Mangifera indica L.) is respiratory climacteric fruit that ripens and decomposes quickly following their harvest. 1‐methylcyclopropene (1‐MCP) is known to affect the ripening of fruit, delaying the decay of mango stored under ambient conditions. The objective of this study was to clarify the role of 1‐MCP in the regulation of ethylene biosynthesis and ethylene receptor gene expression in mango. 1‐MCP significantly inhibited the 1‐aminocyclopropane‐1‐carboxylic acid (ACC) content. The activity of ACC oxidase (ACO) increased on days 6, 8, and 10 of storage, whereas delayed ACC synthase (ACS) activity increased after day 4. The two homologous ethylene receptor genes, ETR1 and ERS1 , were obtained and deposited in GenBank®  (KY002681 and KY002682). The MiETR1 coding sequence was 2,220 bp and encoded 739 amino acids (aa). The MiERS1 coding sequence was 1,890 bp and encoded 629 aa, similar to ERS1 in other fruit. The tertiary structures of MiETR1 and MiERS1 were also predicted. MiERS1 lacks a receiver domain and shares a low homology with MiETR1 (44%). The expression of MiETR1 and MiERS1 mRNA was upregulated as the storage duration extended and reached the peak expression on day 6. Treatment with 1‐MCP significantly reduced the expression of MiETR1 on days 4, 6, and 10 and inhibited the expression of MiETR1 on days 2, 4, 6, and 10. These results indicated that MiETR1 and MiERS1 had important functions in ethylene signal transduction. Treatment with 1‐MCP might effectively prevent the biosynthesis of ethylene, as well as ethylene‐induced ripening and senescence. This study presents an innovative method for prolonging the storage life of mango after their harvest through the regulation of MiETR1 and MiERS1 expression.Mango ( In this study, role of 1‐Methylcyclopropene in the regulation of ethylene biosynthesis and ethylene receptor gene expression in mango fruits were studied. The work reports the role of ethylene receptor family members MiETR1 and MiERS1 in response to senescence related signaling in postharvest mango fruit. Amornputti, Ketsa, and Doorn  reportedand Ünlü  confirmeETR1 and ERS1 receptors in mango is currently limited. The shared common function and specific roles of ETR1 and ERS1 remain unknown. The role of 1‐MCP in the regulation of ethylene receptor gene expression has not been investigated in mango. Therefore, it is necessary to elucidate the mechanisms of ethylene receptor gene expression in response to treatment with 1‐MCP at the molecular level.Mango is a fruit with a great market value. However, the storage life of mango is short  of MiETR1 and MiERS1 was amplified, and analyses of the structures and sequences of the two genes were conducted. The biochemical characteristics of the MiETR1 and MiERS1 isoforms were studied in order to determine specific roles in ethylene signaling and investigate biochemical similarities and differences. In addition, the role of 1‐MCP in the regulation of the physiological characteristics and ethylene biosynthesis in mango was investigated in order to decipher the underlying mechanism of 1‐MCP in delaying senescence. This study presents an innovative method for prolonging the storage life of mango after their harvest through the regulation of the expression of MiETR1 and MiERS1.Regulation of ethylene biosynthesis has an impact on ripening and senescence of mango fruit. The aim of this study was to clarify the role of 1‐MCP in the regulation of ethylene biosynthesis and ethylene receptor gene expression in mango after their harvest. In particular, the role of 1‐MCP in the regulation of 22.1Mangifera indica L.) were harvested from an orchard in Tianyang, Baise City, Guangxi Province, China), in July 2016. Fruits with a similar shape, size, and physiological maturity were selected as experimental materials. These fruits were transported to the Guangxi Key Laboratory of Fruits and Vegetables Storage‐Processing Technology  immediately after harvest and were randomly classified into two groups (60 fruits per group). 1‐MCP (Beijing PLM Biosciences Co.) was purchased in a powder form and dissolved in sterile distilled water at a final concentration of 1 μl/L. One group was placed in polyethylene bags (0.03 mm thick) containing 1 μl/L 1‐MCP. The other group was treated with the same volume of sterile water only (control group). The fruits were treated with 1‐MCP for 24 hr at 25°C and subsequently stored at 25°C and were then collected every two days, frozen in liquid nitrogen, and stored at −80°C until analysis. Three replicates per analysis were used for all measurements.Experimental cultivar “Tainong 1” mango fruit  with an 8 mm diameter probe. Following the removal of a small piece of fruit skin, the firmness from three slices extracted from three different parts of the mango was recorded (means are presented in newtons [N]). The total soluble solid (TSS) content of the fruit was examined on the basis of the AOAC method  using a handheld refractometer . Mango fruit juice was titrated with 0.1 N NaOH to pH 8.2 in order to determine the titratable acidity (TA) using an automatic titrator .2.32 and subsequently stored at 20°C with 90% relative humidity. Three fruits were placed in a 4.2  L airproof glass jar for 2 hr at 25°C to determine the rates of ethylene production at different storage stages. A headspace gas sample (1 ml) was collected from each jar and analyzed using a GC‐2014C gas chromatograph . Subsequently, a thermal conductivity detector (Shimadzu TCD‐2014) with Porapak N column was used to detect the concentration of carbon dioxide in the samples. The levels of ethylene were determined using a flame ionization detector and an OV‐17 capillary column (Zhonghuida Co.) . The remaining pellets were resuspended in distilled water, and the ACC content was determined on the basis of the method described by Boller, Herner, and Kende  ethanol at 4°C. The homogenates were centrifuged for 10 min at 10,000nd Kende  and the nd Kende . The extnd Kende .2.5® ND‐1000 UV‐Vis spectrophotometer (NanoDrop Technologies Inc.) at 260 nm. A 2,100 Bioanalyzer system (Agilent Technologies) was used to evaluate RNA integrity.Total RNA was isolated from 100 mg of fresh mango tissue pulverized using liquid nitrogen. RNA was isolated on the basis of the method described by López‐Gómez and Gómez‐Lim . An RNAp2.6MiETR1 and MiERS1 were acquired from the combination of RT‐PCR and rapid amplification of cDNA ends (RACE) cloning. RNA (10 µg) was used to synthesize first‐strand cDNA. All primers and reagents in cloning were purchased from the PowerScript™ MMLV reverse transcriptase (Clontech). The degenerate primers of the MiETR1 conservative region, PD‐cons1 (5′‐GCCCTGATGCTGGTGCAYATHATHCC‐3′) and PD‐cons1 (5′‐GGTTCATCACGGCCARRAARTCRTT‐3′), were designed according to the homologous sequence of Arabidopsis thaliana (accession no. NP_176808.3). The specific primers of the MiERS1 conservative region, PD‐cons1 (5′‐ATGCCTTCAAGAACTGGTATCAC‐3′) and PD‐cons1 (5′‐CAAGTCCTGCTCCACCTGTA‐3′), were also designed on the basis of the homologous sequence of Arabidopsis thaliana (accession no. NP_181626.1). The PCR conditions were as follows: 94°C for 4 min; 94°C for 30 s, 53°C for 30 s, and 72°C for 90 s following 35 cycles; and 72°C for 6 min. The PCR products were used for isolation, cloning, and sequencing (Invitrogen). The 5′‐ and 3′‐RACE fragments were cloned using the SMART™ RACE cDNA Amplification Kit (Clontech) according to the manufacturer's instructions. The cDNA was directly utilized in 5′‐ and 3′‐RACE PCR, whereas second‐strand synthesis and adaptor ligation were not required. The 5′‐ and 3′‐RACE PCR specific primer pairs of MiETR1  were designed as the forward primer according to the sequence of the conservative region of MiETR1. The 5′‐ and 3′‐RACE PCR specific primer pairs of MiERS1  were designed as the forward primer according to the sequence of the conservative region of MiERS1. The conditions of RACE PCR were as follows: 94°C for 3 min; 94°C for 30 s, 72°C for 3 min following 5 cycles; 94°C for 30 s, 70°C for 3 min following five cycles; 94°C for 30 s, 68°C for 3 min following 25 cycles; and 68°C for 3 min. The products of 5′‐ and 3′‐RACE PCR were purified and cloned into a pMD18‐T vector (Takara Bio) for sequencing . The putative domains of MiETR1 and MiERS1 were predicted using the SWISS‐MODEL (http://swissmodel.expasy.org/) with first approach and project (optimize) modes with default value. The Swiss‐PdbViewer  was used for the visualization of structures. ClustalX v.1.81  was used to align the amino acid sequences on the basis of the default settings and then refined through visual inspection. Subsequently, the minimum evolution method of the MEGA software  was used to generate a phylogenetic tree based on the result of the alignment. Poisson's correction metric and pairwise deletion option were used for the analysis. The reliability of the tree branches was inspected by bootstrap statistical method, which created from 1,000 replicates.Sequence alignment, translation of open reading frame (ORF), and calculation of the molecular mass of the predicted proteins were conducted using the DNAMAN v. 6.0.3.99 software and upload to the server of NCBI‐NIH (2.8MiETR1 gene‐specific primers were as follows: MiETR1F (5′‐CCTACAACTTCAACTCGGAACTT‐3′) and MiETR1R (5′‐TTCATCACCAACAGCATACTCAG‐3′). The sequences designed for the MiERS1 gene‐specific primers were as follows: MiERS1F (5′‐GTATTCTGCCACAAGACATTCCA‐3′) and MiERS1R (5′‐TCAAGACCTTCACTCTCAATCCA‐3′). The mango actin gene was used as an internal control with the following primers: MiactF (5′‐GTGGCTGTTAACGATCCCTT‐3′) and MiactR (5′‐GTGACTGGCTTCTCATCGAA‐3′). The synthesized cDNA was amplified as described earlier. RT‐qPCR was performed using the UltraSYBR Mixture  in a SmartCycler® system (Cepheid). Relative gene expression was calculated using the comparative Ct method (ΔΔCt)  assay. The sequences designed for the 2.9n = 3) with a completely randomized design. All data are presented as mean ± standard error (SE). Fisher's least significant difference test was performed using one‐way analysis of variance by SPSS software  with a significant value of p < .05.All experiments were performed in triplicate (33.1p < .05) in the 1‐MCP‐treated group compared with that observed in the control group. The TSS content in the pulp tissues of both groups was shown to increase as the storage duration extended . The TA content in the pulp tissues of both groups decreased during the 16‐day storage. However, the TA content in the 1‐MCP‐treated group decreased more slowly than that observed in the control group after four days of storage . Production of ethylene in the 1‐MCP‐treated group peaked on day 10 of storage, two days later compared with the control group. These results indicated that treatment of fruit with 1‐MCP after harvest may suppress the climacteric production of ethylene. An increased respiration rate may be associated with fruit senescence and the development of disease during fruit storage. In this study, the respiration rate of mango showed a typical climacteric pattern under ambient storage . In the 1‐MCP‐treated group, peak respiration was reached more slowly than in the control group, indicating that treatment with 1‐MCP reduced the respiration of mango.Ethylene is naturally produced in postharvest mango and quickly leads to overripening. Initially, the rates of