonas aeruginosa, all of which are the most frequently found bacteria in CF lungs. In addition, biofilm formation was inhibited in all bacteria tested using PRMs at minimal inhibitory concentration (MIC). These results suggest that PRMs from the Scedosporium and Lomentospora surface seem to play an important role in Scedosporium colonization in CF patients, helping to clarify how these pathogens interact to each other in CF lungs. Cystic Fibrosis (CF) is an autosomal recessive disease originated from a mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated epithelial chloride channel . It resuStaphylococcus aureus and Haemophilus influenza are frequent in children and Burkholderia cepacia complex and Pseudomonas aeruginosa are found especially in adults , glycoconjugates commonly exposed on ic cells . PRMs halificans , as wellantiacum . The PRMB. cepacia (American Type Culture Collection ATCC 25416), E. coli (ATCC 11229), Methicillin Resistant S. aureus—MRSA (ATCC 9393) and P. aeruginosa (ATCC 27853) were studied in this work. Strains were maintained in Luria-Bertani (LB) broth medium  under refrigeration at 4°C. For recent overnight cultures, an aliquot of each strain was spread on LB agar and incubated for 24 h at 37°C, and bacterial suspensions were prepared from scrapings.L. prolificans (FMR3569 strain), S. apiospermum (RK107-0417 strain) and S. aurantiacum (IHEM21147 strain) were supplied by Dr. J. Guarro, Unitat de Microbiologia, Facultat de Medicina e Institut d`Estudis Avançats, Réus, Spain. S. boydii (HLPB strain) was supplied by Dr. Bodo Wanke, Hospital Evandro Chagas, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil. Strains were maintained at room temperature on Sabouraud  agar slants as stock culture. Mycelia were obtained by growing cells in SAB liquid culture medium for seven days at room temperature with shaking.L. prolificans, S. apiospermum, S. boydii, and S. aurantiacum with 0.05M phosphate buffer, pH 7.2, at 100°C for 2 h. After filtration, the solution was dialyzed, evaporated to a small volume, and lyophilized. Peptidorhamnomannans were purified by hexadecyltrimethylammonium bromide  fractionation, according to The crude glycoprotein was extracted from L. prolificans, S. apiospermum, S. boydii, and S. aurantiacum was determined through broth microdilution method in LB broth against bacteria, according to 8 CFU/ml) were added in each well and incubated at 37°C for 24 h. Streptomycin/penicillin was used as reference compounds (8–0.015 µg/ml) and cultures in LB broth without antibiotics were used as control. After determining MIC values, MBC  was determined by subculturing an aliquot of 10 µl from each well that showed complete growth inhibition in LB agar medium without addition of PRM and evaluation of bacterial growth (Minimum inhibitory concentration (MIC) of the PRM isolated from l growth . After 2B. cepacia (ATCC 25416) and MRSA (ATCC9393) bacterial solutions (prepared as described in MIC section) were added in a 96-well plate and mixed with 1/4, 1/2 and 1 MIC of each PRM. After 24 h of incubation at 37°C, the formed biofilm was gently washed with PBS to remove planktonic cells, air-dried for 10 min and stained for 10 min with 0.5% crystal violet  or 1% safranin (biofilm matrix). The staining solutions were discarded and biofilms were gently rinsed twice with sterile distilled water. Crystal violet impregnated in the biofilm was dissolved in 200 µl of ethanol , and the colored solution was read at an absorbance of 595 nm using a spectrophotometer . Safranin was dissolved in water (100 µl), and the absorbance read at 492 nm.For biofilm formation, 50 µl of B. cepacia and MRSA biofilms were prepared as described in the previous section, and after incubation for 24 h at 37°C, the supernatant was removed and 1/2, 1 and 2 MICs of each PRM were added to the formed biofilm. After 24 h at 37°C, the biofilm was gently washed with PBS and crystal violet and safranin staining were performed as in the previous section.The B. cepacia cells were grown in LB broth in the presence of ¼ MIC of all four PRMs for 24 h at 37°C. Furthermore, fluorescence staining was performed using DCFDA , Nile Red and JC-1  , according to In order to evaluate the effect of fungal PRMs on oxidative stress and membrane integrity and potential, Staining with DCFDA was performed at a final concentration of 10 μM in PBS for 45 min at room temperature in the dark. After washing with PBS, fluorescence  was measured using a SpectraMax plate reader .Nile Red staining at a final concentration of 8 μg/ml was done for 45 min at room temperature in the dark. After washing with PBS, fluorescence  was measured as above .JC-1 stain was used at 2.5 μg/ml in PBS 0.01 M, pH 7.2, for 45 min at 37°C in the dark. After washing with PBS, fluorescence  and 590 nm (red) was measured as above . The memAll statistical analyses were performed using GraphPad Prism 5.0 software . A variance two-way ANOVA was performed using Tukey’s and Bonferroni’s comparisons tests to evaluate biofilm formation and inhibition of preformed biofilm.L. prolificans, S. apiospermum, S. boydii, and S. aurantiacum against B. cepacia, E. coli, MRSA, and P. aeruginosa , which provoke oxidative stress, was measured using DCFDA staining. DCFDA is oxidized by ROS inside the cells to form fluorescent 2’, 7’-dichlorofluorescein (DCF). In this context, S. boydii PRM decreased Nile Red fluorescent intensity, suggesting that it affects lipid integrity in B. cepacia, whereas the other PRMs did not alter bacterial lipids when compared to the control , common prokaryotic storage compounds of carbon and energy present in intracellular lipid droplets . S. boydB. cepacia , indicating that PRMs induce a lower membrane polarization in Cystic fibrosis patients exhibit a reduced clearance of mucus from the lungs which leads to chronic infections caused by bacteria and fungus, being the primary cause of mortality . The stuP. aeruginosa, S. aureus, and B. cepacia. The presence of B. cepacia is considered a threat to CF patients, due to its high patient-to-patient transmissibility and antibiotic resistance, as well as a poorer prognosis of the disease outcome , followed by S. boydii (19.3%), S. aurantiacum (10.0%), and L. prolificans (3.6%)  . Other spatients .L. prolificans , a component of the cell wall of several Aspergillus species as well of a diverse range of fungi, are released during early logarithmic growth   , that miS. boydii and S. apiospermum are involved in the adhesion to an epithelial cell line and in intracellular survival within macrophages, respectively  α-Manp-(1→2)- α-Manp —Finance Code 001; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do estado do Rio de Janeiro (Faperj).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.


