Right atrial electroanatomical mapping may be combined with SoundStar 3D diagnostic ultrasound catheter (EAM‐ICE) as a zero‐fluoroscopy procedure for radiofrequency catheter ablation (RFCA). We aimed to evaluate the efficiency and safety of zero‐fluoroscopy transseptal puncture guided by EAM‐ICE and fluoroscopy combined with intracardiac echocardiography (F‐ICE) in patients with paroxysmal atrial fibrillation (PAF).Zero‐fluoroscopy transseptal puncture is an effective and safe procedure.This study had a prospective design. A total of 57 patients with PAF were enrolled and assigned to two groups. Twenty‐seven patients were enrolled in the EAM‐ICE group, and 30 patients were enrolled in the F‐ICE group.P = 0.000; 118.4 ± 19.7 vs 70.5 ± 13.5 minutes, P = 0.000). There was no use of fluoroscopy in the EAM‐ICE group (0 mGy vs 70.5 ± 13.5 mGy); the duration of fluoroscopy in the EAM‐ICE group was negligible (0 minutes vs 5.4 ± 1.9 minutes). No procedural complication occurred in either group.There were no statistically significant differences in baseline patient characteristics between groups. Transseptal puncture was successful in all patients . Total procedure time and duration of transseptal puncture were lower in the F‐ICE group (199.4 ± 26.0 minutes vs 150.7 ± 22.1 minutes, EAM‐ICE guided zero‐fluoroscopy transseptal puncture is an effective and safe procedure. However, radiation exposure can threaten the health of both doctors and patients.The foramen ovale is a unique anatomical structure located in thick muscle. During EAM, electrography yields low‐voltage readings for the foramen ovale. Whether this information can increase the success of transseptal puncture remains to be determined. No relevant studies have been conducted in Chinese populations. In this study, we developed a method for the combination of EAM with SoundStar 3D diagnostic ultrasound catheter (EAM‐ICE) to perform circumferential pulmonary vein isolation (CPVI). The efficacy and safety of EAM‐ICE‐guided transseptal puncture and catheter ablation are discussed below.22.1A prospective, controlled trial was conducted at Beijing Anzhen Hospital. During the period from June 2019 to October 2019, 57 consecutive patients with paroxysmal atrial fibrillation were enrolled in the study. Patients were divided into two groups.This study was approved by the Beijing Anzhen Hospital Medical Ethics Committee. All participants evaluated in this trial provided written informed consent.The inclusion criteria were as follows: (a) adults aged 18 to 80 years; (b) previous diagnosis with paroxysmal atrial fibrillation (PAF), defined as AF terminating spontaneously or AF onset <7 days before RFCA, with verification of AF using 12‐lead electrography or Holter monitoring; (c) symptomatic PAF patients intolerant or refractory to antiarrhythmic medication or amenable to RFCA treatment.The exclusion criteria were as follows: (a) left atrial appendage thrombosis; (b) abnormal cardiac structure disease ; (c) heart failure; (d) history of RFCA, cryoballoon ablation, and/or cardiac surgery; (e) mental disorder; (f) eGFR <30 mL/min; (g) septic shock; (h) advanced malignant tumor; (i) pregnancy; (j) cardiac tamponade or major hydropericardium; and (k) left atrial anteroposterior diameter > 50 mm.2.2Procedures were performed under local anesthesia. Electrode placement, transseptal puncture, and CPVI were performed according to standard protocols in each patient.2.2.1PreparationSubjects in the EAM‐ICE group underwent transseptal puncture directed with use of the 3D EAM system  and ICE  without the use of fluoroscopy.2.Mapping the right atrium and foramen ovaleBilateral femoral venous puncture was performed. An 8‐F introducer sheath  was inserted in the right femoral vein; a 6‐F introducer ; and an 11‐F introducer sheath  were inserted in the left femoral vein. An ablation catheter  was inserted into the right atrium (RA) via the 8‐F introducer sheath.3.Left atrial mappingInferior vena cava (IVC), superior vena cava (SVC), His bundle, tricuspid valve annulus, coronary sinus (CS), and the low‐voltage area in the atrial septum  were carefully marked Figure . Using t4.Advancing the transseptal sheath into the SVCUnder the direction of ICE and EAM, a three‐dimensional (3D) left atrium (LA) map was created to visualize the anatomy of the LA and pulmonary veins. Not only the pulmonary veins and their ostia but also the left atrial appendage (LAA) was carefully mapped. We also checked the LAA for thrombosis.5.Transseptal punctureBefore inserting the J‐type guide‐wire into the SVC, we measured the distance from the site of puncture to the junction where the sternum met the second rib. We also marked the same distance on the non‐steerable SL1 sheath . A clip‐pin cable was used to connect the J‐type guide‐wire to the Carto system for navigation. One side of each connector was clipped onto the guide‐wire; the other side was connected to the pinbox. The tip of the guide‐wire could thus be visualized on the Carto system as a bipolar electrode. Under the direction of ICE, the guide‐wire was inserted into the SVC. A non‐steerable SL1 sheath . When slightly withdrawing the guide‐wire, bipolar electrode would disappear on Carto system, this position was marked as the tip of the sheath. After completely withdrawing the guide‐wire, a bolus of physiological solution was injected to confirm the position of the SVC. After rotation of the ICE probe, it was placed in a section facing the atrial septum.A clip‐pin cable was used to connect the transseptal needle  to the Carto system. One side of each connector was clipped onto the transseptal needle; the other side was connected to the pinbox. Then the transseptal needle was placed inside the long sheath. The needle indicator was one thumb's width (approximately 1.5 cm) away from the inner sheath. The sheath was withdrawn slightly until the tip of the needle could be visualized as a bipolar electrode on the Carto system.Under the direction of the 3D EAM system, the sheath‐dilator‐needle assembly was pulled out by rotating it in a clockwise direction until it faced the RA septum. Tenting of the interatrial septum by the sheath‐dilator‐needle was noted on ICE. The transseptal needle was then pulled rapidly through the dilator to puncture the atrial septum. The routine of sheath‐dilator‐needle assembly can be visualized on 3D‐EAM  settings. The ablation index (AI) targeted at the anterior LA wall was 500, and the AI targeted at the posterior wall was 400. During ablation, an ICE probe was placed in the right ventricle to monitor the pericardium. Heparin was given during the procedure to maintain an activated clotting time between 300 and 350.Point‐by‐point CPVI was performed using irrigated ablation catheters  in power control mode at 35 W (irrigation flow 17 mL/min). Radiofrequency applications were performed with Visitag  guidance with catheter stability (2.5 mm for 3 seconds) and contact force  were assessed. Echocardiographic findings  were also recorded.2.4Subjects fitting the inclusion and exclusion criteria were assigned to the EAM‐ICE or F‐ICE group. The primary outcome of this trial was the rate of successful transseptal puncture. The secondary outcomes of this trial were the rates of major complications .2.5t test for normally distributed data and by Mann‐Whitney U test for non‐uniformly distributed data. A P‐value <0.05 was considered statistically significant.Data analysis was performed with SPSS statistical software . Normally distributed continuous variables are expressed as means ± SD; non‐uniformly distributed data are expressed as medians (Q1 and Q3). Between‐group comparisons of means were analyzed by independent‐samples 33.1This study included 57 patients, 42 (73.7%) of whom were male. A total of 27 (47.4%) patients were enrolled in the EAM‐ICE group. Patients' detailed information is shown in Table 3.2We divided the procedure of transseptal puncture into several steps to evaluate the differences between groups.3.3P = 0.000; 118.4 ± 19.7 vs 70.5 ± 13.5 minutes, P = 0.000), there was no use of fluoroscopy in the EAM‐ICE group (0 mGy vs 70.5 ± 13.5 mGy), and fluoroscopy time was negligible in the EAM‐ICE group (0 minutes vs 5.4 ± 1.9 minutes). No procedural complication  occurred in either group . We also evaluated total procedure time and trans‐septal duration in the EAM‐ICE and F‐ICE groups. Total procedure duration was defined as the duration of the patient's stay in the operating room. In the EAM‐ICE group, the duration of trans‐septal puncture was considered to be the time required for mapping the RA and foramen ovale with EAM, mapping the LA and foramen ovale with ICE, advancing the long wire and sheath into the SVC, and transseptal puncture. In the F‐ICE group, the duration of transseptal puncture included LA mapping, advancing the long wire and sheath into the SVC, and transseptal puncture. Although total procedure time and transseptal duration were lower in the F‐ICE group . There are several reasons for this prolonged duration. First, we used high‐density voltage mapping to locate the foramen ovale, which required approximately 25 minutes. The foramen ovale was recognized as a low‐voltage zone in the RA. Because we wanted to test the theory that fluoro‐less trans‐septal puncture may be performed without the guidance of ICE, the foramen ovale was initially mapped only with voltage mapping. We enrolled the first few cases treated in this manner in the study. Prolonged trans‐septal durations may therefore indicate unskilled or extremely cautious attempts to map the foramen ovale. For these reasons, the duration of trans‐septal puncture was greater than the duration of RA mapping alone. We also established a special technique for transseptal puncture. By using a clip‐pin cable, we were able to connect the guide‐wire and transseptal needle to the Carto system, which allowed us to visualize the tip of this assembly as a bipolar electrode. This procedure, along with the necessary adjustments, required 15 minutes. During the procedure, an ICE probe was used to verify that the technique described was feasible for mapping the anatomy of the foramen ovale, coronary sinus, and LA and to ensure that the procedure was safe. In contrast, in the F‐ICE group, “trans‐septal duration” was defined as the time required for trans‐septal puncture guided by fluoroscopy, added to the time required for LA mapping guided by ICE. All members of our team are proficient at this technique. Procedure time is likely to decrease as operators become more skilled in performing the required techniques.Procedure time was longer in the EAM‐ICE group (199.4 ± 26.0 minutes vs 150.7 ± 22.1 minutes, To evaluate the differences between procedures, we divided transseptal puncture into several steps. First, we evaluated the accuracy with which the anatomical structures of the RA, LA, SVC, and foramen ovale were visualized. With EAM‐ICE, anatomical structures can be displayed from various angles; however, with F‐ICE, anatomical structures can only be displayed with fluoroscopy, which requires that a specific position be deduced from bone landmarks. The foramen ovale is particularly difficult to recognize with F‐ICE because it can only be evaluated in one ICE sector at a time. We also evaluated whether the tip of the sheath‐dilator‐needle assembly could carry a pin. EAM‐ICE traced the needle tip in three dimensions, while F‐ICE traced the needle tip in two dimensions. EAM‐ICE provided more information than ICE by providing details pertaining to anatomical structures, rendering the procedure effective and safe. Third, when withdrawing the transseptal needle and long sheath together, movement can be evaluated dynamically using either technique. In both groups, two episodes of jumping were discernible.EAM‐ICE upgrades the function of ICE by providing 3D visualization of anatomical structures, thus making the accompanying procedure more effective and safer.5We only analyzed acute outcomes. AF recurrence and long‐term success rates were not analyzed in this trial, which limits the strength of our findings. Patients were not grouped in a random fashion, which limits the strength of the conclusions made from comparisons between EAM‐ICE and F‐ICE.6It is effective and safe to perform the EAM‐ICE procedure. Thus, the use of a zero‐fluoroscopy approach for PAF is feasible.The authors declare no potential conflict of interests.