ethylene production in both groups showed an increasing trend, followed by a slow decline. Treatment with 1‐MCP significantly prevented the production of ethylene after two days of storage  inhibited the increase in the ACC content on days 6, 8, and 10. In the 1‐MCP‐treated group, the ACC content peaked on day 12 of storage, two days later compared with the control group. The activity of ACO increased during the first ten days of storage and declined afterward . The same trend was observed in the activity of ACS in both groups . These results confirmed that treatment with 1‐MCP inhibited the production of ethylene. This effect was closely related to the ACC content and the activities of ACO and ACS.In both groups, the ACC content was initially increased, followed by a decrease throughout the entire storage duration Figure a. Treatmd Figure b in boths Figure c. In the3.3MiETR1 gene was 2,570 bp in length with a 113 bp 3′‐untranslated region, a poly‐A tail, and an ORF of 2,220 bp encoding 739 aa. The deduced protein had a molecular mass of 86,385 Da and a 9.09 isoelectric point. The 5′‐ and 3′‐noncoding sequences were 164 and 309 nucleotides in length, respectively. The MiERS1 gene was 2,171 bp in length and encoded 629 aa, with a predicted molecular weight of 80,518 Da and an isoelectric point of 6.34. The 5′‐ and 3′‐noncoding sequences were 102 and 179 nucleotides in length, respectively, with a poly‐A tail and 1,890 bp ORF. The sequences of MiETR1 and MiERS1 were uploaded to GenBank® (NCBI‐NIH) with accession numbers KY002681 and KY002682.The full‐length cDNAs of two ethylene receptor genes in mango were obtained Figure . The MiE3.4://www.expasy.org). The results revealed that the putative MiETR1 peptide was composed of 24.37% alpha helix, 9.97% beta turn, and 42.93% random coil. The MiERS1 peptide consisted of 43.45% alpha helix, 7.52% beta turn, and 29.94% random coil. The major part of the secondary structure of MiETR1 was random coil, whereas the alpha helix was the major segment of the MiERS1 secondary structure. In addition, both the N‐ and C‐terminal parts were alpha helices. The tertiary structural models of MiETR1 and MiERS1 were built on the basis of the homology of proteins with known crystalline structures using protein modeling software (SWISS‐MODEL and 3D‐JIGSAW) , belonged to the ETR1 subgroup Figure . All proMiETR1 and MiERS1 sequences with other related ETRs and ERSs deposited in GenBank® (NCBI‐NIH)  with ETRs from Citrus sinensis (KDO50509.1) and Citrus clementina (XP006442239.1). A relatively high similarity was found for DNA sequences of ETRs from Arabidopsis thaliana (NP176808.3) (85%) and Glycine max (XP006592983.1) (83%). MiERS1 shared the highest homology (91%) with ERSs from Citrus sinensis (AAC99435.1) and Citrus clementina (XP006444412.1). A relatively high similarity was found for ERS DNA sequences from Arabidopsis thaliana (NP181626.1) (73%) and Glycine max (XP006604726.1) (75%).A phylogenetic tree was generated using ) Figure . As show3.5MiETR1 and MiERS1 mRNA in postharvest mango was further analyzed. Real‐time PCR using gene‐specific primers was used to explore the expression patterns of MiETR1 and MiERS1 genes. In both groups, the expression of MiETR1 mRNA was shown to be upregulated as the storage duration extended  . Similar to MiETR1, the MiERS1 mRNA levels were found to be upregulated as the storage duration extended and reached its maximum on day 6 (53.2‐fold higher levels than those recorded in the control group on day 0) . These results indicated that MiETR1 and MiERS1 may have the ability to combine the putative ethylene receptors with ethylene. Treatment with 1‐MCP prohibited the binding of ethylene to the ethylene receptor site and protein.The relationship between the climacteric production of ethylene and the levels of d Figure a. The ex) Figure b. The Mi4In climacteric fruit, ethylene regulates various and complex responses during growth and development, including ripening and senescence. 1‐MCP is an inhibitor of ethylene perception in plant tissues. Treatment with 1‐MCP delayed senescence in mango by inhibiting enzymatic activity (ACS and ACO) involved in ethylene biosynthesis. In the present study, treatment with 1‐MCP markedly delayed the reduction in firmness and TSS content Figure b as wellMiETR1 and MiERS1, were extracted from mango and found to be similar to those identified in the corresponding genes of Citrus sinensis, Citrus clementina, Arabidopsis thaliana, and Glycine max  expression in mango. This study presents an innovative method for prolonging the storage life of harvest mango through the regulation of expression. Further research is warranted to understand the roles of each member of the ETR family in the process of senescence in mango. The molecular mechanism of ethylene response to regulation through ethylene receptor isoforms in mango remains unclear. In addition, the genetic and physiological characterization of the ETR family is necessary to better understand its function and role in regulating senescence in mango.Treatment with 1‐MCP was effective in delaying the evolution of mango postharvest ripening, including firmness, TSS and TA. 1‐MCP treatment inhibited the production of ethylene in fruit, which was correlated with the ACC content and the activity of ACO and ACS. In addition, MiETR1 and MiERS1 have important functions in ethylene signal transduction. Altogether, these results indicated that treatment with 1‐MCP effectively prevented ethylene‐induced ripening and senescence by regulation of ethylene biosynthesis and ethylene receptor gene (The authors notify that there are no conflicts of interest.This study does not involve any human or animal testing.


We aimed firstly to evaluate knowledge, attitudes, and practices about epilepsy among primary and secondary school teachers in the city of Niamey and secondly to formulate targeted sensitization messages for these teachers.A descriptive cross‐sectional survey was carried out among primary and secondary school teachers in the city of Niamey, capital of Niger, using a self‐administered questionnaire including questions related to knowledge, attitudes, and practices about epilepsy.One hundred and forty‐five (145) teachers aged 27–69 (mean age: 39.57 ± 8.304 years) had answered the questionnaire (52 men and 93 women). In 62.1% of cases, respondents had a bachelor degree in education signifying a high level of education. Sixty‐one respondents (42.1%) considered epilepsy as a brain disease, whereas it was considered as a psychiatric illness by 15.9% of respondents and as an impurity by 38.9%. Sixteen respondents (11%) considered it as a hereditary disease. Epilepsy was considered as a contagious disease by 46.2% of teachers, and the main transmission routes reported were contact with places of crisis (26.2%), physical contact with an epileptic person (16.6%), and contact with saliva (6.9%). It was considered as an incurable disease by 6.9% of teachers. Of the 115 respondents (79.3%) who considered epilepsy as a treatable condition, 46 of them believe that epilepsy is treated by traditional medicine. When someone has an epileptic seizure, 28.3% of respondents think that physical contact with him should be avoided and that the places of the crisis should be avoided.Considering that epilepsy is common in school‐age, the study results suggest the need to train teachers with respect to epilepsy to change misconceptions about epilepsy and to promote positive attitudes toward epileptic people to avoid the rejection of these people by the society responsible for problems of social integration. The study evaluates knowledge, attitudes, and practices about epilepsy among school teachers of primary and secondary schools in the city of Niamey, capital of Niger. The study results revealed a low level of general knowledge about epilepsy among respondents, of whom only 42.1% think that epilepsy is a brain disease. 15.9% and 46.2% of the respondents considered epilepsy to be a psychiatric illness and a contagious disease, respectively. 6.9% of the respondents considered epilepsy to be an incurable disease. The qualitative variables were expressed as percentages and the quantitative variables as mean ± standard deviation. The chi‐square test of Pearson was used to compare the proportions of the qualitative variables. 33.1Table 3.23.2.1All participants had previously ever heard about epilepsy, among whom 19 (13.1%) had already attended at least an epileptic seizure. Twenty‐two (15.2%) had heard about epilepsy from a parent or relative of an epileptic person. Thirty‐eight participants (26.2%) had heard about epilepsy of their colleagues or friends, sixteen (11%) of their elders , sixteen (11%) through the media, and five (3.4%) through health workers. Twenty‐nine participants (20%) did not specify their information source.3.2.2Epilepsy means in Hausa: “Falfadiya” or “Borin jaki” or “Mabougui” or “Shigan sandaji.” These terms vary according to the regions of Niger and signify: “sickness who makes fall with the loss of consciousness” or “insanity of the donkey.” Epilepsy means in Zarma: “Tchouro‐tchouro” or “Karga‐fourou” signifying “insanity of the guineafowl” or “agony crisis of the bird.”3.2.3One hundred and seven participants (73.8%) had given at least one sign suggestive of an epileptic seizure. The most frequent answers were as follows: “the person falls and makes tremors crisis”; “the person falls and only gets up after urinating”; “the person falls and becomes stiff”; “violent convulsions accompanied by coma”; “convulsions”; “the saliva comes out of the mouth of the patient in quantity, and it throws a little everywhere on the ground”; “tremors and shaking with loss of the consciousness”; “loss of the consciousness and convulsions”; “crisis of the movements of the members of the whole body”; “loss of the consciousness, rejection of gas and urine with the bite of the tongue”; “the person falls and faints”; “loss of the consciousness.”Eighteen participants (12.4%) had reported signs that did not evoke an epileptic seizure. The most frequent answers were as follows: “high fever”; “fever and headaches”; “strong agitation”; “agitation and mental disorders”; “loss of balance and fall”; “faintness.”Twenty participants (13.8%) did not answer the question.3.2.4Table p = .024) and women (p < .001) (Table p = .006). Teachers aged 40–49 years believe that epilepsy is transmitted by breathing the gas emitted during the seizure (p = .014) and men think that transmission is done by eating foods touched by an epileptic person (p = .008). We found no relationship between teachers' religious beliefs and their knowledge about epilepsy (Table p = .003), while primary school teachers believe that epilepsy is transmitted by contact with the places of the crisis (p < .001). Primary school teachers considered epilepsy as a brain disease (p < .001) while college teachers thought of witchcraft (p = .003) and lyceum teachers an impurity (p < .001).Epilepsy is essentially considered to be a brain disease by teachers aged 30–39 years p = .02 and wome3.33.3.1The most frequent answers are presented.“I'm afraid I prefer to get away from the patient (14.5%).” “Pour him the water on the head (3.4%).” “Nothing, I observe (11%).” “Burn the places after the crisis (2.1%).” “Maintain the person on the side until the crisis passes (14.5%).” “Avoid to approach him and not touch the places of the crisis (2.1%).” “Withdrawal dangerous objects around the person (17.9%).” “Call for help (3.4%).” “Transport the person to the hospital (3.4%).”3.3.2The most frequent answers are presented.