By contrast, there were no significant associations between BCd and thyroid hormones in either men or women. In conclusion, nonlinear associations may exist between BCd and free thyroxine in smokers. Our study provides empirical support for the future formulation of an acceptable concentration range of BCd and offers a new concept for preventing thyroid problems.Research on the association between blood cadmium (BCd) exposure and thyroid hormone levels in the general population has been inconclusive. Therefore, we examined the associations between BCd and thyroid hormones according to smoking status in Korean adults  using multiple linear regression and restricted cubic splines analysis with data from the Korean National Health and Nutrition Examination Survey (2013). The geometric mean of BCd was 0.74 μg/L in all study participants and was higher in smokers (1.01 μg/L) than in nonsmokers (0.65 μg/L). Restricted cubic splines analysis revealed nonlinear trends between BCd and free thyroxine in smokers ( Cadmium (Cd) is a toxic heavy metal and one of the main endocrine disruptors (ED) that disturbs endogenous hormone action ,2. CadmiThe toxicity of Cd has cancer-causing effects , and affIn previous studies, blood cadmium (BCd) values were 3.5 times higher in smokers’ blood compared to that of nonsmokers in Serbia . BCd wasMany studies have examined the relationship between BCd and thyroid hormones ,20,21,22This cross-sectional study was based on data from the Korea National Health and Nutrition Examination Survey (KNHANES), 2013. KNHANES is a cross-sectional, population-based, nationwide survey that is regularly conducted by the Korea Centers for Disease Control and Prevention. Detailed information on the data collection design and process of KNHANES has been published previously . Of the To determine the concentrations of BCd (μg/L) in venous whole blood, blood samples (3 mL) were collected from each participant into blood collection tubes containing ethylenediaminetetraacetic acid (EDTA) for determination of trace elements . BCd was measured by graphite furnace atomic absorption spectrometry using the model AAnalyst 600 .Analyses of all blood metals were conducted by the Neodin Medical Institute, a laboratory certified by the Korea Ministry of Health and Welfare. Internal quality assurance and control were established using commercial reference materials . For external quality assurance and control, the Neodin Medical Institute fulfilled the requirements of both the German External Quality Assessment Scheme (operated by Friedrich-Alexander University) and the Quality Assurance Program . The institute was also certified by the Ministry of Labor as a designated laboratory for the analysis of specific chemicals, including heavy metals. The method detection limit for Cd was 0.016 μg/L. All samples were above this detection limit.We collected approximately 15 mL of blood for the analysis of serum TSH, fT4, and TPOAb levels. Within 30 min after separation of the serum, the sample was transferred to the testing facility. The specimens were analyzed by an electrochemiluminescence immunoassay within 24 h. Serum TSH, fT4, and TPOAb levels were measured with an electrochemiluminescence immunoassay method with a Cobas8000 E-602 . The reported results of TSH, fT4, and TPOAb measurements met the specifications regarding the accuracy, general chemistry, special immunology, and ligand measurements of the quality control and quality assurance program of the College of American Pathologists [We considered the following as covariates: age, sex, educational level, monthly household income, marital status, BMI, smoking status, alcohol consumption, the urine iodine-to-creatinine ratio (UI/Cre), and other heavy metal (Pb and Hg) levels in the blood. Information on education level, monthly household income, marital status, smoking status, and alcohol consumption was collected through a health interview. Educational level was classified as: less than elementary school, middle school, high school, and college or more. Monthly family income was calculated using standardized classification by sex, residence, and five-year age groups and compared with the standard income level of Korean citizens. Monthly family income was then classified into quartiles.2 for underweight, 18.5–23 kg/m2 for normal weight, 23–27.5 kg/m2 for overweight, and ≥27.5 kg/m2 for obese [Smoking status was classified as never or ever-smoker (former or current). A never smoker was defined as a person who reported never having smoked 100 cigarettes. Pack-years were calculated by multiplying the number of packs of cigarettes smoked per day by the number of years the person has smoked (one pack has 20 cigarettes). Alcohol consumption was classified as never or ever-drinker (moderate or heavy). BMI was calculated as the participant’s weight in kilograms divided by their height in meters squared. BMI cut-point categories were recommended by World Health Organization (WHO) experts in Asian populations as follows: <18.5 kg/mor obese . We examined differences in the following characteristics between men and women: age, educational level, monthly household income, marital status, smoking status, BMI, alcohol consumption, other heavy metal (Pb and Hg) levels in the blood, and the urine iodine-to-creatinine ratio (UI/Cre). The geometric means and 95% confidence intervals (CIs) of BCd concentrations were calculated according to age group, educational level, monthly household income, marital status, smoking status, BMI, and alcohol consumption. We further calculated the means and CIs of thyroid function according to sex.BCd concentrations were natural-log-transformed due to their right-skewed distribution. We performed crude and adjusted multiple linear regression analyses for TSH, fT4, and TPOAb levels in terms of natural-log-transformed BCd concentrations. We also applied five-knot restricted cubic spline analysis  to evaluate the nonlinear, inverted U-shaped associations between natural-log-transformed BCd concentrations and TSH, fT4, and TPOAb levels estimated by the adjusted multiple linear regression analyses. Likelihood ratio tests were performed to compare the linear terms with the cubic spline terms to test for nonlinearity. In order to determine the appropriateness of a linear or non-linear relationship between a dependent variable and independent variables, likelihood ratio tests (LRT) were performed.t-test) for continuous variables. PROC SURVEYREG analyses with and without covariates were conducted to evaluate the relationship between BCd and thyroid function. There statistical analyses accounted for the complex sampling design. SAS software, version 9.4 , was used for data analysis. We carried out the restricted cubic splines analysis for graphical displays using the ‘rms’ package (Frank E Harrell Jr (2022), rms: Regression Modeling Strategies, R package version 6.3-0, (https://CRAN.R-project.org/package=rms (accessed on 1 January 2023). All statistical analyses were two-sided with a significance level of p < 0.05.Differences in basic characteristics between the two groups (men vs. women) were explored using the PROC SURVEYFREQ procedure (Rao-Scott Chi-square test) for categorical variables and the PROC SURVEYREG procedure (Independent p < 0.001). Men were significantly more likely to be current smokers and to consume alcohol compared with women. The lower BMI group (<23 kg/m2) had a higher proportion of women than men, and the proportion of men was greater than that of women in the higher BMI group (≥23 kg/m2).The baseline characteristics of the study participants according to sex are presented in p < 0.05). Smokers had significantly higher mean BCd concentrations than nonsmokers. However, BCd concentration did not significantly differ according to household income in women, BMI in men and women, and alcohol consumption in the overall population, men, and women.β = −0.039, p < 0.0001), TSH , and fT4  in non-smoking group. After adjusting for covariates, there were no significant results.In addition, the restricted cubic splines  show theEndocrine disruptors (ED) are defined as chemicals that disrupt hormone function. Among several chemical substances, Cd is representative. Thyroid hormone is among the many hormones affected by exposure to Cd. The reason for the high accumulation of Cd in the thyroid is that metallothioneins (MT), which are proteins rich in cysteine, exist in the thyroid, and Cd binds well to molecules rich in sulfhydryl groups such as MT or glutathione. When humans are exposed to Cd, it can become toxic to the endocrine system, resulting in problems such as hyperthyroidism or hypothyroidism. As such, thyroid toxicity can be confirmed by glandular synthesis, secretion, disease, etc. above T4, T3, and TSH levels. Also, one of the causes of exposure to Cd is smoking. That is why it is so important to look at the difference between smokers and nonsmokers . TherefoPrevious studies on the relationship between Cd and thyroid hormones reported conflicting results. Thyroid-related hormones such as T3, T4, and TSH and thyroid antibodies could be increased, decreased, or not changed by increasing blood or urinary Cd level . The U.SThese previous studies focused on either urinary Cd or BCd levels ,21,22,26Our study found significant relationships between BCd and thyroid hormones in Model 1 before adjustment , but Model 2, which adjusted for various variables, showed no significant associations. Similar results were obtained when the groups were analyzed separately by sex . HoweverThis study has some limitations. We used cross-sectional data, which could not explain the casual relationship. Also, we only included BCd levels, which indicate the recent Cd level. We could not consider indirect smoking status in this study. However, despite these limitations, our study is the first to consider the nonlinearity in the relationship between BCd and thyroid hormones by smoking status. Also, fT4 was measured, which is a more effective means of assessing thyroid dysfunction compared to T3 and T4 levels .This study investigated the associations between BCd and thyroid hormones according to smoking status in Korean adults. Examining the nonlinear associations revealed significant results for BCd and fT4 in smokers. Further prospective studies are required to confirm the nonlinear associations between BCd and thyroid hormones. Our study provides empirical support for the future formulation of an acceptable concentration range of BCd and provides evidence of the need for strategies to reduce exposure to BCd.