Xenotoca variata is representative of the viviparous Goodeinae subfamily (Goodeidae) from central Mexico. The mitogenome of the X. variata consist of 37 genes in 16,462 bp. Comparing with X. eiseni, the most related of the mitogenomes included, an identity of 91.1% was found and trna-met duplication in X. eiseni is absent in X. variata. The mitogenome provide important information for future studies in evolution of the live-bearing subfamily.The live-bearing fish  Xenotoca variata , Cuitzeo basin, and were storage in the Colección de Peces de la Universidad Michoacana-UMSNH, Mexico (Voucher specimen: CPUM-7031). Briefly DNA was sheared using a Covaris S2  ultrasonicator, and Illumina  adapters were ligated on using methods derived from Fisher et al.  of r et al. , but usir et al. . Genomicr et al. , adapterr et al.  to assemr et al. . Genome r et al.  but the r et al. . Phyloger et al. .Xenotoca variata (GenBank accession: KY471393) consists of 37 genes in 16,511bp  with 12 intergenic spacer sequences (of 1–37bp). The base composition of the genome was as follow: A = 28.7%, C = 26.8%, G = 15.3% and T = 29.2% (GC-rich = 42.1%). Comparing with X. eiseni, the most related of the mitogenomes included in Genbank (trna-met gene in X. eiseni is absent in X. variata. The number of nucleotide differences between both mitogenomes is 1 206bp corresponding to 7.3% of divergence. The complete mitochondrial genome of X. variata provides relevant information to posterior genetic and evolutionary studies of Xenotoca genus and the Goodeidae family.The circular mitogenome of  Genbank , an iden


Prism adaptation (PA) is both a model for visuomotor learning and a promising treatment for visuospatial neglect after stroke. The task involves reaching for targets while prism glasses horizontally displace the visual field. Adaptation is hypothesized to occur through two processes: strategic recalibration, a rapid self-correction of pointing errors; and spatial realignment, a more gradual adjustment of visuomotor reference frames that produce prism aftereffects . While aftereffects can ameliorate neglect, not all patients respond to PA, and the neural mechanisms underlying successful adaptation are unclear. We investigated the feedback-related negativity (FRN) and the P300 event-related potential (ERP) components as candidate markers of strategic recalibration and spatial realignment, respectively. Healthy young adults wore prism glasses and performed memory-guided reaching toward vertical-line targets. ERPs were recorded in response to three different between-subject error feedback conditions at screen-touch: view of hand and target (Experiment 1), view of hand only (Experiment 2), or view of lines to mark target and hand position . Conditions involving a direct view of the hand-produced stronger aftereffects than indirect hand feedback, and also evoked a P300 that decreased in amplitude as adaptation proceeded. Conversely, the FRN was only seen in conditions involving target feedback, even when aftereffects were smaller. Since conditions producing stronger aftereffects were associated with a phase-sensitive P300, this component may index a “context-updating” realignment process critical for strong aftereffects, whereas the FRN may reflect an error monitoring process related to strategic recalibration. Prism adaptation (PA) is a visuomotor task that demonstrates the brain’s adaptation to changes in visually perceived coordinates of objects in space  to investigate event-related potential (ERP) components evoked by the onset of error feedback during prism exposure. Vocat et al.  identifiWhile Vocat et al.  examinedAn early hypothesis proposed by MacLean et al.  was thatexternal environment, which is what was manipulated in the present study; by contrast, the ERN has been described as a measure of error evaluation based on the internal environment  exposure. During PA, participants wore an EEG cap that was used to measure ERPs time-locked to visual feedback at screen touch. Aftereffects were measured using proprioceptive visual straight-ahead pointing . Across experiments, participants were seated at an adjustable chair in front of a 28” touchscreen monitor . A chinrest was locked to the edge of the table to maintain participants at a distance of 48 cm from the screen, and at a height that kept their gaze in line with the center of the monitor. A keyboard (used to record response onset) was placed 10 cm in front of the chinrest with the spacebar in line with the center of the monitor. Speakers (used to emit a go-cue) were placed on the left and right of the monitor. A black horizontal occlusion board extending from the chinrest prevented participants from viewing their reaching movement until the last 3 cm immediately before screen touch, a set-up known as terminal exposure . The EEG was recorded with an average reference, sampled at 500 Hz, amplified , and filtered online through an 8 kHz anti-aliasing filter. The impedance at each electrode was kept below 20 kΩ.N = 20), participants saw their hand and a re-appearance of the target line with terminal exposure; in Experiment 2 (N = 22), participants saw their hand only with the same terminal exposure; and in Experiment 3 (N = 25), participants could not directly view their hand  and instead saw re-appearance of the black target line and a second light gray vertical line that referred to the hand position below the board , which provided a measure of participants’ reaching accuracy excluding any PA aftereffects. Participants then completed a series of PA blocks with either left- or right-shifting prisms. There were four 60-trial prism exposure blocks in total per experiment, two with left-shifting prisms and two with right-shifting prisms. In Experiments 1 and 3, two prism blocks had immediate visual feedback , and two blocks had delayed feedback . These four blocks were presented in a randomized order for each participant. Only results from the blocks with immediate feedback are presented in this article. The prism blocks for Experiment 2 had a similar structure in terms of left- and right-shifting PA blocks, but two blocks had “hand only” feedback (used in the present study) and two blocks had full target feedback  that prompted them to release the spacebar and reach under the occlusion board “as quickly and accurately as possible” to the remembered location of the target. This memory-guided reaching was implemented to help prevent the early onset of error-related ERPs to premature visual feedback provided by the emergence of the participant’s hand from the occlusion board in comparison with the target , so all results for those experiments were collapsed across this factor. For Experiment 2, the three-way interaction between exposure type, phase, and shift direction was significant (p = 0.003). Although subsequent ANOVAs showed a significant phase-by-shift direction interaction for prism