“Physical contact with the patient, contact with the places of the crisis (28.3%).” “Contact with the patient's saliva (2.7%).” “Abandon the patient (4.8%).” “Put something in the patient's mouth (4.8%).” “Make noises around the patient (6.2%).” “Leave dangerous objects around the patient (7.6%).”3.4The most frequent answers are presented.“Do not let epileptic persons drive, walk around water and fire (2.1%).” “Make a treatment by modern medicine (6.9%).” “Morally support people with epilepsy and avoid stigma (4.1%).” “Do not let the person walk long alone in the city (2.1%).”4p > .2). In the same study, 42% of respondents considered epilepsy to be a contagious disease, whereas in the present study 46.2% of teachers think that epilepsy is contagious (p > .2). In other African studies conducted among school teachers, epilepsy is considered as a contagious disease in 28.2–39% of teachers surveyed and in 22.8–53.4% considered as a brain disease , in Mali (34.8%), in Jordan (52%), in India (21.4%), in South Korea (22%), and to that reported in a previous study from Niger (35%)  and lower than that found in teachers in Burkina Faso (40%) (Maiga et al., At the end of the survey, school teachers' responses demonstrate that they were less knowledgeable about epilepsy. Many school teachers emphasized the impact of negative religious and cultural beliefs about epilepsy in their attitude toward people with epilepsy. Besides, many school teachers had shown their satisfaction with the information provided concerning epilepsy, and some of them would like to have more information on this superstitious and mythical disease. Therefore, this underlines the need to integrate well‐targeted information modules on epilepsy as well as the adapted gestures to perform when a child has an epileptic seizure. School teachers underlined the interest of stopping to send at home the students with epilepsy and helping them in the school learning and social integration by changing the negative attitudes of students without medical conditions and promoting positive attitudes toward students with epilepsy. Considering that epilepsy is a common disease in school‐age, it is very important to minimize the negative consequences of this disease in a school environment because it can lead to problems of schooling for children with epilepsy.5In this study assessing knowledge, attitudes, and practices about epilepsy, we find a low overall level of knowledge about this condition among school teachers in the city of Niamey. The study results encourage special attention in the epileptic children in school‐age to avoid the rejection of these children by the society responsible for problems of social integration. This low level of general knowledge about epilepsy among school teachers, knowing that epilepsy is common among school‐age children, justifies the need to integrate into the training modules of the future school teachers information modules related to epilepsy to enable them to acquire know‐how about epilepsy. It is also necessary to organize continuing education seminars for teachers already in post. The study recommends further research to evaluate knowledge, practices, and attitudes regarding epilepsy among school teachers of other cities of Niger and at the same opportunity to address targeted awareness messages for these teachers. The study also recommends conducting research on knowledge, practices, and attitudes of health workers. The study also recommends public education about epilepsy using possible avenues such as the integration of information modules regarding epilepsy into the national education program, the mass media especially radio and television which are an important source of information in most developing countries, and awareness campaigns to change any misconceptions about epilepsy and to promote positive attitudes toward people with epilepsy.All authors have read and agreed to the content of the manuscript. None of the authors has any conflict of interest to declare. The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. We confirm that this manuscript has not previously been published and is not being considered for publication by another journal. The authors alone are responsible for the content and writing of the paper.


Number of patients suffering from diabetes is also increasing in Asian countries where people consume rice as a staple food, hence generation of practically growable high RS rice line has been anticipated. It is known that suppression of starch branching enzyme (BE) IIb increases RS content in cereals. To further increase RS content and for more practical use, we generated a non-transgenic be1 be2b mutant showed a decrease in intermediate amylopectin chains and an increase in long amylopectin chains compared with be2b. The amylose content of be1 be2b mutant (51.7%) was the highest among all pre-existing non-transgenic rice lines. To understand the effects of chewing cooked rice and cooking rice flour on RS content, RS content of mashed and un-mashed cooked rice as well as raw and gelatinized rice flour were measured using be1 be2b and its parent mutant lines. The RS contents of mashed cooked rice and raw rice flour of be1 be2b mutant  were 3-fold higher than those of be2b mutant. Gel-filtration analyses of starch treated with digestive enzymes showed that the RS in be1 be2b mutant was composed of the degradation products of amylose and long amylopectin chains. Seed weight of be1 be2b mutant was approximately 60% of the wild type and rather heavier than that of be2b mutant.The be1 be2b double mutant rice were enriched with long amylopectin chains. This led to a great increase in RS content in cooked rice grains and rice flour in be1 be2b compared with be2b single mutant. be1 be2b generated in this study must serve as a good material for an ultra-high RS rice cultivar.The endosperm starch in The online version contains supplementary material available at 10.1186/s12284-020-00441-0. Starch is produced from photosynthetic products through biological reactions and serves as an important carbohydrate source for heterotrophs. Starch is utilized not only in food industries but also in technological and medical industries, and the applications of starch are expected to expand further in future. Starch is of two types: one type is easily degraded by digestive enzymes, while the other type, known as resistant starch (RS), is less susceptible to digestive enzymes, travels to the colon as a high molecular weight of partially digested starch, and functions similar to fiber  and maize (Zea mays): BEI, BEIIa, and BEIIb. Among these isozymes, BEIIb play a major role in amylopectin branching and is strongly expressed in endosperm and kernels  seeds  = 6–12  activity is accompanied by the loss of BEIIb in rice   and intermediate chains (DP = 12–21), and a slight increase in short amylopectin chains (DP < 10), suggesting that BEI is involved in the generation of branch points of B1, B2, and B3 chains    has higher amylose content than the rice be2b mutant     and RS (14.6%) than the WT , compared with the down-regulation of BEIIb alone   , its parental single mutants be1 (EM557) and be2b (EM10), and WT cultivars (Taichung 65 and Kinmaze), and BEI and BEIIb proteins were detected by western blotting  lead to the deficiencies of corresponding proteins.Total proteins were extracted from the be1 single mutant were translucent, while those of the be2b single mutant and be1 be2b double mutant were opaque . On the other hand, the seed weight of be2b was 56.9% (11.1 mg) of the WT (Kinmaze), and that of the be1 be2b double mutant (12.2 mg) was 62.6% and 57.0% of Kinmaze and Taichung 65, respectively, which were regarded as WT. The seed weight of be1 be2b was significantly heavier than that of be2b  was added as an internal standard (2θ = 28.3°). There were no significant differences in RSC among rice lines by Tukey-Kramer method (P < 0.05)  Fig. .Fig. 3Pbe1 were 5.4 °C, 4.2 °C, and 3.2 °C lower, respectively, than those of the parental WT cultivar Taichung 65; by contrast, the corresponding values of be2b were 6.8 °C, 16.3 °C, and 23.8 °C higher, respectively, than those of the parental WT cultivar Kinmaze. These values are consistent with previous studies  was slightly less than that of Taichung 65 (11.3 mJ mg− 1), whereas that of be2b (17.6 mJ mg− 1) was considerably greater than that of Kinmaze (9.6 mJ mg− 1). By contrast, the required thermal energy of be1 be2b (7.1 mJ mg− 1) was much less than that of be2b (Table be1 be2b was higher than that of be2b (described below).The thermal properties of starch were analyzed by DSC Table . The onsbe1 be2b, parental mutant lines, and the WT, we analyzed the chain length distribution of amylopectin by capillary electrophoresis  showed an increase in DP 6–10 and DP 24–36, and a minor decrease in DP 37–54  showed a considerable decrease in DP 6–13 and an increase in DP > 14 with broad peaks at DP 19 and DP 45  also showed a great decrease in DP 6–16 and an increase in DP > 17, with broad peaks at DP 21 and DP 47  were compared, that of be1 be2b at DP > 10 shifted toward the longer side. Notably, be1 be2b showed less chains with DP 10–20 but more chains with DP > 21 than be2b  was less than in other lines including Taichung 65 (1.8%), Kinmaze (2.5%), be1 (2.0%), and be2b (2.0%) (Table be1 be2b was extremely high (50.5%). The ratio of short- to long-chain of amylopectin (fraction III/II) was approximately 3.0 (starch or amylopectin) in Taichung 65, Kinmaze, and be1; however, this value was 1.0 (starch) or 0.9 (amylopectin) in be2b and 0.5 (starch) or 0.6 (amylopectin) in be1 be2b, indicating that the content of long-chains of amylopectin was the highest in be1 be2b, followed by be2b.To determine the apparent amylose content, true amylose content, and the extra-long chain content, and the ratio of long- to short-chains of amylopectin, we analyzed the debranched purified starch and isolated amylopectin by gel filtration chromatography Fig. S; the per1 be2b 1.