Biologic agents are emerging as an important treatment option for immune-mediated diseases. Injection site reactions following subcutaneous injection of biologic agents is not well described in the literature.To summarize injection site reaction data in phase 3 trials of all biologic agents.MEDLINE, Embase, and CENTRAL databases were systematically searched on February 8, 2022. Proportional meta-analysis was conducted to summarize injection site reaction prevalence for each biologic.There were 158 articles included in the review. The most common types of injection site reactions were erythema (42.8%), unspecified reaction (23.3%), pain (12.4%), and pruritus (5.7%). No patients discontinued their treatment due to injection site reactions in 39 of the 48 studies that reported on discontinuation data. There were 16 biologics included in meta-analysis across 80 eligible studies. The biologics with the highest point prevalence of patients reporting injection site reactions were Canakinumab , Dupilumab , Etanercept , and Ixekizumab . The biologics with the lowest point prevalence of injection site reactions were Risankizumab , Brodalumab , Guselkumab , Secukinumab .The prevalence of injection site reaction in response to biologics ranges from 0.08 to 15.5%. Canakinumab, Dupilumab, Etanercept, and Ixekizumab had the highest prevalence of injection site reactions. Risankizumab, Brodalumab, Guselkumab, and Secukinumab had the lowest prevalence of injection site reactions. Recommendations are made regarding the improvement of adverse event reporting to better understand the epidemiology of injection site reactions. They are typically administered parenterally as intravenous infusions or subcutaneous injections. The most utilized types of biologics in are tumor necrosis alpha (TNF-α) inhibitors and interleukin (IL) inhibitors, particularly inhibitors of IL-23 and IL-17. Biologics are thought to have improved safety profiles in comparison to systemic immunosuppression due to their selective targeting of specific aspects of the immune system. Biologics are indicated in severe conditions requiring immunotherapy that are unresponsive to more affordable and less invasive treatments. Systemic adverse effects of biologics are rare and include increased risk of serious infection, increased incidence of malignancy, and hematologic disturbances. Injection site reactions are adverse events in reaction to a drug or its excipient injected subcutaneously and often include localized pain, pruritus, or erythema.Biologic agents are becoming increasingly used in the treatment of immune-mediated diseases such as psoriasis and rheumatoid arthritis. Despite the mandatory collection and reporting of adverse events in randomized controlled trials, the methodological and reporting quality surrounding adverse events in contemporary clinical trials have been found to be inconsistent.However, evidence on injection site reactions specifically in biologic treatments is limited and reported incidence rates of injection site reactions are highly variable between different biologics. According to a narrative review conducted in 2019, incidence of injection site reactions vary greatly - between 0.5 to 40% depending on the biologic. The underlying mechanisms of injection site reactions were found to be either irritative reactions caused by the proinflammatory actions of the drugs or allergic reactions to the drug itself.The primary aim of this systematic review and meta-analysis is to pool epidemiological data of biologic injection site reactions to provide better estimates of injection site reaction prevalence. Secondary aims are to summarize the types of injection site reactions encountered and make recommendations for improving injection site reaction reporting in the literature. We also hope to provide future clinicians and patients with the tools to make informed decisions about biologic treatment.Supplemental File 1). The review protocol was not registered.This study was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses 2020 statement .A search was conducted on MEDLINE, Embase, and CENTRAL databases from database inception to February 8th, 2022. We chose to include biologics with and without primarily dermatologic indications to conduct a comprehensive review of all available data on injection site reactions. The search strategy combined [biologic names] with Boolean operator “OR”, which was combined with variations of [phase 3 clinical trial] with Boolean operator “AND” (The inclusion criteria included studies that: (1) were original phase 3 clinical trials; (2) trialed a biologic medication; (3) used injection as the form of medication administration; (4) had complete reporting of injection site reactions as adverse events; (5) were conducted on human participants. Secondary analyses of trials  or trials that administered biologics intravenously were excluded. References of included studies were checked manually to identify entries not included in the search. Grey literature  was also searched to obtain original trial data of secondary trial analyses identified in the search.Two reviewers (P.K. and P.L.) conducted title/abstract and full-text screening and data extraction in duplicate. Conflicts were reached either by discussion or by a third reviewer (R.V.). The following variables were extracted: study characteristics, biologic intervention details , injection site reactions, and comparator group data. Biologic dosing regimens were divided into 2 categories: U.S. Food and Drug Administration (FDA)-approved dosing or non-FDA-approved. Only biologics administered under FDA-approved dosing were included in meta-analysis. Studies were categorized into: (1) studies that reported all injection site reactions under “injection site reactions”; (2) studies with reporting of all high-level injection site reactions and its preferred terms within; (3) studies that reported only the most common preferred terms. Category 3 studies were excluded from meta-analysis to avoid undercounting of injection site reactions due to the exclusion of less common preferred terms.The Medical Dictionary for Regulatory Activities (MedDRA), a dictionary of standardized medical terminology developed for coding of adverse effects, defines “injection site reactions” as a “high-level” grouping term with subclasses of “preferred terms” used to describe the type of reaction.Descriptive statistics were used to summarize study characteristics, biologic indications, and types of injection site reactions. Median values were reported with interquartile ranges (IQR). Meta-analysis was not conducted if there were <2 study arms investigating the biologic.A proportional meta-analysis using a random-effects model was conducted to determine point estimates with 95% confidence intervals (CI) of the pooled prevalence of injection site reactions for each biologic as according to Barker et al.2 model was used to assess heterogeneity, where I2 >50% was significantly heterogenous. P value less than 0.05 was statistically significant. All meta-analyses were conducted using R Studio.The In = 1/158), 2000-2004 , 2005-2009 , 2010-2014 , 2015-2019 , 2020-2022 . Most studies involved investigators from multiple countries . There were 13.3% (n = 21/158) of studies conducted in exclusively in Japan, 3.8% (n = 6/159) conducted in the United States, 3.2% (n = 5/158) conducted in China, 1.3% (n = 2/158) conducted in Canada, and 0.6% (n = 1/158) each conducted in Germany and Russia .There were 9146 studies identified in the search and 118 studies included for the review after screening . An addiBiologics identified in our study were Abatacept, Adalimumab, Amgevita, Briakinumab, Brodalumab, Canakinumab, Certolizumab Pegol, Dupilumab, Etanercept, Golimumab, Guselkumab, Ixekizumab, Mepolizumab, Netakinumab, Omalizumab, Rilonacept, Risankizumab, Sarilumab, Secukinumab, Sirukumab, Tezepelumab, Tocilizumab, Ustekinumab, and Vedolizumab.n = 71/158). The most common injection site reaction was erythema , followed by reaction , pain , and pruritus . A full breakdown of injection site reactions preferred terms is available in n = 48/158) studies that reported on treatment discontinuation due to injection site reactions; 81.3% (n = 39/48) of studies had 0 patients discontinue treatment due to injection site reactions, 12.5% (n = 6/48) had 1 patient each, and 6.3% (n = 3/48) had 2 patients each.Details regarding injection site reaction preferred terms available for 1561 injection site reactions across in 71 studies  eligible for meta-analysis; 22 studies  were not eligible for meta-analysis due to the incomplete reporting of injection site reactions in their results and 56 studies  did not use FDA-approved dosing regimens.There were 80 studies , reaction , pruritus , pain , and irritation . Four patients discontinued adalimumab due to injection site reactions. Management of injection site reactions was not reported in any of the trials.There were 45 trials that investigated Adalimumab, of which there were 53 study arms. The most common preferred terms were erythema , plaque psoriasis (PsO) , Crohn’s disease (CD) , ankylosing spondylitis (AS) , psoriatic arthritis (PsA) , ulcerative colitis (UC) , hidradenitis suppurativa , and noninfectious uveitis . The median follow-up duration was 24 weeks (IQR: 16-26). The pooled prevalence of injection site reactions was 0.090    .2: 0%)  .There were 9 other studies that investigated other Adalimumab biosimilars that were not eligible for meta-analysis.n = 21/41), reaction , erythema , rash , and hematoma . No studies reported on discontinuation or management. Meta-analysis was conducted on 2201 patients across 7 trials and 8 study arms. Indications for Certolizumab pegol were RA  and CD . Median follow-up duration was 24 weeks (IQR: 18-32.5). The pooled prevalence of injection site reactions was 0.025   .n = 79/179), reaction , ecchymosis , and rash . There were 5 patients who discontinued etanercept due to injection site reactions. Management of injection site reactions were not reported.Etanercept was investigated in 20 trials and 28 study arms. Injection site reaction preferred terms were available for 179 reactions; the most common types were erythema , RA  PsA , AS , JIA , and extended oligoarticular juvenile idiopathic arthritis (eoJIA)/enthesitis-related arthritis (ERA)/PsA (1/17). The median follow-up duration was 24 weeks (IQR: 12-24). Pooled prevalence of injection site reactions was 0.114   .There were 5 Etanercept biosimilars across 5 trials not eligible for meta-analysis.n = 229/390), erythema , pain , and hypersensitivity . There were 5 patients who discontinued Ixekizumab as a result of injection site reactions. Management of injection site reactions were not reported. Meta-analysis was conducted for 7 study arms across 7 trials for a total of 2205 patients. Indications for Ixekizumab were PsO , PsA , and axial spondylarthritis . Median follow-up duration was 24 weeks (IQR: 14-24). The pooled prevalence of injection site reactions was 0.112   .n = 7/9), PsA , and UC . The median follow-up duration was 36 weeks (IQR: 12-52). The pooled prevalence of injection site reactions was 0.028   .Supplemental File 3. The forest plots are available in Supplemental File 4.Other biologics with fewer than 2000 patients eligible for meta-analysis were Brodalumab (1365 patients), Canakinumab (294 patients), Dupilumab (1888 patients), Golimumab (875 patients), Guselkumab (1852 patients), Omalizumab (394 patients), Risankizumab (707 patients), Sarilumab (475 patients), Secukinumab (1277 patients), and Tocilizumab (1675 patients). Summary data for all biologics eligible for meta-analysis are available in Biologics that were not eligible for meta-analysis were Abatacept, Briakinumab, Mepolizumab, Netakimab, Rilonacept, Sirukumab, Tezepelumab, and Vedolizumab. Type α reactions are caused by high levels of cytokines and typically cause systemic flu-like symptoms such as fever, fatigue, and myalgias. With subcutaneous injection of biologics locally irritative injection site reactions can occur due to the proinflammatory action of the