exposure but not sham exposure, post hoc analyses did not reveal any significant differences in absolute errors. Thus, subsequent analyses were also collapsed across shift direction for Experiment 2. To determine if there were differences across exposure type and phase for each experiment, the absolute errors were submitted to a 3 × 6 repeated-measures ANOVA with the following factors: exposure type , and phase (P1 to P6). Absolute errors were also compared across experiments in a 3 × 6 × 3 mixed ANOVA of exposure type, phase, and experiment .Pointing error size was recorded as the horizontal distance between the location of the screen touch and the target, measured in pixels and converted to visual degrees. Distances to the left of the target were recorded as negative values, and distances to the right of the target were recorded as positive values. For statistical analyses, blocks were divided into six phases of adaptation , consistent with the bins of phases used to measure ERPs (described below). Absolute errors were then submitted to a 2 × 2 × 6 repeated-measures ANOVA with the following factors: exposure type , shift direction , and phase (P1 to P6). Note that sham blocks were coded according to the shift direction of the preceding prism block, and the baseline block was omitted from this analysis for not having a shift direction. For Experiments 1 and 3, there were no significant effects of shift direction on absolute error magnitude (t-tests. Since aftereffects did not differ by shift direction for any experiment (ps ≥ 0.42), these aftereffects were averaged across shift direction and compared across the three experiments using one-way ANOVA.Aftereffects were measured by subtracting average error size during baseline PVSA from average error size during post-PA PVSA blocks. Absolute values of these baseline-corrected aftereffects were compared across left- and right-shifting prism directions using paired samples Data were processed offline using the EEGLAB toolbox , small miss (within 2.6 cm of the target’s edge on either side), or big miss (beyond 2.6 cm of the target’s edge on either side). Grouping accuracy according to three levels  enabled investigation of ERPs correlating with error magnitude .Analyses were conducted using IBM SPSS Statistics (v.25) and GraphPad Prism (v.6). Experiment 1 was a partial replication of MacLean et al.  in that N = 18 for the analyses below.From an initial sample of 20, two participants were excluded from data analysis because of poor EEG data quality resulting in high artifact rejection (>50%), leaving F = 166.73, p < 0.001, ε = 0.38, partial η2 = 0.91) and exposure type  = 214.67, p < 0.001, ε = 0.56, partial 2η = 0.93), and an interaction between phase and exposure type  = 76.77, p < 0.001, ε = 0.26, partial 2η = 0.82). Post hoc analyses revealed that error size for the baseline block did not differ across phase. For prism blocks, error size decreased from phase 1 through 3 and leveled off at phase 4, indicating adaptation to the visual shift. For sham blocks, error size decreased from phase 1–2 and reached baseline levels by phase 4, indicating successful de-adaptation (t(17) = 13.50, p < 0.001) with an average (baseline-corrected) magnitude of 5.34 visual degrees  = 16.56, p < 0.001, partial 2η = 0.49. Post hoc analyses showed that both “small misses” and “big misses” had more negative amplitudes than “hits” (p < 0.05). Amplitudes also showed a linear trend over accuracy level  = 14.72, p = 0.001, partial 2η = 0.46), likely driven by the decrease in voltage from “hit” to “small miss”  = 3.67, p = 0.005, partial 2η = 0.18. Post hoc analyses showed that phase 1 had a more positive amplitude than phases 3 and 4 (p < 0.05). Contrast analyses suggested that amplitudes decreased linearly from phase 1–6  = 14.72, p = 0.001, partial 2η = 0.46; F = 2.90, p = 0.05, partial 2η = 0.15. Post hoc analyses did not reveal any significant differences, but the linear trend of decreasing amplitude from phase 1–6 approached significance, F = 4.10, p = 0.06, partial 2η = 0.19  was more strongly linked to the ERPs and aftereffects, Experiments 2 and 3 employed each type of feedback separately. In Experiment 2, the participants saw their hand at screen-touch, but no re-appearance of the target line . Because participants could not directly see their pointing accuracy in relation to the target’s position, it was hypothesized that an FRN would not be evoked. By contrast, given the apparent role of viewing the hand in producing successful adaptation  = 85.62, p < 0.001, ε = 0.30, partial 2η = 0.81) and exposure type  = 133.37, p < 0.001, ε = 0.55, partial 2η = 0.87), and an interaction between phase and exposure type  = 36.96, p < 0.001, ε = 0.17, partial 2η = 0.65). Post hoc analyses revealed that error size for the baseline block did not differ across phase. For prism blocks, error size decreased from phase 1–2 and leveled off at phase 3, indicating adaptation to the visual shift. For sham blocks, error size decreased from phase 1–2 and reached baseline levels by phase 4, indicating successful de-adaptation (t(20) = 18.31, p < 0.001), with an average (baseline-corrected) magnitude of 4.95 visual degrees  = 8.36, p = 0.001, partial 2η = 0.28. Post hoc analyses showed that “big misses” had more positive amplitudes than “hits” (p < 0.05). Amplitudes also showed a linear trend over accuracy level  = 14.72, p = 0.001, partial 2η = 0.46), suggesting that the positive amplitude increased concomitantly with error size  = 8.14, p = 0.001, partial 2η = 0.28. Post hoc analyses showed that big misses” had more positive amplitudes than “hits” or “small misses” (p < 0.05), and the contrast analysis suggested that voltage increased with error size, F = 13.61, p = 0.001, partial 2η = 0.39  = 6.92, p < 0.001, partial 2η = 0.25. Post hoc analyses showed that phase 1 had a more positive amplitude than phases 3, 4, and 6 (p < 0.06), and contrast analyses suggested that amplitudes decreased linearly from phase 1–6, F = 14.23, p = 0.001, partial 2η = 0.40  and instead saw re-appearance of the black target line and a second light gray vertical line that referred to the hand position below the board. We hypothesized that this feedback would evoke an accuracy-sensitive FRN due to the provision of explicit feedback about pointing accuracy in relation to the target position. By contrast, based on prior studies suggesting that direct view of the hand is important for successful sensorimotor realignment . Additionally, one participant’s behavioral data were lost due to a recording error, although her EEG data were still available. This participant’s behavior during the experiment was observed to be consistent with other participants, thus her EEG data were kept in the analysis to increase power. As a result, the behavioral analysis reflects 21 participants, whereas the EEG analysis reflects 22 participants.F = 27.76, p < 