Both robots and navigation are effective strategies for optimizing screw placement, as compared to freehand placement. However, few studies have compared the accuracy and efficiency of these two techniques. Thus, the purpose of this study is to compare the accuracy and efficiency of robotic and navigation-assisted screw placement in the spinal vertebrae.The 24 spine models were divided into a robot- and navigation-assisted groups according to the left and right sides of the pedicle. The C-arm transmits image data simultaneously to the robot and navigates using only one scan. After screw placement, the accuracy of the two techniques were compared using “angular deviation” and “Gertzbein and Robbins scale” in different segments . In addition, operation times were compared between robot- and navigation-assisted groups.p < 0.001). At the C1–7 and T1–4 segments, the robot-assisted group had a higher rate of acceptable screws than the robot-assisted group. However, at the T5–8, T9–12, and L1–S1 segments, no significant difference was found in the incidence of acceptable screws between the two groups. Moreover, robot-assisted screw placement required less operative time than navigation (p < 0.05).Robots and navigation systems can simultaneously assist in screw placement. The robot-assisted group had significantly less angular deviation than the navigation-assisted group from C1 to S1 (The robot is more accurate and efficient than navigation in aiding screw placement. In addition, robots and navigation can be combined without increasing the number of fluoroscopic views.The online version contains supplementary material available at 10.1007/s00264-022-05638-0. The pedicle screw technique is an important technique in the field of spinal surgery . ConventIn 1995, Amiot . With thHowever, to the best of our knowledge, few studies have compared the accuracy and efficiency of these two techniques. Therefore, this study investigated the accuracy and efficiency of robot-assisted versus navigation-assisted pedicle screw implantation in spinal models.The robot-assisted system  consists of a robotic arm, optical tracking system, robotic workstation, and a navigation toolkit. The navigation-assisted system consists of a workstation with navigation software  and specific surgical instruments. The C-arm type is the Arcadis Orbic 3D  Fig.  Supplem.Fig. 1PoTwenty-four simulation spine models  made up of high-resolution foam cortical shells with cancelli were all radiolucent to optimize image quality and minimize artifacts. The 24 spine models were divided into two groups according to the left and right sides of the pedicle . Each spine model was embedded in a sponge by an assistant, and the bilateral transverse processes were completely covered by colored sand, exposing only the same operative field as that in clinical practice Fig. . The spiImage acquisition of the spinal vertebrae was performed using a C-arm. The image data were transferred to the robot workstation via a network cable. Screw planning, including diameter, length, and trajectory, was carried out in the robotic workstation. First, the sleeve was placed into the screw guide. Then, after the foot pedal was pressed, the robot arm automatically adjusted its direction according to the trajectory planned for the surgery until it exactly matched the planned trajectory. Next, K-wire was drilled into the vertebrae. The cannulated tap was used to prepare the screw channel first; then, the pedicle screw was implanted Fig.  Supplem.The SpineMap 3D 2.0 was selected in the navigation workstation. The instrument tracker, patient tracker, and C-arm tracker were activated. The navigation system and C-arm were connected using a network cable. The image data from the C-arm, which was transmitted to the robot workstation, was sent to the navigation workstation. Next, a screw design was performed. The screw view model of navigation, which is an accurate and easy-to-use model, was selected in the assisted screw placement mode to aid the surgeon in establishing the screw channel with the tap in hand, followed by screw placement Fig.   was recorded by an assistant, including the preparation time (robot/navigation boot time to C-arm scan), screw design time, and robot/navigation-guided screw placement time.t-test were used. Statistical significance was set at p < 0.05.To investigate whether a difference exists between the robot-assisted and navigation-assisted techniques at different segments of the spine, Fisher’s exact test and independent-sample Robots and navigation systems can simultaneously assist in screw placement. A total of 1200 screws were inserted into the spine model from C1 to S1, including 600 screws in the robot group and 600 screws in the navigation group. In each group, the number of implanted screws was 168 in C1–7, 96 in T1–4, 96 in T5–8, 96 in T9–12, and 144 in L1–S1.p < 0.001)  and 98.96% vs. 91.67% (p < 0.05), respectively]. However, at the T5–8, T9–12, and L1–S1 segments, no significant difference was found in the incidence of acceptable screws between the robot-assisted group and navigation-assisted group   Fig.  (Table 1p > 0.05)]. However, the preparation time and guided-screw insertion time in the robot-assisted group were significantly shorter than those in the navigation-assisted group  . We analyzed the reason for this result: the robotic group established the screw path by placing the Kirschner needle through the robotic arm, whereas the navigation group manually established the screw channel using an opener under image guidance. In this difference, the robotic arm is much more capable of maintaining a fixed direction in space than the manual arm, so the angular deviation of the robot-assisted screw placement would be closer to the trajectory of the designed screws than the navigation.Regarding screw angle deviation, to the best of our knowledge, we pioneered this evaluation method for assessing the deviation between the actual and designed screw positions. The accuracy of the robotic and navigational screw placement in a total of six parts C1–7, T1–4, T5–8, T9–12, and L1–S1 was evaluated separately, and it was found that angular deviation existed in both device-assisted screw placements. However, the screw angular deviation in the robotic group was smaller than that in the navigational group in all segments (p < 0.05), while no significant difference was detected between the two groups in segments T5–8, T9–12, and L1–S1 (p > 0.05), indicating that robotic assistance is safer and more valuable than navigation for screw placement in the C1–C4 segment. We analyzed the reasons for this as follows: T5–S1 has a larger mean diameter of the pedicle than C1–C4. Although our angular deviation results showed a slight angular deviation in T5–S1, it was not severe enough to penetrate the bone cortex.Regarding the rate of the acceptable screw (grade A + B), we used the Gertzbein and Robbins scale proposed by Gertzbein et al. to observe the severity of screw penetration through the bone cortex . We founp < 0.05). We compared the following three time periods: preparation time, screw planning time, and guided screw placement time. No statistical difference was found in screw planning time between the robot and navigation groups (p > 0.05). However, the robot had a shorter preparation time and guided-screw-placement time than navigation (p < 0.05). We believe that there are two reasons for this result. First, the surgical instruments of the robot do not require manual registration, whereas those of the navigation require activation and calibration. Second, for guided-screw placement, the robotic arm automatically moved to the designed screw trajectory direction after stepping on the foot pedal during robot-assisted screw placement, and the time used was significantly lower than that for navigation-assisted screw placement, when the direction was manually adjusted intra-operatively according to the images.The second important finding of this study was that the robot had a shorter screw placement time than that of the navigation system Supplementary file2 (TIF 9695 KB)Below is the link to the electronic supplementary material.


Ice cream consumers (n = 92) rated their overall liking, attribute liking, and sweetness and bitterness intensities and described the aftertastes of each sample using check-all-that-apply. The liking scores of Reb D- and M-sweetened ice creams were significantly higher than those of Reb A-sweetened ice cream. Among the three glycosides, only Reb M showed a sweetness intensity comparable with that of sucrose. Consumers perceived the aftertastes of Reb D and M ice creams as being more sweet, pleasant, creamy, and milky, while Reb A was more artificial and chemical. Reb D and M ice creams were also plotted close to sucrose in the correspondence analysis graph, meaning that their aftertaste characteristics were similar to those of sucrose. The present study clearly highlights that Reb D and M have better tastes and provide better perceptions to consumers than Reb A, which is the most widely used glycoside in food industry. There has been a challenge in overcoming the bitter aftertaste of stevia, a natural non-caloric sweetener. Recent research focuses on investigating various types of steviol glycosides, the sweet compounds in stevia leaves, as they exhibit different sensory characteristics. This study determined the sensory properties and acceptability of ice cream sweetened solely with three steviol glycosides, rebaudioside (Reb) A, D, and M (0.09%  The average American adult consumes twice the daily amount of recommended sugar ,2, leadiMany food companies have started to introduce stevia into their food and beverage products to promote healthy options to consumers ,18,19. SStevia rebaudiana) is a South American plant which has up to 300 times the sweetness of sucrose [w/v). They also found that the aftertaste descriptors of Reb D and M were close to sucrose. However, no study to date has investigated the sensory properties of Reb D and M compared to Reb A in food matrices.Stevia  and the major glycoside (Reb A) were compared to sucrose-sweetened controls. Furthermore, we investigated whether Reb A, D, and/or M could be used as sole sweeteners in high-sucrose applications, without compromising sensory quality.The materials used to produce ice cream samples were purchased from a local grocery store: heavy cream , non-fat dry milk , vanilla extract , polydextrose , and sucrose . The steviol glycosides used in the ice cream for the study were high purity (95%) Reb A, Reb D, and Reb M from Sweegen .w/v) in the ice cream formulation. The 0.09% concentration was chosen because it was found to have a similar sweetness level as the 14% sucrose (w/v) from the previous study by Tao and Cho [Reb A, D, and M were used at 0.09%  and zero-calorie sweeteners (at least once a month) were recruited from Auburn University (18–65+ years old). The pre-survey was performed using Qualtrics online survey software , including the consumption behavior of HISs and the frequency of ice cream consumption.All ice cream samples were made two days before the test. One day before the test, a scoop (~30 g) of ice cream was transferred into a 2 oz plastic soufflé cup labeled with random 3-digit coded numbers. They were stored in a walk-in freezer (−20 °C).This study was approved by the University Institutional Review Board of Auburn University  (Protocol #: 21-204 EX 2104). RedJade sensory science software  was used to collect data during the entire testing.artificial, metallic, milky, buttery, chemical, bitter, spicy, vanilla, honey, minty, pleasant, tart, sweet, and spicy. This study used 11 terms from a previous study conducted by Tao and Cho [buttery, creamy, and milky were added to describe the flavor attributes of ice cream. Lastly, the term spicy was used as an attention check. The purchase intent question, using a 5-point Likert scale , was asked at the end of each sample. A 30 s break was enforced before receiving the next sample. During the break, water and unsalted crackers were provided as palate cleansers. After evaluating all four samples, consumer behavior and demographic questions were asked, including low/no-sugar product consumption behavior, familiarity with zero-calorie sweeteners , health-related questions , and demographic questions .After confirming that the code of the