injected substance causing elevated local cytokine concentrations.This systematic review and meta-analysis summarized adverse event data of injection site reactions following biologic use across 158 phase 3 clinical trials. Adverse reactions to biologics have been categorized into 5 distinct types  according to Pichler et al., of which injection site reactions can be either type α or β.T cell-mediated). Local subcutaneous injection of biologics can cause a rapid IgE-mediated allergic reaction, which presents as a local wheal and erythema, or urticaria/anaphylaxis in more severe reactions. Delayed IgG-mediated reactions present hours to days after administration and lead to inactivation of the biologic, causing patients to require greater doses. The most common types of injection site reactions in our review were erythema (42.8%), reaction , pain (12.4%), and pruritus (5.7%). One trial of Adalimumab in CD patients reported 5 accounts of injection site hemorrhage, but this was not elaborated upon in the results.Type β reactions can be categorized into immediate (IgE-mediated) and delayed , Brodalumab , Guselkumab , Secukinumab . Again, this is mostly in line with the literature .incidence as a misnomer; this is a commonly made mistake. Incidence defines the number of occurrences over a specified period of time , while prevalence is a proportion of patients affected by an event. Given the nature of adverse event reporting in clinical trials, which usually reports the number of patients affected rather than number of unique events, incidence data can rarely be calculated. Therefore in our meta-analysis we conducted a proportional meta-analysis to determine the pooled prevalence of injection site reactions. Unfortunately, this introduced a limitation to our review, as included studies had varying follow-up durations which affected our ability to pool accurate prevalence data. Studies with a longer duration of follow-up may have had greater prevalence compared to those with shorter follow-up due to the increased number of exposures to the biologic. However, there is evidence that suggests incidence of injection site reactions decrease over time, which may decrease the significance of this limitation.It is worth noting that most studies that report data on injection site reactions use the term 2 >50%), which suggests that prevalence data may be inappropriately pooled. However, I2 statistics in proportional meta-analysis are usually high due to the low variance of proportional data, but no specific measure of heterogeneity has been developed for this type of analysis. Therefore Barker et al. recommends conservative interpretation of heterogeneity in that it does not necessarily indicate inconsistency in the data.Another limitation is the heterogeneity of our meta-analysis findings. All estimates of prevalence were found to be significantly heterogenous  of studies reported preferred terms for injection site reactions. Additionally, among the preferred terms, “reaction” was the second most common, accounting for 23.3% of all reaction types. While “reaction” is a preferred term designated by MedDRA, describing injection site reactions as “reactions” gives no relevance to the extent, timing, or clinical relevance of the adverse event.The prevalence of injection site reaction in response to biologics varies widely from 0.08 to 15.5%. In our review, the biologics with the highest prevalence of injection site reactions were Canakinumab, Dupilumab, Etanercept, and Ixekizumab, although this may improve in the future with changes to biologic formulations. The biologics with the lowest prevalence of injection site reactions were Risankizumab, Brodalumab, Guselkumab, and Secukinumab.We recommend future clinical trials investigating biologics implement more complete reporting of injection site reactions in terms of type, number of events, and changes in occurrence over time to facilitate a better understanding of its epidemiology and how it impacts patients who receive biologic medications.Click here for additional data file.Supplemental material, online supplementary file 1, for A Systematic Review and Meta-Analysis of Injection Site Reactions in Randomized-Controlled Trials of Biologic Injections by Patrick J. Kim, Rafael Paolo Lansang and Ron Vender in Journal of Cutaneous Medicine and SurgeryClick here for additional data file.Supplemental material, Online supplementary file 2, for A Systematic Review and Meta-Analysis of Injection Site Reactions in Randomized-Controlled Trials of Biologic Injections by Patrick J. Kim, Rafael Paolo Lansang and Ron Vender in Journal of Cutaneous Medicine and SurgeryClick here for additional data file.Supplemental material, Online supplementary file 3, for A Systematic Review and Meta-Analysis of Injection Site Reactions in Randomized-Controlled Trials of Biologic Injections by Patrick J. Kim, Rafael Paolo Lansang and Ron Vender in Journal of Cutaneous Medicine and SurgeryClick here for additional data file.Supplemental material, Online supplementary file 4, for A Systematic Review and Meta-Analysis of Injection Site Reactions in Randomized-Controlled Trials of Biologic Injections by Patrick J. Kim, Rafael Paolo Lansang and Ron Vender in Journal of Cutaneous Medicine and Surgery


Trichinella spiralis is a helminth that induces protection through IgE antibodies. The present study examined T. spiralis susceptibility in high and low IgE responder mice, with a specific focus on the inheritance of IgE responsiveness, which controls IgE production specific for the IgE isotype and non-specific for antigens. Furthermore, low IgE responsiveness is inherited as a recessive trait under a single gene, which is not linked to the H-2 gene. This study determined the total IgE and anti-T. spiralis IgE antibody levels after T. spiralis infection in low IgE responder SJL/J mice were several times lower than those in high IgE responders, such as the BALB/c mice. An IgE-dependent susceptibility to T. spiralis, evaluated in mice treated with anti-IgE antibodies and in control mice, was observed in high IgE responder mice but not in low IgE responder mice. The inheritance of IgE responsiveness and susceptibility to T. spiralis was investigated using crosses of SJL/J with high IgE responders. All of the (BALB/c × SJL/J) F1 and half of the (BALB/c × SJL/J) F1 × SJL backcross progenies were high IgE responders after T. spiralis infection. Total IgE and antigen-specific IgE antibody levels were correlated and not linked to H-2. It should be noted that high IgE responders always exhibited low susceptibility, suggesting that the trait of IgE responsiveness functions as a trait of susceptibility to T. spiralis.IgE antibodies are likely involved in host protection.  I have Helminth infections are characteristically associated with type 2 immune responses, including T helper type 2 (Th2) cells and type 2 innate lymphoid cells, resulting in IgE production, eosinophilia, and intestinal mastocytosis. The type 2 immune response has been considered to be responsible for protection against helminths in a complex manner , 9. AlthTrichinella spiralis is a representative helminth showing IgE–dependent protection in rodent hosts  were purchased from the indicated sources.Pepsin , bovine serum albumin (BSA) , Evans blue (Sigma–Aldrich), streptavidin–peroxydase , azino–bis (3–ethylbenz–thiazoline–6–sulfonic acid) (Sigma–Aldrich), and H2.2T. spiralis antigen was a soluble extract of muscle larvae of T. spiralis. The larvae obtained from mice infected with 400 T. spiralis larvae for 2 months were homogenized and dialyzed against borate–buffered saline (pH 8.0) for 1 day. The antigen was a supernatant obtained after centrifugation at 10,000 rpm for 30 minutes at 4˚C .Rat anti–mouse IgE monoclonal antibodies  were derived from hybridomas established by us, and biotinylated anti–mouse IgE monoclonal antibody (HMK–12) was prepared . An anti2.4The BALB/c, C3H/He, and (C57Bl/6 × DBA/2) F1 mice were purchased from Japan SLC . The SJL/J mice were purchased from the Ohmura Institute for Laboratory Animals . The ASW mice were purchased from The Jackson Laboratory , and (ASW × SJL/J) F1, (BALB/c × SJL/J) F1, and (BALB/c × SJL/J) F1 ×SJL/J backcross mice were bred and maintained in accordance with the institutional guidelines at the Animal Facility of The Jikei University. IgE responsiveness was determined according to a previous study . SJL/J m2.5T. spiralis. As we have previously reported, 100 muscle larvae were used to infect mice in experiments of anti–IgE treatment 3 4 weeks after infection to identify IgE responsiveness in each mouse by anti–DNP IgE antibody production 1 – 3 weeks after immunization. The IgE responsiveness of each backcross mouse was also examined by anti–T. spiralis IgE antibody production 3 weeks after infection. In backcross mice experiments, larval recovery was performed 8 weeks after infection to confirm the IgE responsiveness by anti–DNP IgE production and anti–T. spiralis IgE production.Mice were inoculated orally with 100 or 200 muscle larvae of reatment . To indueriments . High lelculated . In cros2.6T. spiralis muscle larvae. Total IgE levels in the plasma were measured 2 – 4 weeks after infection. Mice were sacrificed 4 – 5 weeks after infection for T. spiralis larval recovery.In experiments of anti–IgE treatment, mice were treated with anti–IgE or control IgG before and during experiments. Mice (five per group) were infected with 100 T. spiralis muscle larvae. Total IgE levels in the plasma were measured 2 – 4 weeks after infection. Anti–T. spiralis IgE antibody was examined 3 weeks after infection. To determine IgE responsiveness in each backcross mouse, mice were immunized with DNP–T. spiralis antigen 4 weeks after infection. Anti–DNP IgE levels were examined 1 – 3 weeks after DNP–T. spiralis antigen immunization. Mice were sacrificed 8 weeks after infection (4 weeks after immunization) for T. spiralis larval recovery.In backcross mice experiments, 24 or 18 backcross mice were infected with 200 T. spiralis muscle larvae. Mice were immunized with DNP–T. spiralis antigen 4 weeks after infection. Anti–DNP IgE antibodies were measured 1 – 3 weeks after immunization. Mice were sacrificed 8 weeks after infection (4 weeks after immunization) for T. spiralis larval recovery. Next, (ASW × SJL/J) F1, ASW, and SJL/J mice (five per group) were infected with 200 T. spiralis muscle larvae. Anti–T. spiralis IgE antibody was examined 3 weeks after infection. Mice were immunized with DNP–T. spiralis antigen 4 weeks after infection to confirm IgE responsiveness 1 and 2 weeks after immunization. Mice were sacrificed 6 weeks after infection (2 weeks after immunization) for T. spiralis larval recovery.In FI experiments, (BALB/c × SJL/J) F1. BALB/c and SJL/J mice (five per group) were infected with 200 2.72O2) was added, and the reaction was stopped with citrate and was read at 490 nm with an ELISA plate reader. In the experiments of anti–IgE treated mice, microtiter plates were coated with an anti–mouse IgE monoclonal antibody (P2B10). These anti–mouse IgE monoclonal antibodies recognized different epitopes of IgE molecules  were injected intradermally into the shaved dorsal skin of Wistar rats. After a 24–hour–sensitization period, 1 mg of 2.9d antibody and analyzed using a flow cytometer cell sorter .White blood cells in peripheral blood from (SJL/J ×BALB/c) F1 × SJL/J backcross mice were stained with fluorescein–conjugated anti–H–22.10t–test using R statistical software version 4.2.3 . A p–value< 0.05 was considered to be statistically significant.Statistical analysis was performed with an unpaired Student’s 33.1T. spiralis and peaked 3 weeks after. From an IgE level of 0.1 μg/mL in an uninfected mouse, total IgE levels in infected BALB/c mice increased by 40 – 60 times  in the plasma of all strains of mice Table 1.In BALB/c mice treated with anti–IgE antibody, the number of muscle larvae 4 – 5 weeks after infection was significantly greater than in control mice Table 1.3.3T. spiralis antigen 4 weeks after infection with 200 T. spiralis muscle larvae. In all individual BALB/c