0.001, ε = 0.42, partial 2η = 0.58) and exposure type  = 45.00, p < 0.001, ε =.52, partial 2η = 0.69), and an interaction between phase and exposure type  = 20.80, p < 0.001, ε = 0.21, partial 2η = 0.51). Post hoc analyses revealed that error size for the baseline block did not differ across phase. For prism blocks, error size decreased from phase 1–2 and leveled off, indicating adaptation to the visual shift. For sham blocks, error size decreased from phase 1–2 and leveled off, indicating some de-adaptation. Error size of sham blocks was significantly lower than baseline across all six phases, suggesting some practice effects (t(21) = 6.79, p < 0.001), with an average (baseline-corrected) magnitude of 1.92 visual degrees  = 27.85, p < 0.001, partial 2η = 0.57. Post hoc analyses showed that all three accuracy levels differed from each other (p < 0.05); namely, contrast analyses suggested that amplitudes became increasingly negative as error size increased, F = 40.40, p = 0.017, partial 2η = 0.66  and electrode site  predicting mean peak voltage 200–350 ms post-screen touch, which found no effect of phase or interaction between phase and electrode site , ε = 0.22 = 4.12, p = 0.003, partial 2η = 0.13 . To explore the nature of the interaction, a series of two-way ANOVAs of phase and experiment were conducted. In the baseline block, there was a main effect of experiment  = 10.52, p < 0.001, partial 2η = 0.27), whereby errors were significantly larger in Experiment 3 than in Experiments 1 or 2 (ps ≤ 0.002). In the prism blocks, there was an interaction between phase and experiment , ε = 0.36 = 4.01, p = 0.006, partial 2η = 0.12), whereby errors were significantly larger in Experiment 3 than in Experiments 1 or 2, but only in phase 6 (ps ≤ 0.004). In the sham blocks, there was also an interaction between phase and experiment , ε = 0.46 = 57.88, p < 0.001, partial 2η = 0.67): for phases 1 through 4, errors were generally largest in Experiment 1, followed by Experiment 2 and then Experiment 3; however, for phases 5 and 6, errors did not significantly differ across experiments (ps ≥ 0.14).With error size during the exposure phase as the outcome, analyses revealed a three-way interaction between exposure type, phase, and experiment, F = 72.25, p < 0.001, partial 2η = 0.72. Post hoc analyses revealed that the aftereffects in Experiment 3 were smaller than the aftereffects in Experiments 1 and 2  and spatial realignment (the P300) by employing different types of visual feedback during PA exposure. We hypothesized that: visual feedback involving direct view of the hand together with a re-appearance of the target position would evoke a phase-sensitive P300, an accuracy-sensitive FRN, and strong aftereffects (Experiment 1); visual feedback involving direct view of the hand only with no explicit feedback comparing the hand position to target position would not evoke an FRN, but would still evoke the P300 and produce strong aftereffects (Experiment 2), and visual feedback involving view of the target position and indirect hand position would evoke an FRN, but not a P300 and would produce weaker aftereffects than the other two experiments (Experiment 3). Overall, our results generally supported these hypotheses with some differences, all of which are discussed in further detail below.As predicted, participants experienced significantly larger aftereffects when they received visual feedback involving a direct view of the hand (Experiments 1 and 2) than when they received only indirect feedback regarding hand position (Experiment 3). This finding is consistent with prior work documenting larger aftereffects when participants viewed their own hand during PA exposure than when they viewed a “symbolic” hand  evoked a negative-going component at electrode site FCz and ~225–375 ms post-screen touch that was sensitive to pointing accuracy. This electrode site, timing, and evoking stimulus  are consistent with the properties of an FRN  did not evoke an FRN, it did evoke positive-going component at electrode site Cz and ~155–205 ms post-screen touch that increased in amplitude with pointing error size. This component may represent a Pe, which typically peaks at 100–200 ms post-response onset over central scalp electrodes and is thought to represent subjective error evaluation  evoked positive-going components that decreased in amplitude as adaptation proceeded. While visual feedback in Experiment 2 evoked a phase-sensitive component with latency and topography that were broadly consistent with the P300 identified by MacLean et al. , visual Taken together, our results provide evidence for the proposal that the processes underlying the FRN and P300 contribute to the PA processes of strategic recalibration and spatial realignment, respectively .While novel in identifying promising neural correlates of PA, our study is not without limitations. First, the present study focused on the FRN and P300 as markers of dissociable PA processes (i.e., recalibration and realignment; Redding and Wallace, Second, we used terminal exposure during PA to minimize the potential for premature neural responses to visual feedback from viewing the hand during the reaching trajectory (MacLean et al., The present study sought to identify ERP correlates of strategic recalibration and spatial realignment during PA. Participants reached for vertical line targets on a touch screen in alternating prism and sham blocks under terminal exposure, and we manipulated visual feedback provided at screen touch. Results showed that a direct view of the reaching hand at screen touch was both critical to obtaining large aftereffects and associated with a P300-like component that decreased in amplitude throughout the adaptation. This ERP component may reflect a context-updating process in the parietal cortex that is important for the realignment of sensorimotor reference frames and strong aftereffects (Donchin and Coles, These findings not only advance our understanding of the neural processes engaged by PA, but they can also inform future studies of neural responses to PA in healthy older adults and stroke patients with visuospatial neglect. While PA has been deemed one of the most promising treatments for neglect, the neural mechanisms of PA-induced neglect recovery require further investigation (Yang et al., The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The studies involving human participants were reviewed and approved by Nova Scotia Health Authority Research Ethics Board. The patients/participants provided their written informed consent to participate in this study.SM, OK, and GE contributed to designing the study and collecting the data. JA and SM analyzed the data. All authors interpreted the results. JA and SM wrote a draft of the manuscript. All authors reviewed and revised the manuscript and confirmed its final version.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.