served sample matched the code on the screen, the panelists were asked to taste a spoonful of the ice cream sample (less than 1/2 of the serving cup) to evaluate their overall liking and attribute liking  of the sample using a 9-point hedonic scale . Next, they were instructed to take another spoon of the same sample and swallow it to evaluate the aftertaste of each sample. For the aftertaste, they were asked to rate the intensities of sweetness and bitterness using a 15 cm-line scale  and then to choose aftertaste descriptors using check-that-all-apply (CATA) analysis. The listed attributes for CATA included  and Cho , who evap < 0.05), and Tukey’s (HSD) tests, treating ice cream samples as a fixed effect and the consumer panel as a random effect. To determine if there was an interaction effect between the overall liking score of each ice cream and gender, data were also analyzed using two-way ANOVA with one interaction effect (fixed effects: ice cream sample and gender). Cohran’s Q test was used to analyze the check-all-that-apply (CATA) responses to determine any significant differences between ice cream samples. Correspondence analysis (CA) was used to show the relationship between sensory attributes and the samples.Data analysis was performed using XLSTAT . The sensory evaluation questions and the sweetness and bitterness intensities were determined by two-way analysis of variance (ANOVA), with a 95% confidence level  of 26.0 ± 5.3 kg/mn = 92).p < 0.05) and every attribute liking (p < 0.001) except for appearance and texture/mouthfeel liking. There were no differences between minor steviol glycosides (Reb D and M) and sucrose ice cream on the appearance liking . In the texture/mouthfeel liking, Reb M and sucrose ice cream were found not to be significantly different from one another (p = 0.052), but the p-value was close to the significance level (p < 0.05). Although Reb M ice cream received slightly higher liking scores than Reb D ice cream in every category, they were not significantly different (p < 0.001), both of which were close to three ‘Might or might not purchase’ . Reb M ice cream received the highest purchase intent score among the steviol glycoside ice creams, although there was no significant difference between Reb D and M (p = 0.49).Sucrose ice cream received scores of over 7.0 (moderately like) on a nine-point hedonic scale and was significantly higher than all three steviol glycosides for overall liking . Reb A and D ice cream samples were both significantly less sweet than sucrose and Reb M ice cream (p < 0.001). Moreover, Reb M and sucrose ice cream samples received bitterness intensity ratings that showed no significant difference (p = 0.175), while the intensity score of Reb M ice cream was higher than that of sucrose ice cream . Reb A ice cream received the highest bitterness score among all the samples (p < 0.001). There was no significant difference in the bitterness intensity between Reb D and M ice cream (p = 0.853). The participants rated the bitterness intensity of Reb D and M at 2.9 and 2.6 on a 15-cm line scale, respectively, while Reb A received 5.4.Among all three steviol glycosides, Reb M ice cream received the highest sweetness intensity and showed comparable sweetness to that of sucrose ice cream (n = 92).bitter, metallic, milky, and tart were used to describe Reb D, Reb M, and sucrose ice creams. Interestingly, for the term artificial, Reb D ice cream was chosen by 12 more panelists than Reb M , but there was no significant difference between them. However, the term artificial was used to describe Reb A significantly more than Reb M  (p < 0.0001), but there was also no significant difference between Reb A and Reb D . The results for the term sweet across all ice cream samples was complementary to a 15-cm line intensity scale in that the sucrose and Reb M ice creams were chosen by most panelists  and Reb A and D ice creams were chosen the least frequent with this term . The term bitter was chosen the most with Reb A ice cream by a consumer panel (37), which was similar to the bitterness intensity scale  was described similarly to both sucrose and Reb M ice creams . Reb A ice cream was described more frequently with negative terms than other ice cream samples, including bitter, metallic, and tart. However, all three steviol glycosides shared metallic, vanilla, and tart terms with no significant difference (p < 0.05). Three terms which were not significantly different from each other were buttery, honey, and minty.Reb D and M ice cream samples received no significant difference from each other for each aftertaste term . The terty scale . Howeverpleasant, vanilla, sweet, and creamy were associated with and chosen for sucrose more than for all three steviol glycosides. Reb A was associated to more negative terms, including metallic, bitter, chemical, and tart, while Reb D and M were mostly associated with positive words. Moreover, both minor steviol glycosides were plotted close to each other and were closer to sucrose when compared to Reb A.The sensory attributes of the sweeteners were summarized visually in n = 37) and female panelists (n = 55) rated their overall liking of the ice creams differently from each other. In this study, it was later found that male  in ice cream and compared them against sucrose ice cream (14% w/v) as a control. Stevia has different physiochemical properties than sucrose [This study examined the sensory quality of three different steviol glycosides  and sucrose ice cream samples at 14% (w/v). When comparing Reb D and M (the minor steviol glycosides) with Reb A (the most widely used steviol glycoside in the food industry), Reb A ice cream was least preferred by panelists; it was given the lowest score among samples in all hedonic liking scores. The consumer panel preferred minor steviol glycosides over the major steviol glycoside, except in appearance and texture/mouthfeel attributes (p < 0.001). Reb D and Reb M ice creams scored at around six points (Like slightly) in all hedonic liking scores except aftertaste liking, which were both at around five points (Neither like nor dislike). They both shared similar scores and showed no significant difference, but the consumer panel showed a slightly higher preference for Reb M over Reb D ice cream. Moreover, Reb M and sucrose ice creams shared more similar attributes than other steviol glycosides . According to Everitt [The results from  Everitt , a mean  Everitt , we founp < 0.05). Thus, a high-purity stevia extraction method could mitigate negative aftertastes  found in stevia, especially Reb A at a high concentration. This could be the potential reason explaining the consumer panel’s preference for minor steviol glycoside ice cream over Reb A ice cream.While many studies have developed ice cream formulations with different ratios of stevia and other sweeteners, few studies have incorporated formulations using purely stevia. Alizadeh et al.  used fivp = 0.220 and p = 0.175, respectively). This could explain why Reb M received the highest hedonic and Likert scores among all steviol glycoside samples, even though Reb D and M ice cream samples received bitterness intensity scores of 2.9 and 2.6, with no significant difference (p < 0.05). A study done by Jung et al. [p < 0.05). In addition, Tao and Cho [p = 0.05) at the same concentrations as this study. However, their consumer panelists were able to distinguish Reb D from Reb M because the Reb M solution provided the highest immediate sweet taste among other samples. Thus, in this study, Reb M ice cream was found to be sweeter than either Reb A or D ice creams. The sweet taste attribute of steviol glycosides is dependent on the functional group (R-groups) at positions C-13 and C-19 of the steviol core, on which different types of sweet molecules are attached [On the sweetness and bitterness intensity scales , the cong et al.  found th and Cho  found thattached ,36. The attached ,55. Thisattached  and foodmilky, vanilla, and pleasant, and they were also plotted close to sucrose in the corresponding analysis. Reb A ice cream was on the opposite side of sucrose ice cream, with more negative attributes such as bitter and metallic  and the sucrose solution. In this study, the term artificial was selected to describe all three steviol glycoside ice creams more than sucrose ice cream but was used to describe Reb M ice cream significantly less than Reb A and D ice creams (p < 0.0001). On the other hand, Tao and Cho [p <0.001). In the CATA analysis , consumemetallic . All ste and Cho  found th and Cho  found thbitter corresponded to the bitterness intensity rating on a 15-cm line scale . This supports previous findings suggesting that Reb M has the highest sweetness intensity compared to other steviol glycosides [Our CATA analysis revealed that consumer panelists associated Reb M and sucrose with the term ycosides ,42,44.n = 37 and n = 55, respectively). The p-value of the interaction effect was 0.06, which is slightly greater than 0.05, the significance level, even though the p-value could be changed with the unequal sample size of the panelists for each gender. However, it is important to note that female participants clearly showed a higher preference for the Reb D and M ice creams than male participants did (There was no interaction between male and female preferences for ice creams sweetened with steviol glycosides versus sucrose (ants did . This maOne possible limitation of this study could be the participants’ household incomes. There are many factors that affect the consumer’s purchase intent, one of which is the price . The majThis study has confirmed that minor steviol glycosides, Reb D and M, had positive effects on the acceptability of zero-sugar ice cream when compared with Reb A, the major steviol glycoside. Although sucrose ice cream received the highest liking scores among ice cream samples, these minor steviol glycosides received overall liking scores between ‘like slightly’ and ‘like moderately’, which were significantly higher than those of Reb A. Furthermore, the aftertaste characteristics of Reb D and M were comparable to sucrose ice cream. Interestingly, only Reb M was found to provide a sweetness taste profile similar to sucrose, but there was no significant difference in flavor liking of Reb D and M ice creams. Thus, it is suggested that Reb D and M can be used as natural non-caloric sweetener options to replace sucrose without adding a bitter aftertaste, even in high-sugar applications such as ice cream or frozen desserts. However, further studies are needed to determine the commercial applications for Reb D and M, as their quantities in stevia leaves are small. Breeding stevia plants for increased concentrations of these minor glycosides would be beneficial for increasing the supply of the desired glycosides. It would also be beneficial to identify an optimal combination that uses both major and minor steviol glycosides to accommodate the bitter aftertaste issue from Reb A and the small extractable quantities of Reb D and M.