% was lesbe1 be2b accumulated high levels of amylose and long-chains of amylopectin, further experiments were conducted to analyze the content and structure of RS in various rice genotypes. RS contents were measured using raw and gelatinized rice flour as well as mashed and un-mashed cooked rice  but considerably higher in raw flour prepared from be2b and be1 be2b . The RS contents of gelatinized rice flour prepared from WT cultivars and be1 were 0.9% and 0.5%, respectively, which were slightly higher than the values obtained from the raw rice flour of these genotypes. In be2b, the RS content of gelatinized rice flour was one-third (3.9%) of that of raw rice flour, whereas in be1 be2b, the RS content of gelatinized rice flour was three quarters (26.6%) of that of raw rice flour. The content of RS in un-mashed cooked rice prepared from Taichung 65, Kinmaze, and be1 was low  but considerably higher in un-mashed cooked rice prepared from be2b and be1 be2b . The RS content of be2b (EM10) in un-mashed cooked rice in the previous report  were higher than those of purified starch, with a shift toward higher molecular weight. In the elution profile of RS obtained from mashed cooked rice of be2b, the first peak was slightly higher than that of RS from raw rice flour but much lower than that of purified starch. The elution profile of RS obtained from mashed cooked rice of be2b did not show a clear second peak but rather a broad elution pattern. The third peak of RS obtained from mashed cooked rice of be2b was higher than that of purified starch but lower than that of RS from raw rice flour, and was obtained at a higher molecular weight at 158 min. In contrast to be2b, the elution pattern of be1 be2b purified starch showed a very high amylose peak at 107 min, broad peak at 120–140 min, low peak of amylopectin long chains at 144 min, and very low peak of amylopectin short chains at 160 min. The elution profile of RS obtained from be1 be2b raw rice flour showed three peaks; the area of the first peak was 30–40% of the area of purified starch, while the second and third peaks were higher than those of purified starch. The elution profile of RS obtained from be1 be2b mashed cooked rice showed only two peaks; the first peak was considerably reduced compared with that of purified starch, while the second peak (at 146 min) was high and appeared to be amalgamated.To determine RS structure, RS was purified from raw rice flour and mashed cooked rice of high RS lines, rch Fig. . In be2bBEI and BEIIb genes has been suppressed using anti-sense RNA . Less amylopectin branches in be1 be2b result in less non-reducing ends to serve as a substrate for SS. This leads to less consumption of ADP-glucose by SS isozymes including SSI. Then, an abundance of ADP-glucose in amyloplasts enhances amylose biosynthesis by granule-bound starch synthase I (GBSSI). The second possibility is that be1 be2b may have inherited some seed weight traits from Taichung 65. The seed weight of Taichung 65, the parental WT genotype of be1, was slightly (1.1-fold) heavier than that of Kinmaze, the parental WT genotype of be2b  flowered in late August to early September, whereas those of be1 (Taichung 65 background) and be1 be2b flowered in mid-August in Akita, Japan. Therefore, it is possible that the be1 be2b double mutant inherited flowering loci from be1 (Taichung 65 background). Earlier flowering time guarantees higher temperature during the seed development, which results in higher activity of starch biosynthetic enzymes, thus enhancing starch synthesis.The be1 be2b plants  + (be2b – WT)] were less chains with DP 10–20 and more chains with DP ≥ 40 . This indicates a synergistic effect due to the loss of both BE isozymes, suggesting that in the absence of BEIIb, the remaining BE enzymes (BEI and/or BEIIa) may generate amylopectin branches with DP 10–20; however, when both BEI and BEIIb are absent, BEIIa alone may not be able to compensate for their absence, and the number of branches with DP 10–20 may also be diminished. The average and median values of amylopectin chain length distribution , suggesting that amylopectin possessed extremely long double-helical branch chains in the absence of BEI and BEIIb.It is clear that the loss of BEIIb, among the three BE isozymes, drastically affects starch properties by decreasing amylopectin short chains  and be1 be2b likely resulted in an extremely high gelatinization temperature due to the significant increase in DP 25–36 in be1 be2b compared with that of be2b   gene  (Table be1 be2b harbors the Wxb (gbss1L) gene; this gene was inherited from either Kinmaze or Taichung 65 and contains a thymine instead of a guanine at the first nucleotide position of intron 1, resulting in a low level of GBSSI protein  lines, in which intermediate components, i.e., glucan molecules, are smaller than normal amylopectin but are enriched with longer average branch chains  and extra-long chain content (20–26.8%) of maize GEMS-0067 lines were much higher than those of rice be1 be2b, as measured by the gel filtration method  in transgenic barley resulted in a further increase in amylose content (99.1%), as measured by the iodine method . Strikingly, the be1 be2b double mutant showed 2.6–6.8-fold higher RS content than be2b , and the amylose may have formed long double helices. Of the true amylose content, 30–40% could not be degraded by digestive enzymes and therefore maintained the original molecular weight of amylose, while 60% was partially degraded to lower molecular weight amylose; hence, the peaks at 145 and 160 min, corresponding to amylopectin long chains and amylopectin short chains, respectively, were higher than those of purified starch. It is also possible that the partially degraded amylose, which was of a similar molecular weight as the long amylopectin chains, formed double helices, thus avoiding degradation by digestive enzymes. The RS sample of be1 be2b rice flour showed a small peak at 160 min. This peak is thought to represent the remaining long amylopectin chains within the size of one amylopectin cluster, and corresponds to RS2. The first peak of the RS structure of mashed cooked rice from be1 be2b was diminished similar to that of be2b. This suggests that almost all gelatinized amylose can be degraded to lower molecular weight amylose. The peak at 120–130 min corresponding to low molecular weight amylose from purified starch was lower in RS prepared from mashed cooked rice of be1 be2b than those of purified starch. It can be speculated that the low molecular weight amylose can also be degraded to an even lower molecular weight. The RS prepared from mashed cooked rice of be2b showed a clear peak at 158 min, which corresponds to long amylopectin chains within the size of one amylopectin cluster, as described above. By contrast, the RS prepared from mashed cooked rice of be1 be2b showed a large peak at 130–170 min, with the tallest peak at 144 min. We speculate that this peak represents the degradation products of amylose, long amylopectin chains spanning two or more clusters, and chains within one cluster. The gelatinized starch was incubated with digestive enzymes at 37 °C. It is possible that starch retrograded and formed double helices with a variety of different sized glucans, thus transforming into RS3, which is more resistant to degradation by digestive enzymes. The type of glucan molecules that form double helices in raw rice flour and retrograded starch can only be speculated at this moment, and further detailed analyses of RS structure are required. In addition, the actual RS content and structure in processed foods should also be determined in future studies.Detailed analyses of RS structure have been performed only in maize GEMS-0067 and be1 be2b rice mutant completely lacking both enzymes at the protein levels was generated. Seed weight of be1 be2b mutant was approximately 60% of the wild type and rather heavier than that of be2b mutant over the past three consecutive years. The be1 be2b mutant showed a decrease in intermediate amylopectin chains with DP10–20 and an increase in long amylopectin chains with DP > 21 compared with be2b. The amylose content of be1 be2b mutant (51.7%) was the highest among all pre-existing non-transgenic rice lines. The RS contents of mashed cooked rice and raw rice flour of be1 be2b mutant were 3-fold higher than those of be2b mutant. Gel-filtration analyses of starch treated with digestive enzymes showed that the RS in be1 be2b mutant was composed of the degradation products of amylose and long amylopectin chains. be1 be2b generated in this study must serve as a good material for an ultra-high RS rice cultivar that expected of lifestyle-related diseases such as diabetes all over the world.The Oryza sativa L.) be1 and be2b single mutant lines, EM557(be1)   possesses a single nucleotide mutation at the end of exon 10 of BEI gene, which introduces a stop codon into exon 11, resulting in no BEI protein (unpublished data). be2b possesses a single nucleotide mutation at the end of intron 9 of BEIIb gene, which produces incorrect mRNA . The be1 and be2b single mutants were crossed, and the F2 population was screened. Opaque seeds were analyzed by western blotting  at room temperature for 10 mins. The supernatant of each sample was loaded onto a 7.5% polyacrylamide gel and separated by SDS-polyacrylamide gel electrophoresis (PAGE). Proteins in the gel were blotted onto a polyvinylidene fluoride (PVDF) membrane and detected by anti-BEI , 8 M urea, 4% (w/v) sodium dodecyl sulfate (SDS), 5% (v/v) β-mercaptoethanol, and 0.05% (w/v) bromophenol blue. Samples were continuously agitated for 3 h, and then centrifuged at 15,000 rpm  varied from 5.0° to 40.0°, with a scanning speed of 0.3° s− 1  and analyzed by gel filtration chromatography  , according to the method of Fujita et al. . AmylopeRS contents were analyzed in cooked rice (mashed and un-mashed) and rice flour (raw and gelatinized). To prepare cooked rice, 1 g polished rice was washed twice with distilled water and cooked with 1.5 volumes of water (w/w of dry polished rice) in 15 mL plastic test tubes using a rice cooker . Then, 250 mg mashed and un-mashed cooked rice were transferred to new tubes. To prepare rice flour, polished rice was ground to a fine powder using a mortar and pestle, and sieved through a 100 μm colander. For the raw rice flour test, 100 mg rice flour was transferred to a tube. For the gelatinized rice flour test, 100 mg rice flour was boiled with two volumes of water (w/w) for 10 mins and the resulting rice pastes were ground again with a pestle and mortar. Samples were digested in 4 mL digestive enzymes using the RS assay kit  containing porcine pancreatic α-amylase (120 U) and amyloglucosidase (12 U) at 37 °C for 16 h by continuous shaking. RS contents were measured as described previously  at room temperature for 10 mins. The precipitate containing RS was washed with 40 mL of 50% ethanol. The precipitation and centrifugation steps were repeated once again. The RS pellet was dissolved in 100% dimethyl sulfoxide (DMSO) and boiled for 1 h, while shaking every 10 mins. The RS solution was centrifuged at 3000 rpm (937 g) at room temperature for 10 mins. The RS samples were precipitated by mixing with three volumes of 100% ethanol and incubated at − 30 °C for 1 h. The RS materials were centrifuged at 3000 rpm (937 g) at 4 °C for 10 mins, and the resulting pellet was suspended in 5 mL of 100% ethanol. After centrifugation at 3000 rpm (937 g) at 4 °C for 10 mins, the RS pellets were dried in a speed vacuum. The dried samples were ground to a fine powder, and 3–5 mg of the powdered RS material was gelatinized in 1 N NaOH and neutralized with 1 N HCl. Then, the gelatinized RS was debranched using Pseudomonas isoamylase  to perform gel filtration analysis, as described above.Mashed cooked rice and raw rice flour (1 g each) were digested in 40 mL digestive enzyme solution, as described above, and precipitated with an equal volume of ethanol. Samples were then centrifuged at 3000 rpm (937 p < 0.05). Significant difference of seed weight between be2b and be1 be2b was tested by Student’s t-test (p < 0.05).DSC, carbohydrate contents of each fraction by gel-filtration chromatography, RS contents, RSC by X-ray diffraction and percentages and average of chain length of amylopectin by capillary electrophoresis data were statistically analyzed by a one-way variance (ANOVA) and Tukey-Kramer method  in Kinmaze were due to difference of the lot of the column.