Third-space endoscopy is a novel, safe, and effective method for treating different gastrointestinal conditions. However, several failed endoscopic procedures are attributed to incomplete myotomy. Lighting devices are used to prevent organic injuries. We aimed to investigate the feasibility of using a hand-made LED-probe (LP) in third-space procedures.This prospective study was conducted in a tertiary-care center in Mexico between December 2016 and January 2019. We included peroral endoscopic myotomy (POEM) and gastric peroral endoscopic myotomy(G-POEM) procedures. Pseudoachalasia, peptic ulcer, normal gastric emptying scintigraphy (GES) and prepyloric tumors were excluded. LP was used to guide or confirm procedures. Clinical and procedural characteristics were recorded and analyzed.p < 0.001); retention percentage at 4 h also improved. LP was successfully placed and adequate myotomy confirmed including 14.2 and 17.8% of POEM and G-POEM difficult patients.Seventy third-space procedures were included , with an average patient age of 46.7 ± 14.3 and 43.7 ± 10.1 years, respectively. For the POEM and G-POEM groups, respectively, 18/42(42.9%) and 13/28(46.7%) patients were males; median procedure times were 50 (interquartile range [IQR]: 38–71) and 60(IQR: 48–77) min, median LP placement times were 5(IQR: 4-6) and 6(IQR: 5-7) min, mild adverse events occurred in 4(9.4%) and 4(14.2%) of cases, and clinical success at 6 months occurred in 100 and 85.7% of cases. Integrated relaxation pressure (IRP) improved from 27.3 ± 10.8 to 9.5 ± 4.1 mmHg (Using an LP is promising and allows guiding during third-space procedures either for submucosal tunnel creation or myotomy confirmation, with excellent safety and efficacy in clinical practice. Third-space endoscopy has emerged as a novel and effective method for the treatment of different gastrointestinal disorders such as achalasia and refractory gastroparesis –4. HowevLighting devices for surgical guidance have been investigated for years with good results. In laparoscopic colorectal and gynecological surgeries, the use of lighted ureteral stents has successfully prevented iatrogenic injuries , 15. In This prospective study was conducted in a tertiary care center in Mexico City, Mexico between December, 2016 and January, 2019. We included patients between the ages of 18 and 90 years with naïve or previously-treated achalasia or severe refractory gastroparesis and who were treated with POEM or G-POEM, respectively, with 6 months of follow-up. Patients with pseudoachalasia were excluded from the POEM procedure. Active peptic ulcer disease, normal gastric emptying scintigraphy (GES) and prepyloric tumor lesions were excluded from G-POEM. Patients with severe clinical conditions that could be contraindications to any of these procedures were also excluded . This protocol was approved by the Local Ethics Committee . Informed consent was obtained from all patients.The diagnosis of achalasia was based on the Chicago Classification , using hThe esophagus was cleaned 24 h before the POEM procedure and antibiotic prophylaxis with third-generation cephalosporines or quinolones was administered. All procedures were performed under general anesthesia. A 9. 8-mm outer diameter with a 2. 8-mm working channel endoscope was used , along with a transparent cap model . An electrosurgical unit , and an I-type hybrid knife  were also used. Closure was performed using hemoclips .The LP system was created using a sterile polyurethane non-weighted 127 cm × 20 Fr nasogastric feeding tube  that was cut at the middle. A thin strip of 150cms long of an ultrabright slim LED strip light system of 1/8 in [3.5 mm] in diameter, with a capability of 112 lm/m , was inserted throughout this catheter and attached to it with one 127 cm long strip of Scotch black tape . This LP was coupled to a power supply system of 12 V that used conventional alkaline AA batteries . After LP creation, it was cleaned with soapy water, rinsed and then dried. Finally, we used an antiseptic wipe , which has 2% w/v chlorhexidine gluconate and 70% v/v isopropyl alcohol, in order to clean the catheter before use .The POEM technique was based on Inoue’s technique . The tecFig. .Fig. 1AnTwenty-four hours after POEM, upper GI endoscopy and upper GI series were performed to rule out adverse events. Antibiotics were continued by intravenous and then oral route for 7 days. A proton pump inhibitor (PPI) was administered for 4 weeks. Diet progressed from a liquid diet up to a normal one over the following 3 weeks. Follow-up continued for 6 months, with HRM, GI endoscopy, pHmetry, TBE, reflux questionnaires, and Eckardt scores evaluated at 3 and 6 months. Success was defined as an integrated relaxation pressure (IRP) < 15 mmHg, Eckardt score < 3, and TBE demonstrating adequate passage of contrast (≥ 80% at 5 min) 3 and 6 months postoperatively.Gastroparesis was diagnosed based on clinical evaluations and scintigraphy. Severe refractory gastroparesis was based on the presence of delayed gastric emptying-related symptoms, including nausea, retching, vomiting, abdominal pain, post-prandial fullness, early satiety and/or bloating. Patients who had failure or recurrence after receiving optimal pharmacological therapies and a Gastroparesis Cardinal Symptom Index (GCSI) score > 2.3   with a rProcedures