and (BALB/c × SJL/J) F1 progeny mice, anti–DNP IgE PCA titers were 160 after immunization. Titers in all parental SJL/J mice were 20  F1 mice and ASW mice than in SJL/J mice  F1 mice and ASW mice and 20 in SJL/J mice. These data confirm that the low IgE trait is recessive. Moreover, low IgE responses again resulted in high susceptibility to T. spiralis.Six weeks after infection with 200 3.4T spiralis antigen immunization. This work has identified 11 high IgE responders and 13 low IgE responder mice. Total IgE levels in backcross mice classified as high IgE responders, owing to anti–DNP IgE PCA titers >160, were approximately twice as high as those in low IgE responder mice, with anti–DNP IgE PCA titers <40  F1 × SJL/J backcross mice were classified as high and low IgE responders by anti–DNP IgE PCA titer 1 – 3 weeks after DNP–ters <40 Table 2.ddH–2 and that of SJL/J mice is ssH–2. Therefore, the H–2 genotype of backcross mice is ssH–2 or sdH–2. Approximately half of the backcross mice expressed dH–2. Comparable numbers of T. spiralis were recovered from sdH–2 and ssH–2 mice. Total IgE levels were equivalent in these sdH–2 and ssH–2 mice. (In addition, the involvement of the H–2 haplotype on susceptibility was evaluated in the backcross mice. The H–2 genotype of BALB/c mice is T. spiralis IgE PCA titers 3 weeks after infection. Total IgE levels in high IgE responders, classified on the basis of anti–T. spiralis IgE PCA titers > 80, were approximately twice as high as those in low IgE responders, with anti–T. spiralis IgE PCA titers < 40 (T. spiralis infection was significantly greater from low IgE responders than from high IgE responders. Approximately 50% of backcross mice in these experiments were high IgE responders (A similar experiment was performed in backcross mice classified as high and low IgE responders by anti–ers < 40 Table 3.4T. spiralis was demonstrated in high IgE responder strains of mice except in the C3H/HeJ mice by comparing anti–IgE–treated mice with IgE–producing control mice. In low IgE responder SJL/J mice, anti–IgE treatment did not have a significant effect on susceptibility (T. spiralis is a result of a difference in IgE responsiveness. The advantage of the protocol used in the present study with the repeated injection of anti–IgE antibodies is that IgE suppression can be induced in every strain of mice. It is not easy to establish each strain of IgE– knockout mice. Some parasites are susceptible to limited strains of mice. This protocol is applicable to these cases to analyze the effect of IgE. In contrast to IgE–suppressed mice with anti–IgE treatment, I reported the effect of high levels of IgE in mice with repeated injections of anti–hapten IgE monoclonal antibody on the protective activity against T. spiralis (T. spiralis was impaired by high levels of IgE in high IgE– responder mice but not in low IgE– responder mice, thereby, suggesting that protective activity is controlled by IgE responsiveness.In the present study, IgE–dependent susceptibility to tibility Table 1.tibility  and IgE–tibility . Moreovespiralis . The proT. spiralis. The present crossing experiments demonstrated that F1 mice were high IgE responders and that backcross mice were segregated into high and low IgE responder groups at a 1:1 ratio (T. spiralis IgE antibody, produced high total IgE levels. Moreover, high IgE responders produced high levels of both anti–DNP IgE antibody and anti–T. spiralis IgE antibody. Major characteristics of this trait are the control restricted to IgE production and the control not restricted to antigen immunization (T. spiralis, suggesting that the genetic trait of IgE responsiveness functions as a trait of susceptibility to T. spiralis.The genetic trait of IgE responsiveness also controls the susceptibility to :1 ratio 3. These:1 ratio . Moreovenization . It shouIL4 gene (IL4 gene or the IFNG gene. In addition, the amounts of IL–4 and interferon–γ in the culture supernatant of spleen cells from immunized mice were not correlated to IgE responsiveness (To understand the control mechanism of IgE production, it is important to identify the locus of IgE responsiveness. I was unable to determine the gene locus in this work. In humans, the genetic control of total serum IgE concentration has been investigated with an antigen non–specific fashion as the IgE responsiveness in mice demonstrated in the present study. A gene controlling total serum IgE concentration in humans is linked to chromosome 5q31.1, especially to the IL4 gene . Similarsiveness . CytokinT. spiralis and H–2 to confirm the lack of genetic linkage between IgE responsiveness and H–2 (sH–2 haplotype, SJL/J mice were low IgE responders and ASW mice were high IgE responders (ssH–2 and sdH–2 backcross mice, neither total IgE levels nor susceptibility to T. spiralis differed significantly, suggesting H–2–independent regulation (T. spiralis, namely Ts–1 and Ts–2 (T. spiralis IgE antibody levels and the number of T. spiralis larvae in mouse strains including SJL/J mice were reported to be under the control of H–2–linked and non–H–2–linked genes (This study focused on  and H–2 , 24, 25.and Ts–2 , have beed genes . GeneralT. spiralis IgE antibodies were detected 2 weeks after infection but not 1 week after infection (T. spiralis. In IgE–knockout BALB/c mice, the number of muscle larvae is reportedly twice that in control BALB/c mice, and the expulsion of adult worms from the intestine is slower than that in control mice (The target of IgE–dependent protection was not specified in the present study. However, anti–nfection Figure 1nfection . This perol mice . Althougrol mice .T. spiralis. High IgE responsiveness results in high protective activity as a dominant trait. IgE antibody is one of the factors responsible for susceptibility. In Schistosoma infection, IgE has protective roles and relates to pathogenesis in humans (Schistosoma egg–granuloma formation in mice (Schistosoma infection is a significant issue in humans and domestic animals. The development of vaccines for the prevention of infection and inhibition of pathogenesis is required. The genetic control of IgE responsiveness may be an important factor for vaccine development.The genetic control of IgE production by an atopy gene has been investigated, and several candidate genes have been reported , 28. Aton humans . IgE als in mice . SchistoThe original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The animal study was reviewed and approved by the Animal Care and Use Committee of The Jikei University.The author confirms being the sole contributor of this work and has approved it for publication.


Tenderness is an important characteristic of meat quality. Calpastatin and calpain play important roles in meat tenderization. However, it is not clear how phosphorylation affects the regulation of calpastatin on μ-calpain and, consequently, meat tenderness. Calpastatin with high and low phosphorylation levels were obtained in vitro corresponding to the treatments by protein kinase A (PKA) and alkaline phosphatase. Then, calpain was incubated with calpastatin with different phosphorylation levels, and the effect of calpastatin on calpain activity under different phosphorylation levels was analyzed. The results showed that PKA promoted the phosphorylation of calpastatin, and a high phosphorylation level was maintained during incubation. The degradation rate of μ-calpain in AP group was higher than that in the other groups, meaning there was lower inhibition of calpastatin on calpain activity. The degradation of calpastatin was lower and its structure was more stable after phosphorylation. One more serine 133 site of calpastatin was identified in PKA group compared with the other groups. Phosphorylation at serine 133 of calpastatin enhanced its inhibition on calpain activity by maintaining its structural stability, thus inhibiting the tenderization of meat. Autolysis of calpain reduces the concentration of Ca2+ required to activate calpain. Autolysis of calpain is associated with activation of calpain and degradation of proteins at early postmortem. Calpain damages the ultrastructure of muscle by degrading proteins in muscle fibers and promotes fragmentation of myofibrillar fibers and tenderization of meat [Tenderness is the primary consideration for consumers when purchasing meat products . Factors of meat . The ear of meat . The deg of meat . Calpain of meat .2+, calpastatin rapidly binds to the activated calpain and inhibits its activity [Calpastatin is an endogenous inhibitor of calpain that regulates the degradation of myofibrillar protein in vivo. Calpastatin regulates protein conversion, muscle differentiation, and growth in living animals . The genactivity . The conactivity . The inhactivity . The degactivity . The degactivity .Protein phosphorylation, a common post-translational modification of proteins, is involved in regulating many important physiological processes  and playHowever, how the phosphorylated calpastatin exerts its inhibitory effect on calpain remains unclear. A hypothesis is that phosphorylation changes the inhibition of calpastatin on calpain activity by changing the structure of calpastatin, which may be due to the binding of phosphate group on calpastatin varying the affinity of calpastatin and calpain. Therefore, models of calpastatin with different phosphorylation levels in vitro were constructed to explore the effects of phosphorylated calpastatin on the activity of calpain in order to understand the mechanism of meat tenderness from a new point of view.μ-calpain (208712) and calpastatin (208900) were obtained from Calbiochem  and Merck-Millipore , respectively. Protein kinase A (P2645), alkaline phosphatase (P0114), ATP (A7699), and primary antibody against calpastatin  were obtained from Sigma-Aldrich . Primary antibody against calpain , Pro-Q Diamond (MPP33300), and SYPRO Ruby (S12000) were obtained from Thermo Fisher Scientific .2, pH 6.8). All groups were incubated at 30 °C for 30 min. Part of each group (50 μL) was mixed with the same volume of loading buffer and incubated at 100 °C for 5 min.Equal amounts of calpastatin (200 μg) were incubated with kinase or phosphatase to regulate its phosphorylation levels as follows: (1) adding protein kinase A (PKA) as a high phosphorylation level group; (2) adding alkaline phosphatase (AP) as a low phosphorylation level group; (3) control group (control) without addition of PKA or AP . The con2 solution was added to all groups to adjust the final concentration of Ca2+ in the system to 0.05 mM and the final volume to 1125 μL. Samples were collected at 4 °C for 1, 2, 12, and 24 h during incubation.The other part of treated calpastatin (800 μL) with different phosphorylation levels was divided into 4 groups, mixed with pure μ-calpain at the ratio of 1:2.5, 5, 10 and 15, respectively. CaClTM MP Imaging System  was applied for scanning, and Quantity One software  was used to quantify and evaluate the phosphorylation levels of calpastatin.Samples (15 μg) were loaded onto polyacrylamide gels . After electrophoresis, gels were stained with Pro-Q Diamond and SYPRO Ruby for phosphorylated and total calpastatin, respectively. The ChemiDocSDS-PAGE electrophoresis and western blotting were used to determine the degradation of μ-calpain and calpastatin. The polyacrylamide gels for calpain comprised 8% separation gel and loading volume was 30 μg. After electrophoresis, the bands were transferred to the PVDF membrane and performed at 100 V for 100 min. After western blotting, PVDF membrane was incubated with antibodies of μ-calpain and calpastatin, respectively. The degradation rate of the 80 kDa large subunit of μ-calpain was used to represent the activity of μ-calpain.4HCO3). Cycles of dehydration with acetonitrile and cleaning with NH4HCO3 fellow were performed. It was then incubated and digested on ice and 37 °C, respectively. A proportion of 0.1% trifluoroacetic acid was added to stop the reaction after digestion overnight. Finally, LC-MS/MS  was used to detect and Mascot 2.2 search engine was used to identify proteins.The proteins for LC-MS/MS identification