B, and taste receptors. Amiloride analogs with lipophilic substituents tend to be more potent modulators than amiloride itself. Adenosine, α‐adrenergic and dopamine receptors are most strongly modulated by amiloride analogs. In addition, for a few GPCRs, more than one binding site for amiloride has been postulated. Interestingly, the nature of the allosteric effect of amiloride and derivatives varies considerably between GPCRs, with both negative and positive allosteric modulation occurring. Since the sodium ion binding site is strongly conserved among class A GPCRs it is to be expected that amiloride also binds to class A GPCRs not evaluated yet. Investigating this typical amiloride‐GPCR interaction further may yield general insight in the allosteric mechanisms of GPCR ligand binding and function, and possibly provide new opportunities for drug discovery.The function of G protein‐coupled receptors (GPCRs) can be modulated by compounds that bind to other sites than the endogenous orthosteric binding site, so‐called allosteric sites. Structure elucidation of a number of GPCRs has revealed the presence of a sodium ion bound in a conserved allosteric site. The small molecule amiloride and analogs thereof have been proposed to bind in this same sodium ion site. Hence, this review seeks to summarize and reflect on the current knowledge of allosteric effects by amiloride and its analogs on GPCRs. Amiloride is known to modulate adenosine, adrenergic, dopamine, chemokine, muscarinic, serotonin, gonadotropin‐releasing hormone, GABA In molecular dynamics simulations, Gutiérrez‐de‐Terán et al2.50 is highly stable in the receptor's inactive conformation. The presence of the ion also avoids rotamer changes in two other highly conserved residues, Trp2466.48 and Asn2807.45. Interestingly, an active receptor conformation caused the site to contract to expel the sodium ion from this allosteric binding site. These calculations agree very well with radioligand binding studies on A2AAR  the receptor is predominantly in an inactive state.Solving the crystal structure of the adenosine Ae Figure A.13 The Figure 2.0 which iFigure 2.0 which i2.50 in a manner similar to sodium ions, as inferred from docking studies  or reduce  the negative allosteric effect of sodium ions on agonist binding. The D52A2.50 mutation also decreased the potency of amilorides with respect to ligand displacement, for example, an 18‐fold reduction in HMA's IC50 value for [3H]ZM‐241,385 binding. Conversely, a big potency gain was observed on the W246A6.48 mutant. HMA's IC50 value increased 25‐fold from 8.9 to 0.36 µM; a similar gain was observed for amiloride, from 63 to 2.6 µM. Apparently, this tryptophan residue, part of a so‐called activation micro‐switch,2AR (and possibly other GPCRs). Indeed, at the adenosine A3 receptor, the mutation of Trp2436.48 into Ala increased the affinity of HMA as well.In a subsequent study, Massink et al12 in Massink et al3H]ZM‐241,385 binding to both the wild‐type and W246A6.48 mutant receptors compared to other substituents and carbon chain elongations. Further derivatization of the phenylethyl moiety yielded 4‐ethoxyphenylethyl derivative 12l . Derivative 12l also showed an increased potency compared to that of HMA for the W246A6.48 mutant receptor, 19‐fold compared to WT for HMA in this study and 76‐fold for 12l.These findings fueled the ambition to design and synthesize novel amiloride/HMA derivatives. The 5′‐substitution of amiloride with phenylethyl , still different from the S3′ active state induced by full agonists.1‐2), as well as the partial‐agonist, stabilized state (S3). HMA competed with the sodium ions, reflected in its effects on both line broadening and chemical shift perturbations in the 23Na NMR binding isotherm.The conformational flexibility of the adenosine A2A receptors, through a number of orthogonal approaches. They all hint in the same direction, that is the amilorides compete with sodium ions at the allosteric sodium ion binding site in which Asp2.50 is the central amino acid. The evidence for other GPCRs is less exhaustive but suggests similar conclusions, which will be discussed below.In conclusion, the effects of amiloride and derivatives have been most extensively studied on adenosine A31A‐adrenergic receptor amiloride and its analogs benzamil, DMA, 5‐(N‐ethyl‐N‐isopropyl)‐amiloride (EIA), MIBA, and HMA increased the dissociation rate of antagonist [3H]prazosin, and the analogs with bulky lipophilic 5′‐moieties were more potent in doing so.1‐adrenergic receptors in which amiloride only showed a competitive interaction with antagonist [3H]prazosin binding but did not influence its dissociation rate.One of the first indications that amiloride inhibited the binding of orthosteric ligands at α‐ and β‐adrenergic receptors were found in 1987 by Howard et al,2‐Adrenergic receptors are allosterically modulated by amilorides as well. At rat, human, bovine, and porcine α2A‐adrenergic receptors amiloride increased the dissociation rate of the antagonists [3H]rauwolscine3H]yohimbine.2A‐adrenergic receptor, which was found for (N‐2‐aminoethyl‐N‐isopropyl)amiloride‐N‐ at the porcine receptor,3H]yohimbine, [3H]rauwolscine, and [3H]RX‐821,002 dissociation.+/H+ exchange protein, making it a GPCR selective amiloride. EIA, HMA, and MIBA were exceptionally strong negative allosteric modulators of antagonist binding, being 50‐ to 80‐fold more potent than amiloride in increasing the dissociation rate of [3H]yohimbine, showing that bulky lipophilic moieties at the 5′‐position of amiloride increase the allosteric potency at the α2A‐adrenergic receptor considerably. The apparent affinities of these amilorides were not correlating at all with their derived allosteric potencies in this study, cautioning to not confuse these two different pharmacological properties with each other.α3H]UK‐14 304 at the human α2A‐adrenergic receptor, with HMA having the largest effect.2A receptor agonist activation, because amiloride, DMA, and HMA decreased the potency of norepinephrine and UK‐41,304 in [35S]GTPγS binding experiments. This paradoxical behavior was in line with previous findings that amilorides displace the orthosteric ligand competitively from the α2A receptor in addition to their allosteric effects.2A‐adrenergic receptors amilorides bind to two different sites, namely the orthosteric site and an allosteric sodium ion site. Howard et alIn contrast to their effect on antagonists, amiloride, DMA, and HMA decreased the dissociation rate of agonist rauwolscine binding, whereas the guanidino‐substituted amiloride CBDMB decreased it.At the α1‐ and β2‐adrenergic receptors amiloride displaced the antagonist [125I]iodocyanopindolol competitively, because their binding was mutually exclusive.1‐adrenergic receptor.1‐adrenergic and the adenosine A2A receptor are the most similar of the solved GPCR crystal structures with such a site.The interaction of amiloride with β‐adrenergic receptors has only been studied by Howard et al in 1987. At both the βtagonist 5Iiodocya43H]INCB3344 binding to the orthosteric site and antagonist [3H]CCR2‐RA‐[R] binding to the intracellular site.125I]CCL2. Amiloride, benzamil, MCGMA, and phenamil did however not displace any of these radioligands.Amiloride interactions with the chemokine receptor family have only been studied by Zweemer et al3H]INCB3344, the intracellular antagonist [3H]CCR2‐RA‐[R], and the orthosteric agonist [125I]CCL2 induced by HMA indicate a noncompetitive allosteric interaction. Remarkably, the dissociation rate of the agonist [125I]CCL2 increased more (9.7‐fold) than of the antagonists (1.25‐ and 1.36‐fold) in the presence of HMA. Saturation binding assays revealed that HMA had a mixed competitive/noncompetitive interaction with the orthosteric antagonist [3H]INCB3344, because the radioligand's Bmax value decreased and KD value increased. HMA had a purely noncompetitive interaction with the intracellular antagonist [3H]CCR2‐RA‐[R], causing a decrease in this radioligand's Bmax value only.The increased dissociation rates of the orthosteric antagonist SCH‐23,390 at the human D1 dopamine receptor and [3H]spiperone at the human D2(short), D2(long), D3, and D4 dopamine receptors. As with other GPCRs, the analogs with lipophilic moieties at the 5′‐position were more potent than amiloride itself. At the D1, D2(short), D2(long), and D3 dopamine receptors the amilorides displaced the orthosteric antagonist [3H]spiperone in both a noncompetitive and competitive manner. This may indicate binding of the amilorides to both the orthosteric and allosteric sites. The authors suggested a positive homotropic cooperativity due to a high Hill coefficient of the effect curves, i.e. amilorides binding at the allosteric site enhance the binding of amilorides to the orthosteric site.The general trend amongst the dopamine receptor subtypes is an increase of the dissociation rate of orthosteric ligands by amiloride and its analogs, as found in a comprehensive study of the effect of amiloride, benzamil, and MIBA.2 receptor complemented results from other studies, in which similar dissociation rate‐increasing effects and mixed competitive/noncompetitive behavior were found. Amiloride competed with and increased the dissociation rate of antagonists [3H]spiperone and [125I]epidepride binding.3H]spiperone at both the rat2 dopamine receptors, and of these amilorides HMA was the most potent amiloride  of GnRH receptor activation by triptorelin and the endogenous ligand GnRH. Furthermore, it was demonstrated that the GnRH receptor harbors a second allosteric site other than the amiloride binding site, because HMA did not compete with FD‐1, another allosteric modulator of the GnRH receptor with a distinct chemical structure.The gonadotropin‐releasing hormone (GnRH) receptor, also known as luteinizing hormone‐releasing hormone receptor, is targeted by various drugs in the market for the treatment of sex‐hormone‐dependent diseases such as breast or prostate cancer.73H]pirenzepine binding at the muscarinic M1 and [3H]N‐methylscopolamine binding at the muscarinic M2 and M3 receptors.3 receptor‐mediated smooth muscle contractionmax) was reduced.3 receptor,3H]N‐methylscopolamine in a competitive manner.2 and M3 receptors sodium ion binding was not detected,2.50 in the muscarinic M3 receptor was suggested, keeping the receptor in an inactive state.2.50 in the muscarinic M2 receptor into the cytosol was also simulated in molecular dynamics calculations.Amiloride effects have been observed on muscarinic receptors in rat tissue preparations. Benzamil and HMA inhibited [83H]8‐OH‐DPAT binding at the rat 5‐HT1A receptor.3H]5‐carboxamidotryptamine binding at the human 5‐HT1B receptor.50 value (200 µM) compared to its Ki in inhibiting [3H]5‐carboxamidotryptamine binding (13 µM).3H]serotonin binding was inhibited by HMA at the rat 5‐HT1C receptor and by benzamil and HMA at the rat 5‐HT2 receptor.1B receptor2B receptor,Amiloride and analogs have been found to inhibit orthosteric ligand binding to serotonin receptors. Benzamil inhibited agonist [92) receptor in complex with the subtype‐selective antagonist N‐ethyl‐2‐[(6‐methoxy‐pyridin‐3‐yl)‐(toluene‐2‐sulfonyl)‐amino]‐N‐pyridin‐3‐yl‐methyl‐acetamide (EMPA) at 1.96 Å resolution.2.50, better than in an earlier crystal structure