were performed in the endoscopic unit under general anesthesia. Forty-eight hours before the procedure, all patients were administered a liquid diet and antibiotic prophylaxis with third-generation cephalosporines or quinolones. Endoscopic instruments were the same as in POEM patients, including the LP..The G-POEM procedure was based on Khashab’s technique . The tecFig. .Fig. 2AnPatients were admitted to the hospital after the procedure and given intravenous antibiotics, and then changed to oral route, in order to complete 7 days. An upper GI series and upper endoscopy were performed at 24 h to rule out adverse events. Diet progressed from a liquid diet to a normal diet over the next week. PPI was administered for 4 weeks. Both GCSI and GES were performed 3 and 6 months postoperatively.P < 0.05 was considered to be statistically significant. SPSS v.23.0  was used for all statistical analyses.Sample size was calculated based on an assumption that there would be at least 90% of efficacy in the completion of third-space procedures when LP is used, with a significance level of 0.05 (type I error of 5%) and a beta of 0.20 (type II error of 20%). Using an online statistically-validated program for sample size calculation , we calculated a minimum of 25 patients for POEM and G-POEM procedures. Quantitative data were expressed as mean (standard deviation [SD]) or median with interquartile range (IQR); qualitative data were expressed as frequencies and percentages. Bivariate comparisons were done using the Friedman test, chi-squared test and a one-way analysis of variance, as appropriate. There were 70 third-space procedures performed between December 2016 and January 2019 (42 POEM and 28 G-POEM).Forty-two patients were included with a mean age of 46.7 ± 14.3 years and 18 (42.9%) were male. Esophagus type grade II (16 [38%]) and achalasia subtype II (20 [47.6%]) were the most common. Thirty-two (76.2%) were naïve, while 10 (23.8%) had been previously treated, . No Chagas disease was found.The median total POEM time was 50 min (IQR 38–71). The mean tunnel and myotomy lengths were 12.9 ± 3.6 cm and 10.5 ± 3.1 cm, respectively. The most common mild adverse event was minor bleeding in 2 cases (4.7%), and the median length of stay (LOS) was 3 days (IQR 1-4).P < 0.001) and did not change at the 6-month follow-up (P < 0.001). The IRP decreased from 27.3 ± 10.8 mmHg to 9.8 ± 3.8 mmHg at the 3-month follow-up (P < 0.001) and 9.5 ± 4.1 mmHg at the 6-month follow-up (P < 0.001); TBE showed emptying of < 50% in 100% of patients to emptying of > 50% in 100% (p > 0.001) after 6 months. Furthermore, 57% presented positive pHmetry, 15% had esophagitis and 12% had clinical symptoms of reflux disease . The most common etiology was diabetes in 12 (42.9%) and the mean duration of disease before G-POEM was 22.2 ± 5.5 months. The most predominant symptoms and previous therapy were: nausea and vomiting in 15 (53.5%), and medical therapy in 22 (78.7%), respectively. The median number of hospitalizations preoperatively was 2 (IQR 2-5).The median total G-POEM time was 60 min (IQR 48–77). The mean tunnel and myotomy lengths were 5.2 ± 0.96 cm and 3.2 ± 0.82 cm, respectively. The most common mild adverse event was capnoperitoneum in 2 patients (7.1%); it required abdominal decompression with a Veress needle and 1 mucosal tear 24 h after procedure and was solve endoscopically with clips. The median LOS was 2 days (IQR 1-6).P < 0.001), and 1.2 ± 0.43 after 6 months (P < 0.001). GES test showed a decrease in RP4H from 35.3 ± 11.6 to 11.1 ± 4.2 after 3 months (P < 0.001), and 9.3 ± 3.2 after 6 months (P < 0.001). The half-emptying time improved from 260.2 ± 66.9 min to 165.9 ± 31.2 min after 3 months (P < 0.001), and 152.7 ± 23.1 min after 6 months (P < 0.001). Clinical response was observed in 24 patients (85.7%) at the 6-month evaluation. Resolution of the predominant symptoms were as follows: resolved in 16 (57.1%), 18 (64.3%) and 9 (32.1%); improved in 10 (35.8%), 5 (17.8%) and 11 (39.2%); not changed in 1 (3.6%), 3 (17.8%) and 7 (25%) and worsened in 1 (3.6%), 2 (7.2%) and 1 (3.6%), for nausea/vomiting, abdominal pain and gastric fullness, respectively. GES was normalized in 17 (60.7%) and partially improved in 8 (28.5%) of patients at the 6-month evaluation (Table The GSCI score decreased from 3.5 ± 0.64 points to 1.8 ± 0.61 after 3 months (P = 0.2). In G-POEM cases, inadequate submucosal direction was found in 5/28(17.8%) of cases and LP use helped to correct it and confirm myotomy of the PMR in all cases. (video The median placement time for POEM and G-POEM were 5 (IQR 4-6) and 6 (IQR 5-7) min, respectively. All probes were successfully placed without adverse events and no adverse events or technical failures were observed after procedures secondary to their use. There was no damage to the submucosal space or at the intraluminal mucosal level in both POEM and G-POEM cases, and we didn’t have any associated infections, neither when LP was placed in the mediastinal space at EGJ level in POEM cases. In general, LP use helped to adequately complete POEM and G-POEM in 11/70 (15.7%) of cases. After initial classic POEM procedure, LP on was placed and not observed in 6/42(14.2%) of cases. Therefore, submucosal tunnel and myotomy were extended up to an adequate LP confirmation. In POEM cases, placement of LP in posterior approach was relatively easier than anterior approach, but without significant differences in placement times .Third-space endoscopy was first described by Sumiyama et al. ; it was The lumen is considered as the first space, while the peritoneum is considered the second space and the intentionally-created tunnel is the third space (space between the mucosa and muscularis propria) –4. DiffeThe use of lighting devices has been explored in medicine for years. In colorectal and gynecological surgeries, for example, iatrogenic ureteric injury is a serious complication with a variable incidence between 0. 