should undergo electrophoresis, Coomassie brilliant blue staining and cleaning with ultrapure water and gel digestion bleaching solution . All results were presented as means and standard deviations.The data were analyzed using SPSS Statistic 21.0 . Generalize linear model (GLM) was used to analyze the variations of phosphorylation level of calpastatin and the degradation of μ-calpain. The differences among the means were compared using the Fisher’s Protected Least Significant Difference (LSD) test (p < 0.05). The phosphorylation level of calpastatin in AP group was significantly lower than that in the control group. These results indicated that AP and PKA catalyzed dephosphorylation and phosphorylation of calpastatin, and calpastatin with different phosphorylation levels were obtained.In order to determine the effect of phosphorylated calpastatin on the activity of μ-calpain, calpastatin with different levels of phosphorylation were obtained by adding PKA and AP. The phosphorylation level of calpastatin after phosphorylation and dephosphorylation treatment is shown in Calpastatin with different phosphorylation levels were mixed with μ-calpain at ratios of 1:2.5, 1:5, 1:10 and 1:15. The phosphorylation levels of calpastatin in each system were detected at 1, 2, 12, and 24 h during incubation . In geneThe degradation of the large subunits represents μ-calpain activity . The degThere was obvious degradation of calpastatin in the calpastatin:μ-calpain = 1:2.5 incubation system, and the degradation of calpastatin was significantly lower in PKA group than that in other treatment groups . The degTwo phosphorylation sites of calpastatin were identified in the PKA group : ThreoniAfter incubating at 30 °C for 30 min, calpastatin with high, medium, and low phosphorylation levels were obtained, corresponding to PKA, control and AP groups. It indicated that calpastatin with different phosphorylation levels were successfully constructed in this study, and PKA and AP could effectively catalyze the phosphorylation and dephosphorylation of calpastatin. After mixing with different ratios of calpain and incubating at 4 °C, the high phosphorylation level of calpastatin in the PKA group was maintained. There was no significant difference between the AP group and the control group in the phosphorylation level of calpastatin, which might be due to the decrease of the phosphorylation level of calpastatin in the control group or the increase of the phosphorylation level of calpastatin in the AP group. The amount of ATP in this study could only catalyze phosphorylation of up to 200 μg protein, and the amounts of PKA and AP in different groups were added according to the standard of 200 μg protein. Therefore, the phosphorylation level of calpastatin should be effectively inhibited by AP in the AP group, and the phosphorylation level of calpastatin would not increase. The reason why there was no significant difference in the phosphorylation level of calpastatin between the control group and AP group was that the phosphorylation level of calpastatin in the control group decreased.2+, which induced conformational changes and revealed the catalytic site [2+ concentration required to induce the formation of the calpain-calpastatin complex, which lead to a significant decrease in the inhibitory efficiency of calpastatin. Therefore, the phosphorylation of calpastatin represents a mechanism that can balance the actual amount of active calpastatin with the level of calpain to be activated [Calpain performs its proteolytic activity through autolysis degradation, so the degradation degree of μ-calpain is often used to indicate its activity. The faster the calpain degradation rate, the faster the activation rate, and the higher the calpain activity . In the tic site . This retic site . The addctivated . In thisctivated .2+ [2+ increase, calpain proteolysis becomes excessive and its targets undergo extensive degradation. The transformation from physiological function to pathological function of calpain is due to the insufficient regulation of calpain proteolysis by calpastatin [2+ regulation, strategies to limit the effects of calpain activation are primarily based on calpastatin [2+ promoted the activity of calpain and also promoted the formation of calpain-calpastatin complex and inhibited the activity of calpain [2+ to promote calpain activation and proteolysis, which is necessary for cell fusion. In general, protein phosphorylation can promote the inhibitory activity of calpastatin by changing the structure of calpastatin from the following two aspects: (1) Protein phosphorylation promoted the binding of calpastatin and calpain, which helped the inhibition domain of calpastatin to better recognize and bind to calpain, thus inhibiting its activity. (2) Protein phosphorylation exerted its inhibitory activity by reducing the degradation by calpain. The degradation of unphosphorylated calpastatin by calpain may also be one of the reasons for the decreased phosphorylation level of calpastatin in the control group.In the incubation process, the structure of calpastatin was relatively stable in the PKA group, and obvious degradation was observed in the AP group. It was speculated that the phosphorylation of calpastatin exerted high inhibitory activity by maintaining its structural stability. Protein post translational modifications affected the specificity and inhibition of calpastatin . The int2+ . Howeverpastatin . Thus, ipastatin . Phospho calpain . In rat  calpain . The dec calpain . TemporaThe primary structure of calpastatin showed that there were four repeated calpain-inhibitory domains (I-IV). Each inhibitory domain contains three subdomains, and each subdomain can bind to one calpain molecule . They haPrevious studies have shown that μ-calpain activity positively regulated meat tenderness and that both phosphorylation and dephosphorylation can promote calpain activity ,26. As aProtein kinase A significantly catalyzed the phosphorylation of calpastatin. The structure and phosphorylation level of phosphorylated calpastatin were well maintained during incubation with calpain. The phosphorylation calpastatin at serine 133 of promoted its inhibition on calpain activity. There may be two ways for phosphorylation to promote the inhibition of calpastatin on the activity of calpain. One is that phosphorylation exposes the domain on the calpastatin that plays an inhibitory role, promoting the binding of calpastatin and calpain and directly inhibiting its activity. The other is that phosphorylation keeps the structural stability of calpastatin, reduces the hydrolysis by calpain, and thus better displays its inhibitory activity. In general, phosphorylation of calpastatin negatively affects meat tenderization by inhibiting calpain activity. Inhibiting the phosphorylation level of calpastatin may be a new way to improve meat tenderness.


However, although the SK channel blocker apamin enhanced LTP induction, it did not fully mimic the effects of isoproterenol. We therefore searched for potential alternative mechanisms using liquid chromatography-tandem mass spectrometry to determine how β-AR activation regulates phosphorylation of postsynaptic density (PSD) proteins. Strikingly, β-AR activation regulated hundreds of phosphorylation sites in PSD proteins that have diverse roles in dendritic spine structure and function. Moreover, within the core scaffold machinery of the PSD, β-AR activation increased phosphorylation at several sites previously shown to be phosphorylated after LTP induction. Together, our results suggest that β-AR activation recruits a diverse set of signaling pathways that likely act in a concerted fashion to regulate LTP induction.Activation of β-adrenergic receptors (β-ARs) not only enhances learning and memory but also facilitates the induction of long-term potentiation (LTP), a form of synaptic plasticity involved in memory formation. To identify the mechanisms underlying β-AR-dependent forms of LTP we examined the effects of the β-AR agonist isoproterenol on LTP induction at excitatory synapses onto CA1 pyramidal cells in the ventral hippocampus. LTP induction at these synapses is inhibited by activation of SK-type K The noradrenergic regulation of memory formation is especially striking, as essentially every aspect of learning and memory, including acquisition, consolidation, retrieval, re-consolidation, and extinction is potently modulated by NE3. Although NE can modulate neuronal activity via activation of both α and β-adrenergic receptor (β-AR) subtypes, noradrenergic regulation of learning and memory importantly  involves β-ARs5. For example, β-AR antagonists inhibit the acquisition and/or consolidation of fear conditioning7, spatial learning8, and other forms of associative learning14. Inhibition of β-ARs also disrupts memory retrieval16, re-consolidation19, and extinction21. β-adrenergic receptor activation also enhances the induction and maintenance of activity-dependent forms of synaptic plasticity thought to be involved in memory formation, such as NMDA receptor (NMDAR)-dependent long-term potentiation (LTP)5. At many excitatory synapses, NE appears to act in strictly modulatory fashion. For example, β-AR activation can increase the potentiation induced by patterns of synaptic activity that are near threshold for LTP induction28 and facilitate the induction of long-lasting, protein synthesis-dependent forms of LTP by patterns of synaptic stimulation that normally induce shorter-lasting LTP30. However, at other synapses β-AR activation does not serve a strictly modulatory role but instead acts as an essential factor (in addition to coincident pre and postsynaptic activity) required for LTP induction. For example, β-AR activation is required for the induction of spike-timing-dependent LTP at excitatory synapses onto pyramidal cells in visual cortex33 and medial prefrontal cortex33. Moreover, the β-AR antagonist propranolol blocks high-frequency stimulation-induced LTP at excitatory synapses onto dentate gyrus granule cells35 and inhibits LTP induction in the CA1 region of the ventral hippocampus36.Noradrenergic neurons in the locus coeruleus project widely throughout the central nervous system and, through the release of the modulatory neurotransmitter norepinephrine (NE), regulate crucial aspects of brain function, including attention, emotional arousal, sensory processing, and memory formation5. Whether these same mechanisms also underlie the role of β-AR signaling at synapses where activation of these receptors is required for LTP induction is less well understood. Notably, in contrast to CA1 pyramidal cells in the dorsal hippocampus, the induction of LTP at Schaffer collateral (SC) synapses onto pyramidal cells in the ventral hippocampus is highly dependent on β-AR activation36. Thus, to investigate the signaling mechanisms underlying plasticity at synapses where activation of β-ARs has an especially prominent role in LTP induction we examined LTP induction and β-AR signaling at SC synapses in the ventral hippocampus. Importantly, at some synapses, including SC synapses in the ventral hippocampus37, LTP induction is suppressed by activation of SK-type calcium-activated potassium channels39. Because activation of β-ARs and PKA inhibits SK channel activity at excitatory synapses40, we initially investigated whether SK channels are a key synaptic target where β-adrenergic receptor signaling can act to facilitate LTP induction. Consistent with previous results36, we find that theta-frequency stimulation induced-LTP at SC synapses in the ventral hippocampus requires β-AR activation. However, the selective SK channel blocker apamin only partially mimicked the ability of β-AR activation to enable LTP induction at these synapses. Thus, to identify potential alternative mechanisms underlying β-AR-dependent forms of LTP we used liquid chromatography-tandem mass spectrometry (LC/MS–MS) to measure changes in phosphorylation of postsynaptic density (PSD) proteins induced by the β-AR selective agonist isoproterenol (ISO) in isolated CA1 regions obtained from ventral hippocampal