Agrimonia pilosa L. (AP) showed potent α-glucosidase inhibitory (AGI) activity, but it is uncertain what phytochemicals play a key factor. The phytochemical study of AP based on AGI activity led to the isolation of four isocoumarins; agrimonolide (1), agrimonolide-6-O-β-d-glucopyranoside (2), desmethylagrimonolide (3), desmethylagrimonolide-6-O-β-d-glucopyranoside (4), and four flavonoids; luteolin (5), quercetin (6), vitexin (7), and isovitexin (8). The four isocoumarins were isolated as α-glucosidase inhibitors for the first time. Isocoumarins, compound 1 (agrimonolide) and 3 (desmethylagrimonolide) showed strong α-glucosidase inhibitory activities with IC50 values of 24.2 and 37.4 µM, respectively. Meanwhile, isocoumarin and flavonoid glycosides showed weak AGI activity. In the kinetic analysis, isocoumarins, compounds 1 and 3 showed non-competitive inhibition, whereas flavonoid, compound 6 showed competitive inhibition. Diabetes is a common metabolic disease in modern society associated with the development of insulin resistance, peculiar glucose and lipid metabolism, impaired insulin signaling and β-cell dysfunction, sub-clinical inflammation, and increased oxidative stress . It is cAgrimonia pilosa L. (AP) is a perennial plant from the Rosaceae family, which is distributed widely in the temperate regions of East Asia, including Korea. The aerial parts of AP have been used as a wild vegetable mainly in the mountain villages of Korea. The diverse biological activities of AP, including anticancer  = 100 μM), was pre-mixed with 100 μL of the sample (15.6–500 μM) in 100 mmol-phosphate buffer (pH 6.9), in a 96 well plate and incubated at 25 °C for 3 min. Then, 47–50μL of α-glucosidase (0.76 unit/mL in 100 mmol-phosphate buffer (pH 6.9), keep on the ice) was added and we set the final volume to 200 μL with 100 mmol-phosphate buffer (pH 6.9) and mixed well, and absorbance was measured in kinetic mode at 405 nm for 5 min with a spectrophotometer. The AGI activity the samples were calculated using the equation: c represents the initial absorbance, As represents the absorbance in the presence of the sample and Ab represents the absorbance in the absence of sample after the reaction. Acarbose, represents the inhibitor, was used as a positive control.The preparation of the AP extract is presented in IK) values were calculated using the following equation: 50 was the half-maximal inhibitory concentration, [S] was the substrate concentration, and Km was the substrate concentration at the half of Vmax. The inhibition constant  activity was inhibited by the compounds (I) in the presence of the substrate (4-mmol p-nitrophenyl-α-d-glucopyranoside in 100-mmol phosphate buffer [pH 6.9]). The inhibition modes  of isocoumarins 1–4 and flavonoids 5–8 were examined using nonlinear regression Michaelis–Menten enzyme kinetics. The Lineweaver–Burk plot methods were used to determine kinetic parameters at increasing substrate and compound concentrations, and the parameters were calculated using a nonlinear regression program . The obtained results represent three independent experiments.Four isocoumarins 18 column  maintained at 40 °C. The separation solvent A and B comprised of water and methanol with 0.5% H3PO4. The gradient solvent system was programmed: 0 min, 15% B; 0–5 min, 15–30% B; 5–12 min, 30–40% B; 12–18 min, 40–50% B; 18–25 min, 50–55% B; 25–35 min, 55–100% B; wash to 38 min with 100% B; and a 5 min recycle time. The flow rate was 1 mL/min. The peak detection was observed with a PDA detector at 200–800 nm.The purity of the obtained compounds was analyzed using high performance liquid chromatography with photodiode array detector  analysis. Sample aliquots were filtered through a 0.2 µm membrane filter. The separations were performed on a YMC Triart C1H- NMR, 13C-NMR and mass spectrometry]. The 1H NMR and 13C NMR spectra were recorded on a Bruker Avance Ⅲ 500 and the chemical shift was referenced to the solvent peak (Hd 2.5 and Cd 39.5 in DMSO-d6).Structure elucidation of the eight compounds from the APE was carried out by spectral techniques [p < 0.01 were considered statistically significant.Data are presented as mean ± standard deviation (triplicate experiments) unless otherwise indicated. Statistical analyses were performed using the general linear model procedure (GLM) in the SAS statistical software . Differences at 1, 3, and 6. With regard to α-glucosidase inhibition, isocoumarin compounds 1 and 3 show non-competitive inhibition, whereas flavonoid compound 6 shows competitive inhibition. Moreover, we found that aglycones of isocoumarin and flavonoid showed more powerful AGI activities than their glycosides. Additionally, the desmethylagrimonolide with a hydroxyl moiety at the C-4′ exhibited strong AGI activity compared to agrimonolide with a methoxy group at the C-4′. This result suggested that AGI activity was increased when the hydroxyl groups attached on the C-4′.Eight compounds with AGI potential were identified from AP, and their AGI activities were successfully determined. Our results suggest that the glucose-lowering effect of AP is attributable to the strong AGI activities of compounds In the present study, although the enzyme inhibitory activities of the isolated compounds were analyzed in vitro, we believe that the results are relevant to the action mechanisms of hyperglycemia prevention in vivo. Our findings provide scientific support for the use of AP extract in traditional medicine for the prevention and treatment of T2DM. AP and its active compounds might be helpful in the development of medications and functional foods for T2DM.