Deep-sea cold seeps are one of the most productive ecosystems that sustained by hydrocarbons carried by the fluid. Once the seep fluid ceases, the thriving autotrophic communities die out, terming as the extinct seep. But heterotrophic fauna can still survive even for thousands of years. The critical role of prokaryotes in active seeps are well defined, but their functions in extinct seeps are poorly understood to date. Here, we clarified the diversity, taxonomic specificity, interspecies correlation, and metabolic profiles of sediment prokaryotes at an extinct seep site of Haima cold seep, South China Sea. Alpha diversity of archaea significantly increased, while that of bacteria remained unchanged in extinct seep compared to active seep. However, archaea composition did not differ significantly at extinct seep from active or nonseep sites based on weighted-unifrac dissimilarity, while bacteria composition exhibited significant difference. Distribution of archaea and bacteria showed clear specificity to extinct seeps, indicating the unique life strategies here. Prokaryotes might live chemolithoautotrophically on cycling of inorganic carbon, sulfur, and nitrogen, or chemoorganotrophically on recycling of hydrocarbons. Notably, many of the extinct seep specific species and networked keystone lineages are classified as Proteobacteria. Regarding the functional diversity and metabolic flexibility of this clade, Proteobacteria is supposed to integrate the geochemical cycles and play a critical role in energy and resource supplement for microbiome in extinct seep. Collectively, our findings shed lights on the microbial ecology and functional diversity in extinct seeps, providing new understanding of biogeochemical cycling after fluid cessation.IMPORTANCE This research paper uncovered the potential mechanisms for microbiota mediated geochemical cycling in extinct cold seep, advancing our understanding in deep sea microbiology ecology. Cold seeps are first discovered in 1983 and considered one of the most productive deep-sea ecosystems –4. Metha–5–pmoA gene responsible for aerobic methane oxidation  coupled sulfate reduction (SR)  , 18. Othion (SR) , 19, sugdch gene in benzoyl-CoA pathway for aromatic hydrocarbon degradation  were also abundant. Gammaproteobacteria (7.17 to 38.25%), Acidimicrobiia (2.19 to 32.56%), Deltaproteobacteria (1.18 to 6.30%), Anaerolineae (2.89 to 17.71%), Bacilli (2.21 to 15.21%), Microgenomatia (0.88 to 9.96%), and NC10 clade (1.09 to 12.55%) dominantly comprised the bacteria community of NS site . While that of bacteria community did not change among the three sites  and permutational multivariate analysis of variance . However, significant difference was not found between the archaea communities of ES site and AS or NS site by PERMANOVA , an extinct seep site (ES), a nonseep site (NS) in Haima cold seep, SCS . For the NS site . ShannonIt was clearly illustrated in the ternary plotting on species level that most archaea and bacteria species were plotted at the corners or edges, showing clear site-specific distribution pattern. Most of them were specific to one or two site, and only 11 archaea amplicon sequence variants (ASVs) and 17 bacteria ASVs were present at all three sites. Compared to 32 archaea ASVs and 85 bacteria ASVs specifically inhabited at AS site, 799 archaea ASVs and 477 bacteria ASVs at ES site, and 762 archaea ASVs and 145 bacteria ASVs at NS site. ES and NS sites shared 196 archaea ASVs and 94 bacteria ASVs, which is much more than they shared with the AS site , B. Ternn = 18). A total of 117 nodes representing prokaryotic species and 257 links representing the node-between correlations were in the network, displaying scale-free, small-world, and modular properties (Table S3). We found that the ASVs with higher relative abundance (indicated by darker color) are less connective (indicated by node size). ANME-2c, Desulfobulbaceae, Milano-WF1B-44, Lokiarchaeia, and Nitrosopumilaceae were highly connective  with samples from all stages  and nitrogen cycling . The proportion of ASVs involved in carbon cycling showed decline at ES and NS sites, while obviously increased in nitrogen cycling. Bacteria participated in carbon cycling , sulfur cycling , and nitrogen cycling . The proportion of ASVs involved in carbon and nitrogen cycling showed obviously increased at ES site compared to AS site, while that of sulfur cycling declined .mcrA) was all belonging to Euryarchaeota, while the benzoyl-CoA degradation pathway genes  and β-oxidation pathway gene (fadA) were dominantly affiliated with Proteobacteria. The bssA gene encoding benzylsuccinate synthase, initiating oxidation of aromatic hydrocarbons, were belonging to Chloroflexi and Archaea  were dominantly belonging to Proteobacteria, Planctomycetes, and archaea, while the aclA/B genes for rTCA pathway were belonging to archaea , denitrification, nitrogen fixation, and nitrification were identified, suggesting a complete nitrogen cycle at ES site. nitrate reduction genes  were carried by Campylobacterota, Proteobacteria, Nitrospinae, and Chloroflex. Further reduction via DNRA (nirB and nirD) and denitrification  was probably mediated by Proteobacteria and Planctomycetes. Nitrogen fixation genes  were dominantly carried by Proteobacteria and Euryarchaeota. Nitrification genes  were dominantly affiliated with Planctomycetes and Thaumarchaeota , and the benzol-CoA degradation pathway and β oxidation pathway for aromatic or chain hydrocarbon oxidation were identified. The key gene for AOM  were replaced by the heterotrophic ones  . A drama32–sqr gene, implying the conversion of hydrogen sulfide into polysulfide  or unchanged (bacteria) in extinct seep. They can either live chemolithoautotrophically on cycling of inorganic carbon, sulfur, and especially nitrogen that is not existing in active seeps, or chemoorganotrophically. Accordingly, we proposed a model for prokaryotic metabolic profiles in extinct seep . It is wSediment samples were obtained from Haima cold seep in the north of South China Sea  for the AS site pushcore were used. So, six samples for each site were used for DNA extraction. Total DNA was extracted from 2 g sediments using the TGuide S96 Magnetic Soil/Stool DNA kit  according to manufacturer’s instructions. The DNA concentration of the samples was measured with the Qubit dsDNA HS assay kit and Qubit 4.0 Fluorometer .Bacterial and archaeal 16S rRNA genes were amplified using the 27F/1492R primer pairs , and 20FRaw data were primarily filtered and demultiplexed using the SMRT Link software (version 8.0) with the minPasses ≥5 and minPredictedAccuracy ≥0.9, and the chimeras were removed by lima (v1.7.0) software to obtain the circular consensus sequencing (CCS) reads. Reads without primers and over length range  were discarded and quality filtering using the Cutadapt (version 2.7) . AmplicoFunctional prediction of prokaryotic communities was conducted with the Faprotax, a manually constructed database mapping prokaryotic taxa to metabolic or other ecologically relevant functions based on the literature on cultured representatives . The pren = 18), and only ASVs present in at least 6 samples were included for correlation calculation. The topological indices were calculated in the MENAP interface  were constructed on the basis of Pearson correlations of bacteria and archaea ASVs abundances, followed by an RMT-based approach that determines the correlation cutoff threshold in an automatic fashion –79 with –nterface . Modulesnterface . Random nterface  in the Mnterface , 29. Othhttps://app.biorender.com) and modified manually.10 nanogram of DNA was used to produce library via VAHTS Universal Plus DNA Library Pren kit for Illumina and the pooled libraries were sequenced on Illumina Novaseq 6000 platform. A total of 7,103,829,330 bases of 18,195,418 raw reads was primarily filtered by Trimmomatic   version. The SchPRJNA846870 for the AS samples (SAMN28906174-SAMN28906179 and SAMN28906134-SAMN28906139) and PRJNA847028 for ES and NS samples. The metagenome sequencing data were deposited at the NCBI Sequence Read Archive under the BioProject PRJNA847028.The amplicon sequencing data were deposited at the NCBI Sequence Read Archive under the BioProject


Mast cells (MCs) play key roles in IgE-mediated immunoresponses, including in the protection against parasitic infections and the onset and/or symptoms of allergic diseases. IgE-mediated activation induces MCs to release mediators, including histamine and leukotriene, as an early response, and to produce cytokines as a late phase response. Attempts have been made to identify novel antiallergic compounds from natural materials such as Chinese medicines and food ingredients. We herein screened approximately 60 compounds and identified salicylaldehyde, an aromatic aldehyde isolated from plant essential oils, as an inhibitor of the IgE-mediated activation of MCs. A degranulation assay, flow cytometric analyses, and enzyme-linked immunosorbent assays revealed that salicylaldehyde inhibited the IgE-mediated degranulation and cytokine expression of bone-marrow-derived MCs (BMMCs). The salicylaldehyde treatment reduced the surface expression level of FcεRI, the high affinity receptor for IgE, on BMMCs, and suppressed the IgE-induced phosphorylation of tyrosine residues in intercellular proteins, possibly Lyn, Syk, and Fyn, in BMMCs. We also examined the effects of salicylaldehyde in vivo using passive anaphylaxis mouse models and found that salicylaldehyde administration significantly enhanced the recovery of a reduced body temperature due to systemic anaphylaxis and markedly suppressed ear swelling, footpad swelling, and vascular permeability in cutaneous anaphylaxis. Mast cells (MCs) and basophils are hematopoietic cells that specifically express the high affinity receptor for IgE, FcεRI, which is essential for IgE-mediated allergic responses . The croIn the present study, we performed a screening to identify candidates for novel inhibitors of the activation of MCs by monitoring the extent of the IgE-mediated degranulation of mouse bone-marrow-derived MCs (BMMCs). As components of a library, we selected compounds that were proven to be present in and/or added as supplements to food, drink, detergents, and perfume as flavors and fragrances, because the biological safety of these compounds has been established under the conditions of their application limits; however, their activity on immunoresponses remains unknown. Through a screening, we identified salicylaldehyde, an aromatic aldehyde giving a fruity flavor, which is a natural compound contained in plant essential oils, as the most effective inhibitor of the IgE-mediated activation of MCs. In vitro experiments showed that salicylaldehyde suppressed IgE-mediated degranulation, IgE-induced cytokine release, and FcεRI expression in MCs. We investigated the effects of salicylaldehyde on IgE-mediated anaphylaxis using a mouse model and found that systemic and cutaneous anaphylactic reactions were significantly inhibited in salicylaldehyde-treated mice.v/v of each compound for 48 h, 5 compounds were found to suppress the extent of IgE-induced degranulation by up to 60%, whereas 42 compounds did not exert apparent suppressive effects and 16 compounds decreased the viability of BMMCs. Sixteen compounds showing toxicity at 0.01% were added to the culture media of MCs under a further 1/10 diluted condition , and two compounds were found to inhibit degranulation without affecting cell viability, eleven had no effect at 0.001%, and three were still toxic. To confirm the inhibitory effects of the candidates, we investigated the degranulation of MCs after a 48 h pretreatment with various concentrations of seven compounds , Ms4a2 (FcεRIβ), and Fcer1g (FcεRIγ) in BMMCs by PCR and found that the pretreatment with 500 μM salicylaldehyde did not decrease any transcripts of the three subunits  model, in which a rapid decrease in body temperature was caused in mice just after an intravenous (i.v.) injection of Ag peaked 20 min after the Ag injection, and then, the body temperature was gradually recovered as observed in control A. By usiBased on these results, we demonstrated that salicylaldehyde exerted beneficial effects on IgE-mediated allergic responses in vivo.The IgE-mediated activation of MCs plays key roles in various allergic diseases, including food allergy, anaphylaxis, and pollinosis. Previous studies examined immunomodulatory natural materials derived from plants and bacteria ,13, and 2+ ionophore-induced (IgE-independent) degranulation of BMMCs and LPS-induced IL-6 release by BMMCs were moderately suppressed by salicylaldehyde-treatment  will contribute to the development of novel antiallergic drugs.C57BL/6J and Balb/c mice were purchased from Japan SLC . All animal experiments were performed in accordance with the guidelines of the Institutional Review Board of Tokyo University of Science. The present study was approved by the Animal Care and Use Committees of Tokyo University of Science: K21004, K20005, K19006, and K18006. BMMCs were generated from mouse (C57BL/6) BM cells by cultivation under IL-3-supplemented conditions as previously reported .BMMCs (5 × 105) sensitized with 200 ng of anti-TNP mouse IgE  in 1 mL of culture medium for 2 h were resuspended in Tyrode’s buffer containing 3 ng/mL TNP-BSA  after washing with Tyrode’s buffer twice. The culture supernatant was harvested at 30 min after the TNP-BSA stimulation to assess β-hexosaminidase activity . The extFlow cytometry was performed to measure the cell surface expression levels of FcεRI and c-kit with FITC antimouse FcεRIα , and APC antimouse CD117 , and to analyze cell viability with DAPI . A