slices. This analysis indicates that β-AR activation has a surprisingly widespread effect on PSD proteins, bi-directionally regulating phosphorylation at several hundred sites found in a wide variety of PSD proteins. Increases in phosphorylation triggered by β-AR activation are predicted to be mediated by multiple families of protein kinases and occur at sites within the core scaffold machinery of the PSD that partially overlap with sites phosphorylated following the induction of LTP39. Our results thus suggest that the ability of β-AR activation to regulate protein phosphorylation of the PSD protein interaction network (PIN) in a way that partially recapitulates changes that occur during LTP induction may importantly contribute to LTP induction in the ventral hippocampus.The modulation of LTP induction by β-AR activation is thought to importantly involve activation of protein kinase A (PKA) and the extracellular signal-regulated kinases ERK1/2 followed by phosphorylation-dependent changes in the activity of postsynaptic glutamate receptors, voltage-activated ion channels, and signaling pathways controlling protein synthesis and gene expression44 . Consistent with this, activation of SC synapses with a 30-s-long train of theta pulse stimulation (TPS) induced LTP at SC synapses in dorsal hippocampal slices maintained in-vitro but had no lasting effect on synaptic transmission at SC synapses in slices from the ventral hippocampus  and prolonged (3 min) trains of TPS delivered in the presence and absence of ISO. Both of these patterns of TPS had no lasting effect on synaptic strength when delivered in the absence of ISO but induced significant LTP when delivered in the presence of ISO  not only enabled the induction of LTP by 30 s of TPS  β2 and β4 subunits, decreasing phosphorylation of three sites in the α subunits of R-type channels (Cav2.3), and bidirectionally regulating multiple sites (increased phosphorylation at three sites and decreased phosphorylation at four sites) in α subunits of P/Q-type channels (Cav2.1).To gain insights into alternative signaling mechanisms that might contribute to β-AR-dependent forms of LTP, we used LC–MS/MS to identify changes in phosphorylation of PSD proteins induced by the β-AR agonist ISO in CA1 mini-slices obtained from the ventral hippocampus. We identified 3321 phosphorylation sites in PSD proteins of which 561(16.9%) were regulated following β-AR activation  have been implicated in the β-AR modulation of LTP inductionhttps://www.phosphosite.org). However, we found that only a very low percentage (10%) of quantitated p-sites have kinase-substrate pairs that have been previously assigned. Thus, we next used the Eukaryotic Linear Motif ELM resource49 and Networkin50 to predict protein kinases for each individual phosphorylation site, followed by manual curation of each phosphorylation site, to identify protein kinase consensus sequences surrounding the phosphorylation sites upregulated by β-AR activation , members of the SHANK family of proteins (Shank1-3), and the disk large-associated guanylate-associated (DLGAP) protein family (DLGAP1-4), along with the associated proteins SynGAP1 and Lrrc7 (Densin-180) . This suggests that β-adrenergic receptor activation might be regulating protein phosphorylation within a subset of phosphorylation sites previously found to be regulated by the induction of LTP, and specifically being modulated by CamKIIα activation.Previously, we found that the induction of LTP by high-frequency synaptic stimulation prominently regulates, and primarily increases, phosphorylation of scaffolding proteins in the PSD41. This increase in phosphorylation was mainly due to the CAMK and AGC families of protein kinases, in particular CaMKIIα/β as well as PKC, PKA, and AKT141. However, except for PKA, these protein kinases are not activated following β-AR activation. Indeed, β-AR activation induced changes in phosphorylation associated with decreased activity in multiple PKC isoforms as well as AKT1 , indicating CaMKIIα activity is not altered by β-AR activation. This suggests that PKA activation might be replacing CaMKII as a kinase for core components of the PSD scaffold machinery in β-AR-dependent forms of LTP.Previously, we found that the induction of LTP by high-frequency synaptic stimulation increases protein phosphorylation preferentially on highly connected components within the PSD protein interaction network (PIN)PIN Fig. A, B. MorPIN Fig. C. This sPIN Fig. . Interes36, suggesting that release of endogenous NE supports HFS-induced LTP at ventral SC synapses. Thus, NE release and activation of β-ARs appears to be an essential factor that, along with coincident pre- and postsynaptic activity, is required for LTP induction at SC fiber synapses onto CA1 pyramidal cells in the ventral hippocampus. In contrast, β-AR facilitates LTP induction in a more conventional, modulatory fashion at SC fiber synapses in the dorsal hippocampus36.Our results indicate that the induction of LTP by a diverse set of induction protocols, ranging in frequency from 5 to 100 Hz and in duration from a few seconds to 3 min, is highly β-AR dependent at SC fiber synapses onto CA1 pyramidal cells in the ventral hippocampus. Although HFS alone did induce some LTP at ventral SC fiber synapses, the amount of potentiation was smaller compared to that induced by HFS of SC fiber synapses in dorsal hippocampus Fig. C, D. Not37, we find that the facilitation of LTP induction by the SK channel blocker apamin is highly activity-dependent. Thus, in the presence of apamin LTP induction exhibits a pronounced, inverted-U shape dependence on TPS train duration  trains of TPS at SC fiber synapses in the dorsal hippocampus22. Unlike apamin, ISO enables the induction of LTP by all three TPS protocols at SC fiber synapses in the ventral hippocampus. This, and our analysis of PSD protein phosphorylation, suggests that β-AR signaling at excitatory synapses in the ventral CA1 region involves multiple downstream pathways that are able to interact with and modulate the distinct mechanisms underlying the induction of LTP by different patterns of TPS.Although activation of SK-type potassium channels suppresses TPS-induced in the ventral hippocampus53, AMPARs48, L-type voltage-gated Ca2+ channels54, and features important roles for the PKA adaptor protein AKAP555 as well as regulators of protein phosphatase signaling27. Consistent with these findings, we find that phosphorylation of all of these proteins is regulated by β-AR at excitatory synapses in the ventral CA1 region. However, the majority of phosphorylation sites in these proteins identified in our experiments do not correspond to sites previously implicated in the modulation of LTP induction by β-AR activation. For example, in addition to increasing phosphorylation of AMPAR GluA1 subunits at S845, a site thought to have a crucial role in the β-AR modulation of LTP induction48, β-AR activation also increased phosphorylation at sites in the cytoplasmic C-terminal tails of AMPAR GluA2 and GluA3 subunits . Interestingly, quantitative biochemical measurements of the AMPAR subunit phosphorylation have found that basal levels of GluA1 subunit phosphorylation at S845 in the hippocampus are vanishingly low, raising doubts about the role of GluA1 S845 phosphorylation in synaptic plasticity57 . Moreover, the potentiation of excitatory postsynaptic currents induced by β-AR activation in hippocampal pyramidal cells is disrupted in GluA3, but not GluA1, null mutant mice59. Thus, future experiments investigating the functional effects of the GluA2/3 subunit phosphorylation at the sites identified in our experiments may help clarify the role of AMPAR phosphorylation in β-AR-dependent forms of LTP. Although a β-AR-mediated increase in phosphorylation of L-type (Cav1.2) calcium channels at S1928 is thought to have a crucial role in theta-frequency stimulation-induced LTP54, our MS analysis did not detect this site. β-AR activation did, however, increase phosphorylation at numerous sites in L-type calcium channel β2 and β4 subunits. Moreover, β-AR activation induced extensive changes in phosphorylation of R-type (Cav2.3) and P/Q-type (Cav2.1) voltage-dependent calcium channels. Indeed, amongst all the ligand-gated and voltage-activated channels regulated by β-AR activation, voltage-activated calcium channels exhibited the most pronounced changes in phosphorylation. Thus, our findings are not only consistent with the notion at L-type calcium channels have an important role in β-AR-dependent forms of LTP54 but also suggest that modulation of other types of calcium channels may be important as well.Results from previous studies suggest that the facilitation of LTP induction by β-AR agonists involves phosphorylation-dependent modulation of NMDARs2+ influx via NMDARs61 and/or N-type voltage-dependent Ca2+ channels63 activates BK channels in neurons. Thus, during theta-frequency trains of synaptic activity, BK channel activation may oppose the strong dendritic spine depolarization needed to relieve the voltage-dependent Mg2+ block of NMDAR ion channels and induce LTP. Although the functional effects of the β-AR-regulated phosphorylation sites we identified in BK channels are unknown, the potential role of these channels in β-AR-dependent forms of LTP is an interesting question for future experiments. The bi-directional changes in phosphorylation induced at multiple sites in SynGAP1 and Lrrc7 (Densin-180) following β-AR activation are also intriguing. SynGAP1, a synaptic Ras GTPase activating protein, has a crucial role in synaptic plasticity65, as does the PSD scaffolding and CamKII binding protein Densin-18066. Interestingly, β-AR activation increased SynGAP1 phosphorylation at S1105. Phosphorylation of this site by CamKII during the induction of LTP is thought to trigger the translocation of SynGAP out of synapses, thereby enabling activation of small G proteins that have a crucial role in promoting changes in AMPAR trafficking and dendritic spine structure required for LTP51. β-AR activation also regulated phosphorylation of several proteins involved in small G protein signaling pathways responsible for plasticity of dendritic spine structure69, such as the NMDAR-associated Rho GTPase Arhgap32 (p250GAP)71 and the protein kinase PAK172. Our identification of these, and other, novel targets of β-AR signaling suggest that the mechanisms underlying the facilitation of LTP induction by β-AR activation are surprisingly diverse and regulate molecular processes involved in the plasticity of both dendritic spine structure and function. Moreover, these results are in line with the diversity of molecular and cellular functions thought to be involved in the induction and maintenance of LTP.Notably, we identified a number of novel phosphorylation sites regulated by β-AR activation. For example, although we did not detect phosphorylation of SK channels, our MS analysis did identify multiple phosphorylation sites in large-conductance BK-type potassium channels. Interestingly, Ca+ channels. This “single target”, PKA signaling-centric approach has merit, as it has provided several important insights into underlying mechanisms. Our MS analysis indicates, however, that β-AR activation regulates a large number of phosphorylation sites in PSD proteins with diverse functional and structural roles at synapses. Thus, rather than acting primarily via modulation of a single downstream target, it seems likely that changes in phosphorylation of multiple synaptic proteins collectively contribute to the ability of β-AR activation to enable LTP induction at SC fiber synapses onto ventral CA1 pyramidal cells. Moreover, β-AR activation not only leads to activation of PKA but also triggers activation of multiple PSD protein kinases, including PAK1, ERK1/2 and JNK3. β-AR activation also decreased phosphorylation sites that regulate SIK3, GSK3α, PKCα/β and PKG protein kinase activity. Thus, bidirectional regulation of multiple protein kinases may importantly contribute to the facilitation of LTP induction by β-AR activation. Notably, β-AR activation did not