The influenza B virus (IBV) causes seasonal influenza and has accounted for an increasing proportion of influenza outbreaks. IBV mainly causes human infections and has not been found to spread in poultry. The replication mechanism and the determinants of interspecies transmission of IBV are largely unknown. In this study, we found that the host ANP32 proteins are required for the function of the IBV polymerase. Human ANP32A/B strongly supports IBV replication, while ANP32E has a limited role. Unlike human ANP32A/B, chicken ANP32A has low support activity to IBV polymerase because of a unique 33-amino-acid insert, which, in contrast, exhibits species specific support to avian influenza A virus (IAV) replication. Chicken ANP32B and ANP32E have even lower activity compared with human ANP32B/E due to specific amino acid substitutions at sites 129–130. We further revealed that the sites 129–130 affect the binding ability of ANP32B/E to IBV polymerase, while the 33-amino-acid insert of chicken ANP32A reduces its binding stability and affinity. Taken together, the features of avian ANP32 proteins limited their abilities to support IBV polymerase, which could prevent efficient replication of IBV in chicken cells. Our results illustrate roles of ANP32 proteins in supporting IBV replication and may help to understand the ineffective replication of IBV in birds. Influenza B viruses infect humans and few other mammals, but fairly rare in birds. Here we found that IBV requires the involvement of host ANP32 proteins in the replication process, in which ANP32A and ANP32B play major roles and can fully support polymerase activity independently, while ANP32E gives only limited support to IBV polymerase because of certain substitutions compared with ANP32A/B. Chicken ANP32A has a 33-amino-acid insert not present in mammals and provides better support to avian IAV polymerase, but this insert impairs its support for IBV polymerase activity. Chicken ANP32B and ANP32E have even lower support to IBV polymerase due to specific inactive mutations at sites 129/130. Our findings reveal an important role for ANP32 proteins in IBV polymerase activity and suggest the possible molecular basis of adaptation and restriction of IBV infection in different species. The influenza A virus (IAV) and influenza B virus (IBV) belong to the group orthomyxoviridae, which are characterized by a single-stranded segmented RNA genome and enveloped spherical or filamentous particles with studded surface proteins , 2. InflMammalian cells have been commonly observed to have a restriction effect on infection by avian IAV and the mechanism of this interspecific infection restriction of IAV has been long studied. Some H1, H5 or H7 subtype IAV viruses can overcome the restriction by evolving certain mutations of polymerase, such as PB2 E627K or G590S and Q591R, and gain ability to replicate in mammals . One of IBVs are believed to be stably adapted to humans and are continually circulating in the human population. IBVs have also occasionally been isolated from other mammals, including dogs, pigs, and harbor seals –22, and Here we created different ANP32 protein knockout cell lines, and demonstrated that human ANP32A and ANP32B proteins are required for the polymerase activity of the influenza B virus. Unlike the polymerase of IAV, the IBV polymerase can use ANP32E, albeit with limited activity. Intriguingly, we found that chicken ANP32A has a much lower ability to support IBV polymerase activity than does mammalian ANP32A, mainly because it harbors a 33-amino-acid insert not present in mammals, which is required to support avian IAV replication. These 33 amino acids give an advantage to avian IAV, but in IBV, they become a barrier. We also found that although chicken ANP32B and ANP32E are all in “short-form” like those proteins from humans, they both have functionally inactive mutations at sites 129/130, resulting in the loss of support to the IBV polymerase. These findings reveal an important role for ANP32 proteins in IBV polymerase activity and suggest the molecular basis of restriction of IBV infection in chickens.AH13 had similar levels of activity in AKO cells, BKO cells, and wild type 293T cells. However, in DKO cells, the H7N9AH13 viral polymerase activity decreased sharply by about 5000-fold, which was comparable to the background value of polymerase activity (293T del PB2)  . The actdel PB2) . We specdel PB2) . The viadel PB2) . We foundel PB2) . These rdel PB2) , was usedel PB2) . Reconstdel PB2) . Dose-dedel PB2) , while edel PB2) . These rIBV mainly infects humans and a few other mammals, and there is no strong evidence of infection in birds, indicating a species specific evolution pattern of IBV. Recently, ANP32 proteins have been reported as key host factors limiting the spread of IAV from birds to mammals. Whether ANP32 proteins from different species give differing supports to IBV, and whether they can act as a barrier limiting the spread of IBV between species, are unclear. We compared the ability of human and chicken ANP32 proteins to support IBV polymerase activity in TKO cells, and we found that human ANP32A (huANP32A) and ANP32B (huANP32B) gave strong support to IBV polymerase, but chicken ANP32A (chANP32A), chicken ANP32B (chANP32B), and chicken ANP32E (chANP32E) gave only weak support to IBV replication, levels which were comparable to human ANP32E (huANP32E) . Similar+33) reduced the polymerase activity in TKO cells, giving it an activity similar to chicken ANP32A. Conversely, deletion of the 33 amino acids from chicken ANP32A (chANP32AΔ33) increased the polymerase activity to a level similar to that of huANP32A  and ostrich, that are all in “short form” without 33-amino-acid insert , supporthuANP32A . This suThe 33 extra amino acids comprise 27 amino acids identical to those in the section neighboring the insert, together with 6 specific amino acids. Previous research has shown that 4 of these 6 amino acids (VLSL) make up a SUMO-interaction motif, and substitution or deletion of these four amino acids can weaken the replication of the avian influenza virus , 13. ChiAll ANP32B proteins from different mammalian species lack the 33-amino-acid insert compared with chANP32A, although the chANP32B has a longer C-terminal of LCAR region than that of huANP32B. We showed that huANP32B gives strong support to IBV replication, while chANP32B gives only limited support to the IBV polymerase . Our preth amino acid, presenting a consecutive ten acidic amino acids deletion in birds; the second one was located downstream of the 200th amino acid, with eight consecutive amino acids differences between mammals and birds  insertion region. To verify this, we conducted our transfection experiments using four mutants: 10-amino-acid deletion in huANP32E (huANP32E_Δ10aa), 10-amino-acid insert in chANP32E (chANP32E_10aa+), and an 8-amino-acid replacement between huANP32E and chANP32E (huANP32E_8aamut and chANP32E_8aamut). The results showed that huANP32E_Δ10aa had similar ability to support IBV polymerase to that of chANP32E; chANP32E_10aa+ had similar activity to huANP32E; and that the 8-amino-acid replacement did not change the activity of huANP32E and chANP32E at all  assay to evaluate the binding kinetics and affinity between polymerase and ANP32 proteins. HuANP32A, chANP32A, huANP32A+33, and huANP32A_165T were fusion expressed at downstream of GST-HRV3C peptide in a pCAGGS vector and purified using Glutathione Sepharose 4B and then digested by PreScission Protease. The purity of ANP32 proteins was checked using SDS-PAGE analysis and western blotting . IBV pol+33) at concentrations of 0.15625–5 μM had valid binding stability to the viral polymerase immobilized on the CM5 chip in a dose-dependent manner , the association rate constants (ka) were distinctly different. The ka of huANP32A was twice as high as those from the other two proteins. This result indicated that huANP32A has a stronger binding affinity to IBV polymerase than chANP32A, and that the 33-amino-acid insert in chANP32A reduces the binding affinity of ANP32A to IBV polymerase. These results may help to explain why chANP32A gives only low support to the IBV polymerase.Consistent with our co-IP results, all these three ANP32A proteins , while that in the corresponding position of ANP32A or ANP32B is Asparagine (129N). An E129N mutation in ANP32E can completely restore support of IBV polymerase activity , indicatUnlike IAV, which comprises a heterogeneous group of different subtypes, IBV forms a more homogeneous cluster with two main lineages, Victoria and Yamagata. IAV can infect many animal species but IBV can only be identified clinically in humans, seals, and pigs. Infection by IBV in avian species has not been confirmed. Taking into consideration that IBVs have the ability to bind to α2,6-linked or α2,3-linked sialyl-glycans , it is pIt is known that the IAV polymerase complex binds to ANP32A or ANP32B for its normal function. The chANP32A shows stronger binding ability to avian IAV strains than do mammal ANP32As or ANP32Bs, which is consistent with its ability to support chicken IAV replication. The 33-amino-acid insert is responsible for this stronger binding ability . SurprisTaking the roles of ANP32 proteins in IAV and IBV together, we summarized the current understanding of interaction between ANP32 proteins and influenza viral polymerase. HuANP32A and huANP32B play major role in supporting both human IAV and IBV replication, but huANP32E has mild support to IBV and no support to human IAV. None of the human ANP32A, ANP32B, or ANP32E supports avian IAV because they do not have a 33-amino-acid insert as chANP32A. Chicken ANP32A harbors a 33-amino-acid insert which enables supporting to both avian and mammal IAV, but not IBV. Chicken ANP32B is a nature inactive molecule to IAV and IBV polymerase because of 129I/130N substitution. Chicken ANP32E has no function in supporting IAV polymerase, but shows a weak support to IBV. The low level support to IBV of ANP32E is due to a 129E mutation. Thus, none of chicken ANP32 proteins show good support to IBV . The difAH13) were kind gifts from Dr. Hualan Chen. The plasmids carrying the genes of influenza Bvirus B/Yamagata/1/73 were kindly provided by Dr Yoshihiro Kawoaka. B/Yamagata/PJ/2018  was used to infect 293T and knockout cells. pCAGGS plasmids containing ANP32A, ANP32B from different species are kept in our lab  Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Martin SchwemmleGuest EditorPLOS PathogensRon FouchierSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064***********************Reviewer Comments :Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: I am still very positive about