of this receptor.2AR.Suno et alThe receptors discussed above all belong to the class A family of GPCRs. Finally, we should like to discuss the evidence, admittedly limited and inconclusive, of amiloride interaction with two class C receptors.10B receptor is activated by γ‐aminobutyric acid (GABA) and it's derivative, baclofen (β‐4‐chlorophenyl‐GABA). This receptor is coupled to potassium and calcium channels through Gi/Go proteins.2+‐free medium. The effect of baclofen (10 µM) was blocked by amiloride (200 µM), which increased the frequency of discharges and slightly reduced their amplitude when applied alone. These effects persisted upon wash‐out and baclofen remained ineffective on the discharges until 30 to 60 minutes after a switch to amiloride‐free medium. Analogs of amiloride, DMA and MIBA, showed a similar mode of action, whereas they were at least twice as potent than amiloride in preventing the effect of baclofen on neocortical spontaneous discharges. DMA alone increased the discharge frequency and slightly reduced the amplitude in a concentration of 100 µM. Analogs lacking the guanidine moiety were ineffective. The authors explicitly stated, however, that an indirect effect of the amilorides via functional antagonism of coactivated adenosine A1 receptors cannot be ruled out.The GABA1150 value of 0.87 ± 0.20 mM. A study of the specificity towards the response mediated by the human sweet taste receptors showed that the suppression of receptor activity by amiloride is specific for hT1R2/hT1R3. Inhibitory effects of lactisole, a known hT1R2/hT1R3 inhibitor, and amiloride on the cellular response to aspartame were examined in cells expressing hT1R3 mutants (hT1R2/hT1R3‐A733V and hT1R2/hT1R3‐F778A). Lactisole was less active on the mutants, whereas amiloride did not show such a differential effect. These results suggest that the binding site of amiloride is distinct from that of lactisole.The heterodimeric T1R2 and T1R3 taste receptor acts as a sweet taste sensor with multiple binding sites for sweeteners.12It is increasingly realized that GPCRs have multiple binding sites that may influence each other in allosteric ways. The surge in crystal structures over the last decade has taught that ligands, including marketed drugs and clinical candidates, may have very different binding sites indeed. From this review, it has become obvious that the sodium ion binding site is yet another receptor domain to tune the ligand response, and that amiloride and its derivatives are prototypic small molecules that intervene with that site.Does this offer options for future drug discovery? One might argue that the generic nature of the site and the evolutionary conservation of the amino acids aligning it are a drawback rather than an opportunity. In that view amilorides are another class of chemical probes that serve to unveil the complexities of GPCR functioning. A recent development, however, may prove this hypothesis wrong.4 (LTB4) receptor BLT1 in complex with antagonist/inverse agonist BIIL260 has recently been reported.2.50, with which it forms a salt bridge. Hydrogen bonds are present with the hydroxyl groups of Ser1063.39 and Ser2767.45  binding , and, less convincingly, on class C receptors (GABAB and T1R2/3 receptors).This review summarizes the current knowledge of the allosteric effects of amiloride and its analogs on GPCRs. Allosteric effects of amilorides have been found on class A GPCRs  effect on the dissociation of orthosteric ligands.2.50 for amiloride binding, just as for sodium ions. In the docking studies performed, the binding mode of amiloride and HMA was predicted in the sodium ion site of the adenosine A2A receptor crystal structure and a CCR2 chemokine receptor homology model. The positively charged guanidinium group has a strong salt bridge interaction with Asp2.50, underlining the great importance of this residue for amiloride binding as found before in mutagenesis studies. Trp6.48 interacts with amilorides as well, in some cases hampering and in other cases accommodating amiloride binding. These interactions of amilorides with the amino acids of the sodium ion site are of interest because these have been shown to be important in receptor functionality, with Asp2.50 and Trp6.48 as most noticeable examples. Mutation of Asp2.50 silences receptor activation in many GPCRs.6.48 is noteworthy as part of an “activation micro‐switch” between the active and inactive states of GPCRs,2A receptor amiloride and HMA seem to toggle this amino acid from one rotamer to another. Although not very likely, amilorides may also influence the oligomerization of class A receptors. The interface for receptor dimerization often involves transmembrane domains 4 and 5 that are not part of the sodium ion binding site. In some cases, however, other domains such as TM6, which also flanks the sodium ion binding site, play a role.Another general “rule” is the importance of Asp50 or Ki), their  effect on the dissociation of orthosteric ligand (koff/koff(control)), and their potency for these dissociation effects (EC50). This information also helps to understand whether the interaction of a particular amiloride with an orthosteric ligand is competitive or noncompetitive. If amiloride inhibits orthosteric ligand binding but does not affect its dissociation rate, the binding is mutually exclusive and the interaction is defined as competitive. If the dissociation rate is changed though, both the orthosteric ligand and amiloride can bind to the receptor at the same time and the interaction is deemed noncompetitive. Another way to confirm a noncompetitive interaction is by showing insurmountability of the inhibiting effect in radioligand saturation (Bmax decrease) or functional assays (Emax decrease), as discussed for the chemokine CCR2, muscarinic M3, and gonadotropin‐releasing hormone receptor. However, these assays have been conducted far less than dissociation assays in amiloride research so we did not include these in Table In contrast with these general “rules,” differences in the affinities, potencies, and modulatory behaviors of amilorides can be quite outspoken, even between receptors where the sodium ion site harbors the same amino acids . To appreciate these differences it is important to discern between the different properties by which the allosteric effect of amilorides on orthosteric ligand binding may be described. In Table In some cases, amilorides behave only as purely competitive inhibitors, whereas in other cases they behave as noncompetitive negative modulators, and a mixed behavior has also been observed. For some receptors the cause for mixed competitive/noncompetitive behavior was explained by a tendency of amilorides to bind both orthosteric and allosteric sites, but also in these cases the observed effect may be caused by binding in the sodium ion site only, where the competitive “fraction” of the allosteric effect is caused by either an overlap of binding with the orthosteric site or a conformational change of the receptor by amiloride binding. The latter option is quite likely from the structural evidence provided by the recently elucidated crystal structures.2A‐adrenergic and adenosine A3 receptors. Thus, in some cases, amilorides may also influence receptor signaling after agonist activation with consequences for effector bias or functional selectivity, for instance between G protein and β‐arrestin signaling.2B‐adrenergic receptor different amilorides even exhibit both positive and negative modulatory effects on the same orthosteric probe. Some of the differences in affinity and modulatory effect may be caused by differences in the sodium ion site itself, but the substantial conservation of the sodium ion site residues amongst GPCRs makes it more likely that these differences are caused by variations in receptor conformations.At some of the discussed receptors, the modulatory effect by amilorides is probe‐dependent, which has been described in other cases of allosteric modulation as well.4 receptor in complex with BILL260 (Figure Clinical application of amilorides targeting GPCRs is not self‐evident due to their micromolar affinities and lack of selectivity. However, it may be feasible to synthesize amiloride analogs with variations on the 5′‐position to improve their affinity and selectivity for GPCRs. In that sense, the recent structure elucidation of the BLT1/leukotriene B0 Figure  is notew


E‐diaminoalkene and vice versa. Remarkably, the forward and the reverse reaction, which are both reversible, follow two completely different reaction pathways. Establishing such unprecedented property in this type of processes was possible by developing a novel synthetic route towards the starting dication. All intermediates involved in both the forward and the backward reactions were comprehensively characterized by a combination of spectroscopic, crystallographic, electrochemical, spectroelectrochemical, and theoretical methods. The presented synthetic route opens up new possibilities for the generation of multi‐pyrrolinium cation scaffold‐based organic redox systems, which constitute decidedly sought‐after molecules in contemporary chemistry.This work presents a stepwise reversible two‐electron transfer induced hydrogen shift leading to the conversion of a bis‐pyrrolinium cation to an  N,N′‐Ethylene bridged bis‐2‐aryl pyrrolinium cations are reported in this work, which (a) undergo stepwise reversible two‐electron reduction coupled two hydrogen transfer to form E‐diaminoalkenes; and (b) have non identical pathways for reduction and oxidation. In compN,N′‐ethylene‐bridged bis‐2‐aryl pyrrolinium cations R3, through the reaction of ethylenediamine with aryl‐isopropyl‐ketone R1, to get first N,N′‐ethylene‐bridged bis‐imine R2 . Subsequent sequential one‐pot reactions of R2 with freshly prepared lithium diisopropylamide (LDA), isobutylene oxide, and triflic anhydride afforded N,N′‐ethylene‐bridged bis‐2‐aryl pyrrolinium cations, R3, with triflate counter anions, as white crystalline solids  in CH3CN/0.1 M NBu4PF6 . Upon one‐electron reductions to compounds H4 and Me4 new bands appear in the visible region at 346 and 478 nm and at 347 and 492 nm, respectively . TD‐DFT calculations confirm the origin of these bands from radical‐containing species . No other bands were observed beyond 500 nm for the one‐electron reduced forms of either compound. This fact likely points to a class I nature of these formally mixed‐valent compounds according to the Robin–Day classification,H4 and Me4 display featureless isotropic electron paramagnetic resonance, EPR spectra at 295 K in fluid solution with g‐values of 2.0028 and 2.0027  and six 1H nuclei of the CMe2 moiety with splittings ranging from 5 to 10 MHz for H4, and similar values for Me4. Spin density calculations at the PBE0/def2‐TZVP level of theory showed that the spin is largely located on one side of the molecule , thus also indirectly corroborating the class I nature of these mixed‐valent compounds. In the absence of a crystal structure, UV/Vis/EPR spectroelectrochemistry provides unequivocal evidence for the proposed chemical and electronic structures of the one‐electron reduced species H4 and Me4, and proves that no H‐atom abstraction takes place in the one‐electron reduced species. The second reduction step