increase CamKIIα protein kinase activity in the ventral hippocampus. Thus, phosphorylation of AGC-type kinase sites likely primarily relies on PKA activation. Our results thus suggest that activation of β-ARs in the ventral hippocampus uses a distinct set of protein kinases to regulate core components of the PSD PIN. A number of these sites are localized within the core signaling machinery of the PSD PIN and many of them have been described to be essential for the induction of LTP. Therefore, the ability of β-AR activation to partially recapitulate changes in phosphorylation in the PSD PIN that occur during the induction of in LTP41 might importantly contribute to the crucial role of these receptors in the induction of β-AR-dependent forms of LTP. Notably, prenatal and juvenile stress induce long-lasting alterations in LTP induction and its modulation by β-AR activation in both the dorsal and ventral hippocampus74. In addition, dysregulation of noradrenergic signaling has been described in a number of psychiatric and neurodegenerative disorders, such as schizophrenia75, ADHD76 and Alzheimer’s disease77. Thus, the novel pathways and targets described here may also represent potential therapeutic targets for treating these disorders.Previous studies investigating the mechanisms underlying β-AR modulation of LTP induction have largely focused on examining the role of individual downstream targets modulated by PKA activation. Indeed, this was the approach we initially used to test the hypothesis that β-AR activation enables LTP induction by inhibiting SK-type KHippocampal slices were obtained from male, C57Bl/6N mice . Littermates were housed with no more than four mice/cage in a 12/12 light/dark cycle with food and water available ad libitum. Experiments and procedures were done in accordance with guidelines and regulations in the U.S. Public Health Service’s Policy on Humane Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the University of California, Los Angeles. Methods and experimental results are reported in accordance with ARRIVE guidelines.3, 1 mM NaH2PO4, 2 mM CaCl2, 1.2 mM MgSO4, and 10 mM glucose . After allowing 4 to 5 min for the brain to cool, hippocampi from both hemispheres were dissected from the rest of the brain and 400-µm-thick slices were cut using a manual tissue slicer. Slices were then transferred to an interface-type chamber perfused (2–3 ml/min) with warm (30 °C) ACSF and allowed to recover for a least 2 h. Slices obtained from the ventral third of the hippocampus were used in all experiments except for those shown in Figs. 42, TPS trains consisted of single pulses of presynaptic fiber stimulation delivered at 5 Hz. In some experiments we also induced LTP using a high-frequency stimulation (HFS) protocol consisting of two, one-second-long trains of 100 Hz stimulation delivered with an inter-train interval of 10 s. The average slope of fEPSPs recorded 40–45 min post-TPS or 55–60 min post-HFS  were used for statistical comparisons. Statistical significance for results from electrophysiological experiments were analyzed using Student t-tests or, where appropriate, one-way or two-way ANOVAs with Bonferroni post hoc comparisons. Statistical tests were performed using SigmaPlot 12.5  or Microsoft Excel. Results are reported as mean ± SEM and full results from statistical tests are provided in the figure legends.Mice were deeply anesthetized with isoflurane and, following cervical dislocation, the brain was removed and rapidly placed in cold (~ 4 °C), oxygenated 95% O2/5% CO2) artificial cerebrospinal fluid (ACSF) containing 124 mM NaCl, 4 mM KCl, 25 mM NaHCO/5% CO2 a41, we prepared isolated PSD fractions from these samples and, following enrichment of phosphopeptides with titanium dioxide (TiO2) chromatography, samples were analyzed using LC–MS/MS. Samples reconstituted in LC buffer A (0.1% formic acid in water), randomized, and then injected onto an EASY-nLC 1200 ultra-high-performance liquid chromatography coupled to a Q Exactive Plus quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). Peptides were separated by a reverse phase analytical column . Flow rate was set to 300 nL/min at a gradient from 3% LC buffer B  to 38% LC buffer B in 110 min, followed by a 10-min washing step to 85% LC buffer B. The maximum pressure was set to 1,180 bar, and column temperature was maintained at 50 °C. Peptides separated by the column were ionized at 2.4 kV in positive ion mode. MS1 survey scans were acquired at the resolution of 70,000 from 350 to 1800 m/z, with a maximum injection time of 100 ms and AGC target of 1e6. MS/MS fragmentation of the 14 most abundant ions were analyzed at a resolution of 17,500, AGC target 5e4, maximum injection time 65 ms, and normalized collision energy of 26. Dynamic exclusion was set to 30 s, and ions with charge + 1, + 7 and > + 7 were excluded.Slices from the ventral hippocampus were prepared as described above with the exception that following slicing the dentate gyrus, CA3 region, and the subiculum were removed to produce “mini-slices” containing just the CA1 region. CA1 mini-slices were then transferred to interface-type chambers perfused with ACSF. After allowing the slices to recover for 2 h, half of the mini-slices from each hippocampus were snap-frozen by transferring them into a pre-frozen microcentrifuge tube placed on a bed of crushed dry ice to serve as untreated control tissue. The remaining slices were collected in the same manner after a 10-min bath application of ACSF containing 1.0 µM ISO. For each experiment (n = 3), we pooled 10 CA1 mini-slices  for each condition. Using techniques described elsewhereMS/MS fragmentation spectra were searched with Proteome Discoverer SEQUEST  against in silico tryptic digested Uniprot all-reviewed Homo sapiens database . The maximum missed cleavages was set to two. Dynamic modifications were set to phosphorylation on serine, threonine, or tyrosine (+ 79.966 Da), oxidation on methionine (+ 15.995 Da), and acetylation on protein N-terminus (+ 42.011 Da). Carbamidomethylation on cysteine (+ 57.021 Da) was set as a fixed modification. The maximum parental mass error was set to 10 ppm, and the MS/MS mass tolerance was set to 0.02 Da. The false discovery threshold was set strictly to 0.01 using the Percolator Node. Individual phospho-site localization probabilities were determined by the ptmRS node, and phospho-sites with < 0.75 localization probability were removed. The relative abundance of phospho-peptides was calculated by integration of the area under the curve of the MS1 peaks using the Minora LFQ node in Proteome Discoverer. No data imputation was performed for missing values. Phospho-peptides were filtered so that each condition had at least two quantified values. Phospho-peptide intensities were then normalized by log2-transformation and sample median subtraction.Supplementary Tables.Supplementary Table 10.


Aristotelia chilensis), is pertinent. Being an understudied species, the biotechnological potential of maqui proteins is little understood. In the present study, we constructed a transcriptome of maqui plantlets using next-generation sequencing, in which we found six cystatin sequences. Five of them were cloned and recombinantly expressed. Inhibition assays were performed against papain and human cathepsins B and L. Maquicystatins can inhibit the proteases in nanomolar order, except MaquiCPIs 4 and 5, which inhibit cathepsin B in micromolar order. This suggests maquicystatins’ potential use for treating human diseases. In addition, since we previously demonstrated the efficacy of a sugarcane-derived cystatin to protect dental enamel, we tested the ability of MaquiCPI-3 to protect both dentin and enamel. Both were protected by this protein , suggesting its potential usage in dental products.Phytocystatins are proteinaceous competitive inhibitors of cysteine peptidases involved in physiological and defensive roles in plants. Their application as potential therapeutics for human disorders has been suggested, and the hunt for novel cystatin variants in different plants, such as maqui ( The cystatin superfamily of proteins is composed of reversible inhibitors of cysteine proteinases, mainly from the family C1A. They present a competitive mechanism, acting as a pseudo-substrate ,3. Its hCompared to animal cystatins, phytocystatins show high homology with members of the cystatin family, although they lack disulfide bonds as stefins. They also present an additional conserved motif, with an unknown function, in an N-terminal α-helix with the following sequence: [LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-X-[EDQV]-[HYFQ]-N . Some phPlant cystatins are thought to play a number of physiological roles, such as regulating endogenous cysteine peptidases during seed development and germination , senescePlasmodium falciparum , respectively. The assays were performed in triplicate, and the Ki parameters were obtained from the following equation -[AG]-[RE]-F-[AS]-V-[EDQ]-[EA]-[HYF]-N. The central loop motif (QxVxG) diverged between proteins with and without C-terminal extension, displaying the QVVAG sequence for the former and QVVSG for the latter. The dipeptide motif from the C-terminal loop (PW) is conserved in most cystatins, although MaquiCPI-2 has an alanine residue instead of a proline preceding the tryptophan.In all protein sequences, the GG dipeptide mot if is conserved in the N-terminal region. The typical phytocystatin motif in A. chilensis , and purification was successfully performed in a single step using affinity chromatography. SDS PAGE analysis (The ORFs from five of six cystatins (MaquiCPI-1 to MaquiCPI-5) were successfully obtained by PCR using specific primers and subcloned in a pET28a vector in frame with an N-terminal His-tag coding sequence. Expression was induced in analysis  revealedA. chilensis cystatin tested against the cys-proteases are presented in MaquiCPIs were tested against papain  and humap < 0.05), which show no significant differences between Elmex (22.30% ± 10.06), CaneCPI-5 (19.37% ± 10.41), 0.1 MaquiCPI-3 (19.93% ± 10.26), 0.25 MaquiCPI-3 (23.72% ± 9.74) and 1.0 MaquiCPI-3 (19.23% ± 12.01) (p < 0.05). In addition, the 0.50 MaquiCPI-3 treatment demonstrated the best protective effect against initial dental erosion (significantly), with the least surface microhardness change (2.90% ± 3.35) compared to all the other treatments (p < 0.05) (The %SMC results on the enamel surface show that the Control group presented a significantly greater surface microhardness change (37.56% ± 13.98) compared to the other treatments ( < 0.05) .p < 0.05), without a significant difference between each other . The treatments CaneCPI-5 (10.49% ± 5.57), 0.1 MaquiCPI-3 (14.87% ± 10.18), 0.25 MaquiCPI-3 (18.52% ± 10.33) and 0.50 MaquiCPI-3 (9.76% ± 10.93), which also did not differ significantly between each other (p < 0.05), presented the lowest surface microhardness change  and tested against different cysteine proteases. Additionally, one of them was tested to protect tooth dentin and enamel, a possible application of this protein.In this work, we identified six different maquicystatins sequences in a transcriptome of maqui plantlets cultivated in a Temporary Immersion Bioreactor (TIB). Five of them were cloned and recombinantly expressed in All deduced proteins share three conserved motifs that help them to interact with the active site cleft of cysteine proteases. These are a conserved glycine and a LARFAV motif in the N-terminal region, a very conserved QxVxG motif in a central loop of the protein and a second loop close to the C-terminal region presenting a tryptophan residue ,49. MaquArabidopsis thaliana event expressing OC-I targeted to the chloroplast. They evaluated its effect on photosynthesis and the accumulation of leaf pigments was evident, suggesting a retardation in senescence. This indicates that the cystatin targets in the chloroplasts might be related to pigment degradation and/or biosynthesis [The presence of putative signal peptides was also investigated. It was clear that MaquiCPI-1, MaquiCPI-3 and MaquiCPI-6 presented this classic