this manuscript, and believe that it adds significantly to the field and our understanding of species-specific functional interactions between ANP32 proteins and influenza viruses. The data are novel, experiments well-executed, and the manuscript provides new detailed insights into the molecular basis of ANP32E usage, as well as avian versus mammalian ANP32A/B interplay with the polymerases of both influenza A and influenza B viruses. The findings have clear implications for trying to understand host restriction. Importantly, all of my previous comments, which were very minor, have been addressed satisfactorily.Reviewer #2: The authors have addressed my major concerns. I appreciate their attention to each of the points raised and thorough responses.Reviewer #4: The results presented in this manuscript advance our understanding of the important role of ANP32 proteins in the regulation and adaptation of the polymerase activity of Influenza A but most importantly on Influenza B viruses. It provides compelling and solid evidence, that ANP32 proteins promote IBV polymerase activity in human cells in a protein specific manner, which is in some parts divergent from the effects seen on the IAV polymerase.Many of the primary research questions in this manuscript have been addressed before by Staller et al. 2019, Journal of Virology using similar methodology. Thus the originality and novelty of the subject is limited. In contrast to Staller et al. who reporter a certain degree of functional diversity between ANP32A and B proteins on the IBV Polymerase activity, the results in this manuscript show functional redundancy of both proteins in human cells. These differences may be attributed to the choice of IBV strain, cell type and method of generating KO clones and are in general interesting but not ground breaking.The investigation of ANP32E, which shows limited support for the IBV polymerase is new and interesting, however, compared to ANP32A/B it appears to be of minor importance and its role in the species restriction of IBV to humans remains uncertain. The chosen methods mostly assess protein functions under non-natural  conditions and only very few assays verify results in the context of a viral infection, which overall is a weak point.In the light of these shortcomings I would suggest submission of this manuscript to a more specialized journal.In addition, I would like to pronounce my concerns about the response of the authors to reviewer #3, which is in parts rude and inappropriate. In a peer review process authors and reviewers should adopt a professional and supportive attitude even if the outcome is not as preferred.To avoid any misinterpretation: My evaluation of the presented scientific work is by no means affected by this statement.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: NoneReviewer #2: (No Response)Reviewer #4: (No Response)**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: NoneReviewer #2: (No Response)Reviewer #4: General remarks:1. What is the rational of using a H7N9 IAV derived polymerase? This subtype has only recently jumped to humans but is not stably adapted (not circulating) to the human population in comparison to the H1N1 and H3N2 subtypes. Please include some information on why this strain was chosen as a comparison to IBV, which is a seasonal strain and much more stable adapted to humans.2.The labeling of the y-axis in all figures displaying IAV polymerase activity using the polymerase reconstitution assay is wrong and needs to be changed to:Firefly Luciferase activity instead of Firefly Polymerase.Specific remarks:1. lines 76-78: What means “the ANP32 family MAINLY includes ANP32A, ANP32B and ANP32E.”? Does this refer to in most species? Or expression levels? Are there other ANP32 proteins present? Please specify this statement.2. lines 78: This sentence is misleading. Please acknowledge that also other groups have contributed to this finding.3. Figure 1: Please indicate the subtype of the FluA polymerase as this is a relevant information for this assay.4. Lane 123: what means: the last “functional member”? What function does this refer to? Please describe this in more detail. Also lane 124: what means “considered to be an important member of the ANP32 family”? Are there other non-important members? What is this hypothesis based on? Please provide more details.5. Line 149: what means ANP32As? Is that s a typo? Please correct.6. Figure 2: The title of this figure is misleading as it states that only ANP32As from different animals are investigated which is not true7. Figure 2B: why is there no ANP32 protein band in the empty vector control lane? These are wild type cells and should express detectable levels of endogenous chicken ANP32 proteins. Please explain and interpret this result.8. Figure 2C: Why were virus growth curves performed at 37°C? For IBV 33°C is commonly used as it also support much higher titers. I wonder whether overexpression of human ANP32B also has a positive effect at 33°C? Can you provide data on this?9. lane 174: Please correct the sentence to …”that ARE all in short form…” and remove the are in lane 176.10. Are there any other aa mutations between the different mammalian and avian type ANP32A proteins beside the 33 aa insertion? Please state so clearly in the primary text (in the respective section) as these mutations can also affect the protein function on the polymerase.11. Which cells were used in figure 3d? Please also include this information in the text and legend.12. lane 187: correct IVB to IBV13. lane 204: I understand that chicken ANP32B also lacks the 33 aa insert. Is that correct? But there is a size shift in the westernblot bands compared to human ANP32B indicating addition amino acids. Please add this information to the beginning of this paragraph.14. line 306: please change mutant to mutations15. figure 7D: What happens if the LCAR region of ANP32A is removed? Is binding to IBV polymerase then abolished? Is that a common mechanism for all ANP32 proteins? Why was the ANP32A_165T mutant not tested in the co-IP assay but in the SPR?16. line 360: I assume that the authors meant write morbidity instead of mobility? Please correct.17: line 362: “polymerase replication” is a confusing description. Please substitute with “polymerase activity” Please substitute18. overall the grammar, spelling and wording in the discussion part is of lower quality than the previous parts of the manuscript and needs major improvement (e.g. lines 417-425)19. lines 420-425: The authors claim that the amount of mammalian-like ANP32A isoform in DF1 cells is not enough to support IBV replication. But in Figure 2C a growth kinetic of B/Yamagata/PJ/2018 is presented. So in principle, IBV replication seems to be supported in theses cells. Please rephrase this part of the discussion accordingly.20. line 427: “chicken IAV”? does this mean avian IAV strains or do you really mean IAV from chicken? Please be precise.**********what does this mean?). 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All press must be co-ordinated with PLOS.Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.Best regards,Martin SchwemmleGuest EditorPLOS PathogensRon FouchierSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064***********************************************************Reviewer Comments : 6 Oct 2020Dear Dr Wang,We are delighted to inform you that your manuscript, "Selective usage of ANP32 proteins by Influenza B Virus Polymerase: implications in determination of host range," has been formally accepted for publication in PLOS Pathogens.We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. 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Sarcoptes scabiei into the stratum corneum. Currently, diagnosis via routine skin scraping is very difficult, and information on the allergenic identification of S. scabiei remains limited.Scabies is caused by burrowing of the mite S. scabiei chitinase-like protein-5 (rSsCLP5) and recombinant S. scabiei chitinase-like protein-12 (rSsCLP12) by measuring the levels of serum-specific IgG and IgE antibodies (Abs) as diagnostic markers. In addition, the allergenic characteristics of rSsCLP5 and rSsCLP12 were evaluated using IgE-binding experiments and skin tests.We performed comparative analysis of the serological diagnostic potential of recombinant The IgE Abs-based indirect enzyme-linked immunosorbent assay (ELISA) methods showed high sensitivity and specificity: the rSsCLP5-based assay had 93.5% sensitivity and 94.4% specificity; the rSsCLP12-based assay had 100% sensitivity and 98.1% specificity. The specific IgE Abs in infested mouse sera could bind rSsCLP5 and rSsCLP12. In skin tests, rabbits in the rSsCLP5 and rSsCLP12 groups and positive control (histamine) groups exhibited allergic reactions. Most test sites in the rSsCLP12 group had edema, bleeding spots, and even ulcers or scabs, but such allergy symptoms were rare in the rSsCLP5 group. Moreover, the allergic history rabbit group had more severe allergic reactions and lower levels of IgE Abs compared to the healthy rabbit group in the same protein group.These findings validate the use of IgE Abs to rSsCLP5 and rSsCLP12 as potentially useful markers for diagnosing scabies. Moreover, both rSsCLP5 and rSsCLP12 have allergenic properties, and the potential allergen rSsCLP12 is a stronger allergen than rSsCLP5. The online version contains supplementary material available at 10.1186/s13071-021-04654-0. IgE against rSsCLP5 and rSsCLP12 could be a useful diagnostic marker of scabies.Specific IgE could bind to rSsCLP5 and rSsCLP12.RSsCLP5 and rSsCLP12 could induce an allergic reaction.The potential allergen rSsCLP12 has a stronger allergenic effect than rSsCLP5.Sarcoptes scabiei. Scabies is an unusually contagious disease that affects nearly 200 million people worldwide [Scabies has been reported in humans and different animal species since the early 1900s [orldwide , and itsorldwide , 5. The orldwide , 7.S. scabiei cannot be cultured in vitro and must be cultivated in suitable hosts. Although new techniques were used to collect a large number of mites from red foxes [S. scabiei and produce specific IgE and IgG antibodies (Abs) to mite allergens and antigens, which means that potential allergens/antigens might have high specificity and sensitivity in serological tests [Ordinary scabies (OS) and CS are two forms of scabies. OS is usually caused by a few mites, which makes the diagnosis very difficult, while CS is easy to diagnose based on the large number of mites and obvious clinical symptoms , 9. The ed foxes  and dogsed foxes , the weied foxes  or host al tests , 15. In al tests .Dermatophagoides spp. can cause severe asthma and other