MACS Quant Analyzer  and FlowJo  were used to detect fluorescence and to analyze data, respectively.The concentrations of IL-6 and TNF-α in the culture supernatant of MCs were measured by ELISA kits purchased from BioLegend (ELISA MAX series). The mouse IL-13 DuoSet ELISA kit  was used to measure the concentration of IL-13. BMMCs were stimulated with anti-TNP IgE and TNP-BSA as the degranulation assay and culture supernatants were harvested at 3 h after the addition of TNP-BSA.Fcer1a forward; 5′-GAGTGCCACCGTTCAAGACA-3′, Fcer1a reverse; 5′-GTAGATCACCTTGCGGACATTC-3′, Ms4a2 forward; 5′-TGGTTGGTTTGATATGCCTTTGT-3′, Ms4a2 reverse; 5′-CACTGCACCCCAGAATGGATA-3′, Fcer1g forward; 5′-ATCTCAGCCGTGATCTTGTTCT-3′, Fcer1g reverse; 5′-ACCATACAAAAACAGGACAGCAT-3′, Hprt forward; 5′-TCCATTCCTATGACTGTA GATTTTATCAG-3′, Hprt reverse; 5′-AACTTTTATGTCCCC CGTTGACT-3′.The preparation of total RNA from BMMCs and reverse transcription to synthesize cDNA were performed using the ReliaPrep RNA Cell Miniprep System  and ReverTra Ace qPCR RT Master Mix , respectively. The mRNA levels of FcεRIα, β, and γ subunits were measured by quantitative PCR using the Step-One Real-Time PCR system  with THUNDERBIRD SYBR qPCR Mix (Toyobo) and the following primers: 6) were collected 10 min after the stimulation with anti-TNP IgE and TNP-BSA. Subsequent steps, including the electrophoresis of cell lysates and transfer of proteins from acrylamide gels to membranes, were performed as previously described [BMMCs  every 10 min after the TNP-BSA injection for 1 h.To establish PCA, 20 μg of TNP-BSA and 0.5% Evans blue  were i.v. injected into mice s.c. preinjected with anti-TNP IgE or saline as a control into the ear , footpad , or back . The thicknesses of the ear and footpad were measured at 1 h after the TNP-injection and back skin was collected to calculate blue staining caused by permeabilized Evans blue using a densitometric analysis with Image J  as previously reported .p values <0.05 were considered to be significant.The two-tailed Student’s t-test (to compare two samples) and a one-way ANOVA-followed by Tukey’s multiple comparison test or Dunnett’s multiple test (for more than three samples) was used, and


R-enantiomers (GAT1665 and GAT1667) displayed mixed allosteric agonist-PAM activity at CB1R while the S-enantiomers (GAT1664 and GAT1666) showed moderate activity. Furthermore, we observed that the R and S-enantiomers had distinct binding sites on CB1R, which led to their distinct behavior both in vitro and in vivo. The R-enantiomers (GAT1665 and GAT1667) produced ago-PAM effects in vitro, and PAM effects in the in vivo behavioral triad, indicating that the in vivo activity of these ligands may occur via PAM rather than agonist-based mechanisms. Overall, this study provides mechanistic insight into enantiospecific interaction of 2-phenylindole class of CB1R allosteric modulators, which have shown therapeutic potential in the treatment of pain, epilepsy, glaucoma, and Huntington’s disease.Positive allosteric modulation of the type 1 cannabinoid receptor (CB1R) has substantial potential to treat both neurological and immune disorders. To date, a few studies have evaluated the structure-activity relationship (SAR) for CB1R positive allosteric modulators (PAMs). In this study, we separated the enantiomers of the previously characterized two potent CB1R ago-PAMs GAT591 and GAT593 to determine their biochemical activity at CB1R. Separating the enantiomers showed that the  The endogenous cannabinoid system (ECS) is composed of endogenous ligands (e.g. anandamide [AEA] and 2-arachidonoylglycerol [2-AG]), anabolic and catabolic enzymes, and receptors  . The ECSCannabis represent a major limitation to its use as medicine. The intoxicating effects of Cannabis are thought to be due to ∆9-tetrahydrocannabinol (THC) binding to the orthosteric site of CB1R -GAT228 was an allosteric-agonist and the opposite enantiomer, (S)-GAT229, showed PAM activity at CB1R within the assays and cell line used  and Sigma-Aldrich , respectively. All other tested compounds were obtained from Dr. Ganesh Thakur, Northeastern University. All compounds were initially dissolved in dimethylsulfoxide (DMSO) and diluted in a 10% DMSO solution in phosphate-buffered saline (PBS). Compounds were added directly to cell culture at the time and concentrations indicated at a final concentration of 0.1% DMSO.2 in DMEM/F-12 containing 10% fetal bovine serum (FBS) and 5 U × 104 U penicillin/streptomycin . In addition, 800 μg/ml geneticin was used for CHO-K1 hCB1R HitHunter cAMP cells and 800 μg/ml geneticin and 300 μg/ml hygromycin B was used for CHO-K1 hCB1R PathHunter βarrestin2 cells.HitHunter (cAMP) and PathHunter (βarrestin2) Chinese hamster ovary (CHO)-K1 cells stably-expressing human CB1R (hCB1R) from DiscoveRx  were maintained at 37°C, 5% CO2. Opti-MEM media was removed and replaced with cell assay buffer (DiscoveRx) and then cells were simultaneously treated with 10 µM FSK and experimental compounds (0.10 nM–10 µM) for 90 min cAMP antibody solution and working detection solutions were added according to the manufacturer’s protocol (DiscoveRx), and cells were incubated for 60 min at room temperature. cAMP solution A was added according to the manufacturer’s protocol (DiscoveRx) and cells were incubated for an additional 60 min at room temperature before chemiluminescence was measured on a Cytation5 plate reader . Data are presented as percent maximal CP55,940-dependent inhibition of cAMP accumulation.cAMP inhibition was performed in the presence of 10 µM forskolin (FSK) using the DiscoveRx HitHunter assay in hCB1R CHO-K1 cells. Cells  were incubated overnight in Opti-MEM (Invitrogen) containing 1% FBS at 37°C and 5% CO2. Cells were then simultaneously treated with experimental compounds (0.10 nM–10 µM) for 90 min. Detection solution was added to cells according to the manufacturer’s protocol (DiscoveRx), and cells were incubated for 60 min at room temperature. Chemiluminescence was measured on a Cytation5 plate reader . Data are presented as percent maximal CP55,940-dependent stimulation.βarrestin2 recruitment was determined using the hCB1R CHO-K1 cell PathHunter assay (DiscoveRx). Cells  were incubated overnight in Opti-MEM (Invitrogen) containing 1% FBS at 37°C and 5% CO3H]CP55,940 in Tris buffer , 1% bovine serum albumin (BSA), pH 7.4) with a total assay volume of 200 µl. The assay began with the addition of transfected hCB1R CHO-K1 cell membranes (25 µg protein per well). The assays were left to equilibrate at room temperature for 2 h before vacuum filtration using a Millipore Sigma 12-well sampling manifold and filter paper that had been soaked in wash buffer. Each reaction well was washed 3 times with a 2 ml aliquot of Tris-binding buffer. The filters were removed then submerged in 5 ml of scintillation fluid . Radioactivity was quantified by liquid scintillation spectrometry. Specific binding was defined as the difference between binding that occurred in the presence and in the absence of 1 µM unlabeled CP55,940. Data are presented as percent [3H]CP55,940 bound.Previous work from our group has shown that CB1R PAMs enhance orthosteric agonist binding  . RadioliThis assay was conducted as described in previous reports . To summad libitum access to food and water, and environmental enrichment. Compounds administered intraperitoneally (i.p.) were prepared in vehicle [ethanol and cremophor in saline (1:1:8)]. Catalepsy was assessed in the bar holding assay 5 min after drug administration. Mice were placed so that their forepaws clasped a 0.7 cm ring clamp positioned 4.5 cm above the surface of the testing space  between 4 and 7 months of age were used for these studies. Animals were group housed at the Laboratory Animal Services Unit (LASU) at the University of Saskatchewan  with a standard 12:12 light-dark cycle, ng space . The lenng space .A conformational analysis was performed on GAT1664, GAT1665, GAT1666, and GAT1667 using Spartan’18 V1.4.5 . A confovia exclusion spheres placed on each residue. The Generalized Born/Surface Area (GB/SA) continuum solvation model for water was used with the dielectric set to 80 outside of the implicit membrane region and 2 within. A truncated Newton conjugate gradient minimization was performed using the OPLS3e force field for one iteration up to a maximum of 1,000 steps and with a 0.1 kcal/mol gradient endpoint. Constraints of 1 kcal/mol were placed on the C-alpha atoms of residues R3.50, Y5.58, L6.33, and Y7.53 to prevent the intracellular opening present in the R* structures from closing during the minimization.The CB1R model has been described in detail in To identify potential binding site(s) for GAT1664, GAT1665, GAT1666 and GAT1667 at CB1R, we used the Forced-Biased Metropolis Monte Carlo simulated annealing program (MMC) as the first step . The MMCOf particular interest in MMC calculations are fragments that persist at particular sites on the protein throughout the annealing schedule, since these fragments clearly have high affinity for those sites. Receptor regions at which all molecular fragments of a studied ligand collect are identified from MMC output. This set of sites is then refined to include only those sites at which the order of the fragments reflects the structure of the entire molecule.GAT1664 and GAT1665 were broken into fragments that included a 6-fluoro-indole ring, a 2-fluoro-phenyl ring and a 3-fluoro-phenyl ring. GAT1666 and GAT1667 were broken into fragments that included 7-fluoro-indole ring, 2-fluoro-phenyl ring and 3-fluoro-phenyl ring. All fragments were prepared with partial charges using the Amber 2002 force field, a point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum MMC . Six MMCDocking: Once the two sites were confirmed, the structures of GAT1664-GAT1667 were docked in the intracellular TMH1/2/4 site with H2.41, F4.46, and W4.50 used as direct interaction sites. GAT1664 and GAT1666 were docked in the TM2/3/EC1 site with Y2.59 and D2.63 as direct interaction sites. In addition, after extensive previously published molecular dynamics calculations on unfluorinated versions of the GAT compounds (GAT1600-3), R (148) was modeled to interact directly with the carbonyl oxygens of the last turn of TMH1 and not the nitro group within the compounds  continuum solvation model for water was used with the dielectric set to 80 outside of the implicit membrane region and 2 within. A truncated Newton conjugate gradient minimization was performed using the OPLS3e force field for 1 iteration, up to a maximum of 1,000 steps and with a 0.1 kcal/mol gradient endpoint. Constraints of 1 kcal/mol placed on the c-alpha atoms of residues R3.50, Y5.58, L6.33, and Y7.53 were set to prevent the intracellular opening from closing during the minimization. The resulting docks were refined with the Induced Fit protocol . The Glide box size was set to 12 Å3 centered on the ligand and the SP docking algorithm employed. Residues within 5 Å of the docked ligand were included in the Prime refinement stage, except in the case of the TM2/3 PAM site where S2.60 and K3.28 were excluded based on mutation data . As the MMGBSA binding energies are approximate free energies of binding, a more negative value indicated stronger binding. For this computation, the VSGB solvation model was employed while the chosen force field OPLS4 was used.Each of the GAT1664-GAT1667 ligand-receptor complexes were evaluated 50 and Emax in 3H]CP55,940 bound  displayed greater PAM potency and efficacy than the S-enantiomers (GAT1664 and GAT1666), (ex vivo model systems (All enantiomers displayed G protein-mediated agonist activity at hCB1R as shown by their ability to inhibit cAMP production in the absence of an orthosteric agonist . The abiAT1666), . InitialAT1666), . ImportaAT1666), , includi systems . These o systems .R-enantiomers) recruit βarrestin2.For consistency with cAMP inhibition experiments and previous studies , compounThe operational model described by 3H]CP55,940 binding, consistent with their proposed PAM activity, with the R-enantiomers GAT1665 and to a greater extent GAT1667 increasing [3H]CP55,940 binding (50 = 28 [15–52] nM). Enantiomers were also tested for their ability to promote G protein coupling in the GTPγS assay (R-enantiomers GAT1665 and GAT1667 being more potent than the S-enantiomers and GAT1667 in particular displaying the greatest potency (EC50 = 8.0 [3.4–17] nM)  . When 1 –17] nM) . Therefoin vivo response to be modulated by our compounds of interest. It was found that GAT1665 and GAT1667 displayed significant cataleptic, hypothermic, and anti-nociceptive effects at 3 and 10 mg/kg i.p. compared to vehicle  compared to vehicle . Both GA vehicle . Therefoed alone .2.41 (4.46 (4.03Å) while the other fluorobenzyl has a T-shaped π-π interaction with each ring on W2414.50   . Upon bikcal/mol . GAT1666kcal/mol . This caate CB1R , 7 (Hursate CB1R .2 group on GAT1665 has a H-bond between 1 of the oxygens shown in red and a hydrogen atom on Y1722.59 (O–H distance 2.17 Å) (2.63 (H–O distance 2.29 Å)  (2.59 (4.05 Å) (2 rotates in order to H-bond with Y1722.59 (R-enantiomers (GAT1665 and GAT1667) undergo a smaller conformational cost than the S-enantiomers (GAT1664 and GAT1666) upon binding to their respective sites on hCB1R (R-enantiomers (GAT1665 and GAT1667) bind to the ago-PAM site  significantly weaker than the S-enantiomers (GAT1664 and GAT1666) bind to the PAM site . Of note, binding of enantiomers to either site was not absolute but was more energetically favourable for the S-enantiomers to bind near the intracellular surface and the R-enantiomers to bind near the first extracellular loop.GAT1665 and GAT1667 displayed the greatest modelled affinity to a distinct allosteric site on hCB1R relative to GAT1664 and GAT1666 , 8. The  2.17 Å) . Another 2.29 Å) . A catio 3.66 Å) . One of (4.05 Å) . Upon biY1722.59 . The conon hCB1R . The R-e3H]CP55,940 at hCB1R. Enhancing the binding of an orthosteric ligand is a characteristic of a PAM and these observations are consistent with all compounds operating in part via positive allostery and/or non-competitive allosteric agonism  acted as ago-PAM’s while GAT1664 and GAT1666 (S-enantiomers) showed little to no activity as either an agonist or PAM. In the G protein coupling assay, the R-enantiomers GAT1665 and GAT1667 also showed higher potency and efficacy than S-enantiomers as agonists and PAMs. As for the in vivo studies, no compounds tested produced catalepsy, hypothermia at or antinociceptive effects at 0.1, 1, 3 or 10 mg/kg i.p. compared to vehicle when tested alone, but GAT1665 and GAT1667 did potentiate the effects of THC as ago-PAMs. These data are congruent with previous observations that CB1R ago-PAMs are inactive or minimally active in vivo in wild-type, otherwise healthy animals under non-pathological and acute conditions , with GAT1665 and GAT1667 displaying the greatest potency and efficacy as CB1R PAMs in the presence of 100 nM CP55,940. In the βarrestin2 assay, GAT1665 and GAT1667 (nditions . In plannditions . In addiing site . GAT1664ing site . Howevering site . Moreoveing site . TherefoThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). 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Sublingual immunotherapy (SLIT) is used worldwide to treat house dust mites (HDM) allergy. Epitope specific immunotherapy with peptide vaccines is used far less, but it is of great interest in the treatment of allergic reactions, as it precludes the drawbacks of allergen extracts. The ideal peptide candidates would bind to IgG, blocking IgE-binding. To better elucidate IgE and IgG4 epitope profiles during SLIT, sequences of main allergens, Der p 1, 2, 5, 7, 10, 23 and Blo t 5, 6, 12, 13, were included in a 15-mer peptide microarray and tested against pooled sera from 10 patients pre- and post-1-year SLIT. All allergens were recognized to some extent by at least one antibody isotype and peptide diversity was higher post-1-year SLIT for both antibodies. IgE recognition diversity varied among allergens and timepoints without a clear tendency. Der p 10, a minor allergen in temperate regions, was the molecule with more IgE-peptides and might be a major allergen in populations highly exposed to helminths and cockroaches, such as Brazil. SLIT-induced IgG4 epitopes were directed against several, but not all, IgE-binding regions. We selected a set of peptides that recognized only IgG4 or were able to induce increased ratios of IgG4:IgE after one year of treatment and might be potential targets for vaccines. Allergen specific immunotherapy (AIT) is the only treatment capable of modifying the course of allergic diseases and altering its natural history, by stimulating allergen-specific immune tolerance . It aimsDermatophagoides pteronyssinus, Dermatophagoides farinae, and Blomia tropicalis [Dermatophagoides pteronyssinus, Der p 1, Der p 2, Der p 5, Der p 7, Der p 23 and Der p 21 seem to represent the most clinically relevant allergens. On the other hand, although Der p 10, a tropomyosin, is considered a major allergen in sources such as shrimp and cockroaches [Blomia tropicalis is a mite that was first described as a storage mite and it is now considered a house dust mite in tropical and sub-tropical areas, such as Brazil. It is less extensively studied and Blo t 5 is its major allergen [House dust mites (HDM) are among the most important allergen sources containing many different allergenic molecules that are identified as etiological agents of allergic rhinitis in the Brazilian population . The mosopicalis ,9. For Dkroaches , it is callergen .There is a plethora of literature that evaluates molecular profiles of IgE and IgG4 recognition during AIT ,14, espeDue to advances in molecular allergology, new allergy vaccines have been developed, among those are peptide vaccines. Immunotherapy with a recombinant B-cell epitope-based, hypoallergenic, vaccine for grass pollen allergy has demonstrated its safety and efficacy ,30, and Until recently, dynamic epitope profiles during immunotherapy were not well defined. In this study, we present a first approximation analysis of IgE and IgG4 epitope binding to ten HDM allergens: Der p 1 (cysteine protease), Der p 2 (NPC2 family), Der p 5, Der p 7 , Der p 10 (Tropomyosin), Der p 23 (Peritrophin-like protein domain), Blo t 5, Blo t 6 (Chymotrypsin), Blo t 12, and Blo t 13 (Fatty acid-binding protein) using sera from ten patients undergoing sublingual immunotherapy (SLIT) for allergic rhinitis. Immunotherapy treatments are immune modulatory, and increasing tolerance is associated with increases in IgG4:IgE ratios . TherefoTo analyze IgE and IgG4 peptide recognition we selected ten relevant HDM allergens  for epitope mapping. A serum pool of 10 patients undergoing SLIT for house dust mite allergy was used for testing before and 1 year after treatment. All patients reported a self-perception of improvement of rhinoconjunctivitis symptoms. Demographical data of individuals included together with clinical symptoms and IgE titers are provided in Peptides with Median Fluorescence Intensity (MFI) higher than 100 were considered positive. Diversity in peptide reactivity was higher post-1-year for both antibodies, going from 46 IgE-reactive to 60 and from 19 to 66 IgG4-reactive peptides a.Peptide diversity for IgE varied among molecules and timepoints without a clear pattern, similar to what was observed for total and specific IgE levels .Blomia tropicalis, the molecule with the highest IgE peptide diversity detected at pre-treatment was Blo t 12  to individual allergen peptides, IgE reactivity to  Der p 1 a. As for Der p 1 b. In linTo better understand the dynamic profile of peptide recognition by IgE and IgG4, patterns of bindings before and after 1-year SLIT were compared . Some peTo better understand whether the IgG4 that was produced during immunotherapy has similar specificity to the original sensitization profile, post-immunotherapy IgG4 binding patterns were compared to baseline IgE binding patterns. We therefore used three-dimensional models of the individual allergens . IdentifBesides the dynamics of peptide recognition during SLIT, we aimed to find potential targets for peptide-based vaccines. We focused on regions that exclusively elicited IgG4 during treatment without co-localization to pre-existing or post-IgE reactivity and also on peptides that induced higher ratios of IgG4:IgE after one year of therapy compared to pre-treatment . For uniFor allergic rhinitis, subcutaneous immunotherapy (SCIT) has been the gold standard. However, in recent years, sublingual immunotherapy (SLIT) has emerged as an effective and safer alternative. There are many studies on immunological profiles developed during SCIT, but for SLIT there is still little information available .Although several studies have mapped IgE and/or IgG epitopes for HDM allergens, ,23,24,25To our knowledge this is the first study to evaluate epitope binding for HDM allergens before and after one year of immunotherapy. To do so, we used a peptide microarray and investigated the diversity and amplitude of IgE and IgG4 peptide repertories against ten HDM allergens. Pooled sera from ten patients undergoing allergen immunotherapy were used at a basal timepoint and after 1-year of SLIT. From the 544 peptides analyzed in the microarray, 46 and 60 IgE peptides as well as 19 and 66 IgG4 peptides were identified before and after 1-year SLIT, respectively. Overall, half of the allergens presented a decreased or similar number of IgE peptides after one year, while all allergens, except Der p 1, presented a higher number of peptides being recognized by IgG4 after 1-year SLIT. Same as total and specific IgE levels, degrees of recognition varied among molecules, antibodies, and timepoints without a clear tendency, probably due to the short interval of immunotherapy.While for most Der p allergens IgE reactivity after one year was found to be similar to baseline levels, several relevant allergens including Der p 1, Der p 2, and Der p 23 showed enhanced reactivity to several peptides. Given the short time span of SLIT, considering the full treatment lasts three years, an increase in IgE reactivity can be anticipated at least for some allergens. A previous study comparing SCIT and SLIT showed that clinical and immunological improvements started after one year of SCIT but only after two years of SLIT . SimilarDermatophagoides farinae allergens, using methodology similar to our work, identifying four peptides for Der f 1, three for Der f 2, two for Der f 5, and three for Der f 7. Similar to our study, all these epitopes were in exposed areas on the surface of the molecules, which would allow the epitopes to recognize the IgE antibody [One study analyzed IgE epitopes in antibody .Besides IgE, we were particularly interested in the identification of IgG4 epitopes induced during AIT. The observed increase in IgG4 binding to HDM peptides/epitopes as well as in IgG4 amplitude during SLIT in our study is in line with previous observations of increased allergen-specific IgG4 levels during immunotherapy for house dust mite as well as other allergen sources ,14,38.IgG4 antibodies are considered to compete with specific IgE for binding to the allergen. This might inhibit IgE-mediated complex formation on sIgE receptor-expressing cells, such as mast cells and basophils .Immunotherapy was shown to alter epitope binding patterns, causing a polyclonal increase in IgG4 levels, with a simultaneous reduction in intensity of IgE epitopes . AccordiBlomia tropicalis [Interestingly, Der p 10, a tropomyosin, was the allergen with most IgE and IgG4 peptides being recognized. This protein is typically described as a minor allergen in the temperate climate areas but is considered a major allergen in other sources such as shrimps, helminths, and cockroaches ,40. Epidopicalis ; thus, fSome major HDM allergens, such as Der p 2 and Der p 23, presented only few linear IgE epitopes. Der p 2, one of the most clinically important allergens, was one of the molecules with few IgE and IgG4 epitopes being detected. This may be explained by the fact that many Der p 2 epitopes are mostly defined by structural features formed from discontinuous regions of the allergen, rather than by a continuous peptide sequence. IgE binding is known to be vulnerable to reduction and alkylation, showing the presence of many conformational epitopes, especially for Der p 2 .It has been postulated that the use of whole extract for allergen immunotherapy could induce neo sensitizations during treatment , similarAs in our study we were highly interested in the therapy-induced IgG4 antibodies, we have chosen the 1-year timepoint to identify the first IgG4 epitopes in investigated HDM allergens. We aim to follow this