sequence for secretion, suggesting they are extracellular. On the other hand, MaquiCPI-2 did not present a typical signal for secretion. However, after cloning and expression of the entire MaquiCPI-2 sequence, the expression was unsuccessful. This question led us to a deeper investigation of the first residues of the MaquiCPI-2 N-terminal sequence. Another analysis was made using the prediction software iPSORT , and thiynthesis . These rynthesis . A priorynthesis . TherefoIt is well known that signal peptides usually negatively interfere with recombinant protein expression . When thin tandem duplication, resulting in a carboxy-extended form, which is present only in plants. This extension underwent a process of neofunctionalization, culminating in the ability to inhibit legumain-like proteases (from the C13 family). In A. chilensis, these cystatins are represented by MaquiCPI-5 and MaquiCPI-6. Parallelly, the evolution of the remaining single-domain phytocystatin resulted in part of the current phytocystatins with introns. Genes with the same characteristics also originated from recent losses of the second domain of carboxy-extended phytocystatins. MaquiCPI-4 is an example that presents these attributes. Finally, a single-domain cystatin from the MRCA of flowering plants was retroduplicated. It resulted in intron loss and, consequently, in the emergence of intronless phytocystatins. Duplication and diversification events culminated in two distinct clusters. In maqui plants, one group is represented by MaquiCPI-1, and the other by MaquiCPI-2 and MaquiCPI-3.A phylogenetic analysis among MaquiCPIs was carried out, and three groups were identified. They share characteristics related to amino acid sequence extensions and the plant cystatin exclusive N-terminus alpha helix motif. The analysis was complemented with amino acid sequences from 12 rice cystatins, which are well characterized. It was observed that, despite the evolutionary distance among the species, only three groups were formed. This supports the evidence of three groups formed by MaquiCPIs, which is what happens in other species such as citrus, barley and turnip, as well as rice ,67,68,69E. coli culture. Although there are divergences in the methods of production, such as culture media, IPTG concentration and E. coli strain, it is clear that the quantity of MaquiCPI-3 produced is superior to most phytocystatins produced with this expression system . Maquicystatins 4 and 5 belong to the same phylogenetic group . This grThe difficulty of inhibiting cathepsin B is related to the existence of an occlusion loop in the enzyme that blocks the catalytic cleft, hampering their interaction . When cyA. chilensis cystatins were able to inhibit cathepsin L with satisfactory Ki values of 0.34, 0.33, 0.38, 0.57 and 1.25 nM, from MaquiCPI-1 to 5. These values indicate that all MaquiCPIs have the potential to control pathologies resulting from deregulated function or cellular quantities of this protease. As cathepsin L is associated with neurological problems such as Parkinson’s disease, urological issues such as proteinuria and even cancers [All of the  cancers ,80,81, i cancers .® and CaneCPI-5. The first is a highly effective commercial solution for controlling erosive tooth wear, containing fluoride and tin [® for enamel erosion in different protocols [® and CaneCPI-5). This is an important finding since, so far, Elmex® is the commercial product with the best results to protect against erosion. It is based on the combination of three inorganic components (Elmex®—SnCl2/NaF/AmF) [This study brought important results regarding the use of MaquiCPI-3 in the field of dentistry. Our in vitro protocol was designed to evaluate the protective effect and the best concentration of MaquiCPI-3 against an initial dental erosion process. In this sense, we used bovine enamel and dentin samples, which demonstrate a similar structure to human tooth samples . Additio and tin . The secrotocols ,88,89,90rotocols ,87,88,89rotocols ,59 and irotocols ,91, thisrotocols . Thus, wrotocols  and thatrotocols . It is iNaF/AmF) ,93, whil®. Although this commercial product is effective against enamel and dentin erosion, its protection is usually evidenced in protocols involving more severe erosive and abrasive challenges [® to occur.When evaluated for protection against dentin erosion, MaquiCPI-3 led to distinct results. This might be explained by the different composition of the dentin tissue. This layer, opposite to enamel, is composed of a large organic content (collagen) that, when demineralized, slows down the progression of erosion. However, this layer can be degraded by matrix metalloproteinases (MMPs) and cysteine cathepsins (CCs) , allowinallenges ,89,96. Mallenges . This, aAristotelia chilensis cultivated in Temporary Immersion Bioreactors. They present the three motifs that form the tripartite wedge as well as the alpha helix phytocystatin exclusive motif. Phylogenetically, they are distributed into three distinct groups, following the same pattern as rice and other species. Recombinantly expressed maquicystatins presented inhibitory activity in nanomolar order against papain and human cathepsins B (except MaquiCPIs 4 and 5) and L. Considering the limitations of an in vitro design of initial erosion, MaquiCPI-3 seems to be a promising agent for inclusion in dental products to protect against enamel (at 0.5 mg/mL) and dentin (at 0.1 mg/mL) erosion. Future studies employing protocols that more closely resemble the clinic, such as in situ design, shorter treatment times and prolonged erosive challenges, also associated with abrasive ones, are necessary to pave the way for the use of MaquiCPI-3 in preventive dentistry.In summary, we identified six different cystatins from a transcriptome of


As of 2022, 36 anti‐seizure medications (ASMs) have been licensed for the treatment of epilepsy, however, adverse effects (AEs) are commonly reported. Therefore, ASMs with a wide margin between therapeutic effects and AEs are preferred over ASMs that are associated with a narrow margin between efficacy and risk of AEs. E2730 was discovered using in vivo phenotypic screening and characterized as an uncompetitive, yet selective, inhibitor of γ‐aminobutyric acid (GABA) transporter 1 (GAT1). Here, we describe the preclinical characteristics of E2730.3H]E2730 binding assay. The GAT1‐selectivity over other GABA transporters was examined by GABA uptake assay of GAT1, GAT2, GAT3, or betaine/GABA transporter 1 (BGT‐1) stably expressing HEK293 cells. To further investigate the mechanism for E2730‐mediated inhibition of GAT1, in vivo microdialysis and in vitro GABA uptake assays were conducted under conditions of different GABA concentrations.Anti‐seizure effects of E2730 were evaluated in several animal models of epilepsy: corneal kindling, 6 Hz–44 mA psychomotor seizure, amygdala kindling, Fragile X syndrome, and Dravet syndrome models. Effects of E2730 on motor coordination were assessed in accelerating rotarod tests. The mechanism of action of E2730 was explored by [3H]E2730 binding on brain synaptosomal membrane was abolished in GAT1‐deficient mice, and E2730 selectively inhibited GAT1‐mediated GABA uptake over other GABA transporters. In addition, results of GABA uptake assays showed that E2730‐mediated inhibition of GAT1 positively correlated to the level of ambient GABA in vitro. E2730 also increased extracellular GABA concentration in hyperactivated conditions but not under basal levels in vivo.E2730 showed anti‐seizure effects in the assessed animal models with an approximately >20‐fold margin between efficacy and motor incoordination. [E2730 is a novel, selective, uncompetitive GAT1 inhibitor, which acts selectively under the condition of increasing synaptic activity, contributing to a wide margin between therapeutic effect and motor incoordination. E2730 was discovered by in vivo phenotypic screening as an ASM candidate with a wide margin between therapeutic and adverse effects.3H]E2730 binding assay in GAT1‐deficient mouse brain.GAT1 was identified as the target of E2730 according to E2730 for 120 minutes at 4°C. Non‐specific binding was defined as the residual binding observed in the presence of 1 mM unlabeled E2730. At the end of the incubation period, the synaptosome‐bound radioligand was recovered by rapid filtration through Whatman GF/B glass fiber filters pre‐soaked in 0.15% polyethyleneimine using a 24‐well cell harvester (Brandel). The filters were washed three times with ice‐cold 50 mM Tris–HCl buffer (pH 7.4) and incubated overnight in a scintillator (PerkinElmer), then the radioactivity was measured by a liquid scintillation counter (PerkinElmer).Synaptosomal membranes were prepared using the whole brain without cerebellum, brainstem, or olfactory bulb from Sprague Dawley rats  or human cerebral cortex based on Fuks et al.2.2.23H]E2730 and the following ASMs: carbamazepine ; lamotrigine ; diazepam ; gabapentin ; ethosuximide ; perampanel , retigabine ; levetiracetam ; acetazolamide ; sodium valproate ; zonisamide ; and topiramate . Unlabeled E2730 (0.1–10 μM) was also assessed to confirm a representative displacement. Non‐specific binding was defined as the residual binding observed in the presence of 1 mM unlabeled E2730. Radioactivity of the synaptosome‐bound radioligand was measured in the same way as described above.For the competitive displacement assay, rat synaptosomal membrane extract (0.2 mg protein/tube) was incubated for 120 minutes at 4°C with 20 nM [2.2.33H]E2730 for 120 minutes at 4°C. Non‐specific binding was defined as the residual binding observed in the presence of 1 mM unlabeled E2730. The radioactivity of the synaptosome‐bound radioligand was measured in the same way as described above.Synaptosomal membrane extract was prepared from homozygous γ‐aminobutyric acid (GABA) transporter 1 (GAT1) KO (Homo), heterozygous GAT1 KO (Hetero), and the littermate wild‐type (WT) mice  using the same approach as described above. Synaptosomal membrane extract (0.4 mg protein/tube) was incubated with 200 nM GABA, cells were incubated for 5 minutes at 37°C in a 5% CO2 atmosphere and then washed with cold HBSS. Washed cells were incubated in a scintillator for 1 hour at room temperature, followed by radioactivity measurements using a liquid scintillation counter.HEK293 cells stably expressing human GAT1 (hGAT1), GAT2 (hGAT2), GAT3 (hGAT3), or betaine/GABA transporter 1 (hBGT‐1) were plated onto 96‐well microtiter plates at a density of 4.0 × 102.3.24 cells/well in DMEM and incubated overnight at 37°C in a 5% CO2 atmosphere. After washing the cells with assay buffer, E2730 (0.0128–1000 μM), tiagabine , or vehicle was added to the plate in the presence of 1% DMSO. After adding [3H]GABA (0.01–500 μM), the plate was incubated for 5 minutes at 37°C in a 5% CO2 atmosphere. The radioactivity of the treated cells was assessed following the same method described above.hGAT1 stably expressing HEK293 cells were plated onto 96‐well microtiter plates at a density of 4.0 × 102.44, 2.5 mM CaCl2, 5.0 mM HEPES, pH 7.4) at a flow rate of 1.5 μL/min. After ≥160 minutes of perfusion, collection of the perfusate (30 μL/20 min/fraction) was initiated, and 12 fractions per animal were collected for 240 minutes. E2730 (10–100 mg/kg), tiagabine (3–30 mg/kg), or vehicle was orally administered 72 minutes after initiating sample collection. Tiagabine was used as a reference ASM of a non‐competitive GAT1 inhibitor. Perfusate was switched from Ringer solution to high‐potassium Ringer solution  after 127 minutes following the collection. GABA concentration in each sample was measured by a high‐performance liquid chromatography with tandem mass spectrometry method.A guide cannula was implanted in the left hippocampus  of ddY mice . After 5–7 days of recovery, a microdialysis probe (Eicom) was inserted into the left hippocampus through the guide cannula. The tip of the microdialysis probe reached 4.8 mm below the bregma. The next day, the probe was connected to a perfusion tube and perfused with Ringer solution  or SAS (SAS Institute Japan) was used for statistical