7-10% . The ideBecause of safety and efficiency, we decided to use a white LED-probe and orally insert it into a conventional 127 cm × 20 Fr nasogastric feeding tube. We spent between 10 to 15 min for LP building and disinfection process. Excellent visualization was obtained with the 112 lm/m of the probe; this was enough to be visualized under the submucosal tunnel or over the intraluminal space when inserted into the tunnel. We didn’t have technical problems during assembly or during procedures, neither when insertion into the patient was performed or after LP was used and procedures were finished. Therefore, based on these results, we confirmed the safety and efficacy of this device, that had a median insertion time of 5 min (4-6) for POEM and 6 (5-7) for G-POEM, without compromising total procedural times, and being similar to those observed in previous studies , 24.P = 1.0), without differences in reflux disease incidence, but more cases with grade B esophagitis were presented in treatment group ; they hypothesize that this is because a longer myotomy is performed in them (1.6 ± 1.2cms) [POEM was performed as described by other groups , 6, 21,  1.2cms) . In our  1.2cms) , 13, 21.P < 0.03). However, this was not clinically expressed, with a non-statistically significant difference between GCSI and GES. In our group, LP provided a better orientation towards PMR identification and myotomy confirmation in all cases, including 5/28 patients where the endoscopist was “lost”, during tunnel creation and PMR was not identified, where the LP allowed the completion of G-POEM procedures, representing a 17.8% benefit in them. However, besides the fact that the G-POEM outcomes were slightly better in our study, when compared with other centers, with a general clinical success (85.7% vs 69–81%) in GCSI and GES (89.2% vs 69–84.2%) at 6-month evaluations, we can’t assume that this could be explained because of the 100% PMR identification (similar to the fluoroscopy-guided group from Xue’s study). However, as stated by other authors, different gastroparesis subtypes with their corresponding physiopathology could explain the real heterogeneous mid-term results more than the simple direct effect of the PMR cutting, inclusive with LP guidance, as in our patients [We performed the G-POEM and LP placement in all cases. Demographic characteristics were similar to other groups –11, 24. The strengths of our study include the use of LP in the two most common and important third-space procedures, the sample size that was reached in both and calculated for statistical significance, adequate and strict procedural and follow-up protocols, technical confirmation of all steps in all cases, and excellent safety without adverse events associated with LP use. Our study also has limitations that should be addressed. First, LP is not yet commercially available. Second, LP has to be made before each procedure by the medical doctor, which, in spite of the fact that it takes only between 10 and 15 min, could be time-consuming. Third, different LED and nasogastric feeding tube brands exist around the world, which limits the availability of the system we used. Fourth, only POEM and G-POEM cases were included; pediatric and other third-space procedures were not included, and fifth, LP was used in 76.2% of naïve POEM cases, which represent a subgroup of non-difficult cases in which LP could have been useless, especially when performed by highly-experienced endoscopists in third-space procedures; therefore, we think that the best advantage of LP use could be found in early-experience endoscopists in third-space procedures. However, we think that LP is a useful device for POEM and G-POEM procedures because of its simplicity, innovation, low costs, safety and the ability to make difficult procedures potentially easier.In conclusion, we have confirmed the feasibility of using LP in third-space endoscopy as a new alternative to performing POEM or G-POEM cases, being specifically useful when classic anatomical landmarks are not completely reliable, in difficult cases, low-volume POEM and G-POEM centers, limited third-space procedures experience and when no other confirming methods are available. However, the real clinical relevance of LP use must be confirmed with longer evaluations and comparative studies. Commercialization and evaluation in other third-space procedures are necessary to elucidate potential advantages of the LP system.Additional file 3. POEM files. This is the POEM files from our cohort of patients with POEM that underwent to LP procedure.Additional file 4. G-POEM files. This is the G-POEM files from our cohort of patients with G-POEM that underwent to LP procedure.