allergies, and in-depth research has increased awareness of their molecular mechanism for causing allergies [S. scabiei causes varying degrees of allergic and inflammatory reactions [Dermatophagoides pteronyssinus, D. farinae, S. scabiei and other allergens and is termed molecular-based allergy diagnosis [llergies –18. Folleactions . Howevereactions . Due to eactions , 21, reciagnosis .S. scabiei chitinase-like protein-12 (rSsCLP12) as a potential allergen via skin testing [S. scabiei chitinase-like protein-5 (rSsCLP5) and rSsCLP12 antigens by detecting IgG and IgE Abs and compared and analyzed allergic reactions to rSsCLP5 and rSsCLP12 via skin tests, pathological tests and change in IgE antibody levels.Previously, our team initially measured the sensitization of recombinant  testing . In the Oryctolagus cuniculus, 3 months old) and Kunming mice  were purchased from Chengdu Tatsuo Biological Technology Co., Ltd. . The live S. scabiei mites were provided by the Department of Parasitology, Sichuan Agricultural University . Two male KM mice were each infested with a large number of S. scabiei total three times at 7-day intervals, and then sera were separated from blood samples obtained from tail-clip blood in mite-allergic mice. Serum samples were collected from 46  New Zealand White rabbits after 2-month infestation with S. scabiei. Twenty-four  sera from New Zealand White rabbits that had been identified as free from S. scabiei were collected as negative control samples to determine the cut-off values for the enzyme-linked immunosorbent assay (ELISA). Serum samples were also collected from rabbits that had been infested with Eimeria spp.  for 2 weeks when feces could be detected in oocysts, Psoroptes ovis var. cuniculi , for 4 weeks with clinical symptoms in ears or Cysticercus pisiformis  for 6 weeks with many cysts in the abdominal cavity. All serum samples were stored at − 20 °C until used.All New Zealand White rabbits  three times for 5 min each and incubated with blocking buffer  at 37 °C for 1.5 h. Then, the plates were washed and incubated with 100 μl diluted sera from mite-infested rabbits, sera from the rabbits infested with other parasites, sera from naïve rabbits (1:5 dilution) and serum-free buffered PBS for 1 h at 37 °C. The plates were washed and incubated with 100 μl biotinylated anti-rabbit IgE Abs  for 1 h at 37 °C. Then, the plates were washed, followed by 30-min conjugation with HRP (horseradish peroxidase)–streptavidin  at 37 °C. Color development was performed by adding 100 μl substrate 3, 3′, 5, 5′-tetramethylbenzidine  for 15 min at room temperature (RT). Then, 100 μl 2 M H2SO4 was used for the stop reaction, and the optical densities were determined at 450 nm (OD450 nm) using an ELISA plate reader  according to the manufacturer’s instructions.For IgG Ab detection, ELISA was performed as previously described . ELISA fS. scabiei-infested rabbit serum samples to determine the sensitivity of the IgG- and IgE-based indirect ELISA. Antigen specificity was determined using cross-reactivity tests using serum samples from the rabbits infested with Eimeria spp. (n = 10), P. ovis var. cuniculi (n = 10) and C. pisiformis (n = 10). The percentages of sensitivity and specificity were calculated as previously described [The cut-off values were calculated and determined as the arithmetic mean of the OD450 nm values + 3 standard deviations (SD) of 24 naïve rabbit serum samples . IgG andescribed , 25.The antigens were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane as described previously . The memS. scabiei after ivermectin treatment. One day before injection of the test components, the back hair of the rabbits was shaved, and four points were marked on the back as the antigen injection sites; the points were > 5 cm apart. Each site was injected intradermally with 0.1 ml volume containing 100 μg antigen, 100 μg histamine or saline. After the injection, the development and changes in wheal reaction, flush reaction and erythema were observed and recorded until 6 h after injection. The sera were separated from blood samples obtained from marginal ear veins (MEV) of experimental rabbits to evaluate the change in IgE Abs, and then the skin from the injection site was obtained for pathological analysis by hematoxylin–eosin (H&E) staining after killing the rabbits. Briefly, rabbits were killed by intravenous injection of 100 mg/kg barbiturate. Then, a 0.5-cm-diameter skin sample centered on the injection point was collected by skin punch. The skin was fixed with 4% paraformaldehyde for 48 h and dehydrated with 75, 85, 95, 100% ethanol and xylene for 15 min (until transparent). The tissues were paraffin-embedded and cut into thin sections (5 μm). The sections were baked in a 60 °C oven for 1.5 h and dewaxed in xylene twice for 10 min each, in 100% ethanol twice for 5 min each, in 95% ethanol for 5 min, in 85% ethanol for 5 min and in 75% ethanol for 5 min; then, they were rinsed with distilled water three times for 5 min each. The sections were stained with hematoxylin solution for 5 min, lightly washed with distilled water and then differentiated with 5% acetic acid and covered with 1% ammonia solution for 20 s. They were lightly washed with distilled water and stained with eosin for 1 min. Finally, the sections were dehydrated with 75, 85, 95 and 100% ethanol for 10 s each and xylene for 1 min. After that, pathological views were photographed under light microscopy and analyzed.Each component group . The pathological analyses were classified based on the different types and severity of pathological injuries: class 0, normal epidermis and dermis structure, no eosinophil infiltration in dermis and subcutaneous tissues or deep subcutaneous near muscle tissue; class 1, intact epidermis, a small amount of eosinophil infiltration in the dermis and subcutaneous tissues or deep subcutaneous near muscle tissue; class 2, intact epidermis, medium quantity eosinophil infiltration in the dermis and subcutaneous tissues or deep subcutaneous near muscle tissue; class 3, damaged epidermis or scabs or hemorrhages, a large amount of eosinophil infiltration in the dermis and subcutaneous tissues or deep subcutaneous near the muscle tissue.Graphs were prepared using GraphPad Prism 6.0. All statistical tests were performed with SPSS 16.0, and all data were analyzed by treating the injected group and time as fixed factors. The rabbits were considered random factors to account for repeated measures variability. The mean ± SD of the data are presented in the appropriate sections. The Duncan multiple range test with the alpha value set as 0.05 was used to compare the IgG and IgE Ab levels and the diameters of the allergic reaction at different time points within the same group and at the same time point between different groups.S. scabiei, C. pisiformis, Eimeria spp. or P. ovis var. cuniculi. The assay sensitivity was 93.5%  and P. ovis var. cuniculi (three samples). Consequently, the specificity of the rSsCLP5-based indirect ELISA was 90.7% . Therefore, an OD450 nm ≥ 0.2873 was deemed positive, and an OD450 nm < 0.2873 was deemed negative  and 94.4% specificity (51/54)  serum, and the results showed 93% sensitivity, 97% specificity and a high degree of repeatability[Eimeria spp. or C. pisiformis. These results show that mite infestation can be distinguished from other parasitic infestations of rabbits by present ELISAs. In general, rSsCLP5 and rSsCLP12 show promise as diagnostic antigens for detecting specific IgE Abs from S. scabiei infestation. Potential allergens can stimulate the production of specific IgG and IgE Abs after animals are stimulated, but indirect ELISA-based specific IgG Ab detection has cross-reactivity with sera from infestation with other parasites [Here, rSsCLP5-based indirect ELISA detection of specific IgG Abs  was better than that of some previous works, such as that using recombinant cofilin protein   and recoatability. In the arasites , 37. Fouarasites . The AHSarasites . In the S. scabiei; it can be detected in the skin of the rabbit after mite infestation [S. scabiei infestation and colonization of the host.Scabies mites can quickly dig burrows into rabbit skin after being placed on the skin . Human sestation . When miAllergens can bind to specific IgE Abs, which is one of the characteristics of allergens . AllergiS. scabiei. We found that both proteins could cause wheals and flushing at the test site, and allergic reactions occurred rapidly, suggesting that both proteins can induce immediate allergic reactions. With the development of allergic reactions, we found that the average diameters of wheals and flushing in the allergic history rabbit group were larger than those in the healthy rabbit group in the same protein group. The allergic reactions occurred when the animals first came into contact with the allergens rSsCLP5 or rSsCLP12. Then, the animals produced more serious allergic reactions when exposed again to the same allergen. In the skin test, the rSsCLP5-injected skin rarely had edema, bleeding spots and ulcers, but most of the test site skin in the rSsCLP12 group had edema, bleeding spots, and even ulcers or scabs, indicating that the potential allergen rSsCLP12 is a stronger allergen than rSsCLP5. In the pathology of allergic skin, both proteins could cause the aggregation of a large number of eosinophils, but the rSsCLP12 group had more cortical hemorrhage cases than the rSsCLP5 group, which also suggests that rSsCLP12 sensitization is stronger than that of rSsCLP5.At present, skin testing is one of the traditional methods for identifying allergens . PreviouS. scabiei, the serum IgE Ab levels rise rapidly in a short time and then decline and are maintained at a particular level [IgE Abs are important mediators of allergic reactions . After aar level , 45. AftS. scabiei interactions but also identifying novel allergens. Carrying out research on the clinical immunotherapy and drug targets of scabies will be of great significance.In conclusion, both rSsCLP5- and rSsCLP12-based indirect ELISAs have high sensitivity and specificity to detect the specific IgE Ab, which validates the use of IgE Abs against rSsCLP5 and rSsCLP12 as potential useful diagnostic markers of scabies. This provides a theoretical reference for finding more effective diagnostic methods for scabies mites in the clinic. In addition, both rSsCLP5 and rSsCLP12 could induce allergic reactions in rabbits, which indicates that these two recombinant proteins have allergenic properties. The present study performed here provides a foundation for not only understanding the host-Additional file 1. The diameter and score of wheals and flush, and pathological damage grades caused by allergy.