evaluation in later timepoints of treatment to better understand the dynamics of epitope recognition. Indeed, even after this first period, the IgG4 diversity and reactivity level was already higher in most allergens, in line with previous observations . The majTo investigate regions that could potentially be used for peptide immunotherapy, we used two approaches to obtain a list of candidate targets to be further evaluated. First, we focused on IgG4 peptides that did not co-localize with IgE epitopes. An engineered vaccine made of non-IgE binding peptides fusioned to the hepatitis B preS protein was able to induce allergen-specific IgG, improve clinical symptoms of seasonal grass pollen allergy, and was well tolerated .Regions that exclusively elicited IgG4 during treatment showed epitopes from 12 up to 33 residues. That length is in the middle of existing data for peptide vaccines, where IgE-mediated peptides are longer, being 20–40 amino acids and typically dependent on a folded tertiary structure ,50. Shor62–94, comprehends a region which was previously predicted as a conformational B cell epitope and potential target for a vaccine design for Der p 5: 77EKPTKEMKD85 [One of the identified IgG4 exclusive epitopes, Der p 5TKEMKD85 . AlthougTKEMKD85 .Next, based on the known ability of allergen immunotherapy to induce IgE-blocking antibodies, we aimed to further evaluate regions recognized by both antibodies at each timepoint. IgG4 monoclonal antibodies directed against IgE epitopes of major allergens have proved successful in inhibiting human nasal allergen challenge responses after injection of cocktails of passive immunotherapy . Since ihttps://www.iedb.org/, accessed on 2 February 2023) [All peptides identified in our study were tested against predicted T and B cell epitopes using the Immune Epitope Database (ry 2023)  and noneWe are now beginning to understand the mechanisms of standard and novel approaches to immunotherapy. We suggest that peptide immunotherapy will significantly improve the safety and management of allergies. The challenges arising from current approaches, including the risk of side-effects, onerous duration of treatment, poor adherence, and high cost can be overcome by application of peptides based on T or B cell epitopes, rather than whole allergens . While wIn summary, results herein presented revealed that IgE profile is variable, with increased levels detected for several peptides of some molecules after one year of treatment. IgG4 recognition was always increased showing that mite allergic patients receiving SLIT can elicit IgG4 to several epitopes from ten mite allergens partially overlapping with IgE after one year of treatment. Some unexpected results revealed that Der p 10 and Blo t 12 were the molecules with the highest number of peptides being recognized, indicating those allergens may play an important role in the Brazilian population. We show that the repertoire for linear IgE and IgG4 epitopes is not identical, and some peptides could be selected as potential vaccine candidates aiming to circumvent IgE-related side effects. For this, we have chosen peptides that are IgG4 exclusive or that induced higher IgG4:IgE ratios, resulting in a set of 29 epitopes. Although this study consists of only a small number of patients, this is the first report considering a panel of different allergens from two different mites in two timepoints of SLIT. Considering the paucity of information on epitope profile during SLIT, the results shown here can pave the way for future studies on specific peptides that can be useful as vaccine candidates.Dermatophagoides pteronyssinus and Blomia tropicalis ).Based on the clinical history and samples available, 10 patients with persistent allergic rhinitis (>4 days per week and >4 weeks) and moderate–severe symptoms (impairment of daily activities) who were undergoing a SLIT  protocol study with IPI-ASAC  extract for Der p and Blo t were selected at the Ambulatório de Imunologia Clínica e Alergia do HCFMUSP  . InclusiThe treatment of allergic rhinitis was standardized for all participants, according to their clinical condition. The following medications were used: budesonide nasal spray as control medication and loratadine 10 mg/day per ors as rescue medication. As for the patients who presented with associated allergic conjunctivitis, the use of olopatadine eye drops was indicated.Pooled sera before treatment (pre-SLIT) and after one year of immunotherapy (post-1-year) were used in this study. This research was approved by the “Comissão de Ética para Análise de Projetos de Pesquisa do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo” (CAAE: 64005516.1.0000.0068).® Analyzer 2.0. A software algorithm breaks down fluorescence intensities of each spot into raw, foreground, and background signal, and calculates average median foreground intensities and spot-to-spot deviations of spot duplicates. A maximum spot-to-spot deviation of 40% was tolerated, otherwise the corresponding intensity value was zeroed. To increase specificity, values above 100 were considered positive for categorical analysis.Peptide microarray was produced by laser-printing . The amino acid sequences of 10 allergens were identified: Der p 1 (P08176.2), Der p 2 (CAK22338.1), Der p 5 (P14004.2), Der p 7 (P49273.1), Der p 10 (O18416.1), Der p 23 (L7N6F8.1), Blo t 5 (O96870.1), Blo t 6 (AAQ24544.1), Blo t 12 (Q17282.1), and Blo t 13 (Q17284.1). GSGSGSG linkers at the C- and N-terminus were used to avoid truncated peptides. The linked and elongated antigen sequences were translated into 15 amino acid peptides with a peptide-peptide overlap of 12 amino acids. The resulting linear peptide microarrays contained 544 different peptides  printed p < 0.05. Analyses were performed using GraphPad Prism 9.4.1. The cutoff value for IgE and IgG4 was established as the average of all measurements above zero at the initial time. To avoid disturbances from outliers, this average was a trimmed mean including the central 95% of the sample with value 100.35, considered 100.The comparison of peptide intensity was conducted using the non-parametric paired samples Wilcoxon test. All statistical analyses were two-tailed, and the significance was set at Dermatophagoides pteronyssinus and Blo t 5 (2JMH) from Blomia tropicalis were taken from the RCSB Protein Data Bank.Tridimensional structures of the allergens Der p 1 (5VPG), Der p 2 (1A9V), Der p 5 (3MQ1), Der p 7 (3H4Z), and Der p 23 (4ZCE) from https://yanglab.nankai.edu.cn/trRosetta/, accessed on 14 October 2022 ) [The 3D structure of Blo t 12 (2MFK) of the RCSB Protein Data Bank only showed the chitin-binding domain corresponding to residues 59–124. To complete the structure of Blo t 12, the N-terminal region (1–58) was modelled using the trRosetta de novo structure prediction server (r 2022 ) .https://saves.mbi.ucla.edu, accessed on 5th December 2022) was used to validate our models. All five models were run through Procheck to check the stereochemical quality of the structure by analyzing residue-by-residue geometry and overall structure geometry. The best model was chosen by Ramachandran plot results. Superimposition of query and template structure and visualization of generated models were performed using UCSF Chimera  software package 1.16 [The structures of the remaining allergens, Der p 10, Blo t 6, and Blo t 13 were obtained by homology modeling using the MODELLER 10.3 software  and 7KO4, 2F91, 2A0A as templates, respectively. Saves 6.0 (age 1.16 .


This is set by the interplay of the cation chloride cotransporters NKCC1 , a main Cl– uptake transporter, and KCC2 , the principle Cl– extruder in neurons. Accordingly, their dysfunction is associated with severe neurological, psychiatric, and neurodegenerative disorders. This has triggered great interest in understanding their regulation, with a strong focus on phosphorylation. Recent structural data by cryogenic electron microscopy provide the unique possibility to gain insight into the action of these phosphorylations. Interestingly, in KCC2, six out of ten (60%) known regulatory phospho-sites reside within a region of 134 amino acid residues  between helices α8 and α9 that lacks fixed or ordered three-dimensional structures. It thus represents a so-called intrinsically disordered region. Two further phospho-sites, Tyr903 and Thr906, are also located in a disordered region between the ß8 strand and the α8 helix. We make the case that especially the disordered region between helices α8 and α9 acts as a platform to integrate different signaling pathways and simultaneously constitute a flexible, highly dynamic linker that can survey a wide variety of distinct conformations. As each conformation can have distinct binding affinities and specificity properties, this enables regulation of [Cl–]i and thus the ionic driving force in a history-dependent way. This region might thus act as a molecular processor underlying the well described phenomenon of ionic plasticity that has been ascribed to inhibitory neurotransmission. Finally, it might explain the stunning long-range effects of mutations on phospho-sites in KCC2.Inhibitory neurotransmission plays a fundamental role in the central nervous system, with about 30–50% of synaptic connections being inhibitory. The action of both inhibitory neurotransmitter, gamma-aminobutyric-acid (GABA) and glycine, mainly relies on the intracellular Cl A and glycine receptors (GABAAR and GlyR), correspondingly (AR are widely expressed in these tissues [reviewed in – influx due to a low intracellular Cl– ([Cl–]i.) concentration and thus to hyperpolarizing inhibitory post-synaptic potentials  and KCC2 (potassium chloride cotransporter 2) . Both tr [Cl–]i. . In matu– influx . NKCC1 i– influx . In glia– influx  and modu– influx .Slc12a2 encoding both NKCC1 splice variants (NKCC1a and NKCCb) are viable, but suffer from deafness, pain perception, and male infertility (Slc12a5 that encodes both splice variants of KCC2 (KCC2a and KCC2b) die shortly after birth due to severe motor deficits that also affect respiration , Ca2+ activated Cl– channels , the pH sensitive Cl– channels and transporters of the SLC4 family [Na+- independent Cl–/HCO3– exchangers  and Na+-dependent Cl–/HCO3– exchangers ], and SLC26 family  and glutamate-activated Cl– channels (EAAT4)  . In thisA or glycine receptors is somewhat unique in that its function can be relatively easily modified via changes to the ionic driving force. In mature neurons, a low [Cl–]i results in ECl being slightly hyperpolarized with respect to the neuronal resting membrane potential Vrest  by only ∼2.5 mM (∼ to 5 mV from −75 to −70 mV) results in an increase in action potential firing by 39%  and 1049 (Arg1049His) that are associated with febrile seizures and/or idiopathic generalized seizure and decreased KCC2 activity  were identified in children of two unrelated families, which are associated with epilepsy of infancy with migrating focal seizures  that are associated with this disorder  that are linked to this disorder and intellectual disabilities  have also been linked to it , spastic quadriparesis (His186fs17 frameshift mutant), spastic paraparesis (Asn376Ile) and minor developmental delay (W892*)  and hearing loss   . Finally-2A > G) . The mut-2A > G) . All of -2A > G) . The humpairment . These sTo sum up, dysregulation of NKCC1 and KCC2 result in an imbalance of excitation/inhibition that is associated with several neurological and psychiatric disorders.