analyses. A 33.150) of E2730 was 7.9 mg/kg in the corneal kindling mice, and the 50% toxic dose (TD50) was 350 mg/kg in the accelerating rotarod test (Table 50 to ED50) of E2730 was 44.3.We characterized the pharmacologic profile of E2730 Figure  in cornest Table . The pro3.2Fmr1 KO mice, and Scn1a+/− mice. E2730 was associated with dose‐dependent decreases in seizure frequency in these models. The ED50 of E2730 in the 6 Hz‐44 mA psychomotor seizure mice was 17 mg/kg  compared with vehicle (40.7°C)  mice at a maximum temperature of 42.5°C, irrespective of treatment with vehicle or E7230 20 mg/kg.The anti‐seizure effect of E2730 against multiple seizure types was further investigated in other epilepsy animal models, such as 6 Hz‐44 mA psychomotor seizures in mice, amygdala kindling rats, g Figure . In the g Figure . The ED5e Figure . In Scn1) Figure . No seiz3.3Bmax) and equilibrium dissociation constant (KD), using the radiolabeled [3H]E2730. The Bmax and KD of E2730 on synaptosomal membranes were 3419 fmol/mg protein and 553.4 nM, respectively, in rats  or voltage‐gated sodium channel (Nav) 1.2 (Nav1.2), both of which are major targets for approved ASMs  and 8.0% (Homo) relative to WT mice  values of E2730 were 1.1 μM for hGAT1, >1000 μM for hGAT2, >1000 μM for hGAT3, and 890 μM for hBGT‐1  receptor, N‐methyl‐D‐aspartate (NMDA) receptor, synaptic vesicle protein 2A (SV2A), or carbonic anhydrase.3H]E2730 in GAT1‐deficient mice were significantly lower than that of WT mice, suggesting that GAT1 could be the target of E2730. As high potassium levels, a known ictogenic stimulation, could induce excessive activation of GABA neurotransmission,50 in animal epilepsy models (7.9–17.1 mg/kg), did not affect the extracellular GABA concentration under basal level in the naïve mouse hippocampus but significantly increased the GABA concentration under high‐potassium‐induced hyper‐activation . In contrast, tiagabine increased the extracellular GABA concentration under both basal and hyper‐activated conditions. These unique characteristics of E2730 could explain its high protective index: E2730 selectively inhibited GAT1 under hyper‐activated condition without disrupting the physiological excitation and inhibition (E/I) balance, which could lead to undesired sedation. These results also suggest that E2730 inhibits GAT1 in an uncompetitive manner, while tiagabine inhibited GAT1 with similar potency irrespective of ambient GABA concentration  receptor.50 of 17 mg/kg. Amygdala kindling rats are a clinically relevant model of temporal lobe epilepsy.Notably, the uncompetitive inhibitor, which is defined as an inhibitor preferentially acting under the condition of higher levels of ligand, is unique among approved drugs with non‐competitive inhibition mode, which is defined as an inhibition pattern showing comparable potency no matter how much ligand is present. For instance, memantine, which is indicated for the treatment of moderate‐to‐severe dementia in Alzheimer's disease (AD), is an uncompetitive antagonist of the Fmr1 KO mice exhibit increased susceptibility to audiogenic seizures,Fmr1 KO mice, and our results showed that E2730 suppressed tonic–clonic seizures in this mouse model with an ED50 of 17.1 mg/kg. DS is a severe genetic epilepsy syndrome caused by loss‐of‐function mutations in SCN1A, a gene encoding the α1 subunit of the Nav1.1.Scn1a+/− mice recapitulate the characteristics of patients with DS.v1.1 is predominantly clustered in the axon initial segments of parvalbumin‐positive inhibitory interneurons, and loss of function of Nav1.1 in inhibitory interneurons results in E/I imbalance with the reduced inhibitory tone, leading to a greater seizure susceptibility in patients with DS compared with healthy people.AR, is effective in both patients and a mouse model of DS.Scn1a+/− mice, supporting our hypothesis that E2730 may be effective against DS by increasing GABAergic inhibitory tone.The anti‐seizure effect of E2730 for two orphan epilepsy syndromes, FXS and DS, was also evaluated in relevant animal models. In FXS, E/I imbalance caused by functional impairment of inhibitory GABAergic neurons has been reported.There is a technical limitation in the microdialysis assay, which measures extracellular concentrations and cannot distinguish synaptic concentrations from extrasynaptic concentrations. E2730 increases synaptic GABA concentration given that GAT1 is concentrated in presynaptic terminals.In conclusion, E2730 is a novel, selective, uncompetitive GAT1 inhibitor, and may be a potential ASM candidate with a wide margin between anti‐seizure effect and AEs  against multiple types of epilepsy, including focal epilepsy, pharmaco‐resistant epilepsy, and orphan epilepsy syndromes. Since the cause of epilepsy is not homogeneous, precision medicine based on causes of each patient is needed. For the precision medicine, a single MOA drug would be an advantage over a drug with multiple MOA because it is easy to customize a combination of drugs each of which has a different MOA related to one of the causes of a patient. Thus, E2730 would potentially provide an additional therapeutic option to treat epilepsy with impaired GABAergic functions.Kazuyuki Fukushima, Yuji Kazuta, Keisuke Hashimoto, Naoto Watanabe, Yoshiaki Furuya, Ting Wu, Takahisa Hanada, and Katsutoshi Ido are all employees of Eisai Co., Ltd. Hiroyuki Higashiyama, Yoshimasa Ito, and Takashi Kosasa were employees of Eisai Co., Ltd. during these works and now are retired. Delia M. Talos, Yeri Song, Nicholas S. Roberts, and Frances E. Jensen contributed to a part of these studies as sponsored research. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.Figure S1Click here for additional data file.Table S1Click here for additional data file.


Dear Editor,The clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) systems, including type II Cas9 and type V Cas12 systems, which serve in the adaptive immunity of prokaryotes against viruses, have been developed into genome-editing tools . Compareex vivo and in vivo via ribonucleoprotein (RNP) and lipid nanoliposomes (LNP), respectively, suggesting the excellent potential for therapeutic genome-editing applications.To address these limitations, we screened 10 natural Cas12i variants and found 1, xCas12i, with robust high activity in HEK293T cells. Engineering of xCas12i by arginine substitutions at the PAM-interacting (PI) REC and RuvC domains led to the production of a variant, high-fidelity Cas12Max (hfCas12Max), with significantly elevated editing activity and minimal off-target cleavage efficiency. In addition, we assessed the base editing efficiency of xCas12i-based base editor, and thus expanded the genome-editing toolbox. We further demonstrated that hfCas12Max could be an effective genome-editing tool In order to identify more Cas12i variants, we developed and employed a bioinformatics pipeline to annotate Cas12i proteins, CRISPR arrays, and predicted PAM preferences, and found 10 new CRISPR/Cas12i systems. To evaluate the activity of these Cas12i variants in mammalian cells, we designed a fluorescent reporter system that detected the increased enhanced green fluorescent protein (EGFP) signal intensity activated by Cas-mediated dsDNA cleavage or double-strand breaks . This syTTR  formation at these loci was measured 48 h after transfection using FACS and targeted deep sequencing  was shown to enhance the activity of the type V system . Combine xCas12i . To furt xCas12i .TTR   . Taken tTTR for various Cas nucleases. In total, editing activity was monitored 43 sites for hfCas12Max with TTN PAMs, 43 sites for ABR001 with TTN PAMs, 43 sites for Cas12i2HiFi with TTN PAMs, 45 sites for SpCas9 with NGG PAMs, 12 sites for LbCas12a with TTTN PAMs, 12 sites for Ultra AsCas12a with TTTN PAMs, and 20 sites for KKH-saCas9 with NNNRRT PAMs. Indel analysis showed that hfCas12Max exhibited an average efficiency of 70%, higher activity than other Cas nucleases, and comparable activity with Cas12Max . This was done by first introducing single mutations  in the conserved active site of xCas12i based on alignment to Cas12i1 and Cas12i2  and S10Bectively . The iniectively  and 1K. ectively . Among ta-TadA8e . At targSK9 site  and S13BSK9 site . We thenSK9 site . We nameSK9 site . In addiSK9 site . These r+ T cells  gene by gene and base editing  delivery and lipid nanoliposomes (LNP) delivery, respectively, demonstrating the great potential for therapeutic genome-editing applications.Through engineering, our Cas12i system has achieved high editing activity, high specificity, and a broad PAM range, comparable to SpCas9, and better than other Cas12 systems. Given its smaller size, short crRNA guide, and self-processing features , the typIn addition, we have confirmed that the type V-I Cas12i system can be used in base editing applications. For base editor, the dCas12i system shows high A-to-G editing at A9-A11 sites even A19 of KLF locus, and C-to-T editing at A7-A10 sites, which is similar to the dCas12a system but is distinct from the dCas9/nCas9 system. Comparable to dCas12a, dCas12i-BE exhibited higher base editing activity at KLF4, PCSK9, and DYRK1A loci , suggestin vivo or ex vivo therapeutic applications.In summary, the Cas12i system described here, which has robust editing activity and high specificity, is a versatile platform for genome editing or base editing in mammalian cells and could be useful in the future for https://doi.org/10.1093/procel/pwac052.The online version contains supplementary material available at pwac052_suppl_Supplementary_MaterialClick here for additional data file.pwac052_suppl_Supplementary_TablesClick here for additional data file.


Inhibition is a core component of executive functions but is not a unitary construct. Instead, different inhibitory processes have specific behavioral effects and neural bases. Three important inhibitory functions explored by the literature are 1) interference control ; 2) inhibition of prepotent responses and 3) inhibition of ongoing responses. These functions were described in the self-regulation theory as the possible main impairment in attention-deficit hyperactivity disorder (ADHD) and since then they have shown an association with several psychiatric disorders.The current study investigated the neural bases of interference control, inhibition of prepotent responses, and inhibition of ongoing responses as they were assessed by a Stroop-matching/stop-signal task developed by our group.The Stroop-matching/stop-signal employs different conditions to create the demands for each inhibition which allows the assessment of these functions using a single protocol. Brain activations were acquired using fNRIS in a block-design method. The concentration of oxygenated hemoglobin (HbO). The first level analysis of HbO signals used a general linear model (GLM) to estimate individual brain activations. The second level analysis was performed using a linear mixed model to generate brain activations at the group level. Alpha level = 0.05 and the false discovery rate was applied when necessary. The sample was composed of 25 young adults .task Interference control showed activation in the left and right temporoparietal junction (TPJ), the right dorsolateral prefrontal cortex (DPLFC), and inferior frontal gyrus (IFG); inhibition of prepotent responses showed increased activity in the right IFG and left DLPFC; the suppression of ongoing responses showed a deactivation of the IFG and DLPFC bilaterally.These results indicate that the three inhibitory functions assessed present distinct brain patterns of function. The lateralization role was evident in DLPFC and IFG activities and recruitment of parietal areas seems to be limited to interference control in this protocol. Also, the stop-signal demand led to the deactivation of areas associated with the resolution of the primary Stroop-matching task. This study elucidates the role of brain mechanisms associated with specific inhibitory processes that are impaired in psychiatric disorders such as ADHD.Financial support: FAPESP ; CAPES Proex ; Mackpesquisa; CNPq [grant 307443/2019-1]None Declared