In addition, it was recently demonstrated that colchicine directly bind to the α3 subunit of GlyRs. Interestingly, other studies have shown a main role of α3GlyRs on chronic inflammatory pain. Nevertheless, the functional effects of colchicine on the α3GlyR function are still unknown. Here, by using electrophysiological techniques and bioinformatics, we show that colchicine inhibited the function of the α3GlyRs. Colchicine elicited concentration-dependent inhibitory effects on α3GlyRs at micromolar range and decreased the apparent affinity for glycine. Single-channel recordings show that the colchicine inhibition is associated with a decrease in the open probability of the ion channel. Molecular docking assays suggest that colchicine preferentially bind to the orthosteric site in the closed state of the ion channel. Altogether, our results suggest that colchicine is a competitive antagonist of the α3GlyRs.Colchicine is a plant alkaloid that is widely used as a therapeutic agent. It is widely accepted that colchicine reduces the production of inflammatory mediators mainly by altering cytoskeleton dynamics due to its microtubule polymerization inhibitory activity. However, other lines of evidence have shown that colchicine exerts direct actions on the function of ion channels, which are independent of cytoskeleton alterations. Colchicine is able to modify the function of several pentameric ligand-gated ion channels, including glycine receptors (GlyRs). Previous electrophysiological studies have shown that colchicine act as an antagonist of GlyRs composed by the  Colchicine is a tricyclic alkaloid known for their effects over cytoskeleton, acting as a microtubule depolymerizing agent . Colchicγ-aminobutyric acid receptor type A (GABAARs) . Similar to GlyRs and GABAARs, the colchicine inhibition of 5-HT3AR function occurred in the absence of pre-incubation through microtubule-independent mechanisms  by altering the microtubule organization . For exaGABAARs) . Nonetheof GlyRs  and of GyRs  to identify the transfected cells  were cultured using standard methods . The celed cells . All rec2, 0.5 GTP, and 2 ATP. The external solution contained (in mM) 140 NaCl, 5.4 KCl, 2.0 CaCl2, 1.0 MgCl2, 10 HEPES (pH 7.4), and 10 glucose. Whole-cell recordings were performed with an Axoclamp 200B amplifier  and acquired using Clampex 10.1 or Axopatch 10.0 software. Data analysis was performed off-line using Clampfit 10.1 . Exogenous glycine-evoked currents were obtained using a manually applied pulse (3–4 s) of the agonist and an outlet tube (200 μm ID) of a custom-designed gravity-fed microperfusion system positioned 50–100 μm from the recorded cell. The methodologies employed for the single channel recordings of α3GlyRs in the cell-attached configuration have been previously published (Glycine-evoked currents were recorded from EGFP-positive transfected HEK 293 cells in the whole-cell voltage-clamp configuration at room temperature (20–24°C) at a holding potential of −60 mV . Patch eublished . The patα3GlyRs in open/closed conformations from the Protein DataBank   using a receptor grid centered on the amino acids that form the binding site defined in the previous step and an extra-precision (XP) configuration. Analysis of the interface GlyR-molecules included structural and energetic parameters performed by the same software. Additionally, a theoretical ΔG bind was calculated by an energy calculation MM-GBSA using Prime . All images were created using PyMol .Protein-ligand docking was performed using the structures of B, 5TIO) . The strB, 5TIO) . InitialB, 5TIO)  in whichAll values were expressed as mean ± s.e.m. of normalized agonist-activated currents. P < 0.05 was considered statistically significant. For statistical analyses, at least six cells were analyzed per condition. All the statistical analyses and plots were performed with MicroCal Origin 8.0 .α3GlyRs. All these experiments were performed in the absence of any pre-incubation with colchicine. The co-application of colchicine produced a rapid inhibition of glycine-activated currents . The alkaloid exerted inhibitory effects in concentrations ranging from 1 to 200 μM (50) was 24 ± 7 μM, with a maximal inhibition of 39 ± 2% at 200 μM (n = 8). Interestingly, the colchicine potency observed in the α3GlyRs was not different from the IC50 obtained in the α1GlyRs . Altogether, these results support a direct inhibition of the channel function by colchicine. Moreover, these data are consistent with a competitive antagonism of colchicine on the α3GlyRs.To test the hypothesis that colchicine modulates directly the function of GlyRs, concentration response curves were generated from HEK293 expressing equation . To invein silico analysis of the interaction of α3GlyRs with colchicine using the crystal structure of α3GlyRs (PDB: 5CFB) as a template  and T204 (−), together with a π-cation interaction with R65 (+) that contributes to the stabilization of the complex . The glycine-activated current inhibition was not significantly different to those obtained using α1GlyRs, in which colchicine was described as a competitive antagonist in a previous study    and the α3GlyRs. Our results provide novel functional information regarding the direct interaction of colchicine with GlyRs, suggesting a molecular mechanism associated with a competitive inhibition at the orthosteric site. Although our experiments does not rule out a possible relevance of α3GlyRs on the analgesic actions of colchicine in inflammatory pain , our results at least confirm that GlyRs are targets of the alkaloid colchicine at the functional level. Further behavioral and functional experiments are necessary to clarify whether the effects of colchicine on GlyRs are relevant players on the beneficial effects of the alkaloid in inflammatory pain conditions.The present work defines the modulatory effects of colchicine on homomeric All datasets generated for this study are included in the article/CM-M, COL, CRR,DF and VPSM performed the experiments and data analysis. LGA, JF, PAC, LG, GEY and GM-C designed the research and contribute with analytical tools. CFB and CRR performed in silico analysis. CM-M, GEY and GM-C wrote the paper. All authors read and approved the submitted version.This work was supported by grant FONDECYT 1160851 and VRID 219.033.111-INV. (to GM-C), FONDECYT 1170252 (to GEY) and FONDECYT 3170108 (to CFB). COL and VPSM were supported by CONICYT doctoral fellowship 21171549 and 21201722 respectively. CRR was supported by CONICYT master fellowship 22200510.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.