– extrusion constitute promising new strategies for treating these debilitating diseases. Phosphorylation has emerged as the major means to rapidly and reversibly modulate intrinsic transport activity, cell surface stability, and plasma membrane trafficking of NKCC1 and KCC2  (6 in KCC2a) and nine phospho-sites in the C-terminus   or which have not yet been functionally investigated   . For KCCTyr1087) . In addi Ser988) . The dif Ser988) . This lo Ser988) . The out Ser988) . Althoug Ser988) .The high number of regulatory phospho-sites enables the transporters to sample across a multitude of signaling pathways, including with-no-lysine kinase (WNK) with their downstream kinase targets STE20/SP1-related proline/alanine rich kinase (SPAK) and oxidative stress response kinase (OSR1), protein kinase C (PKC), Src-tyrosine kinases, brain type creatine kinases and protein phosphatases . The higvia WNK1, phosphorylates Thr6 and Thr1007 of KCC2  results in activation of KCC2 as shown in cultured hippocampal neurons, cultured cortical neurons and slices, and HEK293 cells  and KCC2 (inactivation) and thus their reciprocal regulation  show an accelerated D/H shift due to increased KCC2 function (906E/Thr1007E mice (mimicking phosphorylated states) showed a delayed D/H shift in CA3 pyramidal neurons and hippocampal slices  and phosphorylation of Thr203/Thr207/Thr212 in NKCC1 (activation)  . This isxtrusion .dephosphorylation that of KCCs is true for N(K)CCs and KCC1, KCC3, and KCC4. Phospho-regulation in KCC2 is more complex since phosphorylation and dephosphorylation can both enhance its activity. Dephosphorylation of the following phospho-sites increases KCC2 activity: Thr6 (present only in KCC2a) and Thr906, Thr1007, Thr1009, and Tyr1087 (present in both splice variants)  does not alter KCC2 activity (1087 into aspartate (mimicking the phosphorylated state) abolishes KCC2 activity  intrinsically increases KCC2 activity without affecting cell surface expression CCs and ariants) . The mectability  and mutaactivity . In contactivity . This inpression . The Thr932, Thr934, Ser937, and Ser940  and dephosphorylated via protein phosphatase 1 (PP1)  , 2011. P of KCC2 , with mo of KCC2 . Accordi of KCC2 . Consequ of KCC2 . Mutatioactivity . These dactivity .940 increases concomitantly with KCC2 activity  intrinsically increases KCC2 activity in HEK293 cells without affecting cell surface expression  to affect ion translocation  or by alanine (KCC3-PKO). Analysis by cryo-EM revealed that the “dephosphorylated” KCC3-PKO is more dynamic in the scissor helix region and exhibits a greater rotational flexibility of the C-terminal dimer (wt and KCC3-PM ((De)phosphorylation dependent conformational differences were also reported for KCC3. To examine the effect of phosphorylation on structural organization, two different KCC3 mutants were generated with triple substitutions of Seral dimer . The KCCal dimer . Multiplal dimer . Cryo-EMal dimer . The fir KCC3-PM . The sec KCC3-PM . Thus,  between α8 and α9 helices according to the cryo-EM reconstruction of KCC2 (919 to Ala1053 in hsKCC2b)   agrees whsKCC2b) . In lineα8 helix .1007 forms main chain hydrogen bonds with Trp1008, that itself has side chain interactions with His1051 (pi stacking), and Tyr903 forms a main chain hydrogen bond with Ser899  abrogates stimulation, whereas substitution by the other amino acid residue  maintains sensitivity to staurosporine phosphorylation of phospho-sites most likely results in conformational reorganization as observed for other CCC family members. Many of the phospho-sites in the C-terminus of KCC2 are localized in an unstructured area. Due to biophysical properties of such areas, this part of KCC2 might serve a dual role. It might represent a platform for integrating different signaling pathways and simultaneously constitute a flexible, highly dynamic linker that can survey a wide variety of distinct conformations . As eachA-MH and HN equally wrote the manuscript. A-MH generated all of the figures. Both 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We report on the appearance of multiple tense blisters surrounding the exit site of a Tenckhoff catheter in a 79-year-old woman with end-stage renal disease in peritoneal dialysis. The differential diagnoses included a contact allergic or irritative dermatitis to peritoneal dialysis catheter material and antiseptic agents, bacterial infection, and herpes virus infection, but milia were a clue for a subepidermal blistering disease and lead to appropriate investigations. The laboratory findings, the histopathological examination and the direct immunofluorescence assay confirmed the diagnosis of localized bullous pemphigoid. The disorder typically occurs in elderly people and may be related to drugs, hematological malignancies or neurological conditions but it can also be a complication of hemodialysis or peritoneal dialysis. Bullous pemphigoid (BP) is mostly thought of as a generalized blistering eruption affecting the trunk and the proximal extremities. Although this is a correct picture of the disease, a number of patients with BP may have localizedcutaneous disease either persisting over a variable period of time or initially localized and followed by dissemination later on in the clinical course . We repo3/µL, 6.9%), a high IgE value (520.4 IU/mL) and normal RCP. Despite the change in the products used for the catheter care and dressings, as suggested by the dialysis service, the lesions had not improved. No bacteria or fungi were isolated from repeated cultures of the PD exit site and bullous lesions. Milia led us to suspect a subepidermal blistering disease.A 79-year-old woman with a previous medical history of hypertension, hypercholesterolemia, gout and end-stage renal disease presented to our Clinic with a 6-month history of a worsening pruritic vesico-bullous rash around her peritoneal dialysis (PD) catheter exit site. On examination, there were tense blisters with sero-hemorrhagic content over non-erythematous skin . Crops oA skin biopsy revealed focal epidermal spongiosis, subepidermal clefts and a predominantly eosinophilic dermal infiltrate a. Salt-sThe patient was treated with a two-week course of low-dose prednisone therapy (0.5 mg/kg/die) and topical 0.05% clobetasol propionate ointment once daily, with resolution of the lesions and residual slight hyperpigmentation a,b. DapsBullous pemphigoid (BP) is an autoimmune blistering disease most commonly arising in elderly individuals. The classical clinical findings are widespread, tense, fluid-filled blisters on an erythematous or non-inflammatory base. The disease may start as a localized eruption in 15% to 30% of patients  althoughThe onset of BP in patients receiving hemodialysis has been reported ,9,10,11,The damaged epithelium at the site of chronic irritation or contact dermatitis , an inflThe differential diagnosis of localized BP at the PD catheter exit site mainly includes contact allergic or irritative dermatitis to PD catheter material , antibioBacterial infection (mostly due to Staphylococcus aureus and Pseudomonas aeruginosa) at the PD exit site typically appears with purulent and/or bloody drainage from the PD catheter exit site, with surrounding erythema, tenderness, and swelling. Bullous impetigo is more common in children and histologically the blisters are subcorneal. Prompt diagnosis with isolation of the pathogen by culture and treatment are essential to prevent sepsis and PD-associated peritonitis . A polymEpidermolysis bullosa acquisita (EBA) may be considered in the differential diagnosis of our case of peristomal BP as it shows a predilection for trauma sites and the tendency to heal with milia. Secondary milia are benign keratinous cysts that result from the regeneration process of sweat glands or hair follicles and may appear on sites of previous blistering . The exaIn conclusion, this case documents the second case of localized BP around the peritoneal dialysis catheter exit site. Current estimates suggest that approximately 11% of the global dialysis population patients receive peritoneal dialysis, with an annual global growth rate of 8% . Thus, a


The phosphorylation of nucleosides and their polymerization are crucial issues concerning the origin of life. The question of how these plausible chemical processes took place in the prebiotic Earth is still perplexing, despite several studies that have attempted to explain these prebiotic processes. The purpose of this article is to review these chemical reactions with respect to chemical evolution in the primeval Earth. Meanwhile, from our perspective, the chiral properties and selection of biomolecules should be considered in the prebiotic chemical origin of life, which may contribute to further research in this field to some extent. Various ideas have been proposed throughout this time, the most prominent of which are cosmic life theory , naturalA well-known statement by Nobel Laureate Professor Todd is ‘where there is life, there is phosphorus' . PhosphoAs early as 1871, Darwin recognized the relevance of phosphate in protein synthesis in the origin of life ,14. PhosNucleosides are the basic components of nucleic acids (RNA and DNA) and are important biopolymers in life's chemistry . The pho. 2Although thermodynamically anhydrous circumstances were more favourable to nucleoside phosphorylation and polymerization in the early Earth environment , given i. 2.12+ or Mg2+ or imidazole to the reaction system could accelerate the reaction or increase the phosphorylation of nucleosides [A commonly used activator of nucleoside phosphorylation is diamidophosphate (DAP). The reaction of 0.1 M nucleosides and DAP in an aqueous solution at 50°C pH 5.5–10 produced 2′-NMP, 3′-NMP, 5′-NMP and 2′,3′-cNMP, while the addition of Znleosides ,34  generates a variety of phosphorylation products including 5′-UMP, 2′-UMP, 3′-UMP and cyclic 2′,3'-cUMP, as well as the dimerization products [Pyrophosphate (PPi) is a well-known prebiotic phosphorylation agent that is thought to be a ‘possible ancestor’ of the ATP molecule ,37. Afteproducts  (figure 3m) is a water-soluble phosphate that enables the phosphorylation and polymerization of tiny biomolecules like amino acids and nucleotides [3m, according to subsequent research [3m in the presence of boronate [3m with adenosine in neutral aqueous solutions mediated by Mg2+ yields mostly 2′,3′-cNMP [3m in an aqueous solution in a wet and dry cycle , catalysed by metal ions (particularly Ni(II)), yields a variety of phosphorylation products, including 5′-ATP, 5′-AMP, 3′-AMP, 2′-AMP and 2′,3′-cAMP [Trimetaphosphate teorites ,58, seemteorites , and alsteorites ,59. In g3P and Fe2NiP) can react with adenosine or uridine to generate corresponding phosphorylation products by the simple mixing and mild heating of reactants 3OH is typically a low-solubility mineral, but studies in recent years have shown that it could be dissolved in a semi-aqueous solvent UAFW which could improve the solubility of some insoluble phosphates [5(PO4)3OH in semi-aqueous solvent eutectic was observed, generating 5′-AMP and 2′,3′-cAMP 3OH [2B4O7 · 10H2O) to an adenosine-inorganic phosphate-pyrophosphate reaction system in the presence of formamide and incubating at 90°C yielded the only phosphorylation product −5′-AMP [Formamide has been proposed as a significant component of nucleic acid formation . Several(PO4)3OH , etc. 2′ −5′-AMP ,73.2+, could help to enhance nucleoside phosphorylation [2+ also aids in the conversion of insoluble phosphates (apatites) into more soluble minerals  [Various approaches have been tried to overcome above two types of nucleoside phosphorylation  in water problems. One of which is heating orthophosphates , nucleosides and condensates  to dryness or wet and dry cycles ,66,74–78rylation ,80,81. FO4·6H2O) . As a reO4·6H2O) , lunar sO4·6H2O)  are also. 2.3Even though the water was the most appropriate solvent on the early Earth, phosphorylation was unfavourable to occur in aqueous conditions . Many anAlthough diamidophosphate (DAP) was used to phosphorylate several prebiotic compounds  under aqueous circumstances ,89, the Phosphate can also be released from insoluble minerals by replacing the aqueous environment with eutectic and other low-water solvents ,64,72. F. 3During the phosphorylation of nucleosides to create nucleotides, nucleotide oligomers can also be formed ,38. Even. 3.12+, Zn2+ and Lu3+ accelerated the creation of pentamers, tetramers and trimers [Under prebiotic conditions, the polymerization of nucleotides was investigated using phosphorimidazoles as reaction substrates. Adenosine S'-phosphorimidazolide (ImpA), as an example , underwe trimers –110. Ura. 3.2et al. [Montmorillonite, a clay mineral, is also a relatively effective catalyst –116 for et al.  investiget al. ,118.Fig. 3.3In addition, some scientists have demonstrated that RNA polymerization analogues can be synthesized non-enzymatically from single nucleotides in a lipid environment ,120. The. 3.4et al. reported the condensation of deoxythymidine-5′-phosphate using cyanamide as an activator [As early as 1971, Oro ctivator . In subsctivator –124. The. 4Thanks to those tenacious practitioners, great progress has been accomplished in this field –127, who