The aim of the present study was to describe and measure the occipital-cervical distance by a novel method utilizing the occiput-C4 distance (OC4D) in normal subjects, as a proposed tool to guide restoration of vertical dislocations of the occipitocervical region in patients with basilar invaginations and for performing standardized testing of occipitocervical constructs.We analyzed neutral, flexion, and extension lateral cervical spine radiographs of 150 asymptomatic subjects  that were judged to be normal. The mean age of the included asymptomatic subjects was 48.0 ± 8.4 years old . The OC4D was defined as the shortest distance from the center of the C4 vertebral body to the McGregor’s line. Occipitocervical distances (OCDs) were measured and analyzed its correlation with OC4Ds. Two spine surgeons each performed three measurements of the OC4D and OCD from each asymptomatic subject, from which our reported average values were derived. The height, weight, and body mass index (BMI) of each subject were recorded and analyzed for their correlations with the OC4D and OCD.P >  0.05). The neutral, flexion, and extension OCDs were 23.0 ± 4.8, 27.6 ± 6.0, and 13.8 ± 4.7 mm, respectively. In particular, the neutral OCD was significantly different from those in flexion and extension lateral cervical spine radiographs (P <  0.001). There was no significant correlation between OC4D and OCD in neutral, flexion, and extension . There were positive correlations between OC4D and height, as well as OC4D and weight, in neutral, flexion, and extension lateral cervical spine radiographs . Furthermore, the intra-class correlation coefficients for inter- and intra-observer reliabilities of OC4Ds in neutral, flexion, and extension lateral cervical spine radiographs were significantly higher than those for OCDs (P <  0.001).The OC4Ds from neutral, flexion, and extension lateral cervical spine radiographs were 69.0 ± 6.9, 68.9 ± 6.8, and 68.1 ± 6.9 mm, respectively. There was no significant difference in the OC4D values among neutral, flexion, and extension lateral cervical spine radiographs  for patients with altered occiput-cervical anatomy. Craniocervical joint instability caused by a congenital anomaly , trauma, inflammatory disease, or a tumor may be an indication for occipitocervical fusion (OCF) . During In the present study, we introduced and evaluated the occiput-C4 distance (OC4D) measurement method using lateral cervical spine radiographs from asymptomatic subjects. Additionally, we measured and compared the OC4Ds in different cervical positions and obtained normal value ranges, and further analyzed the correlation of OC4Ds with weight, height, and body mass index (BMI) in order to more comprehensively and accurately define the occipitocervical neutral position. Our findings may support the future use of our practical OC4D measurement method and its reference values for defining occiput-cervical vertical reduction during OCF.In this study, we included 150 sagittal-balanced cervical spine lateral radiographs, which were interpreted to be normal by two spine surgeons , from a radiographic database at the Affiliated Hospital of Southwest Medical University (China). Sagittal-balanced asymptomatic subjects were evaluated via both whole spine radiographs and cervical radiographs.The study population consisted of 73 males and 77 females with an average age of 48.0 ± 8.4 years . All subjects in the present study were from Southwest China, with an average height of 162.4 ± 8.2 cm  and average weight of 62.4 ± 10.3 kg . Of the 150 participants included in the study, 79.3% (119/150) were of Han ethnicity. The proportions of the Yi, Zhuang, Manchu, Hui, Miao, and Mongolian ethnicities were 6.0% (9/150), 4.7% (7/150), 4.7% (7/150), 2.0% (3/150), 2.0% (3/150), and 1.3% (2/150), respectively. Approval for this study was obtained from the Ethics Committee of the Affiliated Hospital of Southwest Medical University (China).Standard lateral cervical radiographs were obtained from each subject. The X-ray tube was centered at C4, and the radiographs were taken from a distance of 2 m from the subject’s left side. Analysis of lateral neutral, flexion, and extension radiographs was performed using our novel OC4D measurement method. The OC4D was defined as the shortest distance from the center of the C4 vertebral body to the McGregor’s line Fig. . In contTo evaluate errors in these measurements, the differences among the means of the first, second, and third tracings for each of the variables were tested using paired t tests. No statistically significant differences were found. Therefore, the average measured values were used for subsequent comparisons. The height, weight, and BMI of each subject were recorded by the co-authors in this study, and BMI was calculated as follows:post-hoc tests. Pearson’s coefficient was used to analyze correlation between OC4D and OCD, and correlations of OC4D/OCD with height, weight, and BMI. Inter- and intra-observer reliabilities of OC4Ds and OCDs were assessed by calculating intra-class and inter-class correlation coefficients (ICCs). The calculated ICCs were interpreted according to the following standard convention: 0.90–1.0, excellent agreement; 0.70–0.89, good agreement; 0.50–0.69, fair/moderate agreement; 0.25–0.49, low agreement; and ≤ 0.24, poor or absent agreement [P <  0.05. All reliability estimates are presented with a 95% confidence interval (CI).SPSS software version 19.0  was used for all statistical analyses. Comparisons of the mean differences between groups that have been split on two independent variables  were performed using Two-Way ANOVA and P >  0.05). In contrast, the mean neutral, flexion, and extension OCDs were 23.0 ± 4.8 mm, 27.6 ± 6.0 mm and 13.8 ± 4.7 mm, respectively; the neutral OCD was significantly greater than that in flexion, but was significantly smaller than that in extension  or extension . However, there was no significant gender differences for OCDs in neutral, flexion, or extension positions between males and females , but there was no significant difference  , and there was also a moderate correlation between OC4D and weight . In contrast, there was no significant correlation between OC4D and BMI . However, there was no significant correlation between OCD and weight, or between OCD and BMI, in neutral, flexion, and extension positions  and extension (14.8 ± 1.48 mm) .Fig. 3TWe found that our novel OC4D measurement has unique advantages compared with those of the OCD in previously reported studies , 13. FirLimitations of the present study included the demographic data not being matched for age, as well as our sample size being relatively small. In spite of these limitations, our study presented a new method for measurement of the occipital-cervical distance, which may have practical valuable for guiding and testing the restoration condition of the occipital-cervical region. Another limitation of this study is that only cervical spinal radiographs were analyzed, as there were no data regarding the overall sagittal alignment of the spine. Although previous studies have reported that cervical curvature can be affected by overall spinal sagittal imbalance –19, onlyIn this study, we proposed and introduced a new OC4D method for estimating the occipital-cervical distance via the shortest distance from the center of the C4 vertebral body to the McGregor’s line (i.e. the OC4D). We found that OC4Ds were not significantly different across neutral, flexion, or extension positions in males or females, and that OC4Ds were significantly positively correlated with both height and weight. Hence, our findings suggest that the OC4D may represent a valuable parameter and intra-operative tool for guiding vertical restoration during OCF for patients with altered occiput-cervical anatomy.