Conclusions from previously reported articles have revealed that many commonly used pharmaceutical excipients, known to be pharmacologically inert, show effects on drug transporters and/or metabolic enzymes. Thus, the pharmacokinetics  of active pharmaceutical ingredients are possibly altered because of their transport and metabolism modulation from the incorporated excipients. The aim of this review is to present studies on the interaction of various commonly-used excipients on pre-systemic metabolism by CYP450 enzymes. Excipients such as surfactants, polymers, fatty acids and solvents are discussed. Based on all the reported outcomes, the most potent inhibitors were found to be surfactants and the least effective were organic solvents. However, there are many factors that can influence the inhibition of CYP450, for instance type of excipient, concentration of excipient, type of CYP450 isoenzyme, incubation condition, etc. Such evidence will be very useful in dosage form design, so that the right formulation can be designed to maximize drug bioavailability, especially for poorly bioavailable drugs. The most popular route for drug delivery is oral administration because of pain avoidance, ease of ingestion, patient compliance and versatility of drug candidates. Moreover, the manufacturing for oral drug delivery systems is less expensive as the production process is simple and there are no requirements for sterile conditions . The groCYP1A or CYP2C) and are distinguished by a number for the individual enzyme or isoforms  [CYP3A4/5 [The principle enzymes responsible for first-pass metabolism or biotransformation of drug molecules are cytochrome P450 (CYP450). CYP450 includes a superfamily of hemeproteins which is categorized into families and subfamilies based on amino acid sequence homology . They arCYP2C19) . AccordiCYP3A4/5 .Several novel, as well as traditional, methods are being investigated to circumvent drug metabolism. Prodrugs, enzyme inhibitors, polymeric excipients, self-emulsifying drug delivery systems (SEDDS) and liposomes have been investigated over the years as inhibitors to metabolic enzymes.Prodrug is one of the common approaches investigated, according to the literature. Approximately 10% of the drugs in the pharmaceutical market are categorized as prodrugs and one third of small molecular weight (Mw) drugs are classified as prodrugs ,36,37,38CYP3A4, using felodipine as a substrate [CYP1A2, CYP1B1, CYP 2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5. The dihydroxybergamottin (DHB) and paradisins inhibit CYP1A2, CYP1B1, CYP2C9, CYP2C19, 2D6 and CYP3A4 [Another promising strategy to reduce the pre-systemic metabolism is the co-administration of CYP450 inhibitors with orally administered drugs . Probablubstrate ,52,53,54d CYP3A4 .Besides grapefruit juice, many drugs in the market such as ketoconazole, Troleandomycin, saquinavir, diltiazem and fluconazole exhibit an enzyme inhibitory effect resulting in improved bioavailability of many drugs ,56. For These include polymeric excipients, self-emulsifying drug delivery systems (SEDDS) and liposomes. Normally, polymers are used as enteric coatings for different types of drugs to overcome hydrolytic instability . Some poThe addition of commonly-used pharmaceutical excipients can be a potential solution to these problems. Pharmaceutical excipients are ingredients other than the active pharmaceutical ingredient (API) present in a finished pharmaceutical drug formulation. These are frequently used as lubricants, diluent, binders, flavorings, coating and coloring agents for the formulation. These substances are often therapeutically inert .The functional roles of pharmaceutical excipients include modulating bioavailability and solubility of APIs, increasing the stability of APIs in the dosage form, maintaining the osmolarity and/or pH of the liquid formulations, preventing dissociation and aggregation, etc. Recently, addition of pharmaceutical excipients in drug formulations have gained attention which can alter the pharmacokinetics of drugs, resulting in improved bioavailability . The aimCYP2E1 [CYP2E1, using 15 healthy adults as volunteers. As a result, the measured AUCs for the metabolites were 16% lower than solid formulation. This is because propylene glycol is a competitive antagonist to CYP2E1; hence, it shows the protective effect in liquid formulation [Substances that can enhance or inhibit cytochrome P450 activity can alter the rate of drug metabolism, leading to an increase or decrease in drug bioavailability. There are many studies on new materials as drug delivery vehicles, including vesicles, block copolymer micelles, degradable polymer particles, dendrimers, polymer prodrugs and lipid nanoparticles; however, the effect of excipients used is often ignored. For instance, liquid acetaminophen, which contains propylene glycol, is less toxic than solid preparations with no propylene glycol. The main reason for acute hepatic failure in Europe and the United States is acetaminophen ,63. Its CYP2E1 . Liquid CYP2E1 . Since cmulation . One method by which excipients may alter the drug metabolism is by inhibiting CYP450 enzymes present in cellular microsomes .Surfactants, also known as surface-active agents, possess hydrophilic (polar) and hydrophobic (non-polar) characteristics. The hydrophobic (non-polar) part is referred to as the tail group and the hydrophilic (polar) part as the head group. Surfactants are normally used to increase the solubility of the drugs and to decrease the interfacial tension between the drug and the medium . SurfactCYP3A4 and CYP2C9 mediated metabolism of testosterone and diclofenac, respectively [50) of the CYP3A4- and CYP2C9-mediated metabolism were determined as 0.60 and 0.03 mM, respectively. Cremophor EL is a heterogeneous non-ionic surfactant made up of castor oil and ethylene oxide with a molar ratio of 1:35 . Cremophectively . CremophCYP3A4: 20.7% and 21.8%, in situ and in vitro at 47.5 μg/mL, respectively [CYP3A4 function.Mudra et al. further showed that solubilizing agents inhibited verapamil-N-demethylase activity in vitro and in situ. The rate of verapamil-N-demethylation was reduced in the presence of Cremophor EL, suggesting moderate inhibition of ectively . The inhectively ,71,72,73w/v). Various enzymes are responsible for metabolism of ethoxycoumarin, but the major enzymes are CYP450 1A2, CYP450 1A1 and CYP450 2B. Increasing the concentration of Cremophor EL decreased the metabolic activity of these enzymes [To support this hypothesis, another study on Cremophor EL presented results consistent with the previous studies. In vitro metabolism of 7-ethoxycoumarin was studied at three different concentration of the surfactant  by determining the intrinsic clearance (Clint) of MDZ in rat hepatocyte and rat microsome systems [In 2004, Gonzalez et al. investigated the effect surfactants have on the metabolism of the  systems . In the  systems .Taxol is an active ingredient used for refractory ovarian cancer, lung cancer and breast cancer. It is readily metabolized by the CYP450 system in the liver. A study conducted by Carlos et al. (1994) illustrated that Cremophor EL had the most significant effect on taxol metabolism, by the CYP450 system, compared to other co-administered drugs . In human and rat liver microsomes, the formation of 6α-hydroxytaxol was completely prevented by Cremophor EL at 20 µL/mL. In human liver slices, Cremophor EL reduced the formation of 6α-hydroxytaxol as well as the ratio of metabolite to parent drug at 20 µL/mL . HoweverCYP3A4 and CYP2C9 enzymes [50 = 0.60 mM) for CYP3A4, whereas stronger inhibition was reported with a similar IC50 value (0.03 mM) for CYP2C9. To understand the action of Cremophor RH40 in biotransformation, it was examined for its inhibitory effect on CYP3A4 in vitro as well as in vivo [CYP3A4 by Cremophor RH40 was 99.40%. Furthermore, single and multiple doses of Cremophor RH40 were examined in male Sprague-Dawley rats for its impact on MDZ metabolism. In comparison to control saline, Cremophor RH40 increased the area under curve (AUC0-∞) of MDZ up to 1.1-fold and decreased the production of 1′-Hydroxymidazolam (1′-OH-MDZ) up to 0.44-fold in a single dose. The MDZ AUC0-∞ was increased by 1.69-fold and 1’-OH-MDZ AUC0-∞ was decreased by 0.9-fold in a multiple-dose regimen [Cremophor RH-40 is found in many oral drug formulations, which is mainly used to improve solubilization of the drugs, such as Tegretol, Anafranil and Sandimmun Neoral. Cremophor RH-40 has a different molar content of ethylene oxide (45 mol) than Cremophor EL . The inh enzymes . The inh in vivo . Accordi regimen .p < 0.05), when the concentration of Cremophor RH40 was increased from 0.05% to 3% w/v. Studies have revealed that chemotherapeutic substances can downregulate the CYP3A gene expression [CYP3A activity. As a result, Cremophor RH40-based SMEDDS reduced the CYP3A protein expression levels at dilutions ranging from 1:50 to 1:100. Another study reported a similar significant inhibition of CYP3A4 and CYP2C19 enzymes in vitro by Cremophor RH40 when rabeprazole was used as a probe drug (p < 0.05) [These results are consistent with the recent study on a Cremophor RH40-based self-micro emulsifying drug delivery system (SMEDDS) . A signipression ,80. Ther < 0.05) . It was CYP3A4 and CYP2C9 by Tween 80 was determined. The IC50 value for CYP3A4 and CYP2C9 was found to be 0.40 and 0.04 mM, respectively. Christiansen et al. and Rao et al. illustrated the inhibition of CYP450 to be a concentration-dependent manner. For example, when the concentration of Tween 80 in SMEDDS was increased from 0.05% to 3% (w/v), the 1′-OH-MDZ production