turned out to be irreversible on the spectroelectrochemical timescale for both compounds and we were not able to generate spectra that can be readily assigned to any particular species. Due to singlet–triplet population equilibria and an increased number of resonances. A spin‐coupled dinuclear EPR species has an intrinsically weaker signal than a mononuclear species at equal concentration. Furthermore, if the dinuclear species takes on different conformations in solution, the spectra could be further broadened, as both exchange and magnetic dipolar interactions heavily depend on interspin distance. Thus, it is much harder to detect a spin‐coupled species than it is to observe an S=1/2 species during an EPR SEC experiment, particularly if the spin‐coupled species is unstable. This fact supports a double H‐atom transfer reaction from the initially formed unstable diradical, that is, [H5] and [Me5] species .In order to isolate the one‐electron as well as the two‐electron reduced products, compounds H3/Me3 and KC8 led to the formation of E‐diaminoalkenes H6 and Me6, in 65 and 73 % yields, respectively  and 1.338(3) Å long, respectively, which falls in the range of a typical C=C double bond distance , the influence of the substituents on the redox potentials of H6 and Me6 are also marginal. The difference in the redox potentials for H3/Me3 (ΔE1/2=0.21 V for H3 and ΔE1/2=0.19 V for Me3) is lower compared to the difference for H6/Me6 (ΔE1/2=0.67 V for H6 and ΔE1/2=0.65 V for Me6) pointing to a larger thermodynamic stability for the one‐electron oxidized forms of H/6Me6 as compared to the one‐electron reduced forms of H/3Me3. The difference is likely related to the presence of a conjugated bridge in H/6Me6. The redox potentials of H6/Me6 clearly point towards an exceptionally electron‐rich nature of these E‐diaminoolefins. Chemical oxidations were carried out with AgOTf in a 1:1 reaction affording the corresponding radical cations H7 and Me7, respectively, in 72 and 79 % yields  and 1.380(3) Å, which are lengthened compared to the parent E‐diaminoalkenes H6 (1.332(2) Å) and Me6 (1.338(3) Å) , and two sets of two equivalent 1H nuclei (AH1=32.3 MHz and AH2=10.76 MHz). Spin‐density calculations  show the spin‐density to be predominantly located on the vinylic part of the bridge and the two connected nitrogen atoms, with a smaller but significant contribution from the H atoms at the C5 carbons. As mentioned above, the second oxidation steps for both H6 and Me6 appear irreversible in their cyclic voltammograms. This transformation was studied in more detail for Me6. Following the second electrochemical oxidation, two new reduction signals are observed in the voltammograms at dramatically shifted negative potentials, which appear to match the redox potentials of Me3 . These observations point to the operation of either an EC  or a more complex mechanism. The peaks at the large negative potentials are semi‐reversible and account for the significantly decreased cathodic current in the redox transition at 0.06 V (for a fully reversible redox process the anodic and cathodic peak currents have the same height). These new redox responses arise from reductions of the dicationic species formed from [Me8] . Notably, two distinct sets of spectroscopic changes were observed on monitoring the second oxidation step of this compound via UV/Vis spectroelectrochemistry. The final spectrum generated after the complete second oxidation looks somewhat similar to Me3. Upon moving the applied potential back to the starting potential, the initial spectrum of Me6 was not regenerated. However, applying more negative potentials which cover also the newly generated reduction steps, and then moving the potential back to the starting point recovered a spectrum corresponding to Me6. These experiments, thus, point to the operation of rather fast chemical reactions during the redox‐induced conversion of Me6 to Me3 via Me7 on timescales which can be monitored via UV/Vis spectroelectrochemistry. The aforementioned signals provide the first evidence for the operation of bistability in these systems. Without any direct experimental evidence for the presence of [H8] and [Me8], the former was investigated computationally. The calculations for [H8] show that the bridge's C−C bond length increased from 1.388 in H7 to 1.466 Å, while the bridge's C−N bond lengths shortened from 1.330 in H7 to 1.270 Å in [H8] , which supports the bis‐iminium resonance structure of [H8] . The calculations show that H3/Me3 are slightly more stable than [8H]/[8Me] species, while H6/Me6 are slightly more stable than [H5]/[Me5]. When H3/Me3 and H6/Me6 were mixed in a 1:1 ratio in THF no reaction took place, which is in contrast to NHC‐CAAC derived triazaalkenes, which undergo comproportionation reactions.H3/Me3 and H6/Me6 can be considered globally reversible even though the second redox events in each direction are individually electrochemically irreversible.Thus, the conversion of N‐centers are bridged by a ‐CH2CH2‐ linker. This novel synthetic route provides a very convenient access to multi‐CAAC‐scaffold systems without the need of using free CAACs as starting materials. Notably, with this synthetic route an extensive substrate scope can be realized with substitution derivatives of both the aryl moieties and the diamine bridge. The reported procedure, thus, holds exceptional promise for synthesizing next generation multi‐CAAC scaffold containing compounds. N,N′‐Ethylene‐bridged bis‐2‐aryl‐pyrrolinium cations undergo a stepwise two‐electron reduction, yielding the E‐diaminoalkenes. The first one‐electron reduction affords the radical cation, which undergoes a second one‐electron reduction to a diradical. The latter, subsequently, and immediately abstracts two hydrogen atoms from the ethylene bridge forming the electron‐rich E‐diaminoalkenes. Upon a two‐electron oxidation, the starting bis‐2‐aryl‐pyrrolinium salts can be recovered. This process is also sequential: the first oxidation leads to a crystalline radical cation and subsequently the corresponding dication. The latter undergoes a 1,3‐hydrogen shift yielding the bis‐2‐aryl‐pyrrolinium salts. Overall, a total of four C−H bonds are cleaved, and four C−H bonds are formed reversibly in response to redox processes. All intermediates in the forward and the reverse cycle have been thoroughly studied by experiment and theory. Unlike many reversible electron transfer‐induced bond activation processes, which occur through common chemical intermediates, the events described in this study do not follow this standard paradigm. Instead, the chemical pathways in the reduction and oxidation events are completely non‐identical. The results unequivocally show that such multi‐CAAC‐scaffold systems are capable of multi‐electron transfer involving chemical reactivity that is traditionally the realm of transition metal complexes. To the best of our knowledge, this is an unprecedented observation in electron transfer induced bond activation reactions. The novel systems presented here can therefore not only mimic transition metal complexes in undergoing reversible multi‐electron transfer‐induced chemical reactions (EEC mechanisms), but also do so in a completely unique way.In conclusion, we have synthesized bis‐2‐aryl‐pyrrolinium salts, where the The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Cerebrospinal fluid (CSF) flow in disease has been investigated with two‐dimensional (2D) phase‐contrast magnetic resonance imaging (PC‐MRI) in humans. Despite similar diseases occurring in dogs, PC‐MRI is not routinely performed and CSF flow and its association with diseases is poorly understood.To adapt 2D and four‐dimensional (4D) PC‐MRI to dogs and to apply them in a group of neurologically healthy dogs.Six adult Beagle dogs of a research colony.Prospective, experimental study. Sequences were first optimized on a phantom mimicking small CSF spaces and low velocity flow. Then, 4D PC‐MRI and 2D PC‐MRI at the level of the mesencephalic aqueduct, foramen magnum (FM), and cervical spine were performed.CSF displayed a bidirectional flow pattern on 2D PC‐MRI at each location. Mean peak velocity (and range) in cm/s was 0.92 (0.51‐2.08) within the mesencephalic aqueduct, 1.84 (0.89‐2.73) and 1.17 (0.75‐1.8) in the ventral and dorsal subarachnoid space (SAS) at the FM, and 2.03 (range 1.1‐3.0) and 1.27 (range 0.96‐1.82) within the ventral and dorsal SAS of the cervical spine. With 4D PC‐MRI, flow velocities of >3 cm/s were visualized in the phantom, but no flow data were obtained in dogs.Peak flow velocities were measured with 2D PC‐MRI at all 3 locations and slower velocities were recorded in healthy Beagle dogs compared to humans. These values serve as baseline for future applications. The current technical settings did not allow measurement of CSF flow in Beagle dogs by 4D PC‐MRI. The choroid plexus as the sole source of CSF is debated, but its constant production creates a pressure that dictates the direction of the fluid flow through the ventricular system to the subarachnoid space (SAS).Phase‐contrast magnetic resonance imaging (PC‐MRI) is a noninvasive method to quantify flow velocities and to characterize flow patterns. In two‐dimensional (2D) PC‐MRI, a bipolar gradient with equal magnitude induces a net phase shift of moving protons, which is proportional to their velocity. Cardiac gating encompasses bidirectional flow measurements during a cardiac cycle and velocity data are presented as coded grayscale values with negative values displayed as dark and positive values as bright pixels on the phase images, depending on flow direction and velocity.Toy breed dogs and brachycephalic dogs in particular show abnormalities of CSF spaces in MRI similar to humans.The aim of this prospective study is to optimize 2D and 4D PC‐MRI for the smaller CSF spaces and slower flow velocity in dogs, to provide normative values from neurologically healthy dogs with normal appearance of CSF spaces on MRI.22.1The 2D and 4D PC‐MRI sequences were adapted for the anatomical differences in dogs compared to humans on a phantom mimicking the smaller CSF spaces and slower CSF flow velocity. A 20 × 15 × 15 cm plastic box was filled with gelatin, embedding 3 infusion lines with a diameter of 2.5 mm, 1.5 mm, and 1.0 mm as straight segments with a U‐turn. The infusion lines were connected to a medical perfusion pump outside the MRI room . The perfusion syringes were filled with water with a similar viscosity to CSF. The liquid flowed from the small to the large diameter infusion lines to reduce the effect of higher resistance in the small lines. Flow velocity was calculated with a linear stream‐profile conversion table (volume to flow velocity). Measurements were repeated when the velocity was gradually reduced.The phantom was placed in a 3T magnetic resonance system  with a SENSE‐head and neck coil. A cardiac frequency with a heartbeat of 100 bpm was simulated by a physiological simulation setting on the Philips software. 3D T1‐ and 3D T2‐weighted images were performed as planning sequences. The 2D and 4D PC‐MRI sequences were aligned perpendicular to the flow in the perfusion lines. First to be tested were sequence parameters for better signal‐to‐noise ratio (SNR) with a voxel size of 1 × 1 × 5 mm with different numbers of acquisitions (NSA 1 and 2), field of view , and flip angle (15° and 20°). The measurements were performed once for each parameter and subjectively evaluated for good SNR. Finally, to find the closest velocity encoding the speed in the infusion lines, without getting aliasing but still having good SNR, different velocities encoding from 10 cm/s to 1 cm/s were tested. Additionally, for the 4D PC‐MRI sequence, both available accelerating techniques, kt‐BLAST and SENSE, were tested for their effect on scan time and signal.The image acquisition and assessment of image quality was performed by the first author, a PhD student with a Master's degree in veterinary medicine under supervision and advice of a senior radiologist (ECVDI Diplomate). Technical support concerning the 4D sequence was provided by a senior scientist in biomedical engineering with expertise in motion encoding.Concerning the 2D sequences, further support was provided by a Professor of Radiology and Biomedical Engineering with a research focus on blood and CSF flow dynamics using flow‐sensitive MRI techniques.A decision of sufficient image quality was made based on the velocity‐to‐noise ratio, that is, the ability to detect a change of gray scale value in time of a pixel undergoing a phase shift. The final decision on acquisition parameters was made in consensus between first and last authors.2.2The study was conducted with 6, adult, purpose‐bred, research Beagle dogs. Sex was equally distributed. The dogs were also used in another parallel research study, which did not interfere with our research aims and allowed the reduction of the number of experimental animals used (3R reduce). The study population had a mean age of 7.42 years  and a mean bodyweight of 13.6 kg . All dogs were declared healthy on clinical neurological examination and showed no structural brain abnormalities on MRI  titrated to effect. The tracheae of the dogs were intubated and sevoflurane  in O2 and air (Fi O2 = 60%) was administered to effect, depending on the plane of anesthesia, with an end‐tidal concentration between 1.8 and 3.3 vol%. The dogs were mechanically ventilated  with a pressure‐controlled mode (8‐11 cm H2O). The respiratory rate was adjusted to achieve an end‐tidal CO2 of 35 to 42 mm Hg (4.66‐5.59 kPa). Acetated Ringer's solution  5 mL/kg/h) was administered during anesthesia. Body temperature was maintained between 37.5°C and 39°C and heart rate was maintained between 70 and 110 beats per minute. If cardiac frequency increased to over 110 bpm, a fentanyl  bolus of 2 μg/kg IV was administered and repeated, if necessary. Anesthesia monitoring included cardiovascular and respiratory variables, which were measured continuously and recorded by a multiparameter monitor  that included pulsoxymetry, noninvasive blood pressure measurement, capnography, inhalant gas analysis, spirometry, and vectorcardiography. Mean blood pressure was kept above 65 mm Hg while heart rate was kept below 110 beats per minute. Anesthesia depth was adapted as necessary.The dogs were fasted prior to anesthesia and premedicated with 0.2 mg/kg methadone  intramuscularly. An intravenous (IV) catheter  was placed aseptically in the cephalic vein. Dogs were preoxygenated with a facemask with a flow of 4 L/min O2.3All dogs were scanned in sternal recumbency with the head slightly extended  and elevated so that no pressure developed on the jugular veins.MRI acquisition was performed by the first author under supervision of a board certified senior radiologist. To exclude intracranial morphological abnormalities and for alignment of PC‐MRI sequences, 3D T1‐weighted  and 3D T2‐weighted  sagittal sequence were acquired.The first PC‐MRI sequence performed was a mid‐sagittal PC‐MRI sequence to visualize pulsation of the CSF flow and to identify possible obstructions of CSF flow. The parameters for CSF measurements in the sagittal plane were as follows: TR, 21 ms; echo time, 6.9; flip angle, 10°; FOV, 250 × 226 mm; slice thickness, 10 mm; voxel size, 0.98 × 1.4 mm; cardiac gating, retrospective; heart phases, 30; and velocity encoding, 5 cm/s. Velocity encoding was reduced if the signal was low or increased if aliasing occurred.Further 2D PC‐MRI sequences were acquired at 3 locations perpendicular to the flow direction Figure . The fir2.4The postprocessing of the 2D data set was performed on an external workstation . The 4D PC data sets were processed by the GT‐flow software .Image analysis was performed by a single investigator (MC) who was instructed and supervised by a board certified senior radiologist. For all 3 locations, regions of interest (ROIs) were placed for each dog into CSF spaces using magnitude and phase‐contrast images in parallel in order to match information of flow with the anatomy. ROIs were propagated and manually adjusted to each of the 32 resulting phase images. One ROI was drawn at the level of the mid‐ampulla of the mesencephalic aqueduct. On images of the caudal aspect of the occipital condyles and the cranial cervical spine, 3 ROIs were placed in the SAS surrounding the spinal cord: 1 ROI in the dorsal SAS and 2 ROIs in the ventral SAS to avoid areas where no flow signal was present and to exclude the basilar artery or spinal artery from the velocity measurements  since average flow underestimates velocity measurements.On the acquired data from the 6 dogs, all ROIs were placed 3 times at least 3 days apart and the mean of the peak velocity is reported.Postprocessing of the 4D data set could not be performed due to high velocity noise and low SNR on the phase images, which led to undetectable velocity in CSF spaces.2.5Descriptive analysis was performed with all values obtained. The data were expressed as mean ± SD or median with range.P ≤ .05, R 1.0.143 2009‐2016).Intraobserver variability was assessed by calculating the SEM based on 3 measurements on each location of the 2D phase images. The statistical distributions of the velocities were visually examined using histograms and quantile plots and tested for normality using the Shapiro‐Wilk test ; echo time, shortest ; flip angle, 15°; FOV, 150 x 150 mm; slice thickness, 5 mm; voxel size, 0.58 × 0.83 mm; NSA, 1; heart phases, 32; cardiac gating, retrospective. Sequence acquisition time was no longer than 3 to 4 minutes. With these parameter settings, flow was visible on the phase images in all infusion line diameters (2.5‐1 mm) with velocities under 1 cm/s. In the phantom test, the calculation of the theoretical estimated velocities in tubes of different diameter corresponded with the measured 2D PC‐MRI velocities. Sufficient velocity‐to‐noise ratio for the volumetric measurements with the 4D PC‐MRI sequence was achieved using the following parameters: TR, shortest; echo time, shortest; flip angle, 5°; FOV, 140 × 140 × 40 mm; voxel size, 1 × 0.99 × 1 mm; stacks, 1; slices, 40; slice orientation, coronal; NSA, 1; heart phases, 16; cardiac gating, retrospective, and acceleration technique SENSE. Velocity encoding was selected at 10 cm/s in all spatial directions (RL‐AP‐FH). Alteration of these parameters to improve velocity‐to‐noise ratio and SNR, such as reduction of voxel sizes for detection of flow in small spaces or lowering velocity encoding, increased the time of the sequence to >14 minutes despite the use of SENSE acceleration. Flow signal could not be obtained on the phase images with acceleration of kt‐BLAST. A flow rate of >3 cm/s was visible with a low SNR within the 2 larger infusion lines (2.5 mm and 1.5 mm) but could not be detected in the smallest line (1 mm). Flow rates below 3 cm/s could not be measured with the 4D PC‐MRI sequence.3.2The 2D PC‐MRI imaging enabled the assessment of CSF flow in every dog. The acquisition time of CSF flow measurements at 3 locations was between 3 and 4 minutes, and heart rate was between 70 and 110 bpm. A bidirectional flow pattern was visible on the sagittal phase images in the dorsal and ventral SAS from the level of the FM to the cervical spine, but not cranial to it. On transverse sequences, a bidirectional flow pattern was visible in all ROIs except for the dorsal SAS of C1‐C2 of 2 dogs, where no signal could be detected. Velocity encoding for measurements at the level of the FM and the cervical spine was set at 5 cm/s for all dogs and then reduced to 3 cm/s at the level of the aqueduct for better SNR. The shortest TR and TE were calculated by the scanner according to the velocity encoding and was for all transverse 2D PC‐MRI sequences 16 to 17 ms and 10 ms, respectively. The acquisition time for T1 weighted, T2 weighted, and 2D PC‐MRI was 32 minutes; the 4D PC‐MRI sequences took additional 14 minutes.P ≤ .05). The mean peak velocity of all 6 dogs for the different locations is listed in Table The CSF peak velocities resulting from all 3 measurements among all dogs were normally distributed (Shapiro‐Wilk test Intraobserver variability for the manual placement and adjustment of the ROI on the 2D phase images was ≤0.68 cm/s for the SAS of FM and cervical spine and ≤0.13 cm/s at the level of the mesencephalic aqueduct. The 4D PC‐MRI images showed marked velocity noise, to the point that CSF spaces were not detectable anymore. Hence, CSF flow velocity measurements and visualization of flow were not possible with the 4D PC‐MRI sequence in any of the dogs.4The present study provides normative data of CSF flow in a group of middle‐aged neurological healthy dogs as baseline for comparison in future studies. Ventricular and subarachnoid CSF space abnormalities are common findings in dogs; however, their relationship to alterations of CSF flow is not well understood and poorly investigated.PC‐MRI is routinely used as a method to quantify CSF flow in humans but needs adaptation for use in veterinary patients, because of the small CSF spaces,Therefore, the results presented here are limited to 2D PC‐MRI with CSF flow velocities measured at 3 different locations. These locations play an important role in the pathomechanism of common disease processes such as stenosis, hydrocephalus and syringomyelia.At each of the 3 locations, the peak velocities measured in the study population of middle aged healthy Beagle dogs were lower than those measured in humans. This is especially true for the level of the mesencephalic aqueduct, where values ranging from 3 to 7 cm/s were obtained in healthy human participants.The anesthesia protocol for this study was chosen because of the relatively low effects on the cardiovascular system. Of note, inhalational anesthetic agents could have a cerebral vasodilating effect and might increase cerebral blood volume and hence intracranial pressure, if the systemic blood pressure is below 60 mm Hg.The diagnostic value of PC‐MRI in dogs needs to be investigated in future studies. Currently, it is unclear if the method will help to identify sites of stenosis or explain other alterations of CSF spaces. Further future applications of CSF flow measurements are the assessment of the intracranial compliance.Authors declare no conflict of interest.Authors declare no off‐label use of antimicrobials.Approved by the Cantonal Veterinary Office of Zurich according to the Swiss national animal protection law with the animal permission number ZH2016/17.Authors declare human ethics approval was not needed for this study.