was reduced from ~80% to 30%. The in vitro metabolism of 7-ethoxycoumarin was reduced from 85% to 65% when the concentration of Tween 80 was increased from 0.03% to 0.10% [Recently, Tween 80 has been reported to inhibit different isoenzymes of CYP450: 3A4, 2C9, 1A1, 1A2 and 2B. Tween 80 (polysorbate 80) is the most commonly-used hydrophilic non-ionic surfactant with the ability to improve the solubility of compounds . Tween 8to 0.10% .p < 0.05), whereas the inhibitory effect of Tween 80 was reported below its CMC with rat liver microsomes  [CYP3A4 by approximately 20% with a Tween 80 concentration of 50 mM [CYP3A activity [The incubation of Tween 80 with rat hepatocytes reduced Clint of MDZ significantly, above its CMC  . A similof 50 mM . Mudra eactivity .v/v [Tween 20 (Polysorbate 20) and Tween 80 are composed of the same hydrophilic group but different hydrophobic groups. Tween 20 has a mixture of palmitic, stearic, lauric and myristic acids, whereas Tween 80 contains oleic, linoleic and stearic acids . Accordiv/v ,74.CYP1A1 by TX-100 was reported far below its CMC value (250 μM) [Triton X-100 (TX-100) is an octylphenol polyethoxylated non-ionic surfactant. Its structure consist of poly(ethylene glycol) with a 4-phenyl group . TX-100 (250 μM) .Furthermore, multiple studies pointed out the ability of other surfactants to interfere with CYP450, which can lead to improved bioavailability of drugs. The surfactants that were reported to show inhibitory actions are listed in Polymers are macromolecular compounds and constitute a large and diverse group of substances, including synthetic polymers, semi-synthetic polymers, natural polymers and fermentation products . These pCYP2D2, 64.6% for CYP2C6, 40.4% and 33.5% for CYP3A2/1, 38.1% for 2B1, 26.2% for 2C11 and 9.4% for 1A2 by mPEG2k-PCL2k at 1000 μg/mL. The extent of inhibition, ranked downwards, was as follows: mPEG2k-PCL2k > mPEG2k-PCL3.5k > mPEG2k-PCL5k > and mPEG2k-PCL10k [Lihui Qiu et al. evaluated the in vitro inhibition of six CYP isoforms by different mPEGx-PCLx (methoxy poly(ethylene glycol)-poly(ε-caprolactone)) amphiphilic copolymer micelles: mPEG2k-PCL2k, mPEG2k-PCL3.5k, mPEG2k-PCL5k and mPEG2k-PCL10k. The inhibitory effect was found to be concentration-dependent in manner . All CYPk-PCL10k . Similarly, Martin and co-workers investigated the effect of 10 commonly-used polymers on seven CYP isoforms . As shown in CYP3A4. The dose-dependent inhibition was reported with the Ki and IC50 values for F68 averaged 0.16 and 0.11 mg/mL, respectively [Huang et al. used the Hill equation and Lineweaver–Burk plots to determine the inhibitory effect of pluronic F68 (F68) on ectively . The mecectively .CYP3A4 activity strongly. On the other hand, the outcomes of this study were limited to CYP3A4. As previously mentioned, there are many families and subfamilies of CYP450. There is a possibility that the least effective polymer (PEG2000) could be the most effective on other CYP450 isoenzymes.Another study tested 22 commonly used excipients, which included sodium alginate, PEG4000, PEG1000, PEG6000 and PEG 2000. All the listed polymers inhibited 1′-OH-MDZ formation in the following order (from strong to weak): Sodium alginate > PEG4000 > PEG1000 > PEG6000 > PEG2000 . The phyCYP3A in a concentration-dependent manner [v/v. Compared to other polymers in the study, P85 reduced the formation of norverapamil by around 25% and 50% at 0.01% and 0.1% v/v, respectively [Pluronic P85 (P85) is a block copolymer consisting of two equal polyoxyethylene chains joined by a polyoxypropylene chain. A study revealed that P85 strongly inhibited norverapamil formation by t manner . The stuectively .S. aureus and N. gonorrhoeae [Fatty acids (FA) are the main components of phospholipids, triacylglycerols and many complex lipids. FAs are present in the majority of dietary fat in humans. Different foods provide a variety of fatty acids. In addition, the human body can synthesize them, either from other fatty acids or nonlipid precursors, e.g., glucose. In recent years, studies have revealed that various FAs have a wide range of microbicidal activity against several Gram-positive and Gram-negative bacteria, as well as enveloped viruses, including orrhoeae ,90. For orrhoeae ,93,94,9550 = 15.8 μM), 2B6 (IC50 = 10.7 μM), 2C8 (IC50 = 13.3 μM) and 2C9 (IC50 = 36.1 μM). One of the mono-unsaturated fatty acids, oleic acid, inhibited all the CYP isoforms except for CYP2E1, CYP2C19 and CYP2A6. Similarly, arachidonic acid inhibited activities of CYP3A4, CYP2C19, CYP2C9, CYP2C8, CYP2B6 and CYP1A2. Other FAs also showed a distinct inhibitory effect on different isoforms: gondoic acid inhibited all except 2C8; linoleic acid inhibited CYP2B6, CYP2C8 and CYP2C9; linolenic acid inhibited CYP1A2, CYP2B6, CYP2C8 and 2C9; and timnodonic acid inhibited 2C8 . Hence, the unsaturated fatty acids are potent inhibitors for CYP enzymes than saturated fatty acids [Besides their potent microbicidal activity, FAs can also reduce pre-systemic metabolism of drugs by inhibiting CYP450 enzymes. The effect of saturated and unsaturated fatty acids on CYP3A4, 2E1, 2D6, 2C19, 2C9, 2C8, 2B6, 2A6 and 1A2 were studied using midazolam 1-hydroxylation, chlorzoxazone 6-hydroxylation, dextromethorphan O-demethylation, mephenytoin 4-hydroxylation, diclofenac 4-hydroxylation, amodiaquine N-deethylation, bupropion hydroxylation, coumarin 7-hydroxylation and phenacetin O-deethylation, respectively. As a result, out of all the saturated fatty acids, lauric acid showed potential inhibition of 2B6. Myristic acid inhibited 1A2  and five polyunsaturated fatty acids  were examined for their effect on six isoforms of CYPs . As shown in CYP2D and >40% inhibition of CYP2E (except acetonitrile) was reported at 1% v/v. Overall, the organic solvents showed concentration-dependent inhibition. The inhibitory effect of organic solvents is summarized in In the last few decades, the effect of organic solvents (such as acetonitrile) on CYP-mediated metabolism has been reported by many researchers ,108,109.rCYP3A4 activity was inhibited in a mixed-type or competitive manner [rCYP3A4 was concluded to be concentration- and time-dependent. The DMSO inhibitory constant (Ki) value for the formation of temazepam and nordiazepam was found to be 24 and 6 mM, respectively.Another study demonstrated similar results with DMSO. When diazepam was dissolved in DMSO, the e manner . The inhCYP2C9 was studied using ethanol [v/v), whereas it required 510 mM (3.0% v/v) of ethanol to competitively inhibit CYP2C9. Ethanol also presented concentration-dependent inhibition for both substrates: S-warfarin and diclofenac. In 2009, the effect of co-solvent on diclofenac and S-warfarin metabolism by  ethanol . The inhCYP3A4 activity using pooled human microsomes [CYP3A4. In contrast, only DMSO and polyethylene glycol showed potential inhibitory effect on midazolam 1′-hydroxylation activity. Similar to this study, DMSO showed strongest inhibition for both metabolic activities. Moreover, the inhibition of testosterone 6β-hydroxylation activity by DMSO was in a concentration-dependent manner: from 10% to 50% inhibition at from 0.1% to 1% v/v concentration. The summary of all the reported organic solvents is shown in Eight organic solvents have been studied for their effects on crosomes . The hydCYP3A4 expression in human liver and colon cells [CYP3A4 is a highly inducible isoenzyme and is mainly regulated by a xenobiotic receptor named Pregnane X receptor (PXR), at the transcription level [CYP3A4 expression. As a result, all the excipients failed to activate the CYP2B6 promoter used for PXR activation assay. Instead, few excipients  showed a decrease in the multi-drug resistance gene (MDR1) and CYP3A4 expression , acetyl triethyl citrate, tributyl citrate, triethyl citrate, diethyl phthalate, dibutyl phthalate, triacetin and dibutyl sebacate. As a result, ATBC, dibutyl phthalate and acetyl triethyl citrate activated PXR. ATBC being the most potent transcription inducer showed dose-dependent activation. However, ATBC induced only intestinal CYP3A4 expression and not hepatic expression. This could be due to the PXR splice variants that vary in their gene targets, expression patterns, ligands, biological functions, subcellular localization and protein interactions [Based on a previous study, Takeshita et al. evaluated the effect of plasticizers on PXR-mediated transcription by the luciferase receptor, PXR-coactivator interaction, PXR knockdown, pression . Tompkinractions P-gp inhibition and CYP450 inhibition. In this review, we present recent research concerning the effects of common pharmaceutical excipients on pre-systemic metabolism by phase I metabolic enzymes (CYP450). According to our review, more than 40 commonly-used excipients were revealed to interfere with different isoforms of CYP450 in vitro, although very few have been assessed in humans. Based on the evidence, the mechanism of action was mainly found to be direct inhibition of the enzymes. Out of all the various excipients, surfactants were the most potent inhibitors due to their ability to cause perturbation of the enzyme’s microenvironment. Despite many similarities in the results from different articles, there appears to be a need for a robust approach to integrate the in vitro data that can predict pharmacokinetic changes in humans. Further research investigations are warranted to shed light on this issue.Pharmaceutical excipients play an important role in pharmaceutical products and are often presumed to be pharmacologically inert. However, there is growing evidence that they can change the pharmacokinetics of APIs through various mechanisms, such as